Synthesis of phosphorus isosters of β-amyloid peptides fragments

Article abstract of DOI:10.1134/S1070363215090121

We have developed a synthetic route to pseudo dipeptides, analogs of certain fragments of β-amyloid peptides (products of the APP protein hydrolysis). These compounds can be used for preparation of phosphinic acidic oligopeptides representing the peptide sequence of the β-amyloid but containing the phosphorus isoster peptide fragment. Synthesis of pseudo ornityl-glutamate, pseudo arginyl-glutamate, pseudo glycyl-leucine, and pseudo isoleucyl-glycine via amino- and amidoalkylation of phosphonic acids containing the structural isoster of the corresponding amino acid is described.

Full text of DOI:10.1134/S1070363215090121

ISSN 1070-3632, Russian Journal of General Chemistry, 2015, Vol. 85, No. 9, pp. 2091–2098. © Pleiades Publishing, Ltd., 2015.
Original Russian Text © M.E. Dmitriev, V.V. Ragulin, 2015, published in Zhurnal Obshchei Khimii, 2015, Vol. 85, No. 9, pp. 1511–1519.
To the 85th Anniversary of birthday of late Yu.G. Gololobov
Synthesis of Phosphorus Isosters
of β-Amyloid Peptides Fragments
M. E. Dmitriev and V. V. Ragulin
Institute of Physiologically Active Compounds, Russian Academy of Sciences,
Severnyi proezd 1, Chernogolovka, Moscow oblast, 142432 Russia
e-mail: rvalery@dio.ru
Received August 3, 2015
Abstract—We have developed a synthetic route to pseudo dipeptides, analogs of certain fragments of β-
amyloid peptides (products of the APP protein hydrolysis). These compounds can be used for preparation of
phosphinic acidic oligopeptides representing the peptide sequence of the β-amyloid but containing the
phosphorus isoster peptide fragment. Synthesis of pseudo ornityl-glutamate, pseudo arginyl-glutamate, pseudo
glycyl-leucine, and pseudo isoleucyl-glycine via amino- and amidoalkylation of phosphonic acids containing
the structural isoster of the corresponding amino acid is described.
Keywords: β-amyloid, phosphorus isoster, peptide, phosphonic acid, amino acid, amidoalkylation,
hydrophosphoryl compound, methylenebis(benzylcarbamate)
DOI: 10.1134/S1070363215090121
Application of endogenous peptides as well as their
fragments and analogs as neuroprotective agents,
stimulators of cognitive functions, and memory
modulators [1–3] has been recognized among the
promising approaches in neuropharmacology. The high
specificity of the action has been found for a series of
neuropeptides, and their receptor as well as non-
receptor intracellular targets have been discovered
[4, 5]. The neurodegenerative action of β-amyloid
peptides (the products of proteolysis of amyloid
precursor protein APP) has been well studied; however,
the neuroprotective and cognitive-stimulating peptides
isolated from APP have been reported as well [6, 7].
Study of functions and targets of the mentioned
compounds is a topical fundamental issue opening the
possibilities to develop the neuroactive drugs. Unfor-
tunately, such peptides are metabolically unstable.
Design of the peptidomimetics (pseudo peptides, the
bioisosters of natural peptides) is an efficient approach
towards synthesis of metalloproteinases and aspertyl-
proteinases inhibitors [8–10] and can potentially lead
to discovery of new neuroprotective agents.
[C(O)NH2] in the dipeptide molecule with the
methylenephosphoryl fragment [P(O)(OH)CH2], inert
towards hydrolysis; this might improve the enzymatic
(metabolic) stability of the peptides and even result in
appearance of new practically important properties
[8–10].
We synthesized the pseudo dipeptides, analogs of
short fragments of the β-amyloid peptides Arg-Glu,
Gly-Leu, and ILeu-Gly, products of APP hydrolysis.
The prepared compounds can be further used in
synthesis of more complex peptide fragments, for
example, oligopeptides reproducing the sequence of
the β-amyloid but containing the phosphorus isoster of
the peptide group (Scheme 1).
The known approach to the preparation of the
phosphinic analogs 1 of dipeptides 2 [11, 12] is based
on the synthesis of the N-protected aminoalkyl-
phosphonic component of the pseudo peptide (phos-
phonic analog of the amino acid) A followed by its
addition to the α-R*-substituted acrylate (B) (Scheme 2).
Recently, a synthesis of pseudo peptide fragments has
been performed via addition of silyl esters of phos-
phonic isoster of the amino acid A to the cor-
Here we suggest a method to prepare pseudo
dipeptides [8–10] via substitution of the amide bond
2091

2092
DMITRIEV, RAGULIN
Scheme 1.
O
O
O
NH2
O
N
OH
H
H
H
H2N
N
H2N
OH
N
N
OH
H
NH2
O
NH
O
HO
O
NH2-Ile-Gly-COOH
NH2-Arg-Glu-COOH
NH2-Gly-Leu-COOH
H2N
OH
P
OH
H2N
O
O
P
OH
OH
O
H
N
O
H2N
HO
HO
H2N
O
O
NH
1а
1b
O
OH
O
O
O
P
P
OH
H2N
H2N
OH
1d
OH
1c
Scheme 2.
B
B
*
R
OY
R
R
O
O
R*
O
R*
H
N
O
P
P
HN
H2N
ZHN
OH
N
H
H
HO
XO
R
O
O
А
А
1
2
responding acrylates B (the Michael–Pudovik reaction)
[10].
the phosphinic acids 3 containing the structural isoster
of the amino acid C, followed by addition of the amino
acid fragment or construction of the α-aminoalkyl-
phosphoryl fragment via the Kabachnik–Fields reac-
tion and the formation of the phosphinic acid isoster of
the peptide 1 (Scheme 3) [14, 15].
According to that method, design of the α-amino-
alkylphosphoryl fragment A of the peptide comprises
four or more stages including protection and deprotec-
tion at nitrogen (N-Cbz) and phosphorus [for example,
РCH(OEt)2] atoms [11, 12]. In turn, addition of the
silyl ester of the component A to acrylates is seriously
limited (or fails) as far as the building of complex
aminoalkylphosphinic building blocks is concerned
[13].
We synthesized pseudo ornityl-glutamate 1a, pseudo
arginyl-glutamate 1b, pseudo glycyl-leucine 1c, and
pseudo isoleucyl-glycine 1d via amino- or amido-
alkylation of phosphonic acids 3 containing the
structural isoster of the corresponding amino acid
(Scheme 3).
In this work we extended the alternative method of
the pseudo peptides synthesis with the reversed order
of building the target molecule: initial addition of the
hypophosphite to the corresponding acrylates to form
Basing on the results obtained during the study of
addition of bis(trimethylsilyl)hypophosphite 4 (gene-
rated in situ from the hypophosphorous acid salts) to
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SYNTHESIS OF PHOSPHORUS ISOSTERS
2093
Scheme 3.
C
C
O
O
R
H
P
H
*
R
*
R
OAlk
O
OAlk
O
H
NH2
O
+
O
*
R
OAlk
O
N
OH
O
H
P
P
O
−H2O
R
OH
OH
3
А
1
Scheme 4.
OH
P
OSiMe3
OSiMe3
EtO
O
EtO
H
P
а
c
b
H2POOX
[HP(OSiMe3)2]
O
R
O
R
4
5а−5c
3а−3c
a, (Me3Si)2NH or (Me3Si)2NH, NH4Cl; b, EtO(O)C–C(R)=CH2; c, EtOH(H2O); X = NH4 or K; R = H (a), i-Bu (b),
CH2CH2COOEt (c).
Scheme 5.
COOEt
O
EtOOC
NPhth
N
O
O
4
OH
EtOOC
+ H2N-CBz
P
Ac2O/AcCl
P
O
O
O
O
H
N
H
EtOOC
OH
3c
Ph
7
HCl
COOEt
COOEt
HN
NH2
N
H
NH2
NH4OH
S
O
O
HOOC
+
HOOC
P
P
H2N
NH
NH2
NH2
1а
OH
OH
1b
acrylates [16–19], we succeeded in synthesis of
phosphonic acids containing structural isosters of
glycine 3a, leucine 3b, and glutamic acid 3c via
alcoholysis of the addition products (silylphosphonites
5a–5c) (Scheme 4).
1d. Amidoalkylation of acid 3c with benzylcarbamate
and 4-(phthalyl)aminobutyraldehyde 6 in a mixture of
acetic anhydride and acetyl chloride at cooling (cf.
Scheme 5 and [20]) afforded the phosphinic acid 7.
Acid hydrolysis of compound 7 followed by ion-
exchange chromatography on a cationite yielded the
aminophosphinic acid, pseudo ornityl-glutamate 1a. In
The phosphonic acids 3a–3c were used as key
intermediates in the synthesis of pseudo peptides 1a–
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 9 2015

2094
DMITRIEV, RAGULIN
Scheme 6.
Ph
O
Ph
Ph
EtOOC
P
Ph
Ph
N
Ph
Ph
OH
+ 1/3
N
H
N
EtOOC
P
OH
N
O
H
Ph
3b
8
9
(1) Ac2O(H+)
HBr, Δ
Dowex(H+)
Ph
O
O
(2)
HN
H
N
Ph
O
O
O
Ph
10
OH
H
OH
HCl, Δ
Dowex(H+)
EtOOC
(3) H2O
HO
P
N
O
P
NH2
O
O
O
11
1c
order to obtain the phosphinic acid analog of arginyl-
glutamate 1b, 1,4-diaminobutyl-2,4-di(hydroxycar-
bonyl)butylphosphinic acid 1a was treated with S-
ethylisothiourea in aqueous ammonia solution (cf.
Scheme 5 and [11]).
22], N,N'-alkylidenebis(alkylcarbamates) in the acetic
anhydride medium act as a source of iminium cations
generated in situ under conditions of the acid catalysis;
these cations are the reactive intermediates in the
amide (carbamate) version of the Kabachnik–Fields
reaction, capable of the interaction with the
nucleophilic РIIIOAc derivative (generated in situ in
the acetic anhydride medium as well) to form the P–C
bond via the Arbuzov reaction.
The construction of the aminophosphoryl fragment
of pseudo glycyl-peptides and the synthesis of
phosphonic isoster of glycine (the pseudo-C-glycyl
component) were complicated by the limited ap-
plicability of formaldehyde (the latter existed in the
polymeric form or in aqueous solution, inappropriate
for the usual synthetic procedure). In view of that, we
used the purposefully prepared imine trimer, 1,3,5-tris-
Methylenenbis(benzylcarbamate) 10 was prepared
as described elsewhere [23]. The two-component
version of amidoalkylation of the phosphonic acid 3b
with compound 10 in acetic anhydride under con-
ditions of acid catalysis [20–23] yielded the target
phosphinic acid 11. Hydrolysis of the latter followed
by chromatography on a cationite gave the pseudo
glycyl-leucine 1c (Scheme 6).
(diphenylmethyl)-hexahydro-S-triazine
8
[11], its
interaction with the phosphonic component 3b
affording the phosphinic acid 9. Hydrolysis of the
latter with hydrobromic acid followed by
chromatography on a cationite yielded the phosphinic
pseudo glycyl-leucine 1c (Scheme 6). On top of that,
we pioneered at performing the alternative synthesis of
the pseudo peptides containing the glycine structural
isoster, using methylenebis(benzylcarbamate) 10
(Scheme 6).
The pseudo isoleucyl-glycine 1d was synthesized
via the three-component Kabachnik–Fields reaction
(Scheme 7) [20–23]. The interaction of the phosphonic
acid 3a with benzylcarbamate and α-methylbutyr-
aldehyde in a 2 : 1 acetic anhydride–acetyl chloride
mixture upon cooling yielded the N-protected amino-
phosphinic acid 12 combining the structural isosters of
isoleucine and glycine. Hydrolysis of compound 12
According to the earlier suggested mechanism of
amidoalkylation of hydrophosphoryl compounds [20–
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SYNTHESIS OF PHOSPHORUS ISOSTERS
2095
Scheme 7.
O
OH
O
OH
P
H2N
O
O
P
O
O
Cbz-NH2
H
O
HCl/H2O
H
P
N
Ac2O/AcCl
O
O
H
OH
OEt
OH
OEt
Ph
3а
12
1d
followed by chromatography on a cationite gave the
pseudo isoleucyl-glycine 1d.
acetal with phthalic anhydride in benzene. The formed
water was eliminated using a Dean–Stark trap, and the
obtained 4-(phthalyl)aminobutyraldehyde dimethyl
acetal was treated with 0.5 mol/L solution of
hydrochloric acid. Yield 57%. 1H NMR spectrum
(CDCl3), δ, ppm: 1.55 m (2H, CH2), 2.08 m (2H, CH2),
To conclude, we suggested and performed the
synthesis of certain phosphorus isosters of four
dipeptides. The prepared compounds may be further
used as building blocks to produce the pseudo peptides
following the β-amyloid sequence.
3
3.26 t (2H, CH2, JHH 7.3 Hz], 7.2–7.40 m (4H, Ar),
9.31 br.t [1H, C(O)H].
N,N'-Methylenebis(benzylcarbamate) (10) was
prepared via the interaction of diethoxymethane with
2 eq. of benzylcarbamate in acetic anhydride medium
EXPERIMENTAL
1H, 31P, and 13С NMR spectra were recorded using
a Bruker DPX-200 spectrometer using TMS or 85%
H3PO4 as references. Melting points were determined
using a Boetius PHMK device or in an open capillary.
TLC analysis was performed using Silufol plates,
Merck glass plates (the silica gel UV-254 thickness of
0.2 mm), and Alufol plates (Kavalier) developing with
iodine vapor or UV irradiation. Dowex 50WX8-200
(H+) (Lancaster), KU IKhT (H+) (Russian Institute of
Chemical Technology, Moscow), and Diasorb (Sulfo)
(Н+) cationites were used for ion-exchange column
chromatography purification. Silpearl and L100/160
(Chemapol) silica gels were used for column
chromatography purification.
1
[23]. Yield 43%, mp 142–144°С. H NMR spectrum
3
3
(CDCl3), δ, ppm: 4.53 d.d (2H, NСН2N, JHH 6.4, JHH
6.9 Hz), 5.07 br.s (4Н, СН2О), 5.77 m (2Н, 2NH), 7.33
br.s (10Н, Ph). 13C NMR spectrum (CDCl3), δС, ppm:
48.1 (NCN), 66.9 (CH2O), 128.1, 128.2, 128.5, 136.0,
156.6 [NC(O)].
Phosphonic acids 3a–3c (general procedure). A
mixture of 0.08 mol of ammonium hypophosphite (or
0.08 mol of potassium hypophosphite and 0.08 mol of
ammonium chloride) and 0.12 mol of hexamethyl-
disilazane was stirred at boiling during 3 h. The
mixture was cooled to 5–10°С, and 0.02 mol of the
corresponding ester (ethyl acrylate, diethyl α-
methyleneglutarate, or ethyl α-isobutyracrylate) was
added dropwise at that temperature. The mixture was
stirred at room temperature during 2–3 h and left
overnight under an argon atmosphere. 30 mL of a 1 : 1
water–ethanol mixture was added dropwise upon
cooling and vigorous stirring, and the formed mass
was evaporated in a vacuum. The residue was
dissolved in 50 mL of chloroform and washed with
aqueous HCl (0.2 mol/L, 3 × 10 mL). The organic
phase was dried over magnesium sulfate, evaporated
under reduced pressure, and thoroughly dried in a high
vacuum.
Benzylcarbamate, formaldehyde diethyl acetal, di-
phenylaminomethane, phthalic anhydride, S-ethyl-
isothiourea hydrobromide, 4-aminobutyraldehyde di-
methyl acetal, hexamethyltriamidophosphite, ethyl
acrylate, acetic anhydride, acetyl chloride, and hypo-
phosphoric acid were commercial products (Reakor,
Alfa Aesar, or Acros Organics). 1,3,5-Tris(diphenyl-
methyl)hexahydro-S-triazine 8 was prepared via the
interaction of diphenylaminomethane with aqueous-
alcoholic formaldehyde solution as described
elsewhere [11]. Diethyl ester of α-methyleneglutaric
acid was prepared from ethyl acrylate in the presence
of hexamethyltriamidophosphite as catalyst [24]. Ethyl
ester of α-isobutylacrylic acid was prepared as
described in [25].
2-(Ethoxycarbonyl)ethylphosphonic acid (3a).
Yield 57% (with respect to the ester, from a mixture of
potassium hypophosphite and ammonium chloride). 1Н
NMR spectrum (CDCl3), δ, ppm: 1.16 t (3Н, СН3, 3JHH
7.3 Hz), 1.90 m (2Н, СН2), 2.48 m (2Н, СН2), 4.28 q
4-(Phthalyl)aminobutyraldehyde (4) was prepared
via the interaction of 4-aminobutyraldehyde dimethyl
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2096
DMITRIEV, RAGULIN
(2Н, СН2О, 3JHH 7.3 Hz), 7.05 d (1H, PH, 1JPH 563.0 Hz),
12.70 br.s (1Н, РООН). 31Р NMR spectrum (D2О): δP
26.10 ppm. Found, %: C 36.63, 36.72; H 6.75, 6.87.
C5H11O4P. Calculated, % C 36.15; H 6.67.
25.2* d (3JPС 19.8 Hz), 27.8, 28.6, 29.6, 31.3, 37.4,
38.2, 49.4 d (1JPС 105.0 Hz), 50.0* d (1JPС 105.0 Hz),
60.5, 61.0, 67.2, 123.3, 128.2, 128.5, 132.0, 134.0,
136.3, 156.4 d (3JPС 4.4 Hz), 168.5, 172.7, 173.9 (3JPС
7.7 Hz). 31P NMR spectrum (CDCl3), δP, ppm: 54.1, 54.4*.
2,4-Bis(ethoxycarbonyl)butylphosphonic acid (3d).
Pseudo ornityl-glutamate (1a). A mixture of 2.0 g
(3.2 mmol) of compound 7 and 0.2 g of hydrazine
hydrate was refluxed in 5 mL of ethanol during 2 h,
cooled and acidified with HCl (6 mol/L) to pH ≈ 1.
The precipitate (phthalic anhydride) was separated
immediately and then after cooling. The reaction
mixture was evaporated almost to dryness, then 18 mL
of 8 mol/L HCl was added, and the mixture was
refluxed during 17 h. The reaction mixture was cooled,
extracted with chloroform (2 × 5 mL), and evaporated.
The oily residue was twice co-evaporated with water
and purified via chromatography on a KU IKhT (H+)
cationite (eluent: 0.1–0.5 mol/L HCl). The eluate (the
positive ninhydrin reaction) was evaporated, the
residue slowly solidified at storage. Yield 0.65 g
(57%), mp 117–123°C, Rf ~ 0.20 (BuOH : AcOH :
Yield 63% (with respect to the ester, from ammonium
1
hypophosphite). H NMR spectrum (CDCl3), δ, ppm:
3
3
1.17 t (3H, CH3, JHH 7.3 Hz), 1.21 t (3H, CH3, JHH
7.3 Hz), 1.75–2.10 m [4H, CH2C(О)], 1.96 m (2H,
CH2P), 2.68 m [1H, CHC(O)], 3.98 q (2Н, CH2O, 3JHH
3
7.3 Hz), 4.05 q (2Н, CH2O, JHH 7.3 Hz), 6.97 d (1Н,
1
РН, JPH 560.0 Hz), 12.65 br.s (1Н, РООН). 31Р NMR
spectrum (CCl4–CD3OD): δP 28.70 ppm. Found P, %:
11.94, 11.90. C10H19O6P. Calculated P, %: 11.63.
2-(Ethoxycarbonyl)-4-methylamylphosphonic acid
(3c). Yield 71% (with respect to the ester, from ammo-
nium hypophosphite). 1H NMR spectrum (CCl4–
CD3OD), δ, ppm: 0.97–1.05 br.t (3H, CH3, 3JHH 7.0 Hz),
3
1.35 d (6H, CH3, JНH 7.0 Hz), 1.48 m (1H, CH), 1.65
m (2Н, СН2СН), 2.10 m (2H, СН2Р), 2.83 m [1Н,
1
1
СНС(O)], 4.17 m (2Н, CH2O), 7.05 d (1H, PH, JPH
H2O = 4 : 2 : 1). H NMR spectrum (D2O), δ, ppm:
560.0 Hz), 12.68 br.s (1Н, РООН). 31Р NMR spectrum
(CCl4–CD3OD): δP 31.10 ppm. Found P, %: 13.63,
13.43. C9H19O4P. Calculated P, %: 13.94.
1.50–2.10 m (8H, CH2CH2CHN, CH2CHCH2P), 2.20–
2.40 br.t (2H, CH2CO), 2.64 m (1H, CHCO), 2.94 m
(2H, CH2N), 3.08 m (1H, CHN). 31P NMR spectrum
(D2O): δP 33.31 ppm. Found, %: C 33.87, 33.71; H
7.23, 7.30; Cl 9.87, 9.67. C10H21N2O6P·HCl·H2O.
Calculated, %: C 34.25; H 6.90; Cl 10.11.
2,4-Bis(ethoxycarbonyl)butyl-1-(benzyloxycar-
bonylamino)-4-(phthalylamino)butylphosphinic acid
(7). 5.7 g (26 mmol) of 4-(phthalyl)aminobutyralde-
hyde 6 was slowly added to a stirred solution of 7.0 g
(26 mmol) of compound 3c and 4.0 g (26 mmol) of
benzylcarbamate in 40 mL of a 1: 1 acetic anhydride–
acetyl chloride mixture. The reaction mixture was
stirred during 30 h, concentrated via co-evaporation
with toluene, poured into 50 mL of ice water, and
evaporated. The residue was dissolved in 30 mL of
chloroform, washed with water (2 × 10 mL), and
evaporated in a vacuum. The residue was crystallized
from petroleum ether and recrystallized from diethyl
ether. Yield 5.3 g (33%), white powder, mp 81–83°C,
Rf 0.23 (CHCl3 : MeOH : AcOH = 10 : 2 : 1). 1H NMR
spectrum (CDCl3), δ, ppm: 1.21 m (6H, CH3), 1.50–
2.00 m (1H, PCH2; 4Н, CH2CH2CHN; 2Н, CH2CH2CO),
2.00–2.40 m (1H, PCH2; 2Н, CH2CO), 2.80 m (1H,
PCH2CH), 3.68 m (2H, NCH2), 3.95 m (1H, РCHN),
Pseudo arginyl-glutamate (1b). 0.5 g (2.8 mmol)
of S-ethylisothiourea hydrobromide was added to a
solution of 0.5 g (1.4 mmol) of compound 1a in 3 mL
of 25% aqueous ammonia at pH ≈ 11. The reaction
mixture was stirred at room temperature during 7 days,
adding aqueous ammonia to maintain pH > 10. After
the reaction was complete, the mixture was evaporated,
co-evaporated with water, and purified via chromato-
graphy on a Dowex 50WX8-200 (H+) cationite (eluent:
water to remove bromine anions, then 0.5% ammonia
solution). The eluate was evaporated, the residue was
additionally purified via chromatography on a Diasorb
(Sulfo) (Н+) cationite (eluent: water); the fractions
showing positive ninhydrin reaction were evaporated,
and the residue was crystallized from acetone. Yield
0.2 g (37%), mp 114–116°C, Rf ~ 0.23 (BuOH : AcOH :
2
1
4.08 q (4H, CH2O, JРH 6.8 Hz), 5.03 m (2H,
H2O = 4 : 1 : 1). H NMR spectrum (D2O), δ, ppm:
3
3
ОCH2Ph), 5.82 d.d (1H, NH, JРH 9.8, JНH 4.7 Hz),
7.2–7.4 m (5H, C6H5), 7.55–7.70 m (2H, Phth), 7.70–
7.90 m (2H, Phth). 13C NMR spectrum (CDCl3), δС,
ppm (hereafter the asterisk marks the signals of the
other diastereomer): 14.1, 14.2, 25.1 d (3JPС 20.5 Hz),
1.40–2.00 m (8H, NHCH2CH2CH2, PCH2CHCH2),
2.11 m (2H, CH2CO), 2.43 m (1H, CHCO), 2.90–3.10
m (1H, CHN), 3.14 m (2H, CH2N). 13C NMR spectrum
(D2O), δС, ppm: 24.0*, 24.5, 25.0, 30.5, 31.7 d (1JPС
97.3 Hz), 34.0, 38.8*, 40.4, 42.1, 49.7 d (1JPС 91.1 Hz),
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SYNTHESIS OF PHOSPHORUS ISOSTERS
2097
50.1* d (1JPС 89.7 Hz), 156.7 (NCN), 181.0 (CO),
182.7 d (3JPС 15.7 Hz, CO). 31P NMR spectrum (D2O),
δP, ppm: 35.1*, 35.5. Found: C 36.85, 36.73; H 7.19,
7.23; N 15.57, 15.54. C11H23N4O6P·H2O. Calculated,
%: C 37.08; H 7.07; N15.72.
1.58 m (1Н, СН, СНСН2СН), 1.85 m (1Н, СН, РСН2),
2.17 m (1Н, СН, РСН2), 2.95 d (2Н, PCH2N, JРH
10.0 Hz), 3.08 m (1Н, СНСO), 3.70 br. q (2Н, СН2О,
JHH ≈ 7.5 Hz), 5.10 m (2Н, PhСН2О), 6.90–7.20 m
(5Н, Ph). 31Р NMR spectrum: δP 40.30 ppm.
2
Pseudo glycyl-leucine (1c). a. A mixture of 2.2 g
(10 mmol) of compound 3b and 2.0 g (3.3 mmol) of
compound 7 in 10 mL of toluene was refluxed during
7–10 h and then evaporated almost to dryness. The
residue (≈3 g), phosphinic acid 9 (δP 43.50 ppm), was
refluxed in 10 mL of 45% aqueous HBr during 10 h.
The reaction mixture was evaporated in a vacuum, and
the residue was purified via chromatography on a KU
IKhT (H+) cationite (eluent: water, then 1 mol/L HCl).
The eluate was evaporated, and the oily residue was
treated with excess of propylene oxide in aqueous
ethanol. Yield 1.1 g (50%), mp 222–224°C (decomp.),
Rf 0.12 (i-BuOH : AcOH : H2O = 20 : 5 : 3). 1H NMR
spectrum (D2O), δ, ppm: 0.62 d (3Н, СН3, JHH 6.4 Hz),
0.68 d (3Н, СН3, JHH 6.4 Hz), 1.20 m (1Н, СН), 1.35
m (2Н, СН2СН), 1.68 m (1Н, РСН2), 1.84 m (1Н,
РСН2), 2.46 m [1Н, СНС(О)], 2.91 d (2Н, PCH2N, JРH
9.4 Hz). 13C NMR spectrum (D2O), δC, ppm: 21.6
(CH3), 22.1 (CH3), 25.5 (CHCH3), 30.82 d (PCH2С,
3 g of the crude acid 11 was hydrolyzed with
20 mL of 8 mol/L of HCl during 15 h. The reaction
mixture was evaporated in a vacuum, co-evaporated
with water, and the residue was purified via
chromatography on a KU IKhT (H+) cationite (eluent:
1 mol/L HCl). The eluate was evaporated in a vacuum,
and the oily residue was treated with excess of pro-
pylene oxide in aqueous ethanol. Yield 1.3 g (65%).
Pseudo isoleucyl-glycine (1d). 6 mL of acetyl
chloride was added to a solution of 1.7 g (10 mmol) of
acid 3a and 1.5 g (10 mmol) of benzylcarbamate in
12 mL of acetic anhydride, and then 1.0 g (12 mmol)
of 2-methylbutyraldehyde was slowly added dropwise.
The reaction mixture was stirred at room temperature
during 1 d, then poured into 50 mL of ice water, and
evaporated in a vacuum. The oily residue (≈ 3 g) was
dissolved in 15 mL of chloroform, washed with water
(2 × 10 mL), and dried over sodium sulfate. The
organic layer was evaporated; the residue was
crystallized from petroleum ether and recrystallized
from diethyl ether. We isolated 1.8 g (47%) of 1-[(N-
benzyloxycarbonyl)amino]-2-methylbutyl-2-(ethyl-
oxycarbonyl)ethylphosphinic acid 12; mp 92–94°С,
Rf 0.25 (CHCl3 : MeOH : AcOH = 10 : 2 : 1). 1Н NMR
1
1JРС 95.6 Hz), 37.5 d (PCH2N, JРС 94.4 Hz), 37.7 d
3
2
(PCCCH2, JРС 12.4 Hz), 42.81 d [СНС(О)], JРС 4.4
Hz), 176.8 d [C(O), JРС 5.0 Hz]. 31P NMR spectrum
3
(D2O–DCl, pH ≈ 1): δP 40.90 ppm. Found: C 42.89,
42.83; H 8.19, 8.23; N 6.17, 6.23. C8H18NO4P.
Calculated, %: C 43.05; H 8.13; N 6.28.
3
spectrum (CDCl3), δ, ppm: 0.92 br.t (3H, СН3, JНH
3
b. 3 mL of acetyl chloride was added to a solution
of 2.2 g (10 mmol) of compound 3b and 3.1 g
(10 mmol) of compound 10 in 12 mL of acetic
anhydride. The mixture was stirred at room temperature
during 17 h, poured into 50 mL of ice water, and
evaporated. The residue was treated with 30 mL of
saturated solution of sodium hydrogen carbonate and
10 mL of diethyl ether. The crystalline benzyl-
carbamate was filtered off. The aqueous layer was
additionally washed with diethyl ether and acidified to
pH ≈ 1–2. The oily product was separated, and the
aqueous layer was additionally extracted with ethyl
acetate (2 × 10 mL). The extract was combined with
the oily product, washed with water, dried over sodium
sulfate, and evaporated in a vacuum. The oily residue
was the phosphinic acid 11 (Rf1 ~ 0.30, CHCl3 :
i-PrOH : AcOH = 20 : 4 : 1). Н NMR spectrum
(CDCl3 + a drop of CF3COOH), δ, ppm: 0.83 m (3H,
СН3), 0.88 m (3Н, СН3), 1.17 br.t (3Н, СН3, JHH ≈ 7.5 Hz),
1.45 m (1Н, СНСН3), 1.51 m (1Н, СН, СНСН2СН),
7.0 Hz), 0.98 d (3Н, СН3, JНH 6.8 Hz), 1.23 br.t (3H,
3
СН3, JНH 7.5 Hz), 1.37 m (1Н, СНСН3), 1.71 m (1Н,
СН2СН), 2.02 m (2Н, РСН2; 1Н, СН2СН), 2.55 br.t
3
(2Н, СН2СО, JНH 7.5 Hz), 3.95 m (1Н, РСНN), 4.12
3
q (2Н, СН2О, JНH 7.5 Hz), 5.11 m (2Н, PhСН2О),
5.23 m (1Н, NH), 7.33 m (5Н, Ph), 8.0 br.s (1Н,
1
РОН). Н NMR spectrum (CD3OD), δ, ppm: 0.87 d
(3H, СН3, 3JНH 6.5 Hz), 0.97 t (3H, СН3, 3JНH 7.0 Hz),
3
1.21 t (3H, СН3, JНH 7.1 Hz), 1.34 m (1Н, СНСН3),
1.64 m (1Н, СН2СН), 1.96 m (2Н, РСН2; 1Н,
СН2СН), 2.52 m (2Н, СН2СО), 3.81 d.d (1Н, РСНN,
3
2
2JPH 10.6, JНH 5.3 Hz), 3.97* d.d (1Н, РСНN, JPH
3
3
10.6, JНH 2.8 Hz), 4.10 q (2Н, СН2О, JНH 7.0 Hz),
5.08 br.s (2Н, PhСН2О), 7.30 m (5Н, Ph). 31Р NMR
spectrum (CDCl3), δР, ppm: 55.8, 55.9*. 31Р NMR
spectrum (CD3OD), δР, ppm: 50.5, 50.8*.
A solution of 1.6 g (4 mmol) of acid 12 in 15 mL of
6 mol/L of HCl was refluxed during 15 h and then
evaporated in a vacuum. The residue was co-
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 9 2015

2098
DMITRIEV, RAGULIN
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and Yiotakis, A.A., Tetrahedron, 1999, vol. 55,
p. 14635. DOI: 10.1016/S0040-4020(99)00910-2.
14. Ragulin, V.V., Rozhko, L.F., Saratovskikh, I.V., and
Zefirov, N.S., Japan Patent 2000, 18568; C. A., 2001;
AN 2001:552806; Ragulin, V.V., Rozhko, L.F.,
Saratovskikh, I.V.,and Zefirov, N.S., Japan Patent 2000,
18581; C. A., 2001, AN 2001:574226.
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Ragulin, V.V., Russ. J. Gen. Chem., 2007, vol. 77,
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SSSR, Ser. Khim., 1988, no. 11, p. 2652.
evaporated with water and purified via chromato-
graphy on a Dowex 50WX8-200 (H+) cationite (eluent:
0.5 mol/L HCl). The fractions exhibiting positive
ninhydrin reaction were combined and co-evaporated
with water, and the residue was treated with propylene
oxide in aqueous ethanol. The formed crystalline
precipitate was recrystallized from aqueous ethanol.
Yield 0.7 g (76%), mp 174–176°С, Rf 0.15 (BuOH :
AcOH : H2O = 20 : 5 : 2). 1Н NMR spectrum (D2О), δ,
3
ppm: 0.86 t (3Н, СН3, JНH 7.3 Hz), 0.99 d (3Н, СН3,
3
3JНH 6.6 Hz), 1.04* d (3Н, СН3, JНH 6.0 Hz), 1.39 m
(2Н, СН2СН), 1.60* m (2Н, СН2СН), 1.87 m (2Н,
РСН2), 2.01 m (1Н, СНСНN), 2.55 m (2Н, СН2СО),
3.08* d.d (1Н, *РСНN, 2JPH 8.8, 3JНH 5.9 Hz), 3.16 d.d
(1Н, РСНN, 2JPH 8.4, 3JНH 4.0 Hz). 13С NMR spectrum
3
(D2О), δС, ppm: 10.4*, 10.8 (СН3), 14.2 d (СН3, JPC
3.3 Hz), 15.8* d (СН3, 3JPC 5.9 Hz), 24.3 d (РСН2, 1JPC
1
95.9 Hz), 24.5* d (РСН2, JPC 95.5 Hz), 24.6 d (СН,
3
2JPC 4.8 Hz), 26.7 d (СН2, JPC 4.0 Hz), 26.8* d (СН2,
3JPC 5.9 Hz), 32.9 (СН2СО), 33.5* (СН2СО), 53.3 d
1
1
(РСN, JPC 91.8 Hz), 54.8* d (РСN, JPC 91.1 Hz),
177.4 d (СО, 3JPC 15.0 Hz). 31Р NMR spectrum (D2О):
δР 44.40 ppm. Found: C 42.76, 42.57; H 8.20, 8.34; N
6.17, 6.23. C8H18NO4P. Calculated, %: C 43.05; H
8.13; N 6.28.
ACKNOWLEDGMENTS
This work was financially supported by the Russian
Foundation for Basic Research (project 15-03-06062)
and the Russian Academy of Sciences in the frame of
the “Fundamental Sciences to Medicine” program.
17. Ragulin, V.V., Kurdyumova, N.R., and Tsvetkov, E.N.,
Zh. Obshch. Khim., 1994, vol. 64, no. 3, p. 419.
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Tsvetkov, E.N., Russ. J. Gen. Chem., 1997, vol. 67,
no. 12, p. 1852.
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