Cyclohexenonic long-chain fatty alcohols as neuronal growth stimulators

Article abstract of DOI:10.3390/51201439

Neurotrophic factors play an important role in the development and maintenance of neurons, thus providing a suitable therapeutic approach for the treatment of neurodegenerative diseases. However, their clinical use has revealed problematic because of a number of technical and biological disadvantages. Among the different strategies proposed to overcome such difficulties, the search for non-peptide substances with neurotrophic potential is giving promising results. Here we will expose major findings in this field, drawing special attention to cyclohexenonic long-chain fatty alcohols, a novel family of compounds that promote neuronal survival and neurite outgrowth.

Full text of DOI:10.3390/51201439

Molecules 2000, 5, 1439–1460
molecules
ISSN 1420-3049
http://www.mdpi.org
Cyclohexenonic Long-Chain Fatty Alcohols
as Neuronal Growth Stimulators
Bang Luu1*, José-Luis González de Aguilar1,2 and Céline Girlanda-Junges1
1Laboratoire de Chimie Organique des Substances Naturelles, UMR CNRS 7509, Centre de
Neurochimie, Université Louis Pasteur, 5, rue Blaise Pascal, 67084 Strasbourg cedex, France.
Tel.: +33-(0)3-88-411672. Fax: +33-(0)3-88-607464.
2Laboratoire de Neurophysiologie Cellulaire et Integrée, UMR CNRS 7519, Université Louis Pasteur,
Strasbourg, France.
* Author to whom correspondence should be addressed. E-mail: luu@chimie.u-strasbg.fr
Received: 13 November 2000; in revised form: 7 December 2000 / Accepted: 7 December 2000 /
Published:22 December 2000
Abstract: Neurotrophic factors play an important role in the development and maintenance
of neurons, thus providing a suitable therapeutic approach for the treatment of
neurodegenerative diseases. However, their clinical use has revealed problematic because of
a number of technical and biological disadvantages. Among the different strategies proposed
to overcome such difficulties, the search for non-peptide substances with neurotrophic
potential is giving promising results. Here we will expose major findings in this field,
drawing special attention to cyclohexenonic long-chain fatty alcohols, a novel family of
compounds that promote neuronal survival and neurite outgrowth.
Keywords: cyclohexenonic long-chain fatty alcohol, neurite outgrowth, neuronal survival,
neurotrophic compound, neurotrophic factor, NGF, synthesis
© 2000 MDPI. All rights reserved.

Molecules 2000, 5
1440
1. Introduction
2. Neurotrophic compounds as therapeutic agents for neurodegenerative diseases
2.1. Natural neurotrophic factors
2.2. Non-peptide neurotrophic factor analogues
3. Long-chain fatty alcohols
4. Cyclohexenonic long-chain fatty alcohols
4.1. Synthesis of cyclohexenonic long-chain fatty alcohols
4.2. Biological activity of cyclohexenonic long-chain fatty alcohols
5. Conclusions
6. Experimental section: synthesis of t-CFAs
6.1. Materials and Methods
6.2. Synthesis of 1-(12-hydroxydodecyl-1-phenylsulfonyl)-2,6,6-trimethyl-1-cyclohexene
6.3. Synthesis of 1-(12-hydroxydodecyl)-2,6,6-trimethyl-1-cyclohexene
6.4. Synthesis of 1-(12-acetoxydodecyl)-2,6,6-trimethyl-1-cyclohexene
6.5. Synthesis of 3-(12-acetoxydodecyl)-2,4,4-trimethyl-2-cyclohexen-1-one
6.6. Synthesis of 3-(12-hydroxydodecyl)-2,4,4-trimethyl-2-cyclohexen-1-one
1. Introduction
Since the discovery of the nerve growth factor (NGF) in the 50’s [1], numerous neurotrophic factors
(NFs) have been proven to play a crucial role in the survival and differentiation of discrete neuronal
populations. The origin of these naturally occurring factors is diverse, including the neurotrophin,
fibroblast growth factor, epidermal growth factor, insulin-like growth factor and transforming growth
factor β families, cytokines, proteases, and protease inhibitors [2]. The fact that such substances are
able to restrain neuronal loss has generated over the past several years a great interest in their
therapeutic potential for the treatment of neurodegenerative diseases. However, the clinical use is at
present restricted because of a number of technical and biological disadvantages. In this respect, the
production of synthetic compounds that mimic the biological actions of natural NFs represents an
attractive approach to bypass these difficulties. In this review, we will outline the limitations of NF
therapies, and the effects of various non-peptide NF analogues. Specifically, we will focus on the
chemistry and biological activity of cyclohexenonic long-chain fatty alcohols, a new family of
compounds generated in our laboratory.

Molecules 2000, 5
1441
2. Neurotrophic compounds as therapeutic agents for neurodegenerative diseases
2.1. Natural neurotrophic factors
As mentioned above, the ability of naturally occurring NFs to promote the survival and development
of neurons provides strong evidence for their therapeutic utilisation. Thus, an increasing number of
preclinical and clinical studies have concerned the application of different NFs to the treatment of
neurodegenerative disorders. In some cases, clinical trials have been conducted with variable success
(Table 1). The main obstacle for using natural NFs as therapeutic agents resides in their chemical
structure. They are hydrophilic, high-molecular weight proteins that can be rapidly degraded by
endogenous peptidases, thus limiting to a large extent oral, subcutaneous or intravenous administration.
In addition, they cannot cross the blood-brain barrier, which impedes severely the treatment of central
nervous system (CNS) disorders. It should be also noted that systemic administration may provoke
adverse side effects (i.e. injection site reactions, cough, asthenia, nausea, anorexia, weight loss) that
worsen the situation of the patients, as has been illustrated by injecting recombinant human ciliary
neurotrophic factor in patients affected of amyotrophic lateral sclerosis [3, 4].
To overcome these problems, diverse strategies have been considered. Intracranial injection of NFs
allows them to reach directly specific brain areas. However, studies that used this aggressive,
uncommon method to treat Alzheimer’s disease patients have reported uncertain results [5, 6].
Table 1. Use of natural NFs in the treatment of neurodegenerative diseases.
Diseases and affected neurons
Alzheimer’s disease:
Tested Neurotrophic Factors
NGF (Neurotrophic Growth Factor)
BDNF (Brain-Derived Neurotrophic Factor)
NT-4/5 (Neurotrophin-4/5)
Cholinergic neurons
bFGF (basic Fibroblast Neurotrophic Factor)
GDNF (Glial-Derived Neurotrophic Factor)
BDNF
Parkinson’s disease:
Dopaminergic neurons
NT-4/5
Ischemia:
TGF-β1 (Transforming Growth Factor)
Striatum, hippocampus and cortex IGF-1 (Insulin-like Growth Factor)
bFGF
NT-4/5
Amyotrophic lateral sclerosis:
Motor neurons
IGF-1
BDNF
CNTF (Ciliary Neurotrophic Factor)

Molecules 2000, 5
1442
In contrast, the local implantation of polymer-encapsulated cells genetically modified to produce
and secrete NFs minimises the incidence of negative immune reactions, and makes possible a
continuous delivery of the therapeutic agent [7, 8]. Likewise, injection into the affected area of
recombinant adenoviruses that encodes for NFs also provides a local and constant delivery system,
although strong inflammatory reactions can be expected [9, 10]. At present, these gene therapies,
already tested in experimental models of neurodegenerative disorders, need further improvements to
gain in applicability and reduced costs. An additional strategy to transport NFs into the CNS consists of
fusing the therapeutic agent with a transferrin receptor antibody, thus forming a conjugate capable of
crossing the blood-brain barrier [11]. Finally, small peptide sequences that contain the active site of
NFs should be able to mimic their biological actions, and solve many of the difficulties resulting from
the use of protein macromolecules [12, 13]. However, these techniques are still at a basic research
level.
2.2. Non-peptide neurotrophic factor analogues
The synthesis or isolation from biological specimens of non-peptide organic compounds is emerging
as a novel and promising approach in the treatment of neurodegenerative disorders. These substances,
that either stimulate NF production or act directly on neurons to promote survival and/or neurite
outgrowth, present a number of advantageous properties. In principle, they are low-molecular weight
compounds that can be chemically manipulated to cross the blood-brain barrier, and to ameliorate their
tolerance and pharmacokinetic parameters. In addition, large-scale production technologies may be
applied. Over the last decade, diverse studies have reported the neurotrophic activity of numerous non-
peptide molecules in vitro (Fig. 1). In some cases, they have also been tested in animal models of
neurodegeneration, and in clinical trials. We will expose now the major results in this field.
A first group of substances possessing a direct neuritogenic action were originally isolated from
microbial extracts. Thus, lactacystin, a sulphur-containing γ-lactam from Streptomyces sp. OM6519
[14], and PS-990, a metabolite of Acremonium sp. KY12702 [15], induce neurite sprouting in mouse
neuroblastoma Neuro 2A cells. Moreover, epolactaene, an epoxide-containing γ-lactam from
Penicillium sp. BM1689P, stimulates neuritogenesis in human neuroblastoma SH-SY5Y cells [16].
Interestingly, recent studies have suggested that the differentiating neuritogenic effect elicited by these
compounds could result from their ability to prevent cell cycle progression, through as yet unclear
mechanisms that include inhibition of either the multicatalytic protease complex proteasome [17] or
DNA replication enzymes [18]. Plants commonly used in traditional medicine constitute another
source of molecules with NF-like activity. Examples of these substances inducing neurite extension in
primary cultures of foetal rat neurons are caryolanemagnolol, a sesquiterpene neolignan from the bark
Magnolia obovata [19], and S-allyl-L-cysteine, an organosulphur compound with a thioallyl group
from the garlic Allium sativum [20]. Finally, Triap (1,1,3-tricyano-2-amino-1-propene), a small
molecule largely studied in the past, stimulates survival of sympathetic ganglion neurons of chick

Molecules 2000, 5
1443
embryos, and neurite outgrowth in rat pheochromocytoma PC12 cells [21]. In vivo, Triap promotes the
differentiation of cholinergic neurons after systemic administration [22]. At present, little is known
about the mechanisms of action of all these compounds, and in particular further efforts must be done
to evaluate their specific actions and toxic side effects under pathological conditions.
Astroglial cells participate actively in the homeostasis and neuronal plasticity of the CNS, by
maintaining an appropriate extracellular medium, and producing a series of metabolic precursors,
immunological mediators and NFs. Thus, substances that induce the synthesis and release of
endogenous NFs by astroglial cells may present a valuable therapeutic potential. In this respect, the
non-peptide compound SR57746A [1-[2-beta-naphthylethyl]-4-(3-trifluoromethylphenyl)-1,2,5,6-
tetrahydropyridine hydrochloride] has been shown to exhibit both NF-like and NF-releasing
properties. On the one hand, SR57746A stimulates, by itself or through potentiation of NGF activity,
neuritogenesis and/or survival in several cell culture systems [23-25].
Figure 1. Structure of non-peptide molecules with NF-like and/or NF-releasing activity.

Molecules 2000, 5
1444
On the other hand, it increases NGF mRNA and protein contents in astrocytoma cells [26], and
cultured astrocytes from neonatal rat cortex [27]. In vivo, SR57746A, either orally administered or
injected, has demonstrated protective effects in a variety of experimental models of neurodegenerative
disorders, such as transient global ischaemia, vincristine-induced septohippocampal lesion, sciatic
nerve crushing, acrylamide-induced neuropathy [28], β-amyloid pepdide-induced memory deficit [29],
progressive motor neuropathy [30], and motor neurone axotomy [31]. 4-Methylcatechol, a non-amine
catechol analogue, has also been shown to induce the synthesis and secretion of NGF in mouse
astroglial cells [32], and to protect neurons against experimental injury [33-37].
Alzheimer’s disease, the main cause of senile dementia, is a neurological disorder characterised by
the presence of β-amyloid pepdide-containing plaques in specific brain areas, and the degeneration of
cholinergic neurons, which are thought to be responsible for cognitive impairments [38]. Because early
studies suggested that NGF may ameliorate this cholinergic dysfunction [39, 40], the use of NGF
stimulators as potential therapeutic agents constitute a very valuable strategy in the treatment of
Alzheimer’s disease. In this respect, two organic compounds, the coenzyme Q analogue idebenone
[2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone], and the alkylated xanthine
derivative propentofylline [1-(5’-oxohexyl)-3-methyl-7-propylxanthine] have been the focus of basic
and clinical research. These substances induce the synthesis and secretion of NGF in cultured mouse
astroglial cells [41, 42], and numerous studies have reported their protective effects in experimentally-
induced neurodegenerative disorders [43-45]. Specifically, both idebenone [46] and propentofylline
[47] ameliorate learning and memory deficits provoked by β-amyloid pepdides. In addition, their
ability to improve cognitive impairments, and delay the progression of Alzheimer’s disease has been
evaluated in a number of clinical trials with promising results [48-50]. At present, however, growing
evidence supports additional neuroprotective mechanisms distinct from stimulation of NGF synthesis
and release. These mechanisms include inhibitioin of adenosine uptake by propentofylline [51], and a
remarkable antioxidant activity of both molecules [52, 53].
Finally, other compounds of diverse nature such as hericenones [54], fellutamide A [55],
sesterterpenes [56], pyrroloquinoline quinone [57], erinacines [58], dictyophorines [59], and
scabronines [60] also stimulates NGF production, and therefore their therapeutic potential should be
explored.
3. Long-chain fatty alcohols
The curative capacity of plants is known for centuries, and people over the world uses commonly
many of them for the treatment of diverse illnesses. Nowadays, research laboratories and
pharmaceutical industries make continuous efforts to characterise novel vegetable substances of
pharmacological application. Our early interest in this field led us to the search for non-peptide NF
analogues in plants reported to heal wounds. Interestingly, the process of cutaneous wound healing

Molecules 2000, 5
1445
requires the participation of a variety of growth factors that contribute actively to the repair and
remodeling of the injured tissue [61]. In particular, NGF has been shown to be locally released in
response to skin injury, and its topical application accelerates the rate of wound healing in both normal
and diabetic mice [62]. Thus, one can expect that the ability of certain plants to cure wounds may
reside in the existence of compounds with NF-like effects.
In 1987, we reported the in vitro neuritogenic activity of long-chain fatty alcohols isolated from the
tropical plant Hygrophila erecta Hochr. (Acanthaceae), which is used in Vietnam and India to treat
wounds. Among these alcohols, n-hexacosanol (Fig. 2; 1), containing 26 carbon atoms, was the most
potent in inducing neuronal maturation. In contrast, n-tetracosanol and n-triacontanol were not
effective, while n-octacosanol presented a slight effect. Similarly, saturated fatty acids were ten times
less potent than the corresponding alcohols, thus supporting the specificity of the molecular structure in
determining the biological activity [63]. In vivo, n-hexacosanol has been shown to promote the
survival of cholinergic neurons after fimbria-fornix transection [64], and to reduce kainic acid-
induced hippocampal damage [65].
Figure 2. Structure of n-hexacosanol (1), retinol (2), and its elongated analogues (3-7).

Molecules 2000, 5
1446
Although these findings predict a potential use of n-hexacosanol as a therapeutic agent, an
important limitation must be taken into account. Due to its highly lipophilic nature, n-
hexacosanol is hardly soluble in both aqueous and organic solvents, which brings about considerable
technical and biological difficulties.
In an attempt to improve the physicochemical properties of n-hexacosanol, we undertook the
synthesis of new molecules that, resembling the structure of long-chain fatty alcohols, could manifest a
neurotrophic activity. Because vitamin A (or retinol; Fig. 2; 2), which is involved in visual function,
cell morphogenesis, differentiation and proliferation [66], bears a long-chain fatty alcohol in its
structure, we used this molecule as starting-point for developing a series of compounds with longer,
saturated hydrocarbon chains. These elongated analogues of perhydroretinol (Fig. 2; 3-7) have been
proved to exhibit NF-like activity in different experimental paradigms. In vitro, 3, 4 and 5 promote the
survival of cultured CNS neurons, and, in addition, 4 and 5 stimulate the proliferation of newborn rat
astrocytes. In vivo, intraperitoneal administration of 3 and 4 reduce memory deficits provoked by lesion
of the basal magnocellular nucleus. From a structural point of view, configuration studies revealed that
the four diastereomers 7a, 7b, 7c and 7d present a similar neurite outgrowth-stimulating effect.
Moreover, removal of the asymmetric methyl groups in the hydrocarbon chain showed that non-
methylated perhydroretinols are as active as their methylated counterparts, which strongly suggests that
the presence of methyl groups is not essential for the biological activity [67-69].
4. Cyclohexenonic long-chain fatty alcohols
4.1. Synthesis of cyclohexenonic long-chain fatty alcohols
From our findings on the neurotrophic action of perhydroretinol analogues, we considered the
possibility of modifying the cyclohexene ring in order to evaluate structure/activity relationships, and
further improve the efficiency of the molecules. Thus, a series of novel, neurotrophic compounds,
known as cyclohexenonic long-chain fatty alcohols (CFAs; Fig. 3), were synthesised.
Figure 3. Structure of diverse cyclohexenonic long-chain fatty alcohols.

Molecules 2000, 5
1447
These CFAs contain basically a cyclohexenonic ring, and an ω-alkanol side chain ranging from 10
to 18 carbon atoms (Fig. 3; 8). In addition, the cyclic moiety may present one (2m- or 4m-CFA; Fig. 3;
9 and 10, respectively), two (dm-CFA; Fig. 3; 11), or three (t-CFA; Fig. 3; 12) methyl groups. The
synthesis of the CFA series 8, 9, 10 and 11 was accomplished using the Umpolung strategy, which
allows the inversion of the polarity in the cyclohexenone ring to introduce the hydrocarbon side chain
by nucleophilic displacement (Fig. 4) [70, 71].
Figure 4. Synthesis of CFA (8), 2m-CFA (9), 4m-CFA (10), and dm-CFA (11). Reagents:
(a) PhSO2Na, HCl, H2O, room temperature (r.t.), 24 hours. (b) HO-(CH2)n-OH, pTsOH,
benzene, reflux, 4 hours. (c) n-BuLi, THF, HMPA, CHPh3, -78°C to r.t., 1 hour;
Br(CH2)nOH; THF, -78°C to -20°C, 2 hours. (d) CHCl3-acetone (5-1), pTsOH, 50°C, 24
hours.
The synthesis of t-CFAs [3-(ω-hydroxy-alkyl)-2,4,4-trimethyl-2-cyclohexen-1-one; 12], according
to the retrosynthetic analysis shown in Fig. 5, was carried out in seven steps from commercially
available geranylbromide 13 (see Section 5 for details) [72]. Briefly, 13 reacted with
benzenesulfinic acid sodium salt in methanol medium
to
give
the
corresponding
geranylphenylsulfone 14, which was cyclised as described by Krischna et al. [73] and Torii et al. [74]
to afford a mixture of 15 and 16 (85/15) in 90% yield (Fig. 6). These two isomers were separated by
successive recrystallizations in hexane, and the desired 15 was obtained in 74% yield with a purity
over 99%. Using a one-pot procedure, and n-butyllithium in the presence of hexamethylphosphoramide
(HMPA), 15 was coupled with the unprotected ω-bromoalkanol to give 17 (79-88%). After reductive
desulfonation by means of 6% Na/Hg amalgam [75] (88-93%), the resulting alcohol 18 was protected
by an acetate to give 19 (97-98%), which was oxidized to the corresponding α,β-unsaturated ketone 20

Molecules 2000, 5
1448
using RuCl3 as catalyst (0,7% mol), and 70% tBuOOH [76] (49-53%). Deprotection of the alcohol by
K2CO3 in methanol gave 12 in 81-96% yield [77] (Fig. 7).
Figure 5. Retrosynthetic analysis of t-CFAs (12).
Figure 6. Synthesis of sulfones 15 and 16 from geranylbromide 13. Reagents: (a) PhSO2Na, MeOH,
0°C, 1 hour (80%). (b) H2SO4 32 eq., AcOH, 12°C, 30 min (90%).

Molecules 2000, 5
1449
Figure 7. Synthesis of t-CFAs (12) from sulfone 15. Reagents: (a) n-BuLi, THF, HMPA, CHPh3, -
78°C to r.t., 1 hour; Br(CH2)nOH, THF, -78°C to –20°C, 2 hours, (79-88%). (b) Na/Hg 6%, MeOH,
Na2HPO4, 0°C to r.t., 3 hours (88-93%). (c) Ac2O, pyridine, r.t., 1 hour (97-98%). (d) RuCl3 0.7%
mol, tBuOOH 70%, cyclohexane, H2O, r.t., 6 hours (49-53%). (e) K2CO3, MeOH, H2O, r.t., 2 hours
(81-96%).
4.2. Biological activity of cyclohexenonic long-chain fatty alcohols
The neurotrophic action of the synthesised CFAs is first assessed by in vitro studies that serve as a
means to evaluate their potential, and to establish the most appropriate structure/activity relationships.
To this aim, and based on previous findings [78], we developed a low-density primary culture that uses
neurons from foetal rat cerebral hemispheres, and allows analysis of both neuronal survival and neurite
outgrowth. Fig. 8 illustrates an example of the protective action of two CFAs, 4m-CFA (n=14) and t-
CFA (n=15), on the survival of cultured CNS neurons.
Survival (fold induction)
*
*
1.5
1.0
0.5
0.0
control
4m-CFA t-CFA
(n=14) (n=15)
Figure 8. Effect of 4m-CFA (n=14), and t-CFA (n=15) on the survival of cultured CNS neurons.
Substances were tested at 100 nM. The data represent means ± SE of three independent experiments.
*p<0.05 vs Control; one-way ANOVA, followed by Student-Newman-Keuls test.

Molecules 2000, 5
1450
Concerning neuritogenesis, important considerations must be taken into account that support the
existence of specificity in the stimulation of neurite sprouting by different CFAs. Thus, comparative
studies on the neuritogenic effect elicited by the series of compounds 8, 9, 10, and 11 (Table 2)
revealed that monomethylated CFAs with 13 or 14 carbon atoms in the aliphatic side chain were the
most potent molecules, while non-methylated CFAs provoked toxicity. Similarly, in the series of t-
CFAs (12), the hydrocarbon chains n=14 and n=15 seem to be the most active (Fig. 9). It is noteworthy
that other studies dealing with the neurotrophic potential of non-peptide compounds have postulated
that aliphatic side chains may potentiate both NF-like [79] and NF-releasing [41, 54, 80] activities. At
present, the intracellular mechanisms underlying these structure requirements are still unknown, and
further investigation is needed. Besides, the in vitro actions of CFAs provides evidence for their
utilisation as neurotrophic factors in vivo, and suggest possible applications in the treatment of
neurological disorders. Our preliminary findings on in vivo administration of CFAs in a transgenic
mouse model of amyotrophic lateral sclerosis [81, 82] have shown that treated animals respond more
rapidly to behavioural tasks (Fig. 10). New experiments are currently in progress.
Cells (%)
70
type 1
type 2
type 3
60
50
40
30
20
10
0
control
t-CFA
t-CFA
t-CFA
t-CFA
t-CFA
t-CFA
(n=12)
(n=13)
(n=14)
(n=15)
(n=16)
(n=18)
Figure 9. Effect of t-CFAs on neurite outgrowth. Six t-CFAs bearing an aliphatic side chain that
ranged from 12 to 18 carbon atoms were tested at 100 nM. After morphometric analysis, neurons
were divided into three categories according to their total neurite length (Type 1 ”ꢀꢁꢁꢂµm; Type 2
101-200 µm; Type 3 201-300 µm). Note that t-CFA (n=14) and (n=15) are the most potent
molecules in stimulating neurite sprouting. The data correspond to two pooled independent
experiments (50-70 cells / experiment).

Molecules 2000, 5
1451
Table 2. Structure/activity relationships of the CFA series 8, 9, 10, and 11 according to their
neuritogenic effect in cultured CNS neurons.
Stimulation of neurite sprouting by 10 ng/mL bFGF was used as positive control. Compounds were
tested at 10 nM. (0) No effect; (+) Stimulatory effect; (-) Toxic effect. The table summarises data from
at least two independent experiments.

Molecules 2000, 5
1452
Time (seconds)
25
20
15
10
A
*
5
0
-12
-9
-8
-7
-6
-5
-3
-2
-1
Time (days)
25
20
15
10
B
***
***
***
***
5
0
-23 -19 -16 -14 -11
-9
-7
-5
-2
-1
Time (days)
Figure 10. Effect of 4m-CFA (n=14) (A), and t-CFA (n=15) (B) on the ability of mice to perform an
exercise. The mice used in these experiments overexpress a mutant Cu/Zn superoxide dismutase gene
associated with the neurodegenerative disease amyotrophic lateral sclerosis. The task consisted in
determining the time (in seconds) necessary to cross a metallic bar of 45 cm long. Substances were
injected intraperitoneally at 8 mg/kg 4m-CFA (n=14) or 32 mg/kg t-CFA (n=15) three times per week
during 50-60 days, and meaurements were taken at days indicated in the abscissas before disease onset
and death of the animals through motor paralysis. Note that treated mice (filled circles in both graphs)
are more active than control animals. *p<0.05, ***p<0.001 vs corresponding control; one-way
ANOVA, followed by Student-Newman-Keuls test.

Molecules 2000, 5
1453
5. Conclusions
In summary, the findings presented herein provide strong evidence that cyclohexenonic long-chain
fatty alcohols exhibit a very promosing NF-like activity, thus establishing the basis for future
investigations. Further research by testing this neurotrophic potential in experimental and transgenic
models of neurodegenerative diseases is currently in progress, in order to evaluate possible therapeutic
effects.
6. Experimental section: synthesis of t-CFAs
6.1. Materials and Methods
Tetrahydrofuran (THF) was distilled from sodium/benzophenone under argon atmosphere prior to
use. Methanol, pyridine, benzene and HMPA were distilled from calcium hydride. All reactions
involving moisture-sensitive reactants were executed under argon atmosphere using oven- and/or
1
flame-dried glassware. H NMR spectra were recorded on a Bruker SY 200 spectrometer at 200 MHz
as solutions in deuterochloroform (CDCl3). Chemical shifts are expressed in parts per million (ppm, ꢃꢄ
downfield from tetramethylsilane (TMS), and are referenced to CHCl3 (7.26 ppm) as internal standard.
Splitting patterns are designed as s (singlet), d (doublet), t (triplet), q (quartet), qn (quintuplet), m
13
(multiplet), or br (broad). Coupling constants are given in hertz (Hz). C NMR spectra were recorded
on the Bruker SY 200 spectrometer at 50 MHz as solutions in CDCl3. Chemical shifts are reported in
ppm downfield from TMS, and are referenced to the centre line of CDCl3 (77.0 ppm) as internal
standard. The attribution of the different carbons (C, CH, CH2, and CH3) was determined by 13C to 1H
polarisation transfer (DEPT). IR spectra were recorded on sodium chloride using a Perkin-Elmer 881
FT-IR spectrometer, and are reported in wave numbers (cm-1). UV spectra were obtained in acetonitrile
solution using a Kontron-Uvikon 810 UV-Vis spectrometer. Mass spectra (MS) were measured on a
Trio 2000 apparatus by direct introduction [ionisation potential 70 eV; M/z relative intensities (in %)
are noted in brackets]. GC chromatograms were obtained by dissolving the sample in ethyl acetate
using a Carlo-Erba apparatus. Microanalyses were performed by the Service Central de Microanalyse
du CNRS (Strasbourg). Routine chromatography monitoring of reactions was performed using 60F254
silica gel TLC plates, which were dipped in a solution of vanillin (1 g) in EtOH/H2SO4 (95/5), and
heated on a hot plate.
6.2. Synthesis of 1-(12-hydroxydodecyl-1-phenylsulfonyl)-2,6,6-trimethyl-1-cyclohexene
To a solution containing sulfone 15 (1 g, 3.5 mmol, 2 eq.), and 4 mg triphenylmethane in dry THF
(8 mL) was added n-butyllithium (4 mL, 1.4 M in hexane, 3 eq.). After stirring for 10 min at -78°C
under argon atmosphere, the mixture was further stirred at room temperature. Then, HMPA (1.5 mL)

Molecules 2000, 5
1454
was added, and after 90 min at room temperature, the mixture was recooled at -78°C. 11-Bromo-
undecanol (439 mg, 1.75 mmol, 1 eq.) was added slowly, and the mixture was stirred for 180 min at -
20°C. After pouring into saturated NH4Cl (40 mL), the solution was extracted with ether. The organic
layer was then washed with brine, dried with MgSO4, and concentrated in vacuo. The residue was
purified by silica-gel chromatography, and eluted with hexane-AcOEt (8-2 to 6-4) to give 17 (n=12) as
1
a white solid (622 mg, 79%). TLC: (hexane-AcOEt: 5-5) Rf=0.43; H-NMR (200 MHz), δ: 0.87 (s,
3H, H-19); 0.97 (s, 3H, H-20); 1.16 (s br, 14H, H-10 to H-16); 1.2-1.57 (m, 8H, H-4, 5, 9, 17); 1.94 (s,
3H, H-21); 1.98-2.25 (m, 4H, H-8, 3); 3.61 (t, J=6.8 Hz, 1H, H-18); 3.71 (t, J=6.8 Hz, 1H, H-7); 7.48-
7.65 (m, 3H, H ar-3', 4'); 7.86-7.92 (m, 2H, H ar-2'); 13C-NMR (50 MHz), δ: 19 (C-4); 23 (C-21); 25.7
(C-16); 28.5 (C-8); 28.9 (C-19, 20); 29.4 (C-9 to C-15); 31.2 (C-17); 32.7 (C-3); 35.9 (C-6); 39.8 (C-
5); 63 (C-18); 67.9 (C-7); 128.4 (C ar-2'); 130.5 (C-2); 133 5 (C ar-4'); 137.8 (C-1); 141.8 (C ar-1');
m.p.: 77-78°C; microanalysis (%): calcd for C27H44O3S (448.7028) C: 72.27, H: 9.88, O: 10.7; found
C: 72.19, H: 9.92, O: 11.09. Coupling with the other hydrocarbon side chains from n=13 to 18 is
shown in Table 3.
Table 3. Analysis of compounds 17 bearing an aliphatic side chain that ranged from 13 to 18 carbon
atoms (TLC: hexane-AcOEt: 5-5).
Product
(n)
Yield
(%)
m.p
(°C)
R.f*
Analysis
Microanalysis (%): C28H46O3S (462.7296)
Calcd: C: 72.68; H: 10.02; O: 10.37
Found: C: 72.49; H: 10.06; O: 10.62
Microanalysis (%): C28H46O3S (462.7296)
Calcd: C: 73.06; H: 10.15; O: 10.07
Found: C: 72.83; H: 10.18; O: 9.85
MS (EI): 334.9 (M-SO2Ph, 100); 136.5 (C10H16); 122.5 (C9H15); 108.5;
94.5; 80.5
(13)
7
.46 56-57
(14)
4
.47 75-76
Microanalysis (%): C30H50O3S (490.7832)
Calcd: C: 73.42; H: 10.27; O: 9.78
(15)
(16)
(18)
6
6
8
.46 58-59
.48 85-87
.56 93-95
Found: C: 73.34; H: 10.34; O: 9.92
Microanalysis (%): C31H52O3S (504.810)
Calcd: C: 73.76; H: 10.38; O: 9.51
Found: C: 73.56; H: 10.37; O: 9.63
Microanalysis (%): C33H56O3S (532.8636)
Calcd: C: 74.38; H: 10.59; O: 9.01
Found: C: 73.90; H: 10.74; O: 9.15

Molecules 2000, 5
1455
6.3. Synthesis of 1-(12-hydroxydodecyl)-2,6,6-trimethyl-1-cyclohexene
To a solution of 17 (n=12) (579 mg, 1.29 mmol, 1 eq.) in dry methanol (25 mL) was added
Na2HPO4 (366 mg, 2 eq.), and mercury-sodium amalgam (6% Na, 4 g) at 0°C under argon atmosphere.
The heterogeneous mixture was stirred for 240 min at room temperature, and then quenched with 1N
HCl. After extraction with ether (3 times), the solution was washed with saturated NaHCO3, dried with
MgSO4, and concentrated in vacuo. The residue was purified by silica-gel chromatography, and eluted
with hexane-AcOEt (8-2-) to give 18 (n=12) as a colorless oil (350 mg, 88%). TLC: (hexane-AcOEt:
1
5-5) Rf=0.59; GC: 40-280°C (20°C/min) 10.50 min; H-NMR (200 MHz), δ: 0.96 (s, 6H, H-19, 20);
1.27 (s br, 16H, H-9 to H-16); 1.35-1.54 (m, 8H, H-4, 5, 8, 17); 1.57 (s, 3H, H-21); 1.83-2.03 (m, 4H,
13
H-3, 7); 3.62 (t, J=6.5 Hz, 2H, H-18); C-NMR (50 MHZ), δ: 19.6 (C-4); 19.8 (C-21); 25.7 (C-16);
28.6 (C-19,20); 28.9 (C-8); 29.6 (C-9 to C-15); 30.5 (C-17); 32.8 (C-3*); 32.81 (C-7*); 34.8 (C-6);
39.8 (C-5); 63 (C-18); 126.3 (C-2); 137.8 (C-1); IR ν: 3330 (broad, O-H); 2925, 2852 (w, C-H); 1466
(s, C-H); 1112 (s, C-O).
6.4. Synthesis of 1-(12-acetoxydodecyl)-2,6,6-trimethyl-1-cyclohexene
To a solution of 18 (n=12) (316 mg, 1.026 mmol) was added acetic anhydride (7 mL), and pyridine
(7 mL). The mixture was stirred for 60 min at room temperature, and quenched with 5% HCl. The
mixture was then extracted with ether, washed with water, dried with MgSO4, and concentrated in
vacuo to obtain 19 (n=12) as a colorless oil (353 mg, 98%). TLC: (hexane-AcOEt: 5-5) Rf=0.75; GC:
40-280°C (20°/min) 11.02; 1H-NMR (200 MHz), δ: 0.96 (s, 6H, H-19, 20); 1.27 (s br, 16H, H-9 to H-
16); 1.35-1.54 (m, 8H, H-4, 5, 8, 17); 1.57 (s, 3H, H-21); 1.83-2.02 (m, 4H, H-3, 7); 2.04 (s, 3H, CH3-
COO); 4.04 (t, J=6.6 Hz, 2H, H-18); 13C-NMR (50 MHz), δ: 19.5 (C-4); 19.8 (C-21); 20.9 (CH3-
COO); 25.9 (C-16); 28.6 (C-19, 20); 28.9 (C-8); 29.6 (C-9 to C-15); 30.5 (C-17); 32.7 (C-7*); 32.75
(C-3*); 34.8 (C-6); 39.9 (C-5); 64.6 (C-18); 126.3 (C-2); 137.8 (C-1); 171.2 (CH3-COO); IR ν: 2925,
2854 (w, C-H); 1744 (w, C=O); 1466 (s, C-H); 1238 (w, C-O).
6.5. Synthesis of 3-(12-acetoxydodecyl)-2,4,4-trimethyl-2-cyclohexen-1-one
To a solution containing 19 (n=12) (321 mg, 0.92 mmol, 1 eq.) in cyclohexane (6 mL) was added
water (0.8 mL), ruthenium trichloride hydrate (1.3 mg, 0.7% mol), and 70% t-BuOOH (1.26 mL, 10
eq.). The solution was stirred for 360 min at room temperature, filtered through a pad of Celite, and
poured into 10% Na2SO3. The solution was then extracted with ether, washed with brine, dried with
MgSO4, and concentrated in vacuo. The residue was purified by silica-gel chromatography, and eluted
with hexane-AcOEt (95-15 to 90-10) to give 20 (n=12) as a colorless oil (227 mg, 53%). TLC:
1
(hexane-AcOEt: 7-3) Rf=0.50; GC: 40-280°C (20°C/min) 12.2 min, 99%; H-NMR (200 MHz), δ:
1.13 (s, 6H, H-19, 20); 1.26 (s br, 16H, H-9 to H-16); 1.35-1.69 (m, 4H, H-8, 17); 1.73 (s, 3H, H-21);

Molecules 2000, 5
1456
1.78 (t, J=7.5 Hz, 2H, H-5); 2.02 (s, 3H, CH3-COO); 2.11-2.19 (m, 2H, H-7); 2.43 (t, J=6.8 Hz, 2H, H-
13
6); 4.03 (t, J=6.8 Hz, 2H, H-18); C-NMR (50 MHz), δ: 11.5 (C-21); 20.9 (CH3-COO); 25.8 (C-16);
26.8 (C-19, 20); 28.8 (C-8); 29.1 (C-17); 29.5 (C-9 to C-15); 30.45 (C-7); 34.2 (C-5); 36.2 (C-4); 37.3
(C-6); 64.5 (C-18); 130.5 (C-2); 165 (C-3); 171 (CH3-COO); 199 (C-1); IR ν: 2925, 2854 (w, C-H);
1741 (w, C=O); 1667 (w, C=O); 1607 (s, C=C); 1468 (s, C-H); 1239 (w, C-O).
6.6. Synthesis of 3-(12-hydroxydodecyl)-2,4,4-trimethyl-2-cyclohexen-1-one
To a solution of 20 (n=12) (132 mg, 0.36 mmol, 1 eq.) in dry methanol (8 mL) was added 3 drops of
water, and K2CO3 (74 mg, 0.54 mmol, 1.5 eq.). After stirring for 150 min at room temperature, the
solution was neutralized at pH=7 with 5% HCl. Then, the solution was extracted with ether, dried with
MgSO4, and concentrated in vacuo. The residue was purified by silica-gel chromatography, and eluted
with hexane-AcOEt (8-2 to 7-3) to give 12 (n=12) as a colorless oil (94 mg, 81%). TLC: (hexane-
1
AcOEt: 7-3) Rf=0.2; GC: 40-280°C (20°/min) 13.94 min, 99%; H-NMR (200 MHz), δ: 1.13 (s, 6H,
H-19, 20); 1.26 (s br, 16H, H-9 to H-16); 1.35-1.59 (m, 4H, H-8, 17); 1.73 (s, 3H, H-21); 1.77 (t, J=7.5
Hz, 2H, H-5); 2.11-2.19 (m, 2H, H-7); 2.43 (t, J=6.8 Hz, 2H, H-6); 3.61 (t, J = 6.5 Hz, 2H, H-18); 13C-
NMR (50 Mhz), δ: 11.4 (C-21); 25.7 (C-16); 26.8 (C-19, 20); 28.8 (C-8); 29.5 (C-9 to C-15); 30.45
(C-7); 32.7 (C-17); 34.2 (C-5); 36.2 (C-4); 37.3 (C-6); 62.9 (C-18); 130.4 (C-2); 165.4 (C-3); 199 (C-
1); IR ν: 3440 (broad OH); 2925,2853 (w, C-H); 1666 (w, C=O); 1605 (s, C=C); 1467 (s, C-H). UV
λmax: 244.5 nm (ε 10420); microanalysis (%): calcd for C21H38O2 (322.530) C: 78.02, H: 11.87; found
C: 78.34, H: 12.03.
Acknowledgements:
This study was supported by Meiji Milk Products Co., LTD. J.L.G.A. is the recipient of a grant Aide
aux Etudes from the Association Française contre les Myopathies.
References
ꢀꢁꢂ/HYLꢃ0RQWDOFLQLꢄꢂ5ꢁꢂ6FLHQFH ꢀꢁꢂꢃꢄꢂꢀꢁꢂꢄꢂꢀꢀꢅꢆꢃꢀꢀꢇꢈꢁ
ꢈꢁꢂ/LQGVD\ꢄꢂ5ꢁ0ꢁꢉꢂ:LHJDQGꢄꢂ6ꢁ-ꢁꢉꢂ$OWDUꢄꢂ&ꢁ$ꢁꢉꢂ'L6WHIDQRꢄꢂ3ꢁ6ꢁꢂ7UHQGVꢃ1HXURVFLꢄ ꢀꢁꢁꢄꢄꢂꢅꢂꢄꢂꢀꢊꢈꢃꢀꢋꢌꢁ
ꢍꢁꢂ0LOOHUꢄꢂ5ꢁ*ꢁꢉꢂ%U\DQꢄꢂ:ꢁ:ꢁꢉꢂ'LHW]ꢄꢂ0ꢁ$ꢁꢉꢂ0XQVDWꢄꢂ7ꢁ/ꢁꢉꢂ3HWDMDQꢄꢂ-ꢁ+ꢁꢉꢂ6PLWKꢄꢂ6ꢁ$ꢁꢉꢂ*RRGSDVWXUHꢄꢂ-ꢁ&ꢁ
1HXURORJ\ ꢀꢁꢁꢅꢄꢂꢆꢂꢄꢂꢀꢍꢈꢋꢃꢀꢍꢍꢀꢁ
ꢆꢁꢂ0LOOHUꢄꢂ5ꢁ*ꢁꢉꢂ3HWDMDQꢄꢂ-ꢁ+ꢁꢉꢂ%U\DQꢄꢂ:ꢁ:ꢁꢉꢂ$UPRQꢄꢂ&ꢁꢉꢂ%DURKQꢄꢂ5ꢁ-ꢁꢉꢂ*RRGSDVWXUHꢄꢂ-ꢁ&ꢁꢉꢂ+RDJODQG
5ꢁ-ꢁꢉꢂ3DUU\ꢄꢂ*ꢁ-ꢁꢉꢂ5RVVꢄꢂ0ꢁ$ꢁꢉꢂ6WURPDWWꢄꢂ6ꢁ&ꢁꢂ$QQXꢄꢃ1HXUROꢄ ꢀꢁꢁꢅꢄꢂꢁꢇꢄꢂꢈꢅꢇꢃꢈꢇꢌꢁ
ꢅꢁꢂ 6HLJHUꢄꢂ $ꢁꢉꢂ 1RUGEHUJꢄꢂ $ꢁꢉꢂ YRQꢂ +ROVWꢄꢂ +ꢁꢉꢂ %lFNPDQꢄꢂ /ꢁꢉꢂ (EHQGDOꢄꢂ 7ꢁꢉꢂ $ODIX]RIIꢄꢂ ,ꢁꢉꢂ $PEHUODꢄꢂ .ꢁꢉ
+DUWYLJꢄꢂ3ꢁꢉꢂ+HUOLW]ꢄꢂ$ꢁꢉꢂ/LOMDꢄꢂ$ꢁꢂ%HKDYꢄꢃ%UDLQꢃ5HVꢄ ꢀꢁꢁꢆꢄꢂꢈꢂꢄꢂꢈꢅꢅꢃꢈꢇꢀꢁ

Molecules 2000, 5
1457
ꢇꢁꢂ(ULNVGRWWHUꢄꢂ-ꢁ0ꢁꢉꢂ1RUGEHUJꢄꢂ$ꢁꢉꢂ$PEHUODꢄꢂ.ꢁꢉꢂ%lFNPDQꢄꢂ/ꢁꢉꢂ(EHQGDOꢄꢂ7ꢁꢉꢂ0H\HUVRQꢄꢂ%ꢁꢉꢂ2OVRQꢄꢂ/ꢁꢉ
6HLJHUꢄꢂ $ꢁꢉꢂ 6KLJHWDꢄꢂ 0ꢁꢉꢂ 7KHRGRUVVRQꢄꢂ (ꢁꢉꢂ 9LLWDQHQꢄꢂ 0ꢁꢉꢂ :LQEODGꢄꢂ %ꢁꢉꢂ :DKOXQGꢄꢂ /ꢁ2ꢁꢂ 'HPHQWꢄ
*HULDWUꢄꢃ&RJQꢄꢃ'LVRUGꢄ ꢀꢁꢁꢂꢄꢂꢇꢄꢂꢈꢆꢇꢃꢈꢅꢎꢁ
ꢎꢁꢂ0D\VLQJHUꢄꢂ'ꢁꢉꢂ3LFFDUGRꢄꢂ3ꢁꢉꢂ/LEHULQLꢄꢂ3ꢁꢉꢂ-DOVHQMDNꢄꢂ,ꢁꢉꢂ&XHOORꢄꢂ&ꢁꢂ1HXURFKHPꢄꢃ,QWꢄ ꢀꢁꢁꢄꢄꢂꢀꢆꢄꢂꢆꢋꢅꢃ
ꢅꢌꢍꢁ
ꢊꢁꢂ$HELVFKHUꢄꢂ3ꢁꢄꢂ6FKOXHSꢄꢂ0ꢁꢉꢂ'HJORQꢄꢂ1ꢁꢉꢂ-RVHSKꢄꢂ-ꢁ0ꢁꢉꢂ+LUWꢄꢂ/ꢁꢉꢂ+H\Gꢄꢂ%ꢁꢉꢂ*RGGDUGꢄꢂ0ꢁꢉꢂ+DPPDQJꢄ
-ꢁ3ꢁꢉꢂ=XUQꢄꢂ$ꢁ'ꢁꢉꢂ.DWRꢄꢂ$ꢁ&ꢁꢉꢂ5HJOLꢄꢂ)ꢁꢉꢂ%DHWJHꢄꢂ(ꢁ(ꢁꢂ1DWꢄꢃ0HGꢄ ꢀꢁꢁꢅꢄꢂꢀꢄꢂꢇꢋꢇꢃꢇꢋꢋꢁ
ꢋꢁꢂ%LODQJꢃEOHXHOꢄꢂ$ꢁꢉꢂ5HYDKꢄꢂ)ꢁꢉꢂ&ROLQꢄꢂ3ꢁꢉꢂ/RFTXHWꢄꢂ,ꢁꢉꢂ5REHUWꢄꢂ-ꢁ-ꢁꢉꢂ0DOOHWꢄꢂ-ꢁꢉꢂ+RUHOORXꢄꢂ3ꢁꢂ3URFꢄꢃ1DWOꢄ
$FDGꢄꢃ6FLꢄꢃ86$ ꢀꢁꢁꢃꢄꢂꢇꢆꢄꢂꢊꢊꢀꢊꢃꢊꢊꢈꢍꢁ
ꢀꢌꢁꢂ+DDVHꢄꢂ*ꢁꢉꢂ.HQQHOꢄꢂ3ꢁꢉꢂ3HWWPDQQꢄꢂ%ꢁꢉꢂ9LJQHꢄꢂ(ꢁꢉꢂ$NOLꢄꢂ6ꢁꢉꢂ5HYDKꢄꢂ)ꢁꢉꢂ6FKPDOEUXFKꢄꢂ+ꢁꢉꢂ.DKQꢄꢂ$ꢁ
1DWꢄꢃ0HGꢄ ꢀꢁꢁꢃꢄꢂꢁꢄꢂꢆꢈꢋꢃꢆꢍꢇꢁ
ꢀꢀꢁꢂ)ULGHQꢄꢂ3ꢁ0ꢁꢉꢂ:DOXVꢄꢂ/ꢁ5ꢁꢉꢂ:DWVRQꢄꢂ3ꢁꢉꢂ'RFWURZꢄꢂ6ꢁ5ꢁꢉꢂ.R]DULFKꢄꢂ-ꢁ:ꢁꢉꢂ%DFNPDQꢄꢂ&ꢁꢉꢂ%HUJPDQꢄꢂ+ꢁꢉ
+RIIHUꢄꢂ%ꢁꢉꢂ%ORRPꢄꢂ)ꢁꢉꢂ*UDQKROPꢄꢂ$ꢁ&ꢁꢂ6FLHQFH ꢀꢁꢁꢆꢄꢂꢀꢈꢇꢄꢂꢍꢎꢍꢃꢍꢎꢎꢁ
ꢀꢈꢁꢂ/H6DXWHXUꢄꢂ/ꢁꢉꢂ:HLꢄꢂ/ꢁꢉꢂ*LEEVꢄꢂ%ꢁ)ꢁꢉꢂ6DUDJRYLꢄꢂ+ꢁ8ꢁꢂ-ꢄꢃ%LROꢄꢃ&KHPꢄ ꢀꢁꢁꢇꢄꢂꢀꢂꢉꢄꢂꢇꢅꢇꢆꢃꢇꢅꢇꢋꢁ
ꢀꢍꢁꢂ;LHꢄꢂ<ꢁꢉꢂ7LVLꢄꢂ0ꢁ$ꢁꢉꢂ<HRꢄꢂ7ꢁ7ꢁꢉꢂ/RQJRꢄꢂ)ꢁ0ꢁꢂ-ꢄꢃ%LROꢄꢃ&KHPꢄ ꢈꢉꢉꢉꢄꢂꢀꢂꢈꢄꢂꢈꢋꢊꢇꢊꢃꢈꢋꢊꢎꢆꢁ
ꢀꢆꢁꢂ2PXUDꢄꢂ6ꢁꢉꢂ)XMLPRWRꢄꢂ7ꢁꢉꢂ2WRJXURꢄꢂ.ꢁꢉꢂ0DWVX]DNLꢄꢂ.ꢁꢉꢂ0RULJXFKLꢄꢂ5ꢁꢉꢂ7DQDNDꢄꢂ+ꢁꢉꢂ6DVDNLꢄꢂ<ꢁꢂ-ꢄ
$QWLELRWꢄ ꢀꢁꢁꢀꢄꢂꢆꢆꢄꢂꢀꢀꢍꢃꢀꢀꢇꢁ
ꢀꢅꢁꢂ7RNLꢄꢂ6ꢁꢉꢂ$QGRꢄꢂ.ꢁꢉꢂ<RVKLGDꢄꢂ0ꢁꢉꢂ0DWVXGDꢄꢂ<ꢁꢂ-ꢄꢃ$QWLELRWꢄ ꢀꢁꢁꢄꢄꢂꢆꢂꢄꢂꢀꢀꢎꢅꢃꢀꢀꢊꢀꢁ
ꢀꢇꢁꢂ.DNH\Dꢄꢂ+ꢁꢉꢂ7DNDKDVKLꢄꢂ,ꢁꢉꢂ2NDGDꢄꢂ*ꢁꢉꢂ,VRQRꢄꢂ.ꢁꢉꢂ2VDGD +ꢁꢂ-ꢄꢃ$QWLELRWꢄ ꢀꢁꢁꢇꢄꢂꢆꢊꢄꢂꢎꢍꢍꢃꢎꢍꢅꢁ
ꢀꢎꢁꢂ)HQWHDQ\ꢄꢂ*ꢁꢉꢂ6FKUHLEHUꢄꢂ6ꢁ/ꢁꢂ-ꢄꢃ%LROꢄꢃ&KHPꢄ ꢀꢁꢁꢂꢄꢂꢀꢂꢁꢄꢂꢊꢅꢆꢅꢃꢊꢅꢆꢊꢁ
ꢀꢊꢁꢂ0L]XVKLQDꢄꢂ<ꢁꢉꢂ.RED\DVKLꢄꢂ6ꢁꢉꢂ.XUDPRFKLꢄꢂ.ꢁꢉꢂ1DJDWDꢄꢂ6ꢁꢉꢂ6XJDZDUDꢄꢂ)ꢁꢉꢂ6DNDJXFKLꢄꢂ.ꢁꢂ%LRFKHPꢄ
%LRSK\Vꢄꢃ5HVꢄꢃ&RPPXQꢄ ꢈꢉꢉꢉꢄꢂꢀꢂꢁꢄꢂꢎꢊꢆꢃꢎꢊꢊꢁ
ꢀꢋꢁꢂ)XNX\DPDꢄꢂ<ꢁꢉꢂ2WRVKLꢄꢂ<ꢁꢄꢂ0L\RVKLꢄꢂ.ꢁꢉꢂ1DNDPXUDꢄꢂ.ꢁꢉꢂ.RGDPDꢄꢂ0ꢁꢉꢂ1DJDVDZDꢄꢂ0ꢁꢉꢂ+DVHJDZDꢄꢂ7ꢁꢉ
2ND]DNLꢄꢂ+ꢁꢉꢂ6XJDZDUDꢂ0ꢁꢂ7HWUDKHGURQ ꢀꢁꢁꢈꢄꢂꢆꢊꢄꢂꢍꢎꢎꢃꢍꢋꢈꢁ
ꢈꢌꢁꢂ 0RULJXFKLꢄꢂ 7ꢁꢉꢂ 0DWVXXUDꢄꢂ +ꢁꢉꢂ .RGHUDꢄꢂ <ꢁꢉꢂ ,WDNXUDꢄꢂ <ꢁꢉꢂ .DWVXNLꢄꢂ +ꢁꢉꢂ 6DLWRꢄꢂ +ꢁꢉꢂ 1LVKL\DPDꢄꢂ 1ꢁ
1HXURFKHPꢄꢃ5HVꢄ ꢀꢁꢁꢃꢄꢂꢀꢀꢄꢂꢀꢆꢆꢋꢃꢀꢆꢅꢈꢁ
ꢈꢀꢁꢂ3DXOꢄꢂ-ꢁ:ꢁꢉꢂ4XDFKꢄꢂ7ꢁ7ꢁꢉꢂ'XFKHPLQꢄꢂ$ꢁ0ꢁꢉꢂ6FKULHUꢄꢂ%ꢁ.ꢁꢉꢂ'D9DQ]Rꢄꢂ-ꢁ3ꢁꢂ'HYꢄꢃ%UDLQꢃ5HVꢄ ꢀꢁꢁꢉꢄꢂꢈꢈꢄ
ꢈꢀꢃꢈꢎꢁ
ꢈꢈꢁꢂ3DXOꢄꢂ-ꢁ:ꢁꢉꢂ'D9DQ]Rꢄꢂ-ꢁ3ꢁꢂ%UDLQꢃ5HVꢄꢃ'HYꢄꢃ%UDLQꢃ5HVꢄ ꢀꢁꢁꢈꢄꢂꢋꢂꢄꢂꢀꢀꢍꢃꢀꢈꢌꢁ
ꢈꢍꢁꢂ3UDGLQHVꢄꢂ$ꢁꢉꢂ0DJD]LQꢄꢂ0ꢁꢉꢂ6FKLOW]ꢄꢂ3ꢁꢉꢂ/H )XUꢄꢂ*ꢁꢉꢂ&DSXWꢄꢂ'ꢁꢉꢂ)HUUDUDꢄꢂ3ꢁꢂ-ꢄꢃ1HXURFKHPꢄ ꢀꢁꢁꢇꢄꢂꢋꢆꢄ
ꢀꢋꢅꢆꢃꢀꢋꢇꢆꢁ
ꢈꢆꢁꢂ0DJD]LQꢄꢂ0ꢁꢉꢂ6FKLOW]ꢄꢂ3ꢁꢉꢂ=DFKD\XVꢄꢂ-ꢁ/ꢁꢉꢂ&DYURLVꢄꢂ(ꢁꢉꢂ&DSXWꢄꢂ'ꢁꢉꢂ)HUUDUDꢄꢂ3ꢁꢂ%UDLQꢃ5HVꢄꢃ0ROꢄꢃ%UDLQ
5HVꢄ ꢀꢁꢁꢂꢄꢂꢈꢁꢄꢂꢍꢌꢀꢃꢍꢌꢇꢁ
ꢈꢅꢁꢂ'XRQJꢄꢂ)ꢁ+ꢁꢉꢂ:DUWHUꢄꢂ-ꢁ0ꢁꢉꢂ3RLQGURQꢄꢂ3ꢁꢉꢂ3DVVLOO\ꢄꢂ3ꢁꢂ%Uꢄꢃ-ꢄꢃ3KDUPDFROꢄ ꢀꢁꢁꢁꢄꢂꢅꢀꢊꢄꢂꢀꢍꢊꢅꢃꢀꢍꢋꢈꢁ
ꢈꢇꢁꢂ*DXWKLHUꢄꢂ7ꢁꢉꢂ%RUQLDꢄꢂ-ꢁꢉꢂ5HLWKꢄꢂ<ꢁꢉꢂ.HDQHꢄꢂ 3ꢁ(ꢁꢉꢂ /H )XUꢄꢂ *ꢁꢉꢂ 6RXEULHꢄꢂ 3ꢁꢂ )XQGꢄꢃ &OLQꢄꢃ 3DUPDFROꢄ
ꢀꢁꢁꢆꢄꢂꢂꢄꢂꢍꢅꢋꢁ
ꢈꢎꢁꢂ/DELHꢄꢂ&ꢁꢉꢂ/DIRQꢄꢂ&ꢁꢉꢂ0DUPRXJHWꢄꢂ&ꢁꢉꢂ6DXEXVVHꢄꢂ3ꢁꢉꢂ)RXUQLHUꢄꢂ-ꢁꢉꢂ.HDQHꢄꢂ3ꢁ(ꢁꢉꢂ/Hꢂ)XUꢄꢂ*ꢁꢉꢂ6RXEULHꢄ
3ꢁꢂ%Uꢄꢃ-ꢄꢃ3KDUPDFROꢄ ꢀꢁꢁꢁꢄꢂꢅꢀꢂꢄꢂꢀꢍꢋꢃꢀꢆꢆꢁ

Molecules 2000, 5
1458
ꢈꢊꢁꢂ )RXUQLHUꢄꢂ -ꢁꢉꢂ 6WHLQEHUJꢄꢂ 5ꢁꢉꢂ *DXWKLHUꢄꢂ 7ꢁꢉꢂ .HDQHꢄꢂ 3ꢁ(ꢁꢉꢂ *X]]Lꢄꢂ 8ꢁꢉꢂ &RXGHꢄꢂ )ꢁ;ꢁꢉꢂ %RXJDXOWꢄꢂ ,ꢁꢉ
0DIIUDQGꢄꢂ-ꢁ3ꢁꢉꢂ6RXEULHꢄꢂ3ꢁꢉꢂ/Hꢂ)XUꢄꢂ*ꢁꢂ1HXURVFLHQFH ꢀꢁꢁꢆꢄꢂꢈꢈꢄꢂꢇꢈꢋꢃꢇꢆꢀꢁ
ꢈꢋꢁꢂ7HUUDQRYDꢄꢂ-ꢁ3ꢁꢉꢂ.DQꢄꢂ-ꢁ3ꢁꢉꢂ6WRUPHꢄꢂ-ꢁ-ꢁꢉꢂ3HUUHDXWꢄꢂ3ꢁꢉꢂ/H )XUꢄꢂ*ꢁꢉꢂ6RXEULHꢄꢂ3ꢁꢂ1HXURVFLꢄꢃ/HWWꢄ ꢀꢁꢁꢅꢄ
ꢀꢅꢁꢄꢂꢎꢋꢃꢊꢈꢁ
ꢍꢌꢁꢂ'XRQJꢄꢂ)ꢁ+ꢁꢉꢂ)RXUQLHUꢄꢂ-ꢁꢉꢂ.HDQHꢄꢂ3ꢁ(ꢁꢉꢂ*XHQHWꢄꢂ-ꢁ/ꢁꢉꢂ6RXEULHꢄꢂ3ꢁꢉꢂ:DUWHUꢄꢂ-ꢁ0ꢁꢉꢂ%RUJꢄꢂ-ꢁꢉꢂ3RLQGURQꢄ
3ꢁꢂ%Uꢄꢃ-ꢄꢃ3KDUPDFROꢄ ꢀꢁꢁꢂꢄꢂꢅꢀꢆꢄꢂꢊꢀꢀꢃꢊꢀꢎꢁ
ꢍꢀꢁꢂ,ZDVDNLꢄꢂ,ꢁꢉꢂ6KLRMLPDꢄꢂ7ꢁꢉꢂ.LQRVKLWDꢄꢂ0ꢁꢉꢂ,NHGDꢄꢂ.ꢁꢂ-ꢄꢃ1HXUROꢄꢃ6FLꢄ ꢀꢁꢁꢂꢄꢂꢅꢋꢉꢄꢂꢋꢈꢃꢋꢇꢁ
ꢍꢈꢁꢂ)XUXNDZDꢄꢂ<ꢁꢉꢂ7RPLRNDꢄꢂ1ꢁꢉꢂ6DWRꢄꢂ:ꢁꢉꢂ6DWR\RVKLꢄꢂ(ꢁꢉꢂ+D\DVKLꢄꢂ.ꢁꢉꢂ)XUXNDZDꢄꢂ6ꢁꢂ)(%6ꢃ/HWWꢄ ꢀꢁꢂꢁꢄ
ꢀꢆꢂꢄꢂꢆꢇꢍꢃꢆꢇꢎꢁ
ꢍꢍꢁꢂ +DQDRNDꢄꢂ<ꢁꢉꢂ 2KLꢄꢂ 7ꢁꢉꢂ )XUXNDZDꢄꢂ 6ꢁꢉꢂ )XUXNDZDꢄꢂ <ꢁꢉꢂ +D\DVKLꢄꢂ .ꢁꢉꢂ 0DWVXNXUDꢄꢂ 6ꢁꢂ -ꢄꢃ 1HXUROꢄꢃ 6FLꢄ
ꢀꢁꢁꢄꢄꢂꢅꢀꢀꢄꢂꢈꢊꢃꢍꢈꢁ
ꢍꢆꢁꢂ.DHFKLꢄꢂ.ꢁꢉꢂ,NHJDPLꢄꢂ5ꢁꢉꢂ1DNDPXUDꢄꢂ1ꢁꢄꢂ1DNDMLPDꢄꢂ0ꢁꢉꢂ)XUXNDZDꢄꢂ<ꢁꢉꢂ)XUXNDZDꢄꢂ6ꢁꢂ-ꢄꢃ3KDUPDFROꢄ
([Sꢄꢃ7KHUꢄ ꢀꢁꢁꢇꢄꢂꢀꢂꢀꢄꢂꢀꢍꢌꢌꢃꢀꢍꢌꢆꢁ
ꢍꢅꢁꢂ 6DLWDꢄꢂ .ꢁꢉꢂ 2KLꢄꢂ 7ꢁꢉꢂ +DQDRNDꢄꢂ <ꢁꢉꢂ )XUXNDZDꢄꢂ 6ꢁꢉꢂ )XUXNDZDꢄꢂ <ꢁꢉꢂ +D\DVKLꢄꢂ .ꢁꢉꢂ 0DWVXNXUDꢄꢂ 6ꢁ
1HXURWR[LFRORJ\ ꢀꢁꢁꢇꢄꢂꢅꢋꢄꢂꢆꢌꢍꢃꢆꢀꢈꢁ
ꢍꢇꢁꢂ 6DLWDꢄꢂ .ꢁꢉꢂ 2KLꢄꢂ 7ꢁꢉꢂ +DQDRNDꢄꢂ <ꢁꢉꢂ )XUXNDZDꢄꢂ 6ꢁꢉꢂ )XUXNDZDꢄꢂ <ꢁꢉꢂ +D\DVKLꢄꢂ .ꢁꢉꢂ 0DWVXNXUDꢄꢂ 6ꢁꢂ -ꢄ
3KDUPDFROꢄꢃ([Sꢄꢃ7KHUꢄ ꢀꢁꢁꢅꢄꢂꢀꢂꢋꢄꢂꢈꢍꢀꢃꢈꢍꢎꢁ
ꢍꢎꢁꢂ.LPXUDꢄꢂ1ꢁꢉꢂ1LVKL]DNLꢄꢂ.ꢁꢉꢂ2ULWDꢄꢂ<ꢁꢉꢂ0DVXGDꢄꢂ<ꢁꢂ$FWD 2WRODU\QJROꢄꢃ6XSSOꢄ ꢀꢁꢁꢁꢄꢂꢈꢆꢉꢄꢂꢀꢈꢃꢀꢅꢁ
ꢍꢊꢁꢂ&R\OHꢄꢂ-ꢁ7ꢁꢉꢂ3ULFHꢄꢂ'ꢁ/ꢁꢉꢂ'HORQJꢄꢂ0ꢁ5ꢁꢂ6FLHQFH ꢀꢁꢂꢆꢄꢂꢀꢅꢇꢄꢂꢀꢀꢊꢆꢃꢀꢀꢋꢌꢁ
ꢍꢋꢁꢂ+HIWLꢄꢂ)ꢁꢂ-ꢄꢃ1HXURVFLꢄ ꢀꢁꢂꢅꢄꢂꢋꢄꢂꢈꢀꢅꢅꢃꢈꢀꢇꢈꢁ
ꢆꢌꢁꢂ 2OVRQꢄꢂ /ꢁꢉꢂ 1RUGEHUJꢄꢂ $ꢁꢉꢂ YRQꢂ +ROVWꢄꢂ +ꢁꢉꢂ %lFNPDQꢄꢂ /ꢁꢉꢂ (EHQGDOꢄꢂ 7ꢁꢉꢂ $ODIX]RIIꢄꢂ ,ꢁꢉꢂ $PEHUODꢄꢂ .ꢁꢉ
+DUWYLJꢄꢂ 3ꢁꢉꢂ +HUOLW]ꢄꢂ $ꢁꢉꢂ /LOMDꢄꢂ $ꢁꢉꢂ /XQGTYLVWꢄꢂ +ꢁꢉꢂ /lQJVWU|Pꢄꢂ %ꢁꢉꢂ 0H\HUVRQꢄꢂ %ꢁꢉꢂ 3HUVVRQꢄꢂ $ꢁꢉ
9LLWDQHQꢄꢂ0ꢁꢉꢂ:LQEODGꢄꢂ%ꢁꢉꢂ6HLJHUꢄꢂ$ꢁꢂ-ꢄꢃ1HXUDOꢃ7UDQVPꢄꢃ3DUNꢄꢃ'LVꢄꢃ'HPHQWꢄꢃ6HFꢄ ꢀꢁꢁꢈꢄꢂꢆꢄꢂꢎꢋꢃꢋꢅꢁ
ꢆꢀꢁꢂ7DNHXFKLꢄꢂ5ꢁꢉꢂ0XUDVHꢄꢂ.ꢁꢉꢂ)XUXNDZDꢄꢂ<ꢁꢉꢂ)XUXNDZDꢄꢂ6ꢁꢉꢂ+D\DVKLꢄꢂ.ꢁꢂ)(%6ꢃ/HWWꢄ ꢀꢁꢁꢉꢄꢂꢀꢋꢅꢄꢂꢇꢍꢃꢇꢇꢁ
ꢆꢈꢁꢂ6KLQRGDꢄꢂ,ꢁꢉꢂ)XUXNDZDꢄꢂ<ꢁꢉꢂ)XUXNDZDꢄꢂ6ꢁꢂ%LRFKHPꢄꢃ3KDUPDFROꢄ ꢀꢁꢁꢉꢄꢂꢁꢇꢄꢂꢀꢊꢀꢍꢃꢀꢊꢀꢇꢁ
ꢆꢍꢁꢂ5HJRꢄꢂ$ꢁ&ꢁꢉꢂ6DQWRVꢄꢂ0ꢁ6ꢁꢉꢂ2OLYHLUDꢄꢂ&ꢁ5ꢁꢂ)UHHꢃ5DGLFꢄꢃ%LROꢄꢃ0HGꢄ ꢀꢁꢁꢁꢄꢂꢀꢋꢄꢂꢀꢆꢌꢅꢃꢀꢆꢀꢎꢁ
ꢆꢆꢁꢂ)XMLꢄꢂ.ꢁꢉꢂ+LUDPDWVXꢄꢂ0ꢁꢉꢂ.DPH\DPDꢄꢂ7ꢁꢉꢂ1DEHVKLPDꢄꢂ7ꢁꢂ(XUꢄꢃ-ꢄꢃ3KDUPDFROꢄ ꢀꢁꢁꢆꢄꢂꢀꢁꢋꢄꢂꢆꢀꢀꢃꢆꢀꢎꢁ
ꢆꢅꢁꢂ+DDJꢄꢂ3ꢁꢉꢂ6FKQHLGHUꢂ7ꢁꢉꢂ6FKlELW]ꢄꢂ:ꢁ5ꢁꢉꢂ+DFNHꢄꢂ:ꢁꢂ-ꢄꢃ1HXUROꢄꢃ6FLꢄ ꢈꢉꢉꢉꢄꢂꢅꢂꢈꢄꢂꢅꢈꢃꢅꢇꢁ
ꢆꢇꢁꢂ<DPDGDꢄꢂ.ꢁꢉꢂ7DQDNDꢄꢂ7ꢁꢉꢂ+DQꢄꢂ'ꢁꢉꢂ6HQ]DNLꢄꢂ.ꢁꢉꢂ.DPH\DPDꢄꢂ7ꢁꢉꢂ1DEHVKLPDꢄꢂ7ꢁꢂ(XUꢄꢃ-ꢄꢃ1HXURVFLꢄ
ꢀꢁꢁꢁꢄꢂꢅꢅꢄꢂꢊꢍꢃꢋꢌꢁ
ꢆꢎꢁꢂ<DPDGDꢄꢂ.ꢁꢉꢂ7DQDNDꢄꢂ7ꢁꢉꢂ6HQ]DNLꢄꢂ.ꢁꢉꢂ.DPH\DPDꢄꢂ7ꢁꢉꢂ1DEHVKLPDꢄꢂ7ꢁꢂ(XUꢄꢃ-ꢄꢃ3KDUPDFROꢄ ꢀꢁꢁꢂꢄ
ꢁꢆꢇꢄꢂꢀꢅꢃꢈꢈꢁ
ꢆꢊꢁꢂ*XW]PDQQꢄꢂ+ꢁꢉꢂ+DGOHUꢄꢂ'ꢁꢂ-ꢄꢃ1HXUDOꢃ7UDQVPꢄꢃ6XSSOꢄ ꢀꢁꢁꢂꢄꢂꢈꢆꢄꢂꢍꢌꢀꢃꢍꢀꢌꢁ
ꢆꢋꢁꢂ5RWKHUꢄꢂ0ꢁꢉꢂ(UNLQMXQWWLꢄꢂ7ꢁꢉꢂ5RHVVQHUꢄꢂ0ꢁꢉꢂ.LWWQHUꢄꢂ%ꢁꢉꢂ0DUFXVVRQꢄꢂ-ꢁꢉꢂ.DUOVVRQꢄꢂ,ꢁꢂ'HPHQWꢄꢃ*HULDWUꢄ
&RJQꢄꢃ'LVRUGꢄ ꢀꢁꢁꢂꢄꢂꢇꢄꢂꢍꢇꢃꢆꢍꢁ
ꢅꢌꢁꢂ0LHONHꢄꢂ5ꢁꢉꢂ0ROOHUꢄꢂ+ꢁ-ꢁꢉꢂ(UNLQMXQWWLꢄꢂ7ꢁꢉꢂ5RVHQNUDQ]ꢄꢂ%ꢁꢉꢂ5RWKHUꢄꢂ0ꢁꢉꢂ.LWWQHUꢄꢂ%ꢁꢂ$O]KHLPHUꢃ'LVꢄ
$VVRFꢄꢃ'LVRUGꢄ ꢀꢁꢁꢂꢄꢂꢅꢀꢄꢂꢈꢋꢃꢍꢅꢁ

Molecules 2000, 5
1459
ꢅꢀꢁꢂ3DUNLQVRQꢄꢂ)ꢁ(ꢁꢉꢂ3DWHUVRQꢄꢂ$ꢁ5ꢁ3ꢁꢉꢂ<RXQJꢄꢂ-ꢁ'ꢁꢉꢂ&DVVꢄꢂ&ꢁ(ꢁ %LRFKHPꢄꢃ3KDUPDFROꢄ ꢀꢁꢁꢆꢄꢂꢆꢋꢄꢂꢊꢋꢀꢃ
ꢊꢋꢇꢁ
ꢅꢈꢁꢂ0RUGHQWHꢄꢂ$ꢁꢉꢂ0DUWRUDQDꢄꢂ*ꢁ(ꢁꢉꢂ0LQRWWLꢄꢂ*ꢁꢉꢂ*LDUGLQDꢄꢂ%ꢁꢂ&KHPꢄꢃ5HVꢄꢃ7R[LFROꢄ ꢀꢁꢁꢂꢄꢂꢅꢅꢄꢂꢅꢆꢃꢇꢍꢁ
ꢅꢍꢁꢂ%DQDWLꢄꢂ5ꢁ%ꢁꢉꢂ6FKXEHUWꢄꢂ3ꢁꢉꢂ5RWKHꢄꢂ*ꢁꢉꢂ*HKUPDQQꢄꢂ-ꢁꢉꢂ5XGXOSKLꢄꢂ.ꢁꢉꢂ9DOHWꢄꢂ*ꢁꢉꢂ.UHXW]EHUJꢄꢂ*ꢁ:ꢁꢂ-ꢄ
&HUHEꢄꢃ%ORRGꢃ)ORZꢃ0HWDEꢄ ꢀꢁꢁꢄꢄꢂꢅꢆꢄꢂꢀꢆꢅꢃꢀꢆꢋꢁ
ꢅꢆꢁꢂ.DZDJLVKLꢄꢂ+ꢁꢉꢂ$QGRꢄꢂ0ꢁꢉꢂ6DNDPRWRꢄꢂ+ꢁꢉꢂ<RVKLGDꢄꢂ6ꢁꢉꢂ2MLPDꢄꢂ)ꢁꢉꢂ,VKLJXURꢄꢂ<ꢁꢉꢂ8NDLꢄꢂ1ꢁꢉꢂ)XUXNDZDꢄ
6ꢁꢂ7HWUDKHGURQꢃ/HWWꢄ ꢀꢁꢁꢀꢄꢂꢁꢀꢄꢂꢆꢅꢇꢀꢃꢆꢅꢃꢇꢆꢁ
ꢅꢅꢁꢂ<DPDJXFKLꢄꢂ.ꢁꢉꢂ7VXMLꢄꢂ7ꢁꢉꢂ:DNXULꢄꢂ6ꢁꢉꢂ<D]DZDꢄꢂ.ꢁꢉꢂ.RQGRꢄꢂ.ꢁꢉꢂ+LJHPRULꢄꢂ+ꢁꢉꢂ.RED\DVKLꢄꢂ-ꢁꢂ%LRVFLꢄ
%LRWHFKꢄꢃ%LRFKHPꢄ ꢀꢁꢁꢆꢄꢂꢈꢂꢄꢂꢀꢋꢅꢃꢀꢋꢋꢁ
ꢅꢇꢁꢂ 'RLꢄꢂ <ꢁꢉꢂ 6KLJHPRULꢄꢂ +ꢁꢉꢂ ,VKLEDVKLꢄꢂ 0ꢁꢉꢂ 0L]REHꢄꢂ )ꢁꢉꢂ .DZDVKLPDꢄꢂ $ꢁꢉꢂ 1DNDLNHꢄꢂ 6ꢁꢉꢂ .RED\DVKLꢄꢂ -ꢁ
&KHPꢄꢃ3KDUPꢄꢃ%XOOꢄ ꢀꢁꢁꢆꢄꢂꢆꢅꢄꢂꢈꢀꢋꢌꢃꢈꢀꢋꢀꢁ
ꢅꢎꢁꢂ<DPDJXFKLꢄꢂ.ꢁꢉꢂ6DVDQRꢄꢂ$ꢁꢉꢂ8UDNDPLꢄꢂ7ꢁꢉꢂ7VXMLꢄꢂ7ꢁꢉꢂ.RQGRꢄꢂ.ꢁꢂ%LRVFLꢄꢃ%LRWHFKꢄꢃ%LRFKHPꢄ ꢀꢁꢁꢆꢄꢂꢈꢂꢄ
ꢀꢈꢍꢀꢃꢀꢈꢍꢍꢁ
ꢅꢊꢁꢂ .DZDJLVKLꢄꢂ +ꢁꢉꢂ 6KLPDGDꢄꢂ$ꢁꢉꢂ +RVRNDZDꢄꢂ6ꢁꢉꢂ0RULꢄꢂ +ꢁꢉꢂ 6DNDPRWRꢄꢂ +ꢁꢉꢂ ,VKLJXURꢄꢂ <ꢁꢉꢂ 6DNHPLꢄꢂ 6ꢁꢉ
%RUGQHUꢄꢂ-ꢁꢉꢂ.RMLPDꢄꢂ1ꢁꢉꢂ)XUXNDZDꢄꢂ6ꢁꢂ7HWUDKHGURQꢃ/HWWꢄ ꢀꢁꢁꢅꢄꢂꢁꢂꢄꢂꢎꢍꢋꢋꢃꢎꢆꢌꢈꢁ
ꢅꢋꢁꢂ .DZDJLVKLꢄꢂ +ꢁꢉꢂ ,VKL\DPDꢄꢂ 'ꢁꢉꢂ 0RULꢄꢂ +ꢁꢉꢂ 6DNDPRWRꢄꢂ +ꢁꢉꢂ ,VKLJXURꢄꢂ <ꢁꢉꢂ )XUXNDZDꢄꢂ 6ꢁꢉꢂ /Lꢄꢂ -ꢁ
3K\WRFKHPLVWU\ ꢀꢁꢁꢃꢄꢂꢆꢈꢄꢂꢀꢈꢌꢍꢃꢀꢈꢌꢅꢁ
ꢇꢌꢁꢂ.LWDꢄꢂ7ꢁꢉꢂ7DND\Dꢄꢂ<ꢁꢉꢂ2VKLPDꢄꢂ<ꢁꢂ7HWUDKHGURQ ꢀꢁꢁꢂꢄꢂꢈꢆꢄꢂꢀꢀꢊꢎꢎꢃꢀꢀꢊꢊꢇꢁ
ꢇꢀꢁꢂ0DUWLQꢄꢂ3ꢁꢂ6FLHQFH ꢀꢁꢁꢃꢄꢂꢀꢂꢋꢄꢂꢎꢅꢃꢊꢀꢁ
ꢇꢈꢁꢂ0DWVXGDꢄꢂ+ꢁꢉꢂ.R\DPDꢄꢂ+ꢁꢉꢂ6DWRꢄꢂ+ꢁꢉꢂ6DZDGDꢄꢂ-ꢁꢉꢂ,WDNXUDꢄꢂ$ꢁꢉꢂ7DQDNDꢄꢂ$ꢁꢉꢂ0DWVXPRWRꢄꢂ0ꢁꢉꢂ.RQQRꢄ
.ꢁꢉꢂ8VKLRꢄꢂ+ꢁꢉꢂ0DWVXGDꢄꢂ.ꢁꢂ-ꢄꢃ([Sꢄꢃ0HGꢄ ꢀꢁꢁꢂꢄꢂꢅꢊꢂꢄꢂꢈꢋꢎꢃꢍꢌꢇꢁ
ꢇꢍꢁꢂ%RUJꢄꢂ-ꢁꢉꢂ7RD]DUDꢄꢂ-ꢁꢉꢂ+LHWWHUꢄꢂ+ꢁꢉꢂ+HQU\ꢄꢂ0ꢁꢉꢂ6FKPLWWꢄꢂ*ꢁꢉꢂ/XXꢄꢂ%ꢁꢂ)(%6ꢃ/HWWꢄ ꢀꢁꢂꢃꢄꢂꢀꢅꢁꢄꢂꢆꢌꢇꢃꢆꢀꢌꢁ
ꢇꢆꢁꢂ%RUJꢄꢂ-ꢁꢉꢂ.HVVODNꢄꢂ3ꢁ-ꢁꢉꢂ&RWPDQꢄꢂ&ꢁ:ꢁꢂ%UDLQꢃ5HVꢄ ꢀꢁꢁꢉꢄꢂꢈꢅꢊꢄꢂꢈꢋꢅꢃꢈꢋꢊꢁ
ꢇꢅꢁꢂ%RUJꢄꢂ-ꢁꢂ-ꢄꢃ1HXURVFLꢄꢃ5HVꢄ ꢀꢁꢁꢀꢄꢂꢀꢇꢄꢂꢇꢈꢃꢇꢎꢁ
ꢇꢇꢁꢂ'DZVRQꢄꢂ0ꢁ,ꢁ &XUUꢄꢃ3KDUPꢄꢃ'HVꢄ ꢈꢉꢉꢉꢄꢂꢋꢄꢂꢍꢀꢀꢃꢍꢈꢅꢁ
ꢇꢎꢁꢂ%RUJꢄꢂ-ꢁꢂ,QWHUQDWLRQDOꢂ3DWHQWꢂ:2ꢋꢀꢏꢌꢅꢎꢅꢆꢄꢂꢀꢁꢁꢀꢉꢂ3&7ꢏ)5ꢋꢌꢏꢌꢌꢎꢆꢈꢂꢐꢀꢅꢏꢀꢌꢏꢋꢌꢑꢁ
ꢇꢊꢁꢂ /XXꢄꢂ %ꢁꢉꢂ %RUJꢄꢂ -ꢁꢉꢂ %HFNꢄꢂ $ꢁꢉꢂ /DDELFKꢄꢂ $ꢁꢉꢂ &UHVWDQLꢄꢂ )ꢁꢉꢂ 1HYHX[ꢄꢂ 2ꢁꢂ ,QWHUQDWLRQDOꢂ 3DWHQW
:2ꢋꢆꢏꢀꢀꢍꢆꢍꢄꢂꢀꢁꢁꢄꢉꢂ3&7ꢏ)5ꢋꢍꢏꢌꢀꢌꢅꢋꢂꢐꢈꢊꢏꢀꢌꢏꢋꢍꢑꢁ
ꢇꢋꢁꢂ.H\OLQJꢃ%LOJHUꢄꢂ)ꢁꢉꢂ6FKPLWWꢂ*ꢁꢉꢂ%HFNꢄꢂ$ꢁꢉꢂ/XXꢄꢂ%ꢁꢂ7HWUDKHGURQ ꢀꢁꢁꢅꢄꢂꢈꢀꢄꢂꢀꢆꢊꢋꢀꢃꢀꢆꢋꢌꢆꢁ
ꢎꢌꢁꢂ*LUODQGDꢃ-XQJHVꢄꢂ&ꢁꢉꢂ.H\OLQJꢃ%LOJHUꢄꢂ)ꢁꢉꢂ6FKPLWWꢂ*ꢁꢉꢂ/XXꢄꢂ%ꢁꢂ7HWUDKHGURQ ꢀꢁꢁꢂꢄꢂꢈꢆꢄꢂꢎꢎꢍꢅꢃꢎꢎꢆꢊꢁ
ꢎꢀꢁꢂ /XXꢄꢂ %ꢁꢉꢂ 6FKPLWWꢂ *ꢁꢉꢂ .H\OLQJꢄꢂ )ꢁꢉꢂ *LUODQGDꢄꢂ &ꢁꢉꢂ <DPDGDꢄꢂ 0ꢁꢉꢂ 6XPDꢄꢂ <ꢁꢂ ,QWHUQDWLRQDOꢂ 3DWHQW
:2ꢋꢋꢏꢌꢊꢋꢊꢎꢄꢂꢀꢁꢁꢁꢉꢂ3&7ꢏ-3ꢋꢊꢏꢌꢍꢅꢇꢌꢂꢐꢀꢀꢏꢌꢊꢏꢋꢊꢑꢁ
ꢎꢈꢁꢂ/XXꢄꢂ%ꢁꢉꢂ6FKPLWWꢂ*ꢁꢉꢂ.H\OLQJꢄꢂ)ꢁꢉꢂ*LUODQGDꢃ-XQJHVꢄꢂ&ꢁꢉꢂ&KDEHUWꢄꢂ3ꢁꢉꢂ/RHIIOHUꢄꢂ-ꢁ3ꢁꢉꢂ/XW]ꢃ%XFKHUꢄꢂ%ꢁꢉ
*RQ]iOH]ꢂ GHꢂ $JXLODUꢄꢂ -ꢁ/ꢁꢉꢂ <DPDGDꢄꢂ 0ꢁꢉꢂ 6XPDꢄꢂ <ꢁꢂ ,QWHUQDWLRQDOꢂ 3DWHQWꢂ :2ꢌꢌꢏꢆꢎꢀꢋꢋꢄꢂ ꢈꢉꢉꢉꢉ
3&7ꢏ-3ꢌꢌꢏꢌꢌꢎꢆꢈꢂꢐꢀꢌꢏꢌꢈꢏꢌꢌꢑꢁ
ꢎꢍꢁꢂ.ULVKQDꢄꢂ+ꢁ-ꢁ9ꢁꢉꢂ-RVKLꢄꢂ%ꢁ1ꢁꢂ-ꢄꢃ2UJꢄꢃ&KHPꢄ ꢀꢁꢇꢃꢄꢂꢀꢀꢄꢂꢈꢈꢆꢃꢈꢈꢅꢁ
ꢎꢆꢁꢂ7RULLꢄꢂ6ꢁꢉꢂ8QH\DPDꢄꢂ.ꢁꢉꢂ,VLKDUDꢄꢂ0ꢁꢂ&KHPꢄꢃ/HWWꢄ ꢀꢁꢃꢇꢄꢂꢆꢎꢋꢃꢎꢊꢈꢁ
ꢎꢅꢁꢂ7URVWꢄꢂ%ꢁ0ꢁꢉꢂ$UQGWꢄꢂ+ꢁ&ꢁꢉꢂ6WUHJHꢄꢂ3ꢁ(ꢁꢄꢂ9HUKRHYHQꢄꢂ7ꢁ+ꢁ 7HWUDKHGURQꢃ/HWWꢄ ꢀꢁꢃꢅꢄꢂꢁꢇꢄꢂꢍꢆꢎꢎꢃꢍꢆꢎꢊꢁ

Molecules 2000, 5
1460
ꢎꢇꢁꢂ0LOOHUꢄꢂ5ꢁ$ꢁꢉꢂ/Lꢄꢂ:ꢁꢉꢂ+XPSKUH\ꢄꢂ*ꢁ5ꢁꢂ7HWUDKHGURQꢃ/HWWꢄ ꢀꢁꢁꢅꢄꢂꢁꢂꢄꢂꢍꢆꢈꢋꢃꢍꢆꢍꢈꢁ
ꢎꢎꢁꢂ%DUXDꢄꢂ1ꢁ&ꢁꢉꢂ6FKPLGWꢄꢂ5ꢁ5ꢁꢂ6\QWKHVLV ꢀꢁꢂꢅꢄꢂꢊꢋꢀꢃꢊꢋꢍꢁ
ꢎꢊꢁꢂ%RUJꢄꢂ-ꢁꢉꢂ6SLW]ꢄꢂ%ꢁꢉꢂ+DPHOꢄꢂ*ꢁꢉꢂ0DUNꢄꢂ-ꢁꢂ'HYꢄꢃ%UDLQꢃ5HVꢄ ꢀꢁꢂꢇꢄꢂꢅꢊꢄꢂꢍꢎꢃꢆꢋꢁ
ꢎꢋꢁꢂ.DNH\Dꢄꢂ+ꢁꢉꢂ2QR]DZDꢄꢂ&ꢁꢉꢂ6DWRꢄꢂ0ꢁꢉꢂ$UDLꢄꢂ.ꢁꢉꢂ2VDGDꢄꢂ+ꢁꢂ-ꢄꢃ0HGꢄꢃ&KHPꢄ ꢀꢁꢁꢃꢄꢂꢆꢉꢄꢂꢍꢋꢀꢃꢍꢋꢆꢁ
ꢊꢌꢁꢂ)XUXNDZDꢄꢂ<ꢁꢉꢂ)XUXNDZDꢄꢂ6ꢁꢉꢂ,NHGDꢄꢂ)ꢁꢉꢂ6DWR\RVKLꢄꢂ(ꢁꢉꢂ+D\DVKLꢄꢂ.ꢁꢂ)(%6ꢃ/HWWꢄ ꢀꢁꢂꢅꢄꢂꢀꢉꢊꢄꢂꢈꢅꢊꢃꢈꢇꢈꢁ
ꢊꢀꢁꢂ5LSSVꢄꢂ0ꢁ(ꢁꢉꢂ+XQWOH\ꢄꢂ*ꢁ:ꢁꢉꢂ+RIꢄꢂ3ꢁ5ꢁꢉꢂ0RUULVRQꢄꢂ-ꢁ+ꢁꢉꢂ*RUGRQꢄꢂ-ꢁ:ꢁꢂ3URFꢄꢃ1DWOꢄꢃ$FDGꢄꢃ6FLꢄꢃ86$
ꢀꢁꢁꢇꢄꢂꢇꢀꢄꢂꢇꢊꢋꢃꢇꢋꢍꢁ
ꢊꢈꢁꢂ*RQ]iOH] GHꢂ$JXLODUꢄꢂ-ꢁ/ꢁꢉꢂ*RUGRQꢄꢂ-ꢁ:ꢁꢉꢂ5HQpꢄꢂ)ꢁꢉꢂ'Hꢂ7DSLDꢄꢂ0ꢁꢉꢂ/XW]ꢃ%XFKHUꢄꢂ%ꢁꢉꢂ*DLGGRQꢄꢂ&ꢁꢉ
/RHIIOHUꢄꢂ-ꢁ3ꢁꢂ1HXURELROꢄꢃ'LVꢄ ꢈꢉꢉꢉꢄꢂꢂꢄꢂꢆꢌꢇꢃꢆꢀꢅꢁ
Samples Availability: Available from the authors.
© 2000 by MDPI (http://www.mdpi.org).