Synthesis and anti-HIV studies of 2- and 3-adamantyl-substituted thiazolidin-4-ones

Article abstract of DOI:10.1016/j.ejmech.2008.02.039

A series of novel thiazolidin-4-ones bearing a lipophilic adamantyl substituent at position 2 or 3 were synthesized. A majority of them showed a modest anti-HIV-1 activity, whereas 2-adamantan-1-yl-3-(4,6-dimethylpyrimidin-2-yl)-thiazolidin-4-one (8) was endowed with a remarkable antiviral potency (EC₅₀ = 0.67 μM). The new series of compounds (22-29) with an adamantyl moiety at the 3-position of the thiazolidinone ring showed good to modest anti-HIV-1 activity (EC₅₀ = 1.0-11 μM) but also pronounced cytostatic activity. For example 24, 26 and 29 showed an EC₅₀ of 1.0-2.0 μM, while the 50% effective concentrations for 23 and 28 were 7.8 and 11.0 μM, respectively. X-ray studies and quantum chemical calculations revealed that the anti-HIV activity of the compounds strongly depends on their dipole moments and conformation of the thiazolidinones.

Full text of DOI:10.1016/j.ejmech.2008.02.039

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European Journal of Medicinal Chemistry 44 (2009) 303e311
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anti-HIV studies of 2- and 3-adamantyl-substituted
thiazolidin-4-ones
a
Jan Balzarini , Barbara Orzeszko-Krzesinska , Jan K. Maurin , Andrzej Orzeszko
b
c,d
e,f,
*
´
a Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
b Warsaw University of Technology, Department of Chemistry, ul. Koszykowa 75, 00-662 Warsaw, Poland
c
´
Institute of Atomic Energy, 05-400 Otwock-Swierk, Poland
d National Medicines Institute, Chełmska 30/34, 00-725 Warsaw, Poland
e Warsaw University of Life Sciences, Institute of Chemistry, ul. Nowoursynowska 159c, 02-787 Warsaw, Poland
f Military University of Technology, ul. Kaliskiego 2, 00-908 Warsaw, Poland
Received 30 November 2007; received in revised form 25 February 2008; accepted 26 February 2008
Available online 10 March 2008
Abstract
A series of novel thiazolidin-4-ones bearing a lipophilic adamantyl substituent at position 2 or 3 were synthesized. A majority of them
showed a modest anti-HIV-1 activity, whereas 2-adamantan-1-yl-3-(4,6-dimethylpyrimidin-2-yl)-thiazolidin-4-one (8) was endowed with a re-
markable antiviral potency (EC50 ¼ 0.67 mM). The new series of compounds (22e29) with an adamantyl moiety at the 3-position of the thia-
zolidinone ring showed good to modest anti-HIV-1 activity (EC50 ¼ 1.0e11 mM) but also pronounced cytostatic activity. For example 24, 26 and
29 showed an EC50 of 1.0e2.0 mM, while the 50% effective concentrations for 23 and 28 were 7.8 and 11.0 mM, respectively. X-ray studies and
quantum chemical calculations revealed that the anti-HIVactivity of the compounds strongly depends on their dipole moments and conformation
of the thiazolidinones.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Thiazolidin-4-ones; Adamantane derivatives; Anti-HIV activity; NNRTIs
1. Introduction
this thiazolidinone. X-ray studies enabled us to ascribe the R
configuration to (þ)-2-adamantan-1-yl-3-(4,6-dimethyl-pyri-
din-2-yl)-thiazolidin-4-one. Furthermore, it was found that the
(þ)-isomer shown in Fig. 1, was predominantly responsible
for the potent anti-HIV-1 activity (EC50 value of 0.178 mM),
while the levo isomer was more than 60-fold less active. These
findings confirm the general rule, that only one defined NNRTI
enantiomer inhibits RT while the other one is usually markedly
less potent or even inactive [2,3].
Moreover we found that all thiazolidinones investigated
showing antiviral activity have similar pharmacophores i.e.
an amide nitrogen atoms connected via one, two or three car-
bon chains to nitrogen atoms in the substituted amines or
pyridines.
We previously reported on several 2-adamantyl-substituted
thiazolidin-4-ones showing modest anti-HIV-1 activity [1]. In
the case of racemic (ꢀ)-2-adamantan-1-yl-3-(4,6-dimethylpyr-
idin-2-yl)-thiazolidin-4-one, a remarkable antiviral potency
(EC50 ¼ 0.35 mM) was observed. The presence of the adaman-
tane moiety played an important role in the eventual antiviral
activity of the compound. This compound behaved as a typical
non-nucleoside reverse transcriptase (RT) inhibitor (NNRTI)
with non-competitive inhibition of RTwith respect to the nucle-
otide substrate (Ki ¼ 12 mM; Ki/Km ¼ 5.5). Separation of the
enantiomers via diastereoisomeric salts was performed for
Prompted by the above observations, we decided to extend
this group of novel non-nucleoside reverse transcriptase inhib-
itors bearing a bulky lipophilic adamantyl entity. We designed
and synthesized three series of thiazolidinone derivatives.
* Corresponding author. Warsaw University of Life Sciences, Institute of
Chemistry, ul. Nowoursynowska 159c, 02-787 Warsaw, Poland.
E-mail address: andrzej_orzeszko@sggw.pl (A. Orzeszko).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.02.039

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J. Balzarini et al. / European Journal of Medicinal Chemistry 44 (2009) 303e311
method. The general pathway of obtaining 3-adamantylmethyl
and 3-adamantylethylthiazolidin-4-ones is presented in Fig. 3.
The structural formulas of the obtained compounds are
given in Table 1.
N
N
O
Structures and purity of novel compounds were confirmed
by elemental analysis, NMR and FTIR spectroscopy, as well
as by chromatographic methods. In some cases we performed
X-ray studies for structure and conformation determination of
the obtained thiazolidinones.
S
Fig. 1. 2-Adamantan-1-yl-3-(4,6-dimethylpyridin-2-yl)-thiazolidin-4-one (7).
Compounds 8e11 represent additional 2-adamant-1-yl-
substituted thiazolidin-4-ones not presented previously. Com-
pounds 12e21 are 2-adamant-1-yl-methylthiazolidin-4-ones.
The thiazolidinones 22e29 have fluorine or chlorine
substituted phenyl ring at the 2-position instead of the ada-
mantane moiety and consist of the adamantyl group connected
via methylene or ethylene bridges at the nitrogen atom of the
thiazolidinone ring. It should be mentioned that such haloaro-
matic substituents were present in the most active thiazolidi-
nones described by Monforte et al. [4e8]. Many of these
thiazolidinones had the pyridyl or pyrimidyl groups as N-sub-
stituents [7,8]. Results obtained by these authors showed that
compounds with a pyrimidine nucleus were mostly 2e3 times
less active against HIV-1. The most active thiazolidinones con-
tained MeO or dihalo substituents attached to the aromatic
ring and a methyl group at the pyridyl nucleus.
2.2. Biological testing
Compounds 8e29 were evaluated for their inhibitory activity
against HIV-1(IIIB) and HIV-2(ROD)-induced cytopathicity in
CEM cell cultures. The results are displayed in Table 2. The
parent pyridine leading derivative 7, the non-nucleoside RT
inhibitor nevirapine and the nucleoside RT inhibitor ddI
(didanosine) were included as reference compounds (Table 2).
Compounds 8e11 belong to the same group as the most
active agent described in our previous publication, i.e. 2-
adamantan-1-yl-3-(4,6-dimethylpyridin-2-yl)-thiazolidin-4-one
(EC50 ¼ 0.35 mM) (7) [1]. The structural difference between
compounds 8e11 and compound 7 consists only in the replace-
ment of a pyridin-2-yl by a pyrimidin-2-yl or pyrimidin-4-yl
nucleus in 11, as the N-substituent. The thiazolidinone 8
(EC50 ¼ 0.67 mM), bearing a 4,6-dimethylpyrimidinyl group,
is only wtwo times less potent than the parent reference com-
pound 7. Its antiviral potency and selectivity was close to that
of the clinically-used compound nevirapine. The replacement
of the 4,6-dimethylpyrimidinyl by the 4-methylpyrimidynyl in
10 (EC50 ¼ 4.50 mM) results in a 6-fold reduction in antiviral
potency. A similar tendency was observed in our previous
work on thiazolidinones containing 4,6-dimethylpyridinyl-
and 4-methylpyridinyl-rings. Thus, 4,6-dimethyl-substituents
connected to a heterocycle seem to be crucial for the activity.
It was also striking to notice that minor modifications such as
the inclusion of a methyl and/or chloro group at the pyrimidine
ring may have a dramatic effect on the eventual antiviral activ-
ity of compounds (Table 2).
2. Results and discussion
2.1. Chemistry
The compounds 2-adamantyl-substituted thiazolidin-4-ones
8e11 and 2-adamantylmethyl thiazolidin-4-ones 12e21 were
prepared according to the scheme shown in Fig. 2 by a one-pot
two-step methodology described previously [1]. The starting
reagents for the synthesis of the title thiazolidinones were ada-
mantane-1-carbaldehyde (1) and adamantan-1-yl-acetaldehyde
(2), respectively. These aldehydes were prepared by Swern ox-
idation from adamantanemethanol and 2-adamantaneethanol
[9]. The general pathway of the 2-adamantylthiazolidin-4-
one syntheses is presented in the scheme given in Fig. 2.
Compounds 22e29 were obtained from commercially
available 2,6-dihalobenzaldehydes as well as 3,4,5-trimethox-
ybenzaldehyde. As amine components 1-aminomethylada-
mantane (5) and 2-aminoethyladamantane (6) were used.
These reagents were synthesized from the respective adaman-
tane alcohols by the Mitsunobu condensation with phthalimide
[10] and consecutive hydrolysis of imides by the Gabriel
The second group of compounds studied belonged to the
thiazolidinones 12e21. In contrast to 8e11, these compounds
contain an adamantylmethylene instead of an adamantyl sub-
stituent connected to the heterocyclic ring at the 2-position.
This little modification of structure invariably causes reduction
of antiviral activity (EC50 ¼ 8.6e53 mM). For example, when
a methylene bridge was inserted between the 1-adamantyl and
the heterocyclic ring in
8
giving compound 12
(EC50 ¼ 8.60 mM), a 13-fold reduction of potency was ob-
served. This tendency is also evident when comparing, for in-
stance, 10 (EC50 ¼ 4.50 mM) and 17 or 18 (completely
inactive), with their respective anti-HIV-1 agents reported pre-
viously [1]. These data point to crucial and rather fixed inter-
action points of adamantane and other parts of the inhibitory
molecule with the HIV-1 RT enzyme, a property which is often
observed for many other non-nucleoside reverse transcriptase
inhibitors. It is interesting that compounds 8 and 12 having
a pyrimidin-2-yl ring at the N-3 position showed activity
R
a
5-53
N
CHO
O
X
X
S
1
X =
2 X = CH2
8-11 X =
12-21 X = CH2
Fig. 2. Synthesis of adamantylthiazolidin-4-ones 8e21: (a) ReNH2,
HSCH2COOH, toluene, reflux.

J. Balzarini et al. / European Journal of Medicinal Chemistry 44 (2009) 303e311
305
X
N
b
O
H2N
X
CHO
37-86
R1,R2,R3
R1,R2,R3
S
5 X = CH2
6 X = CH2CH2
22-29
Fig. 3. Synthesis of adamantyl-substituted thiazolidin-4-ones 22e29: (b) HSCH2COOH, toluene, reflux.
against HIV-1 whereas 11 and 21 with a pyrimidin-4-yl sub-
stituent lost their antiviral properties.
kidney cells, and influenza virus A (H1N1, H3N2) and B in
MDCK cell cultures). None of the compounds showed apprecia-
ble activity at subtoxic concentrations. Since adamantadine and
heterocyclic rimantadine analogues recently obtained display
the anti-influenza virus activity, the inactivity of the adaman-
tanyl derivatives against this virus is most likely due to the bulky
moieties that are linked to the adamantane [11,12].
The third series of compounds (22e29) is structurally
entirely new. These compounds showed good to modest anti-
HIV-1 activity (EC50 ¼ 1.0e11 mM) but also pronounced cyto-
static activity. For example 24, 26 and 29 showed EC50’s of
1.0e2.0 mM, while the 50%-effective concentration for 23
and 28 were 7.8 and 11.0 mM, respectively. In this series the
effect of the alkylene bridge length is not unambiguous, since
the ethyl results in a more pronounced anti-HIV-1 activity than
methyl in compounds 23 and 29 (compare with 22 and 28),
whereas the presence of a methyl resulted in more active
agents than ethyl in compounds 25 and 27 (compare with 24
and 26). It should also be noticed that these thiazolidinones ex-
hibit a certain antiviral activity against HIV-2 (EC50 ¼ 6.5e
46 mM), but it is currently unclear whether this activity reflects
a specific antiviral effect rather than (and most likely) a toxic
effect of the compounds. It is indeed evident from the data in
Table 2 that the antiviral activities against HIV-2 were at com-
pound concentrations that are close to the cytostatic concentra-
tions (CC50) obtained in mock-infected cell cultures.
Four 3-adamantyl-substituted thiazolidin-4-ones from this
series, as well as R enantiomer of 7, were also evaluated for their
inhibitory activity against recombinant HIV-1 and HIV-2 RT.
They showed pronounced inhibition of the HIV-1
RT-catalysed reaction but no appreciable activity against HIV-
2 RT, pointing to the selectivity of the compounds that seem to
behave as NNRTIs. The IC50 for each test compound are pre-
sented in Table 3. Moreover, there was fair correlation between
the log IC50 HIV-1 RT and the log EC50 HIV-1 (r ¼ 0.89).
It would have been obvious to synthesize also 1-adaman-
tanyl derivatives without a methylene group between the nitro-
gen atom and the thiazolidine ring (see Fig. 3). Unfortunately,
1-aminoadamantane does not react at all with aromatic alde-
hydes. An amino-group attached via a tertiary carbon atom
to an adamantyl skeleton is completely inactive in condensa-
tion with aromatic aldehydes, so obtaining of expected com-
pounds was impossible.
2.3. X-ray studies
X-ray measurements are only possible for high-quality
monocrystals with proper dimensions. Only few thiazolidi-
nones from the three compound series could form suitable
crystals. The majority of the compounds obtained are fine-
crystalline, amorphous or liquid. So, for a better understanding
of the differences between the antiviral activities of the studied
compounds, we performed both X-ray structural studies and
quantum chemical calculations only for a number of selected
compounds (i.e. 8, 12, 18, 21 and 26 (for 8 only calculations)).
The molecular geometry of 26 is shown in Fig. 4. The plots
revealed the ‘‘butterfly-like’’ conformation typical for most
NNRTIs [13,14]. The geometries of the remaining three struc-
tures (12, 18, 21) are visualized in Fig. 5 together with the re-
sults of potential density calculations.
2.4. Quantum chemical calculations
Quantum chemical calculations were performed using the
Spartan’04 program. Single point RHF calculations using 6-
31G** basis set were performed for molecular structures taken
from crystallographic studies. The results served as a basis for po-
tentialdensityanddipolemomentcalculationsforthegivenstruc-
tures. In Table 4 volumes, surfaces as well as dipole moments
calculated for thiazolidinones 8, 12, 18, 21, 26 are presented.
For comparison, calculations for 2-adamantan-1-yl-3-(4,6-dime-
thylpyridin-2-yl)-thiazolidin-4-one (7) were also performed.
The results are shown graphically in Fig. 5. The molecules
12, 18 and 21 are topologically non-distinguishable, what can
be seen from values of both volumes and surfaces of the mol-
ecules. Slightly smaller is molecule 7 (see Table 4). In our
opinion the dipole moment might be treated as a global mea-
sure of electrical properties of molecules. From Fig. 5 one can
see that the four molecules have distinct electro-negative
regions (shown in red to yellow) and electro-positive ones
(shown in blue to green). The molecules in the upper row of
All compounds were also evaluated against a broad variety of
DNA and RNAviruses (i.e. herpes simplex virus type 1 and type
2, vaccinia virus and vesicular stomatitis virus (VSV) in HEL
cell cultures; parainfluenza-3 virus, reovirus-1, Sindbis virus,
Coxsackie virus B4 and Punta Toro virus in Vero cell cultures;
VSV, respiratory syncytial virus and Coxsackie virus B4 in
HeLa cell cultures; feline corona virus (FIPV) in Crandel feline

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J. Balzarini et al. / European Journal of Medicinal Chemistry 44 (2009) 303e311
Table 1 (continued)
Table 1
Chemical structures of thiazolidinones
Compound
R
Compound
R
R
N
16
21
N
CH3
N
O
S
CH3
N
General Structure of 8-11
CH3
Compound
R
Compound
R
8
10
CH3
N
N
N
N
X
N
R1R2R3
CH3
O
CH3
Cl
S
General Structure of 22-29
Compound
X
R1
R2
R3
9
11
CH3
N
22
23
24
25
26
27
28
29
CH2
CH2CH2
CH2
3-OCH3
3-OCH3
2-Cl
4-OCH3
4-OCH3
6-Cl
5-OCH3
5-OCH3
N
N
N
CH2CH2
CH2
2-Cl
2-Cl
6-Cl
6-F
CH3
CH3
CH2CH2
CH2
CH2CH2
2-Cl
2-F
6-F
6-F
2-F
6-F
R
N
O
Fig. 5 are endowed with higher dipole moments with the
higher electrostatic potential localized in the groove region be-
tween the carbonyl oxygen and the aromatic nitrogen atoms.
The major difference between molecules in the upper and
those in the bottom row of Fig. 5 is that the latter have hydro-
gen atoms of the pyridine/pyrimidine ring in the close vicinity
of the carbonyl oxygen of the thiazolidinone whereas 7 and 12
have on these sites nitrogen atoms and hence a more negative
potential. One can also see that the thiazolidinone fragment of
both molecules is more co-planar with the aromatic ring than it
is for the upper-row molecules. These facts might correlate
with the anti-HIV activity of compounds listed in Table 4.
Compound 26 belongs to a completely different class of mol-
ecules but from the topological point of view it is very similar
to the others and it has a very similar volume and surface. This
molecule has the dipole moment similar to 7 and 12. The crys-
tal structure of 8 hasn’t been studied. However, because of the
similar activity to the previously studied molecule 7 [1], it was
essential to compare values such as the dipole moment and the
electrostatic potential distribution for this molecule. We there-
fore performed simple quantum-mechanical calculations to
evaluate these values. As can be derived from Table 4 and
Fig. 4 compounds having higher dipole moments and twisted
aromatic rings in relation to the heterocyclic one showed anti-
HIV activity whereas the remaining thiazolidinones are com-
pletely inactive.
S
General Structure of 12-21
Compound
R
Compound
R
12
17
CH3
N
N
N
CH3
CH3
13
18
Cl
CH3
CH3
N
N
N
CH3
14
19
N
N
CH3
N
CH3
CH3
15
20
CH3
CH3
N
N
3. Experimental section
3.1. Chemistry
All commercial reagents and solvents were used without
further purification. Reactions were monitored by GC and

J. Balzarini et al. / European Journal of Medicinal Chemistry 44 (2009) 303e311
307
Table 2
Anti-HIV-1 and anti-HIV-2 activity and cytostatic properties of the compounds
in human T-lymphocyte (CEM) cells
Compound
EC50 (mM)a
CC50 (mM)b
SIc
HIV-1(IIIB)
HIV-2(ROD)
8
0.67 ꢀ 0.49
ꢃ54.9
ꢃ58
122 ꢀ 5.2
>275
182
<5
9
120 ꢀ 21.0
>60.7
10
11
4.5 ꢀ 0.6
ꢃ58.2
136 ꢀ 4.2
30
<1.6
ꢃ58.2
94 ꢀ 10.1
12
13
14
15
16
17
18
19
20
21
8.6 ꢀ 3.6
30 ꢀ 11
27 ꢀ 2.8
3
>53
ꢃ53
102 ꢀ 4.0
27.3 ꢀ 1.8
77 ꢀ 4.0
<1.9
<2.3
1.6
ꢃ11.6
23.5 ꢀ 4.0
ꢃ57
48.7 ꢀ 14
18.1 ꢀ 0.2
17.8 ꢀ 0
>54
21.9 ꢀ 2.8
21.9 ꢀ 5.8
ꢃ54
29.3 ꢀ 3.8
35 ꢀ 2.0
1.6
1.9
115 ꢀ 1.9
77 ꢀ 2.2
<2
53.0 ꢀ 15.2
30.8 ꢀ 5.7
ꢃ11.2
ꢃ62
<1.4
<1.4
<1.9
46 ꢀ 20.3
43 ꢀ 20.1
Fig. 4. ‘‘Butterfly-like’’ conformation of the 26 molecule and the numbering
scheme. The non-H atoms are shown as 30% probability ellipsoids.
ꢃ11.2
22.1 ꢀ 3.1
22
23
24
25
26
27
28
29
ꢃ10.9
ꢃ10.9
6.5 ꢀ 3.9
ꢃ10.1
ꢃ9.7
18 ꢀ 1.0
<1.6
1.4
21
7.8 ꢀ 2.1
1.0 ꢀ 0.6
3.4 ꢀ 1.2
2.0 ꢀ 1.6
3.8 ꢀ 0.8
11.0 ꢀ 2.7
2.0 ꢀ 0.36
11.3 ꢀ 0.97
21.1 ꢀ 2.3
19.0 ꢀ 1.9
20.2 ꢀ 1.3
18.5 ꢀ 3.6
>275
DMSO-d6, chemical shifts (d) were expressed in ppm relative
to tertramethylsilane used as an internal standard, J values are
in hertz, and the splitting patterns were designed as follows: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
Elemental analysis measured on a CHN/S PerkineElmer
2400 element analyzer, were within ꢀ0.4% of the theoretical
values.
X-ray measurements were performed on an Oxford Diffrac-
tion Xcalibur k-axis diffractometer with a Ruby ccd-detector.
The Cu Ka characteristic radiation was used to collect the data.
5.5
10
ꢃ10.5
ꢃ10.1
35.7 ꢀ 4.4
ꢃ4
4.7
>25
4
7.9 ꢀ 0.28
7d
Nevirapine
ddIe
0.35 ꢀ 0.175
0.12 ꢀ 0.11
4.6 ꢀ 2.6
>12
>50
e
42 ꢀ 8.1
120
417
>54
>50
>250
a
EC50: effective concentration or concentration required to protect CEM
cells against the cytopathogenicity of HIV by 50%.
b
CC50: cytotoxic concentration or concentration required to reduce mock-
infected CEM cell viability by 50%.
3.2. Adamantane-1-carbaldehyde (1)
c
SI: selectivity index; ratio CC50/EC50.
Data taken from Ref. [1]. Results of 7 represent the biological activity no-
d
Adamantane-1-carbaldehyde (1) was obtained in the same
way as it was described in our previous publication [1].
ticed for the racemic mixture.
e
Didanosine.
3.3. Adamantan-1-yl-acetaldehyde (2)
TLC. Analytical thin-layer chromatography was performed on
Kieselgel 60 F254 plates (Merck); the spots were located by
UV (254 nm) or iodine. Preparative flash-column chromatog-
raphy was performed using silica gel (Merck) 230e400
mesh. FTIR spectra were recorded on a PerkineElmer 2000
spectrometer in dichloromethane. Melting points (uncor-
rected) were measured in open capillary tubes on a Gallen-
kamp-5 apparatus. 1H NMR spectra were recorded on
a Varian Mercury (400 MHz) spectrometer in CDCl3 and
This compound was synthesized according to the method
published for aldehyde 1. The crude product of the Swern re-
action was purified by flash-column chromatography using
CHCl3 as an eluent. Colourless oil (80%) [15]. FTIR
(CH2Cl2) (cmꢁ1): 1716, 2851, 2908.
3.4. 2-Adamantan-1-yl-methylphthalimide (3) and 2-(2-
adamantan-1-yl-ethyl)phthalimide (4)
Table 3
Anti-HIV-1 and anti-HIV-2 RT activity using polyrC.dG as the template/
Adamantanemethanol (3.33 g, 20 mmol) or adamantanee-
thanol (3.60 g, 20 mmol) were dissolved in 100 mL of dry
THF. Next, phthalimide (2.95 g, 20 mmol), triphenylphos-
phine (5.25 g 20 mmol) and diethyl azodicarboxylate
(3.48 g, 20 mmol) were added to the solution. The mixtures
were stirred for 18 h at room temperature. Then THF was
carefully evaporated and residues were crystallized from
methanol. White crystals of imide 3 (4.5 g, 76%); mp
primer and [3H]dGTP as the radiolabeled substrate
a
IC50 (mM)
Compound
HIV-1 RT
HIV-2 RT
7 (R enantiomer)
13.4 ꢀ 13.6
42.9 ꢀ 3.2
226 ꢀ 58
>600
ꢃ600
>600
>600
ꢃ600
24
25
28
29
a
495 ꢀ 77
1
137 ꢂC; H NMR (CDCl3) d (ppm): 7.84 (m, 4H), 3.23 (s,
31.7 ꢀ 17.7
2H), 1.86 (s, 3H), 1.51e1.59 (m, 12H); FTIR (CH2Cl2)
(cmꢁ1): 1353, 1395, 1710, 1771, 2850, 2908. White crystals
Fifty percent inhibitory concentration, or compound concentration re-
quired to inhibit the HIV RT reaction by 50%.

308
J. Balzarini et al. / European Journal of Medicinal Chemistry 44 (2009) 303e311
Fig. 5. The results of potential density calculations. The values of the electrostatic potential coded in colours are displayed on the ‘‘water accessible’’ surfaces of the
molecules. The geometry of the molecules shown in the drawing was taken from the X-ray structural studies. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
of imide 4 (4.4 g, 70%); mp 155 ꢂC; 1H NMR (CDCl3)
d (ppm): 7.80 (m, 4H), 3.23 (t, J ¼ 7.6 Hz, 2H), 2.40 (t,
J ¼ 7.6 Hz, 2H), 1.85 (s, 3H), 1.48e1.59 (m, 12H); FTIR
(CH2Cl2) (cmꢁ1): 1350, 1395, 1712, 1770, 2852, 2908.
oils HCl in methanol (15 mL) was added and the solvents
were evaporated. Crude products were crystallized from dry
methanol. White crystals of adamantan-1-yl-methylamine hy-
drochloride (1.5 g, 50%); mp 325 ꢂC [16] and white crystals of
2-adamantan-1-yl-ethylamine hydrochloride (1.9 g, 65%); mp
>330 ꢂC [16] were obtained, respectively.
3.5. Adamantan-1-ylmethylamine (5) and 2-adamantan-
1-yl-ethylamine (6) hydrochlorides
3.6. General procedure of preparation of adamantyl-
substituted thiazolidin-4-ones (8e29)
2-Adamantan-1-yl-methylphthalimide (4.1 g, 14 mmol) or
2-(2-adamantan-1-yl-ethyl)phthalimide (4.3 g, 14 mmol)
were heated for 1 h in 40 mL of dry ethanol with hydrazine
hydrate (0.7 g, 14 mmol). After that 20 mL of 10% HCl was
added and the mixture was refluxed for 30 min. Then white de-
posits were filtered off and filtrates were concentrated and fil-
tered again. These solutions were extracted with three portions
of diethyl ether (3 ꢄ 50 mL). The organic layers were dried
over MgSO4 and ether was evaporated. Next, to colourless
All thiazolidin-4-ones were synthesized using the same
method described below. A mixture of appropriate aldehyde
(2 mmol) and respective amine (2 mmol) was refluxed for
0.5e2.5 h in toluene (10 mL) with simultaneous azeotropic re-
moval of water. Then mercaptoacetic acid (0.28 mL, 4 mmol)
was added and the mixture was refluxed further for 6e28 h.
After cooling, the solution was washed two times with a satu-
rated solution of NaHCO3 and brine. The mixture was dried
over magnesium sulphate and the solvent was evaporated.
The residue (oil or solid) was purified by flash-column chro-
matography using chloroform or chloroform:methanol mixture
as eluent. For X-ray studies single crystals were obtained by
recrystallization of respective thiazolidinones from heptane.
Table 4
Geometrical and electrical properties of chosen compounds
3
2
Molecule Volume (A ) Surface (A ) Dipole moment (D) EC50 (mM)a
˚
˚
7
335.59
e
334.22
e
4.38
3.79
4.87
2.87
2.65
4.31
0.35
0.67
8.6
8
12
18
21
25
a
347.86
354.22
348.65
343.48
353.01
354.99
351.59
339.00
nab
na
3.6.1. 2-Adamantan-1-yl-3-(4,6-dimethylpyrimidin-
2-yl)-thiazolidin-4-one (8)
2.0
EC50: effective concentration or concentration required to protect CEM
The reaction was performed for over 22 h. White crystals
were obtained (350 mg, 51%); mp 205 ꢂC; 1H NMR
cells against the cytopathogenicity of HIV by 50%.
b
na: no activity.

J. Balzarini et al. / European Journal of Medicinal Chemistry 44 (2009) 303e311
309
(CDCl3): 6.88 (s, 1H), 5.97 (s, 1H), 3.80 (d, J ¼ 15.8 Hz, 1H),
3.48 (d, J ¼ 15.8 Hz, 1H), 2.43 (s, 3H), 2.32 (s, 3H), 1.92e
1.44 (m, 15H); Anal. (C19H25N3OS) C, H, N.
1H), 3.82 (m, 1H), 3.46 (m, 1H), 3.25 (s, 1H), 3.15 (m, 1H),
2.65 (m, 1H), 2.48 (m, 4H), 1.95e1.55 (m, 22H); Anal.
(C20H32N2OS) C, H, N.
3.6.2. 2-Adamantan-1-yl-3-(4-chloro-6-methylpyrimidin-
2-yl)-thiazolidin-4-one (9)
3.6.9. 2-Adamantan-1-ylmethyl-3-(4-methylpyridin-
2-yl)-thiazolidin-4-one (16)
The reaction was performed for over 24 h. White crystals
The reaction was performed for over 28 h. White crystals
were obtained (50 mg, 5.5%); mp 192 ꢂC; 1H NMR
(CDCl3): 6.98 (s, 1H), 5.99 (s, 1H), 3.83 (d, J ¼ 15.4 Hz,
1H), 3.48 (d, J ¼ 15.4 Hz, 1H), 2.32 (s, 3H), 1.94e1.45 (m,
15H); Anal. (C18H22ClN3OS) C, H, N.
1
were obtained (344 mg, 50%); mp 95 ꢂC; H NMR (CDCl3):
8.28 (s, 1H), 7.77 (s, 1H), 7.02 (s, 1H), 5.92 (m, 1H), 3.94
(m, Hz, 1H), 3.60 (m, 1H), 2.30 (s, 3H), 1.93e1.40 (m,
17H); Anal. (C20H26N2OS) C, H, N.
3.6.3. 2-Adamantan-1-yl-3-(4-methylpyrimidin-
2-yl)-thiazolidin-4-one (10)
3.6.10. 2-Adamantan-1-ylmethyl-3-(6-methylpyridin-
2-yl)-thiazolidin-4-one (17)
The reaction was performed for over 20 h. White crystals
were obtained (140 mg, 15%); mp 163 ꢂC; 1H NMR
(CDCl3): 8.61 (m, 1H), 7.37 (dd, J ¼ 6.0 Hz, J ¼ 2.0 Hz,
1H), 5.45 (d, J ¼ 0.9 Hz, 1H), 3.82 (dd, J ¼ 16.0 Hz,
J ¼ 0.9 Hz, 1H), 3.50 (d, J ¼ 16.0 Hz, 1H), 2.48 (s, 3H),
1.88e1.39 (m, 15H); Anal. (C18H23N3OS) C, H, N.
The reaction was performed for over 24 h. White crystals
were obtained (300 mg, 44%); mp 116 ꢂC; 1H NMR
(CDCl3): 8.20 (s, 1H), 7.37 (s, 1H), 7.00 (s, 1H), 5.91 (m,
1H), 3.94 (m, Hz, 1H), 3.60 (m, 1H), 2.30 (s, 3H), 1.93e
1.33 (m, 17H); Anal. (C20H26N2OS) C, H, N.
3.6.11. 2-Adamantan-1-ylmethyl-3-(4,6-dimethylpyridin-
2-yl)-thiazolidin-4-one (18)
3.6.4. 2-Adamantan-1-yl-3-(2,6-dimethylpyrimidin-
4-yl)-thiazolidin-4-one (11)
The reaction was performed for over 24 h. White crystals
were obtained (378 mg, 53%); mp 159 ꢂC; 1H NMR
(CDCl3): 7.64 (s, 1H), 6.92 (s, 1H), 5.84 (m, 1H), 4.04 (m,
1H), 3.66 (m, Hz, 1H), 2.50 (s, 3H), 2.35 (s, 3H), 1.93e1.33
(m, 17H); Anal. (C21H28N2OS) C, H, N.
The reaction was performed for over 22 h. White crystals
were obtained (200 mg, 34%); mp 176 ꢂC; 1H NMR
(CDCl3): 6.99 (s, 1H), 5.97 (s, 1H), 3.80 (d, J ¼ 15.8 Hz,
1H), 3.48 (d, J ¼ 15.8 Hz, 1H), 2.45 (s, 3H), 2.37 (s, 3H),
1.90e1.40 (m, 15H); Anal. (C19H25N3OS) C, H, N.
3.6.12. 2-Adamantan-1-ylmethyl-3-(2-dimethylamino-
ethyl)-thiazolidin-4-one (19)
3.6.5. 2-Adamantan-1-ylmethyl-3-(2,6-methylpyrimidin-
2-yl)-thiazolidin-4-one (12)
The reaction was performed for over 12 h. Colourless oil
was obtained (320 mg, 50%); 1H NMR (CDCl3): 4.40 (s,
1H), 3.82 (m, 1H), 3.46 (m, 2H), 3.25 (s, 1H), 3.15 (m, 1H),
2.65 (m, 1H), 2.29 (s, 6H), 1.95e1.55 (m 1.93e1.41 17H);
Anal. (C18H30N2OS) C, H, N.
The reaction was performed for over 22 h. White crystals
were obtained (220 mg, 31%); mp 160 ꢂC; 1H NMR
(CDCl3): 7.33 (s, 1H), 5.90 (s, 1H), 3.93 (d, J ¼ 15.8 Hz,
1H), 3.66 (d, J ¼ 15.8 Hz, 1H), 2.51 (s, 3H), 2.37 (s, 3H),
1.93e1.41 (m, 17H); Anal. (C20H27N3OS) C, H, N.
3.6.13. 2-Adamantan-1-ylmethyl-3-
3.6.6. 2-Adamantan-1-ylmethyl-3-(4-chloro-6-
methylpyrimidin-2-yl)-thiazolidin-4-one (13)
(2-dimethylaminoethyl)-thiazolidin-4-one (20)
The reaction was performed for over 12 h. Colourless oil
1
was obtained (300 mg, 47%); H NMR (CDCl3): 4.22 (m,
The reaction was performed for over 28 h. White crystals
were obtained (80 mg, 10.5%); mp 168 ꢂC; 1H NMR
(CDCl3): 8.03 (s, 1H), 5.90 (s, 1H), 3.93 (m, 1H), 3.62 (m,
1H), 2.44 (s, 3H), 1.93e1.41 (m, 17H); Anal.
(C19H24ClN3OS) C, H, N.
1H), 3.82 (m, 1H), 3.46 (m, 1H), 3.25 (s, 1H), 3.15 (m, 1H),
2.65 (m, 1H), 1.95e1.55 (m 1.93e1.41 22H), 1.00 (t,
J ¼ 7.2 Hz, 6H); Anal. (C20H34N2OS) C, H, N.
3.6.14. 2-Adamantan-1-ylmethyl-3-(2,6-
3.6.7. 2-Adamantan-1-ylmethyl-3-(4-methylpyrimidin-
2-yl)-thiazolidin-4-one (14)
The reaction was performed for over 28 h. White crystals
dimethylpyrimidin-4-yl)-thiazolidin-4-one (21)
The reaction was performed for over 22 h. White crystals
1
were obtained (36 mg, 5%); mp 118 ꢂC; H NMR (CDCl3):
1
were obtained (70 mg, 10%); mp 103 ꢂC; H NMR (CDCl3):
6.99 (s, 1H), 5.97 (s, 1H), 3.80 (d, J ¼ 15.8 Hz, 1H), 3.48
(d, J ¼ 15.8 Hz, 1H), 2.45 (s, 3H), 2.37 (s, 3H), 1.90e1.40
(m, 17H); Anal. (C20H27N3OS) C, H, N.
8.60 (s, 1H), 7.31 (s, 1H), 5.92 (s, 1H), 3.90 (m, Hz, 1H),
3.60 (m, 1H), 2.44 (s, 3H), 1.93e1.41 (m, 17H); Anal.
(C19H25N3OS) C, H, N.
3.6.15. 3-Adamantan-1-ylmethyl-2-(3,4,5-
3.6.8. 2-Adamantan-1-ylmethyl-3-(2-pyrrolidin-
1-ylethyl)-thiazolidin-4-one (15)
The reaction was performed for over 12 h. Colourless oil
was obtained (180 mg, 26%); 1H NMR (CDCl3): 4.41 (s,
trimethoxyphenyl)-thiazolidin-4-one (22)
The reaction was performed for over 6 h. White crystals
1
were obtained (477 mg, 58%); mp 67 ꢂC; H NMR (CDCl3):
7.11 (s, 2H), 6.30 (m, 1H), 3.74 (s, 2H), 3.71 (s, 9H), 3.33

310
J. Balzarini et al. / European Journal of Medicinal Chemistry 44 (2009) 303e311
(m, 1H), 3.21 (m, 1H), 1.92e1.34 (m, 15H); Anal.
(C23H31NO4S) C, H, N.
3.7. X-ray measurements
The data were collected using Cu Ka radiation. After
mounting and centering the single crystal on the diffractome-
ter reflections were collected up to 2q < 140ꢂ. Structures were
solved by direct methods from SHELXS-97 program [17]
and then refined on F2 using SHELXL-97 software [18].
The detailed structural data have been deposited with CCDC
under the numbers 669067, 669068, 669069 and 669070,
respectively.
3.6.16. 3-(2-Adamantan-1-ylethyl)-2-(3,4,5-
trimethoxyphenyl)-thiazolidin-4-one (23)
The reaction was performed for over 6 h. White crystals
1
were obtained (677 mg, 78%); mp 63 ꢂC; H NMR (CDCl3):
7.10 (s, 2H), 6.30 (m, 1H), 3.71 (s, 9H), 3.33 (m, 1H), 3.21
(m, 1H), 3.14 (s, 2H), 1.92e1.34 (m, 17H); Anal.
(C24H33NO4S) C, H, N.
3.8. Biological methods
3.6.17. 3-Adamantan-1-ylmethyl-2-(2,6-
dichlorophenyl)-thiazolidin-4-one (24)
3.8.1. Cells and viruses
The reaction was performed for over 6 h. White crystals
were obtained (688 mg, 86%); mp 149 ꢂC; 1H NMR
(CDCl3): 7.51 (m, 3H), 6.68 (m, 1H), 3.33 (m, 1H), 3.81
(m, 1H), 3.30 (m, 2H), 1.92e1.34 (m, 15H); Anal.
(C20H23Cl2NOS) C, H, N.
Human immunodeficiency virus type 1 [HIV-1(IIIB)] was
obtained from Dr. R.C. Gallo (when at the National Cancer
Institute, Bethesda, MD). HIV-2(ROD) was provided by
Dr. L. Montagnier (when at the Pasteur Institute, Paris,
France).
3.6.18. 3-(2-Adamantan-1-ylethyl)-2-(2,6-
dichlorophenyl)-thiazolidin-4-one (25)
3.8.2. Activity assay of test compounds against HIV-1 and
HIV-2 in cell culture
CEM cells (4 ꢄ 104) per millilitre were infected with HIV-
1(IIIB) or HIV-2(ROD) at w100 CCID50 (50% cell culture in-
fective dose) per millilitre of cell suspension. Then 100 ml of
the infected cell suspension was transferred to microlitre plate
wells and mixed with 100 ml of the appropriate dilutions of the
tested compounds. Giant cell formation was recorded micro-
scopically in the HIV-infected cell cultures after 4 days. The
50% effective concentration (EC50) of the tested compounds
was defined as the compound concentration required to inhibit
virus-induced cytopathicity (CEM) by 50%. The 50% cyto-
static concentration (CC50) was defined as the compound con-
centration required inhibiting mock-infected CEM cell
proliferation by 50%.
The reaction was performed for over 6 h. Amorphous glass
was obtained (650 mg, 79%); 1H NMR (CDCl3): 7.61 (m, 3H),
6.62 (m, 1H), 3.78 (s, 2H) 3.57 (m, 1H), 2.41 (m, 1H), 1.92e
1.05 (m, 17H); Anal. (C21H25Cl2NOS) C, H, N.
3.6.19. 3-Adamantan-1-ylmethyl-2-(2-chloro-6-
fluorophenyl)-thiazolidin-4-one (26)
The reaction was performed for over 6 h. White crystals
were obtained (688 mg, 86%); mp 136 ꢂC; 1H NMR
(CDCl3): 7.80e7.70 (m, 3H), 6.69 (m, 1H), 3.81 (m, 1H),
3.54 (m, 1H), 3.30 (m, 2H), 1.99e1.30 (m, 15H); Anal.
(C20H23ClFNOS) C, H, N.
3.6.20. 3-(2-Adamantan-1-ylethyl)-2-(2-chloro-6-
fluorophenyl)-thiazolidin-4-one (27)
The reaction was performed for over 6 h. Amorphous glass
was obtained (550 mg, 69%); H NMR (CDCl3): 7.79e7.65
(m, 3H), 6.62 (m, 1H), 3.77 (s, 2H) 3.57 (m, 1H), 2.41 (m,
1H), 1.93e1.10 (m, 17H); Anal. (C21H25ClFNOS) C, H, N.
3.8.3. Anti-reverse transcriptase assays
The source of the reverse transcriptases used was recombi-
nant HIV RT [derived from HIV-1(IIIB) or HIV-2(ROD)],
constructed and prepared as described before [1]. The RT as-
says contained a total reaction mixture volume (50 ml) of
50 mM TriseHCl (pH 7.8), 5 mM dithiothreitol, 300 mM
glutathione, 500 mM EDTA, 150 mM KCl, 5 mM MgCl2,
1.25 mg of bovine serum albumin, labelled substrate
[8-3H]dGTP (1 mCi) (specific radioactivity 11 Ci/mmol)
(2.5 mM) (Moravek Biochemicals, Brea, CA), a fixed concen-
tration of the template/primer poly(C).oligo(dG)12e18
(0.1 mM), 0.06% Triton X-100, 10 ml of inhibitor at various
concentrations, and 1 ml of the RT preparation. The reaction
mixtures were incubated at 37 ꢂC for 30 min, at which time
200 ml of yeast RNA (150 mg/ml) and 1 ml of trichloroacetic
acid (5% v/v) in saturated Na4P2O7 (0.1 M in 1 M HCl) were
added. The solutions were kept on ice for 30 min, after which
the acid-insoluble material was washed and analyzed for ra-
dioactivity. The IC50 for each test compound was determined
as the compound concentration that inhibited HIV RT activity
by 50%.
1
3.6.21. 3-Adamantan-1-ylmethyl-2-(2,6-
difluorophenyl)-thiazolidin-4-one (28)
The reaction was performed for over 6 h. White crystals
were obtained (270 mg, 37%); mp 145 ꢂC; 1H NMR
(CDCl3): 7.77e7.67 (m, 3H), 6.69 (m, 1H), 3.81 (m, 1H),
3.53 (m, 1H), 3.30 (m, 2H), 1.99e1.32 (m, 15H); Anal.
(C20H23F2NOS) C, H, N.
3.6.22. 3-(2-Adamantan-1-ylethyl)-2-(2,6-
difluorophenyl)-thiazolidin-4-one (29)
The reaction was performed for over 6 h. Amorphous glass
1
was obtained (350 mg, 46%); H NMR (CDCl3): 7.74e7.65
(m, 3H), 6.60 (m, 1H), 3.75 (s, 2H) 3.58 (m, 1H), 2.40 (m,
1H), 1.89e1.10 (m, 17H); Anal. (C21H25F2NOS) C, H, N.

J. Balzarini et al. / European Journal of Medicinal Chemistry 44 (2009) 303e311
311
4. Summary
References
[1] J. Balzarini, B. Orzeszko, J.K. Maurin, A. Orzeszko, Eur. J. Med. Chem.
42 (2007) 993e1003.
In the presented publication, as well as in our previous pa-
per, we performed the synthesis of two new subclasses of
NNRTIs. The first one includes the thiazolidine-4-ones with
the adamantyl moiety attached directly, or via methylene or
ethylene bridges, to the thiazolidinone ring. Compounds
with the adamantyl group at the 3-position of the heterocyclic
ring belong to the second subclass of NNRTIs. Compounds 7
and 8 bearing a 4,6-dimethylpyridinyl and 4,6-dimethylpyri-
midinyl nucleus showed the highest anti-HIV activities
(EC50 ¼ 0.35 and 0.67 mM, respectively). The X-ray measure-
ments revealed that these agents exist in a ‘‘butterfly confor-
mation’’ common for many NNRTIs. Quantum chemical
calculations revealed also that the anti-HIV activity of the
compounds strongly depends on their dipole moments.
[2] B.L. de Corte, J. Med. Chem. 48 (2005) 1689e1696.
[3] T.K. Venkatachalam, C. Mao, F.M. Uckun, Bioorg. Med. Chem. 12
(2004) 4275e4284.
[4] M.L. Barreca, A. Chimirri, L. De Luca, A.M. Monforte, P. Monforte,
A. Rao, M. Zappala, J. Balzarini, E. De Clercq, C. Pannecouque,
M. Witvrouw, Bioorg. Med. Chem. Lett. 11 (2001) 1793e1796.
[5] A. Rao, J. Balzarini, A. Carbone, A. Chimirri, E. De Clercq, A.M. Monforte,
P. Monforte, C. Pannecouque, M. Zapalla, Antivir. Res. 63 (2004) 79e84.
[6] M.L. Barreca, J. Balzarini, A. Chimirri, E. De Clercq, L. De Luca,
H.D. Holtje, M. Holtje, A.M. Monforte, P. Monforte, C. Pannecouque,
¨
¨
A. Rao, M. Zappala, J. Med. Chem. 45 (2002) 5410e5413.
[7] A. Rao, J. Balzarini, A. Carbone, A. Chimirri, E. De Clercq,
A.M. Monforte, P. Monforte, C. Pannecouque, M. Zapalla, Farmaco 59
(2004) 33e39.
[8] A. Rao, J. Balzarini, A. Carbone, A. Chimirri, E. De Clercq, A.M. Monforte,
P. Monforte, C. Pannecouque, M. Zapalla, Farmaco 57 (2002) 747e751.
[9] A.J. Mancuso, D. Swern, Synthesis (1981) 165e185.
[10] O. Mitsunobu, Synthesis (1981) 1e28.
Acknowledgment
[11] G. Zoidis, C. Fytas, I. Papanastasiou, G.B. Foscolos, G. Fytas,
E. Padalko, E. de Clercq, L. Naesens, J. Neyts, N. Kolocouris, Bioorg.
Med. Chem. 14 (2006) 3341e3348.
The study was supported from the Flemish Fonds voor Weten-
schappelijk Onderzoek (no. G-0188-07) and the Geconcerteerde
Onderzoeksacties (GOA no. 05/19) of the K.U.Leuven. We thank
Lizette van Berckelaer, Leen Ingels, Leentje Persoons, Vicky
Broeckx and Frieda De Meyer for excellent technical help.
The work was supported in part (for A.O. and J.K.M.) by
Polish Ministry of Science and Higher Education Grant,
PBZ-MIN 014/P05/2004.
[12] N. Kolocouris, G. Zoidis, G.B. Foscolos, G. Fytas, S.R. Prathalingham,
J.M. Kelly, L. Naesens, E. De Clercq, Bioorg. Med. Chem. Lett. 17
(2007) 4358e4362.
[13] A. Basavapathruni, K.S. Anderson, Curr. Pharm. Des. 12 (2006)
1857e1865.
[14] E. De Clercq, Collect. Czech. Chem. Commun. 63 (1998) 449e479.
[15] J.R. Luly, J.F. Dellaria, J.J. Plattner, J.L. Soderquist, N. Yi, J. Org. Chem.
52 (1987) 1487e1492.
[16] P.E. Aldrich, E.C. Hermann, W.E. Meier, M. Paulshock, W.W. Prichard,
J.A. Snyder, J.C. Watts, J. Med. Chem. 14 (1971) 535e543.
[17] G.M. Sheldrick, SHELXS-97, Program for X-ray Structure Solution,
Appendix. Supplementary data
University of Gottingen, Germany, 1997.
¨
[18] G.M. Sheldrick, SHELXL-97, Program for X-ray Structure Refinement,
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.ejmech.2008.02.039.
University of Gottingen, Germany, 1997.
¨