(S)-N-Benzyl-3(6)-methylpiperazine-2,5-diones as chiral solvating agents for N-acylamino acid esters

Article abstract of DOI:10.1016/j.tetasy.2008.06.013

Three closely related diketopiperazines, (S)-1-benzyl-6-methylpiperazine-2,5-dione (S)-1a, (S)-1-benzyl-3-methylpiperazine-2,5-dione (S)-1b, and (S)-6-methyl-1-(pentafluorobenzyl)piperazine-2,5-dione (S)-1c, were prepared and screened as potential chiral solvating agents in NMR spectroscopy. The ¹H NMR spectra of 13 racemic α-amino acid derivatives (RS)-5a-5m were taken in CDCl₃ in the presence of equimolar amounts of enantiopure diketopiperazines (S)-1a-1c at 29 °C, 0 °C, and -20 °C. Compound (S)-1a exhibited the strongest chiral solvating properties for racemic α-amino acid derivatives (RS)-5a-5m and was recognized as a suitable CSA for the determination of their enantiomer composition. Weaker interactions of diketopiperazines (S)-1b and (S)-1c with compounds (RS)-5a-5m indicate that the position and properties of substituents play an important role in the binding affinity of diketopiperazines 1 towards amino acid derivatives 5. Association constants for binding of (S)-1a to each enantiomer of the leucine derivative (RS)-5d in CDCl₃ at -20 °C were also determined by NMR titration.

Full text of DOI:10.1016/j.tetasy.2008.06.013

Tetrahedron: Asymmetry 19 (2008) 1557–1567
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
(S)-N-Benzyl-3(6)-methylpiperazine-2,5-diones as chiral solvating agents
for N-acylamino acid esters
ˇ
*
ˇ
Crt Malavašic, Jernej Wagger, Branko Stanovnik, Jurij Svete
ˇ
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerceva 5, PO Box 537, 1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
Three closely related diketopiperazines, (S)-1-benzyl-6-methylpiperazine-2,5-dione (S)-1a, (S)-1-benzyl-
3-methylpiperazine-2,5-dione (S)-1b, and (S)-6-methyl-1-(pentafluorobenzyl)piperazine-2,5-dione (S)-
1c, were prepared and screened as potential chiral solvating agents in NMR spectroscopy. The 1H NMR
Received 24 April 2008
Accepted 11 June 2008
Available online 14 July 2008
spectra of 13 racemic
molar amounts of enantiopure diketopiperazines (S)-1a–1c at 29 °C, 0 °C, and ꢀ20 °C. Compound (S)-1a
exhibited the strongest chiral solvating properties for racemic -amino acid derivatives (RS)-5a–5m and
a-amino acid derivatives (RS)-5a–5m were taken in CDCl3 in the presence of equi-
a
was recognized as a suitable CSA for the determination of their enantiomer composition. Weaker inter-
actions of diketopiperazines (S)-1b and (S)-1c with compounds (RS)-5a–5m indicate that the position and
properties of substituents play an important role in the binding affinity of diketopiperazines 1 towards
amino acid derivatives 5. Association constants for binding of (S)-1a to each enantiomer of the leucine
derivative (RS)-5d in CDCl3 at ꢀ20 °C were also determined by NMR titration.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
the N-substituents on chiral solvating properties of closely related
diketopiperazines (S)-1a–c.
NMR spectroscopy represents one of the most common ways
for determining the enantiomeric excess and absolute configura-
tion of chiral compounds. In addition to various fluorinated
2. Results and discussion
a
-hydroxymethylidenecamphor-based chiral lanthanide shift re-
First, we decided to take a closer look at the interactions of reg-
ioisomeric diketopiperazines (S)-1a and (S)-1b with a series of 12
agents, which are nowadays commonly used reagents for determi-
nation of ee and absolute configuration,1–3 chiral solvation agents
(CSA) that are not covalently attached to the enantiomers have also
been successfully employed for the determination of enantiomeric
purity and absolute configuration. Examples of CSA are carboxylic
acids and amides,4 1,10-(anthracene-9,10-diyl)bis(2,2,2-trifluoro-
ethanol),5 cyclodextrins,6 crown ethers,7 calixarenes,8 binaphthyls,9
N-benzoylated
a-amino acid methyl esters (RS)-5a–5l in CDCl3
solution. Both (S)-1-Benzyl-6-methylpiperazine-2,5-dione (S)-
1a16–19 and (S)-1-benzyl-3-methylpiperazine-2,5-dione (S)-1b15
were prepared from (S)-alanine methyl ester hydrochloride (S)-2
following literature procedures. Similarly, the model amino acid
derivatives (RS)-5a–5l were prepared in two steps according to
a
-amino acid derivatives,10 porphyrins,11 and diketopipera-
zines.12–14
Within this context, we have recently reported chiral solvating
known general protocols20,21 from the corresponding racemic
a-
amino acids:
(RS)-6c, -leucine (RS)-6d,
phenylglycine (RS)-6f, -methionine (RS)-6g,
(RS)-6h, -glutamic acid (RS)-6i, -serine (RS)-6j,
(RS)-6k, and D,L-tryptophan (RS)-6l. Thus, compounds 6a–l were
D,L
-alanine (RS)-6a,
-b-phenylalanine (RS)-6e,
-aspartic acid
-histidine
D,L-valine (RS)-6b, D,L-norvaline
properties of (S)-1-benzyl-6-methylpiperazine-2,5-dione (S)-1a,12
which was used as a CSA for the determination of the enantiomeric
purity of tert-butyl pyroglutamate12 and tryprostatin B ana-
logues.13,14 In an extension, (S)-1-benzyl-6-methylpiperazine-2,
5-dione (S)-1a, its regioisomer (S)-1-benzyl-3-methylpiperazine-
2,5-dione (S)-1b,15 and its analogue (S)-6-methyl-1-(pentafluorob-
enzyl)piperazine-2,5-dione (S)-1c were prepared and tested as CSA
D,L
D
,L
D,L-a-
D,L
D,L
D
,L
D
,L
D,L
first esterified with thionyl chloride in methanol20 and the crude
esters 7a–l were subsequently benzoylated (Scheme 1).21
Once the racemic amino acid derivatives (RS)-5a–l were pre-
pared, the 1H NMR spectra of equimolar mixtures of (S)-1a and
(RS)-5a–l (1:5 = 1:1, c = 0.073 M) were taken in CDCl3 at three dif-
ferent temperatures: 29 °C, 0 °C, and ꢀ20 °C. In these spectra, only
the signals (doublets) for the NH protons of compounds (RS)-5
were split, while all CH protons and the NH of (S)-1a appeared as
single sets of signals. This observation was in agreement with
for a set of 13 racemic N-acylated a-amino acid esters 5. Herein, we
report the results of this study, which showed a significant effect of
* Corresponding author. Tel.: +386 1 2419 100; fax: +386 1 2419 220.
E-mail address: jurij.svete@fkkt.uni-lj.si (J. Svete).
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.06.013

ˇ
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Crt Malavašic et al. / Tetrahedron: Asymmetry 19 (2008) 1557–1567
1558
H
N
O
COOMe
COOMe
NH2
COOMe
NH
i, ii
iv
iii
Me
Cl
Me
Me
Me
N
O
N
Ref. 17
Ref. 19
Ref. 18
O
Ar
2
(S)-
Ar
Ar
3a
(S)- (Ar = Ph)
4a
(S)- (Ar = Ph)
1a
(S)- (Ar = Ph)
3c
(S)- (Ar = C6F5)
4c
(S)- (Ar = C6F5)
1c
(S)- (Ar = C6F5)
Ph
N
COOMe
O
v
iii
Ref. 15
Me
Cl
HN
Me
N
H
O
Ref. 15
O
4b
(S)-
(S)-1b
COOH
NH2
COOMe
NH2 HCl
COOMe
NH
vii
Ref. 21
vi
Ref. 20
R1
R1
R1
O
(RS)-6a-l
(RS)-7a-l
Ph
(RS)-5a-l
Scheme 1. Reagents and conditions: (i) ArCHO (Ar = phenyl or pentafluorophenyl), HC(OMe)3, MeOH, rt; (ii) NaCNBH3, rt; (iii) ClCH2COCl, CH2Cl2, Et3N, 0 °C; (iv) NH3, MeOH,
rt; (v) BnNH2 (excess), MeOH, rt; (vi) SOCl2, MeOH, 0–20 °C; (vii) PhCOCl, H2O, CH2Cl2, Et3N, rt.
H
O
N
Me
N
O
3
( )-5d
R
(S)-5d
Ph
(S)-1a
(
S
)-1a
-20 ºC
-20 ºC
2
(
S
)-1a
( )-5d
)-5d
S
(
R
0 °C
0 ºC
1
( )-5d
S
( )-5d
R
( )-1a
S
COOMe
29 ºC
8.1
29 ºC
NHCOPh
(RS)-5d
8.2
8.0
7.9
7.8
7.7
7.6
7.5
7.4
7.3
7.2
7.1
7.0
6.9
6.8
6.7
6.6
6.5
f1 (ppm)
Figure 1. Signals for the NH protons in partial 1H NMR spectra of equimolar mixture of (S)-1a and (RS)-5d (c = 0.073 M) taken in CDCl3 at 29 °C, 0 °C, and ꢀ20 °C.
almost exclusive NH splitting in closely related examples, de-
scribed previously.12–14 Thus, splitting of NH doublets was ob-
served in mixtures of (S)-1a and (RS)-5a–e,g,i,l; however, it could
not be established for mixtures (S)-1a and (RS)-5f,h,j due to an
overlap with the signals of the aromatic protons. The only com-
pound which exhibited a single doublet for the NH proton even
at ꢀ20 °C was the histidine ester (RS)-5k. Typically, the 1H NMR
spectra at 29 °C exhibited broad triplets for the NH proton of com-
pounds (RS)-5a–e,g,i,l as a consequence of two partially resolved
doublets. Resolution increased with decreasing temperature and
broad triplets observed at 29 °C evolved into well-resolved dou-
blets at ꢀ20 °C corresponding to each of the enantiomers of race-
mic amino acid derivatives (RS)-5a–e,g,i,l. The best resolution
was observed with leucine derivative (RS)-5d (Fig. 1). Typically,
the difference between the chemical shifts, dR ꢀ dS, for the benz-
amide NH protons of (R)-5 and (S)-5 increased from ꢁ0.02 ppm
(29 °C) to ꢁ0.05 ppm (0 °C) and ꢁ0.08 ppm (ꢀ20 °C). Along with
decreasing temperature, the d chemical shift of the amidic protons
increased, that is, doublets for NH of (R)- and (S)-5a–l and a singlet
for NH of (S)-1a were shifted toward higher d values (Fig. 1, Table 1).
Finally, the 1H NMR spectrum of enantiomerically enriched (S)-5d
was taken in CDCl3 at ꢀ20 °C in the presence of (S)-1a, and the enan-
tiomers of 5d were identified by relative intensities. In the presence
of (S)-1a, the NH proton of the (R)-enantiomer of 5d appeared at a
dR and dS are chemical shifts of NH protons of (RS)-5 in the presence of 1 equiv of
(S)-1.

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Crt Malavašic et al. / Tetrahedron: Asymmetry 19 (2008) 1557–1567
1559
Table 1
Selected 1H NMR data for equimolar mixtures (S)-1a and (RS)-5a–la
Compound 5
T (°C)
Chemical shift of NH of (R)- and (S)-5a–l (ppm)
NH of 1a d (ppm)
dR
dS
dR ꢀ dS
COOMe
Me
25b
6.859b
7.111b
7.282b
6.836b
7.050b
7.190b
0.023b
0.061b
0.092b
7.005b
7.948b
8.403b
ꢀ15b
ꢀ40b
NHCOPh
5a
COOMe
NHCOPh
5b
29
0
ꢀ20
6.750
6.847
6.957
6.728
6.799
6.880
0.022
0.048
0.078
7.060
7.628
8.049
COOMe
29
0
ꢀ20
6.804
6.962
7.098
6.781
6.910
7.021
0.023
0.052
0.077
ꢁ7.2c
7.757
8.129
NHCOPh
5c
COOMe
29
0
ꢀ20
6.750
6.948
7.129
6.717
6.875
7.025
0.033
0.073
0.104
7.150
7.704
8.088
NHCOPh
5d
COOMe
29
0
ꢀ20
6.698
6.783
6.867
6.677
6.823
6.926
0.021
0.060
0.059
7.068
7.645
8.059
NHCOPh
5e
COOMe
c
c
c
c
c
c
c
c
c
29
0
ꢀ20
7.041
7.606
8.034
NHCOPh
5f
COOMe
MeS
29
0
ꢀ20
7.112
7.092
0.020
7.162
7.704
8.100
NHCOPh
c
c
c
c
c
c
5g
MeOOC
COOMe
c
c
c
c
c
c
c
c
c
29
0
ꢀ20
7.171
7.735
8.134
NHCOPh
5h
MeOOC
COOMe
29
0
ꢀ20
7.144
7.128
0.016
7.114
7.655
8.047
c
c
c
NHCOPh
5i
c
c
c
HO
COOMe
c
c
c
c
c
c
c
c
c
29
0
ꢀ20
7.037
7.557
7.925
NHCOPh
5j
HN
N
COOMe
29
0
ꢀ20
8.206d
8.288d
8.549d
8.206d
8.288d
8.549d
0d
0d
0d
6.860
7.631
7.897c
NHCOPh
5k
H
N
29
0
ꢀ20
6.782
6.879
6.958
6.760
6.853
6.927
0.022
0.026
0.031
ꢁ7.01b
ꢁ7.57b
7.990
COOMe
NHCOPh
5l
(continued on next page)

ˇ
ˇ
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Crt Malavašic et al. / Tetrahedron: Asymmetry 19 (2008) 1557–1567
Table 1 (continued)
Compound 5
T (°C)
Chemical shift of NH of (R)- and (S)-5a–l (ppm)
NH of 1a d (ppm)
dR
dS
dR ꢀ dS
H
N
29
0
ꢀ20
6.009
6.248
6.369
6.089
6.219
6.327
0.020
0.029
0.041
6.702
c
COOMe
NHCOMe
7.654
5m
a
b
c
Unless otherwise specified, all spectra were taken in CDCl3 at 29 °C, 0 °C, and ꢀ20 °C with a 300 MHz spectrometer.
Spectrum was taken with a 600 MHz spectrometer. For details see Ref. 12.
Overlapped by the signals for aromatic protons.
d
The signal for the benzamide NH of (RS)-5 was not resolved in the presence of (S)-1a.
COOMe
-isomer
(S)
(S)-isomer
NHCOPh
(S)-5d
major isomer
-isomer
(R)
Ph
COOMe
(R)-isomer
O
N
NHCOPh
(R)-5d
minor isomer
Me
N
H
O
(S)-1b
7.185 7.180 7.175 7.170 7.165 7.160 7.155 7.150 7.145 7.140 7. 135 7.130 7.125 7.120 7. 115 7.110 7.105 7.100 7.095 7.090 7.085 7.080 7. 075 7.070 7.065 7.060 7. 055
f1 (ppm)
COOMe
(S)-isomer
NHCOPh
(S)-5d
major isomer
H
O
N
COOMe
(R)-isomer
NHCOPh
Me
N
O
(R)-5d
minor isomer
Ph
(S)-1a
7.19
7.18
7. 17
7.16
7.15
7.14
7. 13
7.12
7.11
7.10
7. 09
7.08
7.07
7.06
7.05
7.04
7.03
7. 02
7.01
7.00
6.99
6. 98
6.97
f1 (ppm)
Figure 2. Signals for the NH protons in partial 1H NMR spectra (CDCl3, ꢀ20 °C) of enantiomerically enriched (S)-5d in the presence of (S)-1a and (S)-1b.
higher chemical shift (7.118 ppm) than the NH proton of (S)-5d
(7.056 ppm, Fig. 2).
(S)-1a at 29 °C (dR ꢀ dS ꢁ 0.02 ppm, cf. Table 1). The best result in
the (S)-1b series was also obtained with the leucine derivative
(RS)-5d at ꢀ20 °C (dS ꢀ dR = 0.021 ppm), while splitting could not
be established for mixtures (S)-1b and (RS)-5f–i,k due to an over-
lap with the aromatic protons (Fig. 3, Table 2). The 1H NMR spec-
trum of enantiomerically enriched leucine derivative (S)-5d was
also taken in CDCl3 at ꢀ20 °C in the presence of (S)-1b in order
to identify the enantiomers of (RS)-5d by the relative intensity of
the signals. In the presence of (S)-1b, the d chemical shift of the
NH proton of (S)-5d was higher than the chemical shift of the NH
proton of (R)-5d (Fig. 2).
Next, we investigated the chiral solvating properties of (S)-1-
benzyl-3-methylpiperazine-2,5-dione (S)-1b, a regioisomer of (S)-
1a. Again, the 1H NMR spectra of equimolar mixtures (S)-1b and
(RS)-5a–l (1:5 = 1:1, c = 0.073 M) were taken in CDCl3 at 29 °C,
0 °C, and ꢀ20 °C. Somewhat to our surprise, compound (S)-1b
turned out to be a less effective CSA than its regioisomer (S)-1a.
Thus, no signal splitting was observed at 29 °C, while at 0 °C, just
a slight splitting was observed for ‘the most active’ leucine deriva-
tive (RS)-5d. At ꢀ20 °C, however, the spectra of compounds
(RS)-5c–5e,j,l exhibited two sets of signals for the NH protons. A
typical difference between the chemical shifts at ꢀ20 °C,
dS ꢀ dR ꢁ 0.01 ppm, was two times as low than in the presence of
On the basis of these data we concluded that (S)-1a is a more
effective CSA for N-acylamino acid esters 5 than its regioisomer
(S)-1b. However, a minor disadvantage of (S)-1a was, that the sig-

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Crt Malavašic et al. / Tetrahedron: Asymmetry 19 (2008) 1557–1567
1561
Ph
O
N
Me
N
H
O
(R)-5d
( )-5d
S
3
(S)-1b
(S)-1b
-20 °C
-20 °C
(S)-1b
2
(RS)-5d
0 °C
0 °C
(S)-1b (overlapped)
COOMe
1
NHCOPh
(RS)-5d
29 °C
(RS)-5d
29 °C
8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5
f1 (ppm)
Figure 3. Signals for the NH protons in partial 1H NMR spectrum of equimolar mixture of (S)-1b and (RS)-5d in CDCl3 at 29 °C, 0 °C, and ꢀ20 °C.
Table 2
Selected 1H NMR data for equimolar mixtures (S)-1b and (RS)-5a–la
Compound 5
T (°C)
Chemical shift of NH of (RS)-5a–m (ppm)
NH of 1b (ppm)
dR
dS
dS ꢀ dR
c
29
0
ꢀ20
6.895b
7.047b
7.179b
6.895b
7.047b
7.179b
0b
0b
0b
COOMe
Me
7.769
8.135
NHCOPh
5a
COOMe
NHCOPh
5b
c
29
0
ꢀ20
6.741b
6.865b
6.976b
6.741b
6.865b
6.976b
0b
0b
0b
7.784
8.166
COOMe
NHCOPh
5c
c
29
0
ꢀ20
6.808b
6.963b
7.093
6.808b
6.963b
7.103
0b
0b
ꢁ7.84b
0.010
8.196
COOMe
c
29
0
ꢀ20
6.747b
6.933
7.101
6.747b
6.941
7.122
0b
0.008
0.021
NHCOPh
7.780
8.144
5d
COOMe
c
29
0
ꢀ20
6.715b
6.846b
6.950
6.715b
6.846b
6.955
0b
0b
7.789
8.167
NHCOPh
0.005
5e
COOMe
c
c
c
c
c
c
c
c
c
c
29
0
ꢀ20
NHCOPh
7.773
8.159
5f
(continued on next page)

ˇ
ˇ
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Crt Malavašic et al. / Tetrahedron: Asymmetry 19 (2008) 1557–1567
Table 2 (continued)
Compound 5
T (°C)
Chemical shift of NH of (RS)-5a–m (ppm)
NH of 1b (ppm)
dR
dS
dS ꢀ dR
b
29
0
ꢀ20
7.086b
7.086b
0b
COOMe
MeS
c
c
c
7.776
8.150
c
c
c
NHCOPh
5g
MeOOC
COOMe
c
c
c
c
29
0
ꢀ20
7.176b
7.176b
c
c
7.727
8.125
NHCOPh
c
c
5h
c
29
0
ꢀ20
7.154b
7.154b
0b
MeOOC
COOMe
c
c
c
7.790
8.154
c
c
c
NHCOPh
5i
HO
COOMe
c
c
c
c
c
c
29
0
ꢀ20
7.133
7.621
7.965
NHCOPh
7.600
7.625
0.025
5j
HN
N
c
c
29
0
ꢀ20
6.904b
6.904b
0b
COOMe
c
c
c
c
c
c
7.952
NHCOPh
5k
H
N
c
29
0
ꢀ20
6.790b
6.872b
6.939
6.790b
6.872b
6.947
0b
COOMe
0b
7.628
8.008
0.008
NHCOPh
5l
H
N
COOMe
29
0
ꢀ20
6.099
6.236
6.350
6.119
6.265
6.398
0.020
0.029
0.048
6.826
c
NHCOMe
7.736
5m
a
b
c
All spectra were taken in CDCl3 at 29 °C, 0 °C, and ꢀ20 °C.
The signal for the benzamide NH of (RS)-5 was not resolved in the presence of (S)-1b.
Overlapped by the signals for aromatic protons.
nals of the NH proton of the N-benzoylated amino acids 5 were fre-
quently overlapped by the signals of the aromatic protons, espe-
cially in the case of more functionalized amino acids, such as
histidine, serine, aspartic acid, glutamic acid, and methionine (cf.
Tables 1 and 2). In order to circumvent this inconvenience, we pre-
(1:5 = 1:1, c = 0.073 M) in CDCl3 under standard conditions, that
is, at 29 °C, 0 °C, and ꢀ20 °C. With (RS)-5m, both diketopiperazines,
(S)-1a and (S)-1b, acted as reasonably effective CSA with a typical
difference in chemical shifts dR ꢀ dS for the acetamide NH protons
of (RS)-5m increasing from ꢁ0.02 ppm (29 °C) to ꢁ0.03 ppm (0 °C)
and ꢁ0.05 ppm (ꢀ20 °C). The signal of the indole’s NH proton also
appeared as two singlets in the presence of (S)-1a at 0 °C and
ꢀ20 °C and in the presence of (S)-1b at ꢀ20 °C (Figs. 5 and 6, Tables
1 and 2).
As shown previously, diketopiperazine (S)-1a undergoes associ-
ation with various amino acid esters via C@Oꢂ ꢂ ꢂH–N hydrogen
bonded associates in CDCl3 solution.12–14 Within this context, the
binding mode between (RS)-N-benzoylalanine methyl ester (RS)-
5a and CSA (S)-1a in CDCl3 solution has been determined by 1H
NMR, 13C NMR, and ROESY spectroscopy.12 Accordingly, the bind-
ing mode of diketopiperazines (S)-1a–c with N-benzoylated amino
acid esters (RS)-5a-m in CDCl3 solution should be the same. Inter-
actions between diketopiperazines (S)-1a–c and N-acylated amino
acid esters 5a–m should result in a reversible association, that is, in
the reversible formation of the corresponding diastereomeric
associates (R,S)-8, (S,S)-8, (R,S)-9, and (S,S)-9 (Fig. 7), which is
exhibited as the splitting (doubling) of signals in the NMR spectra
pared
(S)-1-(pentafluorobenzyl)-6-methylpiperazine-2,5-dione
(S)-1c as a pentafluoro analogue of (S)-1a, which does not exhibit
aromatic signals in 1H NMR. Compound (S)-1c was prepared from
(S)-alanine methyl ester (S)-2 and pentafluorobenzaldehyde fol-
lowing a three-step literature procedure for the preparation of its
non-fluorinated analogue (S)-1a (cf. Scheme 1).16–19 Then, 1H
NMR spectra of the valine ester (RS)-5b and the leucine ester
(RS)-5d were taken in CDCl3 in the presence of (S)-1c
(1c:5b,d = 1:1, c = 0.073 M) at 29 °C, 0 °C, and ꢀ20 °C. To our disap-
pointment, no splitting of the NH signals or any other signals was
observed in these spectra (Fig. 4).
The above experiments showed that N-substituents in diketopi-
perazine 1 play an important role in the efficacy of diketopipera-
zines 1a–c as CSA in NMR spectroscopy. Since we were
interested in the influence of the N-acyl group as well, 1H NMR
spectra of N-acetyltryptophan methyl ester (RS)-5m20j,21g were ta-
ken in the presence of equimolar amounts of (S)-1a and (S)-1b

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Crt Malavašic et al. / Tetrahedron: Asymmetry 19 (2008) 1557–1567
1563
H
N
O
Me
N
O
C
F
5
(S)-1c
3
6
(RS)-5d
(S)-1c
-20 ºC
-20 °C
2
(S)-1c
(RS)-5d
0 ºC
0 ºC
1
(S)-1c
(RS)-5d
COOMe
29 ºC
8.4
29 ºC
NHCOPh
(RS)-5d
8.5
8.3
8.2
8.1
8.0
7.9
7.8
7.7
7.6
7.5 7.4
7.3
7.2
7.1
7.0
6.9
6.8
6.7
6.6
6.5
f1 (ppm)
Figure 4. Signals for the NH protons in partial 1H NMR spectrum of equimolar mixture of (S)-1c and (RS)-5d in CDCl3 at 29 °C, 0 °C, and ꢀ20 °C.
H
O
N
Me
N
O
3
(S)-5m
Ph
(R)-5m
(S)-1a
(S)-1a
NH of indole
-20 ºC
-20 ºC
(S)-1a (overlapped)
2
(S)-5m
(R)-5m
NH of indole
0 ºC
0 ºC
H
N
1
COOMe
(S)-5m
(R)-5m
(S)-1a
NH of indole
NHCOMe
29 ºC
5m
(RS)-
29 ºC
8.8 8.7 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9
f1 (ppm)
Figure 5. Signals for the NH protons in partial 1H NMR spectrum of equimolar mixture of (S)-1a and (RS)-5m in CDCl3 at 29 °C, 0 °C, and ꢀ20 °C.
(cf. Figs. 1–6, Tables 1 and 2). On the basis of these NMR data, it can
be concluded that (S)-1a is the most effective CSA among the
investigated diketopiperazines (S)-1a–c. The different efficacies of
(S)-1a and (S)-1b can be explained by the distance between the
NH proton of (RS)-5 and the stereogenic center of (S)-1 in the cor-
responding associates 8 and 9. Namely, the distance d1a between
the NH hydrogen atom of (RS)-5 and the chiral center C(6) of (S)-
1a in associates 8 is shorter than the distance d1b between NH of
(RS)-5 and C(3) of (S)-1b (d1a < d1b, Fig. 7). Consequently, (S)-1a is
a more effective CSA than (S)-1b. The distances, d1a ꢁ 0.45 nm
and d1b ꢁ 0.55 nm, could also be estimated on the basis of the X-
ray structure of the enamino analogue of (S)-1a comprising two
C@Oꢂ ꢂ ꢂH–N hydrogen bonded molecules in a crystal cell.12 Unfor-
tunately, we do not have an explanation for the lack of chiral sol-
vating properties of (S)-1c, which might be due to electronic
properties of the pentafluorobenzyl group. In contrast to the N-
substituents in diketopiperazines (S)-1a–c, the
a-C-substituents
of the acylamino acid esters 5 did not significantly influence the
efficiency of 1a–c as CSA in CDCl3 solution. For example, the split-
ting of the NH signals observed for intermolecular cross-interac-
tions of (S)-1a with
a-amino acid esters (RS)-5a–e,g,l,m was
pretty much the same for the whole range of amino acid deriva-
tives (RS)-5a–e,g,l,m. Exceptions were compounds (RS)-5h,j,k,
where the signals for the NH protons appeared at higher chemical

ˇ
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Crt Malavašic et al. / Tetrahedron: Asymmetry 19 (2008) 1557–1567
1564
Ph
O
N
Me
N
H
O
3
(S)-5m
(R)-5m
(S)-1b
(S)-1b
NH of indole
-20 ºC
-20 ºC
(S)-1b (overlapped)
2
(S)-5m
(R)-5m
NH of indole
0 ºC
0 ºC
1
H
N
(R)-5m
(S)-5m
NH of indole
(S)-1b
COOMe
29 ºC
29 ºC
NHCOMe
5m
(RS)-
8.8 8.7 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9
f1 (ppm)
Figure 6. Signals for the NH protons in partial 1H NMR spectrum of equimolar mixture of (S)-1b and (RS)-5m in CDCl3 at 29 °C, 0 °C, and ꢀ20 °C.
MeO
R1
MeO
R1
MeO
R1
H
O
O
H
O
O
H
O
O
O
H
O
H
O
H
CDCl3
KS
CDCl3
KR
N
N
8
N
N
+
N
N
N
N
N
N
O
O
O
Me Ar
Me Ar
Me Ar
R2
R2
R2
8
1a
(R,S)-
(S)-
(S,S)-
(RS)-5
Me
Me
Me
O
MeO
R1
MeO
MeO
R1
H
N
O
CDCl3
KS
CDCl3
KR
H
O
O
H
O
H
H
O
O
H
N
R1
N
O
+
N
N
N
N
O
O
N
O
O
Ph
Ph
Ph
R2
R2
R2
9
1b
(S)-
(R,S)-
5
(RS)-
9
(S,S)-
d1b
Me
MeO
R1
MeO
R1
H
N
O
H
O
O
O
O
H
N
d1a < d1b
N
N
N
H
N
O
O
O
Ph
Ph
Me
Ph
Ph
d1a
9
Compounds
8
Compounds
R3
R3
MeO
R1
H
O
FG
N
H
O
O
O
H
MeOOC
O
+
N
N
N
H
O
N
N
R2 Ar
R2 Ar
O
Ph
Ph
1
(S)-
5h-k
(RS)-
8h-k 9h-k
or
OMe
O
NH
H
O
O
H
O
H
N
MeOOC
MeOOC
or/and
MeOOC
N
N
H
N
H
MeO
N
H
O
O
O
O
Ph
Ph
Ph
Ph
5k
(RS)-
(RS)-5h
5j
(RS)-
Figure 7.

ˇ
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Crt Malavašic et al. / Tetrahedron: Asymmetry 19 (2008) 1557–1567
1565
Table 3
CSA and their testing against various types of chiral substrates is
currently in progress.
1H NMR titration data for mixtures (S)-1a (c = 2.5?100 mM) and (RS)-5d
(c = 5 mM)a,b
[(S)-1a] (mM)
dR (ppm)
dS (ppm)
D
dR (ppm)
DdS (ppm)
4. Experimental
2.5
5
10
25
50
6.625
6.649
6.687
6.783
6.886
6.972
6.610
6.625
6.650
6.714
6.787
6.854
ꢀ0.038
ꢀ0.062
ꢀ0.100
ꢀ0.196
ꢀ0.299
ꢀ0.385
ꢀ0.023
ꢀ0.038
ꢀ0.063
ꢀ0.127
ꢀ0.200
ꢀ0.267
4.1. General methods
Melting points were determined on a Kofler micro hot stage.
The NMR spectra were obtained on a Bruker Avance DPX 300 at
300 MHz for 1H and 75.5 MHz for 13C nucleus, using CDCl3 as sol-
vent with TMS as the internal standard. Optical rotations were
measured on a Perkin-Elmer 241MC Polarimeter. Mass spectra
were recorded on AutoSpecQ and Q-Tof Premier spectrometers
and IR spectra on a Perkin-Elmer Spectrum BX FTIR spectropho-
tometer. Microanalyses were performed on a Perkin-Elmer CHN
Analyzer 2400 II.
100
a
All spectra were taken in CDCl3 at ꢀ20 °C.
d0NH of 5d = 6.587 ppm (c = 5 mM).
b
shift (>7.0 ppm, cf. Table 1). Deshielding of the NH protons in com-
pounds (RS)-5h,j,k with functionalized -substituents can be ex-
a
plained by the preferential formation of intramolecular hydrogen
bonds, either between the NH group and a hydrogen bond acceptor
functional group at the b-position in the case of compounds (RS)-
5h,j,k or between the ester group and the hydroxy group in the
case of the serine derivative (RS)-5j (Fig. 7).
(S)-Alanine methyl ester hydrochloride (S)-2 and (RS)-amino
acids 6a–l are commercially available (Sigma-Aldrich). (S)-1-Benz-
yl-6-methylpiperazine-2,5-dione
(S)-1a,17–19
(S)-1-benzyl-3-
methylpiperazine-2,5-dione (S)-1b,15 and N-benzoylated amino
acid methyl esters (RS)-5a,20a (RS)-5b,20b,21a (RS)-5c,d,20c,21a (RS)-
5e,20d,21a (RS)-5f,20e,21b (RS)-5g,20f,21a (RS)-5h,i,20g,21c (RS)-5j,20h,21d
(RS)-5k,20i,21e (RS)-5l,20j,21f and (RS)-5m,20j,21g were prepared
according to the literature procedures.
Finally, the binding constants for association of the (R)- and the
(S)-enantiomer of N-benzoyl-D,L-leucine methyl ester (RS)-5d with
CSA (S)-1a, KR and KS, were determined. First, 1H NMR spectra of
mixtures of (S)-1a (c = 2.5?100 mM) and (RS)-5d (c = 5 mM) were
taken in CDCl3 at ꢀ20 °C. This NMR titration gave the complexa-
Source of chirality: (i)
(Fluka AG), product number 05200, puriss, P99.0% (dried material
AT),
¼ þ7:5 ꢄ 0:5 (c 2, MeOH), mp 107–110 °C, ee not
specified.
L-alanine methyl ester hydrochloride
dR = d0 ꢀ dR and
D
dS = d0 ꢀ dR,à for the NH
½ ꢃ
a 2D0
tion-induced shifts,
D
protons of each enantiomer of a guest molecule, (R)-5d and (S)-5d,
at different concentrations of a host molecule (S)-1a (Table 3).
Benesi–Hildebrand treatment22 of these data then furnished the
4.2. Synthesis of (S)-1-(pentafluorobenzyl)-6-methyl-
piperazine-1,4-dione (S)-1c
complexation-induced shifts at saturation binding,
D
dmaxR = ꢀ0.362
ppm,
D
dmaxS = ꢀ0.254 ppm, and the binding constants, KR =
45.4 Mꢀ1 for the (R)-5d and KS = 38.6 Mꢀ1 for the (S)-5d (Fig. 8).
This compound was prepared from (S)-alanine methyl ester
hydrochloride (S)-2 and pentafluorobenzaldehyde following the
literature procedure for the preparation of its analogue (S)-1a12,16
via reductive benzylation17 (2?3c), chloroacetylation18 (3?4c),
and cyclization with ammonia19 (3?1c).
3. Conclusion
The present study on the utilization of (S)-1-benzyl-6-methylpi-
perazine-2,5-dione (S)-1a, its regioisomer (S)-1-benzyl-3-methyl-
piperazine-2,5-dione (S)-1b, and its close analogue (S)-1-
(pentafluorobenzyl)-6-methylpiperazine-2,5-dione (S)-1c as CSA
for racemic acylamino acid esters (RS)-5a–m in CDCl3 solution
showed that the position and properties of substituents strongly
influence the efficiency of diketopiperazines (S)-1a–c as a CSA.
With respect to this, compound (S)-1a is a superior CSA for racemic
N-benzoylated amino acid esters (RS)-5a–m. In addition, the asso-
ciation constants, KR = 45.4 Mꢀ1 and KS = 38.6 Mꢀ1, for the forma-
tion of the diastereomeric complexes (R,S)-8d and (S,S)-8d
between (S)-1a and both enantiomers of the leucine ester (R)-5d
and (S)-5d in CDCl3 were determined. In conclusion, (S)-1a is an
easily available and effective CSA, which can be employed for a
4.2.1. Methyl (S)-2-(pentafluorobenzylamino)propanoate (S)-3c
This compound was prepared following a slightly modified lit-
erature procedure for the preparation of its benzyl analogue (S)-
3a.17 Compound (S)-2 (1.40 g, 10 mmol), trimethyl orthoformate
(20 mL), anhydrous methanol (7.5 mL), and triethylamine
(1.4 mL, 10 mmol) were added to a 250 mL flask, the flask was
flushed with argon, and closed with a septum. Then, pentafluoro-
benzaldehyde (1.54 mL, 12.5 mmol) was added via syringe and
the mixture was stirred at room temperature for 1 h. Next, a solu-
tion of NaBH3CN (3.14 g, 50 mmol) in a mixture of trimethyl ortho-
formate (20 mL) and anhydrous methanol (5 mL) was added via
syringe and stirring at room temperature was continued for 2 h.
The reaction mixture was cooled to 0 °C, the septum was removed,
after which the mixture was carefully acidified with 2 M hydro-
chloric acid (20 mL), and extracted with dichloromethane
(2 ꢅ 25 mL). The organic phases were combined and extracted
with 3.8 M hydrochloric acid (2 ꢅ 50 mL). The aqueous phases
were combined, cooled to 0 °C, and made slightly alkaline
(pH ꢁ 8) with 50% aq NaOH, and the product was extracted with
dichloromethane (4 ꢅ 40 mL). The organic phases were combined,
dried over anhydrous Na2SO4, filtered, and the filtrate was evapo-
rated in vacuo to give (S)-3c, which was used in the next step with-
out purification. Yield: 2.574 g (91%) of a yellowish oil. 1H NMR
(300 MHz, CDCl3): d 1.32 (3H, d, J = 7.2 Hz, CH3); 3.37 (1H, q,
J = 7.2 Hz, CH); 3.72 (3H, s, OMe); 3.84 (1H, d, J = 13.2 Hz, 1H of
CH2Ar); 3.94 (1H, d, J = 13.2 Hz, 1H of CH2Ar). 13C NMR
(75.5 MHz, CDCl3): d 19.0, 29.3, 38.8, 51.9, 55.9, 175.4. m/z
simple determination of ee of a-acylamino acid esters 5 and anal-
ogous chiral amides by 1H NMR.12–14 Exceptions are esters of
b-functionalized N-acylamino acids, such as aspartic acid, serine,
and histidine, where intermolecular association with CSA (S)-1a
is diminished by competitive intramolecular hydrogen bonding.
An important advantage of (S)-1a over the lanthanide-based chiral
shift reagents is retained resolution of 1H NMR spectra of the inves-
tigated compounds due to absence of a paramagnetic lanthanide
nuclei. Since the substituents exhibit a significant impact on the
enantioselectivity of diketopiperazine type CSA, a further study
on the preparation and utilization of novel diketopiperazine-based
à
d
0 is chemical shift (ppm) of a NH proton of pure compound (RS)-5d (c = 5 mM) in
CDCl3 at ꢀ20 °C.

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Crt Malavašic et al. / Tetrahedron: Asymmetry 19 (2008) 1557–1567
1566
Figure 8. Benesi–Hildebrand data treatment for a mixture of (R)-5d and (S)-1a. A double reciprocal plot of complexation-induced shifts for the NH proton of (R)-5d and (S)-5d
(1/
D
dR and 1/
D
dS) as a function of 1/[(S)-1a] with slopes 1/K
D
dmaxR = ꢀ63.5 mM ppmꢀ1 and 1/K
D
dmaxS = ꢀ103.5 mM ppmꢀ1 and intercepts 1/
D
dmaxR = ꢀ2.420 ppmꢀ1 and
1/D
dmaxS = ꢀ3.532 ppmꢀ1, respectively.
(ESI) = 284 (MH+). m/z (HRMS) Found: 284.0710 (MH+).
11H10F5NO2 requires: m/z = 284.0720 (MH+). mmax (NaCl) 3457,
3341, 2957, 1739 (C@O), 1656 (C@O), 1520, 1505, 1452, 1203,
1150, 1028, 937, 722 cmꢀ1
CDCl3): d 1.55 (3H, d, J = 7.2 Hz, CH3); 3.86 (1H, q, J = 7.2 Hz, 6–
H); 3.99 (1H, dd, J = 17.7, 3.6 Hz, 1H of CH2NH); 4.08 (1H, d,
J = 17.7 Hz, 1H of CH2NH); 4.09 (1 H, d, J = 14.7 Hz, 1H of CH2Ar);
5.40 (1H, d, J = 14.7 Hz, 1H of CH2Ar); 6.36 (1H, s, NH). m/z
(EI) = 308 (M+). m/z (HRMS) Found: 308.058419 (M+). C12H9F5N2O2
requires: m/z = 308.059520. (Found: C, 46.74; H, 2.88; N, 9.04.
C
.
4.2.2. Methyl (S)-2-[2-chloro-N-(pentafluorobenzyl)-
acetylamino]propanoate (S)-4b
C12H9F5N2O2 requires: C, 46.76; H, 2.94; N, 9.09.); mmax (KBr)
This compound was prepared following a slightly modified lit-
erature procedure for the preparation of its benzyl analogue (S)-
4a.18 Chloroacetyl chloride (0.8 mL, 10 mmol) was added slowly
to a stirred cold solution (0 °C) of the crude (S)-3c (2.8 g, 10 mmol)
and triethylamine (1.75 mL, 12.5 mmol) in dichloromethane
(12 mL). The reaction mixture was allowed to warm to room tem-
perature and stirred at room temperature for 1 h. Hydrochloric
acid (3.8 M, 3 mL) was then added and the mixture was extracted
with dichloromethane (3 ꢅ 12 mL). The organic phases were com-
bined, dried over anhydrous Na2SO4, filtered, after which the fil-
trate was evaporated in vacuo, and the residue was purified by
FC (EtOAc–hexanes, 1:4). Fractions containing the product were
combined and evaporated in vacuo to give (S)-4c, which was used
in the next step without purification. Yield: 1.908 g (53%) of a yel-
lowish oil. 1H NMR (300 MHz, CDCl3): d 1.42 and 1.59 (3H, 2d,
65:35, J = 6.6, 5.4 Hz, CH3CH); 3.63 and 3.71 (3H, 2s, 65:35,
OMe); 3.91 (1H, q, J = 6.6 Hz, CHCH3); 4.20–4.37 (2H, m, CH2);
4.60–4.85 (2H, m, CH2). 13C NMR (75.5 MHz, CDCl3): d 13.9, 29.3,
40.7, 41.1 (B), 52.4, 52.8, 65.1, 166.2, 167.4, 170.6, 170.9. m/z
(ESI) = 360 (MH+). m/z (EI-HRMS) Found: 360.042587 (MH+).
3212, 3078, 2984, 1687 (C@O), 1667 (C@O), 1522, 1509, 1441,
1327, 1302, 1132, 1020, 947, 849, 783 cmꢀ1
.
Acknowledgments
The financial support from the Slovenian Research Agency
through Grants P1-0179 and J1-6689-0103-04 is gratefully
acknowledged. We also thank pharmaceutical companies Krka
d.d. (Novo mesto, Slovenia) and Lek d.d., a new Sandoz company
(Ljubljana, Slovenia) for financial support.
References
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John Wiley & Sons: New York, 1994.
2. Parker, D. Chem. Rev. 1991, 91, 1441–1457.
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13H10F5NO3 requires: m/z = 360.042587 (MH+). mmax (NaCl) 3463,
2995, 2955, 1747 (C@O), 1666 (C@O), 1523, 1504, 1438, 1223,
1123, 1027, 946, 799 cmꢀ1
.
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4.2.3. (S)-1-(Pentafluorobenzyl)-6-methylpiperazine-1,4-dione
(S)-1c
This compound was prepared following a slightly modified lit-
erature procedure for the preparation of its benzyl analogue (S)-
1a.19 Ammonia was bubbled through a cold (0 °C) solution of the
crude (S)-4c (3.59 g, 10 mmol) in anhydrous methanol (12 mL)
for 10 min and the reaction mixture was stirred at room tempera-
ture for 27 h. The precipitate was collected by filtration and
washed with cold (0 °C) aqueous ethanol (50%, 5 mL) to give (S)-
1c. Yield: 1.324 g (43%) of white crystals; mp 186–188 °C (from
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ˇ
12. Wagger, J.; Golic Grdadolnik, S.; Grošelj, U.; Meden, A.; Stanovnik, B.; Svete, J.
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2801–2815.
14. Wagger, J.; Svete, J.; Stanovnik, B. Synthesis 2008, 1436–1442.
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4111–4118.
EtOH–H2O);
½
a 2D8
ꢃ
¼ þ4:2 (c 0.15, CHCl3). 1H NMR (300 MHz,

ˇ
ˇ
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