Synthesis of a "memory tripeptide" (Arg-Glu-Arg, RER) and the Kabachnik-Fields reaction with di-and tripeptides as a method for the synthesis of phosphorus-containing peptide analogs

Article abstract of DOI:10.1007/s11172-010-0063-x

Methods for the synthesis of potential "twin-drugs" containing fragments of the glutamate receptor antagonist and cognitive function enhancing oligopeptides were developed. The "memory tripeptide" Arg-Glu-Arg (RER) containing the tripeptide sequence of a protein APP_328-330, a β-amyloid precursor, was synthesized. A method for the synthesis of α-aminophosphonates with oligopeptides as the amine component of the one-pot three-component Kabachnik-Fields reaction was developed. A method for the synthesis of phosphonopeptides by the introduction of α- aminophosphonates into the peptide chain was proposed.

Full text of DOI:10.1007/s11172-010-0063-x

Russian Chemical Bulletin, International Edition, Vol. 59, No. 1, pp. 200—208, January, 2010
200
Synthesis of a "memory tripeptide" (Arg—Glu—Arg, RER)
and the Kabachnik—Fields reaction with diꢀ and tripeptides as a method
for the synthesis of phosphorusꢀcontaining peptide analogs
E. D. Matveeva,a, T. A. Podrugina,a M. V. Prisyazhnoi,a S. O. Bachurin,b and N. S. Zefirova
aDepartment of Chemistry, M. V. Lomonosov Moscow State University,
1 Leninskie Gory, 119992 Moscow, Russian Federation.
Fax: +7 (495) 939 02 90. Eꢀmail: matveeva@org.chem.msu.ru
bInstitute of Physiologically Active Compounds, Russian Academy of Sciences,
1 Severnyi proezd, 142432 Chernogolovka, Moscow Region, Russian Federation.
Fax: +7 (496) 524 9508. Eꢀmail: ipac@ipac.ac.ru
Methods for the synthesis of potential "twinꢀdrugs" containing fragments of the glutamate
receptor antagonist and cognitive function enhancing oligopeptides were developed. The "memory
tripeptide" Arg—Glu—Arg (RER) containing the tripeptide sequence of a protein APP328—330
,
a βꢀamyloid precursor, was synthesized. A method for the synthesis of αꢀaminophosphoꢀ
nates with oligopeptides as the amine component of the oneꢀpot threeꢀcomponent Kabachꢀ
nik—Fields reaction was developed. A method for the synthesis of phosphonopeptides by the
introduction of αꢀaminophosphonates into the peptide chain was proposed.
Key words: arginyl—glutamyl—arginine, phosphonopeptides, phosphorusꢀcontaining pepꢀ
tides, peptide synthesis, αꢀaminophosphonates, the Kabachnik—Fields reaction, [tetra(tertꢀ
butyl)phthalocyanine]aluminum chloride.
A topical problem of organic synthesis is the design of
studying the mechanism of its action, a specific pentapepꢀ
tide sequence APP328—332, namely, Arg—Glu—Arg—
—Met—Ser (RERMS), was identified as an active domain
responsible for the aboveꢀmentioned functions. Moreover,
analogous synthetic pentapeptides RERMS and SMRER
also eliminate memory deficiency. It is most interesting
that even palindromic tripeptide Arg—Glu—Arg (RER)
containing the tripeptide sequence APP328—330 protects
from memory loss induced by βꢀamyloid.6,7 It was
shown8,9 that both the family of pentapeptide derivatives
corresponding to the amino acid sequence APP328—332 and
the family of tripeptide derivatives corresponding to the
amino acid sequence APP328—330 can be used as enhancers
of cognitive functions for the treatment of Alzheimer´s
disease. Interestingly, the "memory tripeptide" RER
(Arg—Glu—Arg) retains activity upon both inversion of
the configuration of one of the arginine residues and parꢀ
tial acylation.6—8 Note that analogous triꢀ and pentapepꢀ
tides are potential inhibitors of urokinase receptors10 and
angiotensinꢀIꢀconverting enzymes11 and possess antiꢀ
thrombotic9 activity.
substances with specified properties, in particular, newꢀ
generation drugs for the treatment of neurodegenerative
disorders. One of the promising directions for solving this
problem is the development of bioisosteric analogs of the
known biologically active substances1 or the synthesis of
"twinꢀdrugs" on the basis of the soꢀcalled "fragmentꢀbased
design." 2 This area of various classes of physiologically
active substances comprise oligopeptides capable of imꢀ
proving cognitive functions and influencing the central
nervous system. Let us concentrate our attention on two
examples.
An actively studied class of endogenous peptide regulaꢀ
tors is represented by peptides similar to adrenocorticotroꢀ
pic and melatocyte stimulating hormones, which are
known by the general name of melanocortins. At present,
synthetic heptapeptide Semax (Met—Glu—His—Phe—
—Pro—Gly—Pro), being a structural analog of adrenoꢀ
corticotropic hormone devoid of hormonal activity
(ACTH4—10),3 is the single widely used nootropic drug
used in clinical practice.4
In addition, it is known that the protein APP, viz., the
βꢀamyloid precursor (playing, probably, the key role in the
genesis of Alzheimer´s disease), performs important funcꢀ
tions in normal brain tissues and, particularly, plays a speꢀ
cific role in the formation of longꢀterm memory.5,6 When
We have previously12 developed a convenient method
for the synthesis of the antagonist for the first subtype of
glutamate receptors of 1ꢀaminoindaneꢀ1,5ꢀdicarboxylic
acid (AIDA). In continuation of these studies, we decided
to create hybrid molecules containing both the fragments
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 196—204, January, 2010.
1066ꢀ5285/10/5901ꢀ0200 © 2010 Springer Science+Business Media, Inc.

Synthesis of a "memory tripeptide"
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
201
of AIDA and its bioisosteres, aminophosphonates,13,14 and
the oligopeptide chain mimicking this "memory tripepꢀ
tide" RER (Arg—Glu—Arg). At the same time, the develꢀ
opment of a method for the synthesis of phosphorus anaꢀ
logs of peptides, i.e., phosphonopeptides based on αꢀamiꢀ
nophosphonates, is an independent problem.
A possible approach to the synthesis of αꢀaminophosꢀ
phonates is based on the involvement of amino acids and
their esters as the amine component into a new catalytic
process developed by us.14—16 In the present work, we exꢀ
tended the synthetic potential of the described method and
applied it to diꢀ and tripeptides.
Although tripeptide Arg—Glu—Arg is commercially
available (Cambridge Research Biochemicals Ltd; MWGꢀ
Biotech Ltd, UK), we synthesized it using the peptide
synthesis method17—19 adapted for laboratory practice acꢀ
cording to Scheme 1 from the derivatives of ωꢀnitroꢀLꢀ
arginine (1a—c) and αꢀpꢀnitrophenyl γꢀbenzyl NꢀBocꢀLꢀ
glutamate (2). Benzyl NꢀBocꢀωꢀnitroꢀLꢀargininate (1b)
was prepared from commercially available NꢀBocꢀωꢀniꢀ
troꢀLꢀarginine (1a) in 95% yield. The subsequent NꢀBocꢀ
deprotection in compound 1b affords benzyl ωꢀnitroꢀLꢀ
argininate (1c) in nearly quantitative yield. The protecting
groups of the starting amino acids were selected in such
a way that they could be removed in one step by catalytic
hydrogenolysis. Then the coupling of NꢀBocꢀprotected
pꢀnitrophenyl γꢀbenzyl glutamate (2) with Cꢀprotected
nitroarginine (1c) gave dipeptide glutamylarginine (3a)
(93% yield). The NꢀBocꢀdeprotection afforded the salt of
dipeptide 3b with trifluoroacetic acid. In the next step,
NꢀBocꢀprotected nitroarginine (1а) was introduced into
the reaction with dipeptide 3b in the presence of dicycloꢀ
hexylcarbodiimide (DCC) and Nꢀhydroxybenzotriazole
(HOBt). As a result, protected tripeptide 4a was obtained
in 60% yield. The target "memory tripeptide" 4b was obꢀ
tained as a salt upon treatment of compound 4a with triꢀ
fluoroacetic acid followed by hydrogenolysis on palladium.
The introduction of αꢀaminophosphonate as a Cꢀterꢀ
minus of the peptide fragment allows one to synthesize
phosphonopeptides, whose physiological activity is someꢀ
times several orders of magnitude higher than that of pepꢀ
tides.20,21 The synthesis of a phosphonopeptide was exemꢀ
plified by coupling of arginine, glutamic acid, and αꢀamiꢀ
nophosphonate 5 (Scheme 2). In the first step, NꢀBocꢀ
protected arginine (1а), preꢀactivated with DCC, reacted
with αꢀaminophosphonate 5 to give phosphonodipeptide
Scheme 1
Reagents and conditions: i. PhCH2Cl, DIEA, NaI, DMF; ii. TFA, 2 h; iii. DMF, DIEA, HOBt; iv. DCC, CH2Cl2, HOBt, 0 °C, 2 h;
v. 1) TFA, 2 h; 2) HCOONH4, Pd/C, AcOH—MeOH, 48 h.

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Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Matveeva et al.
Scheme 2
Reagents and conditions: 1) i. DCC, HOBt, CH2Cl2, 0 °C, 2 h; 2) 5; ii. 1) TFA, 2 h; 2) DMF, DIEA, HOBt, 2
6 in 60% yield. In the second step, phosphonotripeptide 7
was obtained in 90% yield upon coupling of phosphonoꢀ
dipeptide 6 with Bocꢀprotected αꢀ(pꢀnitrophenyl) γꢀbenꢀ
zyl glutamate (2).
dipeptide in this medium is a zwitterion, an equimolar
amount of triethylamine was added to the reaction mixꢀ
ture to liberate the amino group. In this case, aminophosꢀ
phonate 9b was obtained in 75% yield. The transesteriꢀ
fication of the phosphonate group seems to occur as
a side process.
Analogously we carried out the reactions with glycylꢀ
glycine (12) and glutathione (13). αꢀAminophosphonates
14 and 15 were synthesized in 70 and 40% yields, respecꢀ
tively (Scheme 4).
It is most likely that partial transesterification of the
phosphonate group occurs in trifluoroethanol, which is
detected in the 31Р NMR spectra (additional signals apꢀ
peared at δ 20—24).
We introduced dipeptide 3b into the Kabachnik—Fields
reaction. Indanꢀ1ꢀone, viz., the structural fragment of the
ligand of the AIDA glutamate receptors,22 was used as the
carbonyl component (Scheme 5). Due to the high solubilꢀ
ity of protected dipeptide 3b in organic solvents, the reacꢀ
tion occurs readily in dichloromethane at 30 °C to form
aminophosphonate 16 in 60% yield.
Model dipeptides were primarily used for the evaluaꢀ
tion of a possibility of using peptides as an amine compoꢀ
nent in the catalytic version of the Kabachnik—Fields reꢀ
action developed by us earlier.16 We found that glycylleuꢀ
cine methyl ester (8a) reacts with benzaldehyde and diꢀ
ethyl phosphite using [tetra(tertꢀbutyl)phthalocyanine]ꢀ
aluminum chloride (tPcAlCl) as the catalyst to give the
corresponding αꢀaminophosphonate 9a. The reaction was
carried out in methanol at ambient temperature in the
presence of molecular sieves 4 Å, but the yield of dipeptide
9a was only 15% because of the side formation of comꢀ
pounds 10 and 11 (Scheme 3).
We found that the reaction of nonꢀesterified dipeptide
8b with benzaldehyde catalyzed by phthalocyanines afꢀ
fords the corresponding αꢀaminophosphonate 9b. Since
dipeptide 8b is almost insoluble in methanol, the reaction
was carried out in boiling 2,2,2ꢀtrifluoroethanol. Since the
Scheme 3
8: R1 = Me (a), H (b)
9: R1 = Me, R2 = Et (a); R1 = H, R2 = Et (b)

Synthesis of a "memory tripeptide"
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
203
Scheme 4
The next step to the synthesis of "memory tripeptide"
analogs was the introduction of tripeptide 4a as the amine
component into the catalytic Kabachnik—Fields reaction
(Scheme 6). Indanꢀ1ꢀone and 5ꢀbenzyloxycarbonylindanꢀ
1ꢀone as the carbonyl components and diethyl phosphite
as the P—H component were used in the reaction. The
Boc group was removed from protected arginylglutaꢀ
mylarginine 4a by treatment with trifluoroacetic acid and the
resulting salt was introduced into the Kabachnik—Fields
31P NMR spectroscopy, IR spectroscopy, and mass specꢀ
trometry.
The IR spectra of compounds 6, 7, 9, and 14—19 conꢀ
tain absorption bands at 1250—1260 cm–1 corresponding
to the Р=О group, at 1600—1640 cm–1 corresponding to the
C=N group of the guanidine fragments and the NO2 group
(for compounds 7 and 16—19), and at 1680—1690 and
1720—1740 cm–1 characteristic of C=O of the amide and
carboxylic acid groups . The broad bands corresponding to
t
reaction in the presence of PcAlCl and an equimolar
the OH and NH groups are observed at 3250—3500 cm–1
.
amount of diisopropylethylamine. As a result, aminophosꢀ
phonates 17 and 18 were obtained in 60% yields. The final
step of the synthesis of the phosphorusꢀcontaining "memꢀ
ory tripeptide" analog, namely, removal of protecting
groups, for compound 17 was carried out by catalytic hyꢀ
drogenolysis in the presence of Pd/C in aqueous acetic
acid at ambient temperature. The target aminophosphoꢀ
nate 19 was obtained in 95% yield.
Since in this process we used peptides based on optiꢀ
cally active Lꢀamino acids, aminophosphonates 6, 7, 9,
and 15—19 are mixtures of diastereomers that differ in
configurations of the carbon atom bound to the phosphoꢀ
1
rus atom. Therefore, the 31P, H, and 13C NMR spectra
exhibit doubling or broadening of the majority of signals.
The 31Р NMR spectra of compounds 6, 7, 9, and 14—19
contain signals at δ 20—31 corresponding to the dialkylꢀ
aminophosphonate groups.
The approach developed opens a way to the synthesis
of phosphonopeptides.
1
The H NMR spectra of all synthesized aminophosꢀ
All oligopeptides synthesized and their phosphoꢀ
rus analogs were characterized by 1H, 13C, and
phonates 6, 7, 9, and 14—19 exhibit signals for nonequiꢀ
valent phosphonate ethoxy groups: triplets of the methyl
Scheme 5

204
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Matveeva et al.
Scheme 6
carbon atoms of the peptide fragment appear at δ 51—56.
The chemical shifts of the carbon atoms of the guanidine
groups lie at δ 159—160 (δ 155.6 for the unprotected group
of compound 19). The signals of the carboxylic and amide
carbon atoms are present at δ 169—179. Other signals in
the 1H and 13C NMR spectra of the αꢀphosphorus peptide
analogs correspond to the structure of the carbon skeleton
of the starting carbonyl compound and peptides.
Experimental
The 1H (400 MHz), 13C (100.61 MHz), and 31P (161.98 MHz)
NMR spectra were recorded for solutions in CDCl3, CD3OD,
D2O, and CD3COOD on a Bruker Avance 400 instrument relaꢀ
tive to Me4Si as an internal standard (1H, 13C). Chemical shifts
were measured in the δ scale. IR spectra were obtained on
a URꢀ20 instrument in CCl4. Elemental analysis was carried out
on a VarioꢀII CHN analyzer. Mass spectra (MALDI—TOF) were
measured on an Autoflex II instrument (Bruker Daltonics) using
dihydroxybenzoic acid (DHB) as a matrix.
R = H (17), COOCH2Ph (18)
Reagents and conditions: 1) CF3COOH, 2 ; 2)
,
HP(O)(OEt)2, tPcAlCl, CH2Cl2, DIEA
NαꢀBocꢀNωꢀnitroꢀLꢀarginine (1a) and αꢀpꢀnitrophenyl γꢀbenꢀ
zyl NꢀBocꢀglutamate (2) (Reanal) were used without additional
purification. [Tetra(tertꢀbutyl)phthalocyanine]aluminum chloꢀ
ride was synthesized according to the known23 procedure.
The reactions were monitored, and chromatographic sepaꢀ
ration was carried out, using TLC on plates Merck TLC Silica gel
60 F254 and on columns with silica gel Merck 60 (70—230 mesh
ASTM).
Benzyl NαꢀBocꢀNωꢀnitroꢀLꢀargininate (1b).17 A mixture of
NαꢀBocꢀNωꢀnitroꢀLꢀarginine (1a) (9.6 g, 30 mmol), diisopropylꢀ
ethylamine (DIEA) (7 mL, 40 mmol), benzyl chloride (5.6 mL,
48 mmol), and NaI (0.35 g) in DMF (20 mL) was stirred for 7 h
at 80 °C. The reaction mixture was cooled and poured into ice
water (150 mL). The reaction product was extracted with ethyl
acetate (100 mL). The extract was consecutively washed with
a 1 М solution of NaHCO3 and brine and dried with sodium
sulfate. The solution was concentrated in vacuo. A yellow oily
substance was obtained in a yield of 11.72 g (95%), Rf 0.65
(CHCl3 : CF3CH2OH, 10 : 1). 1H NMR, δ: 1.42 (s, 9 H, But);
1.68 (br.m, 2 H, CHCH2CH2CH2, Arg); 1.87 (br.m, 2 H,
groups at δ 1.0—1.4 and multiplets of the methylene proꢀ
tons at δ 3.6—4.6. The signals for the proton at the
αꢀcarbon atom of the aminophosphonate unit (compounds
9, 14, and 15) are observed at δ 3.3—5.0 as two doublets of
the diastereomeric pair with the spinꢀspin coupling conꢀ
stant 2JH,P = 10.6—14.6 Hz. In compounds 9 and 14, the
signals for the diastereotopic methylene protons appear
as two doublets at δ 3.0—3.7 (JАВ = 17.0—18.8 Hz).
No protons of the hydroxy and amino groups are manifestꢀ
ed due to deuterioexchange. The signals corresponding
to the αꢀprotons of the amino acid fragments appear at
δ 3.5—4.7.
CHCH2CH2CH2, Arg); 3.27, 3.44 (both br.m,
2 H,
CHCH2CH2CH2, Arg); 4.35 (m, 1 H, CH, Arg); 5.16 (m, 2 H,
CH2Ph); 7.34 (m, 5 H, arom.).
Benzyl NωꢀnitroꢀLꢀargininate trifluoroacetate (1c).17 Benzyl
NꢀBocꢀNωꢀnitroꢀLꢀargininate (1b) (8.2 g, 20 mmol) was dissolved
in trifluoroacetic acid (60 mL). The solution was stirred for ~2 h
until the starting compound disappeared from the reaction mixꢀ
ture (TLC monitoring). Trifluoroacetic acid was evaporated
in vacuo. A yellow oily substance was obtained in a yield of 8.45 g
(99%), Rf 0.05 (CHCl3—CF3CH2OH, 10 : 1). 1H NMR, δ: 1.64,
1.74, 1.97 (all br.m, 4 H, CHCH2CH2CH2, Arg); 3.22, 3.24
(both t, 2 H, CHCH2CH2CH2, Arg, 2JH,H = 5.8 Hz); 4.18 (t, 1 H,
CH, Arg, 2JH,H = 5.8 Hz); 5.26 (m, 2 H, CH2Ph); 7.41 (m, 5 H,
arom.).
The 13C NMR spectra of the phosphorus analogs of
peptides 6, 7, 9, and 15—19 contain doubled signals of
diastereomeric mixtures. The signal for the αꢀcarbon atom
Benzyl NαꢀBocꢀ(γꢀbenzyl αꢀLꢀglutamyl)ꢀNωꢀnitroꢀLꢀargiꢀ
ninate (3a).19 A solution of compound 2 (3 g, 6.54 mmol) and
Nꢀhydroxybenzotriazole (HOBt) (0.88 g, 6.54 mmol) in DMF
(11 mL) was added to a solution of HArg(NO2)OBzl•CF3COOH
(1c) (2.77 g, 6.54 mmol) and DIEA (2.3 mL, 13.2 mmol) in DMF
1
at the phosphorus atom appears at δ 53—69 with JP,C
=
= 149—165 Hz. The chemical shifts of the diastereotopic
ethoxy groups at the phosphorus atom lie at δ 15.5—20
(CH3) and δ 62—65 (OCH2). The signals of the chiral

Synthesis of a "memory tripeptide"
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
205
(15 mL) cooled to 4 °C. The mixture was stirred with cooling for
2 h and kept for 12 h at 4 °C. Then the reaction mixture was
poured into water and extracted with ethyl acetate (250 mL).
The organic phase was washed with a saturated solution of
NaHCO3 (3×60 mL), brine (2×60 mL), a 10% solution of
citric acid (2×60 mL), and again with brine (2×60 mL) and dried
with Na2SO4. The solution was concentrated in vacuo, the resiꢀ
due was dissolved in a minimum amount of dichloromethꢀ
ane, and chromatographed on a column with silica gel in diꢀ
chloromethane—methanol (50 : 1) solvent mixture as the eluent.
A solid yellow glassy substance was isolated in a yield of
(m, 10 H, arom.). 13C NMR, δ: 24.08, 24.41 (C(4), 2 Arg); 26.63
(C(3), Glu); 28.14 (CH3, But); 29.15 (C(4), Glu); 30.11 (C(3),
2 Arg); 40.63 (C(5), 2 Arg); 51.51 (C(2), Glu); 52.97, 53.48 (C(2),
2 Arg); 66.79, 67.65 (CH2Ph); 80.78 (C, But); 128.18, 128.38,
128.52, 128.59, 128.66, 134.89, 135.49 (C arom.); 155.36 (C=O,
Boc); 159.07, 159.16 (C=NH, 2 Arg); 171.36, 172.08 (NHC=O,
2 Arg); 173.17, 173.39 (C(O)OBzl, Arg, Glu). MS (MALDI—TOF),
m/z: 829 [М]+.
LꢀArginylꢀαꢀLꢀglutamylꢀLꢀarginine tetraacetate (4b). Comꢀ
pound 4a (415 mg, 0.5 mmol) was dissolved in a mixture of
trifluoroacetic acid (8 mL) and dichloromethane (8 mL). The
resulting solution was stirred for ~3 h until the starting comꢀ
pound disappeared from the reaction mixture (TLC). Trifluoroꢀ
acetic acid was evaporated in vacuo, and the residue was dried to
a constant weight. The obtained glassy substance was dissolved
in a mixture of acetic acid (5 mL) and methanol (5 mL), and
ammonium formate (300 mg) was added; 5% Pd/C (600 mg)
was added in 200ꢀmg portions every 8 h. The mixture was stirred
for 48 h (TLC). After the reaction was over, the catalyst was
filtered off, and the filtrate was concentrated in vacuo. The residue
(oily substance) was dissolved in 50% AcOH, the solvent was evaꢀ
porated, and the residue was triturated with acetonitrile. A light
orange amorphous substance was obtained in a yield of 330 mg
(90%). 1H NMR, δ: 1.58—1.93 (m, 8 H, CHCH2CH2CH2,
2 Arg); 1.98—2.20 (m, 2 H, CHCH2CH2, Glu); 2.29—2.41 (m, 2 H,
CHCH2CH2, Glu); 3.21—3.38 (m, 4 H, CHCH2CH2CH2,
2 Arg); 4.11 (t, 1 H, CH, Arg, J = 6.23 Hz); 4.18 (dd, 1 H, CH,
Arg, J = 5.61 Hz, J = 7.47 Hz); 4.39 (dd, 1 H, CH, Glu, J = 4.98 Hz,
J = 9.34 Hz). 13C NMR, δ: 26.26, 27.22 (C(4), 2 Arg); 30.38,
30.81, 31.47 (C(3), 2 Arg, C(3), Glu); 36.22 (C(4), Glu); 40.18,
40.46 (C(5), 2 Arg); 55.43, 56.84, 57.64 (C(2), Glu, C(2), 2 Arg);
159.52, (br.s, C=NH, 2 Arg); 172.42, 175.28, (NHC=O,
Arg, Glu); 180.88, 184.06 (COOH, Arg, Glu, AcOH). MS
(MALDI—TOF), m/z: 459 [М]+.
1
3.82 g (93%), Rf 0.35 (CHCl3—CF3CH2OH, 10 : 1). H NMR,
δ: 1.38 (s, 9 H, But); 1.60 (br.m, 2 H, CHCH2CH2CH2, Arg);
1.73, 1.87 (both br.m, 2 H, CHCH2CH2CH2, Arg); 1.97, 2.14
(both m, 2 H, CHCH2CH2, Glu); 2.49 (m, 2 H, CHCH2CH2,
Glu); 3.19, 3.41 (both br.m, 2 H, CHCH2CH2CH2, Arg); 4.33
(m, 1 H, CH, Arg); 4.63 (m, 1 H, CH, Glu); 5.10 (m, 4 H,
CH2Ph); 7.33 (m, 10 H, arom.). 13C NMR, δ: 24.25 (C(4), Arg);
27.33 (C(3), Glu); 28.05 (CH3, But); 29.30 (C(4), Glu); 30.15
(C(3), Arg); 40.34 (C(5), Arg); 51.16 (C(2), Glu); 53.64 (C(2),
Arg); 66.39, 67.27 (CH2Ph); 80.08 (C, But); 128.01, 128.11,
128.20, 128.40, 128.47, 134.86, 135.49 (C arom.); 155.81 (C=O,
Boc); 159.09 (C=NH, Arg); 171.23 (NHC=O, Arg); 172.92
(C(O)OBzl, Arg, Glu).
Benzyl (γꢀbenzyl αꢀLꢀglutamyl)ꢀNωꢀnitroꢀLꢀargininate triꢀ
fluoroacetate (3b).19 BocꢀGlu(OBzl)Arg(NO2)OBzl (3a) (1.9 g,
3 mmol) was dissolved in 20 mL of trifluoroacetic acid. The
solution was stirred for ~2 h until the starting compound disapꢀ
peared from the reaction mixture (TLC). Trifluoroacetic acid
was evaporated in vacuo. A yellow oily substance was obtained in
a yield of 1.93 g (99%), Rf 0.07 (CHCl3—CF3CH2OH, 10 : 1).
1H NMR, δ: 1.62 (br.m, 2 H, CHCH2CH2CH2, Arg); 1.71, 1.93
(both br.m, 2 H, CHCH2CH2CH2, Arg); 2.15 (br.m, 2 H,
CHCH2CH2, Glu); 2.58 (m, 2 H, CHCH2CH2, Glu); 3.13, 3.21
(both br.m, 2 H, CHCH2CH2CH2, Arg); 4.41 (m, 1 H, CH,
Arg); 4.60 (m, 1 H, CH, Glu); 5.07 (m, 4 H, CH2Ph); 7.33
(m, 10 H, arom.).
Benzyl NαꢀBocꢀNωꢀnitroꢀLꢀarginylꢀ(γꢀbenzyl αꢀLꢀglutamyl)ꢀ
NωꢀnitroꢀLꢀargininate (4a). A solution of NαꢀBocꢀNωꢀnitroꢀLꢀ
arginine (1a) (0.96 g, 3 mmol) and HOBt (0.54 g, 4 mmol) in
CH2Cl2 (15 mL) was cooled to 0 °C, and DCC (0.62 g, 3 mmol)
was gradually added with stirring at a temperature not higher
than 5 °C. The mixture was stirred with cooling for 2 h and kept
for 12 h at 4 °C. After this, a solution of compound 3b (1.93 g,
3 mmol) and DIEA (1.05 mL, 6 mmol) in CH2Cl2 (15 mL) was
added dropwise with cooling to the mixture. The mixture was
stirred with cooling for 4 h and left for 12 h at 4 °C. Then the
mixture was poured into water and extracted with ethyl acetate
(100 mL). The organic phase was washed with a saturated soluꢀ
tion of NaHCO3 (2×40 mL), brine (1×40 mL), a 10% solution of
citric acid (2×40 mL), and again with brine (1×40 mL) and dried
with Na2SO4. A white crystalline substance was isolated by colꢀ
umn chromatography on silica gel (dichloromethane—methanol
(20 : 1) as the eluent) in a yield of 1.5 g (60%). Found (%):
C, 52.28; H, 6.16; N, 18.55. C36H51N11O12. Calculated (%):
C, 52.10; H, 6.19; N, 18.57. 1H NMR, δ: 1.39 (s, 9 H, But); 1.61
(br.m, 4 H, CHCH2CH2CH2, 2 Arg); 1.74, 1.90 (both br.m, 4 H,
CHCH2CH2CH2, 2 Arg); 1.97, 2.10 (both m, 2 H, CHCH2CH2,
Glu); 2.46 (m, 2 H, CHCH2CH2, Glu); 3.14, 3.62 (both br.m,
4 H, CHCH2CH2CH2, 2 Arg); 4.18, 4.48 (both m, 2 H, CH,
2 Arg); 4.58 (m, 1 H, CH, Glu); 5.12 (m, 4 H, 2 CH2Ph); 7.32
Diethyl (1ꢀaminoꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀyl)phosphonate
(5).24 Ammonium carbonate (0.58 g, 6 mmol), molecular sieves
4 Å (1.5 g), and PctAlCl (0.16 g, 0.2 mmol) were added to
a solution of 2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀone (0.8 g, 6 mmol) in
anhydrous ethanol (9 mL). The reaction mixture was heated to
60 °C and stirred for 3—4 h, and then diethyl phosphite (0.93 mL,
7.2 mmol) was added. The reaction mixture was refluxed with
stirring for 24 h. The molecular sieves were filtered off and
washed with methanol (3×2 mL), and the filtrate was concenꢀ
trated in vacuo. Column chromatography in CH2Cl2—MeOH
(10 : 1) as the eluent afforded an orange oily substance in a yield
1
of 0.57 g (35%), Rf 0.22 (CHCl3—MeOH, 20 : 1). H NMR, δ:
1.15, 1.30 (both t, 6 H, 2 CH3, J = 7.07 Hz); 2.24, 2.80 (both m,
2 H, CН2 cycl.); 3.02 (m, 2 Н, cycl.); 3.10 (br.m, NH2); 3.91,
4.09 (both m, 4 Н, ОCН2); 7.24, 7.62 (both m, 4 Н, arom.).
31P NMR, δ: 23.18. IR, ν/cm–1: 1235 (Р=О); 3290, 3375,
3470 (NH2).
NαꢀBocꢀNꢀ[1ꢀ(Diethoxyphosphoryl)ꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ
1ꢀyl]ꢀNωꢀnitroꢀLꢀargininamide (6). A solution of NαꢀBocꢀNωꢀniꢀ
troꢀLꢀarginine (1a) (0.32 g, 1 mmol) and Nꢀhydroxybenzotriazꢀ
ole (0.32 g, 1.2 mmol) in DMF (5 mL) was cooled to 0 °C. Then
DCC (0.21 g, 1 mmol) was gradually added with cooling at such
a rate that the temperature of the solution did not exceed 5 °C.
The mixture was stirred with cooling for 1 h, and a solution of
diethyl (1ꢀaminoꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀyl)phosphonate (5)
(0.27 g, 1 mmol) and diisopropylethylamine (0.36 mL, 2 mmol)
in DMF (4 mL) was added dropwise. The mixture was stirred

206
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Matveeva et al.
with cooling for 2 h and kept for 12 h at 4 °C, then poured into
water, and extracted with ethyl acetate (60 mL). The organic
phase was worked up as described for the synthesis of 4a. An
orange glassy solid substance was obtained in a yield of 0.35 g
(60%). 1Н NMR, δ: 1.17, 1.34 (both t, 6 H, 2 CH3, J = 7.07 Hz);
1.41 (s, 9 H, But); 1.70 (br.m, 2 H, CHCH2CH2CH2, Arg); 1.92
(br.m, 2 H, CHCH2CH2CH2, Arg); 2.48—2.73 (m, 2 H, CH2
cycl.); 3.05—3.33 (m, 4 H, CH2 cycl., CHCH2CH2CH2, Arg);
4.29—4.40 (m, 4 H, 2 OCH2); 4.67 (m, 1 H, CH, Arg); 7.28
(m, 4 H, arom.). 31P NMR: δ 23.32.
(m, 3 H, CH2, CH(CH3)2); 3.04 (HA), 3.23 (HB) (AB system,
2 H, Gly, 2JH,H = 17.1 Hz); 3.39 (d, αꢀCH, JH,P=10.6 Hz); 3.64,
3.66 (both s, 3 H, OMe); 3.77—4.07 (m, 4 H, 2 OCH2);
4.50—4.55 (m, 1 H, CH (Leu)); 7.19—7.27, 7.40—7.51 (both m,
5 H, arom.). 31P NMR, δ: 21.65, 21.77. IR, ν/cm–1: 990, 1180
(P—O—Alk); 1250 (P=О); 1680 (C=O, amide); 1740 (C=O);
3270 (NH).
Diethyl [hydroxy(phenyl)methyl]phosphonate (10) was isolatꢀ
ed in a yield of 0.17 g (35%), Rf 0.49 (CHCl3—MeOH, 10 : 1).
1Н NMR, δ: 1.22, 1.27 (both t, 6 Н, 2 CН3, J = 7.07 Hz); 4.06,
4.15 (both m, 4 Н, 2 ОCН2); 4.39 (br.s, 1 Н, ОН); 5.03 (d, 1 Н,
Nαꢀ[NꢀBocꢀ(γꢀBenzyl) αꢀLꢀglutamyl]ꢀNꢀ[1ꢀ(diethoxyphosꢀ
phoryl)ꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀyl]ꢀNωꢀnitroꢀLꢀargininamide
(7). NαꢀBocꢀNꢀ[1ꢀ(diethoxyphosphoryl)ꢀ2,3ꢀdihydroꢀ1Hꢀinꢀ
denꢀ1ꢀyl]ꢀNωꢀnitroꢀLꢀargininamide (6) (0.34 g, 0.6 mmol) was
dissolved in trifluoroacetic acid (15 mL). The solution was stirred
for 16 h. Trifluoroacetic acid was evaporated in vacuo. The amine
(0.39 g) was obtained as a salt. This salt and DIEA (0.21 mL,
1.2 mmol) were dissolved in DMF (5 mL). The solution was
cooled to 4 °C, and a solution of compound 2 (0.28 g, 0.6 mmol)
and HOBt ((0.11 g, 0.8 mmol) in DMF (5 mL) was added. The
mixture was stirred with cooling for 3 h and kept for 12 h at
ambient temperature. Then the reaction mixture was poured into
water and extracted with ethyl acetate (50 mL). Workꢀup as deꢀ
scribed above and column chromatography (CHCl3—MeOH,
20 : 1) afforded an orange glassy solid substance in a yield of 0.43 g
(90%). 1Н NMR, δ: 1.23, 1.37 (both t, 6 H, 2 CH3, J = 7.07 Hz);
1.40 (s, 9 H, But); 1.65 (br.m, 2 H, CHCH2CH2CH2, Arg);
1.85—1.99 (m, 2 H, CHCH2CH2CH2, Arg); 2.12—2.21 (m, 2 H,
CHCH2CH2, Glu); 2.34 (m, 2 H, CHCH2CH2, Glu); 2.48—2.53
(m, 2 H, CH2 cycl.); 3.23, 3,42 (both m, 2 H, CH2 cycl.); 3.61
(br.m, 2 H, CHCH2CH2CH2, Arg); 4.09 (m, H, CH, Glu); 4.29,
4.42—4.48 (both m, 4 H, 2 OCH2); 4.64—4.69 (m, 1 H, CH,
Arg); 5.09 (m, 2 H, CH2Ph); 7.32 (m, 9 H, arom.). 31P NMR, δ:
22.42. 13C NMR, δ: 19.86 (CH3, POEt); 24.70, 24.81 (C(4),
Arg); 27.55 (C(3), Glu); 27.83 (C(3)H2, cycl.); 28.00 (C(4), Glu);
28.27 (CH3, But); 30.28, 30.34 (C(3), Arg); 33.82 (2CH2, cycl.);
40.22, 40.29 (C(5), Arg); 51.24, 51.34 (C(2), Arg); 53.53 (C(2),
2
CН, JН,Р = 11 Hz); 7.37—7.50 (m, 5 Н, arom.). 31Р NMR, δ:
21.58. IR, ν/cm–1: 1240 (P=О); 3280 (OH). Found (%): C, 54.29;
Н, 7.00. C11Н17О4Р. Calculated (%): C, 54.09; Н, 6.97.
3ꢀIsobutylpiperazineꢀ2,5ꢀdione (11) was also isolated from the
reaction mixture in a yield of 0.45 g (81%), Rf 0.05 (CHCl3—MeOH,
10 : 1). IR, ν/cm–1: 1675, 1690 (C=O), 3250 (NH). M.p. 255 °C.
Nꢀ{(Diethoxyphosphoryl)phenyl)methyl}glycylꢀLꢀleucine (9b).
GlycylꢀLꢀleucine (8b) (0.42 g, 2.2 mmol), molecular sieves 4 Å
(500 g), PctAlCl (0.08 g, 0.1 mmol), and triethylamine (0.3 mL,
2.2 mmol) were added to a solution of benzaldehyde (0.2 mL,
2 mmol) in 2,2,2ꢀtrifluoroethanol (4 mL). The reaction mixture
was heated to 60 °C and stirred for 3 h, and diethyl phosphite
(0.39 mL, 3 mmol) was added. The reaction mixture was refluxed
with stirring for 120 h. Molecular sieves were filtered off and
washed with methanol (3×2 mL), and the filtrate was concenꢀ
trated in vacuo. After column chromatography (CHCl3—MeOH,
10 : 1), an orange oily substance was obtained in a yield of 0.63 g
(75%), Rf 0.31 (CHCl3 : MeOH, 10 : 1). Found (%): C, 55.15;
H, 7.74; N, 6.60. C19H31N2O6P. Calculated (%): C, 55.06; H,
7.54; N, 6.76. 1Н NMR, δ: 0.94, 0.99 (both d, 6 H, 2 CH3 (Leu),
J = 6.0 Hz); 1.07—1.13, 1.27—1.36 (both t, 6 H, 2 CH3 (Et),
J = 7.07 Hz); 1.57—1.81 (m, 3 H, CH2, CH(CH3)2); 3.13 (HA),
3.40 (HB) (AB system, 2 H, Gly, 2JH,H = 17.0 Hz); 3.30 (d, αꢀCH,
2JH,P = 11.3 Hz); 3.64—3.78, 3.87—3.97, 4.06—4.30 (three m,
4 H, 2 OCH2); 4.50—4.55 (m, 1 H, CH(COOH)); 7.34—7.43
(m, 5 H, arom.). 31P NMR: δ 23.13, 23.41. 13C NMR, δ:
1
Glu); 64.58 (d, C(1), cycl., JC,P =154.4 Hz); 60.39, 61.06
16.23—16.43 (CH3, Et); 21.83, 22,88, 24.85, 29.69 (Leu); 40.61,
(OCH2); 66.5 (CH2Ph); 80.24 (C, But); 125.7, 126.60, 126.95,
127.23, 128.13, 128.30, 128.57, 135.69, 138.36, 142.00 (C arom.);
158.55 (C=NH, Arg); 172.01, 173.09, 173.22, 176.16, 176.21
(NHC=O, Glu, Arg, C(O)OBzl, Glu). MS (MALDI—TOF),
m/z: 789 [М]+, 652 [M – P(O)(OEt)2]+.
40.96 (Leu); 50.80, 50.90 (CH2, Gly); 59.83 (d, αꢀC, JC,P
=
1
= 158.1 Hz); 63.54, 64.07 (both d, 2 OCH2); 128.19—128.91
(C arom.); 174.84, 175.02 (C=O, Gly, Leu). IR, ν/cm–1: 980,
1190 (P—O—Alk); 1250 (P=О); 1690 (C=O, amide); 1745
(C=O, acidic); 3280 (NH).
Methyl Nꢀ{(diethoxyphosphoryl)(phenyl)methyl}glycylꢀLꢀ
leucinate (9a). A solution of methyl glycylꢀLꢀleucinate hydroꢀ
chloride (8a) (0.75 g, 3.2 mmol) in 1 М NaOH (20 mL) was
extracted with ethyl acetate (3×29 mL). The extract was dried
with anhydrous Na2SO4 and concentrated in vacuo. Benzaldeꢀ
hyde (0.2 mL, 2 mmol), molecular sieves 4 Å, and tPcAlCl (0.08 g,
0.1 mmol) were added to a solution of the obtained methyl glyꢀ
cylꢀLꢀleucinate (0.3 g, 1.5 mmol) in methanol (3 mL). The reacꢀ
tion mixture was stirred for 3 h, and an additional portion of the
dipeptide ester (0.3 g, 1.5 mmol) was added. The reaction mixꢀ
ture was stirred for 3 h more, and diethyl phosphite (0.39 mL,
3 mmol) was added. After 72 h (TLC), molecular sieves
were filtered off and washed with methanol (3×2 mL). The filꢀ
trate was concentrated in vacuo. After column chromatography
(CH2Cl2—MeOH, 20 : 1), an orange oily substance was obtained
in a yield of 0.13 g (15%), Rf 0.50 (CHCl3—MeOH, 10 : 1).
1Н NMR, δ: 0.85, 0.88 (both d, 6 H, 2 CH3 (Leu), J = 6.0 Hz);
1.01, 1.21 (both t, 6 H, 2 CH3 (Et), J = 7.07 Hz); 1.49—1.60
Nꢀ{(Diethoxyphosphoryl)(phenyl)methyl}glycylglycine (14).
Glycylglycine (12) (0.29 g, 2.2 mmol), molecular sieves 4 Å (500 g),
tPcAlCl (0.08 g, 0.1 mmol), and triethylamine (0.3 mL, 2.2 mmol)
were added to a solution of benzaldehyde (0.2 mL, 2 mmol) in
2,2,2ꢀtrifluoroethanol (6 mL). The reaction mixture was heated
to 60 °C and stirred for 3 h, and diethyl phosphite (0.39 mL,
3 mmol) was added. The reaction mixture was refluxed with
stirring for 120 h. Molecular sieves were filtered off and washed
with methanol (3×2 mL), and the filtrate was concentrated
in vacuo. After column chromatography (CHCl3—MeOH,
10 : 1), an orange oily substance was obtained in a yield of 0.51 g
(70%), Rf 0.27 (CHCl3 : MeOH, 10 : 1). Found (%): C, 50.05;
H, 6.37; N, 7.90. C15H23N2O6P. Calculated (%): C, 50.28;
H, 6.47; N, 7.82. 1Н NMR, δ: 1.27 (t, 6 H, 2 CH3 (Et), J = 7.07 Hz);
2
3.46 (HA), 3.70 (HB) (AB system, 2 H, Gly, JH,H = 18.8 Hz);
3.43—3.60, 3.66—3.89, 4.00—4.09 (all m, 4 H, 2 OCH2);
2
4.19 (m, 2 H, CH2(COOH)); 4.79 (d, αꢀCH, JH,P=14.6 Hz);
7.34—7.43 (m, 5 H, atom.). 31P NMR, δ: 21.61. IR, ν/cm–1: 980,

Synthesis of a "memory tripeptide"
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
207
1190 (P—O—Alk); 1250 (P=О); 1690 (C=O, amide); 1745
(C=O, acidic); 3280 (NH).
(8 mL), and 2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀone (0.4 g, 3 mmol), moꢀ
lecular sieves 4 Å (500 mg), PcAlCl (0.08 g, 0.1 mmol), and
t
Nꢀ[(Diethoxyphosphoryl)phenyl)methyl]ꢀLꢀγꢀglutamylꢀLꢀcysꢀ
teinylglycine (15). Glutathione (13) (0.68 g, 2.2 mmol), molecuꢀ
lar sieves 4 Å (500 mg), tPcAlCl (0.08 g, 0.1 mmol), and triethylꢀ
amine (0.46 mL, 3.3 mmol) were added to a solution of benzalꢀ
dehyde (0.2 mL, 2 mmol) in 2,2,2ꢀtrifluoroethanol (4 mL). The
reaction mixture was heated to 60 °C and stirred for 3 h, and
diethyl phosphite (0.39 mL, 3 mmol) was added. The mixture
was refluxed with stirring for 120 h. Molecular sieves were
filtered off and washed with methanol (3×2 mL), and the filtrate
was concentrated in vacuo. After column chromatography
(CHCl3—MeOH, 15 : 1), an orange oily substance was obꢀ
tained in a yield of 0.46 g (40%), Rf 0.37 (CHCl3 : MeOH, 10 : 1).
Found (%): C, 47.65; H, 6.27; N, 7.74. C21H32N3O9PS. Calcuꢀ
lated (%): C, 47.27; H, 6.05; N, 7.88. 1Н NMR, δ: 1.20, 1.25
(both t, 6 H, 2 CH3 (Et), J = 7.07 Hz); 1.81—2.04, 2.23—2.50
DIEA (0.35 mL, 2 mmol) were added. The reaction mixture was
stirred for 3 h at 30 °C, diethyl phosphite (0.2 mL, 1.5 mmol) was
added, and the mixture was stirred for 1 day at 30 °C. Then more
diethyl phosphite (0.2 mL, 1.5 mmol) was added. The reaction
was monitored by TLC, this lasted for 120 h. The molecular
sieves were filtered off and washed with methanol (3×2 mL), and
the filtrate was concentrated in vacuo. After column chromatogꢀ
raphy (CHCl3—MeOH, 20 : 1), an orange oily substance was
obtained in a yield of 0.59 g (60%). 1Н NMR, δ: 1.18—1.35 (m, 6 H,
2 CH3); 1.58 (br.m, 4 H, CHCH2CH2CH2, 2 Arg); 1.73, 1.91
(both br.m, 4 H, CHCH2CH2CH2, 2 Arg); 1.98, 2.10 (both m,
2 H, CHCH2CH2, Glu); 2.30—2.60 (m, 4 H, CHCH2CH2, Glu,
C(2)H2, cycl.); 2.87—3.06 (m, 2 H, 3CH2, cycl.); 3.12—3.33
(br.m, 4 H, CHCH2CH2CH2, 2 Arg); 3.57, 3.81 (both m, H,
CH, Arg); 4.01, 4.62 (both m, 6 H, CH, Arg, CH, Glu, 2 OCH2);
5.09 (m, 4 H, 2 CH2Ph); 7.18—7.76 (m, 14 H, arom.). 31P NMR,
δ: 25.17, 25.62. 13C NMR, δ: 15.97—16.21 (Me, POEt); 24.37,
24.48 (C(4), 2 Arg); 26.58 (C(3), Glu); 28.83 (C(4), Glu); 30.08
(C(3), 2 Arg); 32.01 (3CH2, cycl.); 33.28 (2CH2, cycl.); 40.67
(C(5), 2 Arg); 51.86, 52.53 (C(2), Glu); 52.96, 54.49, 55.50,
55.84 (C(2), 2 Arg); 63.21, 64.04 (OCH2); 66.81, 67.62 (CH2Ph);
68.06, 68.40 (both d, C(1), 1JC,P = 158.8 Hz, 1JC,P = 159.5 Hz);
125.14, 126.48, 126.69, 128.19, 128.38, 128.63, 129.12, 134.93,
135.46, 139.33, 144.81 (C arom.); 159.17 (br.s, C=NH, 2 Arg);
171.40, 173.24 (NHC=O, Arg, Glu); 173.50, 176.34 (C(O)OBzl,
Arg, Glu). IR, ν/cm–1: 970, 1190 (P—O—Alk); 1250 (br, N=O,
NO2, P=О); 1600, 1630 (br, N=O, NO2, C=N, amide); 1730
(C=O); 3300 (NH). MS (MALDI—TOF), m/z: 981 [М]+, 844
[M – P(O)(OEt)2]+.
3
(both m, 4 H, CH2CH2, Glu); 3.45—3.87 (m, 3 H, CH2, Cys,
2CH, Glu); 3.93—4.25 (m, 6 H, 2 OCH2, CH2, Gly); 4.31—4.37
(m, H, 2CH, Cys); 5.01 (d, αꢀCH, 2JHP = 11.1 Hz); 7.28—7.39,
7.49 (m, 5 H, arom.). 31P NMR, δ: 21.54.
Benzyl Nꢀ(1ꢀ(diethoxyphosphoryl)ꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀ
yl)ꢀ(γꢀbenzyl)ꢀαꢀLꢀglutamylꢀNωꢀnitroꢀLꢀargininate (16). 2,3ꢀDihyꢀ
droꢀ1Hꢀindenꢀ1ꢀone (0.4 g, 3 mmol), molecular sieves 4 Å
(500 mg), PctAlCl (0.08 g, 0.1 mmol), and diisopropylethylamine
(0.35 mL, 2 mmol) were added to a solution of dipeptide 3b
(1.3 g, 2 mmol) in dichloromethane (7 mL). The reaction mixꢀ
ture was stirred for 3 h at 30 °C, diethyl phosphite (0.39 mL,
3 mmol) was added, and the mixture was stirred for 1 day at 30 °C.
Then more diethyl phosphite (2 mmol) was added. The duration
of the reaction was 72 h (TLC). Molecular sieves were filtered
off and washed with methanol (3×2 mL), and the filtrate
was concentrated in vacuo. After column chromatography
(CHCl3—MeOH, 20 : 1), an orange oily substance was obtained
in a yield of 1.09 g (60%). 1Н NMR, δ: 1.30 (t, 6 H, 2 CH3,
J = 7.07 Hz); 1.57 (br.m, 2 H, CHCH2CH2CH2, Arg); 1.67, 1.83
(both br.m, 2 H, CHCH2CH2CH2, Arg); 1.86, 2.01 (both m,
2 H, CHCH2CH2, Glu); 2.37—2.66 (m, 4 H, CHCH2CH2,
Glu, CH2 cycl.); 3.24 (m, 2 H, CH2 cycl.); 3.41 (br.m, 2 H,
CHCH2CH2CH2, Arg); 3.58 (m, 1 H, CH, Arg); 4.06—4.14
(m, 4 H, 2 OCH2); 4.56 (m, 1 H, CH, Glu); 5.13 (m, 4 H,
CH2Ph); 7.32 (m, 14 H, arom.). 31P NMR, δ: 30.35. 13C NMR,
δ: 16.56 (CH3); 24.39 (C(4), Arg); 29.63 (C(3), Glu); 30.23 (C(4),
Glu); 30.76 (C(3), Arg); 31.41 (C(3)H2, cycl.); 33.18 (C(2)H2,
cycl.); 40.32 (C(5), Arg); 50.71 (C(2), Glu); 53.18 (d, C(1), cycl.,
1JC,P =149.3 Hz); 56.12 (C(2), Arg); 62.40, 62.62 (both d, OCH2,
2JC,P = 7.3 Hz); 66.54, 67.45 (CH2Ph); 125.6, 126.71, 127.05,
127.33, 128.24, 128.51, 128.55, 128.66, 135.00, 135.74, 138.46,
142.10 (C arom.); 159.45 (C=NH, Arg); 171.54 (NHC=O,
Glu); 173.11, 176.18 (C(O)OBzl, Arg, Glu). IR, ν/cm–1: 970,
1190 (P—O—Alk); 1250 br (N=O, NO2, P=О); 1600, 1630 br
(N=O, NO2, C=N, amide); 1730 (C=O); 3300 (NH). MS
(MALDI—TOF), m/z: 780 [М]+, 643 [M – P(O)(OEt)2]+.
Benzyl [Nαꢀ(1ꢀ(diethoxyphosphoryl)ꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ
1ꢀyl)ꢀNωꢀnitroꢀLꢀarginyl]ꢀ(γꢀbenzyl) αꢀLꢀglutamylꢀNωꢀnitroꢀLꢀ
argininate (17). Tripeptide 4a (0.83 g, 1 mmol) was dissolved in
a mixture of trifluoroacetic acid (15 mL) and dichloromethane
(15 mL). The solution was stirred for ~4 h until the starting
compound disappeared from the reaction mixture (TLC). The
solvents were evaporated in vacuo to a constant weight. The
obtained glassy substance was dissolved in dichloromethane
Benzyl [Nαꢀ(5ꢀbenzyloxycarbonylꢀ1ꢀ(diethoxyphosphoryl)ꢀ
2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀyl)ꢀNωꢀnitroꢀLꢀarginyl]ꢀ(γꢀbenzyl) αꢀLꢀ
glutamylꢀNωꢀnitroꢀLꢀargininate (18) was synthesized using benꢀ
zylꢀ1ꢀoxoꢀ2,3ꢀdihydroꢀ1Hꢀindeneꢀ5ꢀcarboxylate as the carbonꢀ
yl component using the method of synthesis of αꢀaminophosꢀ
phonate 17. An orange oily substance was obtained in a yield
of 0.68 g (60%). 1Н NMR, δ: 1.09—1.35 (m, 6 H, 2 CH3);
1.46—1.82 (br.m, 8 H, CHCH2CH2CH2); 1.86—2.12 (m, 2 H,
CHCH2CH2, Glu); 2.34—2.57 (m, 4 H, CHCH2CH2, Glu,
C(2)H2, cycl.); 2.87—3.00 (m, 2 H, C(3)H2, cycl.); 3.10—3.37
(br.m, 4 H, CHCH2CH2CH2, 2 Arg); 3.56, 3.73 (both m, 1 H,
CH, Arg); 4.00—4.65 (m, 6 H, CH, Arg, CH, Glu, 2 OCH2);
5.09—5.17 (m, 6 H, 2 CH2Ph); 7.10—7.78 (m, 19 H, arom.).
31P NMR, δ: 27.66, 27.77. 13C NMR, δ: 15.96—16.20 (Me,
POEt); 24.26, 24.49 (C(4), 2 Arg); 26.62 (C(3), Glu); 28.79 (C(4),
Glu); 29.66 (C(3)H2, cycl.); 30.06 (C(3), 2 Arg); 32.03 (C(2)H2,
cycl.); 40.67 (C(5), 2 Arg); 51.66, 51.77 (C(2), Glu); 52.95, 54.49,
55.50, 55.85 (C(2), 2 Arg); 63.20, 64.00 (OCH2); 66.52, 66.81,
1
67.62 (CH2Ph); 68.70, 69.10 (both d, C(1), JC,P = 157.4 Hz,
1JC,P = 158.5 Hz); 124.23, 125.64, 128.18, 128.37, 128.63, 129.07,
129.73, 134.91, 135.47, 136.41, 144.57, 150.17 (C arom.); 159.18,
(br.s, C=NH, 2 Arg); 171.41, 171.84 (NHC=O, Arg, Glu);
173.22, 173.49, 176.32 (C(O)OBzl, Arg, Glu). IR, ν/cm–1: 970,
1190 (P—O—Alk); 1250 (br, N=O, NO2, P=О); 1600, 1630
(br, N=O, NO2, C=N, amide); 1730 (C=O); 3300 (NH). MS
(MALDI—TOF), m/z: 1115 [М]+, 978 [M – P(O)(OEt)2]+.
{Nαꢀ[1ꢀ(Diethoxyphosphoryl)ꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀyl]ꢀ
Lꢀarginyl}ꢀαꢀLꢀglutamylꢀLꢀarginine (19). A flow of hydrogen was
passed through a solution of αꢀaminophosphonate 17 (0.2 g) in
a mixture of acetic acid (8 mL) and water (2 mL) (300 mL h–1
)

208
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Matveeva et al.
with stirring, the Pd/C catalyst being added portionwise (50 mg
a day, 10 mg with an interval of 1.5 h). The process was moniꢀ
tored by TLC. When the reaction was over, the catalyst was
filtered off, and the filtrate was concentrated in vacuo. The obꢀ
tained oily substance was dissolved in 50% acetic acid, the solꢀ
vent was evaporated, and the residue was triturated with acetoniꢀ
trile until a powderꢀlike light brown amorphous substance was
obtained. The yield was 0.14 g (95%). 1Н NMR, δ: 1.05—1.17
(m, 6 H, 2 CH3); 1.30—1.70 (br.m, 8 H, CHCH2CH2CH2,
2 Arg); 1.78—2.10 (br.m, 2 H, CHCH2CH2, Glu); 2.18—2.56
(m, 4 H, CHCH2CH2, Glu, C(2)H2, cycl.); 2.72—3.26 (br.m,
6 H, C(3)H2, cycl., CHCH2CH2CH2, 2 Arg); 3.77, 3.85 (both
m, 2 H, CH, Arg, CH, Glu); 3.88—4.06 (m, 4 H, 2 OCH2); 4.23
(m, 1 H, CH, Arg); 7.06—7.55 (m, 4 H, arom.). 31P NMR, δ:
27.04, 27.10. 13C NMR, δ: 15.62 (Me, POEt); 24.33, 24.43 (C(4),
2 Arg); 26.70, 26.89 (C(4), Glu); 27.94, 28.39 (C(3), 2 Arg);
29.54, 29.94 (C(3)H2, cycl.); 31.24, 31.41 (C(2)H2, cycl.); 31.73,
31.89 (C(3), Glu); 40.31, 40.57 (C(5), 2 Arg); 52.47, 52.55 (C(2),
Glu); 53.24, 53.58, 54.32, 54.54 (C(2), 2 Arg); 64.33, 64.73 (both d,
5. R. Mileusnic, C. L. Lancashire, A. N. B. Johnston, S. P. R.
Rose, Eur. J. Neurosci., 2000, 12, 4487.
6. R. Mileusnic, C. L. Lancashire, S. P. R. Rose, Ann. N. Y.
Acad. Sci., 2005, 1048, 149.
7. R. Mileusnic, C. L. Lancashire, S. P. R. Rose, Eur. J. Neuroꢀ
sci., 2004, 19, 1933.
8. R. Mileusnic, S. P. R. Rose, US Pat. 20080153759, 2008.
9. R. Mileusnic, S. P. R. Rose, Eur. Pat. EP 1381627, 2004.
10. K. Bifulco, I. LonganesiꢀCattani, L. Gargiulo, O. Maglio,
M. Cataldi, M. De Rosa, M. P. Stoppelli, V. Pavone, M. V.
Carriero, FEBS Lett., 2008, 582, No. 7, 1141.
11. K. Katayama, H. E. Anggraeni, T. Mori, M. A. Abdulatef,
S. Kawahara, M. Sugiyama, T. Nakayama, M. Maruyama,
M. Muguruma, J. Agric. Food Chem., 2008, 56, № 2, 355.
12. E. D. Matveeva, T. A. Podrugina, O. N. Zefirova, A. S.
Alekseev, S. O. Bachurin, R. Pellicciari, N. S. Zefirov, Zh. Org.
Khim., 2002, 38, 1825 [Russ. J. Org. Chem. (Engl. Transl.),
2002, 38].
13. Amonophosphonic and Aminophosphinic Acids: Chemistry and
Biological Activity, Eds V. P. Kukhar and H. R. Hudson, John
Wiley and Sons, 2000.
2
1
OCH2, JC,P = 8.43 Hz); 66.73, 68.64 (both d, C(1), JC,P
=
1
= 164.4 Hz, JC,P = 161.8 Hz); 125.27, 127.06, 128.92, 129.58,
138.14, 143.13 (C arom.); 155.61 (br.s, C=NH, 2 Arg); 169.41,
172.20, 172.28 (NHC=O, Arg, Glu); 176.81, 177.19, 178.50,
178.96 (COOH, Arg, Glu). IR, ν/cm–1: 970, 1190 (P—O—Alk);
1250 (P=О); 1600, 1630 (C=N, amide); 1735 (C=O);
3000—3500 (NH, OH). MS (MALDI—TOF), m/z: 711 [М]+,
574 [M – P(O)(OEt)2]+.
14. N. S. Zefirov, E. D. Matveeva, ARKIVOC, 2008, (i), No. 1, 1.
15. E. D. Matveeva, N. S. Zefirov, Zh. Org. Khim., 2006, 42,
1254[Russ. J. Org. Chem. (Engl. Transl.), 2006, 42].
16. E. D. Matveeva, T. A. Podrugina, M. V. Prisyazhnoi, I. N.
Rusetskaya, N. S. Zefirov, Izv. Akad. Nauk, Ser. Khim., 2007,
768 [Russ. Chem. Bull., Engl. Transl., 2007, 56, 798].
17. H. Otsuka, K. Inoue, M. Kanayama, F. Shinozaki, Bull.
Chem. Soc. Jpn, 1966, 39, 882.
18. G. L. Tritsch, D. W. Woolley, J. Am. Chem. Soc., 1960, 82,
№ 11, 2787.
19. Y. Chen, C. Guohui, M. Zhao, C. Wang, K.Qian, S. Morrisꢀ
Natschke, K. Lee, S. Peng, Bioorg. Med. Chem., 2008,
16, 5914.
This work was financially supported by the Russian
Foundation for Basic Research (Project No. 08ꢀ03ꢀ00611),
the Council on Grants at the President of the Russian Feꢀ
deration (Target Program "Development of the Scientific
Potential of the Higher School," Grant 02.445.11.7468),
and the Russian Academy of Sciences (Grants of Fundaꢀ
mental Research 1ꢀOKhNM RAS and 10ꢀOKhNM RAS,
Grant of the Presidium of the RAS 9P).
20. A. Arendt, M. Hoffmann, A. Kolodziejczyk, A. Sobczak,
T. Sokolowska, C. Wasielewski, Pol. J. Chem., 1979, 53, 447.
21. I. Ntai, B. O. Bachmann, Bioorg. Med. Chem. Lett., 2008,
18, 3068.
22. R. Pellicciary, R. Luncia, G. Constantino, M. Marinozzi,
B. Natalini, P. Jakobsen, A. Kanstrup, G. Lombardi, F. Moꢀ
roni, C. Thomsen, J. Med. Chem., 1995, 38, 3717.
23. S. A. Mikhalenko, S. V. Barkanova, O. L. Lebedev, E. A.
Luk´yanets, Zh. Obshch. Khim., 1971, 41, 2735 [J. Gen. Chem.
USSR (Engl. Transl.), 1971, 41].
References
1. P. Ertl, J. Chem. Inf. Comput. Sci., 2003, 43, N.2, 374.
2. D. C. Rees, M. Congreve, C. W. Murray, R. Carr, Nature
Rev. Drug Discovery, 2004, 3, 660.
3. M. A. PonomarevaꢀStepnaya, V. N. Nezavibat´ko, L. V. Anꢀ
tonova, L. A. Andreeva, L. Yu. Alfeeva, V. N. Potman, A. A.
Kamenskii, I. P. Ashmarin, Khim. Farm. Zh., 1984, 7, 790
[Chem. Pharm. J. (Engl. Transl.), 1984, 7].
24. E. D. Matveeva, T. A. Podrugina, E. V. Tishkovskaya, L. G.
Tomilova, N. S. Zefirov, Synlett, 2003, 15, 2321.
4. M. A. PonomarevaꢀStepnaya, V. N. Nezavibat´ko, I. P. Ashꢀ
marin, A. A. Kamenskii, L. V. Antonova, Author´s Certifiꢀ
cate No. 939 440, USSR, 1981 (in Russian).
Received October 21, 2008;
in revised form September 21, 2009