Stereoselective preparation of pyridoxal 1,2,3,4-tetrahydro-β- carboline derivatives and the influence of their absolute and relative configuration on the proliferation of the malaria parasite Plasmodium falciparum

Article abstract of DOI:10.1016/j.bmc.2014.01.057

We have selectively synthesized by Pictet-Spengler condensation of tryptophan and pyridoxal the four stereoisomers of a pyridoxal β-carboline derivative that was designed to inhibit the proliferation of Plasmodium falciparum. Biological investigation of the four compounds revealed that they all inhibit the growth of P. falciparum. With an IC₅₀ value of 8 ± 1 μM, the highest inhibitory effect on the proliferation of the parasite was found for the 1,3-trans-substituted tetrahydro-β-carboline that was obtained from d-tryptophan. Lower activity was found for its enantiomer, while the two diastereomeric cis-products were markedly less effective. Apparently a distinct spacial orientation of the carboxyl group of the substituted tetrahydropyridine unit of the compounds is needed for high activity, while the absolute configuration of the molecules is of lesser importance.

Full text of DOI:10.1016/j.bmc.2014.01.057

Bioorganic & Medicinal Chemistry 22 (2014) 1832–1837
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Stereoselective preparation of pyridoxal 1,2,3,4-tetrahydro-b-carbo-
line derivatives and the influence of their absolute and relative
configuration on the proliferation of the malaria parasite
Plasmodium falciparum
Renate Brokamp a, Bärbel Bergmann b, Ingrid B. Müller b, Stefan Bienz a,
⇑
a Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
b Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht Str. 74, D-20359 Hamburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
We have selectively synthesized by Pictet–Spengler condensation of tryptophan and pyridoxal the four
stereoisomers of a pyridoxal b-carboline derivative that was designed to inhibit the proliferation of Plas-
modium falciparum. Biological investigation of the four compounds revealed that they all inhibit the
Received 9 December 2013
Revised 30 January 2014
Accepted 30 January 2014
Available online 8 February 2014
growth of P. falciparum. With an IC50 value of 8
of the parasite was found for the 1,3-trans-substituted tetrahydro-b-carboline that was obtained from
-tryptophan. Lower activity was found for its enantiomer, while the two diastereomeric cis-products
1 lM, the highest inhibitory effect on the proliferation
D
Keywords:
Plasmodium falciparum
Malaria
Tetrahydro-b-carboline
Pyridoxal
were markedly less effective. Apparently a distinct spacial orientation of the carboxyl group of the
substituted tetrahydropyridine unit of the compounds is needed for high activity, while the absolute
configuration of the molecules is of lesser importance.
Ó 2014 Elsevier Ltd. All rights reserved.
PLP dependent enzyme
1. Introduction
PT3, however, was available in just a very small amount: it ar-
ose as a minor side product upon the reductive amination of pyri-
Malaria is an infectious disease, widely-spread in tropical and
sub-tropical areas in South America, Asia, and, in particular, sub-
saharan Africa. It is caused by a parasite of the genus Plasmodium,
the most virulent being Plasmodium falciparum. Malaria causes
more than 2 million fatalities per worldwide year, and more than
300 million new infections occur annually. Since there are no vac-
cines against malaria available as yet and because the few existing
antimalarials are progressively losing their efficacy due to the
development of drug resistance, there is a vital need for new and
potent antiplasmodials. As a new strategy to interfere with the par-
asite’s metabolism, poisoning of pyridoxal 5-phosphate-dependent
ornithine decarboxylase (PfODC) was suggested by Müller et al.1
With their initial investigation they showed that a pyridoxyl-
amino acid derivative, 1,2,3,4-tetrahydro-b-carboline derivative
PT3 (Fig. 1), is a promising candidate for further examination.
PT3 showed a distinct antiplasmodial activity, inhibiting the
proliferation of cultured P. falciparum with an IC50 value of
doxal with tryptophan methyl ester. Due to the small amount of
sample, PT3 was only characterized with a mass spectrum. More
detailed data that would have revealed the isomeric purity as well
as the stereochemical identity of the material were not obtained.
Hence, the preliminary biological study could not assign the
observed inhibitory effect of PT3 to a specific isomeric form of
the compound. More material and access to all four stereoisomers
of PT3, compounds cis-1 and trans-1 in both enantiomeric forms
(Fig. 1; the symbols
D and L referring to the configuration of the
tryptophan moiety), was thus needed to be able to complete the
biological evaluation of PT3.
In this report we describe the preparation of all four isomeric
1,2,3,4-tetrahydro-b-carboline derivatives of the type 1 and the
results of their evaluation as antiplasmodial agents.
2. Results and discussion
2.1. Synthesis
14 l
m, while leaving mammalian cells largely unaffected.1
The most direct method to prepare 1,3 disubstituted 1,2,3,4-tet-
rahydro-b-carboline derivatives is the Pictet–Spengler condensation
⇑
Corresponding author. Tel.: +41 44 635 42 45; fax: +41 44 635 68 12.
E-mail address: stefan.bienz@chem.uzh.ch (S. Bienz).
http://dx.doi.org/10.1016/j.bmc.2014.01.057
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

R. Brokamp et al. / Bioorg. Med. Chem. 22 (2014) 1832–1837
1833
L
L
CO2Me
CO2Me
NH
NH
N
H
N
H
OH
Me
OH
Me
HO
HO
CO2Me
N
N
NH
N
H
cis-L1
trans-L1
OH
Me
HO
D
D
CO2Me
CO2Me
N
NH
NH
N
H
N
H
PT3
OH
Me
OH
Me
HO
HO
N
N
cis-D1
trans-D1
Figure 1. Target structures to be investigated with regards to their antiplasmodial activity.
of tryptamine derivatives with aldehydes,2,3 and most possibly,
PT3 arose from just this reaction, which easily could have
proceeded in competition with the intended reductive amination
of tryptophan methyl ester with pyridoxal.
addition of 1.1 equiv of NEt3, was treated with pyridoxal hydro-
chloride in the presence of molecular sieves in CH2Cl2 at reflux for
15–60 min to allow the formation of imine 2. Cyclization to the
tetrahydro-b-carboline products cis-1 and trans-1 was then
effected by the addition of an excess of trifluoroacetic acid (TFA),
while heating was continued for an additional 3–5 h.
The reaction products were found to depend on the exact reac-
tion conditions. If 30 min were provided for the imine formation,
and the subsequent acid treatment was performed for 3.5 h, the
reaction delivered a 75% yield of the cis- and trans-b-carboline
derivatives in a ratio of 93:7. The two compounds, however, only
arose as racemates. If, on the other hand, only a short time was
allowed for imine formation and TFA was added after just 15 min
of mixing the starting materials, only a little racemization occurred
(er >95:5). The desired products, however, arose in the unsatisfac-
tory low chemical yield of 15% (cis/trans ratio of 95:5).
The Pictet–Spengler condensation of tryptophan esters with
aldehydes has been described previously. It was studied in more
detail by Cook4–8 and Bailey9–11 and their co-workers, particularly
also with regards to the stereochemical outcome of the reaction. It
was shown that thermodynamic control of the process leads
preferentially to trans-configured products, in particular when
N-benzyl tryptophan methyl ester was used as the starting mate-
rial. Kinetic control, on the other hand, leads predominantly to
the cis-configured products, however with only moderate selectiv-
ities in most cases. Depending on the reaction conditions, also
partial racemization was observed.
For the synthesis of the target compounds of type 1,
Pictet–Spengler condensation of
D
- and
L
-tryptophan methyl esters
From these initial results we concluded that racemization
occurs during imine formation, prior to the cyclization, and that
imine formation has to be effected as completely as possible prior
to the addition of TFA because it is inhibited by the excess of the
acid. That racemization is a problem that can occur in the course
with pyridoxal was performed (Scheme 1). Following the proce-
dure of Bailey et al. described for the kinetically controlled
transformation,9 tryptophan methyl ester, either dissolved as the
free base or liberated in situ from its hydrochloric salt by the
L
CO2Me
L
CO2Me
NH2
N
H
MS 3Å
CH2Cl2
N
N
H
OH
Me
H
O
HO
OH
N
HO
L2
N
Me
TFA
L
L
CO2Me
CO2Me
NH
NH
N
H
N
H
+
OH
Me
OH
Me
HO
HO
N
N
cis-L1
trans-L1
Scheme 1. Pictet–Spengler condensation of tryptophan methyl ester with pyridoxal, shown with L-tryptophan derivative as the starting material.

1834
R. Brokamp et al. / Bioorg. Med. Chem. 22 (2014) 1832–1837
of the Pictet–Spengler condensation was discovered earlier. It was
studied in more detail with the reaction of tryptophan methyl ester
with benzaldehyde.9 Loss of enantiomeric purity proved particu-
larly prominent when the condensations were performed at
elevated temperatures and in the presence of catalytic amounts
of acid. Mechanistically, the racemization was rationalized by an
imine (or iminium ion) tautomerism by which imine L3 is in equi-
librium with D3 via compound 4 (or their protonated forms,
Scheme 2). Considering the function of pyridoxal in biological pro-
cesses it is readily conceivable that the corresponding imine–imine
isomerisation, the reversible interconversion of L2 and D2, is
favored for the pyridoxyl as compared to the phenyl derivative.
Ungemach, the stereoisomer for which the carbon nuclei 13C(1)
and 13C(3) are more deshielded can be assigned to the cis-config-
ured compound. Accordingly, the second-eluting compound, with
the respective 13C signals found at d 53.9 and 56.8 ppm, was de-
duced to be cis-1 and the first-eluting component, with the corre-
sponding signals registered at d 50.3 and 54.4 ppm, to trans-1. This
assignment was supported by the ROESY spectra of the two com-
pounds. A distinct cross peak for the signals of H-C(1) and H-C(3)
can be recognized in the spectrum of cis-1 but not so in the
spectrum of trans-1. Thus, the cis relationship of the two H atoms
in cis-1 is confirmed. Finally, the calculated IR spectra of cis-1
and trans-1 show distinctive differences in the regions of 1/k
1200 cmꢀ1 and 1250 cmꢀ1, which find their match in the experi-
mental spectra of the two compounds.
Due to the double activation of H-C(a) of the tryptophan moiety
by the carboxyl and the pyridoximine groups, slightly basic condi-
tions may lead to high isomerization rates. However, to circumvent
the problem by working under purely acidic conditions is not
feasible since imine formation is largely blocked when an excess
of acid is present. In fact, when the hydrochlorides of both compo-
nents of the transformation were reacted with each other at ꢀ15 °C
and, to effect cyclization, at 23 °C for a prolonged period of time,
the desired products were obtained in only 32% yield (as a cis/trans
mixture of 56:44). In addition, the enantiomeric ratio of these
products turned out to be only 88:12, indicating that the effect of
avoiding basic conditions was offset by the effect of a longer
reaction time under slightly acidic conditions.
The assignment of the absolute configurations in the several
compounds of the type 1 is based on chemical considerations only.
Since no mechanistic path for the Pictet–Spengler reaction is con-
ceivable that would induce controlled inversion of the configura-
tion at the stereogenic center of the starting amino acid, the
absolute configuration (of the major enantiomer) at C(3) is as-
sumed to be retained. Thus, the dextrorotary products obtained
from l-tryptophan methyl ester, (+)-cis-L1 and (+)-trans-L1, were
assigned to be of (S)-configuration at C(3) of the tetrahydro-b-
carboline frameworks, the levorotary products, (ꢀ)-cis-D1 and
(–)-trans-D1, resulting from the
D-amino acid of (R)-configuration
The best conditions to realize satisfactory chemical yields and
high enantiomeric purities was to perform the imine formation
at this respective center. The enantiomeric purities of the samples
were determined with the cis-isomers, which were base-line sepa-
rated upon HPLC on a chiral stationary phase (Nucleodex beta-PM,
Macherey–Nagel). We were not able to directly determine the
enantiomeric ratio of the trans-isomers because these isomers
did not separate sufficiently upon chiral HPLC. Since, however,
racemization was shown to occur prior to the Pictet–Spengler cycli-
zation, the enantiomeric ratio should be the same for both isomers
deriving from the same batch.
under slightly basic conditions at low temperature over
a
prolonged period of time, followed by cyclization under strongly
acidic conditions. Thus, tryptophan methyl ester was allowed to
react with pyridoxal hydrochloride at ꢀ15°C for 18 h, and subse-
quently with TFA for an additional 18 h until cyclization was
complete. This procedure delivered 79% of a mixture of cis-1 and
trans-1 in a ratio of 75:25 and enantiomeric ratios of >95:5. The
low cis/trans selectivity might appear as a drawback of this proce-
dure. However, it was in fact beneficial for our purpose since we
were interested in obtaining suitable amounts of both isomers
for biological testing.
The diastereoisomeric products cis-1 and trans-1 were sepa-
rated by column chromatography—the minor trans isomer eluting
as the first component. Unfortunately, chromatography was asso-
ciated with a considerable loss of material. The 1H and 13C NMR
spectra of the two compounds show the typical signals reported
for 1,3-disubstituted 1,2,3,4-tetrahydro-b-carbolines as well as
the expected signals for the pyridoxyl portions, proving the
connectivity of the molecules. In the NMR spectra, particularly well
observed in the 13C NMR spectra, duplication of some of the signals
was observed, indicating that the compounds are present as two
conformers. For the stereochemical assignment of the compounds,
several methods were applied. The relative configurations of
the stereogenic centers in the structures were deduced by the
13C NMR method reported by Ungemach et al.,12 by ROESY
spectroscopy, and by comparison of calculated and measured IR
spectra of the two compounds. According to the investigation of
2.2. Biological evaluation
The antiplasmodial activities of the four isomeric tetrahydro-b-
carbolines (+)-cis-L1 (er >95:5), (+)-trans-L1(er >95:5), (ꢀ)-cis-D1
(er = 88:12), and (ꢀ)-trans-D1 (er = 88:12) were investigated as de-
scribed previously for PT3.1 Thus, cultured P. falciparum was trea-
ted with the four compounds, and the growth inhibitory effects on
the parasites was determined with the [3H]-hypoxanthine incorpo-
ration assay.13 The results are shown in Figure 2. As can be recog-
nized from the graph, all four isomers of 1 show antiplasmodial
activity. Strongest inhibition with an IC50 of 8
1
l
M was observed
-tryptophan,
with (ꢀ)-trans-D1, the trans product deriving from
D
followed by (+)-trans-L1 (IC50 = 22
3 lM). The inhibitory effects
of the two cis-configured products (+)-cis-D1 and (ꢀ)-cis-L1 were
almost the same (IC50 = 108 11
l
M and 91
2 lM), but markedly
lower than those of the trans-products. Since PT3 showed an IC50
value of 14
l
M in the original study,1 it has to be assumed that
the respective sample was mainly composed of the trans-config-
ured isomer(s).
L
D
N
CO2Me
CO2Me
CO2Me
N
N
N
H
N
H
N
H
L3
4
D3
Scheme 2. Proposed racemization of tryptophan imines by imine–imine tautomerism.

R. Brokamp et al. / Bioorg. Med. Chem. 22 (2014) 1832–1837
1835
Figure 2. Effect of the enantiomerically enriched compounds of type 1 on cultured P. falciparum. Proliferation was determined after 48 h incubation by the [3H]-hypoxanthine
incorporation assay described in the Section 3. The experiment was conducted in at least three independent analyses, each in triplicate. Error bars and indicated errors in the
IC50 values represent standard deviations.
Figure 3. Overlay of the six lowest energy conformers of trans-1 and cis-1, calculated with Gaussian on the DFT level with B3LYP functional and 6-31(d,p) basis set.
The structural dependency of the inhibitory effects is rather
interesting. Evidently, the overall shape rather than a particular
absolute configuration of a stereogenic center within the com-
pounds is important for the biological activity. While in the
trans-configured compounds the carboxyl group is directed almost
perpendicular to the plane of the tetrahydro-b-carboline frame-
work, the same group is located ‘in-plane’ in the case of the
cis-configured products (Fig. 3). Possibly this characteristic is pre-
ferred for the molecules to fit into the enzymatic pocket. Surpris-
promising. Apparently, since the trans-isomers show higher inhibi-
tions than the cis-compounds, the active sites of the affected PLP
dependent enzymes of P. falciparum presumably accommodate
best molecules that have their carboxylic group located axially at
the heterocycle, standing perpendicularly to the plane of the
tetrahydro-b-carboline framework. This can be regarded as a key
structural feature that should be considered when new analogous
structures are developed with the goal to obtain new antimalarials
that show activieties similar to established drugs with IC50 values
in the nM range.
ingly (ꢀ)-trans-D1, the derivative of
D-tryptophan, is the most
active compound. Since L-ornithine is the substrate for the targeted
3. Experimental
PfODC it would be expected that a derivative of L-tryptophan
would best fit into the active site of the enzyme. But this is not
the case. This suggests that compounds of type 1 may not be
targeting the originally envisioned PfODC but a different enzyme
of the rather large family of PLP-dependent enzymes.14
3.1. General for synthesis and analysis
D
-Tryptophan methyl ester hydrochloride was prepared accord-
ing to the literature,15 and the remaining starting materials and
reagents were purchased from Sigma–Aldrich. The solvents CH2Cl2
and MeOH used for the reactions were of puriss. grade, absolute,
stored over molecular sieve and also purchased from Sigma-
Aldrich. Solvents used for column chromatography and thin layer
chromatography (TLC) were of techn. grade. The reactions were
carried out under N2 in oven-dried (100 °C) glass equipment and
monitored by TLC for completion. Thin layer chromatography
(TLC): Merck tlc plates silica gel 60 on aluminum with fluorescence
indicator F254, with the indicated solvent system; the spots were
visualized by UV light (254 and 366 nm). Column chromatography:
2.3. Conclusion
We have shown that the four isomeric products of the Pictet–
Spengler condensation of tryptophan and pyridoxal can be
prepared in enantiomerically enriched form. However, partial
racemization could not be prevented, even when rather mild
conditions were used. For the preparation of enantiomerically pure
compounds and for the specific synthesis of either the cis or the
trans isomeric forms, more optimization of the reaction conditions
would be necessary. The inhibitory effects on the proliferation of P.
falciparum are, as expected, different for the four stereoisomeric
Fluka silica gel 60 (40–63
lm), containing 0.1% Ca, with the
indicated solvent system. Mp: Büchi 510; heating rate 2 °C minꢀ1
;
compounds, and, with an IC50 value of 8 l
M for trans-D1 rather

1836
R. Brokamp et al. / Bioorg. Med. Chem. 22 (2014) 1832–1837
range 2/3 to fully molten. Specific optical rotation [
a
]D: JASCO
obtained upon evaporation of the solvents after chromatography).
+59.6 (c 0.27, DMSO). On-line UV(DAD) (MeCN/H2O/HCO2H;
P-2000 polarimeter; concentration in g per 100 ml. IR and VCD
spectra: ChiralIR-2X (Bio Tools), 1/k in cmꢀ1, s = strong, m = medium,
w = weak. 1H NMR spectra: Bruker AC-600 (600 MHz), d in ppm rel-
ative to the solvent peak (acetone, d = 2.05), coupling constant J in
Hz. 13C NMR spectra: Bruker AC-600 (150 MHz), d in ppm rel. to the
solvent (acetone, d = 206.7), multiplicities from DEPT-135 and
DEPT-90 experiments. High resolution electrospray ionization
mass spectrometry (HR-ESI-MS): Samples were analyzed with a
Waters Acquity UPLC (Waters, Milford, USA) connected to an Acquity
ek detector and a Bruker maXis QTof high-resolution mass spec-
trometer (Bruker Daltonics, Bremen, Germany). An Acquity BEH
½ ꢂ
a 2D5
in nm): kmin 243, kmax 281, kmin 286, kmax 289, kinfl 319, ksh 331.
IR: 3273m, 3057w, 2736w, 2334w, 1748s, 1388m, 1234s, 1139m,
1021s, 733s, 721s. 1H NMR (CD3COCD3, the sample appears to be
a mixture of atropisomers in a ratio of approx. 53:47; some signals
are broadened or duplicated and overlapping; signal interpretation
supported by a COSY and ROESY spectrum; numbering of the car-
bons according to the b-carboline nomenclature): 11.88/11.86 (2
br. s, ArAOH); 9.64 (br. s, CH2OH); 7.95/7.94 (2s, pyridyl H); 7.50
(d, J = 7.2, arom. C(5)H); 7.22 (d, J = 7.8, arom. C(8)H); 7.05–6.99
(symm. m with 12 lines, AB portion of ABMX system, C(6)H and
C(7)H); 6.03–6.00 (m with broadened lines, C(1)H, couples with
N(2)H and homoallylic with C(4)H2); 4.96 (br. s, N(9)H); 4.94–
4.93 (m with broadened signals, 1 H of CH2OH); 4.76–4.73 (m with
broadened signals, 1 H of CH2OH); 4.18/4.15 (2 br. s, N(2)H);
4.07–4.05 (m with broadened signals, CHCO2); 3.84 (s, CO2CH3);
3.29 (ddd, J = 15.1, 3.5, 1.4, C(4)Hcis); 3.03 (ddd, J = 15.1, 11.7, 2.6,
C(4)Htrans); 2.23 (s, pyridyl CH3). ROESY: crosspeak at 6.03–6.00
(m of C(1)H)/4.07–4.05 (m of CHCO2) indicative for the cis relation-
ship of the respective H-atoms. 13C NMR (CD3COCD3; some signals
are duplicated; numbering of the signals according to the b-carbo-
line (n) and the pyridine (n0) nomenclature): 172.9 (s, COOCH3);
153.0/152.8 (2 s, pyridyl C(50)); 148.93/148.86 (2 s, pyridyl C(40));
141.1/141.0 (2 d, pyridyl C(60)H); 137.6/137.5 (2 s, C(8a));
132.82/132.79/132.7/132.6 (4 s, 2 arom. C); 130.89/130.86 (2 s, 1
arom. C); 127.8/127.7 (2 s, 1 arom. C); 122.4 (d, C(6)H); 119.9 (d,
C(7)H); 118.8 (d, C(5)H); 112.1/112.0 (2 d, C(8)H); 108.32/108.27
(s, C(4)); 61.0/60.9 (2 t, CH2OH); 56.9/56.8 (2 d, C(3)H); 53.9/53.8
(2 d, C(1)H), 52.7 (q, CO2CH3); 26.0/25.9 (2 t, C(4)H2); 19.2 (q, pyr-
C18 HPLC column (1.7
lm, 1 ꢁ 50 mm, Waters) was used with a
mixture of H2O + 0.1% HCOOH (A) and CH3CN + 0.1% HCOOH (B)
solvent (0.1 ml flow rate, linear gradient from 5% to 98% B within
4 min followed by flushing with 98% B for 1 min). UV(DAD) spectra
were recorded between 200 and 600 nm at 1.2 nm resolution and
20 points sꢀ1. The mass spectrometer was operated in the positive
electrospray ionization mode at 4000 V capillary voltage, ꢀ500 V
endplate offset, with a N2 nebulizer pressure of 1.6 bar and dry
gas flow of 8 l minꢀ1 at 200 °C. MS acquisitions were performed
in the mass range of m/z 50–2000 at 20,000 resolution (full width
at half maximum) and 1.5 Hz spectra rate. Prior to analysis, masses
were calibrated between m/z 158 and 1450 below 2 ppm accuracy
with a 2 mm soln. of HCO2Na.
3.2. Methyl (1S,3S)- and (1R,3S)-1-(3-hydroxy-5-hydroxymethyl-
2-methylpyridin-4-yl)-1,2,3,4-tetrahydro-9H-pyrido[3,4-
b]indole-3-carboxylate (cis-L1 and trans-L1)
idyl CH3). HR-ESI-MS: calcd. for C20H22N3O4 ([M+H]+): m/z
+
Procedure with one component used as hydrochloride salt and the
other as free base.
L
-Tryptophan methyl ester (1.91 g, 8.74 mmol,
368.16048; found: 368.16078.
1.0 equiv) and pyridoxal hydrochloride (1.96 g, 9.61 mmol,
1.1 equiv) were added to CH2Cl2 (50 ml) over activated molecular
sieves (MS 3 Å, 8.5 g) at ꢀ15 °C. The mixture was stirred for 18 h
at this temperature, then TFA (2.00 ml, 26.2 mmol, 3.0 equiv) was
added. Stirring at ꢀ15 °C was continued for 1 h, then the mixture
was slowly warmed to 23 °C (over 5 h), and stirring was continued
for another 18 h. The suspension was filtered through a plug of
cotton to remove the molecular sieves, the solids were washed
with MeOH, and the solvent was evaporated in vacuo. The crude
product was passed through a short column of SiO2 (200 g, CH2Cl2/
MeOH 10:1) to afford a material consisting primarily of the two de-
sired products cis-L1 and trans-L1 in a ratio of 75:25 (yield: 2.54 g,
6.92 mmol, 79%). Column chromatography (500 g SiO2, CH2Cl2/
MeOH/NH3 100:5:1) afforded trans-L1 (114 mg, first eluting
isomer), cis-L1 (357 mg), and a mixture of the two isomers
(779 mg). This mixture was re-chromatographed (250 g SiO2) to
afford a second batch of trans-L1 (80 mg) and cis-L1 (322 mg) and
again some of the mixture of the two isomers (237 mg). Mass bal-
ance for the chromatographies: approximately 50% of the material
was lost upon chromatography. Overall yields of the purified prod-
ucts, obtained as off-white amorphous solids: trans-L1 (194 mg,
0.53 mmol, 6%), cis-L1 (679 mg, 1.85 mmol, 21%). The enantiomeric
ratio of cis-L1 (er >95:5) was determined by HPLC on a Nucleodex
Data of trans-L1 (minor isomer; er not determined but possibly
the same as for cis-L1 (>95:5)): mp: 169.0–169.9 °C (precipitate
obtained upon evaporation of the solvents after chromatography).
½
a 2D5
ꢂ
ꢀ10.6 (c 0.26, DMSO). On-line UV(DAD) (MeCN/H2O/HCO2H;
in nm): kmin 246, kmax 281, kmin 286, kmax 290, kinfl 319, ksh 329.
IR: 3305m, 2952m, 2906m, 2358w, 1731m, 1709m, 1348m,
1219s, 1129m, 1015s, 745s. 1H NMR (CD3COCD3, the sample ap-
pears to be a mixture of atropisomers in a ratio of approx. 50:50;
some signals are broadened or duplicated and overlapping; signal
interpretation supported by a COSY and ROESY spectrum; number-
ing of the carbons according to the b-carboline nomenclature)):
12.02/12.00 (2 br. s, ArAOH); 9.73 (br. s, CH2OH), 7.95 (s, pyridyl
H); 7.48 (d, J = 7.5, C(5)H); 7.19, (d, J = 7.9, C(8)H); 7.03–6.97
(well-structured m, AB portion of ABMX system, C(6)H and
C(7)H); 6.26–6.27 (m with broadened lines, C(1)H); 5.12 (br. s,
N(9)H); 4.95–4.92 (m, 5 broadened lines, 1H of CH2OH); 4.77–
4.74 (m, 6 lines, 1H of CH2OH); 4.48–4.47 (m with broadened sig-
nals, CHCO2); 4.36/4.32 (2 br. s, N(2)H); 3.66 (s, CO2CH3); 3.46
(dt, J = 15.6, 1.5, C(4)Hcis); 3.35 (ddd, J = 15.7, 6.2, 2.3, C(4)Htrans);
2.23 (s, pyridyl CH3). ROESY: missing crosspeak at 6.26–6.27 (m
of C(1)H)/4.48–4.47 (m of CHCO2) indicative for the trans relation-
ship of the respective H-atoms. 13C NMR (CD3COCD3; some signals
are duplicated; numbering of the signals according to the b-carbo-
line (n) and the pyridine (n0) nomenclature): 174.0 (s, COOCH3);
153.2/153.0 (2 s, pyridyl C(50)); 149.0/148.9 (2 s, pyridyl C(4’));
141.3/141.2 (2 d, pyridyl C(6’)H); 137.3/137.2 (2 s, C(8a)); 132.6,
131.8/131.7, 131.27/131.25 (5 s, 3 arom. C); 127.8/127.8 (2 s, 1
arom. C); 122.4 (d, C(6)H); 119.8 (d, C(7)H); 118.8 (d, C(5)H);
112.0/111.9 (2 d, C(8)H); 106.9/106.8 (2 s, C(4)); 60.9/60.8 (2 t,
CH2OH); 54.5/54.4 (2 d, C(3)H), 52.4 (q, CO2CH3); 50.3/50.2 (2 d,
C(1)H); 24.00/23.97 (2 t, C(4)H2); 19.2 (q, pyridyl CH3). HR-ESI-
beta-PM column (particle size 5
Düren, Germany) with 40% MeCN, 60% H2O (containing 2% of
Et3NHOAc) as the eluent and using a flow rate of 0.7 ml minꢀ1
l
m, 200 ꢁ 4 mm, Macherey–Nagel,
.
The areas of the signals detected at 224 and 300 nm (UV-DAD) at
Rt = 15.5 min and 18.4 min for cis-L1 and cis-D1, respectively, were
used to calculate the ratio of the two enantiomers. The enantio-
meric trans-L1 and trans-D1 were not separated (not even partially)
under the given conditions.
Data of cis-L1 (major isomer; er >95:5, no signal detected in
HPLC for cis-D1): mp: 193.7–194.3 °C (decomp., precipitate
MS: calcd for C20H22N3O4 ([M+H]+): m/z 368.16048; found:
+
368.16088.

R. Brokamp et al. / Bioorg. Med. Chem. 22 (2014) 1832–1837
1837
3.3. Methyl (1R,3R)- and (1S,3R)-1-(3-hydroxy-5-
were cultivated in 90 mm Petri dishes (Nunc, Denmark) and incu-
bated at 37 °C in the presence of 90% N2, 5% O2, and 5% CO2. The
impact of the four pyridoxyl-adducts of type 1 on the erythrocytic
stages of P. falciparum was determined by using the [3H]-hypoxan-
thine incorporation assay as described previously,13 and the IC50
values were calculated from at least three independent sigmoidal
inhibition curves as shown in Figure 2 using GraphPad Prism 4.0.
Chloroquine was used as positive control to confer optimal assay
conditions. The individual IC50 values including standard devia-
hydroxymethyl-2-methylpyridin-4-yl)-1,2,3,4-tetrahydro-9H-
pyrido[3,4-b]indole-3-carboxylate (cis-D1 and trans-D1)
Procedure with both starting materials used as hydrochloride salts
and without addition of TFA. D-Tryptophan methyl ester hydrochlo-
ride (1.15 g, 4.52 mmol, 1.0 equiv) and pyridoxal hydrochloride
(1.18 g, 5.80 mmol, 1.3 equiv) were added to CH2Cl2 (30 ml) over
activated molecular sieves (3 Å, 7.0 g) at ꢀ15 °C. The mixture was
stirred for 24 h at this temperature and, then it was slowly warmed
to 23 °C (over 5 h), and stirring was continued for another 18 h. The
suspension was filtered through a plug of cotton to remove the
molecular sieves, the solids were washed with MeOH, and the sol-
vent was evaporated in vacuo. The crude product was passed
through a short column of SiO2 (200 g, CH2Cl2/MeOH 10:1) to af-
ford a material consisting primarily of the two desired products
cis-D1 and trans-D1 in a ratio of 56:44 (yield: 537 mg, 1.46 mmol,
32%). Column chromatography (500 g SiO2, CH2Cl2/MeOH/NH3
100:5:1) afforded trans-D1 (129 mg), cis-D1 (198 mg), and a mix-
ture of the two isomers (98 mg). Re-chromatography of this mix-
ture and again of the mixture obtained therefrom resulted in
additional trans-D1 (6 + 7 mg), cis-D1 (10 + 19 mg), and a remaining
mixture of the two isomers (35 mg). Overall yields of the purified
products, obtained as off-white amorphous solids: trans-D1
(142 mg, 0.39 mmol, 9%), cis-D1 (227 mg, 0.62 mmol, 14%). The
enantiomeric ratio of cis-D1 (er = 88:12) was determined by HPLC
as described above for cis-L1.
tions (SD) for the four samples are: 91
2
l
M (cis-L1, er >95:5),
M (cis-D1,
1 l
M (trans-D1, estimated er = 88:12).
22 l
3
l
M (trans-L1, estimated er >95:5), 108 11
er = 88:12), and 8
Acknowledgement
We like to thank the analytical laboratories of the Department
of Chemistry for their measurements and Dr. Jan Helbing for his
calculations.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.01.057.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
References and notes
Data of cis-D1 (major isomer, er = 88:12): mp: 192.0–192.4 °C
(decomp., precipitate obtained upon evaporation of the solvents
1. Müller, I. B.; Wu, F.; Bergmann, B.; Knockel, J.; Walter, R. D.; Gehring, H.;
Wrenger, C. PLoS ONE 2009, 4.
2. Cox, E. D.; Cook, J. M. Chem. Rev. 1995, 95, 1797.
3. Stöckigt, J.; Antonchick, A. P.; Wu, F. R.; Waldmann, H. Angew. Chem., Int. Ed.
2011, 50, 8538.
4. Ungemach, F.; Dipierro, M.; Weber, R.; Cook, J. M. Tetrahedron Lett. 1979, 3225.
5. Ungemach, F.; Dipierro, M.; Weber, R.; Cook, J. M. J. Org. Chem. 1981, 46, 164.
6. Jawdosiuk, M.; Cook, J. M. J. Org. Chem. 1984, 49, 2699.
7. Zhang, L. H.; Cook, J. M. Heterocycles 1988, 27, 2795.
after chromatography). ½a D25
ꢂ
–53.6 (c 0.27, DMSO). UV, IR, and
NMR data identical to those of cis-L1. HR-ESI-MS: calcd. for
C
20H22N3O4 ([M+H]+): m/z 368.16048; found: 368.16120.
+
Data of trans-D1 (minor isomer; er not determined but possibly
the same as for cis-D1 (8:12)): mp: 168.60–169.9 °C (precipitate
obtained upon evaporation of the solvents after chromatography).
a 2D5
+6.0 (c 0.25, DMSO). UV, IR, and NMR data identical to those of
ꢂ
8. Zhang, L. H.; Cook, J. M. Heterocycles 1988, 27, 1357.
½
trans-L1. HR-ESI-MS: calcd for C20H22N3O4 ([M+H]+): m/z
+
9. Bailey, P. D.; Hollinshead, S. P.; Mclay, N. R.; Morgan, K.; Palmer, S. J.; Prince, S.
N.; Reynolds, C. D.; Wood, S. D. J. Chem. Soc., Perkin Trans. 1 1993, 431.
10. Bailey, P. D.; McLay, N. R. J. Chem. Soc., Perkin Trans. 1 1993, 441.
11. Bailey, P. D.; Beard, M. A.; Phillips, T. R. Tetrahedron Lett. 2009, 50, 3645.
12. Ungemach, F.; Soerens, D.; Weber, R.; Dipierro, M.; Campos, O.; Mokry, P.;
Cook, J. M.; Silverton, J. V. J. Am. Chem. Soc. 1980, 102, 6976.
13. Das Gupta, R.; Krause-Ihle, T.; Bergmann, B.; Müller, I. B.; Khomutov, A. R.;
Müller, S.; Walter, R. D.; Lüersen, K. Antimicrob. Agents Chemother. 2005, 49,
2857.
368.16048; found: 368.16092.
3.4. Inhibition assays on cultured P. falciparum
P. falciparum 3D7-strain was maintained in continuous culture
according to Trager and Jensen16 with modification.13 The parasites
were grown in human erythrocytes (O+), RPMI 1640 medium
supplemented with 25 mm HEPES, 20 mm aq NaHCO3 soln, and
0.5% AlbuMAX II (Invitrogen, Germany) at 4% hematocrit. The cells
14. Müller, I. B.; Hyde, J. E.; Wrenger, C. Trends Parasitol. 2010, 26, 35.
15. Song, Q.-H.; Tang, W.-J.; Hei, X.-M.; Wang, H.-B.; Guo, Q.-X.; Yu, S.-Q. Eur. J. Org.
Chem. 2005, 1097.
16. Trager, W.; Jensen, J. B. Science 1976, 193, 673.