Naturally occurring norephedrine oxazolidine derivatives in khat (Catha edulis)

Article abstract of DOI:10.1055/s-0031-1298430

Khat (Catha edulis Forsk.) is a perennial shrub whose young leaves are chewed for their psychostimulating and anorectic properties. The main active principles of khat are believed to be the phenylpropylamino alkaloids, primarily ()-cathinone [(S)-α-aminopropiophenone], (+)-cathine [(1S)(2S)- norpseudoephedrine], and ()-norephedrine [(1R)(2S)-norephedrine]. GC-MS analyses of young leaf extracts indicated the presence of two oxazolidine derivatives, 2,4-dimethyl-5-phenyloxazolidine and 4-methyl-2-(trans-1-pentenyl)-5- phenyloxazolidine. To ascertain the chemical identity of these compounds, we synthesized the putative compounds by condensation of norephedrine and acetaldehyde or trans-2-hexenal, respectively. Spectroscopic analyses (GC-MS, NMR) of the structures of these synthetic compounds showed them to have identical retention indexes and mass spectra characteristic to 2,4-dimethyl-5-phenyloxazolidine and 4-methyl-2-(trans-1-pentenyl)-5- phenyloxazolidine. Marked differences in the ratios between each of these two norephedrine oxazolidine derivatives and total phenylpropylamino alkaloids were found among thirteen different khat accessions further indicating polymorphism in alkaloid ratios and content in C. edulis. Georg Thieme Verlag KG Stuttgart · New York.

Full text of DOI:10.1055/s-0031-1298430

838 Original Papers
Naturally Occurring Norephedrine Oxazolidine
Derivatives in Khat (Catha edulis)
Authors
Raz Krizevski1,2, Einat Bar1, Nativ Dudai1, Asaf Levy1, Efraim Lewinsohn1, Yaron Sitrit2, Shimon Ben-Shabat3
1
Affiliations
Department of Aromatic, Medicinal and Spice Crops, Newe Yaʼar Research Center, Agricultural Research Organization,
Ramat Yishay, Israel
2
The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Beer-Sheva, Israel
Department of Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
3
Key words
Abstract
sized the putative compounds by condensation of
norephedrine and acetaldehyde or trans-2-hexe-
nal, respectively. Spectroscopic analyses (GC‑MS,
NMR) of the structures of these synthetic com-
pounds showed them to have identical retention
indexes and mass spectra characteristic to 2,4-di-
methyl-5-phenyloxazolidine and 4-methyl-2-
(trans-1-pentenyl)-5-phenyloxazolidine. Marked
differences in the ratios between each of these
two norephedrine oxazolidine derivatives and to-
tal phenylpropylamino alkaloids were found
among thirteen different khat accessions further
indicating polymorphism in alkaloid ratios and
content in C. edulis.
"
l Khat (Catha edulis,
!
Celastraceae)
l norephedrine
Khat (Catha edulis Forsk.) is a perennial shrub
whose young leaves are chewed for their psycho-
stimulating and anorectic properties. The main
active principles of khat are believed to be the
phenylpropylamino alkaloids, primarily (−)‑cathi-
none [(S)-α-aminopropiophenone], (+)-cathine
[(1S)(2S)-norpseudoephedrine], and (−)-nor-
ephedrine [(1R)(2S)-norephedrine]. GC‑MS analy-
ses of young leaf extracts indicated the presence of
two oxazolidine derivatives, 2,4-dimethyl-5-
phenyloxazolidine and 4-methyl-2-(trans-1-pen-
tenyl)-5-phenyloxazolidine. To ascertain the
chemical identity of these compounds, we synthe-
"
"
l phenylpropylamino alkaloids
"
l phenyloxazolidine alkaloids
"
l GC‑MS
Introduction
[9–13]. (S)-Cathinone, (1S)(2S)-norpseudoephe-
drine, and (1R)(2S)-norephedrine have been
found in only a few plant species, such as C. edulis
(Celastraceae) and Ephedra gerardiana sikkimen-
sis (Ephedraceae) [14]. Interestingly, the phenyl-
pentenylamine alkaloid merucathine, structurally
resembling cathine, has also been detected in C.
edulis [9]. N‑methylation of (1R)(2S)-norephe-
drine and (1S)(2S)-norpseudoephedrine yields
(1R)(2S)-ephedrine and (1S)(2S)-pseudoephe-
drine, respectively, adrenolergic compounds
found in most, but not all, Ephedra spp. [15]. A re-
lated compound identified in Ephedra species in-
clude 3,4-dimethyl-5-phenyloxazolidone (ephe-
droxane), a heterocyclic oxazolidone derivative
of ephedrine that possesses anti-inflammatory
activity [16]. Other oxazolidine derivatives such
as 2,4-dimethyl-5-phenyloxazolidine and 2,3,4-
!
Khat (Catha edulis Forsk., Celastraceae) is a peren-
nial shrub that is cultivated in parts of Africa and
the Middle East [1,2]. The main active principles
of khat are believed to be (S)-cathinone, (1S)(2S)-
norpseudoephedrine and (1R)(2S)-norephedrine,
three phenylpropylamino alkaloids that are cen-
tral nervous system stimulants, mild euphoriants,
and anorectics [3]. In general, phenylpropylamino
alkaloids mimic the action of adrenaline and
cause the release of endogenous cathecholamines
from post-ganglionic sympathetic fibers [4]. (S)-
cathinone is estimated to be one-third as potent
as amphetamine and ten times more potent than
(1S)(2S)-cathine and (1R)(2S)-norephedrine [1,5].
The metabolic precursors of the phenylpropyl-
amino alkaloids occur mainly in young khat
leaves. Fully developed leaves contain only trace
amounts of these two metabolites but have higher
levels of (1S)(2S)-norpseudoephedrine and its di-
astereomer (1R)(2S)-norephedrine as compared
to young leaves [1,6–8].
received
revised
accepted
Nov. 29, 2011
March 14, 2012
March 15, 2012
Bibliography
DOI http://dx.doi.org/
10.1055/s-0031-1298430
Published online April 11, 2012
Planta Med 2012; 78: 838–842
© Georg Thieme Verlag KG
Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Shimon Ben-Shabat
Department of Pharmacology
Faculty of Health Sciences
Ben-Gurion University of the
Negev
P.O. Box 653
Beer-Sheva 84105
Israel
trimethyl-5-phenyloxazolidine
are
possibly
formed from the spontaneous conjugation of
ephedrine and acetaldehyde [17]. Phenylpropyl-
amino alkaloid-derived oxazolidines are pharma-
cologically active stimulating prodrugs that read-
ily hydrolyze in aqueous systems to release the
pharmacoactive phenylpropylamino alkaloid
Phone: + 97286479354
Fax: + 97286472984
sbs@bgu.ac.il
Phenylpropylamino alkaloids constitute a rela-
tively minor subgroup within the amino alkaloids
Krizevski R et al. Naturally Occurring Norephedrine… Planta Med 2012; 78: 838–842

Original Papers 839
from the conjugated aldehyde [18–20]. The above-described N-
methylation, oxazolidine, and oxazolidone derivatives of phenyl-
propylamino alkaloids found in Ephedra could serve as a second-
ary stabilization mechanism for the highly reactive free amino
group. In L. williamsii, a total of 14 conjugates derived from the
active principle mescaline and various Krebs cycle components
were discovered [12,13]. In A. berlandieri and A. rigidula, two
other unique phenylpropylamino alkaloids have been identified,
amphetamine and its N-methylation derivative, N-methylam-
phetamine [21,22]. However, to date, the chemical mechanism
that stabilizes the amino group of the phenylpropylamino alka-
loids present in khat leaves is still unknown. We have previously
speculated about the existence of such a mechanism [8]. We re-
port here the presence and chemical identification of two nor-
ephedrine oxazolidine derivatives that occur in khat tissues.
GC‑MS
The analysis was performed with an Agilent GC‑MSD system
model 6890 N, interfaced with an Agilent model 5973 N mass
spectrometer. The non-chiral Rtx-5 SIL (cross-linked 95% di-
methyl – 5% diphenylpolysiloxane) was 30-m long with an inner
diameter of 0.25 mm and a stationary phase film thickness of
0.25 µm, directly interfacing the mass spectrometer. The gas
chromatograph was set to splitless injector mode. Helium was
used as the carrier gas with a flow rate of 0.8 mL/min. The in-
jector temperature was 250°C, and the detector temperature
was 280°C. The oven was operated with the following tempera-
ture program: an initial temperature of 70°C held for 1 min; and
10°C/min up to 125°C for 5.5 min, 2.5°C/min up to 140°C for
6 min, and 20°C/min up to 260°C for 6 min. The temperature of
the transfer GC‑MS line was 280°C. A quadrupole mass spec-
trometer scanned masses in the 41–350 m/z range. Compounds
were identified by comparison of their MS and retention times
to authentic standards, to literature retention indexes, and to
the computerized Wiley or PMW TOX2 libraries.
Materials and Methods
!
Plant material and chemical standards
Linearity and detection limit calibration curves were generated
using 10 standard dilutions (with 4 replicates) for each of the
synthesis products, 2,4-dimethyl-5-phenyloxazolidine and 4-
methyl-2-(trans-1-pentenyl)-5-phenyloxazolidine, at the range
of 407–0.4 µg/mL and 335–0.32 µg/mL, respectively. Limit of de-
tection (LOD) values were 0.7 and 0.8 µg/mL for the above two
compounds, respectively.
Ten accessions of khat (Catha edulis, Forsk.) shrubs were culti-
vated in open field conditions under commercial growing practi-
ces, which include drip irrigation and fertilization, at the Newe
Yaʼar Research Center in Northern Israel [8]. These khat plants
were six years old at the beginning of this study and originated
from seeds collected from a single parent shrub. Additionally,
three locally grown cultivars, “Bialik” (Bi), “Lavi” (La), and “Mi-
chael” (Mi), were examined. This plant material was made avail-
able for research by three families of growers, Bialik, Lavi, and Mi-
chael, who employ different growing practices for their plants.
The “Bialik” plants were 40 years old, the “Lavi” plants 120 years
old, and the “Michael” plants 5 years old. A representative vouch-
er specimen (#Cathaedulis0001) was deposited in the National
Herbarium of the Hebrew University of Jerusalem. The plants
were identified by Dr. Nativ Dudai. The standards (1R)(2S)-ephe-
drine, (1R)(2S)-norephedrine, and 1-phenyl-1,2-propanedione
were purchased from Sigma Chem. Co., while the acetaldehyde
standard was purchased from Sigma. Racemic cathinone was syn-
thesized by oxidation of racemic norephedrine with KMnO4 [8].
ESI‑MS
Electrospray ionization-mass spectrometry (ESI‑MS) was per-
formed on a Bruker Esquire 3000 Plus MS instrument. The MS
conditions were optimized as follows: API electron spray inter-
face, positive mode polarity, a drying gas flow of 10 L/min, nebu-
lizer gas pressure of 60 psi, drying gas temperature of 335°C,
fragmentor voltage of 0.4 V, capillary voltage of 4451 V, and scan
range of m/z 25–1000, at 1.15 s/scan. For ESI‑MS/MS analysis, the
analytical parameters were optimized by infusing the sample so-
lution (1 µg/mL in methanol:water 50:50) into the source at a
flow rate of 10 µL/min. The optimized parameters were: declus-
tering potential (DP) 200 eV, focusing potential (FP) 400 eV, en-
trance potential (EP) 12 eV.
Extraction of khat samples
For each of the 13 khat accessions, freshly picked young leaves
and stems (~ 0.5 g) were crushed to a fine powder with a mortar
and pestle under liquid nitrogen. Three milliliters of double dis-
tilled H2O containing 100 µg of (1R)(2S)-ephedrine as the internal
standard (khat does not contain ephedrine) were added to the
fine powder, and the sample was shaken for 30 min at 250 RPM
before filtering through one layer of Miracloth (Calbiochem) into
an 8-mL glass vial. One and a half milliliters of 1 N NaOH were
added to the sample to deionize and retrieve the alkaloids in their
uncharged form. Methyl tert-butyl ether (MTBE), 3 mL, was
added to the sample, which was then vortexed for 30 s and cen-
trifuged at 10 g for 5 min to separate the emulsion into two frac-
tions. The top organic fraction containing the alkaloids was col-
lected, and the aqueous residue was reextracted with an addi-
tional 3 mL of MTBE. The pooled ether extracts were dried by
the addition of anhydrous sodium sulfate and then evaporated
under a gentle stream of nitrogen to a final volume of 0.5 mL.
One microliter of the solution was then injected into the GC‑MS
instrument for analysis (see below) [23,24].
Chemical synthesis of 2,4-dimethyl-5-phenyloxazolidine
and 4-methyl-2-(trans-1-pentenyl)-5-phenyloxazolidine
2,4-Dimethyl-5-phenyloxazolidine and 4-methyl-2-(trans-1-
pentenyl)-5-phenyloxazolidine were chemically synthesized
from norephedrine plus acetaldehyde and trans-2-hexenal, re-
spectively, using a 1:10 equivalent ratio. Norephedrine was dis-
solved in 1:1 v/v aqueous 1 N NaOH and ethanol solution. The re-
action mixture was stirred for 30 min. The NaOH was added to
deionize and retrieve the alkaloids in their uncharged form.
MTBE, 3 mL, was added to the sample, which was then vortexed
for 30 s and centrifuged at 10 g for 5 min. The top organic fraction
containing the alkaloids was collected, and the aqueous residue
was reextracted with an additional 3 mL of MTBE. The pooled
ether extracts were dried by the addition of anhydrous sodium
sulfate and then evaporated to dryness under reduced pressure.
The residue was subjected to silica gel chromatography (eluted
with 50% chloroform in methanol) to give the compound. TLC
solvent system:methanol: NH3OH (98:0.5 v/v) [25].
1H NMR spectra were recorded on a Bruker DMX-500 instrument
operating at 500.1 MHz. Chemical shifts are reported in parts per
million (δ), with TMS as the internal standard.
Krizevski R et al. Naturally Occurring Norephedrine… Planta Med 2012; 78: 838–842

840 Original Papers
Fig. 1 Mass spectra (GC‑MS) of plant extracted
and chemically synthesized 2,4-dimethyl-5-phenyl-
oxazolidine (A) and 4-methyl-2-(trans-1-pentenyl)-
5-phenyloxazolidine (B). The following compounds
were also evident on chromatograms: 1-phenylpro-
pane-1,2-dione (P), cathinone (C), norephedrine
(NE), and norpseudoephedrine (NP).
Fig. 2 Synthesis of norephedrine oxazolidine de-
rivatives present in C. edulis. Spontaneous conjuga-
tions of acetaldehyde to ephedrine alkaloids (reac-
tion S1) forming 2,4-dimethyl-5-phenyloxazolidine
(compound A). Spontaneous conjugations of trans-
2-hexenal to ephedrine alkaloids (reaction S2)
forming 4-methyl-2-(trans-1-pentenyl)-5-phenylox-
azolidine (compound B).
2,4-dimethyl-5-phenyloxazolidine: 1H‑NMR (CDCl3): δ = 7.11–
7.22 (m, 5H), 4.97 (d, 1H), 4.62 (d, 1H), 3.56 (m, 1H), 1.25 (d,
3H), 1.10 (d, 3H). ESI‑MS (positive mode): m/z = 178.1; MS/MS
(178.0): 91.0, 115.1, 117.1. Yield ~ 80%; purity ~ 90%.
we chemically conjugated norephedrine and an aldehyde. Com-
pound A was prepared by reacting norephedrine and acetalde-
hyde to give 2,4-dimethyl-5-phenyloxazolidine (l Figs. 1b and
"
2, compound A). The synthesized compound had an identical re-
tention time and a mass spectrum characteristic to that of the nat-
ural product detected in the chromatogram of the khat samples
4-methyl-2-(trans-1-pentenyl)-5-phenyloxazolidine:
1H‑NMR
(CDCl3): δ = 7.10–7.20 (m, 5H), 5.67 (m, 2H), 4.93 (d, 1H), 4.61 (d,
1H), 3.54 (m, 1H), 1.98 (m, 2H), 1.40 (m, 2H), 1.20 (d, 3H), 1.05 (m,
3H). ESI‑MS (positive mode): m/z = 232.2; MS/MS (232.0): 91,
105, 115, 117, 159. Yield ~ 75%; purity 85–90%.
"
(l Fig. 1d and e). Compound B was synthesized by reacting nor-
ephedrine with trans-2-hexenal to yield 4-methyl-2-(trans-1-
"
pentenyl)-5-phenyloxazolidine (l Figs. 1c and 2, compound B).
The synthetic compound B had an identical retention time and a
mass spectrum characteristic to that of the natural product de-
"
Results
tected in khat samples (l Fig. 1f and g). Although norephedrine
!
and acetaldehyde conjugation is a spontaneous reaction, under
our extraction conditions this conversion was inadvertent if no al-
dehyde was added, suggesting that the plant tissues are the
source of the conjugates or the aldehydes that conjugate with nor-
ephedrine or with norpseudoephedrine present in C. edulis tis-
sues, giving rise to compounds A and B (l Fig. 2).
To verify that 2,4-dimethyl-5-phenyloxazolidine (A) and 4-meth-
yl-2-(trans-1-pentenyl)-5-phenyloxazolidine (B) are not artifacts
GC‑MS analysis of the MTBE extract from khat leaves revealed the
phenylpropylamino alkaloids, cathinone, norpseudoephedrine,
norephedrine and their metabolic precursor 1-phenylpropane-
"
1,2-dione (l Fig. 1a). The chromatograms also showed the pres-
"
ence of two additional khat phytochemicals, designated A and B,
that could possibly be related tothe phenylpropylamino alkaloids.
In order to verify the chemical structure of compounds A and B,
Krizevski R et al. Naturally Occurring Norephedrine… Planta Med 2012; 78: 838–842

Original Papers 841
Fig. 3 Norephedrine derivatives in young leaves of
different C. edulis accessions. Total ephedrine alka-
loids (upper panel). Percentage of oxazolidine de-
rivatives from ephedrine alkaloids (lower panel).
The results shown are averages of five replica-
tes S.E. The numbers in the x-axis represent
different Khat accessions, while the letters indicate
locally grown cultivars.
unintentionally formed during the extraction of alkaloids from
khat tissues, we analyzed, using ESI‑MS, plant extracts employ-
ing two different extraction procedures. Both alkaline extractions
followed by partition with an organic solvent (MTBE) or direct
extraction with methanol resulted in the detection of 2,4-di-
methyl-5-phenyloxazolidine, with m/z 178 [M + H]+, and 4-meth-
yl-2-(trans-1-pentenyl)-5-phenyloxazolidine, with m/z 232 [M +
H]+. We also analyzed, by ESI‑MS/MS, the synthesized com-
pounds analogous to the natural compounds and found them to
be identical to the natural compounds 2,4-dimethyl-5-phenylox-
azolidine and 4-methyl-2-(trans-1-pentenyl)-5-phenyloxazoli-
dine found in khat tissues. The major ions (m/z) 91, 115, 117 cor-
responding to 2,4-dimethyl-5-phenyloxazolidine as well as ions
(m/z) 91, 105, 115, 117, 159 corresponding to 4-methyl-2-
(trans-1-pentenyl)-5-phenyloxazolidine of the natural com-
pounds were identical to those of the synthetic compounds. In
addition, we verified the chemical structure of the synthetic
products by 1H‑NMR (see Materials and Methods).
tent, but also in the oxazolidine derivatives in a seemingly inde-
pendent manner.
Discussion
!
Aldehydes are highly reactive natural plant products that form
covalent bonds with available NH2 and SH groups containing
molecules amino acid residues via a Schiff base [27]. We report
here the occurrence of two norephedrine oxazolidine derivatives
in khat leaves based on identification by GC‑MS and ESI‑MS/MS
and on comparison with their synthetic analogues. Our findings
constitute the first evidence for the conjugation of norephedrine
"
and various aldehydes in khat tissues (l Fig. 1). Under our exper-
imental extraction conditions, norephedrine did not form any
oxazolidine derivatives that could be formed in case of aldehyde
impurities. Interestingly, 4-methyl-2-(trans-1-pentenyl)-5-phe-
nyloxazolidine was only detected in the homegrown accessions
Bialik, Lavi, and Michael and was not present in the accessions
under cultivation at the Newe Yaʼar Research Center (numbered
accessions). The absence of 4-methyl-2-(trans-1-pentenyl)-5-
phenyloxazolidine in these accessions could be due to low levels
of trans-2-hexenal generated in the plant or to different plant
chemotypes selected for their psychostimulant effects, cellular
or subcellular localization from norephedrine within the plant
tissues, or the age of the plants. The data presented here suggests
that (1R)(2S)-norephedrine is not the final product of the phenyl-
propylamino alkaloids pathway in C. edulis as previously thought,
but that the metabolic pathway continues downstream to form
oxazolidine derivatives. The N-methylation step that is promi-
nent in Ephedra spp. seemingly does not occur in C. edulis. There-
fore, it is possible that in this plant most of the downstream flux
in the phenylpropylamino alkaloids pathway proceeds through
an aldehyde-conjugation mechanism in the form of oxazolidine
derivatives. This hypothesis, in turn, suggests that in both C.
edulis and E. sinica, phenylpropylamino alkaloids can serve as
precursors for other more complex alkaloid molecules rather
To determine the abundance and ratio between phenylpropyl-
amino alkaloids and their oxazolidine derivatives in different
khat accessions, leaf extracts from 13 different accessions were
analyzed using GC‑MS. Marked differences in the concentrations
of 2,4-dimethyl-5-phenyloxazolidine and 4-methyl-2-(trans-1-
pentenyl)-5-phenyloxazolidine as a percentage of total phenyl-
"
propylamino alkaloids were found (l Fig. 3). Similarly, all the
plant material analyzed contained 2,4-dimethyl-5-phenyloxazo-
lidine at different ratios with respect to total phenylpropylamino
alkaloids (calculated as percentage of total phenylpropylamino
alkaloids). In some accessions, such as 128 and 91, the levels of
2,4-dimethyl-5-phenyloxazolidine reached 33% of the total
phenylpropylamino alkaloids fraction, while in Mi the level was
"
much lower, being as little as 6% (l Fig. 3). Only three accessions,
Li, Mi, and Bi, accumulated low levels of 4-methyl-2-(trans-1-
pentenyl)-5-phenyloxazolidine, 2.2, 1.8, and 1.3%, respectively
"
(l Fig. 3). These data imply that the different khat accessions dis-
play a marked polymorphism not only in the amino alkaloid con-
Krizevski R et al. Naturally Occurring Norephedrine… Planta Med 2012; 78: 838–842

842 Original Papers
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than being the final accumulated products as previously thought.
Thus the stabilization mechanism of phenylpropylamino alka-
loids in khat might be related to that found in Ephedra species.
Since N-methylation of phenylpropylamino alkaloids does not
occur in khat, oxazolidine or oxazolidone formation might be a
possible stabilization mode of phenylpropylamino alkaloids in
khat tissues. With the advent of better genomic, metabolomic,
and transcriptomic platforms to study alkaloid metabolism [26],
we will gain much of the needed knowledge to elucidate the bio-
synthetic pathways to phenylpropylamino alkaloids and their de-
rivatives.
At present, we can only speculate about the ecological roles of
phenylpropylamino alkaloids and their oxazolidine derivatives
in the plants. It could also be that these compounds may possess
interesting and important pharmacological activities. Further re-
search in the unique phytochemistry of khat is needed to better
understand the therapeutic potential of this plant.
The ephedrine alkaloids have been subjected to frequent phar-
macological examinations, particularly on their actions against
the central and autonomic nervous systems. The pharmacologi-
cal action of the analog of ephedrine, an oxazolidine derivative,
was examined mainly on the central and autonomic nervous sys-
tems, and contrary to ephedrine, exhibited central depressant ac-
tions [28]. It is also shown that a heterocyclic oxazolidone deriv-
ative of ephedrine possesses anti-inflammatory activity [16]. We
hypothesize that the new oxazolidine we have found in Khat has
similar activity to that of ephedrine alkaloid-derived oxazolidine.
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Acknowledgements
!
This work was supported by the Israel Science Foundation (Grant
No. 814/06 to E.L. and N.D.).
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Conflict of Interest
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There are no conflicts of interest among authors or the funding
agency.
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