Biomimetic synthesis and HPLC-ECD analysis of the isomers of dracocephins A and B

Article abstract of DOI:10.3762/bjoc.12.247

Starting from racemic naringenin ((±)-1), a mixture of dracocephin A stereoisomers 6-(2″-pyrrolidinone-5″-yl)naringenin (±)-2a-d and its regioisomer, dracocephin B 8-(2″-pyrrolidinone-5″-yl)naringenin (±)-3a-d originally isolated from Dracocephalum rupestre, have been synthesized in a one-pot reaction. The separation of 2a-d and 3a-d was achieved by preparative HPLC. The four stereoisomers of each natural product were separated by analytical chiral HPLC and their absolute configuration was studied by the combination of HPLC-ECD measurements and TDDFT-ECD calculations. The synthesized flavonoid alkaloids were further characterized by physicochemical and in vitro pharmacological studies.

Full text of DOI:10.3762/bjoc.12.247

Biomimetic synthesis and HPLC–ECD analysis of the isomers
of dracocephins A and B
Viktor Ilkei1, András Spaits1, Anita Prechl2, Áron Szigetvári2, Zoltán Béni2,
Miklós Dékány2, Csaba Szántay Jr2, Judit Müller2, Árpád Könczöl2, Ádám Szappanos3,
Attila Mándi*3, Sándor Antus3, Ana Martins4,5, Attila Hunyadi6, György Tibor Balogh2,
György Kalaus (†)1, Hedvig Bölcskei1, László Hazai*1 and Tibor Kurtán*3
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2016, 12, 2523–2534.
1Department of Organic Chemistry and Technology, Budapest
University of Technology and Economics, Szt. Gellért tér 4, H-1111
Budapest, Hungary, 2Gedeon Richter Plc., Gyömrői út 19-21, H-1103
Budapest, Hungary, 3Department of Organic Chemistry, University of
Debrecen, P. O. Box 400, H-4002 Debrecen, Hungary, 4Department
of Medical Microbiology and Immunobiology, University of Szeged,
Dóm tér 9, H-6720 Szeged, Hungary, 5Permanent address: Synthetic
Systems Biology Unit, Institute of Biochemistry, Biological Research
Centre, Temesvári krt. 62, H-6726 Szeged, Hungary and 6Institute of
Pharmacognosy, Faculty of Pharmacy, University of Szeged, Eötvös
utca 6, H-6720 Szeged, Hungary
doi:10.3762/bjoc.12.247
Received: 26 July 2016
Accepted: 02 November 2016
Published: 24 November 2016
Associate Editor: J. S. Dickschat
© 2016 Ilkei et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
Attila Mándi* - mandi.attila@science.unideb.hu; László Hazai* -
hazai@mail.bme.hu; Tibor Kurtán* - kurtan.tibor@science.unideb.hu
* Corresponding author
Keywords:
absolute configuration; Dracocephalum rupestre; dracocephins A–B;
ECD calculation; flavonoid alkaloids; HPLC–ECD
Abstract
Starting from racemic naringenin ((±)-1), a mixture of dracocephin A stereoisomers 6-(2”-pyrrolidinone-5”-yl)naringenin (±)-2a–d
and its regioisomer, dracocephin B 8-(2”-pyrrolidinone-5”-yl)naringenin (±)-3a–d originally isolated from Dracocephalum
rupestre, have been synthesized in a one-pot reaction. The separation of 2a–d and 3a–d was achieved by preparative HPLC. The
four stereoisomers of each natural product were separated by analytical chiral HPLC and their absolute configuration was studied
by the combination of HPLC–ECD measurements and TDDFT–ECD calculations. The synthesized flavonoid alkaloids were further
characterized by physicochemical and in vitro pharmacological studies.
Introduction
Flavonoid alkaloids are a small subgroup of flavonoids have been reported to exhibit a wide range of pharmacological
possessing a five- or six-membered nitrogen heterocycle at- activities [1]. Dracocephins A and B were isolated as a mixture
tached to C-6 or C-8 of ring A of the flavonoid moiety. They of four stereoisomers by Ren et al. in 2008 from Draco-
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cephalum rupestre [2], which is an herb widely distributed The spontaneous cyclization of the Strecker aldehyde 5 yields
throughout western China and used in folk medicine for the the acylaminocarbinol intermediate 6, which readily loses water
treatment of various conditions including cold, cough, ictero- on protonation, resulting in the N-acyliminium ion 7a/b, a
hepatitis and laryngalgia. Dracocephins A (±)-2a–d and B strong electrophilic reagent.
(±)-3a–d have been found to be conjugates of racemic narin-
genin ((±)-1) with pyrrolidine-2-one with C-6–C-5” and Results and Discussion
C-8–C-5” linkage, respectively. Due to the two chirality The aim of our work was to synthesize the natural flavonoid
centers, four possible stereoisomers exist for each regioisomer, alkaloids 2a–d and 3a–d in a biomimetic scheme in order
and accordingly, the isolated substances were verified to be 1:1 to devise an efficient route to these natural products for struc-
mixtures of two diastereoisomeric racemates (2a, 2c/2b, 2d; 3a, ture–activity relationship studies.
3c/3b, 3d) by HPLC–ECD analysis (Figure 1).
First the N-acylaminocarbinol reagent was prepared in the form
The planar structure and absolute configuration of the first- of racemic 5-ethoxypyrrolidine-2-one ((±)-9) by the partial
eluted stereoisomer of dracocephins A (±)-2a–d was deter- reduction of succinimide (8) with sodium borohydride at 0 °C,
mined by single-crystal X-ray diffraction analysis as (2R,5”S)- as described in the literature [5] (Scheme 2).
2a [2]. The biosynthesis of these flavonoid–pyrrolidone conju-
gates is proposed by Leete [3] and Tanaka et al. [4] to proceed In the next step, dracocephins A (±)-2a–d and B ((±)-3a–d)
via an acylaminocarbinol intermediate, which presumably arises were prepared by reacting racemic naringenin ((±)-1) with the
through the Strecker-degradation of the corresponding amino N-acylaminocarbinol ether (±)-9 in the presence of a catalytic
acids, which is L-glutamine (4) in the case of dracocephins A amount of p-toluenesulfonic acid (PTS) in nitromethane at
and B as shown in Scheme 1.
101 °C for 4 h as shown in Scheme 3.
Figure 1: Structures of (±)-naringenin, (±)-dracocephins A1–A4 and B1–B4 with the indication of the absolute configuration and elution orders for sep-
aration on Chiralpak AS-H* column [2]. The absolute configurations are presented as proposed by Ren et al. [2].
Scheme 1: Proposed biosynthetic route to dracocephins A and B.
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as proven by the separation of the stereoisomers by chiral
HPLC and by the integration or deconvolution of NMR signals
of Hy-3 recorded in CD3OD.
The separation of the stereoisomers of dracocephins A (±)-2a–d
could be achieved by chiral HPLC using an analytical Chiralpak
IC column with the eluent MeCN/2-propanol/TFA 97:3:0.1
(Figure 2).
Scheme 2: Synthesis of (±)-5-ethoxypyrrolidine-2-one ((±)-9).
The product was purified by column chromatography resulting Four peaks with alternating signs of the ECD signal in the
in a mixture of dracocephins A and B as a white crystalline HPLC–UV and –ECD traces were observed, the integration of
solid in 64% yield. Although their mixture could not be separat- which showed a near 1:1:1:1 ratio of the stereoisomers. The
ed on silica gel by column chromatography, the two isomeric HPLC–ECD spectra of the separated stereoisomers were re-
products could be separated by preparative HPLC to result in corded, which revealed that the first and fourth eluted stereoiso-
regioisomers (±)-2a–d and (±)-3a–d as crystalline powders in a mers are enantiomers, as well as the second and third eluted
ratio of 43:57, respectively. These data clearly showed that the ones (Figure 2c,d). On the basis of the reported data [2], the
reaction took place without notable regioselectivity, which was (2S,5”S) and (2R,5”S) absolute configurations were assigned to
in agreement with the observed 58:42 ratio of natural draco- the first- and second-eluted stereoisomers 2b and 2a, respec-
cephins A and B [2].
tively, while the third and fourth eluted 2c and 2d stereoiso-
mers had (2S,5”R) and (2R,5”R) absolute configuration. The
The regioisomeric dracocephins A and B were identified by elution order of stereoisomers 2b and 2a was inverted in our
NMR spectroscopy. The CD3OD solution of compounds analysis with Chiralpak IC column compared to the reported
(±)-2a–d showed a cross-peak between the singlet of H-8 and separation using Chiralpak-AS-H [2].
the multiplet of H-2’ in the 2D NOESY spectrum, which
corroborated the structure of dracocephins A. Moreover, com- In order to confirm the assignment of the absolute configura-
pounds (±)-3a–d showed 2D NOESY cross peaks between tions for the stereoisomers 2a–d independently from the results
multiplets of the pyrrolidine ring and H-2’, corresponding to of Ren et al. and to test the applicability of the ECD calcula-
dracocephins B. Aliquots of sample (±)-2a–d and sample tions for the stereochemical studies of flavanoid alkaloids, the
(±)-3a–d were dissolved in DMSO-d6 in order to allow compar- solution TDDFT–ECD calculation method [6] was applied to
ison with literature data. The 1H chemical shifts of the herein 2a–d and 3a–d. For the validation of our computational ap-
synthesized dracocephins A and B showed good agreement with proach on flavonoids, the ECD spectra of (R)-naringenin ((R)-
those of the isolated flavonoid alkaloids [2]. Similarly to the 1), the building blocks of dracocephins A and B, were calcu-
natural route, no diastereoselectivity occurred in this sequence, lated first with different methods to identify which is able to
Scheme 3: Synthesis of (±)-dracocephins A and B (±)-2a–d and (±)-3a–d and the elution order of stereoisomers on a Chiralpak IC* column with the
eluent hexane/MeCN/TFA 97:3:0.1. Absolute configurations and the names of stereoisomers are shown as proposed by Ren et al. [2].
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Figure 2: a) Chiral HPLC–UV and HPLC–ECD traces of dracocephins A (2a–d) using Chiralpak IC column with the eluent MeCN/2-propanol/TFA
97:3:0.1 monitored at 290 nm. b) HPLC–ECD spectra of the first [black: (2S,5”S)-2b or dracocephin A2] and fourth eluted [red: (2R,5”R)-2d or draco-
cephin A4] stereoisomers of dracocephins A. c) HPLC–ECD spectra of the second [black: (2R,5”S)-2a or dracocephin A1] and third eluted [red:
(2S,5”R)-2c or dracocephin A3] stereoisomers of dracocephins A. The absolute configurations were assigned on the basis of the publication of
Ren et al. [2].
reproduce most precisely the experimental ECD transitions. B3LYP functionals resulted in 4 low-energy conformers over
ECD and VCD calculations of naringenin have been reported 2% Boltzmann population with an equatorial C-2 aryl group
recently by Abbate et al., which could serve as a good basis for near coplanar with the C-2– H-2 bond, the B97D and M06-2X
our computational studies [7]. Naringenin is a flavanone deriva- ones afforded 8 conformers including four high-energy ones
tive, whose negative 327 nm n–π* Cotton effect (CE) and the with an axial C-2 aryl group (Supporting Information File 1,
positive 289 nm π–π* CE were correlated with M helicity of the Figures S2, S4, S6, and S8). The four CAM-B3LYP/TZVP
fused hetero ring and (2R) absolute configuration [8,9]. The (PCM/CHCl3) conformers (Figure 3) differed mostly in the ori-
mirror image HPLC–ECD spectra of (R)- and (S)-naringenin entation of the 7- and 4’-OH protons and the condensed hetero
were recorded after separating the enantiomers on a Chiralpack ring had M helicity with envelope conformation and C-2
IA column with the eluent hexane/2-propanol 80:20 (Support- pointing out of the plane (ωC-8a,O-1,C-2,C-3 = −51.6°,
ing Information File 1, Figure S1).
ωC-8a,C-4a,C-4,C-3 = 0.4° for the lowest-energy conformer).
The initial conformational search of (R)-1 with the Merck Mo- The C-2 aryl group was nearly coplanar with the C-2–H-2 bond
lecular Force Field (MMFF) and CHCl3 solvent model yielded and the ωH-2,C-2,C-1’,C-2’ torsional angle had values in the range
8 conformers within a 21 kJ/mol energy window, which were of –11.0° to –13.7°. The B97D/TZVP PCM/CHCl3 and M06-
reoptimized at five different DFT levels [B3LYP/6-31G(d) in 2X/TZVP reoptimizations produced four high-energy
vacuo, B3LYP/TZVP PCM/CHCl3, B97D/TZVP [10,11] PCM/ conformers having axial C-2 aryl groups with 34.9% and 32.7%
CHCl3, CAM-B3LYP/TZVP [12,13] PCM/CHCl3 and M06- total populations, which apparently overestimated this type of
2X/TZVP [14,15] PCM/CHCl3]. While the B3LYP and CAM- conformers and were not corroborated by the experimental
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Beilstein J. Org. Chem. 2016, 12, 2523–2534.
Figure 3: Structure and population of the low-energy CAM-B3LYP/TZVP PCM/CHCl3 conformers (>2%) of (R)-1.
NMR data. Interestingly, the B97D/TZVP PCM method The results of the ECD calculations of naringenin suggested
achieved good results with the conformationally flexible that the CAM-B3LYP/TZVP (PCM/CHCl3) reoptimization of
12-membered macrolides, dendrolides A–M [11].
the initial conformers and BH&HLYP/TZVP or CAM-B3LYP/
TZVP ECD calculations produce the best agreement and thus
these methods were considered primarily in stereochemical
ECD studies of 2a–d and 3a–d.
The BH&HLYP, CAM-B3LYP, B3LYP and PBE0 functionals
and TZVP basis set were applied for the ECD calculations of
the computed conformers, from which only the BH&HLYP and
CAM-B3LYP could reproduce the highest-wavelength charac- The MMFF conformational search of (2R,5’’R)-2d and
teristic n-π* transition, signs, relative intensities and shapes of (2R,5’’S)-2a resulted in 22 and 23 conformers in a 21 kJ/mol
the other bands, while the other two methods produced much energy window, respectively, the reoptimization of which at
worse agreement (Figure 4).
CAM-B3LYP/TZVP PCM/MeCN level yielded only 2 low-
energy conformers over 2% population (Figure 5 and Figure 6).
In both stereoisomers, hydrogen bonding between the 7-OH and
N–H group fixed the relative orientation of the pyrrolidin-2-one
and the naringenin moieties, and this interaction was also re-
sponsible for the low number of computed conformers. The two
computed conformers had comparable populations and they
differed only in the orientation of the 4’-OH. The flavanone
moiety adopted a conformation similar to that of naringenin.
The Boltzmann-averaged BH&HLYP/TZVP and CAM-
B3LYP/TZVP ECD spectra of (2R,5’’R)-2d and (2R,5’’S)-2a
confirmed the (2R) absolute configuration of the flavanone
units, which was also evident from the negative 330 nm n–π*
CE. The BH&HLYP/TZVP and CAM-B3LYP/TZVP ECD
spectra of (2R,5’’R)-2d showed a better agreement with the
ECD of 2a (second-eluted stereoisomer) on the basis of the
agreement with the 202 nm positive CE but the negative
330 nm n–π* transition was missing from the computed ECDs.
Figure 4: Experimental HPLC–ECD spectrum of (R)-naringenin ((R)-1)
compared with the Boltzmann-weighted ECD spectra computed for the
CAM-B3LYP/TZVP PCM/CHCl3 low-energy conformers of (R)-1 at
various levels.
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Beilstein J. Org. Chem. 2016, 12, 2523–2534.
Figure 5: Structure and population of the low-energy CAM-B3LYP/TZVP PCM/MeCN conformers (>2%) of (2R,5’’R)-2d.
Figure 6: Structure and population of the low-energy CAM-B3LYP/TZVP PCM/MeCN conformers (>2%) of (2R,5’’S)-2a.
The negative 330 nm n–π* transition could be only reproduced
by the B97D/TZVP conformers (Supporting Information File 1,
Figure S15), which, however, contained 28.1% total population
of conformers with an axial C-2 aryl group and the overall
agreement of their computed ECDs were quite bad. The experi-
mental ECD spectra of the epimers (2R,5’’R)-2d and (2R,5’’S)-
2a showed the same negative/positive/negative ECD pattern
from 370 nm to 210 nm and they only had a difference in the
additional positive CE of (2R,5’’S)-2a at 202 nm (Figure 7).
However, it seemed that (2R,5’’R)-2d had also negative CE
below 200 nm, which could not be measured precisely due to
the UV cut-off limitation of the 2-propanol eluent. Thus the ex-
perimental HPLC–ECD spectra of (2R,5’’R)-2d and (2R,5’’S)-
Figure 7: Experimental HPLC-ECD spectra of (2R,5’’S)-2a (second
2a only showed differences in the position, shape and relative
intensities of the bands, while the patterns of the ECD bands
were identical.
eluted stereoisomer) and (2R,5’’R)-2d (fourth eluted stereoisomer)
compared with the Boltzmann-weighted ECD spectra computed for the
CAM-B3LYP/TZVP PCM/MeCN low-energy conformers of (2R,5’’R)-2
at various levels.
The Boltzmann-averaged BH&HLYP/TZVP and CAM-
B3LYP/TZVP ECD spectra of (2R,5’’S)-2a reproduced well the of (2R,5’’R)-2d (fourth eluted stereoisomer). All these calcula-
negative n–π* 330 nm CE and the intense 213 nm negative CE tion results would favor the configurational assignment
of (2R,5’’R)-2d but it changed the sign of CE to positive below (2R,5’’S)-2d (fourth eluted stereoisomer) and (2R,5’’R)-2a
210 nm, which was characteristic of (2R,5’’S)-2a (Figure 8).
(second eluted stereoisomer), which, however, contradicts the
previously published results [2]. From the ECD calculation
Interestingly, the B3LYP/TZVP and PBE0/TZVP ECD spectra point of view, the differences in the HPLC ECD spectra of the
of (2R,5’’S)-2a gave a perfect agreement with the HPLC–ECD epimers (2R,5’’R)-2d and (2R,5’’S)-2a were not sufficient and
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Beilstein J. Org. Chem. 2016, 12, 2523–2534.
the calculation results were not consistent enough to allow the
revision of the previous configurational assignment.
The separation of the stereoisomers of dracocephins B 3a–d
could be performed on an analytical Chiralpak IC column using
the eluent acetonitrile/2-propanol/trifluoroacetic acid 90:10:0.1
(Figure 9a).
Only partial separation could be achieved for the first two
eluting stereosiomers, which was still sufficient to record their
HPLC–ECD spectra. The comparison of HPLC–ECD spectra
showed that the first and fourth eluted stereoisomers were enan-
tiomers as well as the second and third eluted ones. Similarly to
dracochepins A, the elution order was inverted for the first- and
second-eluted stereoisomers compared to that of the reported
data with Chiralpak AS-H [2]. The solution TDDFT-ECD
calculation approach was also applied to the stereoisomers of
dracocephins B 3a–d in order to check the applicability of this
method.
Figure 8: Experimental HPLC–ECD spectra of (2R,5’’S)-2a and
(2R,5’’R)-2d compared with the Boltzmann-weighted ECD spectra
computed for the CAM-B3LYP/TZVP PCM/MeCN low-energy
conformers of (2R,5’’S)-2 at various levels.
Figure 9: a) Chiral HPLC–UV and HPLC–ECD traces of dracocephins B1–B4 3a–d using a Chiralpak IC column with the eluent MeCN/2-propanol/
TFA 90:10:0.1 monitored at 285 nm. b) HPLC–ECD spectra of the first- [black: (2R,5”S)-3b or dracocephin B2] and fourth eluted [red: (2S,5”R)-3d or
dracocephin B4] stereoisomers of dracocephins B. c) HPLC–ECD spectra of the second [black: (2S,5”S)-3a or dracocephin B1] and third eluted [red:
(2R,5”R)-3c or dracocephin B3] stereoisomers of dracocephins B. The absolute configurations were assigned on the basis of the publication of
Ren et al. [2].
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The MMFF conformational search of (2R,5’’R)-3c and
(2R,5’’S)-3b resulted in 41 and 44 conformers in a 21 kJ/mol
energy window, respectively, the reoptimization of which at
CAM-B3LYP/TZVP PCM/MeCN level yielded 2 low-energy
conformers over 2% population for both stereoisomers
(Figure 10 and Figure 11).
Similarly to dracochepins A (2a–d), hydrogen bonding be-
tween the 7-OH and NH groups anchored the orientation of the
pyrrolidinone moiety. The two conformers differed in the orien-
tation of the two 4’-OH and the C-2 aryl moiety adopted equa-
torial orientation with M helicity and envelope conformation of
the condensed heteroring.
Figure 12: Experimental HPLC-ECD spectra of 3b (first eluted stereo-
isomer) and 3c (third eluted stereoisomer) compared with the Boltz-
mann-weighted ECD spectra computed for the CAM-B3LYP/TZVP
PCM/MeCN low-energy conformers of (2R,5’’R)-3 at various levels.
The calculated Boltzmann-averaged ECD spectra of (2R,5’’R)-
3c and (2R,5’’S)-3b verified the (2R) absolute configuration of
the third-eluted and second-eluted stereoisomers on the basis of
the high-wavelength negative n–π* and positive π–π* CEs and
the BH&HLYP and CAM-B3LYP functionals could reproduce B3LYP ECD spectra of (2R,5’’R)-3c had a weak positive CE at
the negative n–π* transition (Figure 12).
about 250 nm, which was missing from the experimental curve
and they rather resembled the HPLC-ECD curve of the first-
The absolute configuration of C-5” could not be unambiguous- eluted stereoisomer. The BH&HLYP and CAM-B3LYP ECD
ly deduced from the calculations. The BH&HLYP and CAM- spectra of (2R,5’’S)-3 reproduced quite well the signs and posi-
Figure 10: Structure and population of the low-energy CAM-B3LYP/TZVP PCM/MeCN conformers (>2%) of (2R,5’’R)-3c.
Figure 11: Structure and population of the low-energy CAM-B3LYP/TZVP PCM/MeCN conformers (>2%) of (2R,5’’S)-3b.
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Beilstein J. Org. Chem. 2016, 12, 2523–2534.
tion of the experimental ECD bands, although the intensity of A plausible mechanism for the formation of dracochepins A and
the 201 negative CE was overestimated (Figure 13).
B involves the electrophilic attack of the N-acyliminium ion
7a,b on the aromatic ring A of racemic naringenin ((±)-1) at
C-6 and C-8 (Figure 14).
The electrophilic reagent arises from the amidocarbinol ether
(±)-9 after protonation and loss of ethanol. The planar structure
of the cation explains the lack of stereoselectivity observed in
the reaction.
Physicochemical measurements
Physicochemical characterization of dracocephins A and B was
also carried out. The relevant calculated (total polar surface
area; TPSA) and measured (pKa, log P/D and Pe) parameters
are shown in Table 1. The lipophilicity of neutral species of
naringenin and dracocephins A and B are 3.39, 2.52 and 2.39,
respectively. Similarly to this lipophilicity trend, the TPSA
value of (±)-naringenin is lower than dracocephins A and B,
however, there is no difference between the calculated TPSA
values of 2a–d and 3a–d. The pKa values of the three phenolic
hydroxy groups of the evaluated compounds are quite similar
Figure 13: Experimental HPLC-ECD spectra of 3b and 3c compared
with the Boltzmann-weighted ECD spectra computed for the CAM-
B3LYP/TZVP PCM/MeCN low-energy conformers of (2R,5’’S)-3 at
various levels.
Similarly to dracocephins A (2a–d), ECD spectra computed for and these are in the range of 6.5 and 12.0. Among these proton-
(2R,5’’S)-3 with B3LYP and PBE0 functionals resembled more dissociation constants, the lowest acidic pKa values are predom-
the experimental HPLC–ECD spectrum of 3c than that of 3b.
inant in physiological environment, which is also indicated by
Figure 14: Proposed mechanism for the formation of dracocephins A and B (2a–d and 3a–d) starting from (±)-9.
Table 1: Calculated and measured medicinal chemical parameters of dracocephins A and B compared to those of naringenin.
Compound
pKa
log P
log D7.4/log D6.5
TPSA
86.99
116.1
116.1
PAMPA-BBB (Pe × 10–6 cm/s)
6.91 ± 0.06
9.20 ± 0.08
11.85 ± 0.06
(±)-1
3.39 ± 0.01
2.52 ± 0.02
2.39 ± 0.02
2.80/3.25
8.8 ± 0.6 (medium)
6.69 ± 0.01
9.47 ± 0.03
12.34 ± 0.18
2a–d
1.74/2.31
1.1 ± 0.1 (poorly permeable)
non-permeable
6.54 ± 0.00
9.54 ± 0.00
11.97 ± 0.02
3a–d
1.51/2.12
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Beilstein J. Org. Chem. 2016, 12, 2523–2534.
the difference of log D values between pH 7.4 and pH 6.5. The could be confirmed, while the C-5” of the pyrrolidinone unit
blood–brain barrier (BBB) specific penetration of dracocephins could not be assigned unambiguously. Dracocephins A (2a–d)
isomers has also been studied by the PAMPA (parallel artificial and B (3a–d) were also characterized by physicochemical and
membrane permeability assay)-BBB model system, which indi- in vitro biological measurements. A plausible mechanism of the
cates passive diffusion of test compounds across the brain capil- synthesis of these natural products was also proposed.
lary endothelium. Although naringenin ((±)-1) showed a medi-
Experimental
um penetration characteristic in this model system, draco-
cephins A (2a–d) and B (3a–d) had been described as poorly General
and practically non-permeable compounds, respectively. These Melting points were measured on a SANYO Gallenkamp appa-
results are in good correlation with a lower lipophilicity ratus and are uncorrected. IR spectra were recorded on a Bruker
(log P/D7.4) and higher TPSA of dracocephins A and B com- FTIR instrument. NMR measurements (1H, 13C, gCOSY, 1D
pared to that of naringenin.
NOESY and 2D NOESY, gHSQCAD, gHMBCAD) were per-
formed on Varian 400 MHz (equipped with 5 mm OneNMR
15N-31P/{1H-19F} PFG Probe), Varian 500 MHz (equipped
Pharmacological studies
In connection with the BBB penetration studies, 2a–d and 3a–d with 1H{13C/15N} 5 mm PFG Triple Resonance 13C Enhanced
were tested for their potential towards CNS cytotoxic activity Cold Probe) and Varian 800 MHz (equipped with 1H{13C/15N}
on undifferentiated SH-SY5Y neuroblastoma cells. None of the Triple Resonance 13C Enhanced Salt Tolerant Cold Probe)
two isomeric compounds exerted cytotoxicity on this cell line spectrometers. 1H chemical shifts are given on the delta scale as
(IC50 > 150 μM for both compounds), which rules out their parts per million (ppm) with tetramethylsilane (TMS) as the
potential CNS antitumor activity and it also suggests them as internal standard (0.00 ppm). 13C chemical shifts are given on
non-neurotoxic substances. On the other hand, the possibility the delta scale as parts per million (ppm) with tetramethylsilane
for neuroprotective activity could not be tested with the applied (TMS) or dimethylsulfoxide-d6 as the internal standard
experimental setup. As an additional bioassay on compounds (0.0 ppm and 39.5 ppm, respectively). MS spectra were re-
2a–d and 3a–d, their potential to interfere with the function of corded on VG-Trio-2 and Finnigan MAT 95SQ instruments
P-glycoprotein (P-gp) was tested. By transporting a wide using EI or ESI techniques. HRMS analyses were performed on
variety of compounds from the CNS back into the blood stream, an LTQ FT Ultra (Thermo Fischer Scientific, Bremen,
P-gp is an important element of the BBB [16]. Naringenin is Germany) system. TLC was carried out on TLC Silicagel 60
known to interfere with the BBB penetration of vincristine, a F254 on 20 × 20 aluminium sheets (Merck) and preparative TLC
P-gp substrate, through modulating this efflux pump [17], and was performed using Silicagel 60 F254+366 (Merck) coated glass
an additional nitrogen-containing group has the chance to sig- plates. Column chromatography was performed using Geduran
nificantly increase P-gp inhibitory activity [18]. Based on the Si 60 (Merck) silica. Racemic naringenin was purchased from
unaltered accumulation of rhodamine 123, a P-gp substrate Sigma-Aldrich and used without further purification.
fluorescent dye, in the P-gp transfected L5178MDR cells, we ob-
served that none of the synthesized compounds inhibits this Preparative HPLC
transporter. The antioxidant activity of dracocephins A and B Chemicals and reagents
was also measured, but these compounds, along with narin- Acetonitrile used in the preparative chromatographic separation
genin were virtually inactive in the DPPH radical scavenging was gradient grade LiChrosolv purchased from Merck. The
test (IC50 > 200 µg/mL), compared to the positive control formic acid was reagent grade from Sigma-Aldrich. Water was
quercetin (IC50 = 9.4 ± 0.6 µg/mL).
purified with a Milli-Q system. The sample solvent was spec-
troscopy grade dimethyl sulfoxide (Uvasol) from Merck.
Conclusion
An efficient one pot synthesis of dracocephins A (2a–d) and B Apparatus
(3a–d) was achieved starting from racemic naringenin ((±)-1), The separation of the isomeric mixtures of flavonoid alkaloids
using the readily accessible N-acylaminocarbinol reagent (+)-9). was performed with a Shimadzu chromatograph equipped with
The regioisomeric dracochepins A and B could be separated by an LC-8A pump unit, SPD-M20A Photodiode detector and
preparative HPLC and their connectivity was determined on the CBM-20A system controller. The samples were introduced via
basis of NMR data. Stereoisomers of dracocephins A (2a–d) an SIL-10AP sample injector and the chromatograms were
and B (3a–d) were separated by chiral HPLC and their stereo- processed using the LCsolution software. The fractions were
chemistry was studied by TDDFT-ECD calculations. By testing collected by an FRC10A fraction collector module. The
different methods for the calculation of conformers and ECD, temperature was controlled by a CTO-20AC prominence
the configurational assignment of C-2 of the flavanone moiety column oven.
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Beilstein J. Org. Chem. 2016, 12, 2523–2534.
Preparative separation procedure
(±)-Naringenin ((±)-1): (2R)-1 (first peak) tR = 5.19 min on a
The sample (500 µL/injection) was loaded to a Kinetex 5 µm Chiralpak IA column (hexane/2-propanol 80:20), λ [nm] (Φ)}:
phenyl-hexyl AXIA packed 21.2 × 150 mm column in the 327 (–6.75), 314sh (–4,02), 289 (28.91), 235sh (–7.96), 215
concentration of 20 mg/mL. The separation was achieved (–28.48); (2S)-1 (second peak): tR = 5.84 min on a Chiralpak IA
with isocratic elution (75% HPLC grade water containing column (hexane/2-propanol 80, λ [nm] (Φ)}: 327 (5.49), 314sh
0.1% formic acid, 25% acetonitrile containing 0.1% (3.49), 289 (–22.65), 235sh (4.69), 215 (21.67).
formic acid) at 25 °C. The flow rate was 21 mL/min. The frac-
Preparation of dracocephins A ((±)-2a–d) and
B ((±)-3a–d)
tions were collected based on UV absorption at 220 nm wave-
length.
To a suspension of racemic naringenin [(±)-1] (200 mg,
Chiral HPLC–ECD analysis
0.735 mmol) in nitromethane (5 mL) was added 5-ethoxypyrro-
Chiral HPLC separations were carried out with a Jasco HPLC lidine-2-one ((±)-9, 114 mg, 0.883 mmol), a catalytic amount of
system on Chiralpak IC column (250 × 4.6 mm i.d.; 5 μm) p-toluenesulfonic acid and the mixture was refluxed for 4 h.
using eluent hexane/acetonitrile 97:3 or 90:10 with 0.1% TFA The solvent was evaporated in vacuo and the solid residue was
additive to set the pH to about 2 at a flow rate of 1.0 mL/min for purified by column chromatography (CH2Cl2/CH3OH = 10:1).
2 and 3. HPLC–UV and OR chromatograms were measured An isomeric mixture of (±)-2a–d and (±)-3a–d (168 mg, 64%)
with Jasco MD-910 multiwavelength and OR-2090Plus chiral was obtained as a white crystalline solid, Rf (CH2Cl2:CH3OH =
detectors, respectively. The HPLC–ECD traces were recorded 10:1) 0.32. A sample of the mixture (47 mg) was subjected to
at the specified wavelength with a Jasco J-810 CD spectropo- further purification by preparative HPLC, which gave title com-
larimeter equipped with a 1 cm HPLC flow cell and the base- pounds (±)-2a–d (16 mg, 99.66% purity) as a white solid, mp
line was zeroed after the start of each run. The on-line ECD and 168–170 °C; IR (KBr) νmax: 3393, 3034, 2970, 1634, 1519,
UV spectra were recorded simultaneously by stopping the flow 1455, 1340, 1310, 1279, 1170, 1090 cm−1; 1H NMR (799.7
at the UV absorption maximum of each peak. ECD ellipticity MHz, CD3OD) δH 2.21–2.27 (m, 1H, Hx-4”), 2.36–2.42 (m,
values (Φ) were not corrected for concentration. For an 1H, Hx-3”), 2.43–2.49 (m, 1H, Hy-4”), 2.59–2.64 (m, 1H,
HPLC–ECD spectrum, three consecutive scans were recorded Hy-3”), 2.71–2.75 (m, 1H, Hx-3), 3.12 and 3.13 [sum 1H, (dd, J
and averaged with 2 nm bandwidth, 1 s response, and standard = 17.0, 12.8 Hz, Hy-3A) and (dd, J = 17.0, 12.6 Hz, Hy-3B), re-
sensitivity. The HPLC–ECD spectrum of the eluent was re- spectively], 5.33–5.36 (buried m, 2H, H-5” and H-2), 5.95 (s,
corded in the same way. The concentration of the injected sam- 1H, H-8), 6.80–6.82 (m, 2H, H-3’ and H-5’), 7.29–7.32 (m, 2H,
ple was set so that the HT (voltage) value did not exceed 500 V H-2’ and H-6’); 13C NMR (201.1 MHz, CD3OD) δC 26.7
in the HT channel.
(C-4”), 32.07 and 32.10 (C-3”), 43.96 and 43.99 (C-3), 49.4
(C-5”), 80.52 and 80.53 (C-2), 96.04 and 96.06 (C-8), 103.1
Dracocephins A1–A4 (2ª–d): 2b (first eluted stereoisomer): (C-10), 109.44 and 109.46 (C-6), 116.4 (C-3’ and C-5’), 129.09
tR = 7.26 min, λ [nm] (Φ)}: 330 (4.43), 288 (–15.34), 249sh and 129.10 (C-2’ and C-6’), 131.0 (C-1’), 159.12 and 159.13
(2.48), 230 (11.60), 213 (27.01). 2a (second eluted stereoiso- (C-4’), 163.47 and 163.50 (C-5), 163.90 and 163.91 (C-9),
mer): tR = 7.49 min, λ [nm] (Φ)}: 329 (–3.95), 289 (13.60), 166.8 (C-7), 181.60 and 181.63 (C-2”), 198.1 (C-4); HRMS [M
250sh (–2.18), 227 (–7.35), 202 (15.98). 2c (third eluted stereo- + H] calcd for C19H18O6N: 356.11286; found: 356.11284;
isomer): tR = 11.49 min, λ [nm] (Φ)}: 329 (1.92), ESI–MS–MS (CID = 35%) (rel. int. %): 339(100); 338(4);
289 (–7.86), 250sh (1.09), 227 (4.56), 202 (–10.49). 2d 311(2); 236(3); and title compounds (±)-3a–d (21 mg, 99.92%
(fourth eluted stereoisomer): tR = 12.38 min, λ [nm] (Φ)}: 330 purity), also a white solid, mp 238–240 °C; IR (KBr) νmax:
(–5.71), 288 (17.65), 249sh (–3.54), 230sh (–17.68), 213 3416, 3034, 2970, 1630, 1519, 1447, 1378, 1343, 1257, 1176,
(–33.89).
1081 cm−1; 1H NMR (799.7 MHz, CDCl3:CD3OD = 2:1) δH
2.15–2.29 (buried m, 2H, Hx-3” and Hx-4”), 2.30–2.37 (m, 1H,
Dracocephins B1–B4 (3a–d): 3b (first peak): tR = 4.87 min, λ Hy-3”), 2.38–2.46 (m, 1H, Hy-4”), 2.73–2.79 (m, 1H, Hx-3),
[nm] (Φ)}: 327 (–2.15), 288 (8.40), 252sh (–0.88), 234sh 3.09 and 3.14 [sum 1H, (dd, J = 17.0, 13.1 Hz, Hy-3A) and (dd,
(–2.24), 221 (–5.07), 201 (19.79). 3a (second peak): tR = 4.87 J = 17.1, 13.2 Hz, Hy-3B), respectively], 5.28–5.37 (buried m,
min, λ [nm] (Φ)}: 332 (0.65), 290 (–2.74), 254 (0.13), 240sh 2H, H-5” and H-2), 6.00 (s, 1H, H-6), 6.87–6.90 (m, 2H, H-3’
(0.90), 203 (18.81). 3c (third peak): tR = 5.53 min, λ [nm] (Φ)}: and H-5’), 7.28–7.31 (m, 2H, H-2’ and H-6’); 13C NMR (201.1
328 (–1.02), 288 (6.25), 254 (–0.42), 235sh (–2.14), 203 MHz, CD3OD) δC 25.8 and 25.9 (C-4”), 31.1 (C-3”), 42.8 and
(–11.47). 3d (fourth peak): tR = 7.56 min, λ [nm] (Φ)}: 328 43.5 (C-3), 48.4 and 48.6 (C-5”), 79.7 and 80.0 (C-2), 96.6
(1.02), 288 (–4.68), 252sh (0.17), 234sh (0.42), 221 (1.46), 201 (C-6), 102.6 (C-10), 107.9 (C-8), 115.9 (C-3’ and C-5’), 128.2
(–9.10).
(C-2’ and C-6’), 129.3 (C-1’), 158.0 (C-4’), 161.9 (C-9), 163.4*
2533

Beilstein J. Org. Chem. 2016, 12, 2523–2534.
12.Yanai, T.; Tew, D. P.; Handy, N. C. Chem. Phys. Lett. 2004, 393,
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(C-5), 165.6* (C-7), 180.2 (C-2”), 196.9 and 197.0 (C-4); (*:
may be reversed); HRMS [M + H]: calcd for C19H18O6N
356.11286; found: 356.11285; ESI–MS–MS (CID = 35%) (rel.
int. %): 339 (100); 338 (4); 311 (3); 236 (6).
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Supporting Information
Supporting Information File 1
16.Schinkel, A. H. Adv. Drug Delivery Rev. 1999, 36, 179–194.
doi:10.1016/S0169-409X(98)00085-4
Details of physicochemical measurements, biology and
computational section, computed ECD spectra, conformers
of compounds 1–3 and NMR spectra of compounds 2 and
3.
17.Mitsunaga, Y.; Takanaga, H.; Matsuo, H.; Naito, M.; Tsuruo, T.;
Ohtani, H.; Sawada, Y. Eur. J. Pharmacol. 2000, 395, 193–201.
doi:10.1016/S0014-2999(00)00180-1
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2003, 28, 203–228. doi:10.1046/j.1365-2710.2003.00487.x
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-12-247-S1.pdf]
License and Terms
Acknowledgements
The authors are grateful to Gedeon Richter Plc. for the finan-
cial support, and to Prof. Joseph Molnár (Department of
Medical Microbiology and Immunobiology, University of
Szeged, Szeged, Hungary) for generously providing cell lines
for the bioassays. Á. Szappanos, A. Mándi, S. Antus and T.
Kurtán gratefully acknowledge the support provided by OTKA/
NKFI (Grant Nos: K-112951, K-105871 and PD 121020) and
the CPU time by the National Information Infrastructure Devel-
opment Institute (NIIFI 10038).
This is an Open Access article under the terms of the
Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
(http://www.beilstein-journals.org/bjoc)
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