SPIROCYCLIC DIPEPTIDES OF 1-AMINO-1-CYCLOHEXANECARBOXYLIC ACID

Article abstract of DOI:10.1135/cccc19940195

Spirocyclic cyclodipeptides with 1-amino-1-cyclohexanecarboxylic acid of the general formula cyclo(-Ach-A-), where A is Gly, L-Ala, D-Ala, L-Val, D-Val, L-Leu, D-Leu, D-Pgl, L-Phe or D-Phe, have been prepared by cyclization of the corresponding linear dipeptide methyl esters.The peptides cyclo(-L- or D-Ala-Ach-), cyclo(-L-Val-Ach-) and cyclo(-D-Leu-Ach-) show higher activity in the Chick Embryotoxicity Screening Test when compared with derivatives containing 1-amino-1-cyclopentanecarboxylic acid or 1-amino-1-cyclobutanecarboxylic acid.

Full text of DOI:10.1135/cccc19940195

Spirocyclic Dipeptides
195
SPIROCYCLIC DIPEPTIDES
OF 1-AMINO-1-CYCLOHEXANECARBOXYLIC ACID
a
b
c
Jarmila VINSOVA , Karel KOSAR and Evzen KASAFIREK
a Department of Organic Chemistry,
Faculty of Pharmacy, Charles University, 501 65 Hradec Kralove, The Czech Republic
b Department of Biological and Medical Sciences,
Faculty of Pharmacy, Charles University, 501 65 Hradec Kralove, The Czech Republic
c Research Institute for Pharmacy and Biochemistry,
130 60 Prague 3, The Czech Republic
Received April 16, 1993
Accepted May 17, 1993
Dedicated to Assoc. Prof. Dr Milan Celadnik on the occasion of his 70th birthday.
Spirocyclic cyclodipeptides with 1-amino-1-cyclohexanecarboxylic acid of the general formula
cyclo(-Ach-A-), where A is Gly, L-Ala, D-Ala, L-Val, D-Val, L-Leu, D-Leu, D-Pgl, L-Phe or D-Phe,
have been prepared by cyclization of the corresponding linear dipeptide methyl esters. The peptides
cyclo(-L- or D-Ala-Ach-), cyclo(-L-Val-Ach-) and cyclo(-D-Leu-Ach-) show higher activity in the
Chick Embryotoxicity Screening Test when compared with derivatives containing 1-amino-1-cyclo-
pentanecarboxylic acid or 1-amino-1-cyclobutanecarboxylic acid.
Recently increased attention has been paid to the study of spirocyclic cyclodipeptide
substances derivated from 1-amino-1-cyclobutane- and 1-amino-1-cyclopentanecarbox-
ylic acids mainly for their ability to influence the memory of experimental animals1 – 3
.
Spirocyclic cyclodipeptide cyclo(-alanyl-1-amino-1-cyclopentanecarbonyl-) (VUFB-
15754; ALAPTIDE) is a potential drug with nootropic activity. It beneficially affects
the growth of the diploid cell structure of human embryonic lungs and stimulates
epidermal regeneration4. To study relations between chemical structure and biological
activity we synthesized a series of spirocyclic cyclodipeptides containing 1-amino-1-
cyclopentanecarboxylic acid5 (Acp)*, 1-amino-1-cyclobutanecarboxylic acid7 (Acb)
* The symbols for amino acids and peptides are according to the suggestions of the IUPAC-IUB
Commission of Biochemical Nomenclature6. Acb, 1-amino-1-cyclobutanecarboxylic acid; Acp, 1-amino-
1-cyclopentanecarboxylic acid; Ach, 1-amino-1-cyclohexanecarboxylic acid; Pgl, phenylglycine; DMSO,
dimethyl sulfoxide; DCCI, N,N′-dicyclohexylcarbodiimide; CMS, caudal morphogenetic system.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

196
Vinsova, Kosar, Kasafirek:
and in the present paper we describe the synthesis of cyclodipeptide derivatives with
1-amino-1-cyclohexanecarboxylic acid (Ach).
The synthesis of these asymmetrically substituted spirocyclic 2,5-piperazinediones
started with N-benzyloxycarbonyl amino acids which were condensed with methyl
ester of 1-amino-1-cyclohexanecarboxylic acid8 by N,N′-dicyclohexylcarbodiimide
methods9. The protected dipeptide methyl esters IIa – IIj listed in Table I were hydro-
genolyzed in the presence of 5% palladium on carbon in methanol. Free dipeptide
methyl esters IIIa – IIIj were cyclized without being isolated in anhydrous conditions in
the presence of 0.1 mol of acetic acid in toluene. The resulting cyclodipeptides Ia – Ij
were isolated and purified by recrystalization. Their characteristics are summarized in
Table II.
The Chick Embryotoxicity Screening Test (CHEST I)10,11 was employed with regard
to the possibility of an ultimate comparison of the antiproliferative effects of newly
synthesized spirocyclic dipeptides.
SCHEME 1
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Spirocyclic Dipeptides
197
EXPERIMENTAL
The melting points were determined on a Koffler block and were not corrected. The melting points
of subliming compounds were determined in a sealed tube. The samples for analyses were dried in
vacuo at 70 Pa over phosphorus pentoxide at 78 °C. The optical rotation measurements were carried
out on Polamat A (Zeiss Jena, Germany) polarimeter in a 10 cm tube at 25 °C. The evaporation of
solvents from samples was carried out generally at reduced pressures. Thin-layer chromatography
was carried out on Silufol UV 254 nm in system petroleum ether–ethyl acetate 1 : 1. The measure-
ment of the inhibition of proliferative activity was read using a stereomicroscope. Dimethyl sulfoxide
of analytical grade was subjected to vacuum fractional distillation before use.
TABLE I
Properties of protected linear dipeptides II
Calculated/Found
M.p., °C
Yield, %
[α]D
Formula
M.w.
b
Compound
RF
ca
% C
% H
% N
IIac,d
IIb
IIc
IId
IIe
IIf
98 – 100
C18H24N2O5
348.4
–
–
–
oil
95
−22.7
C19H26N2O5
362.4
–
–
–
–
–
–
0.449
0.457
0.807
0.828
–
1.0
oil
93
+24.6
0.9
C19H26N2O5
362.4
120 – 122
−20.8
C21H30N2O5
390.5
64.60
64.82
7.74
7.92
7.17
7.28
78
1.1
122 – 126
+21.6
1.1
C21H30N2O5
390.5
64.60
64.58
7.74
8.03
7.17
7.07
95
oil
75
−19.8
C22H32N2O5
404.5
–
–
–
–
–
–
–
–
–
1.2
IIg
IIh
IIi
oil
64
+24.3
1.1
C22H32N2O5
404.5
–
oil
74
−79.0
C24H28N2O5
424.5
–
0.4
97.5 – 98.5
−13.4
C25H30N2O5
438.5
68.47
68.53
6.90
6.92
6.39
6.48
0.816
0.812
75
1.0
IIj
94 – 96
+15.1
0.9
C25H30N2O5
438.5
68.47
68.02
6.90
6.90
6.39
6.49
82
a
b
c
d
In methanol. Petroleum ether–ethyl acetate 1 : 1. Ethyl ester. Refs13,14
.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

198
Vinsova, Kosar, Kasafirek:
N-Benzyloxycarbonylpeptide Methyl Esters IIb – IIj. General Procedure
To a stirred suspension of methyl 1-amino-1-cyclohexanecarboxylate hydrochloride (0.58 g, 3 mmol)
in methylene chloride (15 ml) was added 1-ethylpiperidine (3 mmol) and after 5 min Z-amino acid
(3 mmol). The suspension was cooled to −15 °C and N,N′-dicyclohexylcarbodiimide (3.3 mmol) was
added. The mixture was stirred at −15 °C for 1 h and at 4 °C for 24 h. Excess of DCCI was de-
stroyed by acetic acid (0.2 ml). N,N′-Dicyclohexylurea was removed by filtration and the filtrate was
diluted by methylene chloride (30 ml). The solution was evaporated in a vacuum (at 40 °C max.).
The semi-solid residue was dispersed in ethyl acetate (40 ml) and filtered again to remove the re-
mains of N,N′-dicyclohexylurea. The filtrate was washed with 5% hydrochloric acid (10 ml), 5% sodium
carbonate (10 ml) and water (10 ml) and dried over sodium sulfate. The solution was evaporated and
TABLE II
Properties of spirocyclic cyclodipeptides I
Calculated/Found
M.p.a, °C
Yield, %
[α]D
Formula
M.w.
Compound
cb
% C
% H
% N
Ia c
Ib
Ic
Id
Ie
If
307 – 308
–
C9H14N2O2
182.2
–
–
–
350 – 352
−18.8
C10H16N2O2
196.3
61.20
61.11
8.22
8.31
14.27
13.90
47
0.3
354 – 356
+19.4
0.3
C10H16N2O2
196.3
61.20
61.30
8.22
8.08
14.27
14.47
67
344 – 346
−16.7
C12H20N2O2
224.3
64.26
64.57
8.99
8.76
12.49
12.35
20
0.8
355 –358
+18.0
0.8
C12H20N2O2
224.3
64.26
64.16
8.99
8.97
12.49
12.73
48
323 – 325
−19.2
C13H22N2O2
238.3
65.52
65.62
9.30
8.96
11.75
11.70
38
0.8
Ig
Ih
Ii
323 – 326
+17.3
0.8
C13H22N2O2
238.3
65.52
66.02
9.30
9.36
11.75
11.92
39
293 – 295
−31.4
C15H18N2O2
258.3
69.75
69.68
7.02
7.19
10.84
10.64
35
1.0
279 – 281
+48.6
0.9
C16H20N2O2
272.3
70.56
70.59
7.40
7.30
10.29
10.16
32
Ij
283 – 285
−46.2
C16H20N2O2
272.3
70.56
70.52
7.40
7.35
10.29
10.19
29
0.6
a
b
c
Melting points were measured in sealed capillary tube. In acetic acid. Refs13,14
.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Spirocyclic Dipeptides
199
the residue was recrystallized from ethyl acetate–petroleum ether. The yields and analytical data are
listed in Table I.
In IR spectra of all dipeptides expected maxima were found: 3 395 (NH), 1 705 (CO, ester), 1 660
(CO, amide-I), 1 494 (CO, amide-II).
Spirocyclic Cyclodipeptides Ib – Ij. General Procedure
N-Benzyloxydipeptide methyl ester IIb – IIj (2 mmol) was dissolved in methanol and 5% palladium
on carbon (0.5 g) was added. The suspension was hydrogenated at 40 °C for 2 – 6 h. The catalyst
was removed by filtration and the clear filtrate was evaporated to dryness. The crude oily linear di-
peptides IIIb – IIIj were cyclized by heating for 2 h in dry toluene (120 ml) and acetic acid (0.2 ml),
with continuous removal of toluene–water azeotrope. The crude products were recrystallized from
2-propanol. The yields and analytical data are listed in Tables II and III.
Antiproliferative Activity Test
The experiments were performed on chick embryos of White Leghorn stock (breeding station Do-
brenice, The Czech Republic) incubated for 40 h in a forced-draft incubator at 37.5 °C and at a
relative humidity of 60 – 70%. The substances were administered subgerminally to the embryos which
were all in the Hamburger–Hamilton developmental stages 10 and 11 (ref.12) representing the critical
stage interval of the embryo’s trunk development. By the routine window technique a single dose (3
µl) was administered to 8 – 10 embryos using a calibrated glass micropipette. The solvent used, 10%
aqueous dimethyl sulfoxide, had no effect on chick embryo development. The dose range was selected
on the basis of preliminary tests starting with the usual concentration of most substances tested.
Twenty four hours after the administration and subsequent incubation, the length of the newly
formed portion of the trunk was measured as an indicator of CMS function. The distance between the
vitelline arteries and the tip of the trunk which were measured by an ocular micrometer were trans-
formed to millimeters by a simple algorithm. The lower limit of normal growth of CMS within 24 h
was 2.3 mm. A decrease of the mean value below this level indicated an interaction of the compound
tested with the function of the CMS. This state was regarded as growth retardation, i.e. inhibition of
proliferation.
TABLE III
Characteristic IR frequencies (cm−1) for cyclodipeptides I
Compound
Band A
Band B
Amide-I
Amide-II
Amide-III
CH2
Ib
Ic
Id
Ie
If
3 170
3 165
3 190
3 200
3 180
3 180
3 180
3 175
3 175
3 040
3 035
3 080
3 100
3 060
3 060
3 050
3 040
3 040
1 655
1 650
1 650
1 665
1 655
1 658
1 655
1 650
1 652
1 440
1 438
1 440
1 445
1 440
1 440
1 437
1 440
1 440
1 315
1 315
1 292
1 297
1 310
1 310
1 295
1 315
1 315
1 475
1 475
1 465
1 470
Ig
Ih
Ii
1 490
1 475
1 478
Ij
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200
Vinsova, Kosar, Kasafirek:
RESULTS AND DISCUSSION
The result of the CHEST I for the highest concentration (1 . 10−3 mol l−1) cannot be
considered reliable because of the insolubility of the majority of the dipeptides tested.
The only response obtained for Ia represents the significant antiproliferative effect.
Following the adminitration of the concentration of 1 . 10−4 mol l−1, the strongest inter-
action with the developing CMS was recorded in peptides Ic and Ie. The considerable
antiproliferative effect was shown by peptides Ia, Ib, Id and Ig. The insignificant
growth inhibition was caused by Ih. Compound If showed no antiproliferative effect.
At the lowest concentration tested (1 . 10−5 mol l−1) no antiproliferative effects were
proved for most of the dipeptides used. The very strong antiproliferative effect was
shown by Ie again. Cyclodipeptide Ij cannot be tested in the concentration spectrum
selected for absolute insolubility (see Table IV).
To compare the antiproliferative effects of cyclo(-A-Acb-), cyclo(-A-Acp-) and
cyclo(-A-Ach-) Ia – Ij (Table V), the middle concentration was chosen. It is evident
that the interference with CMS which resulted in a significant antiproliferative effect
was almost identical in all the representatives from the series of cyclo(-A-Ach-). The
only exceptions are cyclo(-L-Leu-Ach-) and cyclo(-D-Pgl-Ach-). The presence of L-Leu
and surprisingly also D-Pgl in spirocyclic cyclodipeptides which contain all three car-
boxylic acids, seems to be favourable and does not inhibit the proliferation of CMS in
cell populations.
TABLE IV
Antiproliferative activity of cyclic dipeptides. Length of embryo trunk (in mm) 24 h after treatment
(mean ± SE)
c, mol l−1
Compound
1 . 10−3
1 . 10−4
1 . 10−5
Ia
Ib
Ic
Id
Ie
If
1.6 ± 0.7
1.75 ± 0.8
1.8 ± 1.0
1.5 ± 1.0
1.6 ± 1.0
1.5 ± 0.9
2.5 ± 0.2
1.6 ± 0.5
1.8 ± 0.85
2.5 ± 0.2
2.4 ± 0.4
2.25 ± 0.4
2.4 ± 0.1
1.6 ± 0.8
2.6 ± 0.25
1.75 ± 0.8
2.1 ± 0.3
a
a
a
a
a
a
a
a
a
Ig
Ih
Ii
2.1 ± 0.4
2.25 ± 0.4
a
a
Ij
a Insoluble.
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Spirocyclic Dipeptides
201
On the basis of our results it can be concluded that at the concentration of 1 . 10−4
mol l−1 the highest antiproliferative activity was observed in those spirocyclic cyclodi-
peptides that contain the amino acids of D-series, D-Phe, D-Val and D-Ala, regardless on
the structure of cycloalkanecarboxylic acid. On the contrary, spirocyclic dipeptides of
all three series containing L-Leu and D-Pgl exhibited none or minimal interference with
proliferation.
TABLE V
Comparison of the length of the trunk 24 h after treatment with cyclopeptides cyclo(-A-Acb-),
cyclo(-A-Acp-) and cyclo(-A-Ach-) in concentration 1 . 10−4 mol l−1
A
Cyclo(-A-Acb-)
Cyclo(-A-Acp-)
Cyclo(-A-Ach-)
Gly
L-Ala
D-Ala
L-Val
D-Val
L-Leu
D-Leu
D-Pgl
L-Phe
D-Phe
2.46 ± 0.4
2.23 ± 0.3
1.93 ± 0.6
1.8 ± 0.6
2.1 ± 0.5
2.3 ± 0.3
2.0 ± 0.5
2.3 ± 0.35
1.8 ± 0.8
1.4 ± 0.95
1.7 ± 0.9
2.6 ± 0.24
1.6 ± 0.9
2.4 ± 0.2
1.23 ± 0.8
2.1 ± 0.3
1.75 ± 0.8
1.8 ± 1.0
1.5 ± 1.0
1.6 ± 1.0
1.5 ± 0.9
2.5 ± 0.2
1.6 ± 0.5
2.25 ± 0.7
a
2.1 ± 0.4
a
2.5 ± 0.2
1.38 ± 0.8
1.8 ± 0.85
a
Insoluble.
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Collect. Czech. Chem. Commun. (Vol. 59) (1994)