Synthesis, in vitro biological activity, hydrolytic stability and docking of new analogs of BIM-23052 containing halogenated amino acids

Article abstract of DOI:10.1007/s00726-020-02915-3

One of the potent somatostatin analogs, BIM-23052 (DC-23-99) d-Phe-Phe-Phe-d-Trp-Lys-Thr-Phe-Thr-NH₂, has established in vitro growth hormone inhibitory activity in nM concentrations. It is also characterized by high affinity to some somatostatin receptors which are largely distributed in the cell membranes of many tumor cells. Herein, we report the synthesis of a series of analogs of BIM-23052 containing halogenated Phe residues using standard solid-phase peptide method Fmoc/OtBu-strategy. The cytotoxic effects of the compounds were tested in vitro against two human tumor cell lines—breast cancer cell line and hepatocellular cancer cell line, as well as on human non-tumorigenic epithelial cell line. Analogs containing fluoro-phenylalanines are cytotoxic in μM range, as the analog containing Phe (2-F) showed better selectivity against human hepatocellular cancer cell line. The presented study also reveals that accumulation of halogenated Phe residues does not increase the cytotoxicity according to tested cell lines. The calculated selective index reveals different mechanisms of antitumor activity of the parent compound BIM-23052 and target halogenated analogs for examined breast tumor cell lines. All peptides tested have high antitumor activity against the HepG2 cell line (IC₅₀ ≈ 100?μM and SI > 5) compared to breast cells. This is probably due to the high permeability of the cell membrane and the higher metabolic activity of hepatocytes. In silico docking studies confirmed that all obtained analogs bind well with the somatostatin receptors with preference to ssrt3 and ssrt5. All target compounds showed high hydrolytic stability at acid and neutral pH, which mimic physiological condition in stomach and human plasma.

Full text of DOI:10.1007/s00726-020-02915-3

Amino Acids
https://doi.org/10.1007/s00726-020-02915-3
ORIGINAL ARTICLE
Synthesis, in vitro biological activity, hydrolytic stability and docking
of new analogs of BIM‑23052 containing halogenated amino acids
Dancho Danalev1 · Desislava Borisova2 · Spaska Yaneva3 · Maya Georgieva4 · Anelia Balacheva4 ·
Tatyana Dzimbova4,5 · Ivan Iliev6 · Tamara Pajpanova4 · Zdravka Zaharieva1,7 · Ivan Givechev1,7
Emilia Naydenova2
·
Received: 13 May 2020 / Accepted: 6 November 2020
© Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract
One of the potent somatostatin analogs, BIM-23052 (DC-23-99) d-Phe-Phe-Phe-d-Trp-Lys-Thr-Phe-Thr-NH2, has established
in vitro growth hormone inhibitory activity in nM concentrations. It is also characterized by high afnity to some somato-
statin receptors which are largely distributed in the cell membranes of many tumor cells. Herein, we report the synthesis of
a series of analogs of BIM-23052 containing halogenated Phe residues using standard solid-phase peptide method Fmoc/
OtBu-strategy. The cytotoxic efects of the compounds were tested in vitro against two human tumor cell lines—breast can-
cer cell line and hepatocellular cancer cell line, as well as on human non-tumorigenic epithelial cell line. Analogs contain-
ing fuoro-phenylalanines are cytotoxic in μM range, as the analog containing Phe (2-F) showed better selectivity against
human hepatocellular cancer cell line. The presented study also reveals that accumulation of halogenated Phe residues does
not increase the cytotoxicity according to tested cell lines. The calculated selective index reveals diferent mechanisms of
antitumor activity of the parent compound BIM-23052 and target halogenated analogs for examined breast tumor cell lines.
All peptides tested have high antitumor activity against the HepG2 cell line (IC50 ≈ 100 μM and SI>5) compared to breast
cells. This is probably due to the high permeability of the cell membrane and the higher metabolic activity of hepatocytes.
In silico docking studies confrmed that all obtained analogs bind well with the somatostatin receptors with preference to
ssrt3 and ssrt5. All target compounds showed high hydrolytic stability at acid and neutral pH, which mimic physiological
condition in stomach and human plasma.
Keywords Peptides · Analogs of BIM-23052 · Somatostatin · Solid-phase peptide synthesis · Hydrolytic stability ·
Cytotoxic efects · Docking
Handling editor: P. Mefre.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00726-020-02915-3) contains
supplementary material, which is available to authorized users.
5
* Emilia Naydenova
South-West University “Neoft Rilski”, Blagoevgrad,
e_naydenova@abv.bg
Bulgaria
6
Institute of Experimental Morphology, Pathology
and Anthropology with Museum, Bulgarian Academy
of Sciences, Acad. G. Bonchev str., bl. 25, 1113 Sofa,
Bulgaria
1
Biotechnology Department, University of Chemical
Technology and Metallurgy, Sofa, Bulgaria
2
Department of Organic Chemistry, University of Chemical
Technology and Metallurgy, Sofa, Bulgaria
7
Testing Center Global Test Ltd, 31 Krushovski vrah Street,
3
Department of Fundamental of Chemical Technology,
Sofa, Bulgaria
University of Chemical Technology and Metallurgy, Sofa,
Bulgaria
4
Institute of Molecular Biology “Roumen Tsanev”, Bulgarian
Academy of Sciences, Sofa, Bulgaria
Vol.:(0123456789)
1 3

D. Danalev et al.
BIM-23052 (DC-23-99) d-Phe-Phe-Phe-d-Trp-Lys-Thr-
Phe-Thr-NH2 is a linear amide analog of somatostatin of
particular interest. This peptide has established in vitro
GH-inhibitory activity in nM concentrations (Coy and
Murphy 1997). BIM 23,052 and the CGP 23,996-like com-
pounds in vitro bind selectively to rat sstr5 versus human
sstr1, mouse sstr2, mouse sstr3, and human sstr4. The lin-
ear compound BIM 23,052 displayed ~ 1000-fold lower
afnity for human sstr5 than for rat sstr5. Compounds
that bind potently to human sstr5, such as L-362, 855 and
BIM 23,052, are relatively less potent in GH inhibition
(O’Carroll et al. 1994). In addition, it is characterized
by high afnity to some sstrs largely distributed into the
cell membranes of many tumor cells (Shimon 2003). In a
good correlation with Coy and Murphy studies, our previ-
ous investigations on a series of BIM-23052 analogs with
general structure d-Phe-Phe-Phe-d-Trp-Lys-Xxx6-Phe-Thr-
NH2, where Xxx is Aib (α-aminoisobutyric acid), Ac5c
(1-aminocyclopentanecarboxylic acid), and Ac6c (1-ami-
nocyclohexanecarboxylic acid) revealed very good activity
of newly synthesized analogs against a panel of tumor cell
lines (Naydenova et al. 2019).
Introduction
Use of the peptides that can directly target cancer cells
without afecting normal cells is evolving as an alternate
strategy to conventional chemotherapy. Peptides possess
many advantages, such as small size, simple synthesis
and modifcation, cell-penetrating ability including tumor
cells, and good biocompatibility (Thayer 2011; Borghouts
et al. 2005). Apart from the use of peptide-based LHRH
agonists and antagonists for treating cancer, somatostatin
analogs are the only approved cancer therapeutic peptides
on the market (Strowski and Blake 2008). Potent analogs
of somatostatin including octreotide (sandostatin) have
been developed for the treatment of acromegaly, gigan-
tism, thyrotropinoma, diarrhea and fushing episodes asso-
ciated with carcinoid syndrome, and diarrhea in patients
with vasoactive intestinal peptide-secreting tumors (Saltz
et al. 1993). Somatostatin (SST) is an endogenous cyclic
tetradecapeptide hormone that plays an important regu-
latory role in a number of cellular functions, including
inhibition of the insulin and endocrine secretion as well
as cell proliferation. It is also a neurotransmitter in the
central nervous system, reducing the amount of gastric
acid, and many others (Pollak and Shally 1998; Pyronnet
et al. 2008). The study of the anti-angiogenic efects of
the somatostatin analogs is also of interest, because of
the presence of somatostatin receptors on the membrane
of endothelial cells (Torre et al. 2002). Somatostatin has
the ability to afect cellular reproduction by binding to the
so-called G-protein coupled somatostatin receptors (sstrs)
subtype 1–5 (Reisine et al. 1995; Patel 1997, 1999; Breder
et al. 1992; Meyerhof 1998). The distribution of sstrs var-
ies widely in some tumor cells such as those present in
the brain, digestive pancreatisc tract, lung, thyroid, gland,
prostate, lymphatic system and ovaries (Susini et al. 2006;
Reubi et al. 2001, 1995; Weckbecker et al. 2003; Patelet
al. 1995; Bruns et al. 1994; Nilsson et al. 1995). Unfor-
tunately, using of somatostatin in the medicinal practice
is limited because of its very short lifetime in the human
plasma < 3 min and poor absorption by the gastrointestinal
tract (Dinnendahl et al. 2010; Haberfeld 2009). To over-
come the disadvantages of somatostatin, a large number
of analogs have been synthesized to increase their bio-
logical activity and stability. They have been shown to
be efective against pancreatic (Paz-Bouza et al. 1987),
breast (Rose et al. 1983), bone (Schally et al. 1984a, b) and
prostate (Schally et al. 1987; Zalatnai et al. 1988) cancers.
Several long-acting analogs of SST have found applica-
tion for treatment of acromegaly and symptoms caused by
neuroendocrine tumors, most notably carcinoid syndrome
and vasoactive intestinal peptide-secreting tumors (Saltz
et al. 1993).
In our previous studies a number of shortened somatosta-
tin analogs have been synthesized and tested for their biolog-
ical activity (Staykova et al. 2012a, b, c, 2015; Naydenova
et al. 2018). The obtained results with the C-amide analogs
of octreotide (SMS 201-995) modifed at positions 5 with
Orn, diaminobutanoic acid and diaminopropanoic acid and
at positions 6 with the unnatural amino acids tert.-leucine,
reveal concentration-dependent antiproliferative effects
against the HT-29, MDA-MB-231, HepG2 and HeLa cell
lines (Staykova et al. 2012a). In addition, all synthesized
peptides demonstrated considerable antioxidant activity
measured by HORAC and ORAC methods (Staykova et al.
2012b). Further, some modified octapeptide analogs of
somatostatin with the general structure d-Phe-c(Cys-Phe-
d-Trp-Xxx-Yyy-Cys)-Thr-NH2, where Xxx is Lys or Orn and
Yyy is α-aminoisobutyric acid, 1-aminocyclopentanecar-
boxylic acid and 1-aminocyclohexanecarboxylic acid, were
prepared. The cytotoxic efects of the compounds were also
tested in vitro against a panel of tumor cell lines and the
compounds were the most efective to the HT-29 tumor cells
(Staykova et al. 2015).
Coy and Murphy in their studies showed that the opti-
mization of hydrophobicity of the analogs of somatostatin
increased their biological activity (Coy and Murphy 1997).
Halogenation is a useful strategy to modulate the properties
of biologically active molecules. Fluorine is extensively used
in the pharmaceutical industry to improve the pharmacoki-
netics of drugs. It is a halogen element of periodic table
with extreme electronegativity. The chemical bond between
carbon and fuorine in organic compounds is polarized in
the opposite direction to carbon–hydrogen bond, and is both
1 3

Synthesis, in vitro biological activity, hydrolytic stability and docking of new analogs of…
more stable, and less polarizable than a C–H bond. The van
der Waals radius of fuorine is only slightly greater than
that of hydrogen, so fuorine is often considered isosteric
with hydrogen. Moreover, introduction of 18F in the place
of hydrogen atom is widely used in nuclear medicine for
manufacturing radiotracers in the diagnosis of cancer (Pająk
et al. 2016). On the other hand, chlorine is also a halogen
element, and similarly to fuorine has several electron pairs
which are able to form strong hydrogen bonds in the active
centers of enzymes as well as to realize stronger interactions
with diferent receptors.
against two human tumor cell lines (MDA-MB-231 and
HepG2). To estimate their selectivity, the non-tumorigenic
epithelial cell line MCF-10A was used. Finally, we also
examined the hydrolytic stability of the resulting peptide
analogs under physiological conditions such as: physiologi-
cal pH value of 2 (stomach), 7.4 (blood plasma) and 9.0
(small intestine) and temperature 37 °C.
Materials and methods
Based on the conclusions of Barrie et al. that inhibition
of angiogenesis by somatostatin and somatostatin-like com-
pounds is structurally dependent (Barrie et al. 1993) as well
as the investigations of Coy and Murphy (1997), and tak-
ing into account some specifc properties of fuorine and
chlorine, the aim of this work was to synthesize analogs of
the potent linear analog of SST, BIM23052, including dif-
ferently halogenated Phe in the position 1, 2 and 3 (Fig. 1).
Thus, peptides with a general formula d-Phe(H or X)-
Phe(H or X)-Phe (H or X)-d-Trp-Lys-Thr-Phe-Thr-NH2,
where X is fuorine or chlorine, were successfully obtained.
In this context, the second objective in this study was in vitro
evaluation of antiproliferative effects of these peptides
Synthesis
The protected amino acids and Fmoc-Rink Amide MBHA
Resin were purchased from Iris Biotech (Germany). All
other reagents and solvents were analytical or HPLC grade
and were purchased from Merck (Germany).
The LC/MC spectra were recorded on an LCMS-8045
triple quadrupole liquid chromatograph mass spectrom-
eter (LC–MS/MS), Shimadzu, (Shimadzu Corporation).
Optical rotations were recorded on an MCP200 modular
circular polarimeter (Anton Paar Opto Tec GmbH, Seelze,
Germany).
Fig.1 Structure of original BIM-23052 and the halogenated phenylalanines used in the current work: a l-4-F-Phe; b d-2-Phe; c l-4-Cl-Phe
1 3

D. Danalev et al.
The conventional solid-phase peptide synthesis
based on Fmoc (9-fluorenylmethoxycarbonyl) chem-
istry was employed to synthesize a series of new ana-
logs of BIM-23052. Rink-amide MBHA resin and
HBTU (3-[Bis(dimethylamino)methyliumyl]-3H-ben-
zotriazol-1-oxide hexafluorophosphate) or DIC (N,N′-
Diisopropylcarbodiimide) were used as solid-phase carrier
and coupling reagents, respectively.
O
O
ZNH
CH3
H2N
O
OH
i)
ii)
X
X
2a,b
1a,b
iii)
The halogenated amino acids Fmoc-Phe(4-Cl)-OH,
Fmoc-Phe(4-F)-OH, and Fmoc-d-Phe(2-F)-OH were syn-
thesized at the Institute of Molecular Biology, BAS, accord-
ing to the procedures previously described by Pajpanova
(Scheme 1) (Pajpanova 2000). Separation of the racemates
2a, b was achieved using alkaline protease from Bacillus
subtilis DY strain according to Aleksiev et al. (1981). As well
as the high level of enantiomeric discrimination involved
with enzymatic processes, the workup procedure after
the use of protease is usually particularly straightforward
since the unchanged d-amino acid derivatives 4a, b can be
extracted from the reaction mixture using a water—immisci-
ble solvent, but the Nα-l-protected enantiomers 3a, b—after
acidifying the reaction mixture. In our present experiments,
we achieved good resolution of the both racemic derivatives.
The Nα-benzyloxycarbonyl protected l-enantiomers 3a, b
recrystallized from appropriate alcohol in good yields (up to
93%) were used for catalytic hydrogenation step to obtain the
fnal halogenated l-phenylalanines 5a, b. The Z-protecting
group was removed by hydrogenolysis with 10% palladium
on charcoal in methanol using formic acid as a hydrogen
donor.
O
O
ZNH
CH3
ZNH
O
OH
X
X
4a,b
3a,b
v)
iv)
O
O
ZNH
H2N
OH
OH
X
X
6a,b
5a,b
iv)
vi)
O
O
H2N
FmocNH
OH
OH
X
X
Further, Fmoc-OSu was used as reagent for the synthe-
sis of the corresponding Fmoc-derivatives 7a, b because of
monitored high yields under mild conditions. The optimum
developed procedure uses a 10% excess of the correspond-
ing halogenated amino acids over Fmoc-OSu, a minimum
volume of dioxane to aqueous phase (~ 1:10 by volume)
and twofold excess of sodium carbonate over the amino
acid component. The reaction was most efcient when the
reactants were stirred vigorously at room temperature. The
preparation of Fmoc-l-4-F-Phe 7a and Fmoc-l-4-Cl-Phe 7b
was accomplished in 84–85% yields and very good purity.
The synthesis of Fmoc-d-2-F-Phe was accomplished in
a similar manner as outlined in Scheme 1. The chiral purity
for all halogenated Phe derivatives was confrmed by LE-CE
(Koidl et al. 2005) and HPLC (Kučerová et al. 2013). The
chiral analysis of halogenated amino acids by LE-CE was
performed using the Cu(II) complexes of l-4-Hypro dis-
solved in 5 mM phosphoric acid and adjusted to pH 4.5 by
adding ammonia. The LOQ (limit of quantitation) for the
minor enantiomer was estimated to be about 0.1%.
7a,b
8a,b
vi)
O
FmocNH
OH
X
9a,b
Scheme 1 Chiral separation of racemic d,l-4-F-Phe 1a and d,l-4-
Cl-Phe 1b: (i) Z-Osu/Na2CO3,H2O/acetone, 3 h; (ii) DMAP/EDCI,
MeOH, 0 °C, 2 h; (iii) alkaline protease from Bacillus subtilis DY
strain, DMF/H2O, NaHCO3, pH 9, 37 °C, 4 h; (iv) 10% Pd/C, MeOH,
HCOOH; (v) 1 N NaOH, MeOH; (vi) Fmoc-Osu, Na2CO3,dioxane/
H2O, RT
pH 4.00, 70/30 (v/v) volume ratios. In the d-forms, 0.05%
of the unwanted enantiomer can be detected.
Three-functional amino acids were protected as follows:
Nα-Fmoc-Thr(tBu)-OH, Nα-Fmoc-Lys(Boc)-OH, Nα-Fmoc-
d-Trp(Boc)-OH. The coupling reactions were performed
using for amino acid/TBTU/HOBt/DIEA/resin a molar
All HPLC measurements were performed on teicoplanin-
based CSPs (Astec Chirobiotic T) column. The mobile phase
was composed of MeOH/20 mM ammonium acetate bufer,
1 3

Synthesis, in vitro biological activity, hydrolytic stability and docking of new analogs of…
ratio 3/3/3/9/1, or amino acid/DIC/HOBt/resin a molar ratio
3/3/3/1, respectively. The Fmoc-αNH-group was deprotected
by treatment with 20% piperidine solution in dimethylforma-
mide. Coupling and deprotection reactions were monitored
by the standard Kaiser and chloranyl tests. Final cleavage
of the synthesized peptide from the resin was done, using a
mixture of 95% trifuoroacetic acid (TFA), 2.5% triisopro-
pylsilane (TIS) and 2.5% water.
did not interfere with the cells’ viability. After 24 h incuba-
tion, MTT solution was added and cells were incubated for
3.5 h. Later, the medium was removed and 100 μl solution
DMSO/ethanol (v/v) was added to each well. The plates
were placed in a plate shaker at room temperature till com-
plete dissolving of purple formazan.
ELISA plate reader (Bio-Tek) was used for reading the
results. Optical density (OD) was determined at a wave-
length of 550 nm and a reference wavelength of 630 nm.
Cell viability determined by MTT assay was calculated as:
The peptide was obtained as a fltrate in TFA and precipi-
tated with cold dry ether. The precipitate was fltered, dis-
solved in water and lyophilized to obtain the crude peptide.
The peptide purity was monitored by RP-HPLC and
ESI-MS analysis on Shimadzu Nexera X2, Column: Agi-
lent Infnitylab Poroshell 120 (100×4.6 mm), 2.7 um parti-
cle size, C 18 stationary phase, elution fow: 0.30 mL/min;
temperature of column: 40 °C using a linear binary gradient
of phase A: H2O (10% AcCN; 0.1% HCOOH) and phase B:
AcCN (5% H2O; 0.1% HCOOH) as follows.
% viability = ((OD sample − OD blank control)∕
(OD control − OD blank control)) × 100.
Cytotoxicity was expressed as IC50 values (the concentra-
tion that causes 50% cell death), calculated using non-linear
regression analysis (GraphPad Prizm 6 Software). The statis-
tical analysis included application of one-way ANOVA, fol-
lowed by Bonferroni’s post hoc test. p<0.05 was accepted
as the lowest level of statistical signifcance. All results are
presented as mean SD.
Time (min)
m.ph. A (%)
m.ph. B (%)
0.01
80
20
10.00
5
95
15.00
5
95
15.50
80
20
22.00
80
20
Docking studies
The optical rotation was measured at c=1 in methanol.
The analytical data for the synthesized peptides are sum-
marized in Table 2.
Docking of ligands
Somatostatin receptors were modeled as previously
described (Naydenova et al. 2019). 3D structures of the
ligands were modeled in Avogadro (https://www.chemc
omp.com). Ligands were protonated at physiological pH 7.4.
Docking was carried out with GOLD 5.2 software. It uses
generic algorithm and considers full ligand conformational
fexibility and partial protein fexibility. The binding site for
sstrs, in literature (Møller et al. 2003), was defned as resi-
dues Ser-Gln-Leu-Ser (305–308) for sstr1, Phe-Asp-Phe-Val
(294–297) for sstr2, Tyr-Phe-Leu-Val (295–297) for sstr3,
Asn-His-Val-Ser (293–296) for sstr4, and Tyr-Phe-Phe-Val
(286–289) for sstr5. For the docking, we use the frst resi-
dues from the sequences and the space within 10 Å radius of
them. ChemScore scoring function of GOLD was used. The
conformations of the ligands with the best scoring functions
were selected and parameters of the scoring function were
used to fnd correlations between them and the obtained
in vitro results. Molegro Molecular Viewer (https://moleg
ro.com/index.php) was used for generating fgures.
Cytotoxic efect
The cytotoxicity was evaluated by colorimetric assay based
on tetrazolium salt MTT. The yellow MTT tetrazolium
salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) is metabolized in viable cells to insoluble pur-
ple formazan crystals, later dissolved in lysis solution. The
assay was performed on MDA-MB-231 (breast cancer cell
line), HepG2 (human hepatocellular cancer cell line) and
MCF-10A (non-tumorigenic epithelial cell line). Cells were
cultured in Dulbecco’s modifed Eagle’s medium (DMEM)
(Gibco, Austria) supplemented with 10% fetal bovine serum
(Gibco, Germany), 100 U/mL penicillin and 0.1 mg/mL
streptomycin (Gibco, USA) under a humidifed 5% CO2
atmosphere at 37 °C. The complete medium for MCF-10
cells was additionally supplemented with insulin, hydrocor-
tisone, and EGF (epidermal growth factor) according to the
protocol recommended by Sigma-Aldrich.
For experiments, cells in exponential phase of growth
were seeded into 96-well flat-bottom plates after treat-
ment with Trypsin–EDTA, at a density of 2 × 104 cells/
well, at fnal volume 100 μl/well. Cells were incubated
overnight and treated with the compounds of investigation
and further incubated for 24 h. The peptides were all dis-
solved in DMSO and tested in a wide concentration range
(400–40–4–0.4–0.04 µM). The fnal concentration of DMSO
To fnd the relationship between sets of data derived from
in vitro assay and docking results, we tried to predict it with
the help of Pearson’s correlation, using GraphPad Prism 3.0.
1 3

D. Danalev et al.
Hydrolytic stability
Results and discussion
Selected pH values mimic human pH in the stomach, blood
plasma and small intestine. Bufers used for determination
of hydrolytic stability of the target compounds are prepared
according to the European Pharmacopoeia, 6th Edition, as
follows:
To optimize the physicochemical properties (stability) and
to enhance the anti-tumor potential of the parent BIM-23052
molecule d-Phe1-Phe2-Phe3-d-Trp4-Lys5-Thr6-Phe7-Thr8-
NH2, a series of halogenated analogs were synthesized. To
study the structure–activity relationships, Phe at position 2
and 3 was successively or simultaneously replaced with Phe,
and halogenated at position 4 in the aromatic ring. The infu-
ence of the type of halogen atom in the aromatic ring of Phe
was determined using Phe (4-Cl) and Phe (4-F). Also, the
d-Phe at position 1 was replaced with Phe (2-F). All analogs
were synthesized as C-terminal amides to increase plasma
half-life (Table 1).
(i) Bufer with pH 2.0—6.57 g KCl is dissolved in water
(CO2 free) and 119.0 mL 0.1 mol/L HCl is added.
The obtained solution is completed to 1000.0 mL
with dH2O.
(ii) Buffer with pH 7.4—2.38 g Na2HPO4, 0.19 g
KH2PO4 and 8.0 g NaCl are dissolved in dH2O. The
obtained solution is completed to 1000.0 mL with
dH2O.
We synthesized the SST analog BIM-23052 (compound
S1) as a standard to compare its in vitro cytotoxic activity
toward the selected cell lines to the other compounds.
Halogenated amino acids Fmoc-Phe(4-Cl)–OH, Fmoc-
Phe(4-F)–OH, and Fmoc-d-Phe(2-F)–OH were synthesized
using self-developed laboratory procedure illustrated in
Scheme 1. The key intermediate 5b was obtained according
to the procedures previously described by Pajpanova (2000).
The peptides were synthesized by standard
solid-phase peptide chemistry methods—Fmoc
(fluorenylmethoxycarbonyl)-strategy, using TBTU
(2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafuoroborate) or DIC (N, N′-diisopropylcarbodiimide)
an efcient peptide coupling reagent. The Fmoc-Rink-amide
MBHA resin was used as solid-phase carrier to obtain the
C-terminal amide derivatives. The cleavage of the synthe-
sized peptides from the resin was done, using TFA. The
yields of each peptide were over 90%. HPLC analysis
revealed a purity of > 95% for all obtained analogs. The
compounds were checked by LC–electrospray ionization
mass spectrometry and the optical rotation was measured in
methanol. The data are summarized in Table 2.
(iii) Buffer with pH 9.0—1000.0 mL of solution I is
mixed with 420.0 mL of solution II. Solution I:
6.18 g H3BO3 is dissolved in 0.1 mol/L KCl and it
is completed to 1000.0 mL with the same solvent;
Solution II: 0.1 mol/L NaOH.
An HPLC gradient method was developed as fol-
lows: HPLC Shimadzu LC20AD chromatographic system
equipped with PDA (photodiode array detectors) detector,
column: Infnity Lab Poroshell 120 EC-C18 150×4.6 mm,
4 μm; mobile phase: A: water:AcCN:TFA=90:10:0.1, and
B: water:AcCN:TFA = 5:95:0.1; elution rate 0.7 mL/min,
column temperature 40 °C; scanning wave length 280 nm,
internal standard Trp is added for quantifcation; injected
volume: 20 µL. The elution was performed in gradient mode
as follows.
Time (min)
m.ph. A (%)
m.ph. B (%)
0
80
20
10
5
95
10.1
80
20
16.5
80
20
Cytotoxic efect
All newly synthesized compounds were tested against two
tumor cell lines of human origin (MDA-MB-231 and Hep-
G2) and one human non-tumorigenic epithelial mammary
Table 1 Structure of the
No.
Code
Amino acid sequence
synthesized analogs
1
2
3
4
5
6
S1
S2
S3
D1
D2
D5
d-Phe1-Phe2-Phe3-d-Trp4-Lys5-Thr6-Phe7-Thr8-NH2 (BIM-23052)
d-Phe1-Phe(4-Cl)2-Phe3-d-Trp4-Lys5-Thr6-Phe7-Thr8-NH2
d-Phe1-Phe(4-Cl)2-Phe(4-Cl)3-d-Trp4-Lys5-Thr6-Phe7-Thr8-NH2
d-Phe1-Phe(4-F)2-Phe3-d-Trp4-Lys5-Thr6-Phe7-Thr8-NH2
d-Phe1-Phe(4-F)2-Phe(4-F)3-d-Trp4-Lys5-Thr6-Phe7-Thr8-NH2
d-Phe(2-F)1-Phe2-Phe3-d-Trp4-Lys5-Thr6-Phe7-Thr8-NH2
1 3

Synthesis, in vitro biological activity, hydrolytic stability and docking of new analogs of…
Table 2 Characteristics of the
Code General formula Mwexact [MH]+
tR (min)
M.p (oC)
Chromato-
graphic purity
(%)
ꢀ546a (°)
20
synthesized analogs
S1
S2
S3
D1
D2
D5
C61H75N11O10
1121.57 1122.57 6.02
132–135 − 20.00 88
140–143 − 26.00 95
C61H74ClN11O10 1155.53 1156.76 7.58
C61H73Cl2N11O10 1189.49 1191.65c 7.27; 7.97 and 8.42 119–123 − 14.00 96d
C61H74FN11O10
C61H73F2N11O10 1157.55 1158.55c 5.87; 6.11 and 6.3b 117–119 − 26.00 96c
C61H74FN11O10 1139.56 1140.55 6.97 117–120 − 14.00 92
1139.56 1140.55 6.07
118–120 − 26.00 87
aOptical rotation in methanol (c=1) at 20 °C
bAll three peaks have the same molecular mass and are isomers of the same compound
c[MH]+ value is for the main peak
dChromatographic purity is calculated as a sum for all three peaks
cell line MCF-10A by using the standard MTT test. After
evaluation on peptide analogs containing halogenated amino
acids, especially diferently fuorinated as well as chlorinated
phenylalanines. It is important to mention that reactions of
condensation of halogenated amino acids into the amino acid
sequence required several repetitions of the procedure, as
well as with changing of activation agent HBTU with DIC.
The activity of newly synthesized compounds was evalu-
ated against two tumor cell lines (MDA-MB-231 and Hep-
G2), and normal human cell line MCF-10A. The obtained
results are collected after 24 h of incubation. The revealed
values for IC50 for compounds S2 and S3 (fuorinated ana-
logues) as well as D1 and D2 (chlorinated analogues) show
that accumulation of more than one halogenated Phe resi-
due does not lead to increase of cytotoxic activity (Table 3).
Monohalogenated Phe(4-F or Cl) analogs have better activ-
ity against HepG-2 cell line than double substituted ana-
logs. In addition, IC50 value for analog containing Phe(2-
F) D5 shows better selectivity against HepG-2 tumor cell
line. According to MDA-MB-231 cell line, chlorinated
analogs do not show activity in the tested concentration
range, but they show good activity against HepG-2 cells.
The fuorinated analogs show also good activity, better
against HepG-2 tumor cell line. The obtained IC50 values
reveal lower efect of newly synthesized compound against
non-tumorigenic epithelial cell line (MCF 10). Selectivity
index (ratio between IC50 of MCF-10/IC50 of tumor cell line)
was observed at MDA-MB-231 treated with compounds S1
and D2. The calculated selectivity index (SI) for S1 and D2
were, respectively, 1.75 and 1.66. In addition, S1 was tested
at MCF-7 breast cancer (luminal adenocarcinoma type A).
The calculated IC50 was 120 6.5 μM and SI=4.9. The big
diference between SI and S1 for MDA-MB-231 and MCF-7
shows a diferent mechanism of antitumor activity for exam-
ined breast tumor cell lines.
incubation of the examined tumor cells with the new com-
pounds for 24 h, the percent of vitality was calculated rela-
tive to untreated controls (100% viability). All compounds
exerted concentration-dependent cytotoxic efects after 24 h
exposure, which enabled the construction of concentra-
tion–response curves (Fig. 2).
Their cytotoxic efect was expressed in μM concentration.
The data are summarized in Table 3.
Our previous investigations on the octreotide analogs
including unnatural amino acids Dap (diaminopropanoic
acid), Dab (diaminobutanoic acid) and Orn (Staykova
et al. 2012a) on the same cell line showed that compound
(d-Phe-c(Cys-Phe-d-Trp-Dap-Tle-Cys)-Thr-NH2 has IC50 at
0.03 mM concentration against MDA-MB-231 cells. In addi-
tion, better activity even in nM range of IC50 shows analogs
of BIM-23052 with sterically restricted amino acids Aib
(α-aminoisobutyric acid), Ac5c (1-aminocyclopentanecar-
boxylic acid), Ac6c (1-aminocyclohexanecarboxylic acid)
and Tle (tert-leucin) (Naydenova et al. 2019). Herein, the
obtained results for IC50 values reveal lower activity but it is
combined with better stability for newly synthesized analogs
of BIM-23052.
Docking
The results from docking were analysed in Molegro
Molecular Viewer, where the total energies of the obtained
ligand–receptor complexes were calculated (Table 4).
In previous our papers, we reported the synthesis of mod-
ifed analogs of BIM-23052 containing unnatural hindered
amino acids (α-aminoisobutyric acid, 1-aminocyclopentane-
carboxylic acid and 1-aminocyclohexanecarboxylic acid) to
elucidate the infuence of the modifcation to the in vitro
cytotoxic activity (Naydenova et al. 2019). To continue
our investigation on the structure–activity relationship of
analogs of BIM-23052, herein we report the synthesis and
All peptides tested have high antitumor activity against
the HepG2 cell line (IC50 ≈ 100 μM and SI>5) compared
to breast cells. This is probably due to the high permeability
1 3

D. Danalev et al.
Fig.2 Comparative graph of
cytotoxic efects of the tested
compounds after 24 h on: a
HepG2 cells; b MDA-MB-231;
c MCF-10
of the cell membrane and the higher metabolic activity of
hepatocytes.
residues for ligand recognition. The binding site was
defned as residues within 10 Å radius of the frst residue
in those sequences. Docking was performed with fve cho-
sen models and six ligands.
With an attempt to explain the results of biological
activity of the analogs of BIM, we performed docking
with previously obtained structures of all fve somatosta-
tin receptors. A review study of Møller et al. (2003) has
shown that residues Ser-Gln-Leu-Ser (305–308) for sstr1,
Phe-Asp-Phe-Val (294–297) for sstr2, Tyr-Phe-Leu-Val
(295–297) for sstr3, Asn-His-Val-Ser (293–296) for sstr4,
and Tyr-Phe-Phe-Val (286–289) for sstr5 are important
Total energies show how strong the ligand binds the
receptor. We assume that they represent the afnity of the
compound to the respective receptor type. The results of
the docking show that the tested analogs bind at difer-
ent degrees to diferent receptor types (one-way ANOVA,
Kruskal–Wallis test, p value 0.0003).
1 3

Synthesis, in vitro biological activity, hydrolytic stability and docking of new analogs of…
Table 3 IC50 values of newly synthesized compounds after 24 h treat-
The majority of tumors express sstr 2, followed by sstr
1, sstr 5 and sstr 3, while sstr 4 is expressed in minority of
tumors (Susini and Buscail 2006; Reubi et al. 2001; Weck-
becker et al. 2003). This determines the need of highly selec-
tive SST analogs to SST receptors. The data in Table 4 show
that all analogs bind most strongly to ssrt3, followed by ssrt5
and ssrt4 and they bound weakly to ssrt1 and ssrt2. The
ligands form a larger number of hydrogen bonds and interact
(electrostatic, hydrophobic) with ssrt3 and sstr5 than with
ssrt1 (Fig. 3). This is due to the fact that in the frst case, the
binding is between the alpha helices and a greater number of
amino acid residues are involved, while in the second case it
is on the surface of the receptor.
ment of cells
Code
IC50 of Mean SD (μM)
MDA-MB-231
HepG2
MCF-10A
S1
S2
S3
D1
D2
D5
334.8 15.4
–
–
356.7 1.11
277.4 1.06
>400
105.6 1.48
79.18 1.56
232 2.91
78.14 1.41
106 1.34
>400
>400
>400
334.3 1.9
>400
85.48 2.00
320.2 15.35
A correlation was found between the biological efect
of the analogs with respect to HepG2 and the total energy
Table 4 Total energies of the complexes between ligands and fve models of sstrs
Code
Total energy of the com- Total energy of the com- Total energy of the com- Total energy of the com- Total energy of the
plex with sstr1, kJ
plex with sstr2, kJ
plex with sstr3, kJ
plex with sstr4, kJ
complex with sstr5,
kJ
S1
S2
S3
D1
D2
D5
− 175.289
− 165.076
− 260.445
− 288.472
− 183.272
− 141.026
− 159.547
− 169.339
− 179.97
− 150.622
− 250.963
− 135.881
− 322.739
− 313.005
− 317.304
− 293.568
− 364.883
− 337.411
− 234
− 283.73
− 178.57
− 232.318
− 263.308
− 259.6
− 150.473
− 209.099
− 214.973
− 205.41
− 249.643
− 215.059
Fig. 3 a Binding of D1 with sstr3—binding is between the alpha
helices and a greater number of amino acid residues are involved. b
Binding of D2 with sstr5—binding is between the alpha helices and a
greater number of amino acid residues are involved. c Binding of D5
with sstr1—binding on the surface of the receptor (picture was gener-
ated by Molegro)
1 3

D. Danalev et al.
values of the complexes with sstr5 (Pearson r 0.85, p value
0.01). Positive dependence has a biological meaning, and
it shows that as a more stable complex is formed with the
receptor, the efect is stronger. Another implication of this
result is that in the cell tested line, the biological efect is
likely to be due to the action of the analogs on sstr5, i.e.
probably the highest percentage of sstr5 is expressed there.
Finally, the hydrolytic stability of newly synthesized BIM
analogs was investigated.
– The analog containing Phe(2-F) residue with good selec-
tivity according tumor cell line of breast cancer (MDA-
MB-231) shows good hydrolytic stability at all pH values
during 96 h.
– Introducing of one Phe(4-Cl) residue does not slow down
the hydrolysis process at neutral and alkaline pH, even in
neutral pH it is faster than with starting compound BIM-
23052.
– Introducing of one Phe(4-F) residue (the most active
analog) slows down the hydrolysis at alkaline pH, but
the incorporation of a second Phe(4-F) residue leads to
acceleration of the hydrolysis process.
The hydrolytic stability of peptides is one of the most
important properties in terms of their application in prac-
tice. Pre-information on stability is essential for the phar-
macokinetic behavior in the body, storage conditions, the
occurrence of toxic efects associated with their degradation
products and others.
– All synthesized analogs are quite stable in acid and neu-
tral pH and they can be hydrolyzed in the organism in the
small intestine.
The work solutions are prepared with a concentration
of 0.04 mg/mL in the corresponding pH bufer. Samples
are incubated at 37 °C for 96 h. Samples are injected every
1 h in the chromatographic system with addition of internal
standard Trp. Detection is done at 280 nm by monitoring the
absorbance of the indole cycle of Trp residue. The obtained
results show that all compounds are stable at 96 h at pH
2.0, except compound S3 (containing two chlorinated Phe),
where minor hydrolysis till 93.55% is monitored. At pH
7.0, all compounds show good hydrolytic stability for 96 h,
except both chlorinated analogs (compounds S2 and S3),
where 85.11% and 69.92%, respectively, of starting product
is monitored for 8 h. All compounds are hydrolyzable at pH
9.0 with diferent rates of hydrolysis except compound D5
(containing 2-F-d-Phe) which is stable till 96 h (Table 5).
The pharmacokinetic investigation on octreotide reveals
that the elimination half-life is 100 min when the compound
is applied subcutaneously; after intravenous injection, the
substance is eliminated in two phases with half-lives of 10
and 90 min, respectively (Dinnendahl et al. 2010; Haberfeld
2009). Our obtained data for hydrolytic stability show the
following structure–stability relationships:
Conclusions
It was shown that changes in any single position of the pep-
tide may afect other residues at all other positions in the par-
ent peptide. Therefore, each single modifed peptide variant
may lead to diferent activity and stability.
Taking into account all obtained results, we can conclude
that introduction of fuorinated Phe has good anti-prolifer-
ative efect against tested cancer cell lines (MDA-MB-231)
and (HepG2) at μM concentration. Moreover it is higher
than those of parent compound BIM-23052. In addition,
these analogs afected weaker the viability of the non-tumor
epithelial cell line (MCF-10A), which also shows certain
selectivity toward the tumor cells.
The analog of BIM-23052 containing Phe(2-F) residue
shows better selectivity according to HepG2 cell line. Chlo-
rinated analogs are also selective for HepG2 and show good
activity, but they do not show activity against breast cancer
cell line. The results obtained for the cytotoxic efect of the
newly synthesized compounds on these cell lines indicate
that increasing number of halogenated residues in the pep-
tide structure does not lead to better activity.
– All newly synthesized halogenated analogs are stable at
pH 2.0. Introduction of second Phe(4-Cl) residue leads
to decrease in hydrolytic stability and the appearance of
hydrolysis at the 96th hour.
Compounds S1 and D2 showed closed SI according to
tumor cell line MDA-MB-231, respectively, 1.75 and 1.66.
In addition, parent compound S1 showed SI=4.9 according
to MCF-7 breast cancer which reveals a diferent mecha-
nism of antitumor activity for the examined breast tumor
cell lines.
Table 5 Hydrolytic stability of newly synthesized peptides at pH 9.0
All peptides tested have high antitumor activity against
the HepG2 cell line (IC50 ≈ 100 μM and SI>5) compared
to breast cells. This is probably due to the high permeability
of the cell membrane and the higher metabolic activity of
hepatocytes.
Code
Hour
% Starting product
S1
S2
S3
D1
D2
D5
24
24
24
24
8
19.43
21.84
9.89
77.73
56.40
100.00
In silico calculations show that the best complex, newly
synthesized molecules form with sstr3 and sstr5. Hydrolytic
stability investigations demonstrate good stability at acid
96
1 3

Synthesis, in vitro biological activity, hydrolytic stability and docking of new analogs of…
Laskowski RA, MacArthur MW, Moss D, Thornton JM (1993) PRO-
and neutral pH for all newly synthesized BIM analogs. They
are more sensitive to hydrolytic degradation in an alkaline
environment, which showed a diferent hydrolysis rate.
CHECK: a program to check the stereochemical quality of pro-
tein structures. J Appl Cryst 26:283–291
Meyerhof W (1998) The elucidation of somatostatin receptor
functions: a current view. Rev Physiol Biochem Pharmacol
133:55–105
Molecular Operating Environment (MOE), 2012.10; Chemical Com-
puting Group Inc., 1010 Sherbooke St. West, Suite #910, Mon-
treal, QC, Canada, H3A 2R7, 2012. Available at https://www.
chemcomp.com
Funding This research was funded by Bulgarian National Fund of
Scientifc Research at the Ministry of Education and Science, Grant
number DN 19/17, 20.12.2017.
Møller LN, Stidsen CE, Hartmann B, Holst JJ (2003) Somatostatin
receptors. Biochim Biophys Acta 1616:1–84
Compliance with ethical standards
Naydenova ED, Wesselinova DW, Staykova STS, Goshev IG, Vezen-
kov LT (2018) Synthesis, cytotoxicity and antioxidant activity
of new analogs of RC-121 syntheticderivatives of somatosta-
tin. Anti-Cancer Agents Med Chem 18:1417–1424. https://doi.
org/10.2174/1871520618666180417164344
Conflict of interest The authors declare no confict of interest.
Ethical approval This article does not contain any studies with animals
or humans performed by any of the authors.
Naydenova ED, Wesselinova DW, Staykova STs, Danalev DL, Dzim-
bova TA, (2019) Synthesis, in vitro biological activity and dock-
ing of new analogs of BIM-23052 containing unnatural amino
acids. Amino Acids 51(9):1247–1257
Informed consent All authors are aware of the details of their research
work that are published in the current paper and give their consent to
their publication.
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