On differences between racemic and enantiomerically pure forms of aziridine-2-carboxamide

Article abstract of DOI:10.1007/s10593-012-1067-2

Synthesis, X-ray, and cytotoxicity studies of (S)- and (R)-aziridine-2- carboxamide (Leakadine) are described. X-ray data for the enantiomerically pure form are compared with those for racemic aziridine-2-carboxamide in order to explain the 21°C large mel

Full text of DOI:10.1007/s10593-012-1067-2

Chemistry of Heterocyclic Compounds, Vol. 48, No. 6, September, 2012 (Russian Original Vol. 48, No. 6, June, 2012)
ON DIFFERENCES BETWEEN RACEMIC
AND ENANTIOMERICALLY PURE FORMS
OF AZIRIDINE-2-CARBOXAMIDE*
M. Turks1**, I. Rijkure1, S. Belyakov2, D. Zicāne1, V. Kumpiņš1,
Ē. Bizdēna1, A. Meikas3,4, and A. Valkna3,4
Synthesis, X-ray, and cytotoxicity studies of (S)- and (R)-aziridine-2-carboxamide (Leakadine) are
described. X-ray data for the enantiomerically pure form are compared with those for racemic
aziridine-2-carboxamide in order to explain the 21°C large melting point difference between both
series. It was found that despite their overall low cytotoxicity (S)-aziridine-2-carboxamide is slightly
more cytotoxic than (R)-aziridine-2-carboxamide.
Keywords: aziridine-2-carboxamide, chiral aziridines, Leakadine.
Aziridines are rather reactive constrained heterocycles. More than a hundred compounds from the
aziridine class have shown certain biological activity [1]. In many cases, this activity is based on the strong
alkylating properties of aziridines. Therefore, many representatives of the aziridine series possess distinct
cytotoxicity rather than selective biological activity. On the other hand, there are several classes of aziridines
that have selective activity. These are the natural alkaloids mitomycins [2], peptides madurastatin and
miraziridine [3], anticancer drug azinomycin A [4] and others. Additionally, derivatives of aziridine carboxylic
acid may act as neoplasm inhibitors and therefore are considered as useful anticancer drugs [5, 6]. The latter
class includes Azimexon, Imexon, and aziridine-2-carboxamide (Leakadine, 1) [7-9]. In the case of Imexon,
each of its enantiomers has been studied separately [10]. On the other hand, racemic amide 1 is known since
1957 as a chemical entity [11]. The enantiopure forms of compound 1 have been prepared, but their crystal
structures and other properties have not been studied in detail. Thus, Nakajima et al. have obtained isomer (S)-1
via (2S)-aziridine carboxylic acid benzyl ester [12], but Jähnisch et al. produced isomer (R)-1 in a kinetic
resolution experiment using a sophisticated derivative of (R)-carvone [13].
_______
*Dedicated to Professor Ivars Kalvinsh on the occasion of his 65th birthday.
**To whom correspondence should be addressed, e-mail: maris_turks@ktf.rtu.lv.
1Riga Technical University, 14/24 Azenes St., Riga LV-1007, Latvia.
2Latvian Institute of Organic Synthesis, 21 Aizkraukles St., Riga LV-1006, Latvia; e-mail: serg@osi.lv.
3Institute for Gene Technology, Tallinn Technical University, 15 Akadeemia tee, Tallinn 12618, Estonia;
e-mail: andres.valkna@ttu.ee.
4Competence Center for Cancer Research, 15 Akadeemia tee, Tallinn 12618, Estonia; e-mail:
andres.valkna@ttu.ee.
_________________________________________________________________________________________
Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 6, pp. 928-935, May, 2012. Original
article submitted March 13, 2012.
0009-3122/12/4806-0861©2012 Springer Science+Business Media New York
861

We report here a practical synthesis of (S)-Leakadine ((S)-1) and (R)-Leakadine ((R)-1), comparative
X-ray studies of enantiomerically pure isomer (R)-1 and racemic compound (±)-1, and the initial cytotoxicity
results of the aforementioned products. In order to prepare sufficiently large quantities of enantiomerically pure
forms of Leakadine (1), we have based our synthetic plan on the well-established synthesis of (R)- and (S)-N-tri-
tylaziridine-2-carboxylic acid methyl esters ((S)-3 and (R)-3). These latter were obtained from (R)- and
(S)-serine methyl ester hydrochloride (2), respectively [14]. The syntheses occur identically in (R)- and
(S)-series, and only the latter is depicted in the scheme. Ammonolysis of ester (S)-3 with ammonia followed by
exchange of N-protecting group produced N-benzyloxycarbonyl-protected intermediate (S)-5 [15]. It should be
noted that both intermediates (S)-4 [16] and (S)-5 are relatively stable, but deprotection of the latter via
hydrogenation produces crude (S)-1 in a much more convenient way [17]. Moreover, detritylation in the amide
stage is preferable over a similar step in the ester (S)-3 stage as the proton H-2 in the latter compound is more
labile and thus more prone to racemization. The overall process is easily scalable and permits the reproducible
synthesis of both enantiomeric forms of Leakadine (1) on a gram scale. HPLC analysis on the chiral stationary
phase revealed that both isomers (S)-1 and (R)-1 are of excellent enantiomeric purity (ee > 99.8%). HPLC
conditions were developed with racemic Leakadine ((±)-1) as a standard substance, which was prepared by
combining previously published procedures of synthesis of aziridine carboxylic acid methyl ester [9] and its
aminolysis [18].
O
O
O
MeOH–THF
NH3, rt
[14]
OMe
HO
OMe
NH2
80–90%
N
N
NH2·HCl
(S)-2
Tr
Tr
(S)-3
(S)-4
O
O
1) TFA, CH2Cl2–MeOH, –10°C
H2, Pd/C
2) CbzOSuc, K2CO3, H2O–THF, 0°C
MeOH–THF, rt
NH2
NH2
80–85%
80–90%
N
N
H
Cbz
(S)-1
(S)-5
Melting points of enantiomerically pure (S)-1 or (R)-1 crystallized from methanol (153°C) and racemic
Leakadine ((±)-1) (132°C, MeOH) were determined by differential scanning calorimetry. As expected, isomers
(S)-1 and (R)-1 possessed identical melting point values, which differed significantly from that of racemic amide
(±)-1. This prompted us to perform more detailed studies by single crystal X-ray diffraction analysis. Here we
report the first X-ray studies of both racemic Leakadine (1) and (R)-Leakadine ((R)-1). Monocrystals of
excellent quality were obtained also for isomer (S)-1, but, as expected, they revealed a diffraction pattern
identical to that of isomer (R)-1 and, therefore, were not studied further.
The molecular structure of the isomer (R)-1 with atomic numbering scheme is presented in Figure 1.
The crystallographic parameters of both structures (±)-1 and (R)-1 are equal within error limits. However, the
molecular packing in crystals of the racemate (±)-1 differs significantly from the packing of the pure enantiomer
(R)-1. Figure 2 shows the packing diagram in the crystal structure of amide (±)-1. The packing diagram of
compound (R)-1 is illustrated in Figure 3. Molecules of isomer (R)-1 form the axial orthorhombic crystal lattice
(space group P212121), whereas racemate (±)-1 gives the centrosymmetric monoclinic lattice (space group
P21/c). In both crystal structures all hydrogens of the NH groups participate in the intermolecular bifurcated
hydrogen bonds.
The hydrogen bonds form a three-dimensional net in the crystal structures. In the crystal structure of
racemate (±)-1 there are moderate N(6)−H···O and N(6)−H···N hydrogen bonds and weak N(1)−H(1)···O(5)
bonds. In the crystal structures of isomers (R)-1 and (S)-1 there are moderate N(6)−H···O and N(6)−H···N
bonds and very weak N(1)−H(1)···O(5) hydrogen bonds. On the other hand, the intermolecular distance
862

Fig. 1. Molecular structure of the isomer (R)-1 with representations of the atomic thermal
vibration ellipsoids at 50% probability.
Fig. 2. The packing arrangement in
the unit cell of racemate (±)-1 (mp
132°C) showing the intermolecular
hydrogen bonds.
Fig. 3. The packing arrangement in the
unit cell of isomer (R)-1 (mp 153°C)
showing the intermolecular hydrogen
bonds.
C(3)···C(3) equal to 3.366(3) Å in racemate (±)-1 destabilizes the crystal as the sum of Van der Waals' radii for
carbon atoms is 3.4–3.5 Å. The crystal packing of enantiopure forms show the distance C(3)···C(3) equal to
3.652(4) Å. This may explain the different melting points for the racemate and the pure enantiomers.
Additionally, one can assume that the melting points of racemic and enantiomerically pure Leakadine (1)
are determined by entropy factors. The thermodynamic expression T = Q/S, where Q is heat of the phase
transition and S is its entropy, can be written for the phase transition temperature, T. This means that the
relatively low entropy of crystals of isomers (R)- and (S)-1 leads to an increase in melting point. The entropy
value for the monoclinic structure of racemate (±)-1 is higher than for the orthorhombic structure of isomer (R)-1,
and this situation results in the decreased melting point of racemate (±)-1 compared to isomers (R)- and (S)-1.
The cytotoxic activity of isomers (R)-1, (S)-1 and racemate (±)-1 has been studied. The following cell
lines were used in the current study: large-cell lung carcinoma cell line NCI-H460 (ATCC number HTB-177,
Fig. 4), human pancreatic adenocarcinoma BxPC-3 (ATCC number CRL-1687, Fig. 5), and normal human
umbilical vein endothelial cells (HUVEC, ATCC number PCS-100-010, Fig. 6). All cells were propagated under
conditions suggested by the provider. Each compound was tested on three cell lines at least in three
863

Fig. 4. Inhibition of NCI-H460 lung cancer cell proliferation by Leakadine isoforms in
the range of effective doses.
Fig. 5. Inhibition of BxPC-3 pancreatic adenocarcinoma cell proliferation by Leakadine
isoforms in the range of effective doses.
Fig. 6. Inhibition of normal human cells (human umbilical vein cell line
HUVEC)proliferation by Leakadine isoforms in the range of effective doses.
864

concentrations. Effective concentrations were obtained experimentally in preliminary test series where the effect
on ascending concentrations of drugs on cell proliferation (toxicity) was measured using MTT methodology
[19]. Every single test was performed in at least five parallels.
MTT tests determine the toxicity of compounds based on cell proliferation. It measures the
mitochondrial activity of cells in a tested population. It is a widely used effective and inexpensive test in drug
development that measures the effect of tested compounds on mitochondrial metabolism 19. According to
available data Leakadine (1) is an immunostimulator that elevates the levels of antitumor factors of the immune
system – T helper cells and locally synthesized antibodies – and indirectly reduces the activity of T suppressors
that inhibit immunological responses in growing neoplasms [20]. Therefore one can expect a relatively weak
effect of isomers (R)-1, (S)-1, and racemate (±)-1 on cancer cell proliferation (MTT assay). Nevertheless, this is
the first report on comparative cytotoxicity studies of isomers (R)-1, (S)-1, and racemate (±)-1 on the given cell
lines, and experimental results show that different Leakadine (1) forms behave slightly differently on all tested
cell lines. It is interesting to note that isomer (R)-1 shows less toxicity. This means that this Leakadine (1)
enantiomer will produce fewer side effects if applied in the systemic treatment of cancer. Further studies on the
impact of enantiomerically pure forms of Leakadine (1) on the immune system will be reported elsewhere.
In summary, we have developed a straightforward, reproducible, and scalable method for the synthesis
of both enantiomers of aziridine-2-carboxamide (Leakadine). We have found that there are significant
differences between the crystal packing of its enantiomerically pure and racemic forms. At the macroscopic
level this is manifested by a melting point difference as high as 21°C. An initial MMT assay revealed that the
(S)-isomer is slightly more cytotoxic than the (R)-isomer.
EXPERIMENTAL
1
IR spectra were recorded on a Perkin-Elmer Spectrum BX FT-IR instrument in KBr discs. H and
13C NMR spectra were recorded on a Bruker Avance 300 spectrometer (300 and 75 MHz, respectively); solvent
residual signal or MeOH were used as internal standard [21]. HPLC was performed on an Agilent Technologies
1200 Series chromatograph with a UV-Vis diode array detector (220 nm); reverse phase C18, 3.5 μm, 4.6×150 mm,
0.8 ml/min; eluent A: 0.01 M KH2PO4 in water containing 6% MeCN; eluent B: MeCN; gradient: 20 to 90% B in
10 min, 90% B 5 min. Enantiomeric excess of enantiomerically pure Leakadine (1) was determined by HPLC
(Chiralpak IC 46 × 250 mm), eluent system: 2-PrOH–hexane, 1:1, in isocratic mode, flow rate 0.35 ml/min; UV
detector at 210 nm. Melting points of compounds (S)-4, (R)-4, (S)-5, and (R)-5 were determined on a Stuart SMP10
melting point apparatus and are uncorrected. Melting points of compounds (S)-1, (R)-1, and (±)-1 were determined
by an indium-calibrated (mp 156.6°C) differential scanning calorimeter Mettler 300 (Mettler-Toledo AG). Specific
rotation data were recorded on an Atago AP-300 polarimeter. Progress of the reactions was monitored by TLC
using Merck Silica Gel 60 F254 plates.
Reactions were carried out using solvents and reagents dried according to standard procedures. Isolated
yields refer to chromatographically homogeneous substances.
The cytotoxicity measurements were performed following Mossman et al. [19]. MTT indicates
coloration by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. The optical density in the
biological tests in 96-well microtiter plates was determined using a Tetretek Multiscan MC C/340 horizontal
spectrophotometer.
(-)-(2S)-N-(Triphenylmethyl)aziridine-2-carboxamide ((S)-4). Gaseous NH3 was bubbled through a
solution of ester (S)-3 [14] (35.0 g, 0.102 mol) in MeOH (1000 ml) and THF (50 ml) at 0°C for 1 h. The
resulting reaction mixture was stirred for 36 h at ambient temperature followed by evaporation of the solvents.
Et2O (100 ml) was added to the solid residue, and the resulting suspension was stirred for 2 h at ambient
temperature and filtered. Yield 30.5 g (91%). In the series consisting of eight experiments isolated yields of
amide (S)-4 varied from 80 to 90%. Mp 150-153°C. [α]D –63° (c = 2.00, CHCl3). IR spectrum, ν, cm-1: 3453,
22
865

3288, 3155, 3055, 2924, 2853, 1659, 1600, 1445. 1H NMR spectrum (CDCl3), δ, ppm (J, Hz): 7.44-7.39 (6H, m,
3
H Ph); 7.32-7.22 (9H, m, H Ph); 6.67 (1H, br. s) and 5.38 (1H, br. s, CONH2); 2.07 (1H, d, J = 2.7, 3-CHA);
3
3
3
1.98 (1H, dd, J = 6.6, J = 2.7, 2-CH); 1.51 (1H, d, J = 6.6, 3-CHB). 13C NMR spectrum (CDCl3), δ, ppm:
173.8; 143.2; 129.2; 127.8; 127.2; 74.5; 33.9; 29.9. Found, %: C 80.51; H 6.08; N 8.63. C22H20N2O. Calculated,
%: C 80.46; H 6.14; N 8.53.
(+)-(2R)-N-(Triphenylmethyl)aziridine-2-carboxamide ((R)-4) was prepared from ester (R)-3 in a
similar manner to that described above. In the series consisting of six experiments isolated yields of amide (R)-4
22
varied from 80 to 90%. Mp 150-153°C. [α]D 63° (c = 2.00, CHCl3). Other analytical data of amide (R)-4 are
identical to those of isomer (S)-4.
(-)-(2S)-N-Benzyloxycarbonylaziridine-2-carboxamide ((S)-5). Trifluoroacetic acid (96.3 ml, 1.3
mol) was added to a solution of (S)-4 (21.3 g, 64.9 mmol) in CH2Cl2 (85 ml) and MeOH (102 ml) at -10 to
-15°C. The resulting reaction mixture was stirred at -10°C for 10 h (TLC control, eluent CHCl3–MeOH, 19:1).
Crushed ice (300 g) was added and the organic phase was evaporated under reduced pressure (bath temperature
≤ 30°C). The precipitate (Ph3COH + Ph3COMe) was filtered and washed with water (20 ml). The filtrate was
cooled to 0°C and carefully neutralized at this temperature with solid K2CO3 (52 g) until pH 8-9. The resulting
basic cold aqueous solution was diluted with THF (200 ml) and solution of N-(benzyloxycarbonyl-
oxy)succinimide (17.0 g, 68.4 mmol) in THF (130 ml) was added at -5 to 0°C with vigorous stirring. The pH
level of the resulting mixture was controlled at 8-9 during the full course of the reaction. After stirring for 4 h at
0°C the reaction mixture was extracted with CHCl3 (3×200 ml). The combined organic layer was washed with
brine (100 ml), dried over Na2SO4, filtered, and evaporated under reduced pressure. Et2O (80 ml) was added to
the solid residue, and the resulting suspension was stirred for 1 h at ambient temperature and filtered. Yield 12.9
g (91%). In the series consisting of eight experiments isolated yields of isomer (S)-5 varied from 80 to 90%. Mp
125-126°C. [α]D23 -95° (c = 2.0, CHCl3). IR spectrum, ν, cm-1: 3394, 3321, 3272, 3213, 3065, 3028, 3008, 2935,
1712, 1677, 1458, 1433, 1398, 1342, 1306, 1200. 1H NMR spectrum (DMSO-d6), δ, ppm (J, Hz): 7.94 (1H, br.
2
2
s) and 7.46 (1H, br. s, CONH2); 7.39-7.31 (5H, m, H Ph); 5.08 (1H, d, J = 12.5) and 4.99 (1H, d, J = 12.5,
PhCH2); 3.04 (1H, dd, 3J = 5.2, 3J = 3.3, 2-CH); 2.39 (1H, dd, 3J = 5.2, 2J = 1.9, 3-CHA); 2.33 (1H, dd, 3J = 3.3,
2J = 1.9, 3-CHB). 13C NMR spectrum (DMSO-d6), δ, ppm: 168.2; 160.8; 136.0; 128.4; 128.1; 127.9; 67.3; 35.7;
29.8. Found, %: C 60.05; H 5.42; N 12.83. C11H12N2O3. Calculated, %: C 59.99; H 5.49; N 12.72.
(+)-(2R)-N-Benzyloxycarbonylaziridine-2-carboxamide ((R)-5) was prepared from compound (R)-4
in a similar manner to that described above. In the series consisting of six experiments isolated yields of isomer
(R)-5 varied from 80 to 90%. Mp 125-126°C. [α]D20 95° (c = 2.00, CHCl3). Other analytical data of isomer (R)-5
are identical to those of isomer (S)-5.
(-)-(2S)-Aziridine-2-carboxamide ((S)-1). A suspension consisting of isomer (S)-5 (12.60 g, 57.5
mmol), MeOH (350 ml), THF (60 ml), and 10% Pd/C (1.26 g) was stirred under hydrogen atmosphere at
ambient pressure and temperature for 40 min. The resulting mixture was filtered through Celite and evaporated
under reduced pressure. The crude product (S)-1 (4.90 g, 99%) was crystallized from abs. EtOH (16 ml) in the
presence of activated carbon (0.24 g). Yield of pure compound (S)-1 4.09 g (83%). In the series consisting of
seven experiments isolated yields of isomer (S)-1 varied from 80 to 85%. Enantiomeric excess was determined
by HPLC: τS 19.2 min, τR 21.2 min. Mp 153°C (measured by DSC) (mp 141-142°C [12]). [α]D23 -63° (c = 5.50,
MeOH). [α]D23 -45° (c = 3.00, DMF) ([α]D -44.8° (c = 1.07, DMF) [12]). IR spectrum, ν, cm-1: 3300, 2063,
23
1
3
1650, 1522, 1467, 1447, 1318, 1220, 1183. H NMR spectrum (D2O), δ, ppm (J, Hz): 2.60 (1H, dd, J = 6.0,
3J = 3.1, 2-CH); 1.92 (1H, d, 3J = 6.0, 3-CHA), 1.85 (1H, d, 3J = 3.1, 3-CHB). 13C NMR spectrum (D2O+ MeOH),
δ, ppm: 175.8; 29.3; 25.4. Found, %: C 41.96; H 7.03; N 32.68. C3H6N2O. Calculated, %: C 41.85; H 7.02;
N 32.54.
(+)-(2R)-Aziridine-2-carboxamide ((R)-1) was prepared from compound (R)-5 in a similar manner to
that described above. In the series consisting of five experiments isolated yields of isomer (R)-1 varied from 80
to 85%. Mp 153°C (measured by DSC). [α]D23 +63° (c = 5.5, MeOH). Other analytical data of isomer (+)-(R)-1 are
identical to those of isomer (S)-1
866

TABLE 1. Crystallographic Data for the Investigated Crystals of Isomer
(R)-1 and Racemate (±)-1
Parameters
(R)-1
(±)-1
Molecular formula
M
C3H6N2O
86.094
C3H6N2O
86.094
Crystal habit
Crystal size, mm
Crystal color
Crystal system
Unit cell dimensions
a, Å
Prism
Prism
0.17 × 0.28 × 0.33
Colorless
Orthorhombic
0.21 × 0.36 × 0.37
Colorless
Monoclinic
5.0991(4)
5.0932(2)
b, Å
8.2306(6)
8.1643(4)
c, Å
9.9681(7)
9.9526(4)
β, deg.
V, Å3
90.0
93.452(3)
418.35(5)
413.10(3)
Space group
Z
P212121
P21/c
4
4
F(000)
184
184
μ, mm–1
0.105
0.106
dcalc., g/cm3
1.367
1.384
2θmax for data, deg.
Index ranges
Reflection collected
Independent reflections (Rint)
Reflections with I > Nσ(I)
Final R-factor
wR index for all data (on F2)
Number of refined parameters
(Δ/σ)max
60.0
55.0
-7<h<7; -11<k<11, -13<l<14
-6<h<6; -9<k<10, -12<l<12
1235
1677
732 (0.030)
539 (N = 2)
0.0429
945 (0.020)
796 (N = 3)
0.0360
0.1055
0.0921
79
79
0.007
0.005
Δρmax / Δρmin, e·Å
0.170 / –0.169
0.341 / –0.353
X-ray Studies of (R)- and (±)-Leakadine. The crystal structures of the racemate (±)-1 and (R)-1 were
established by X-ray diffraction analysis. A single crystal automatic diffractometer Bruker-Nonius KappaCCD
(MoKα-radiation, λ 0.71073 Å, T 193(2) K) was used for data collection. The crystal structures were determined
by the direct method [22] and refined by full matrix least squares using SHELXL97 and maXus program
packages [23, 24]. All of the hydrogen atoms were located from difference synthesis of electron density
distribution. The non-hydrogen atoms were refined anisotropically, and the hydrogen ones, isotropically. The
main crystallographic data and structure refinement parameters are given in Table 1. For further details, see
crystallographic data for isomer (R)-1 and racemate (±)-1 deposited with the Cambridge Crystallographic Data
Center as Supplementary Publication (CCDC numbers 863993 and 863994, respectively).
Authors thank PharmIdea Ltd. and The Latvian Council of Science (grant 10.0030) for financial support.
Dr. E. Sūna and Mr. A. Kinēns are acknowledged for HPLC analysis. Authors are indebted to Dr. P. Trapencieris
and Dr. M. Svilāns for helpful discussions. Dr. J. Zicāns and M. Bārtule are acknowledged for DSC analysis.
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