Synthesis of glutaryl-containing derivatives of GRGD and KRGD peptides

Article abstract of DOI:10.1007/s11172-019-2705-y

New derivatives of tetrapeptides GRGD (Gly-Arg(Pbf)-Gly-Asp(OMe)-OMe) and KRGD (Boc-Lys-Arg(Pbf)-Gly-Asp(OMe)-OMe) containing a glutaric acid fragment as a linker were synthesized. The linker provides the possibility for binding these derivatives to other biologically active molecules or nanoparticles.

Full text of DOI:10.1007/s11172-019-2705-y

2316
Russian Chemical Bulletin, International Edition, Vol. 68, No. 12, pp. 2316—2324, December, 2019
Synthesis of glutaryl-containing derivatives of GRGD and KRGD peptides*
А. М. Demin,a А. V. Vakhrushev, А. А. Tumashov, and V. P. Krasnova
a
a,b
аI. Ya. Postovsky Institute of Organic Synthesis, Ural Branch of the Russian Academy of Sciences,
2
2 ul. S. Kovalevskoi, 620990 Ekaterinburg, Russian Federation.
Fax: +7 (343) 374 1189. E-mail: demin@ios.uran.ru
b
Institute of Natural Sciences and Mathematics, Ural Federal University,
9 ul. Mira, 620002 Ekaterinburg, Russian Federation
1
New derivatives of tetrapeptides GRGD (Gly-Arg(Pbf)-Gly-Asp(OMe)-OMe) and KRGD
Boc-Lys-Arg(Pbf)-Gly-Asp(OMe)-OMe) containing a glutaric acid fragment as a linker were
(
synthesized. The linker provides the possibility for binding these derivatives to other biologi-
cally active molecules or nanoparticles.
Key words: GRGD, KRGD, peptides, amino acids, coupling agents, peptide synthesis.
The so-called vector molecules (antibodies, aptamers,
family of RGD peptides is considered as one of the most
promising for use as vector molecules in the development
of drugs for the diagnosis and treatment of cancer. The
peptides, etc.) are used in the design of drugs for the
treatment of cancer to ensure their targeted delivery and
retention in the tumor. The mechanism of accumulation
of such compounds can be based, for example, on the
interaction with receptor proteins expressed on the surface
of tumor cells. Antibodies are the most efficient among
vector molecules. However, their isolation, purification,
and storage are complex and very expensive. During iden-
tification of the binding sites of various antibodies or
polypeptides with antigens, short peptide sequences were
identified exhibiting biological activity comparable to that
of antibodies.1 Thus, it was found that peptides contain-
ing the Arg-Gly-Asp amino acid sequence (RGD** motif)
in their structure play a key role in molecular recognition
9
cyclic peptide Cilengitide (c(RGDf-N(Me)V) is currently
undergoing clinical trials as a potential antitumor drug for
the treatment of patients with glioblastoma and unmethyl-
ated MGMT promoter status. Peptide derivatives of the
RGD family are used in design of angiogenesis-imaging
1
0—15
drugs containing in their composition isotopic,
1
6—21
21—23
fluorescent,
or MRI contrast
labels, cytostatic
2
4—26
16,21,27,28
agents,
or agents for photodynamic therapy.
Such materials are synthesized based on both the linear
derivatives of the RGD peptide and the cyclic pep-
tides (c(RGDfK), c(RGDyK), c(RGDfV), c(RGDfE),
c(RGDfС), etc.). The RGD peptide derivatives are either
commercially available or can be obtained by solid-phase
synthesis or by synthesis in solution. The solid-phase
—4
5
during cell adhesion. Peptides of this family specifically
interact with transmembrane receptors, for example, in-
tegrins IIb/IIIa, α β , and α β , which are involved in
synthesis of pentapeptides, as a rule, is carried out accord-
v
3
v 5
6
—8
14,25,28—30
tumor metastasis and angiogenesis.
Therefore, the
ing to the Fmoc strategy, starting from Gly
or
t 27,31,32
L-Asp(OBu )
at the C-terminus. The linear deriva-
tives of the RGD peptide are synthesized in solution using
both the Fmoc and the Boc strategies. In this case, the
synthesis begins from C-protected derivatives of L-Asp at
*
Based on the materials of the 4th Russian Conference on
Medical Chemistry with International participation (June 9—14,
019, Ekaterinburg, Russia).
* In abbreviations of the peptide names c(RGDfK), c(RGDyK),
2
*
2
4,33—37
the C-terminus
or from N-protected derivatives
2
7,38
of L-Arg at the N-terminus.
For the covalent binding
c(RGDfV), c(RGDfE), and c(RGDfС), the letter c stands for
cyclo- (cyclopeptide), all other symbols are the designation of
amino acids in the single-letter system: R is L-Arg, L-arginine;
G is Gly, glycine; D is L-Asp, L-aspartic acid; f is D-Phe,
D-phenylalanine; y is D-Tyr, D-tyrosine; K is L-Lys, L-lysine;
E is L-Glu, L-glutamic acid; C is L-Cys, L-cysteine; V is L-Val,
L-valine; S is L-Ser, L-serine. The following protective group
abbreviations were used: Pbf is 2,2,4,6,7-pentamethyldihydro-
benzofuran-5-sulfonyl; Mds is 4-methoxy-2,6-dimethylphenyl-
sulfonyl; Mtr is 4-methoxy-2,3,6-trimethylphenylsulfonyl; Pmc
is 2,2,5,7,8-pentamethylchroman-6-sulfonyl; Mis is 1,2-di-
methylindole-3-sulfonyl.
of cyclic peptides, for example, RGDyK or RGDfC, to
biomolecules (or to the surface of nanoparticles), the
ε-amino group of L-Lys of the RGDyK fragment or the
thiol group of L-Cys of the RGDfC fragment, respectively,
are used. In this case, the binding can be accomplished
2
8
either directly or through a linker (derivatives of poly-
1
0—13,15,27
ethylene glycol,
tripeptides (GGG, DDD,
1
0,14
20,21
SSS),
caproic,
or maleimide cross-linkers,
glutaric, or adipic acids
RGD peptide is modified at the -amino group of L-Arg.
ε-amino-
2
7,39
35,36
). The linear
2
4
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2316—2324, December, 2019.
066-5285/19/6812-2316 © 2019 Springer Science+Business Media, Inc.
1

GRGD and KRGD peptide derivatives
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
2317
In the case of longer linear peptides, the ε-amino group
Table 1. Optimization of the synthesis of Boc-Gly-
Asp(OMe)-OMe (5)
of L-Lys is most often used.1
5,23,28
The purpose of the present work is to develop and
optimize methods for the synthesis of glutaryl-containing
derivatives of tetrapeptides GRGD (Gly-Arg(Pbf)-Gly-
Asp(OMe)-OMe) и KRGD (Boc-Lys-Arg(Pbf)-Gly-
Asp(OMe)-OMe), which are capable of conjugation with
other biologically active molecules or nanoparticles with-
out involvement in the synthesis of the guanidine group
of Arg and the carboxyl groups of Asp and, therefore, are
able to act as vector molecules in such constructs.
Coupling
agent
Yield*
Purity**
%
CMC
DCC
EDC
PyBOP
TBTU
15
49
52
71
81
96.9
93.6
99.8
94.2
99.1
*
*
After purification by flash-column chromatography.
* HPLC data.
Results and Discussion
The synthesis was carried out using traditional methods
of peptide synthesis in solution by sequential peptide chain
extension, starting from the C-terminus and L-Asp(OMe)-
in pure form, impurities of the starting compound dis-
appeared within 1 h after the beginning of the reaction
(according to TLC). The isolated product 7 was used
without additional purification.
3
5,36
OMe (1) (Scheme 1), similarly to the earlier developed
method for the preparation of linker-containing derivatives
of RGD peptide. Protecting groups in the amino acids
were chosen so that, in the molecules of the target peptides,
the guanidine group of the Arg residue and the β-carboxyl
group of Asp remained uninvolved in the formation of
amide bonds, which is necessary to preserve the specific-
For the synthesis of the RGD derivative, we selected
α
a N-protected L-arginine derivative of 8 containing N -Fmoc
group, which in the next stage of the synthesis of tetra-
ω
peptide can be removed with organic bases, and N -Pbf
group, which is removed with TFA (see Scheme 1).
The protection of the guanidine group of Arg is neces-
sary in order to prevent its elimination in basic media
because of its high nucleophilicity. This can lead to the
formation of ornithine and δ-lactam. At present, the most
frequently used protection is the benzenesulfonyl-type
electron-enriched groups, such as Mds, Mtr, Pmc, and
ity of the peptide binding to α β integrins expressed on
v
3
the surface of tumor cells.10
N-Protected dimethyl glycyl-L-aspartate (7) was ob-
tained at the first stage (see Scheme 1). N-(Fluorenyl-
methoxycarbonyl)glycyl chloride (2) and N-(tert-butyl-
oxycarbonyl)glycine (3) were used as N-protected glycine
derivatives. Chloride 2 was synthesized in quantitative
4
3
Pbf. The latter group is the most suitable because of its
highest lability under acidic conditions. Not so long ago,
3
6,40
43
yield from Fmoc-Gly-OH treated with oxalyl chloride
Mis was proposed as a protective group, which is even
and was further used for the synthesis of dipeptide 4 with-
more labile under acidic conditions, but so far it has not
been widely used and is not commercially available.
The protected tripeptide Fmoc-Arg(Pbf)-Gly-
Asp(OMe)-OMe (9) was synthesized by coupling of
dipeptide 7 with Fmoc-Arg(Pbf)-OH (8) using various
coupling agents: CMC, EDC•HCl, PyBOP, TBTU,
HBTU (Table 2). The best yields and product purity were
achieved in the presence of uronium salts TBTU and
HBTU. The Fmoc protective group in tripeptide 9 was
removed by treatment with a 5 equiv. excess of piperidine
in MeOH, which gave tripeptide 10 with a free amino
group (see Scheme 1). The reaction was completed within
2 h (according to TLC).
out additional purification. The coupling of Boc-Gly-OH
(
3) with Asp(OMe)-OMe (1) was carried out using various
carbodiimide coupling agents (CMC, DCC, EDC•HCl)
and uronium salts (PyBOP, TBTU) most often used
in peptide synthesis.41 The purity and yields of the iso-
lated dipeptide 5 are given in Table 1, from which it fol-
lows that the use of TBTU as a coupling agent was most
efficient.
For further coupling with Fmoc-Arg(Pbf)-OH (8), the
amino groups of dipeptides Fmoc-Gly-Asp(OMe)-OMe (4)
and Boc-Gly-Asp(OMe)-OMe (5) were deprotected (see
Scheme 1).
In the case of dipeptide 4, the Fmoc group was removed
Tetrapeptide derivatives 12 and 14 were obtained by
the coupling of tripeptide 10 with Fmoc-Gly-OH (11) or
Boc-Lys(Fmoc)-OH (13) using TBTU, since this reagent
showed the highest efficiency in the synthesis of peptide
9 (see Scheme 1). Tetrapeptides 12 and 14 were isolated
in pure form by flash-column chromatography. The Fmoc
group in both tetrapeptides was removed by treatment with
5 equiv. excess of piperidine in MeOH (2 h). If in the case
of peptide 12 the removal of the Fmoc protection and the
isolation of the target product 15 with the free amino group
by standard procedures using piperidine (20%) in CH Cl
2
2
or MeOH.3
6,42
However, these conditions led to the for-
mation of dioxopiperazine 6 through the coupling of the
glycine amino group with the α-carboxyl group of aspar-
tic acid.4 According to the H NMR data, the yield of
2
1
the target product was less than 10%.
To remove the Boc group, a solution of Boc-Gly-
Asp(OMe)-OMe (5) in CH Cl was treated with concen-
2
2
trated TFA (see Scheme 1). The target product was formed

2318
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
Demin et al.
Scheme 1

GRGD and KRGD peptide derivatives
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
2319
Table 2. Optimization of the synthesis of Fmoc-Arg(Pbf)-
Gly-Asp(OMe)-OMe (9)
ence of protection of the guanidine group of Arg and the
β-carboxyl group of Asp in the synthesized peptide de-
rivatives allows their conjugation without involvement of
these groups in the synthesis, which is necessary to preserve
the biological activity of the resulting conjugates. Sub-
sequently, the protective groups of these derivatives can
be removed under mild conditions using traditional
methods of peptide synthesis.
Coupling
agent
Yield*
Purity**
%
CMC
10
30
46
96
97
93.6
91.4
95.4
96.5
97.0
EDC
PyBOP
TBTU
HBTU
Experimental
*
*
After purification by flash-column chromatography.
* HPLC data.
4
9
Dimethyl L-aspartate hydrochloride and N-(tert-butyl-
5
0
oxycarbonyl)glycine were obtained according to the known
procedures. The following agents were used: N-(fluorenyl-
ω
methoxycarbonyl)glycine (Sigma—Aldrich, Germany), L-(N -
proceeded quite smoothly, in the case of peptide 16 the
reaction was accompanied by the formation of a large
amount of unidentified byproducts. The presence of free
amino groups in the molecules of the synthesized peptides
α
2
,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-N -fluor-
enylmethoxycarbonyl)-L-arginine (Sigma—Aldrich, Germany),
ε
α
N -fluorenylmethoxycarbonyl-N -tert-butyloxycarbonyl-L-lysine
Sigma—Aldrich, Germany), N-cyclohexyl-N´-{2-(N-methyl-
(
1
0, 15, and 16 allows their further conjugation with other
morpholino)ethyl}carbоdiimide methyl p-toluenesulfonate (CMC,
ICN Biomedical Inc., USA), N,N´-dicyclohexylcarbоdiimide
(DCC, Sigma—Aldrich, Germany), N-ethyl-N´-(3-dimethyl-
aminopropyl)carbоdiimide hydrochloride (EDC•HCl, Alfa
Aesar, UK), 1H-benzotriazol-1-yloxytri(pyrrolidinyl)phosphon-
ium hexafluorophosphate (PyBOP, Alfa Aesar, UK), O-(1H-
benzotriazol-1-yl)-N,N,N´,N´-tetramethyluronium tetrafluoro-
borate (TBTU, Alfa Aesar, UK), O-(1H-benzotriazol-1-yl)-
N,N,N´,N´-tetramethyluronium hexafluorophosphate (HBTU,
Alfa Aesar, UK), N,N-diisopropylethylamine (DIPEA, ICN
Biomedical Inc., USA), glutaric anhydride (Sigma—Aldrich,
Germany).
biologically active molecules or nanoparticles containing
carboxyl groups on the surface (for example, similarly to
4
4—46
the works
).
The coupling of tetrapeptides 15 and 16 with glutaric
anhydride (17) (see Scheme 1) gave protected tetrapept-
ides 18 and 19 containing glutaric acid residues with the
free carboxyl group, which can be involved in the coupling
with amino groups of other biomolecules or surface amino
groups of nanoparticles (for example, similarly to the
works3
5,46—48
) in order to develop diagnostic or thera-
peutic agents for visualization and treatment of malignant
neoplasms.
All the target compounds were purified by flash-column
chromatography. The structure and purity of the obtained
products were confirmed by H NMR spectroscopy, high
IR spectra were recorded on a Nicolet 6700 IR Fourier-
transform spectrometer equipped with a diamond crystal Smart
Orbit attenuated total reflection (ATR) accessory, the scanning
–
1
region of ATR spectra was 400—4000 cm , the number of scans
was 64. NMR spectra were recorded on Bruker DRX400 (400 MHz,
Germany) and Bruker Avance 500 (500 MHz, Germany) spectro-
meters in DMSO-d6 at ~20 C, using SiMe4 as an inter-
nal standard. Optical rotation were measured on a Perkin—
1
resolution mass spectrometry, elemental analysis, IR
spectroscopy, and HPLC on normal and reverse station-
ary phases. The presence of a mixture of diastereomers of
the RGD peptide derivatives cannot always be identified
–
1
Elmer M341 polarimeter (USA) (expressed in (deg mL) (g dm)
,
–
1
solution concentration in g (100 mL) ). Elemental analysis of
the samples was carried out on a Perkin—Elmer PE 2400, series
II automated CHN analyzer (USA). Mass spectra of compounds
1
in the H NMR spectra and in the HPLC chromatograms.
However, it was shown earlier36 that the diastereomers of
(
HRMS) were recorded after chromatographic separation on
the RGD peptide can differ in retention times when HPLC
is performed on a chiral stationary phase (CSP). Therefore,
we additionally evaluated the optical purity of key peptides
a Bruker maXis impact HD instrument (Germany), using electro-
spray ionization (ESI) in the positive ion mode, an operating
voltage on the needle 4.5 kV, carrier gas nitrogen, flow rate
9
, 12, and 14 by HPLC using a (S,S) Whelk-O1 column
–1
4
.5 L min . Reversed-phase HPLC and chiral HPLC were
and detected the presence of only individual peaks in the
chromatograms. This indirectly confirms that the peptides
were obtained as individual stereoisomers. The specific
optical rotation was measured for the target compounds.
In conclusion, we developed and optimized a method
for the synthesis of new protected derivatives of tetrapep-
tides GRGD (Gly-Arg(Pbf)-Gly-Asp(OMe)-OMe) and
KRGD (Boc-Lys-Arg(Pbf)-Gly-Asp(OMe)-OMe) con-
taining a fragment of glutaric acid as a linker, which
provides the possibility to bind these derivatives to other
biologically active molecules or nanoparticles. The pres-
performed on an Agilent 1100 chromatograph (USA) using
Kromasil 100-5C18, Phenomenex Luna C-18, and (S,S) Whelk-O1
(CSP) columns, 250×4.6 mm, 5 μm, flow rate 0.8 mL min–1
.
Normal-phase HPLC was carried out on a Knauer Smartline-1000
chromatograph (Germany) with a Reprosil 2 column, 250×4.6 mm,
–
1
5
2
μm, flow rate 1 mL min . Detection at wavelengths of 220 or
54 nm. Mobile phases and retention times of compounds (τ, min)
are indicated in specific procedures. Preparative HPLC was
performed on a Shimadzu LC-20 chromatograph with a Reprosil-
–1
Pur-C18 column, 250×21.2 mm, 5 μm, flow rate 5 mL min
,
detection at a wavelength of 220 nm. Silica gel 60 0.040—
0.063 mm (Alfa Aesar, UK) was used for flash chromatographic

2320
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
Demin et al.
separation. Sorbfil plates (Imid Ltd., Russia) were used for TLC,
peptides were visualized under UV light, in iodine vapors, and
with a 0.2% solution of ninhydrin in acetone.
N-(Fluorenylmethoxycarbonyl)glycyl chloride (2). Oxalyl
chloride (0.513 mL, 6.072 mmol) and DMF (5 μL) was added
to a pre-cooled to 0 С suspension of Fmoc-Gly-OH (1.500 g,
2.72 (dd, 1 Н, CH (Asp), J = 16.6 Hz, J = 6.8 Hz); 1.38
A 1 2
t
(s, 9 Н, Bu ).
3
Methyl 2-(3,6-dioxopiperazin-2-yl)acetate (6). Dipeptide 4
(0.533 g, 1.210 mmol) was dissolved in 20% piperidine solution
in CH Cl (6 mL) with magnetical stirring. The stirring was
2
2
continued for 4 h at ~20 C. A white precipitate was formed
during the reaction, which further was collected by filtration and
dried. The yield was 0.212 g (94%), a white crystalline powder,
m.p. 199—201 С (decomp.). Found (%): С, 44.85; H, 5.64;
5
.060 mmol) in anhydrous benzene (15 mL). The reaction mix-
ture was heated for 30 min to 50 С (until the precipitate was
completely dissolved) and stirring was continued for another
3
0 min at ~20 С. The solvent was evaporated, the residue was
N, 14.81. C H N O . Calculated (%): C, 45.16; H, 5.41;
7 10 2 4
dried in vacuo to obtain a light yellow crystalline powder acyl
chloride 2 (similarly to the works3 ), which was used further
in the synthesis of dipeptide 4 without additional purification.
The yield was 1.566 g (98%), m.p. 101—108 С.
N, 15.05. HPLC (Kromasil 100-5C18, λ = 254 nm, МеCN—H O
2
6,40
1
(6.3 : 3.7)), τR 6.2. R 0.85 (benzene—EtOAc (1 : 1)). H NMR
f
(500 MHz), δ: 8.12 (s, 1 Н, NH (Asp)); 8.12, 8.06 (both s,
1 H each, NH); 4.15 (ddd, 1 Н, С Н (Asp), J = 5.6 Hz,
α
1
Dimethyl N-(fluorenylmethoxycarbonyl)-glycyl-L-aspartate
J2 = 5.2 Hz, J3 = 1.2 Hz); 3.79 (ddd, 1 Н, СНА (Gly),
(
4). A solution of dimethyl ester hydrochloride 1 (0.980 g, 4.960
J1 = 17.3 Hz, J2 = 1.5 Hz, J3 = 1.5 Hz); 3.71 (ddd, 1 Н,
mmol) and DIPEA (1.728 mL, 9.919 mmol) in CH Cl (7 mL)
СН (Gly), J = 17.3 Hz, J = 2.35 Hz, J = 0.8 Hz); 3.60 (s, 3 Н,
2
2
B
1
2
3
was added to a pre-cooled to 0 С solution of acyl chloride 2
COOMe); 2.78 (dd, 1 Н, СН (Asp), J = 16.8 Hz, J = 5.2 Hz);
A 1 2
(
1.566 g, 4.960 mmol) in CH Cl (7 mL) with stirring. The reac-
2.71 (dd, 1 Н, СН (Asp), J = 16.8 Hz, J = 5.3 Hz).
B 1 2
2
2
tion mixture was allowed to stand for 24 h at ~20 C, then se-
Dimethyl glycyl-L-aspartate trifluoroacetate (7). TFA (4 mL)
was added to a solution of dipeptide 5 (0.180 g, 0.566 mmol) in
quentially washed with 5% aqueous HCl (3×10 mL), brine
(
1×10 mL), 5% aqueous Na CO (3×10 mL), and brine until
CH Cl (2 mL) and the mixture was allowed to stand for 4 h at
2
3
2 2
neutrality. The organic layer was dried with MgSO . The solvent
~20 C. The solvent was evaporated. The residue was dried
in vacuo. The yield was 0.189 g (98%), an oil. Found (%):
4
was evaporated under reduced pressure. The product was dried
in vacuo. The pure target product was obtained either by flash-
column chromatography (benzene—EtOAc, gradient from 100 : 0
to 40 : 60), or by recrystallization from a mixture of hexane—
EtOAc. The yield was 1.573 g (72%), a white crystalline powder,
С, 35.45; H, 4.32; F, 18.02; N, 7.64. C H N O •1.1 CF CO H.
8
14
2
5
3
2
Calculated (%): С, 35.65; H, 4.43; F, 18.24; N, 8.15. HPLC
(Phenomenex Luna C18(2), МеOH—H O—TFA (6 : 4 : 0.1)),
2
1
τR 3.0. R 0.56 (CHCl —MeOH (3 : 1)). H NMR (400 МHz),
f
3
m.p. 127 С, [α]D20 +34.3 (c 1.04, СHCl ). Found (%): С, 62.73;
δ: 8.90 (d, 1 Н, NH (Asp), J = 7.7 Hz); 8.01 (br.s, 1 Н, NH(Gly));
3
H, 2.71; N, 6.31. C H N O . Calculated (%): C, 62.72; H, 5.49;
4.73 (ddd, 1 Н, C H (Asp), J = 7.4 Hz; J = 6.4 Hz, J = 5.9 Hz);
23
24
2
7
α
1
2
3
N, 6.36. HPLC (Kromasil 100-5C18, λ = 254 nm, МеCN—H O
3.65 (s, 3 Н, MeО); 3.62 (s, 3 Н, MeО); 3.60 (m, 2 Н, CH (Gly));
2
2
1
(
(
(
7 : 3)), τR 5.0. R 0.66 (benzene—EtOAc (1 : 1)). H NMR
2.85 (dd, 1 Н, CH (Asp), J = 16.8 Hz, J = 5.8 Hz); 2.79 (dd,
B 1 2
f
t
500 MHz), δ: 8.37 (d, 1 Н, NH (Asp), J = 7.9 Hz); 7.90
1 Н, CH (Asp), J = 16.8 Hz, J = 6.7 Hz); 1.38 (s, 9 Н, Bu ).
A
1
2
3
ω
d, 2 Н arom. (Fmoc), J = 7.5 Hz); 7.71 (d, 2 Н arom. (Fmoc),
Dimethyl L-(N -2,2,4,6,7-pentamethyldihydrobenzofuran-5-
α
J = 7.5 Hz); 7.42 (t, 2 Н arom. (Fmoc), J = 7.3 Hz); 7.33
t, 2 Н arom. (Fmoc), J = 7.3 Hz); 7.56 (t, 1 Н, NH (Gly),
J = 6.1 Hz); 4.67 (dt, 1 Н, С Н (Asp), J = 7.3 Hz, J = 6.9 Hz);
sulfonyl-N -fluorenylmethoxycarbonyl)arginyl-glycyl-L-aspartate
(
(9). HBTU (0.115 g, 0.304 mmol) and DIPEA (0.20 mL,
1.15 mmol) were added to a solution of dipeptide trifluoroacet-
α
1
2
4
.28 (d, 2 Н, СН2 (Fmoc), J = 7.0 Hz); 4.22 (t, 1 Н, СН
ate 7 (Gly-Asp(OMe) •1.1 CF CO H) (0.193 g, 0.562 mmol)
2 3 2
(
Fmoc), J = 7.1 Hz); 3.64 (d, 2 Н, СН (Gly), J = 6.4 Hz);
and Fmoc-Arg(Pbf)-OH (8) (0.197 g, 0.304 mmol) in CH Cl2
2
2
3
1
.62, 3.60 (both s, 3 H each, COOMe); 2.80 (dd, 1 Н, СНA,
(5 mL). The reaction mixture was stirred for 20 h at ~20 C,
J = 16.5 Hz, J = 6.1 Hz); 2.71 (dd, 1 Н, СН , J = 16.5 Hz,
diluted with CH Cl (15 mL), and sequentially washed with 5%
2
B
1
2 2
J2 = 6.9 Hz).
aqueous citric acid (3×15 mL), water (3×15 mL), 5% aqueous
NaHCO (3×15 mL), water (4×15 mL) to neutral рН. The or-
Dimethyl N-(tert-butyloxycarbonyl)glycyl-L-aspartate (5).
DIPEA (2.98 mL, 18.27 mmol) and TBTU (2.013 g, 6.279 mmol)
were added to a solution of Boc-Gly-OH (3) (1.00 g, 5.708 mmol)
and dimethyl ester hydrochloride 1 (1.128 g, 5.708 mmol) in
3
ganic layer was dried with MgSO , the product was purified by
4
flash-column chromatography in the CHCl —MeOH solvent
3
system (gradient from 100 : 0 to 95 : 5). The yield was 0.250 g
20
CH Cl (20 mL). The resulting solution was stirred for 4 h. Then
(97%), m.p. 100—102 С, [α]D +8.0 (c 1.01, MeOH). Found (%):
2
2
the reaction mixture was allowed to stand for 20 h at ~20 C,
C, 58.37; H, 6.34; N, 10.45. C H N O S•0.1 CHCl . Calcul-
42 52 6 11 3
diluted with CH Cl (30 mL), and sequentially washed with 5%
ated (%): C, 58.74; H, 6.10; N, 9.76. HPLC (Kromasil 100-5C18,
МеCN—H O (7 : 3)), τ 7.9; ((S,S) Whelk-О1, МеCN—H O
2
2
aqueous NaHCO (4×20 mL), water (4×50 mL) to neutral рН,
3
2
R
2
5
% aqueous citric acid (10 mL), water (2×10 mL) to neutral рН.
(7 : 3), λ = 220 and 254 nm), τ 15.26. R 0.67 (CHCl —MeOH
R f 3
–
1
The organic layer was dried with MgSO and concentrated, the
(9 : 1)). IR, ν/cm : 3319 (NH); 2930, 2865 (CH , CH); 1737,
4
2
product was dried in vacuo. The yield was 1.266 g (81%), a color-
1728, 1710 (C=O (COOMe)); 1665, 1620 (C=O (CO—NH
(amide 1)); NH—CO—O, C—C arom.); 1546, 1536 (N—C=O
(amide 2), C—C arom.); 1440, 1407, 1369 (C—N (amide 3),
less oil, [α]D20 +45.4 (c 1.16, CHCl ). Found (%): С, 49.12;
3
H, 7.34; N, 8.58. C H N O . Calculated (%): С, 49.05; H, 6.97;
13
22
2 7
N, 8.80. HPLC (Kromasil 100-5C18, МеCN—H O (6.3 : 3.7),
R—SO —N); 1227 (C—H); 1166, 1089, 1031, 993 (C—O
2
2
1
τ 6.72. R 0.55 (benzene—EtOAc (1 : 1)). H NMR (500 MHz),
(COOMe), R—SO —N, N—H, C—C arom.); 851, 807, 782,
R
f
2
1
δ: 8.24 (d, 1 Н, NH (Asp), J = 7.8 Hz); 6.98 (t, 1 Н, NH (Gly),
760, 740 (C—H, N—H, S—O); 660, 641, 620, 566, 506. H NMR
J = 6.0 Hz); 4.67 (dt, 1 Н, C H (Asp), J = 14.3 Hz, J = 4.5 Hz);
(400 МHz), δ: 8.25 (d, 1 Н, NH (Asp), J = 7.9 Hz); 8.21 (t, 1 Н,
NH (Gly), J = 5.6 Hz); 7.89 (d, 2 Н, Ar (Fmoc), J = 7.9 Hz);
7.72 (t, 2 Н, Ar (Fmoc), J = 7.0 Hz); 7.58 (d, 1 Н, NH (Arg),
α
1
2
3
.62 (s, 3 Н, MeО); 3.00 (s, 3 Н, MeО); 3.54 (d, 2 Н, CH (Gly),
2
J = 6.1 Hz); 2.79 (dd, 1 Н, CH (Asp), J = 16.6 Hz, J = 6.1 Hz);
B
1
2

GRGD and KRGD peptide derivatives
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
2321
J = 7.6 Hz); 7.41 (t, 2 Н, Ar (Fmoc), J = 7.5 Hz); 7.31 (t, 2 Н,
Ar (Fmoc), J = 7.5 Hz); 7.05, 6.67, 6.39 (all br.s, 3 Н, NH (Arg));
τR 6.18; ((S,S) Whelk-О1, МеOH), τ 11.35; (Reprosil 2, hex-
R
i
ane—Pr OH—МеOH (3.0 : 0.7 : 0.3)), τ 15.05. R 0.46 (CHCl —
R
f
3
–
1
4
.67 (dt, 1 Н, C H (Asp), J = 7.6 Hz, J = 6.5 Hz); 4.35—4.17
MeOH (7 : 1)). IR, ν/cm : 3321 (NH); 2949, 2870 (CH , CH);
α
1
2
2
(
m, 3 Н, СН, СН (Fmoc)); 3.97 (m, 1 Н, C H (Arg)); 3.72 (d,
1730 (C=O (COOMe)); 1656, 1616 (C=O (CO—NH (amide 1),
2
α
2
Н, СН (Gly), J = 6.0 Hz); 3.61 (s, 3 Н, MeО); 3.57 (s, 3 Н,
NH—CO—O, C—C arom.); 1540 (N—C=O (amide 2),
2
MeО); 3.03 (m, 2 Н, C(5)H (Arg)); 2.94 (br.s, 2 Н, СН (Pbf));
C—C arom.); 1438, 1407, 1369 (C—N (amide 3), R—SO —N);
2
2
2
2
1
.78 (dd, 1 Н, CH (Asp), J = 16.4 Hz, J = 6.2 Hz); 2.69 (dd,
1224 (C—H); 1157, 1089, 1049, 1031, 994 (C—O (COOMe),
B
1
2
Н, CHA (Asp), J1 = 16.4 Hz, J2 = 7.0 Hz); 2.48 (s, 3 Н,
R—SO —N, N—H, C—C arom.); 852, 807, 783, 759, 741
2
1
Me (Pbf)); 2.42 (s, 3 Н, Me (Pbf)); 2.00 (s, 3 Н, Me (Pbf));
(C—H, N—H, S—O); 660, 641, 620, 566, 507. H NMR
1
2
.70—1.30 (m, 4 Н, C(3)H , C(4)H (Arg)); 1.39 (s, 6 Н,
(400 МHz), δ: 8.30 (d, 1 Н, NH (Asp), J = 8.0 Hz); 8.25 (t, 1 Н,
NH (Gly-1), J = 11.4 Hz); 8.03 (d, 1 Н, NH (Arg), J = 7.7 Hz);
7.89 (d, 2 Н, Ar (Fmoc), J = 7.5 Hz); 7.70 (t, 2 Н, Ar (Fmoc),
J = 7.6 Hz); 7.48 (t, 1 Н, NH (Gly-2), J = 6.0 Hz); 7.41 (t, 2 Н,
Ar (Fmoc), J = 7.4 Hz); 7.32 (t, 2 Н, Ar (Fmoc), J = 7.4 Hz);
7.00, 6.65, 6.35 (all br.s, 3 Н, NH(Arg)); 4.65 (dt, 1 Н,
2
2
Me (Pbf)).
Dimethyl (N -2,2,4,6,7-pentamethyldihydrobenzofuran-5-
sulfonyl)-L-arginyl-glycyl-L-aspartate (10). Tetrapeptide 9 (0.416 g,
.490 mmol) was dissolved in MeOH (5 mL) with magnetical
stirring, followed by the addition of piperidine (0.24 mL,
.45 mmol). The stirring was continued for 2 h at ~20 C. The
ω
0
2
C H (Asp), J = 14.1 Hz, J2 = 14.2 Hz); 4.30 (m, 3 Н,
α 1
reaction mixture was concentrated, washed with hexane, and
dried in a vacuum cabinet. The target compound was isolated by
Ar (Fmoc)); 4.22 (dt, 1 Н, C H (Arg), J = 15.6 Hz,
α 1
J2 = 14.1 Hz); 3.72 (t, 2 Н, CH (Gly-1), J = 6.1 Hz); 3.66
2
flash-column chromatography (eluent CHCl —MeOH, gradient
(d, 2 Н, СН (Gly-2), J = 6.1 Hz); 3.61 (s, 3 Н, MeО); 3.59
3
2
from 100 : 0 to 20 : 80). The yield was 0.236 g (76%), a foamed
(s, 3 Н, MeО); 3.02 (dt, 2 Н, C(5)H2 (Arg), J1 = 12.3 Hz,
2
0
oil, m.p. 101—107 С, [α]D +16.1 (c 1.02, MeOH). Found (%):
J = 11.9 Hz); 2.95 (s, 2 Н, СН (Pbf)); 2.78 (dd, 1 Н, CH (Asp),
2 2 A
С, 49.81; H, 6.34, N, 12.39. C27H42N O S•0.3CHCl . Calcul-
J = 16.6 Hz, J = 6.1 Hz); 2.72 (dd, 1 Н, CH (Asp), J = 16.4 Hz,
1 2 B 1
6
9
3
ated (%): C, 49.49; H, 6.44; N, 12.68. HPLC (Kromasil
J2 = 6.7 Hz); 2.47 (s, 3 Н, Me (Pbf)); 2.42 (s, 3Н, Me (Pbf));
1
00-5C18, МеOH—0.1%CF COOH (7 : 3)), τ = 5.0. R 0.62
2.00 (s, 3 Н, Me (Pbf)); 1.70—1.60 (m, 2 Н, C(3)H ); 1.50—1.40
3
R
f
2
–
1
(
CHCl —MeOH (1 : 1)). IR, ν/cm : 3319 (NH); 2931,
(m, 2 Н, C(4)H (Arg)); 1.39 (s, 6 Н, 2 Me (Pbf)). HRMS, found:
3
2
+
2
1
860 (CH , CH); 1738, 1728 (C=O (COOMe)); 1687, 1659,
m/z 906.3698 [M + H] , calculated for C H N O S: 906.3708;
found: m/z 928.3514 [M + Na] , calculated for C H N NaO S:
2
44 56
7 12
+
620 (C=O (CO—NH (amide 1), C—C arom.); 1547, 1536
44 55 7 12
(
(
(
N—C=O (amide 2), C—C arom.); 1440, 1408, 1369 (C—N
928.3527.
ε
α
amide 3), R—SO —N); 1220 (C—H); 1158, 1089, 993 (C—O
COOMe), R—SO —N, N—H, C—C arom.); 851, 808, 782,
Dimethyl N -fluorenylmethoxycarbonyl-N -tert-butyloxy-
2
ω
carbonyl-L-lysyl-(N -2,2,4,6,7-pentamethyldihydrobenzofuran-
2
1
7
33 (C—H, N—H, S—O); 659, 641, 618, 565, 507. H NMR
5-sulfonyl)-L-arginyl-glycyl-L-aspartate (14). DIPEA (0.272 mL,
1.56 mmol) and TBTU (0.251 g, 0.781 mmol) were added to
a solution of tripeptide 10 (0.445 g, 0.710 mmol) and Boc-
(
400 МHz), δ: 8.60 (br.s, 1 Н, NH (Gly)); 8.53 (d, 1 Н,
NH (Asp), J = 7.7 Hz); 8.10—7.10 (br.s, 2 Н, NH2 (Arg),
J = 7.6 Hz); 6.88, 6.45 (both br.s, 3 Н, NH (Arg)); 4.68 (m, 1 Н,
Lys(Fmoc)-OH (13) (0.333 g, 0.710 mmol) in CH Cl (25 mL)
2
2
C H (Asp)); 3.80—3.70 (m, 1 Н, C H (Arg)); 3.80 (d, 2 Н,
with stirring, the stirring was continued for 16 h at ~20 C. The
reaction mixture was diluted with CH Cl (25 mL) and seq-
α
α
СН (Gly), J = 4.9 Hz); 3.62 (s, 3 Н, MeО); 3.61 (s, 3 Н, MeО);
2
2
2
3
.09—3.00 (m, 2 Н, C(5)H2 (Arg)); 3.02—2.93 (m, 2 Н,
uentially washed with 5% aqueous NaHCO3 (15 mL), water
(3×15 mL) to neutral рН, 5% aqueous citric acid (5 mL), and
water (3×10 mL) to neutral рН. The organic layer was dried with
СН (Pbf)); 2.82 (dd, 1 Н, CH (Asp), J = 16.5 Hz, J = 6.1 Hz);
2
B
1
2
2
(
1
1
.72 (dd, 1 Н, CHA (Asp), J = 16.6 Hz, J = 6.8 Hz); 2.48
1 2
s, 3 Н, Me (Pbf)); 2.42 (s, 3 Н, Me (Pbf)); 2.01 (s, 3 Н, Me (Pbf));
MgSO and concentrated. The product was dried in vacuo and
4
.70—1.60 (m, 2 Н, C(3)H ); 1.60—1.50 (m, 2 Н, C(4)H (Arg));
purified by flash-column chromatography in the CHCl —MeOH
2
2
3
.41 (s, 6 Н, 2 Me (Pbf)).
solvent system (gradient from 100 : 0 to 85 : 15). The yield was
ω
20
Dimethyl N-fluorenylmethoxycarbonyl-glycyl-(N -2,2,4,6,7-
0.681 g (93%), a foamed oil, m.p. 106 С, [α]D –4.1 (c 2.86,
pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginyl-glycyl-L-
aspartate (12). DIPEA (0.267 mL, 1.532 mmol) and TBTU
MeOH). Found (%): С, 58.90; H, 6.77; N, 10.79; S, 2.93.
C53H72N O S•0.2Н О. Calculated (%): C, 58.90; H, 6.75;
8
14
2
(
(
0.246 g, 0.766 mmol) were added to a solution of tripeptide 10
0.480 g, 0.766 mmol) and Fmoc-Gly-OH (11) (0.228 g,
N, 10.37; S, 2.97. HPLC (Kromasil 100-5C18, МеCN—H O
2
(7 : 3)), τ 11.64; ((S,S) Whelk-О1, МеOH), τ 12.19; (Reprosil 2,
R
R
i
0
.766 mmol) in CH Cl (25 mL) with stirring. The reaction
hexane—Pr OH—МеOH (3.0 : 0.7 : 0.3)), τ 10.05. R 0.67
2
2
R f
–
1
mixture was allowed to stand for 20 h at ~20 C. Then the reac-
(CHCl —MeOH (1 : 1)). IR, ν/cm : 3319 (NH); 2935, 2865
3
tion mixture was diluted with CH Cl (15 mL) and sequentially
(CH , CH); 1738 (C=O (COOMe)); 1685, 1658, 1616 (C=O
2
2
2
washed with 5% aqueous NaHCO (4×20 mL), water (3×25 mL)
(CO—NH (amide 1), NH—CO—O, C—C arom.); 1536, 1518
3
to neutral рН, 5% aqueous citric acid (5 mL), and water
(N—C=O (amide 2), C—C arom.); 1439, 1408, 1367 (C—N
(
3×10 mL) to neutral рН. The organic layer was dried with
(amide 3), R—SO —N); 1242 (C—H); 1161, 1089, 1049, 1019,
2
MgSO and concentrated, the product was dried in vacuo and
994 (C—O (COOMe), R—SO —N, N—H, C—C arom.); 852,
4
2
purified by flash-column chromatography in the CHCl —MeOH
806, 782, 759, 741 (C—H, N—H, S—O); 660, 641, 620, 564,
3
1
solvent system (gradient from 100 : 0 to 85 : 15). The yield was
507. H NMR (400 МHz), δ: 8.32 (d, 1 Н, NH (Asp), J = 5.2 Hz);
2
0
0
.698 g (98%), a foamed oil, m.p. 125 С, [α]D –2.1; (c 1.54,
8.21 (t, 1 Н, NH (Gly), J = 5.6 Hz); 7.85 (d, 1 Н, NH (Arg),
J = 7.8 Hz); 7.68 (d, 2 Н, Ar (Fmoc), J = 7.5 Hz); 7.40 (t, 2 Н,
Ar (Fmoc), J = 7.6 Hz); 7.32 (t, 2 Н, Ar (Fmoc), J = 7.4 Hz);
MeOH). Found (%): С, 57.22; H, 6.05, N, 10.34; S, 3.47.
C H N O S•H O. Calculated (%): C, 57.19; H, 6.22; N, 10.61;
44
55
7
12
2
S, 3.47. HPLC (Kromasil 100-5C18, МеCN—H O (6 : 4)),
6.90 (d, NH (Lys), J = 8.4 Hz); 6.79, 6.65, 6.35 (all br.s, 3 Н,
2
ε
τR 11.96; (Phenomenex LunaC-18, МеCN—H O (7 : 3)),
NH (Arg)); 4.65 (dt, 1 Н, C H (Asp), J = 11.4 Hz, J = 6.7 Hz);
α 1 2
2

2322
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
Demin et al.
4
2
.28 (m, 3 Н, Ar (Fmoc)); 4.20 (dt, 1 Н, C H (Arg), J = 13.9 Hz,
(C—H); 1163, 1090, 995 (C—O (COOMe), R—SO —N, N—H,
α
1
2
J = 7.0 Hz); 3,87 (m, 1 H, С Н (Lys)); 3.72 (d, 2 Н, CH (Gly),
C—C arom.); 852, 807, 783, 734 (C—H, N—H, S—O); 659, 641,
α
2
1
J = 5.7 Hz); 3.61 (s, 3 Н, MeО); 3.58 (s, 3 Н, MeО); 3.02 (dt,
Н, C(5)H (Arg), J = 12.3 Hz, J = 5.5 Hz); 2.95 (s, 2 Н,
620, 567, 507. H NMR (400 МHz), δ: 8.32 (d, 1 Н, NH (Asp),
2
J = 5.2 Hz); 8.21 (t, 1 Н, NH (Gly), J = 5.6 Hz); 7.85 (d, 1 Н,
2
1
2
СН (Pbf)); 2.92—2.94 (m, 2 H, C(6)H (Lys)); 2.78 (dd, 1 Н,
NH (Arg), J = 7.8 Hz); 6.9 (d, 1 H, NH (Lys), J = 8.4 Hz); 6.85
2
2
ε
CH (Asp), J = 16.6 Hz, J = 6.3 Hz); 2.72 (dd, 1 Н, CH (Asp),
(d, 1 H, NH (Lys), J = 7.7 Hz); 6.79, 6.65, 6.35, (all br.s, 3 Н,
α
A
1
2
B
J = 16.6 Hz, J = 6.8 Hz); 2.47 (s, 3 Н, Me (Pbf)); 2.41 (s, 3 Н,
NH (Arg)); 4.65 (dt, 1 Н, C H (Asp), J = 11.4 Hz, J = 6.7 Hz);
1
2
α 1 2
Me (Pbf)); 2.00 (s, 3 Н, Me (Pbf)); 1.70—1.60 (m, 2 Н, C(3)H );
4.20 (dt, 1 Н, C H (Arg), J = 13.9 Hz, J2 = 7.0 Hz); 3.87
α 1
2
1
.50—1.40 (m, 2 Н, C(4)H (Arg)); 1.50—1.40 (m, 6 H, C(5)H +
(m, 1 H, С Н (Lys)); 3.72 (d, 2 Н, CH (Gly), J = 5.7 Hz); 3.61
2
2
α
2
+
3
C(4)H + C(3)H (Lys)); 1.40 (s, 6 Н, 2 Me (Pbf); 1.35 (s, 9 Н,
(s, 3 Н, MeО); 3.58 (s, 3 Н, MeО); 3.02 (dt, 2 Н, C(5)H (Arg),
2
2
2
+
Me (Boc)). HRMS, found: m/z 1077.4956 [M + H] , calcu-
J = 12.3 Hz, J = 5.5 Hz); 2.95 (s, 2 Н, СН (Pbf)); 2.92—2.94
1 2 2
lated for C53H73N O14S: 1077.4967; found: m/z 1099.4779
(m, 2 H, C(6)H (Lys)); 2.78 (dd, 1 Н, CH (Asp), J = 16.6 Hz,
2 A 1
8
+
[
M + Na] , calculated for C53H72N NaO14S: 1099.4786.
J = 6.3 Hz); 2.72 (dd, 1 Н, CH (Asp), J = 16.6 Hz, J = 6.8 Hz);
8
2 B 1 2
ω
Dimethyl glycyl-(N -2,2,4,6,7-pentamethyldihydrobenzo-
2.47 (s, 3 Н, Me (Pbf)); 2.41 (s, 3 Н, Me (Pbf)); 2.00 (s, 3 Н,
Me (Pbf)); 1.70—1.60 (m, 2 Н, C(3)H ); 1.50—1.40 (m, 2 Н,
furan-5-sulfonyl)-L-arginyl-glycyl-L-aspartate (15). Piperidine
0.294 mL, 2.98 mmol) was added to a solution of tetrapeptide
2 (0.539 g, 0.595 mmol) in MeOH (5 mL). The reaction mix-
2
(
C(4)H (Arg)); 1.50—1.40 (m, 6 H, C(5)H + C(4)H + C(3)
2
2
2
1
H (Lys)); 1.40 (s, 6 Н, 2 Me (Pbf); 1.35 (s, 9 Н, 3 Me (Boc)).
2
+
ture was allowed to stand for 2 h at ~20 C and concentrated,
HRMS, found: m/z 855.4275 [M + H] . Calculated for
C38H63N O12S: 855.4286.
the product was dried in vacuo and purified by flash-column
8
ω
chromatography in the CHCl —MeOH solvent system (gradient
Dimethyl N-carboxybutanoyl-glycyl-(N -2,2,4,6,7-penta-
methyldihydrobenzofuran-5-sulfonyl)-L-arginyl-glycyl-L-aspar-
tate (18). Glutaric anhydride 17 (0.119 g, 0.168 mmol) was added
to a solution of tetrapeptide 15 (0.115 g, 0.168 mmol) in CHCl3
(3 mL) with magnetical stirring. The reaction mixture was allowed
to stand for 16 h at ~20 C, followed by the evaporation of vola-
tile components. The product was dried in vacuo. The target
compound was isolated by flash-column chromatography (eluent
3
from 95 : 5 to 20 : 80), as well as by preparative HPLC (eluent
MeOH—0.2%AcOH (65 : 35)). The yield was 0.321 g (79%),
2
0
a foamed oil, m.p. 126 С, [α]D +4.5 (c 1.67, MeOH). Found
%): C, 47.64; H, 6.40; N, 13.15; S, 4.28. C29H45N O10S•
(
7
•
2H O•CH COOH. Calculated (%): C, 47.74; H, 6.85; N, 12.57;
2 3
S, 4.11. HPLC (Kromasil 100-5C18, МеOH—0.1%CF COOH
3
–
1
(3 : 7)), τR 3.51. R 0.30 (CHCl —MeOH (3 : 1)). IR, ν/cm :
3
f 3
308 (NH); 2940, 2870 (CH , CH); 1737 (C=O (CO—NH
CHCl —MeOH, gradient from 100 : 0 to 0 : 100). The yield was
2
3
2
0
(
(
(
amide 1), C—C arom.); 1658, 1618 (C=O (COOMe)); 1537
N—C=O (amide 2), C—C arom.); 1438, 1406, 1369 (C—N
0.099 g (74%), a crystallized oil, m.p. 100 С, [α]D –1.5
(c 3.22, MeOH). Found (%): С, 51.05; H, 6.31; N, 11.94; S, 3.93.
amide 3), R—SO —N); 1278, 1235, 1202 (C—H); 1165, 1089,
C34H51N O13S. Calculated (%): C, 51.18; H, 6.44; N, 12.29;
2
7
1
033, 993 (C—O (COOMe), R—SO —N, N—H, C—C arom.);
S, 4.02. HPLC (Kromasil 100-5C18, МеОН—0.5%AcOH,
2
8
52, 807, 783 (C—H, N—H, S—O); 659, 641, 618, 566, 507.
gradient from 3 : 7 to 9 : 1), τ 17.86. R 0.40 (CHCl —MeOH
R f 3
1
–1
H NMR (400 МHz), δ: 8.30 (d, 1 Н, NH (Asp), J = 8.0 Hz);
(2 : 1)). IR, ν/cm : 3309 (NH); 2928, 2856 (CH , CH); 1736
2
8
.25 (t, 1 Н, NH (Gly-1), J = 11.4 Hz); 8.03 (d, 1 Н, NH (Arg),
(C=O (COOMe)); 1643 (C=O (CO—NH (amide 1), C—C
J = 7.7 Hz); 7.89 (d, 3 Н, NH(Arg)); 4.65 (dt, 1 Н, C H (Asp),
arom.); 1537 (N—C=O (amide 2), C—C arom.); 1438, 1408,
α
J = 14.1 Hz, J = 14.2 Hz); 4.22 (dt, 1 Н, C H (Arg), J = 15.6 Hz,
J = 14.1 Hz); 3.72 (t, 2 Н, CH (Gly-1), J = 6.1 Hz); 3.66
1370 (C—N (amide 3), R—SO —N); 1222 (C—H); 1156, 1090,
2
1
2
α
1
1026, 994 (C—O (COOMe), R—SO —N, N—H, C—C arom.);
2
2
2
(
(
d, 2 Н, СН (Gly-2), J = 6.1 Hz); 3.61 (s, 3 Н, MeО); 3.59
s, 3 Н, MeО); 3.02 (dt, 2 Н, C(5)H2 (Arg), J1 = 12.3 Hz,
851, 808, 782, 733 (C—H, N—H, S—O); 659, 640, 618, 565,
2
1
506. H NMR (400 МHz), δ: 8.28 (d, 1 Н, NH (Asp), J = 7.7 Hz);
J = 11.9 Hz); 2.95 (s, 2 Н, СН (Pbf)); 2.78 (dd, 1 Н, CH (Asp),
8.24 (t, 1 Н, NH (Gly-1), J = 5.8 Hz); 8.04 (d, 1 Н, NH (Arg),
J = 8.8 Hz); 8.02 (t, 1 H, NH (Gly-2), J = 5.6 Hz); 7.05, 6.67,
2
2
A
J = 16.6 Hz, J = 6.1 Hz); 2.72 (dd, 1 Н, CH (Asp), J = 16.4 Hz,
J2 = 6.7 Hz); 2.47 (s, 3 Н, Me (Pbf)); 2.42 (s, 3 Н, Me (Pbf));
1
2
B
1
6.39 (all br.s, 3 Н, NH (Arg)); 4.66 (dt, 1 Н, C H (Asp),
α
2
(
.00 (s, 3 Н, Me (Pbf)); 1.70—1.60 (m, 2 Н, C(3)H ); 1.50—1.40
J = 13.6 Hz, J = 6.8 Hz); 4.23 (dt, 1 Н, C H (Arg), J = 13.2 Hz,
1 2 α 1
2
m, 2 Н, C(4)H (Arg)); 1.39 (s, 6 Н, 2 Me (Pbf)). HRMS, found:
J = 7.7 Hz); 3.71 (m, 4 Н, 2 СН (Gly-1, Gly-2)); 3.61 (s, 3 Н,
2
2
2
+
m/z 684.3022 [M + H] . Calculated for C H N O S: 684.3027.
MeО); 3.57 (s, 3 Н, MeО); 3.03 (m, 2 Н, C(5)H (Arg)); 2.51
2
29
46
7
10
α
ω
Dimethyl N -tert-butyloxycarbonyl-L-lysyl-(N -2,2,4,6,7-
pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginyl-glycyl-L-
aspartate (16) was obtained similarly to compound 15. The
product was purified by flash-column chromatography in the
(br.s, 2 Н, СН (Pbf)); 2.71 (dd, 1 Н, CH (Asp), J = 16.7 Hz,
2 B 1
J = 7.0 Hz); 2.79 (dd, 1 Н, CH (Asp), J = 16.5 Hz, J = 6.2 Hz);
2
A
1
2
2.48 (s, 3 Н, Me (Pbf)); 2.40 (s, 3 Н, Me (Pbf)); 2.20 (t, 2 Н,
СН (glutaric acid), J = 7.4 Hz); 2.16 (t, 2 H, СН (glutaric
2
2
CHCl —MeOH solvent system (gradient from 100 : 0 to 85 : 15)
acid), J = 7.6 Hz); 2.00 (s, 3 Н, Me (Pbf)); 1.70 (m, 2 Н, CH2
3
and preparative HPLC (eluent MeOH—0.2%AcOH (35 : 65)).
(glutaric acid); 1.60—1.40 (m, 4 Н, C(3)H , C(4)H (Arg)); 1.41
2
2
+
The yield was 0.198 g (68%), a foamed oil, m.p. 111 С,
(s, 6 Н, 2 Me (Pbf)). HRMS, found: m/z 798.3333 [M + H] .
2
0
[
α]D –6.6 (c 0.45, MeOH). Found (%): С, 52.44; H, 7.35,
Calculated for C34H52N O S: 798.3344.
7 13
ε
α
N, 10.70. C38H62N O12S•MeCOOH. Calculated (%):C, 52.50;
Dimethyl N -carboxybutanoyl-N -tert-butyloxycarbonyl-L-
lysyl-(N -2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-
8
ω
H, 7.27; N, 12.25; S, 3.50. HPLC (Kromasil 100-5C18, МеOH—
0
.15%CF COOH (75 : 25)), τ 6.41. R 0.38 (CHCl —MeOH
arginyl-glycyl-L-aspartate (19) was obtained similarly to com-
3
R
f
3
–
1
(
(
1 : 1)). IR, ν/cm : 3302 (NH); 2938, 2870 (CH , CH); 1738
C=O (COOMe)); 1685, 1657 (C=O (CO—NH (amide 1),
pound 18. The product was purified by flash-column chroma-
2
tography in the CHCl —MeOH solvent system (gradient from
3
NH—CO—O, C—C arom.); 1545 (N—C=O (amide 2), C—C
100 : 0 to 75 : 25) and preparative HPLC (eluent MeOH—
0.2%AcOH (65 : 35)). The yield was 0.051 g (23%), a crystallized
arom.); 1439, 1408, 1367 (C—N (amide 3), R—SO —N); 1243
2

GRGD and KRGD peptide derivatives
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
2323
oil, m.p. 99 С, [α]D20 –3.1 (c 0.25, MeOH). Found (%): С, 50.42;
H, 7.39; N, 11.52; S, 3.37. C43H68N O S•2H O•CH COOH.
10. S. Liu, Bioconjug. Chem., 2015, 26, 1413.
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Nucl. Med. Biol., 2017, 48, 1.
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Isotopes, 2019, 148, 168.
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K. Chen, J. Wang, Amino Acids, 2014, 46, 2355.
14. X. Chen, R. Park, A. H. Shahinian, J. R. Bading, P. S. Conti,
Nucl. Med. Biol., 2004, 31, 11.
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Bull., 2016, 65, 363.
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17. Y. Zheng, S. Ji, A. Czerwinski, F. Valenzuela, M. Pennington,
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19. R. Tang, J. Xue, B. Xu, D. Shen, G. P. Sudlow, S. Achilefu,
ACS Nano, 2015, 9, 220.
8
15
2
3
Calculated (%):C, 50.74; H, 7.19; N, 10.52; S, 3.01. HPLC
Kromasil 100-5C18, МеOH—0.15%CF COOH (65 : 35)),
(
3
–
1
τR 10.11. R 0.40 (CHCl —MeOH (2 : 1)). IR, ν/cm : 3297
f
3
(
NH); 2973, 2935, 2860 (CH , CH); 1738 (C=O (COOMe));
2
1
1
659 (C=O (CO—NH (amide 1), NH—CO—O, C—C arom.);
537 (N—C=O (amide 2), C—C arom.); 1439, 1403, 1392, 1367
(
C—N (amide 3), R—SO —N); 1242, 1203 (C—H); 1162, 1090,
2
9
8
5
8
95 (C—O (COOMe), R—SO —N, N—H, C—C arom.); 852,
2
03, 782, 733, 722 (C—H, N—H, S—O); 658, 641, 618, 566,
1
07. H NMR (400 МHz), δ: 8.27 (dt, 1 Н, NH (Asp), J = 5.2 Hz);
.27 (t, 1 Н, NH (Asp), J = 3.5 Hz); 8.25 (t, 1 Н, NH (Gly),
J = 5.4 Hz); 7.85 (d, 1 Н, NH (Arg), J = 7.7 Hz); 7.75 (t, 1 H,
NH (Lys), J = 6.1 Hz); 6.85 (d, 1 H, NH (Lys), J = 7.7 Hz);
ε
α
4
1
.65 (dt, 1 Н, C H (Asp), J = 13.3 Hz, J = 7.5 Hz); 4.20 (dt,
α
1
2
Н, C H (Arg), J = 13.9 Hz, J = 7.0 Hz); 3,87 (m, 1 H,
α
1
2
С Н (Lys)); 3.72 (d, 2 Н, CH (Gly), J = 4.9 Hz); 3.61 (s, 3 Н,
α
2
MeО); 3.58 (s, 3 Н, MeО); 3.02 (dt, 2 Н, C(5)H2 (Arg),
J = 11.6 Hz, J = 6.0 Hz); 2.95 (s, 2 Н, СН (Pbf)); 2.92—2.94
20. B. R. Smith, Z. Cheng, A. De, A. L. Koh, R. Sinclair, S. S.
Gambhir, Nano Lett., 2008, 8, 2599.
21. K. Chen, J. Xie, H. Xu, D. Behera, M. H. Michalski,
S. Biswal, A. Wang, X. Chen, Biomaterials, 2009, 30, 6912.
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24. А. Nortcliffe, I. N. Fleming, N. P. Botting, D. O’Hagan,
Tetrahedron, 2014, 70, 8343.
1
2
2
(
m, 2 H, C(6)H (Lys)); 2.78 (dd, 1 Н, CH (Asp), J = 16.6 Hz,
2
A
1
J = 6.3 Hz); 2.72 (dd, 1 Н, CH (Asp), J = 16.6 Hz, J = 6.8 Hz);
2
B
1
2
2
.47 (s, 3 Н, Me (Pbf)); 2.41 (s, 3 Н, Me (Pbf)); 2.00 (s, 3 Н,
Me (Pbf)); 1.65—1.75 (m, 6 H, 3 CH (glutaric acid)); 1.70—1.60
2
(
(
m, 2 Н, C(3)H ); 1.50—1.40 (m, 2 Н, C(4)H (Arg)); 1.45—1.40
m, 6 H, C(5)H2 + C(4)H2 + C(3)H2 (Lys)); 1.35 (s, 6 Н,
2
2
2
9
Me (Pbf); 1.32 (s, 9 Н, 3 Me(Boc)). HRMS, found: m/z
+
69.4598 [M + H] , calculated for C43H69N O15S: 969.4603;
25. R. Colombo, M. Mingozzi, L. Belvisi, D. Arosio, U. Piarulli,
N. Carenini, P. Perego, N. Zaffaroni, M. De Cesare, V. Cas-
tiglioni, E. Scanziani, C. Gennari, J. Med. Chem., 2012,
8
+
found: m/z 991.4415 [M + Na] , calculated for C H N NaO S:
4
3
68
8
15
9
91.4423.
5
5, 10460.
The authors are grateful to M. I. Kodess for recording
26. X. Wang, X. Qiao, Y. Shang, S. Zhang, Y. Li, H. He, S. Chen,
the NMR spectra, I. N. Ganebnykh for recording the mass
spectra, and E. N. Chulakov for isolation of compounds
by preparative HPLC.
Amino Acids, 2017, 49, 931.
2
7. M. Boisbrun, R. Vanderesse, P. Engrand, A. Olié, S. Hupont,
J.-B. Regnouf-de-Vains, C. Frochot, Tetrahedron, 2008,
6
4, 3494.
The work was performed in the framework of the Russian
state assignment (Theme No. АААА-А19-119011790130-3)
using equipment of the Centre for Joint Use "Spectroscopy
and Analysis of Organic Compounds" at the I. Ya. Pos-
tovsky Institute of Organic Synthesis of the Russian
Academy of Sciences (Ural Branch).
2
8. C. Frochot, B. Di Stasio, R. Vanderesse, M.-J. Belgy,
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2
3
9. H. Vilac, P. M. T. Ferreira, N. M. Micaelo, Tetrahedron,
2
014, 70, 5420.
0. K. Yamada, I. Nagashima, M. Hachisu, I. Matsuo, H. Shimizu,
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Received August 29, 2019;
in revised form November 1, 2019;
accepted November 8, 2019
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