Stereoisomers of 4-amino-3-hydroxy-1-cyclohexanecarboxylic acid and 4-amino-3-oxo-1-cyclohexanecarboxylic acid as mimetics of a twisted cis-amide bond

Article abstract of DOI:10.1016/S0957-4166(01)00062-3

Stereoselective synthesis of the title compounds was performed. The relative configuration of methyl t-4-(tert-butoxycarbonylamino)-c-3-hydroxy-r-1-cyclohexanecarboxylate and methyl trans-4-(tert-butoxycarbonylamino)-3-oxo-r-1-cyclohexanecarboxylate was c

Full text of DOI:10.1016/S0957-4166(01)00062-3

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 455–462
Stereoisomers of 4-amino-3-hydroxy-1-cyclohexanecarboxylic
acid and 4-amino-3-oxo-1-cyclohexanecarboxylic acid as
mimetics of a twisted cis-amide bond
Krzysztof Krajewski, Zbigniew Ciunik and Ignacy Z. Siemion*
Faculty of Chemistry, Wroc*law University, Joliot-Curie 14, 50-383 Wroc*law, Poland
Received 29 December 2000; accepted 6 February 2001
Abstract—Stereoselective synthesis of the title compounds was performed. The relative configuration of methyl t-4-(tert-butoxy-
carbonylamino)-c-3-hydroxy-r-1-cyclohexanecarboxylate and methyl trans-4-(tert-butoxycarbonylamino)-3-oxo-r-1-cyclohexane-
carboxylate was confirmed by X-ray diffraction methods. Analogues of cyclolinopeptide A (CLA) containing these twisted
cis-amide bond mimetics were then synthesised. © 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
eoisomers with trans-arrangement of the amino and
carboxyl groups. Both cAOC and tAOC are mimetics
of the cis/trans isomerisation transition state structures
(torsion angles V are the same—about 50°) but corre-
spond to different peptide chain conformations about
the twisted cis-peptide bond (Fig. 1).
Recently we have presented the synthesis of new mimet-
ics of a twisted cis-peptide bond moiety—cis-4-amino-
3-oxo-1-cyclohexanecarboxylic acid (cAOC) and its
precursor
c-4-amino-t-3-hydroxy-r-1-cyclohexanecar-
boxylic acid (ctAHC).1 We have since expanded our
investigations to study trans-4-amino-3-oxo-1-cyclohex-
anecarboxylic (tAOC) and t-4-amino-c-3-hydroxy-r-1-
cyclohexanecarboxylic (tcAHC) acids—the diaster-
Moreover, the (1S,4S)-enantiomer of the tAOC system
[t(S)AOC] mimics the conformation of the cyclosporin
A (CsA) backbone fragment (residues 11 and 1) in the
complex with cyclophilin A (CyPA, PPI-ase, Fig. 2).2
The position of this fragment in relation to the CyPA
catalytic domain corresponds to the position of Ala-Pro
residues of the peptide substrates in their complexes
with PPI-ases, but the directions of the CsA backbone
and the substrate backbone are opposite.3,4
cAOC (in particular the (1S,4R)-enantiomer) mimics
the Pro-Pro fragment of cyclolinopeptide A [CLA, c-
(Leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe)], a nonapeptide
isolated from linseed,5 which exhibits
a distinct
immunosuppressive activity,6 and has a cis-amide bond
between proline residues. The tAOC residue mimics this
fragment less well if a conformation of CLA, as indi-
cated by the X-ray structure, is considered.7 Both CsA
and CLA affect the same cyclophilin binding site.
Analysis of the dependence of immunosuppressive
activity of CLA analogues on conformational restric-
tions introduced by cAOC and tAOC may well prove
useful in predicting the biologically active conformation
of CLA.
Figure 1. A schematic representation of the twisted cis-pep-
tide bond (A) and its mimetic (B).
* Corresponding author. E-mail: siemion@wchuwr.chem.uni.wroc.pl
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00062-3

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K. Krajewski et al. / Tetrahedron: Asymmetry 12 (2001) 455–462
Figure 2. The stereoview of superimposition of tc(S)AHC moiety (thick line) and the 9-2 fragment of CsA (bonded to CyPA).
(S)AOC-OMe), respectively. X-Ray structures were
obtained for both model compounds 9 and 10.
2. Results and discussion
The synthetic methods used for synthesis of tcAHC and
ctAHC are different, but the method of oxidation is the
same for both diastereoisomers. We obtained methyl
As shown in Scheme 1, compound 8 was prepared from
3-cyclohexene-1-carboxylic acid 1. Bromination of the
double bond of 1 with Br2 in CHCl3 afforded the
dibromide 2 which was subjected to base induced lac-
tonisation to give the bromolactone 3. The bromination
gave a diastereoisomeric mixture of 90% of 2 and 10%
of c-3,t-4-dibromo-r-1-cyclohexanecarboxylic acid, but
only 2 underwent lactonisation (step b) under the con-
ditions employed. The cis-bromolactone 3 ring was
then opened to afford 4 in 95% yield by treatment with
MeOH/NaHCO3. Protection of the hydroxyl group of 4
t-4-amino-c-3-hydroxy-r-1-cyclohexanecarboxylate
8
(tcAHC-OMe) as a racemic mixture and also as the
single enantiomer 8i ((1S,3S,4S)-, tc(S)AHC-OMe).
The Boc-derivatives 9 and 9i were prepared from
tcAHC-OMe and tc(S)AHC-OMe, and then oxidised
to give the racemic methyl trans-4-(tert-butoxycarbonyl-
amino)-3-oxo-1-cyclohexanecarboxylate 10 (Boc-tAOC-
OMe), and the (1S,4S)-enantiomer 10i (Boc-t-
Scheme 1. (a) Br2, CHCl3; (b) NaOH, H2O, D; (c) NaHCO3, MeOH; (d) Ac2O, Pyr, DCM; (e) NaN3, DMF, D; (f) MeONa,
MeOH then amberlite; (g) H2, 10% Pd–C, MeOH; (h) Boc2O, DMAP, acetone; (i) PDC, DMF.

K. Krajewski et al. / Tetrahedron: Asymmetry 12 (2001) 455–462
457
was necessary to avoid any formation of methyl 3-
azido-4-hydroxy-1-cyclohexanecarboxylate,8 and acet-
ate protection of the hydroxyl group followed by reac-
tion of 5 with NaN3 in DMF cleanly afforded the azide
6. The acetate protecting group was then removed from
6 to give methyl t-4-azido-c-3-hydroxy-r-1-cyclohexane-
carboxylate 7, which was hydrogenated in the presence
of catalytic palladium to give the amino alcohol 8.
residue in both of the HPLC fractions of Val-tcAHC,
we performed a synthesis of Val-tc(S)AHC from 8i.
After comparison of retention times (Fig. 4) we found
that the diastereoisomer with the shorter retention time
contained the (1S,3S,4S)-4-amino-3-hydroxy-1-cyclo-
hexanecarboxylic acid residue (tc(S)AHC) and the
diastereoisomer with the longer retention time con-
tained
(tc(R)AHC),
the
(1R,3R,4R)-4-amino-3-
hydroxy-1-cyclohexanecarboxylic acid residue.
Methyl t-4-(tert-butoxycarbonylamino)-c-3-hydroxy-r-
1-cyclohexanecarboxylate 9 was prepared from 8 by
reaction with Boc2O in the presence of N,N-dimethyl-
aminopyridine; the X-ray structure of 9 confirmed its
relative configuration (Fig. 6). Oxidation of 9 with
pyridinium dichromate (PDC)9 afforded Boc-tAOC-
OMe 10. Again, the relative configuration and confor-
mation of 10 were confirmed on the basis of its X-ray
structure (Fig. 7).
The homochiral dipeptides Val-tc(R)AHC-OH and
Val-tc(S)AHC-OH, as well as the Val-ct(R)AHC-OH
and Val-ct(S)AHC-OH synthesised previously,1 were
used in solid-phase syntheses of CLA analogues.
The linear peptides (Val-AHC-Phe-Phe-Leu-Ile-Ile-Leu-
OH) were formed by coupling Boc-Val-AHC-OH with
the Merrifield resin bonded hexapeptide Phe-Phe-Leu-
Ile-Ile-Leu-. The resulting octapeptides were cleaved
from the resin using the ‘low TFMSA’ method11 and
linear CLA analogues were cyclised after purification
by preparative HPLC. The cyclisations were successful
in the case of peptides containing ct(R)AHC and
ct(S)AHC residues, but failed in the case of peptides
containing tc(R)AHC and tc(S)AHC residues. These
results are consistent with the structural properties of
both types of residue: in tcAHC residues the sub-
stituents at the C-(1) and C-(4) atoms are equatorial
and their relative configuration is trans-, so tcAHC
residues induce extended conformations of peptides
containing them, which disfavours the peptide cyclisa-
tion process. In contrast, the ctAHC residues, with cis-
relative configuration of the substituents at C-(1) and
C-(4) are backbone-turn inducers and facilitate
cyclisation.
The synthesis of homochiral compounds 2i–10i was
performed analogously, starting from (S)-3-cyclohex-
ene-1-carboxylic acid 1i, with an e.e. of 93% (prepared
via diastereoselective Diels–Alder reaction and
D-pan-
tolactone ester hydrolysis).10 The 96% e.e. of 8i was
determined by analysis of its dansyl derivative by chiral
HPLC (Fig. 3). Enantiomeric enrichment (from 93 to
96%) was seen only when cis-bromolactone was formed
and then recrystallised from hot water (step b).
Racemic methyl t-4-amino-c-3-hydroxy-r-1-cyclohex-
anecarboxylate was used for the synthesis of
diastereoisomeric dipeptides (Val-tcAHC) by acylation
with Boc-Val and deprotection. These dipeptide
diastereoisomers were then separated (as previously for
Val-ctAHC diastereoisomers)1 by preparative HPLC.
To determine the absolute configuration of the tcAHC
Figure 3. The HPLC chromatograms of (a) DNS-tcAHC-OMe and (b) DNS-tc(S)AHC-OMe.

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K. Krajewski et al. / Tetrahedron: Asymmetry 12 (2001) 455–462
Figure 4. The HPLC chromatograms of (a) Val-tcAHC-OH and (b) Val-tc(S)AHC-OH.
The ct(R)AHC and ct(S)AHC residues in the cyclic
peptides were oxidised with PDC in DMF to c(R)AOC
and c(S)AOC residues. All obtained peptides were
characterised by HPLC analysis (purity ]93%) and
ESIMS spectra.
based on COSY and HETCOR experiments. CD spec-
tra were measured on a Jasco-600 spectropolarimeter in
methanol. ESIMS spectra were recorded on a Finnigan
MAT TSQ700 instrument. HPLC analyses were per-
formed on a reverse phase ODS column (Vydac 218TP)
eluted with increasing linear gradient of CH3CN (1.6%/
min) in the presence of 0.1% TFA, or on a chiral
b-cyclodextrin column (Merck ChiraDex) eluted with a
MeOH:water (40:60) mixture. Melting and boiling
Comparison of the CD spectra of the cyclic analogues
and CLA (the amide bond region, Fig. 5) showed that
the analogue with the c(S)AOC residue is the most
conformationally similar to CLA.
1
points are uncorrected. The H, 13C NMR and ESIMS
data for the racemic 1–10 and the enantiomeric 1i–10i
were identical.
3. Experimental
3.1. General
3.2. Preparation of t-3,c-4-dibromo-r-1-cyclohexanecar-
boxylic acid 2
1H and 13C NMR spectra were recorded on a Bruker
AMX-300 spectrometer in the indicated solvents; chem-
ical shifts are given in ppm. The NMR assignment was
To a solution of 3-cyclohexene-1-carboxylic acid 1 (7.56
g, 60 mmol) in CHCl3 (200 mL) was added a 2 M
solution of Br2 in CHCl3 in small portions until the
Figure 5. Comparison of the CD spectra (an amide region) of the cyclic CLA analogues containing ct(R)AHC, ct(S)AHC,
c(R)AOC, c(S)AOC residues and CLA.

K. Krajewski et al. / Tetrahedron: Asymmetry 12 (2001) 455–462
459
yellow colour persisted (65 mmol Br2). The solution
was stirred at room temperature for 15 min and
extracted with satd NaHCO3 aq. (2×100 mL). To the
aqueous layers was added H2SO4 aq. (0.5 M) to give
pH 1 (oil precipitates). The mixture was extracted with
Et2O (3×100 mL). The organic layers were separated,
dried (MgSO4) and concentrated under reduced pres-
sure. The residual colourless oil crystallised slowly.
This product contained 10% (GC–MS) t-3,c-4-
dibromo-r-1-cyclohexanecarboxylic acid. Yield: 15.25 g
(89%). 1H NMR (300 MHz, CDCl3) l 10.96 (bs, 1-
COOH), 4.70 (dd, J=6.3, 3.2, 1-H3), 4.60 (dd, J=5.6,
3.2, 1-H4), 2.96 (m, 1-H1), 2.62 (m, J=14.9, 11.7, 3.3,
1-H2%), 2.53 (m, 1-H5%), 2.23 (dddd, J=14.9, 5.0, 3.5,
1.5, 1-H2), 2.08–1.95 (m, 3-H5, H6%, H6). 13C NMR
(75 MHz, CDCl3) l 180.97 (CO), 51.83 (C4), 51.79
(C3), 37.36 (C1), 30.55 (C2), 28.09 (C5), 22.93 (C6).
63–64°C. 1H NMR (300 MHz, CDCl3) l 4.60 (m,
1-H4), 3.69 (s, 3-CH3), 3.50 (ddd, J=10.4, 4.2, 3.2,
1-H3), 2.41 (dddd, J=11.0, 11.0, 4.1, 4.1, 1-H1), 2.33–
2.23 (m, 1-H5%), 2.20 (bs, 1-OH), 2.05–1.72 (m, 5-H2%,
H2, H5, H6%, H6). 13C NMR (75 MHz, CDCl3) l
174.41 (CO), 70.03 (C3), 61.12 (C4), 51.84 (CH3), 40.59
(C1), 32.42 (C2), 31.49 (C5), 22.86 (C6). Anal. calcd for
C8H13O3Br: C, 40.53; H, 5.53. Found: C, 40.20; H,
5.51%.
3.7. Preparation of methyl (1S,3S,4R)-4-bromo-3-
hydroxy-1-cyclohexanecarboxylate 4i
Conditions were the same as for synthesis of 4 (except
the reaction scale). Yield: 448 mg (94%). Mp: 67–68°C,
[h]2D0 −33.1 (c 1.0, CHCl3).
3.8. Preparation of methyl c-3-acetoxy-c-4-bromo-r-1-
cyclohexanecarboxylate 5
3.3. Preparation of (1S,3R,4R)-3,4-dibromo-1-cyclohex-
anecarboxylic acid 2i
To a solution of 4 (5.93 g, 25 mmol) in DCM (70 mL)
under nitrogen were added sequentially pyridine (3.0 g,
38 mmol), DMAP (75 mg, 0.61 mmol) and Ac2O (3.8 g,
38 mmol). The solution was stirred for 4 h. After DCM
evaporation, the residue was shaken with water (100
mL) and hexane (100 mL), and the aqueous layer was
extracted with hexane (100 mL). The combined organic
layers were washed with aq. 5% CuSO4 and dried
(MgSO4). Hexane was removed under reduced pressure
Conditions were the same as for synthesis of 2 (except
the reaction scale). Yield: 1.256 g (85%).
3.4. Preparation of endo-4-bromo-6-oxabicy-
clo[3.2.1]octan-7-one 3
A mixture of 2 (14.30 g, 50.0 mmol) and water (80 mL)
was neutralised (phenolphthalein) with 1 M NaOH aq.
(50.8 mL). The solution was then heated for 30 min at
60°C, wherein white crystals of 3 formed. The mixture
was cooled to room temperature and filtered. The crys-
tals were washed with water. The filtrate was again
heated for 30 min at the same temperature, and a
second portion of crystals was collected to give a total
1
affording yellowish oil 6. Yield: 5.95 g (85%). H NMR
(300 MHz, CDCl3) l 4.60 (ddd, J=10.8, 4.3, 3.2,
1-H3), 4.51 (m, 1-H4), 3.60 (s, 3-CH3), 2.45–2.34 (m,
1-H1), 1.99 (s, 3-CH3CO), 2.20–1.70 (m, 6-H5%, H2%,
H2, H5, H6%, H6). 13C NMR (75 MHz, CDCl3) l
173.60 (CO), 169.76 (CH3CO), 71.43 (C3), 53.16 (C4),
51.59 (CH3), 40.23 (C1), 31.44 (C2), 28.52 (C5), 22.52
(C6), 20.75 (CH3CO). Anal. calcd for C10H15O4Br: C,
43.03; H, 5.42. Found: C, 43.08; H, 5.53%.
1
of 7.68 g (75%). Mp: 100–101°C. H NMR (300 MHz,
CDCl3) l 4.91 (d, J=6.5, 1-H3), 4.17 (ddd, J=11.0,
6.1, 0.8, 1-H4), 2.72 (m, 1-H1), 2.55 (ddddd, J=12.2,
6.5, 5.4, 2.8, 0.4, 1-H2%), 2.42 (ddd, J=13.4, 6.9, 6.1,
1-H5%), 2.17 (dddd, J=14.2, 13.1, 11.2, 6.5, 1-H5), 1.99
(ddddd, J=11.0, 9.3, 4.1, 2.7, 1.4, 1-H6%), 1.87 (d,
J=12.2, 1-H2), 1.68 (dddd, J=13.3, 13.3, 5.7, 2.2,
1-H6). 13C NMR (75 MHz, CDCl3) l 177.31 (CO),
81.69 (C3), 47.51 (C4), 37.60 (C2), 37.20 (C1), 30.62
(C5), 27.62 (C6). Anal. calcd for C7H9O2Br: C, 41.00; H
4.42. Found: C, 40.98; H, 4.56%.
3.9. Preparation of methyl (1S,3S,4R)-3-acetoxy-4-
bromo-1-cyclohexanecarboxylate 5i
Conditions were the same as for synthesis of 5 (except
the reaction scale). Yield: 430 mg (91%). [h]2D0 −39.4 (c
1.2, CHCl3).
3.10. Preparation of methyl c-3-acetoxy-t-4-azido-r-1-
cyclohexanecarboxylate 6
3.5. Preparation of (1S,4R,5S)-4-bromo-6-oxabicy-
clo[3.2.1]octan-7-one 3i
To a stirred solution of 5 (5.58 g, 20 mmol) in DMF
(40 mL) was added NaN3 (5.84 g, 90 mmol) and
15-crown-5 (five drops) under nitrogen. The mixture
was stirred under nitrogen at 75°C for 120 h. Half of
the solvent was evaporated under reduced pressure. The
residue was diluted with water (120 mL) and extracted
with Et2O (3×40 mL). The combined organic layers
were washed with water (20 mL) and brine (20 mL),
dried (MgSO4), and evaporated to give a yellow liquid
which was purified by flash chromatography (silica gel,
EtOAc:petroleum 1:6), after which pure 6 was obtained
as a colourless liquid (2.52 g, 53%). 1H NMR (300
MHz, CDCl3) l 4.68 (ddd, J=11.3, 9.8, 4.6, 1-H3),
3.63 (s, 3-CH3), 3.38 (ddd, J=11.3, 9.8, 4.5, 1-H4), 2.42
Conditions were the same as for synthesis of 3 (except
the reaction scale). Yield: 529 mg (59%). Mp: 79–80°C,
[h]2D0 +68.3 (c 1.2, CHCl3).
3.6. Preparation of methyl c-4-bromo-c-3-hydroxy-r-1-
cyclohexanecarboxylate 4
To a solution of 3 (6.15 g, 30 mmol) in MeOH (150
mL) was added NaHCO3 (2.64 g, 31.5 mmol) and the
mixture was stirred for 1 h. The solvent was evaporated
under reduced pressure and CHCl3 (75 mL) was added.
After filtration and evaporation of CHCl3, 4 was
obtained as white crystals. Yield: 6.75 g (95%). Mp:

460
K. Krajewski et al. / Tetrahedron: Asymmetry 12 (2001) 455–462
(dddd, J=12.0, 12.3, 3.7, 3.7, 1-H1), 2.34–2.24 (m,
1-H2%), 2.11–1.98 (m, 2-H5%, H6%), 2.06 (s, 3-CH3CO),
1.56–1.28 (m, 3-H2, H5, H6). 13C NMR (75 MHz,
CDCl3) l 173.84 (CO), 169.97 (CH3CO), 74.33 (C3),
62.34 (C4), 51.78 (CH3), 40.42 (C1), 32.61 (C2), 29.12
(C5), 26.54 (C6), 20.90 (CH3CO).
(dddd, J=12.5, 12.5, 3.5, 3.5, 1-H1), 2.26 (dddd, J=
13.1, 4.0, 3.5, 2.0, 1-H2%), 2.03 (dddd, J=13.0, 3.5, 3.7,
3.3, 1-H5%), 1.93 (dddd, J=13.3, 3.5, 3.7, 3.3, 1-H6%),
1.66–1.54 (m, 2-H6, H2), 1.44 (s, 9-3CH3, Boc; m, 1-H5).
13C NMR (75 MHz, CDCl3) l 174.71 (CO), 157.03
(CꢀO, Boc), 79.98 (C, Boc), 73.71 (C3), 55.91 (C4), 51.69
(CH3), 41.12 (C1), 36.19 (C2), 30.27 (C5), 28.22 (3CH3,
Boc), 27.20 (C6). ESIMS (m/z, CH3Cl:MeOH, 1:1,
1×10−5 M NaCl) 296.4 (100%, M+Na+), 569.8 (10%,
2M+Na+). Anal. calcd for C13H23NO5: C, 57.13; H, 8.48;
N, 5.12. Found: C, 57.31; H, 8.40; N, 4.89%.
3.11. Preparation of methyl (1S,3S,4S)-3-acetoxy-4-
azido-1-cyclohexanecarboxylate 6i
Conditions were the same as for the synthesis of 6
(except the reaction scale). Yield: 177 mg (49%). [h]D20
+25.0 (c 1.2, CHCl3).
3.15. Preparation of methyl (1S,3S,4S)-4-(tert-butoxy-
carbonylamino)-3-hydroxy-1-cyclohexanecarboxylate 9i
3.12. Preparation of methyl t-4-amino-c-3-hydroxy-r-1-
cyclohexanecarboxylate 8
Conditions were the same as for the synthesis of 9
(except the reaction scale). Yield: 21 mg (46%). Mp:
100–105°C, [h]2D0 +11.4 (c 1.9, MeOH).
To a solution of MeONa (prepared by addition of Na
metal (0.12 g, 5.2 mmol) to MeOH (10 mL)) was added
a solution of 6 (2.41 g, 10 mmol) in MeOH (30 mL).
The solution was stirred for 4 h under nitrogen and
then neutralised by the addition of Amberlite IRC-50
(3.6 g, pH 6). After filtration, 10% palladium on carbon
(100 mg) was added and the mixture was stirred under
hydrogen for 1 h. After filtration and evaporation, 8
was obtained as a colourless oil that crystallised very
3.16. Preparation of methyl trans-4-(tert-butoxycar-
bonylamino)-3-oxo-1-cyclohexanecarboxylate 10
To a solution of 9 (41 mg, 0.15 mmol) in DMF (2 mL)
was added PDC9 (339 mg, 0.9 mmol). The solution was
stirred at 23°C for 24 h, diluted with water (30 mL) and
extracted with Et2O (3×10 mL). The combined organic
layers were washed with 5% CuSO4 aq. (10 mL) and
dried (MgSO4). After Et2O evaporation, 10 was
obtained as a white solid. Yield: 30 mg (84%). Mp:
1
slowly (1.66 g, 9.6 mmol, 96%). H NMR (300 MHz,
CDCl3) l 3.66 (s, 3-CH3), 3.28 (ddd, J=11.1, 9.4, 4.4,
1-H3), 2.57 (ddd, J=11.4, 9.4, 3.8, 1-H4), 2.40 (dddd,
J=12.4, 12.4, 3.4, 3.4, 1-H1), 2.26–2.12 (m, 1-H2%),
2.00–1.90 (m, 2-H5%, H6%), 1.46 (ddd, J=11.1, 13.0,
12.4, 1-H2), 1.52–1.37 (m, 1-H6), 1.25 (dddd, J=11.4,
13.6, 11.8, 3.3, 1-H5). 13C NMR (75 MHz, CDCl3) l
174.94 (CO), 73.65 (C3), 56.07 (C4), 51.73 (CH3), 41.56
(C1), 35.81 (C2), 32.20 (C5), 27.40 (C6).
1
128–130°C. H NMR (300 MHz, CDCl3) l 4.21 (ddd,
J=11.8, 5.6, 5.6, 1-H4), 3.69 (s, 3-CH3), 2.76–2.59 (m,
4-H1, H2%, H2, H5%), 2.17 (dddd, J=14.0, 6.9, 3.5, 2.3,
1-H6%), 1.90 (dddd, J=14.1, 13.6, 11.1, 3.4, 1-H6), 1.44
(s, 9-3CH3, Boc; m 1-H5). 13C NMR (75 MHz, CDCl3)
l 205.19 (C3), 173.19 (CO), 155.12 (CꢀO, Boc), 79.71
(C, Boc), 58.43 (C4), 52.05 (CH3), 44.13 (C1), 42.50
(C2), 33.64 (C5), 28.22 (3CH3, Boc), 27.12 (C6). ESIMS
(m/z, CH3Cl:MeOH, 1:1, 1×10−5 M NaCl) 294.2 (M+
Na+). Anal. calcd for C13H21NO5: C, 57.55; H, 7.80; N,
5.16. Found: C, 57.29; H, 7.58; N, 5.06%.
3.13. Preparation of methyl (1S,3S,4S)-4-amino-3-
hydroxy-1-cyclohexanecarboxylate 8i
Conditions were the same as for synthesis of 8 (except
the reaction scale). Yield: 112 mg (88%). [h]2D0 +29.2 (c
0.5, MeOH), 96% e.e. Samples (1.5 mg) of 8 and 8i were
converted to their dansyl (DNS-) derivatives by reaction
(4 h at 40°C) with dansyl chloride (5 mg) in an acetone/
saturated NHCO3 aq. (1:1, 2 mL) solution and extrac-
tion with Et2O. After solvent evaporation the derivatives
were dissolved in MeOH and analysed by chiral HPLC
3.17. Preparation of methyl (1S,4S)-4-(tert-butoxycar-
bonylamino)-3-oxo-1-cyclohexanecarboxylate 10i
Conditions were the same as for synthesis of 10 (except
the reaction scale). Yield: 17 mg (85%). Mp: 109–113°C,
[h]2D0 +1.8 (c 1.5, MeOH). CD (MeOH): 300 nm ([[]=−
581, n“p*).
(b-cyclodextrin stationary phase), eluting with
MeOH:water (40:60) mixture (Fig. 3, 8i—96% e.e.).
a
3.18. General procedure of preparation of dipeptides
Boc-Val-AHC-OMe
3.14. Preparation of methyl t-4-(tert-butoxycarbony-
lamino)-c-3-hydroxy-r-1-cyclohexanecarboxylate 9
To a stirred solution of AHC-OMe (0.951 g, 5.50 mmol)
and Boc-Val-OH (1.196 g, 5.50 mmol) in DCM (10 mL)
cooled to −3°C was added a solution of DCC (1.134 g,
5.50 mmol) in DCM (15 mL). The solution was stirred
at 0°C for 30 min and then for a further 2.5 h at room
temperature. The mixture was then cooled to 0°C and
filtered. The filtrate was diluted with EtOAc (60 mL)
and washed with solutions of NaHCO3, KHSO4,
NaHCO3 and water, dried (MgSO4) and the solvent was
then evaporated to give a white foam. Yields: Boc-Val-
ctAHC-OMe (1.972 g, 97%) and Boc-Val-tcAHC-OMe
(1.911 g, 94%). ESIMS (m/z, CH3Cl:MeOH, 1:1, 1×10−5
To a solution of 8 (52 mg, 0.30 mmol) in acetone was
added (10 mL) Boc2O (100 mg, 0.46 mmol) and N,N-
diisopropylethylamine (0.05 mL, 0.29 mmol). The solu-
tion was stirred at 26°C for 20 h. The solvent was
removed under reduced pressure affording a yellowish
oil which was purified by flash chromatography (silica
gel, CH3OH:CHCl3, 5:95) and crystallisation from Et2O/
hexane, after which pure 6 was obtained as white
1
crystals. Yield: 66 mg (80%). Mp: 162–163°C. H NMR
(300 MHz, CDCl3) l 3.63 (s, 3-CH3), 3.33 (ddd, J=10.5,
9.7, 4.0, 1-H3), 3.27 (ddd, J=9.7, 9.5, 3.7, 1-H4), 2.32

K. Krajewski et al. / Tetrahedron: Asymmetry 12 (2001) 455–462
461
M NaCl) 395.2 (M+Na+), 767.5 (2M+Na+) (identical for
by gel chromatography (Sephadex LH-20, eluent:
MeOH) and preparative HPLC. ESIMS (m/z,
CH3Cl:MeOH , 1:1, 1×10−5 M NaCl) Val-ct(R)AHC-
Phe-Phe-Leu-Ile-Ile-Leu-OH 1027.8 (M+Na+), Val-
both dipeptides).
3.19. General procedure for the preparation of
dipeptides Boc-Val-AHC-OH
ct(S)AHC-Phe-Phe-Leu-Ile-Ile-Leu-OH
1027.7
(M+Na+), Val-tc(R)AHC-Phe-Phe-Leu-Ile-Ile-Leu-OH
1027.9 (M+Na+), Val-tc(S)AHC-Phe-Phe-Leu-Ile-Ile-
Leu-OH 1027.8 (M+Na+).
To a stirred solution of Boc-Val-AHC-OMe (1.860 g,
5.00 mmol) in THF (10 mL) was added LiOH (0.420 g,
10.00 mmol) in water (12 mL). The solution was stirred
at 23°C for 2 h. The reaction mixture was acidified with
KHSO4 aq. to pH 2 and extracted with Et2O (2×50 mL).
The combined organic layers were dried (MgSO4) and the
solvent was evaporated to give a white foam. Yields:
1.701 g (95%, Boc-Val-ctAHC-OH) and 1.736 g (97%,
3.22. General procedure of cyclisation of CLA
analogues
To the solution of the linear octapeptide (100 mg, 0.1
mmol) in a DCM:DMF mixture (4:1, 2.0 L) BOP (0.4
mmol), HOBt (0.4 mmol) and DMAP (0.5 mmol) were
added sequentially and the solution was stirred for 7 days
at room temperature. The solvent was evaporated under
reduced pressure and the residue was washed with water
(2×10 mL) and purified by preparative HPLC.
Boc-Val-tcAHC-OH).
ESIMS
(m/z,
CH3Cl:
MeOH, 1:1, 1×10−5 M NaCl), 381.3 (M+Na+), 739.6
(2M+Na+) (identical for both dipeptides).
3.20. General procedure for the preparation of
homochiral dipeptides Val-AHC-OH
ESIMS (m/z, CH3Cl:MeOH, 1:1, 1×10−5 M NaCl) c-
(-Val-ct(R)AHC-Phe-Phe-Leu-Ile-Ile-Leu-) 1009.6 (M+
To a stirred solution of Boc-Val-AHC-OH (1.074 g, 3.00
mmol) in DCM (10 mL) was added TFA (10 mL). The
solution was stirred for 45 min and the solvent removed
under reduced pressure affording a yellowish oil. The
product was purified by gel chromatography (Sephadex
G-10, eluent: 0.1% AcOH aq.). Yields: 0.763 g (80%,
Val-ctAHC-OH AcOH) and 0.887 g (93%, Val-tcAHC-
OH AcOH).
Na+),
c-(-Val-ct(S)AHC-Phe-Phe-Leu-Ile-Ile-Leu-)
1009.8 (M+Na+).
The linear peptides containing tc(R)AHC and tc(S)AHC
did not give the cyclic peptides under the above indicated
conditions.
3.23. Preparation of CLA analogues containing
The mixtures of diastereoisomeric dipeptides (Val-
ct(R)AHC-OH, Val-ct(S)AHC-OH and Val-tc(R)AHC-
OH, Val-tc(S)AHC-OH) were separated via HPLC on a
preparative reverse phase column (C-18, gradient from
water to 20% acetonitrile in water, with constant concen-
tration of TFA—0.1%).
c(R)AOC and c(S)AOC residues
To a solution of PDC (46 mg, 0.12 mmol) in DMF (0.2
mL) the cyclic octapeptide (10 mg, 0.01 mmol) in DMF
(0.2 mL) was added. The solution was shaken at room
temperature for 46 h, then water (5 mL) was added and
the mixture was extracted with EtOAc (2×10 mL). The
combined organic layers were washed twice with water
and dried (MgSO4). After filtration the solvent was re-
moved in a stream of N2. Yields: 4.5 mg (45%, c-(-Val-
c(R)AOC-Phe-Phe-Leu-Ile-Ile-Leu-)) and 5.0 mg (50%,
Yields: 307 mg (55%, Val-ct(R)AHC-OH·TFA), 245 mg
(44%, Val-ct(S)AHC-OH·TFA), 296 mg (53%, Val-
tc(R)AHC-OH·TFA) and 295 mg (53%, Val-tc(S)-
AHC-OH·TFA). ESIMS (m/z, MeOH:water:AcOH,
1:1:0.05) 259.3 (M+H+) (identical for all dipeptides).
c-(-Val-c(S)AOC-Phe-Phe-Leu-Ile-Ile-Leu-)).
ESIMS
(m/z, CH3Cl:MeOH, 1:1, 1×10−5 M NaCl) c-(-Val-c(R)-
AOC-Phe-Phe-Leu-Ile-Ile-Leu-) 1007.7 (M+Na+), c-
(-Val-c(S)AOC-Phe-Phe-Leu-Ile-Ile-Leu-) 1007.8 (M+
Na+).
3.21. Synthesis of linear CLA analogues containing
ct(R)AHC, ct(S)AHC, tc(R)AHC and tc(S)AHC
residues
The hexapeptide Phe-Phe-Leu-Ile-Ile-Leu- was synthe-
sised on Merrifield resin using standard Boc strategy with
DIC as a coupling reagent. Its purity was checked by
HPLC and ESIMS. The homochiral dipeptides were
coupled with the hexapeptide after Boc protection of the
amine group. The coupling was performed in DMF over
3 h using Boc-Val-AHC-OH (1.5 equiv.), PyBOP (1.5
equiv.), HOBt (1.5 equiv.) and DIEA (3.0 equiv.).
3.24. X-Ray structural analysis of 9 and 10
C13H23NO5 9, Mr=273.32 g mol−1, white, crystal size
0.10×0.10×0.20 mm, a=5.9890(5), b=26.377(2), c=
3
,
,
9.3591(7) A, i=100.33(3)°, V=1454.5(2) A , T=110 K,
monoclinic, P21/c, Z=4, Dcalcd=1.248 g cm−3, v=0.095
mm−1, extinction coefficient 0.008(2) final R1=0.0447,
wR2(F2)=0.1257, S=1.072 for 265 parameters and 3520
unique observed reflections (3.5<q<28.8°) with I]2|(I).
C13H21NO5 (10), Mr=271.31 g mol−1, white, crystal size
Cleavage of the peptide from Merrifield resin was
effected by use of a mixture of TFA (5 mL): TFMSA (1
mL): DMS (3 mL): m-cresol (1 mL) per 1 g of resin at
0°C over 4 h. The resulting mixture was filtered into Et2O
(100 mL). Hexane was added to the solution until the
peptide precipitation, the mixture was kept at
−24°C for 1 h and filtered. The crude peptide was purified
0.10×0.15×0.20 mm, a=14.655(1), b=5.7924(4), c=
3
,
,
17.398(7) A, i=109.60(1)°, V=1391.3(2) A , T=110 K,
monoclinic, P21/c, Z=4, Dcalcd=1.295 g cm−3, v=0.099
mm−1, extinction coefficient 0.004(1) final R1=0.0393,
wR2(F2)=0.0969, S=1.114 for 257 parameters and 3343
unique observed reflections (3.7<q<28.8°) with I]2|(I).

462
K. Krajewski et al. / Tetrahedron: Asymmetry 12 (2001) 455–462
Figure 6. ORTEP14 drawing of the molecular structure of 9 (one enantiomer).
Figure 7. ORTEP14 drawing of the molecular structure of 10 (one enantiomer).
The crystals of both compounds 9 and 10 were grown
from EtOAc/hexane solutions. All measurements of
References
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The data were corrected for Lorentz and polarisation
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solved by direct methods (program SHELXS-9712) and
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F2 data using the SHELXL-9713 program. Non-hydro-
gen atoms were refined with anisotropic thermal
parameters; hydrogen atoms were included from the Dz
maps and refined with isotropic thermal parameters.
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The structure of 9 (Fig. 6) indicates that it possesses a
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indicates only small conformational changes. The rings
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Acknowledgements
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We thank M.Sc. M. Hojniak for GC–MS measure-
ments and Dr. M. Lisowski for CD measurements.