Synthesis and pharmacological evaluation of fluorescent and photoactivatable analogues of antiplasmodial naphthylisoquinolines

Article abstract of DOI:10.1021/jm061464w

The naphthylisoquinoline (NIQ) alkaloids from tropical Ancistrocladaceae and Dioncophyllaceae plants show high antiplasmodial activities in vitro and in vivo, even against chloroquine-resistant strains of the malaria pathogen. For the directed optimization of these activities, an investigation of the mode of action seems most rewarding. We have therefore embarked on the identification of the respective target protein in Plasmodium falciparum. For this purpose, we have developed a flexible pathway for the synthesis of a chemically divergent series of photoactive and fluorescent derivatives of such alkaloids and succeeded in preparing the first functionalized NIQ derivatives, 10, 12, and 35, suited for fluorescence and photoaffinity labeling experiments. Pharmacological investigations ensured that the modified alkaloid derivatives retained their antiplasmodial activity. The work may pave the way for a further improvement of the activity of these natural products and will thus increase their pharmacological potential as a valuable lead structure against the widespread tropical disease malaria.

Full text of DOI:10.1021/jm061464w

6104
J. Med. Chem. 2007, 50, 6104-6115
Synthesis and Pharmacological Evaluation of Fluorescent and Photoactivatable Analogues of
Antiplasmodial Naphthylisoquinolines
Gerhard Bringmann,*,† Christian M. Gampe,† Yanina Reichert,† Torsten Bruhn,† Johan H. Faber,† Martin Mikyna,†
Matthias Reichert,† Matthias Leippe,‡ Reto Brun,∇ and Christoph Gelhaus‡
Institute of Organic Chemistry, UniVersity of Wu¨rzburg, Am Hubland, D-97074 Wu¨rzburg, Germany, Zoological Institute, UniVersity of Kiel,
Olshausenstrasse 40, D-24098 Kiel, Germany, and Swiss Tropical Institute, Socinstrasse 57, CH-4051 Basel, Switzerland
ReceiVed December 21, 2006
The naphthylisoquinoline (NIQ) alkaloids from tropical Ancistrocladaceae and Dioncophyllaceae plants show
high antiplasmodial activities in vitro and in vivo, even against chloroquine-resistant strains of the malaria
pathogen. For the directed optimization of these activities, an investigation of the mode of action seems
most rewarding. We have therefore embarked on the identification of the respective target protein in
Plasmodium falciparum. For this purpose, we have developed a flexible pathway for the synthesis of a
chemically divergent series of photoactive and fluorescent derivatives of such alkaloids and succeeded in
preparing the first functionalized NIQ derivatives, 10, 12, and 35, suited for fluorescence and photoaffinity
labeling experiments. Pharmacological investigations ensured that the modified alkaloid derivatives retained
their antiplasmodial activity. The work may pave the way for a further improvement of the activity of these
natural products and will thus increase their pharmacological potential as a valuable lead structure against
the widespread tropical disease malaria.
Introduction
Extracts of tropical lianas of the plant families Dioncophyl-
laceae and Ancistrocladaceae have been widely used in tradi-
tional medicine for the treatment of infectious diseases like
malaria.1,2 Indeed, the secondary metabolites isolated from these
plants, the naphthylisoquinoline alkaloids (Figure 1),1 have
proven to exhibit remarkable pharmacological activities.3 Thus,
dioncophylline C (1) and dioncopeltine A (2) display excellent
antiplasmodial activities, both in vitro (against P. falciparum,
P. berghei)4,5 and in vivo (P. berghei, mouse model).6 Other
representatives of this class of alkaloids show good antitrypa-
nosomal or antileishmanial activities.7 Also from a stereochem-
ical and biochemical point of view, these alkaloids are unique:
Thus, the rotationally hindered biaryl axis between the two
molecular portions (i.e., the naphthalene and the isoquinoline
part), which, in connection with the stereogenic centers at C-1
and C-3, leads to rotational isomers, has triggered the develop-
ment of efficient new methods for the atropo-selective synthesis
of axially chiral compounds.8 Furthermore, the unusual molec-
ular framework of these secondary metabolites originates from
an unprecedented biosynthetic origin of isoquinoline alkaloids
from acetate units, both for the naphthalene and the isoquinoline
portion.9
Some first hints at a possible mode of action of the
antiplasmodial naphthylisoquinoline alkaloids were already
obtained from NMR, IR, and Raman investigations on drug-
heme interactions: Similar to the antimalarial drug chloro-
quine,10 these biaryls form, albeit weaker, complexes with
ferriprotoporphyrin,11 thus possibly affecting the hemozoin
precipitation, the essential heme detoxification process in
intraerythrocytic stages of P. falciparum.12 The fact that the
naphthylisoquinolines likewise show antiplasmodial activity
Figure 1. Structures of antiplasmodial naphthylisoquinoline alkaloids.
against chloroquine-resistant strains of P. falciparum4 suggested
that either the molecular mechanism of chloroquine-resistance
in P. falciparum is circumvented by these compounds or that
NIQsa have a different mode of action, possibly by selective
inhibition of one of the essential proteins of the pathogen.
Therefore, these secondary metabolites are promising candidates
for the development of new antimalarial drugs.
In this paper, we describe the synthesis of fluorescence-
labeled antiplasmodial naphthylisoquinoline derivatives and the
first investigations aiming at their localization in parasite-
infected cells. For this purpose, a synthetic route to photoacti-
vatable NIQ analogues for photoaffinity studies is described to
identify possible molecular targets of NIQs within the parasite.
Results and Discussion
Basic Considerations. For the localization of NIQs in target
cells, dioncophylline A (3) was chosen as a first representative
of this class of compounds to elaborate the best conditions for
a Abbreviations: NIQ, naphthylisoquinoline; IC50, half-maximal inhibi-
tory concentration; QSAR, quantitative structure-activity relationship;
DMF, dimethylformamide; THF, tetrahydrofuran; LC-MS, liquid chroma-
tography coupled to mass spectrometry; NBS, N-bromosuccinimide; AIBN,
azobisisobutyronitrile; TMSCl, trimethylsilyl chloride; ESI-MS, electron-
spray ionization mass spectrometry; HPLC, high-performance liquid chro-
matography; NBO, natural bond order; HF, Hartree-Fock; HRMS, high-
resolution mass spectrometry; TFA, trifluoroacetic acid; CD, circular
dichroism.
* To whom correspondence should be addressed. Tel.: +49-
931-8885323.
Fax:
+49-931-8884755.
E-mail:
bringman@
chemie.uni-wuerzburg.de.
† University of Wu¨rzburg.
‡ University of Kiel.
∇ Swiss Tropical Institute.
10.1021/jm061464w CCC: $37.00 © 2007 American Chemical Society
Published on Web 11/09/2007

Naphthylisoquinolines for Photoaffinity Labeling
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6105
Scheme 1. Synthesis of the Fluorescent Dioncophylline A
Derivative 10a
Figure 2. Fluorescent derivative A of dioncophylline A (3).
the attachment of a fluorescent probe. Although not reaching
the excellent antiplasmodial activities of dioncophylline C (1)
and dioncopeltine A (2), dioncophylline A (3) still shows an
acceptable activity against P. falciparum (IC50 ) 0.14 µg/mL)4
and is, in particular, easily accessible by isolation from
Triphyophyllum peltatum13 or by total synthesis.14 Furthermore,
the structural requirements of this compound for bioactivity are
particularly well-known from extensive QSAR studies.15,16 Thus,
while the OH function and in particular the NH group in the
isoquinoline part of 3 are crucial for the antiplasmodial activity,16
the 5-position does not belong to the pharmacophore of the
molecule, so that a functionalization at this C-atom should be
possible without substantial loss of activity. For this reason,
dioncophylline A (3) was connected in this particular position,
via a flexible alkyl linker to prevent steric interference, at first
to a fluorophore and then, in addition, to a photolabile group.
For the in vitro and in vivo identification of the compound
(e.g., after photoaffinity experiments), the dansyl residue was
chosen as an easily available and robust fluorophore. In contrast
to other chromophores that emit blue light, it exhibits a green
luminescence, which is detectable in a cellular environment.
Synthesis of a Fluorescent Derivative of Dioncophylline
A (3). In a first, simplified approach, only the dansyl fluorophore
was planned to be attached to 3 via C-5, using a hexamethyl-
enediamine linker as in A (Figure 2). For this purpose,
dioncophylline A (3) was brominated and O- and N-protected
according to a known procedure.17 The resulting N,8-O-
dibenzyl-5-bromodioncophylline A (5) was lithiated and then
formylated with DMF to give the aldehyde 6 in good yields
(Scheme 1). This slight detour led to much better results than
a direct Vilsmeyer formylation of dioncophylline A (3). The
precursor 9 to the dansyl linker was prepared from 1,6-
hexanediamine (8), by treatment with dansyl chloride (7) in the
presence of Et3N.18 Besides the desired monoamine 9, the
respective bisdansylated compound was obtained as a byproduct
(ratio 1:5), which was separable by column chromatography.
The dansyl derivative 9 was attached to the protected 5-formyl-
dioncophylline A 6 by reductive amination,19 yielding the still
O- and N-benzylated fluorescent analogue 10 of dioncophylline
A as the major product (Scheme 1) and traces of a byproduct,
presumably an overreacted tertiary amine, as deduced from LC-
MS data. The last step of the synthesis toward a fluorescent
analogue 12 of dioncophylline A (3) was the cleavage of the
two benzyl groups of 10 by hydrogenation. However, under all
conditions applied, no debenzylation products were isolated in
significant yields. To overcome this difficulty, the reaction
a Reagents and conditions: (a) 1: n-BuLi, THF, -78 °C, 110 min; 2:
DMF, 45 min, 83%; (b) Et3N, CH2Cl2, rt, 3 h, 55%; (c) NaBH(OAc)3,
CH2Cl2, rt, 24 h, 68%.
sequence was changed (Scheme 2). Accordingly, N,8-O-
dibenzyl-5-formyldioncophylline A (6) was first debenzylated
to give compound 11, which was successfully coupled with the
amine 9 to yield the fluorescent derivative 12 of dioncophylline
A (3).
Before submitting 12 to biological testings, its fluorescence
emission spectrum, along with that of free dioncophylline A
(3), was recorded, to exclude quenching effects. From the mea-
sured spectra (Figure 3), it becomes obvious that the respective
emission maxima, 524 nm in the case of 12 and 355 nm for 3,
are clearly separated, thus avoiding mutual interferences.
Biological Investigations with Fluorescent Derivatives of
Dioncophylline A. Given the good physical properties of the
fluorescent derivates 10 and 12 of dioncophylline A (3), their
bioactivities against several parasites were tested (Table 1)
before using these compounds as valuable alternatives to 3 for
investigation of the cellular localization of dioncophyllines, e.g.,
in parasite-infected red blood cells, in order to minimize the
risk of having a loss of activity or a change of the mode of
action due to the synthetic modifications.

6106 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Bringmann et al.
Scheme 2. Synthesis of the Unprotected Fluorescent
Dioncophylline A Derivative 12a
Figure 4. Microscopic visualization of 10 in Plasmodium-infected red
blood cells. Only the infected cell is labeled (A: transmission mode;
B: fluorescence mode).
conclusion, 10 significantly differentiates between the parasite
and the erythrocyte (Figure 4).
A possible explanation of this phenomenon is either that the
plasma membrane of infected erythrocytes is not permeable for
10 or that the substance can freely diffuse but its concentration
in the host cell may be below the detection limit. By contrast,
the accumulation of 10 by the intraerythrocytic parasite leads
to a visible fluorescence. It remains to be solved whether
accumulation of 10 in the parasite cell takes place by an active
transport or by diffusion. Possibly 10 is trapped in the parasite
by an affinity-driven complex formation with an as yet unknown
target molecule.
a Reagents and conditions: (a) Pd/C, H2, THF, 9 h, 73%; (b) 9,
NaBH(OAc)3, CH2Cl2, rt, 24 h, 48%.
As seen in Figure 4B, the parasite is labeled distinctly within
its confines to the host cell cytosol. Although the fluorescence
appears not to be distributed uniformly inside the parasite, the
localization of 10 cannot be assigned to a single subcellular
compartment, like, e.g., to the digestive vacuole. The hemozoin
appears as dark dots, which may indicate that it does not
agglomerate 10 (Figure 4B) or that the fluorescence is quenched
by hemozoin. Our findings hint at a different behavior of 10 in
comparison to chloroquine, which accumulates in the food
vacuole, suggesting that 10 and chloroquine have different
modes of action. Consequently, the exact cellular localization
and molecular target of 10 is restricted to the parasite cell but
remains to be further investigated. Therefore, additional experi-
ments are required and are presently in progress, e.g., time-
dependent effects of 10 against different intraerythrocytic stages
of P. falciparum.
Figure 3. Fluorescence emission spectra of 3 (blue; excitation at 305
nm) and of 12 (red; excitation at 310 nm), together with a fluorescing
sample of 12 (right upper corner).
Table 1. Bioactivity Results for 10 and 12a
P. falciparum
cytotoxicityb
The deprotected labeled compound 12 was also used for
incubation experiments in P. falciparum blood stages, leading
to the same specific fluorescent labeling of infected erythrocytes
as detected for the precursor 10 (for the microscopic visualiza-
tion of 12, see Supporting Information).
standard
3
10
12
0.055c
0.381
0.045
0.602
0.010d
29.1
2.50
16.5
a All values in µM. b Against L-6 cells (rat skeletal myoblast cells).
Synthesis of Photoactive and Fluorescent Derivatives of
Dioncophylline A for Photoaffinity Labeling. In a second,
more comprehensive approach, dioncophylline A (3) was
planned to be equipped with a fluorescent and, in addition, with
a photoaffinity probe. For the assembly of such multifunctional
molecules, which can be used in protein purification, several
different strategies are known.21 In our case a three-functional
core unit was planned to serve as the central module to connect
the required building blocks, for which the hydroxylated amino
acids L-serine (13) and L-tyrosine (14) were chosen (see Figure
5). These were planned to be linked to the dansyl residue as
the fluorescence label via the amino group. To the free hydroxy
function of the amino acid, by contrast, we intended to fix three
different types of photoactive groups. With a variety of
photoactive substances at hand, we wanted to check for an
incorporation of the respective compounds into the cell and
cross-linking to the target protein. The use of different photo-
active chromophores, in this case a benzophenone, a trifluo-
romethyldiazirine, and a tetrafluorophenylazide (to generate
radicals, carbenes, and nitrenes, respectively), is a common
c Chloroquine. d Podophyllotoxin.
As seen from Table 1, the benzylated precursor 10 possesses
almost the same antiplasmodial activity as the standard chlo-
roquine, thus being even more active than dioncophylline A
(3) itself, simultaneously exhibiting a lower toxicity, while the
deprotected target compound, 12, was less active against P.
falciparum. Therefore and because of its initially better synthetic
availability, 10 was chosen as a first fluorescent probe to perform
in vitro labeling of P. falciparum blood stages.
For fluorescence-microscopic examinations, P. falciparum
trophozoites were incubated with 10 (0.5 µM) for 4 h under
normal culture conditions. As a result, substance 10 obviously
affected early blood stages of Plasmodium. Moreover, a
fluorescent labeling and accumulation of 10 was detected in
infected erythrocytes, whereas noninfected erythrocytes were
not labeled20 (Figure 4). The fluorescence was found to be
clearly restricted to the parasite in the host cell. Like noninfected
erythrocytes, the host cell cytoplasm was totally unlabeled. In

Naphthylisoquinolines for Photoaffinity Labeling
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6107
Figure 5. Schematic overview of possible photoactive and fluorescent derivatives of dioncophylline A (3); the numbers indicate the order of
synthetically connecting the different modules.
Scheme 3. Synthesis of the Photosensitive Benzophenone 16,
the Arylazide 19, and the Diazirine 23a
Scheme 4. Synthesis of a Dioncophylline A Derivative 25
Equipped with a C3 Linker for Attachment of Analytical Probesa
a Reagents and conditions: (a) Ag2SO4, I2, CH2Cl2, rt, 82%; (b) BnBr,
Cs2CO3, acetone, reflux, 10 h, 64%; (c) Pd(OAc)2, Cs2CO3, n-Bu4NBr, PPh3,
H2O, MeCN, reflux, 1.5 h, 82%; (d) LiAlH4, THF, -78 °C, 63%; (e) Pd/
C, H2, MeOH, 5 h, 65%.
aldehyde, which was reduced under mild conditions with Me2-
NH·BH3 in acetic acid to give the alcohol 18. In contrast to the
known procedures,23 the final hydroxyl-bromine exchange was
carried out by the use of the improved reagent combination27
PPh3/(CCl2Br)2 in CH2Cl2, which increased the over-all-yield
of the synthesis.28
The synthesis of 23 started with the commercially available
ketone 20, whose oxime (as an E/Z-mixture) was tosylated in
pyridine and then treated with liquid NH3 for 8 h to provide
the diaziridine 21. For the subsequent last two steps in the
synthesis of 23, the oxidation of 21 to the diazirine 22 and its
side-chain halogenation, the introduction of a one-pot procedure
resulted in much higher yields compared to the literature
protocols:23 Oxidation of 21 was carried out with MnO2 in CCl4
followed by the filtration of the heterogeneous oxidation reagent
and immediate radical bromination with NBS/AIBN right from
the crude filtrate. With the oxidation yielding the desired
diazirine 22 as the only product, no purification was necessary
at this level. This is a significant improvement since it avoids
the material-consuming and thus yield-diminishing workup of
the sensitive, and highly volatile, intermediate 22.
a Reagents and conditions: (a) NBS, CCl4, AIBN, reflux, 3 h, 89%; (b)
NaN3, acetone/H2O, reflux, 6 h, 64%; (c) HNMe2‚BH3, AcOH, rt, 1.5 h;
(d) (CCl2Br)2, PPh3, CH2Cl2, 0 °C, 1 h, 68%; (e) H2NOH‚HCl, MS 3 Å,
pyridine, 80 °C, 2 h, 89%; (f) TosCl, pyridine, reflux, 3 h, 98%; (g)
NH3(liq.), Et2O, -35 °C, 8 h, 60%; (h) MnO2, CCl4, rt, 30 min, quant.; (i)
NBS, CCl4, AIBN, reflux, 1 h, 56%.
procedure to meet the a priori unpredictable situation in the
particular biological system.21-23 And, finally, the amino acid
module as the central building block should connect the
fluorophore and the photoactive moiety to the bioactive naph-
thylisoquinoline portion.
The first of the photoactive precursors, 4-(bromomethyl)-
benzophenone (16),24 was readily available by radical bromi-
nation of the cheap 4-methylbenzophenone (15) (Scheme 3).
For the synthesis of the aryl azide 19 and the diazirine 23,
several routes had been reported,23,25,26 starting with the aldehyde
17 and the ketone 20, respectively (Scheme 3). By optimization
of the reaction conditions, better yields were achieved in both
cases (vide infra). A nucleophilic substitution reaction of 17
with NaN3 in acetone/water provided the desired para-substituted
Both the aryl azide 19 and the diaziridine 23 proved to be
highly photoactive by UV spectroscopy. Irradiation at 254 and
356 nm led to rapid decomposition of the starting material.29
For the functionalization of 3 according to the concept
presented in Figure 5, the alkaloid was halogenated at C-5
(Scheme 4). This was readily achieved by the use of Ag2SO4

6108 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Bringmann et al.
Scheme 5. Preparation of the Two-Fold Functionalized Serine
Building Block 29 and Independent Synthesis of the Unexpected
Side Product 30a
Scheme 6. Synthesis of the Building Block 34 with a Central
Tyrosine Unita
a Reagents and conditions: (a) TMSCl, MeOH, rt, 14 h, 70%; (b) dansyl
chloride, pyridine, rt, 24 h, 85%; (c) 16, Cs2CO3, NaI, acetone, reflux, 9 h,
45% of 28; (d) NaOH, THF/H2O, reflux, 76%; (e) Na2SO3, H2O, rt, 3 h,
67%; (f) 16, NaOH, EtOH, 80 °C, 12 h, 50%.
and I2, which produced exclusively 5-iododioncophylline A.
After standard O- and N-benzyl protection in acetone/Cs2CO3,
an optimized Heck reaction with methyl acrylate gave the
desired cinnamate ester 24. Its reduction was achieved with
LiAlH4. While no incomplete reduction to the corresponding
aldehyde occurred, some over-reduction of the enone system
took place, leading to an inseparable mixture. This, however,
did not need to be resolved, since the final hydrogenation of
the exocyclic double bond with simultaneous cleavage of the
N- and O-protecting groups gave the intermediate 25 with the
fully saturated side chain, anyhow.
While the alkaloid and the photoactive groups were now
available, the preparation of the desired N-dansyl-L-serine and
-L-tyrosine-based amino acid linkers turned out to be challeng-
ing. L-Serine (13) itself could not be dansylated in any solvent/
base system, because of its high hydrophilicity and the resulting
low solubility in organic solvents. The corresponding methyl
ester 26, by contrast, prepared in MeOH/TMSCl, was readily
N-functionalized (Scheme 5), the best results being obtained
by using dansyl chloride in pyridine.30 The following reactions
involving photoactive groups were performed, exemplarily, with
the benzophenone derivative 16 as the most stable of these
groups, because their now-required attachment to the hydroxy
function imposed some unforeseen difficulties. Thus, the
scheduled substitution reaction of the benzophenone derivative
16 did not take place in THF with KH as the base, while alkali
metal carbonates, in particular Cs2CO3 in acetone, produced the
N-alkylated serine derivative 28, because of the higher acidity
of the sulfonamide proton as compared to that of the aliphatic
hydroxy group. Besides the unexpected, but in principle usable,
N,N-disubstituted serine derivative 28, the formation of the
4-(dansylmethyl)benzophenone (30) as a side product was
observed in varying quantities, depending on the equivalents
of base used and on the reaction time.31 For a further confirma-
tion of the postulated structure of this undesired product, the
a Reagents and conditions: (a) dansyl chloride, Et2O, NaOH, rt, 20 h,
69%; (b) NaOH, MeOH, reflux, 2 h, 93%; (c) NaH, BF3‚Et2O, THF, reflux,
2 h; (d) 16, Cs2CO3, acetone, reflux, 9 h; then: H2O, reflux, 30 min, 63%.
compound was prepared in a directed two-step synthesis. Thus,
reduction of dansyl chloride (7) to give the corresponding
sulfinic acid and substitution reaction with 4-bromomethylben-
zophenone (16) provided the same compound 30, identical in
all chromatographic and spectral (NMR and ESI-MS) data to
those of the undesired side product.
To avoid the decreased yields resulting from the formation
of this side product and from the following ester hydrolysis of
28, the phenolic, and thus more OH-acidic, amino acid L-
tyrosine (14) instead of L-serine (13) was chosen as the central
module. Although the more lipophilic amino acid 14 even
underwent double dansylation in the two-phase system Et2O/
H2O/NaOH (Scheme 6), the resulting N,O-didansyl-L-tyrosine
(31) was easily cleaved back to the desired mono-N-dansyl
derivative 32 in refluxing NaOH/MeOH, by selective hydrolysis
of the sulfonate ester.
Again the following steps were, exemplarily, optimized for
the benzophenone derivative 16 as the most stable of the
available photoactive groups, so that it should later be possible
to adopt these optimum reaction conditions to the subsequent
synthesis of the more sensitive diazirine and arylazide deriva-
tives. According to LC-MS and NMR, the ‘classic’ conditions
for the O-alkylation of phenolic hydroxy functions with alkali
carbonates in acetone resulted in a complex reaction mixture
of N-,O-, and N,O-alkylated tyrosine derivatives, which was
hard to resolve by column chromatography. For the selective
O-alkylation, an in situ protection-deprotection strategy was

Naphthylisoquinolines for Photoaffinity Labeling
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6109
applied, as previously developed for serine and threonine.32
Thus, reaction of the deprotonated amino acid with BF3 led to
the formation of the oxazaborolidinone 33, which was charac-
terized by isolation and NMR spectroscopy, but could, after
removal of the NaBF4 by filtration, also be immediately
subjected to the selective O-alkylation, right in the crude filtrate.
Eventual addition of an excess of water after completion of the
reaction according to TLC effected the in situ hydrolysis of the
intermediate oxazaborolidinone heterocycle and thus set free
the desired O-alkylated tyrosine 34 as the only regioisomer,
hence completing the efficient one-pot synthesis.
Scheme 7. Eventual Linkage of the Two Portions 34 and 25 by
the Use of Phosgenea
With the photoactive and fluorescent amino acid derivatives
29 and 34 in hand, we focused on their subsequent attachment
to the alkaloid derivative 25 via an ester bridge. Despite the
expectation of regioisomers due to the presence of three possible
reaction sites, viz. the aliphatic and the phenolic OH groups
and the secondary amine, and the hence possible formation of
different products, we tried the esterification reaction directly
on 34. None of the classical reagents used, carbonyldiimidazole,
dicyclohexylcarbodiimide, SOCl2, or (COCl)2, however, led to
any conversion of the starting material, leaving open whether
the activated carboxylic group was too inactive to be attacked
by the nucleophile or whether even the amino acid was not
activated at all, maybe for sterical reasons. Indeed, no reaction
of the acid to any kind of activated species was observed, neither
by TLC nor by HPLC. Under Mitsunobu conditions,33 i.e., with
specific activation of the aliphatic hydroxy group to react, again
no formation of the desired ester occurred. In view of the
assumption that the reaction might have failed because the amino
acid had not been activated, we finally used the strong, but toxic
and more difficult to handle, activation reagent phosgene, which
now, for the first time, did succeed in converting the amino
acid into a reactive species as observed by TLC (more rapidly
eluting spot), eventually leading to the isolation of a product in
which both, the amino acid and the alkaloid, were present
according to MS and NMR, albeit only in a poor yield (Scheme
7).
a Reagents and conditions: (a) 1: phosgene (20% solution in toluene),
pyridine, reflux, 1.5 h; 2: 25, THF, rt, 12 h, 14%.
A more in-depth NMR and MS analysis, however, revealed
that the compound isolated was not the desired ester, but the
carbamate 35. The formation of this unexpected product, which,
expectedly, had retained the phosgene-derived CO2, may be
explained by the structure and reactivity of the activated
carboxylic acid. It is most probable that the reaction of the acid
34 with phosgene yields the respective oxazolidindione 36
(Figure 6), which apparently does not react in the anticipated
way. Thus, instead of the initially expected attack on the lactone
carbonyl function (C-5), to provide a carbaminic acid, which
would then decarboxylate spontaneously to give the desired
ester, the ‘open-chain’ carbamate ester 35 is formed with the
observed retention of the CO2 portion. The actually occurring
attack of the O-nucleophile on the carbamate-CO group (C-2)
rather than on the lactone-CO group (C-5) might be due to the
electron-withdrawing properties of the dansyl moiety and the
sterical properties of the heterocycle, making quantum chemical
calculations on the structure and reactivity of the presumed
intermediate 36 rewarding.
Figure 6. Structure of the assumed intermediate activated amino acid
derivative 36. The substituent below the ring plane rotates easily,
whereas the dansyl moiety has a fixed position due to hydrogen
bondings (dashed lines). The arrows show the possible (full black arrow)
and impossible (disrupted blue arrow) approach vectors of the nucleo-
phile from above the ring plane; the benzophenone-4-methylene portion
is abbreviated as R′ (left) or CH3 (right).
An NBO population analysis of an HF/6-31G(d)34 optimized
structure of 36 revealed a small -I effect of the dansyl moiety
with respect to the carbamate carbonyl. This effect is, however,
not significant and results in a slightly more polarized carbamate
CO double bond in comparison to the unsubstituted analogue.
The calculations also show that the lone pair of the ring nitrogen
interacts with the dansyl moiety; thus the +M effect to the ring
observed in an unsubstituted anhydride is significantly lowered
and the carbamate carbonyl should be more reactive. The
interaction of the ring nitrogen lone pair with the dansyl moiety
also affects the structure: one of the two diastereotopic S-oxygen
atoms, viz. the upper one, is coplanar with the heterocyclic ring.
Therefore and because of hydrogen bondings between the
proximal aromatic protons and the SO2 oxygens, the dansyl
moiety has a fixed position above the plane of the cyclic
carbamate (Figure 6). HF/6-31G(d) calculations of 36 showed

6110 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Bringmann et al.
Table 2. IC50 Bioactivity Results for Dioncophylline A (3) and Its
Derivatives 24, 25, and 35a
derivative should be rewarding and are presently being per-
formed in our laboratories.
P. falciparum
cytotoxicityb
From the fluorescence distribution one might assume the
cysteine protease falcipain-2 to be a possible target. This protein
is one of the principal cysteine proteases in the digestive vacuole
but has also been found in other cell compartments.35 Moreover
falcipain-2 is known to be inhibited by some isoquinoline
derivatives, making it a possible target of the naphthylisoquino-
lines.36 However, the genuine alkaloid 3 did not inhibit re-
combinantly expressed plasmodial falcipain-2 expressed plas-
modial falcipain-2 (data now shown). Consequently, dionco-
phylline A (3) and presumably its derivatives may act on other
plasmodial target molecules of the parasite.
standard
3
24
25
35
0.12c
0.38
0.19
2.42
0.96e
0.007d
29.1
>140
99.0
69.5
a All values in µM. b Against L-6 cells (rat skeletal myoblast cells).
c Chloroquine. d Podophyllotoxin. e In the case of 35 the IC50 value of the
standard was 0.309.
Conclusions. In the search for the target protein(s) of the
highly antiplasmodial naphthylisoquinolines,3 the two first
approaches are described in this paper. The dansyl-functionalized
dioncophylline A derivatives 10 and 12 were shown to be good
candidates for being capable of visualizing the in vitro distribu-
tion of these alkaloids. The remarkable ability of these naph-
thylisoquinoline derivatives to accumulate exclusively in the
pathogen inside infected erythrocytes raised the question if a
transport mechanism exists for these biaryl alkaloids or whether
they exhibit a high affinity to the pathogens proteins. The new
dioncophylline A analogues like 12 constitute a promising basic
structure to develop malaria-specific diagnostics, e.g., for the
sensitive, fluorescence-based detection of parasites in blood
samples of a clinical patient, even with a low degree of
parasitemia. Toward the identification of putative target mol-
ecules of naphthylisoquinolines, a first route to photoactive
fluorescent derivatives of dioncophylline A was developed.
Tyrosine turned out to be a possible central building block to
connect the dansyl and benzophenone moieties to a dionco-
phylline A derivative. Pharmacological investigations and
microscopic studies of the distribution of the photoaffinity
complex 35 indicated that this alkaloid derivative should be
suitable for first exploratory photoaffinity studies. These experi-
ments are presently in progress. After the first access to
photoactivatable antiplasmodial naphthylisoquinolines reported
here, related, further optimized compounds will soon follow,
thus establishing a variety of instruments required for the
identification of the target protein for antiplasmodial naphth-
ylisoquinolines.
Figure 7. Microscopic visualization of 35 in Plasmodium-infected red
blood cells. Only the infected cell is labeled (A: transmission mode;
B: fluorescence mode).
that all local minima found are within 4-20 kJ/mol above the
energetically lowest structure found, and in all these conformers
the dansyl moiety has this fixed position, whereas the large
substituent of the amino acid rotates easily (rotational barrier:
29 kJ/mol), thus shielding the bottom face of the heterocycle
toward an approach of the electrophile from below. On the upper
face, the dansyl moiety blocks the approach vector of the
nucleophilic attack at the lactone carbonyl, leaving only the
trajectory to the carbamate CO double bond unhindered.
Considering these electronic and steric effects the observed
reaction to the product 35 becomes understandable.
Still, despite the poor yield in the last step and the unexpected
structure, compound 35 does fulfill the desired properties of a
molecule that joins together the active alkaloid to a fluorescence
and a photoaffinity probe via an amino acid core. Thus, we
decided to first exemplarily test 35 for its applicability to
photoaffinity labeling before preparing the corresponding di-
azirine and azide substances.
Antiplasmodial Activities and Cellular Biodistribution of
the Fluorescence and Photoaffinity-Labeled Derivative 35
of Dioncophylline A. As for the fluorescent derivatives 10 and
12, the synthetic key precursors 24 and 25 and the final
photoactivatable complex 35 were tested for their antiplasmodial
and cytotoxic activities before investigation of the cellular
distribution of 35 in parasite-infected erythrocytes. The methyl
acrylate derivative 24 of dioncophylline A (3) did show
relatively low IC50 values (Table 2), while attachment of an
n-propanol moiety in the 5-position of 3, as in 25, decreased
the activity against P. falciparum blood stages, thus also
providing valuable data for our ongoing QSAR investiga-
tions.15,16 Most importantly, the designated photoaffinitiy tool
35 itself did not only reveal antiplasmodial activity (IC50 ) 0.96
µM), which is only slightly weaker than that of genuine
dioncophylline A (3), but also entered the parasite cell in the
microscopic studies (Figure 7), showing that it is indeed a
promising first model compound for performing photoaffinity
labeling assays.
Experimental Section
General Information. All used solvents were distilled before
use. THF was predried over CaH2 and freshly distilled from
potassium. Commercially available material was used without
further purification. Reactions with water- or oxygen-sensitive
compounds were carried out in predried glassware under argon.
The reaction vessels in which phosgene solution was handled were
equipped with a washing bottle filled with aqueous NaOH solution
to neutralize gaseous phosgene. Reactions involving aryl azides and
diazirines were carried out in brown flasks preventing the solutions
from direct exposure to light.
Thin-layer chromatography was carried out using silica gel 60
F254 or C-18 F254s aluminum foil. Detection of the compounds was
achieved by fluorescence quenching at 254 nm, fluorescence at 356
nm, or staining with ninhydrin or KMnO4 solutions. Flash chro-
matography was performed using silica gel (20-63 mesh), which
was deactivated with 7.5% NH3 solution (25%) if needed. NMR
spectra were obtained on a Bruker DMX 600, Avance 400, Avance
250, or Avance 200 apparatus and are reported in ppm relative to
internal solvent signal, with coupling constants (J) in Hertz (Hz).
For 19F spectroscopy CFCl3 was used as the internal standard. EI
mass spectrometry was carried out on a Finnigan MAT 8200; ESI-
HRMS was measured on a Bruker Daltonik micrOTOF-focus.
Significantly, its distribution in a culture of P. falciparum
blood stages was the same as observed for the exclusively
dansyl-labeled dioncophylline A derivatives, 10 and 12 (see
above). For this reason, photoaffinity labeling experiments with
this first photoactivatable and fluorescent naphthylisoquinoline

Naphthylisoquinolines for Photoaffinity Labeling
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6111
Analytical HPLC was performed on a Jasco System (DG-1580,
LG-1580, PU-1580, CO-1560, AS-1555, MD-1510) using a Chro-
molith Performance RP-18e (100 × 4.6 mm) with a MeCN (A)/
H2O(B) solvent mixture stabilized with 0.05% TFA: 0.5 min 10%
A, 3 mL/min; 5 min 50% A, 5 mL/min; 9 min 100% A, 5 mL/
min; 13 min 100% A, 6 mL/min; 15 min 10% A, 6 mL/min.
Preparative HPLC was carried out on a Jasco System (PU-2087
Plus, MD-2010 Plus) with a Waters 600 Controller an a Waters
996 Photodiode Array Detector. As a column a Chromolith RP-
18e (100 × 10 mm) was used with a MeCN (A)/H2O(B) solvent
mixture stabilized with 0.05% TFA: 0.5 min 10% A, 12 mL/min;
5 min 50% A, 12 mL/min; 9 min 100% A, 12 mL/min; 13 min
100% A, 20 mL/min; 15 min 10% A, 20 mL/min.
and Ph), 7.44-7.54 (m, 2H, 7′′′ and 3′′′ aromatic H), 8.13 (d, J )
7.3 Hz, 1H, 4′′′ aromatic H), 8.33 (d, J ) 8.7 Hz, 1H, 8′′′ aromatic
H), 8.49 (d, J ) 8.6 Hz, 1H, 2′′′ aromatic H); 13C NMR (62.5
MHz, methanol-d4) δ 19.6 (1-Me), 20.2 (3-Me), 20.9 (2′-Me), 23.7
(4-C), 26.9 (1-C), 27.2 (3-C), 28.1, 30.3, 30.4, 30.5, 30.6, 30.8 (2×),
33.1, 36.5, 43.6 (5-CH2), 45.8 (NMe2), 47.1, 51.3, 52.4 (4′-OMe),
56.9 (NCH2Ph), 57.0 (5′-OMe), 75.7 (OCH2Ph), 106.8, 110.3,
116.4, 117.7, 119.7, 120.6, 124.3, 127.7, 127.8, 128.2, 128.7, 128.8,
129.0 (2×), 129.5, 130.1 (2×), 130.9, 131.0, 131.2, 132.9, 134.6,
135.5, 137.2, 137.9, 138.0, 140.8, 153.2, 158.7, 159.3 (4′-C), 170.8
(5-C); IR (KBr) 3453, 3293, 3060, 3040, 2930, 2853, 1593, 1455,
1393, 1323, 1263, 1155, 1145, 1130, 1070, 793 cm-1; CD
(MeOH): ∆ꢀ218 -10.6, ∆ꢀ238 +6.4, ∆ꢀ262 -2.5, ∆ꢀ342 +1.4, ∆ꢀ362
-1.7; MS (EI): m/z (%): 919 [M]+ (6), 904 [M - CH3]+ (83),
169 [C12H11N]+ (83), 91 [C7H7]+ (100); MS (ESI positive): m/z
(%): 920 [M + H]+; HRMS (ESI): C57H66N4O5S requires for M
+ H at m/z 919.48267; found: 919.48271.
N,8-O-Dibenzyl-5-formyldioncophylline A (6). To a solution
of 517 (100 mg, 0.157 mmol) in absolute THF (10 mL) at -78 °C
was added a solution of n-butyllithium (0.25 mL of 1.6 M in hexane,
0.393 mmol) dropwise. The reaction mixture was stirred at
-78 °C under argon for 110 min. In parallel, a DMF solution (90
µL, 1.17 mmol) in THF (0.5 mL) was cooled down to -78 °C and
was then quickly added into the reaction mixture. The resulting
mixture was stirred at -78 °C for 45 min. Ethanol (0.55 mL) was
added followed by saturated aqueous NH4Cl solution (1 mL). The
suspension was warmed to room temperature and extracted with
diethyl ether. The ether phases were combined and dried over
MgSO4. After column chromatography on deactivated (7.5% NH3)
silica gel (petroleum ether-EtOAc, 8:2 elution), 6 was obtained
as a yellow solid (76 mg, 0.13 mmol, 83%): mp 83-85 °C
5-Formyldioncophylline A (11). N,8-O-Dibenzyl-5-formyldi-
oncophylline A (6) (3 mg, 0.005 mmol) was hydrogenated in dry
THF (1.6 mL) in the presence of Pd/C (10%, 0.8 mg) at room
temperature and 3 bar H2 for 9 h. After filtration and evaporation
of the solvent, the residue was purified by preparative TLC (CH2-
Cl2-MeOH, 95:5 elution) to give 11 (1.5 mg, 0.0037 mmol, 73%)
1
as a white solid: mp 110-112 °C (CH2Cl2-MeOH); H NMR
(400 MHz, methanol-d4) δ 1.57 (d, J ) 6.2 Hz, 3H, 3-CH3), 1.72
(d, J ) 6.8 Hz, 3H, 1-CH3), 2.17 (s, 3H, 2′-CH3), 3.09 (dd, J )
10.9, 18.1 Hz, 1H, 4-Hax), 3.32-3.36 (m, 1H, 4-Heq), 3.89-3.95
(m, 1H, 3-H), 3.92 (s, 3H, 5′-OCH3), 3.98 (s, 3H, 4′-OCH3), 4.12
(q, J ) 6.7 Hz, 1H, 1-H), 6.77 (d, J ) 8.5 Hz, 1H, 6′-H), 6.89 (d,
J ) 8.0 Hz, 1H, 8′-H), 6.94 (s, 1H, 3′-H), 7.21 (t, J ) 8.2 Hz, 1H,
7′-H), 7.57 (s, 1H, 6-H), 9.95 (s, 1H, CHO); 13C NMR (100 MHz,
methanol-d4) δ 18.8 (1-Me), 25.5 (3-Me), 28.8 (2′-Me), 34.6 (3-
C), 39.4 (4-C), 41.1 (4′-OMe), 64.5 (5′-OMe), 105.4, 107.7, 129.2,
129.8, 135.4, 136.0, 167.7, 169.5, 170.7, 176.1 (4′-C), 180.1 (5-
C), 191.2 (CHO); IR (KBr): ν 2962, 2925, 2852, 1686, 1592, 1457,
1394, 1261, 1087; 1025, 802; 698 cm-1; MS (ESI positive): m/z
(%): 406 [M + H]+; HRMS (ESI): C25H27NO4 requires for M +
H at m/z 406.20128; found: 406.20135.
23
1
(petroleum ether-EtOAc); [R]D +13.5 (c ) 0.12, MeOH); H
NMR (400 MHz, CDCl3) δ 1.33 (d, J ) 6.7 Hz, 3H, 3-CH3), 1.40
(d, J ) 6.6 Hz, 3H, 1-CH3), 2.13 (s, 3H, 2′-CH3), 3.00 (dd, J )
11.4, 18.5 Hz, 1H, 4-Hax), 3.39 (dd, J ) 4.6, 18.6, 1H, 4-Heq),
3.52 (d, J ) 13.8 Hz, 1H, N-CH2Ph), 3.57 (mc, 1H, 3-H), 3.91 (d,
J ) 13.8 Hz, 1H, N-CH2Ph), 3.99 (s, 3H, 5′-OCH3), 4.00 (s, 2H,
O-CH2Ph), 4.02 (s, 3H, 4′-OCH3), 4.12 (q, J ) 6.7 Hz, 1H, 1-H),
6.39 (d, J ) 6.9 Hz, 2H, Ph), 6.78 (s, 1H, 3′-H), 6.82 (d, J ) 7.7
Hz, 1H, 6′-H), 6.97-7.08 (m, 3H, Ph), 7.17-7.40 (m, 7H, Ar-
H), 7.54 (s, 1H, 6-H), 10.13 (s, 1H, CHO); 13C NMR (62.5 MHz,
CDCl3) δ 16.9 (1-Me), 22.8 (3-Me), 23.4 (2′-Me), 25.0 (4-C), 29.5
(1-C), 29.8 (3-C), 32.1 (NCH2Ph), 56.6 (4′-OMe), 59.8 (5′-OMe),
70.4 (OCH2Ph), 91.9, 103.4, 107.8, 107.9, 108.9, 128.1, 128.2,
136.1, 138.9, 153.6, 154.2, 156.6, 165.7 (4′-C), 173.4 (5-C), 193.3
(CHO); IR (KBr) 3060, 3040, 2960, 2940, 2860, 2848, 1693, 1593,
1455, 1393, 1360, 1263, 1210, 1130, 1070, 738 cm-1; CD
(MeOH): ∆ꢀ221 -17.6, ∆ꢀ240 +11.3, ∆ꢀ261 -4.8, ∆ꢀ301 +1.8, ∆ꢀ321
-1.5, ∆ꢀ353 +2.5; MS (EI): m/z (%): 585 [M]+ (4), 370 [M-CH3]+
(100), 91 [C7H7]+ (32); MS (ESI positive): m/z (%): 586 [M +
H]+; HREIMS Calcd for C38H36O4N+ (M - CH3)+: 570.26389,
found: 570.26360.
5-[N′-(1′′,6′′-Hexanediamino-N′′-dansyl)]methyldioncophyl-
line A (12). A solution of 5-formyldioncophylline A (11) (2 mg,
4.9 µmol) and N-dansyl-1,6-hexanediamine (9) (2.6 mg, 7.4 µmol)
in CH2Cl2 (1 mL) was cooled to 0 °C for 15 min and then treated
with sodium triacetoxyborohydride (1.6 mg, 7.5 µmol). The mixture
was stirred at room temperature under an Ar atmosphere for 24 h.
After being quenched by addition of aqueous saturated NaHCO3,
the product was exacted with CH2Cl2. The organic phase was
washed with brine and dried over MgSO4, and the solvent was
evaporated. The residue was purified by preparative TLC (CH2-
Cl2-MeOH, 95:5 elution) to give 12 (1.7 mg, 2.3 µmol, 48%) as
a pale-yellow solid: [R]D23 +6.5 (c ) 0.18, MeOH); 1H NMR (400
MHz, methanol-d4) δ 1.26-1.31 (m, 2H, 4′′-CH2), 1.30-1.42 (m,
2H, 3′′-CH2), 1.55-1.65 (m, 4H, 5′′-CH2, 2′′-CH2), 1.59 (d, J )
6.3 Hz, 3H, 3-CH3), 1.70 (d, J ) 6.7 Hz, 3H, 1-CH3), 2.16 (s, 3H,
2′-CH3), 2.80 (t, J ) 6.6 Hz, 2H, 1′′-CH2), 2.84-2.94 (m, 2H,
4-Hax, 4-Heq), 2.86 (s, 6H, (H3C)2-N), 3.00-3.05 (m, 2H, 6′′-CH2),
3.88-3.98 (m, 1H, 3-H), 3.90 (s, 3H, 5′-OCH3), 3.93-3.96 (m,
2H, 1′′′-CH2), 3.96 (s, 3H, 4′-OCH3), 4.20 (q, J ) 6.9 Hz, 1H,
1-H), 6.79 (d, J ) 8.5 Hz, 1H, 6′-H), 6.87 (d, J ) 7.7 Hz, 1H,
8′-H), 6.91 (s, 1H, 3′-H), 7.14 (s, 1H, 6-H), 7.18 (t, J ) 8.2 Hz,
1H, 7′-H), 7.26 (d, J ) 7.6 Hz, 1H, 6′′′-H), 7.50-7.58 (m, 2H, 3′′′
and 7′′′-H), 8.12 (dd, J ) 1.3, 7.3 Hz, 1H, 4′′′-H), 8.32 (d, J ) 8.6
Hz, 1H, 8′′′-H), 8.53 (d, J ) 8.5 Hz, 1H, 2′′′-H); 13C NMR (100
MHz, methanol-d4) δ 17.5 (1-Me), 24.6 (3-Me), 27.0 (2′-Me), 28.4
(4-C), 28.5 (1-C), 30.3 (2×), 30.6 (1-C), 30.8 (3-C), 33.5, 40.9
(5-CH2), 42.5 (NMe2), 50.4, 55.8 (4′-OMe), 58.5 (5′-OMe), 116.1,
120.3, 121.2, 122.6, 124.4, 126.1, 129.3, 130.9, 133.8, 140.4, 141.6,
141.8, 142.3, 145.8, 147.0, 152.2, 153.7, 154.3, 155.6, 157.2, 164.0
(4′-C), 169.0 (5-C); IR (KBr) 2925, 2853, 1656, 1458, 1385, 1261,
1028, 1075, 798, 722, 626 cm-1; MS (ESI positive): m/z (%): 740
[M + H]+; HRMS (ESI): C43H54N4O5S requires for M + H at m/z
739.38877; found: 739.38881.
N,8-O-Dibenzyl-5-[N′-(1′′,6′′-hexanediamino-N′′-dansyl)]-
methyldioncophylline A (10). N-benzyl-8-O-benzyl-5-formyldi-
oncophylline A (6) (13 mg, 0.022 mmol) and N-dansyl-1,6-
hexanediamine (9) (11.65 mg, 0.033 mmol) were mixed in CH2Cl2
(2 mL), cooled to 0 °C for 15 min, and then treated with sodium
triacetoxyborohydride (7 mg, 0.033 mmol). The mixture was stirred
at room temperature under Ar for 24 h. The reaction was stopped
by adding aqueous saturated NaHCO3, and the product was
extracted with CH2Cl2. The organic phase was washed with brine
and dried over MgSO4, and the solvent was evaporated. The residue
was purified by preparative TLC (CH2Cl2-MeOH, 95:5 elution)
to give 10 (13.8 mg, 0.015 mmol, 68%) as a pale yellow solid:
23
1
mp 64 °C (CH2Cl2); [R]D +18.7 (c ) 0.08, MeOH); H NMR
(400 MHz, methanol-d4) δ 1.08-1.10 (m, 4H, 3′′-CH2, 4′′-CH2),
1.28-1.32 (m, 4H, 5′′-CH2, 2′′-CH2), 1.30 (d, J ) 6.7 Hz, 3H,
3-CH3), 1.42 (d, J ) 6.6 Hz, 3H, 1-CH3), 2.09 (s, 3H, 2′-CH3),
2.58 (t, J ) 7.5 Hz, 2H, 1′′-CH2), 2.71 (dd, J ) 11.1, 17.1 Hz, 1H,
4-Hax), 2.80 (s, 6H, (H3C)2-N), 2.78-2.83 (m, 3H, 6′′-CH2 and
4-Heq), 3.51 (d, J ) 13.4 Hz, 1H, N-CH2Ph), 3.62 (mc, 1H, 3-H),
3.80 (s, 2H, 1′′′-CH2), 3.86 (s, 2H, O-CH2Ph), 3.91 (s, 3H, 5′-
OCH3), 3.93 (d, J ) 13.8 Hz, 1H, N-CH2Ph), 3.94 (s, 3H, 4′-OCH3),
4.15 (q, J ) 6.7 Hz, 1H, 1-H), 6.28 (d, J ) 6.9 Hz, 2H, Ph), 6.84-
7.04 (m, 6H, aromatic H and Ph), 7.15-7.38 (m, 8H, aromatic H

6112 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Bringmann et al.
N,8-O-Dibenzyl-5-iododioncophylline A. A mixture of dion-
cophylline A (200 mg, 0.53 mmol) and AgNO3 (182 mg, 0.58
mmol) in CH2Cl2 (15 mL) was cooled to 0 °C, and iodine (148
mg, 0.58 mmol) dissolved in EtOH (2 mL) was added. The mixture
was stirred at 0 °C until completion of the reaction according to
TLC and then treated with an aqueous Na2S2O3 solution (5%, 5
mL) and extracted with CH2Cl2. After evaporation of the solvent,
the purification of the residue by column chromatography on silica
gel (CH2Cl2-MeOH, 99:1 to 95:5) led to N,8-O-dibenzyl-5-
iododioncophylline A (292 mg, 580 µmol, 82%) as a colorless solid,
which was recrystallized from MeOH: mp 154 °C; [R]D25 ) -107.4
(c ) 0.15, EtOH); IR (KBr) 3423, 2923, 2360, 1587, 1452, 1379,
the next step. For analytical purposes the desired compound was
further purified by preparative HPLC: mp 100 °C; [R]D25 ) -39.6
(c ) 0.07, MeOH); IR (KBr) 3416, 2964, 2931, 2870, 2837, 1736,
1
1592, 1454, 1389, 1261, 1128, 1073, 1013 cm-1; H NMR (400
3
MHz, methanol-d4) δ 1.55 (d, JH-H ) 6.32 Hz, 3H, 3-Me), 1,72
3
(d, JH-H ) 6.96 Hz, 3H, 1-Me), 2.14 (s, 3H, 2′-Me), 3.05-3.14
(m, 2H, 4-CH2), 3.94-4.00 (m, 10H, 5′/6′-OMe, NCH2Ph, OCH2-
Ph), 4.21-4.23 (m, 1H, 3-H), 4.25 (dd, 2JH-H ) 5.32 Hz, 3JH-H
1.64 Hz, 2H, CH2OH), 4.56-4.58 (m, 1H, 1-H), 6.22 (dt, 3JH-H
)
)
5.28 Hz, 3JH-H ) 15.56 Hz, 1H, HCdCHCH2OH), 6.42 (d, 3JH-H
) 6.44 Hz, 2H), 6.88 (s, 2H, 6′-H, 3′-H), 6.92-6.94 (m, 1H, HCd
CHCH2OH), 7.03-7.07 (m, 2H), 7.10-7.19 (m, 3H), 7.33-7.41
(m, 5 H); 13C NMR (100 MHz, methanol-d4) δ 19.6 (1-Me), 20.1
(3-Me), 20.9 (2′-Me), 29.0 (4-C), 37.0 (1-C), 40.1 (3-C), 51.3
(NCH2Ph), 56.9 (4′-OMe), 57.1 (5′-OMe), 63.5 (CH2OH), 75.4
(OCH2Ph), 106.9, 110.5, 119.9, 128.0, 128.3, 128.9, 129.2, 130.2,
131.4, 131.9, 133.1, 137.0, 137.1, 137.0, 137.2, 137.8, 150.3, 155.8
(4′-C), 158.7 (5-C); MS (EI, 70 eV): m/z (%): 613 (2) [M]+, 601
(42), 600 (100), 582 (18) [M - CH2OH], 493 (12), 492 (33), 490
(17), 91 (79) [CH2Ph]+. HRMS (ESI): C41H44NO4 requires M +
H at m/z 614.32703; found: 614.32661.
1
1263, 1189, 1099, 963, 806, 760, 578 cm-1; H NMR (600 MHz,
methanol-d4) δ 1.35 (d, 3JH-H ) 6.30 Hz, 3H, 3-Me), 1.48 (d, 3JH-H
) 6.60 Hz, 3H, 1-Me), 2.16 (s, 3H, 2′-Me), 2.76 (dd, 2JH-H ) 17.20
Hz, 3JH-H ) 11.00 Hz, 1H, 4-H), 2.84 (dd, 2JH-H ) 17.30 Hz, 3JH-H
) 4.50 Hz, 1H, 4-H), 3.39 (m, 1H, 3-H), 3.94 (s, 3H, 5′-OMe),
3.98 (s, 3H, 4′-OMe), 4.45 (q, 3JH-H ) 6.70 Hz, 1H, 1-H), 6.76 (s,
3
3
1H, 3′-H), 6.79 (d, JH-H ) 7.50 Hz, 1H, 6′-H), 6.90 (d, JH-H
)
8.4 Hz; 1H, 8′-H), 7.24 (dd, 3JH-H ) 8.2 Hz, 3JH-H ) 8.2 Hz, 1H,
7′-H), 7.44 (s, 1H, 6-H); MS (EI, 70 eV): m/z (%): 503 (17) [M]+,
488 (100) [M - CH3], 376 (2) [M - I], 361 (7) [M - I - CH3];
Anal. (C24H26INO3) C, H, N.
3-(Dioncophylline-A-5-yl)-propan-1-ol (25). To a mixture of
3-(N,8-O-dibenzyldioncophylline-A-5-yl)-2-propen-1-ol and its 3,4-
reduced derivative (164 mg, 267 µmol) in MeOH (20 mL), Pd/C
(10%, 35.0 mg) was added, and the atmosphere was replaced with
H2 (1 bar). After stirring for 5 h, the solution was filtered over
celite and the solvent was removed in vacuo at 20 °C. The crude
product was purified by chromatography on Sephadex LH-20 with
EtOAc-MeOH (1:1) as the eluent to give a brownish solid (92.0
Methyl 3-(N,8-O-Dibenzyldioncophylline-A-5-yl)-acrylate (24).
A mixture of nBu4NBr (189 mg, 586 µmol), Cs2CO3 (478 mg, 1.47
mmol), and PPh3 (15.7 mg, 59.9 µmol) in acetonitrile (20 mL) was
degassed for 10 min in an ultrasonic bath. The N,8-O-dibenzyl-5-
iododioncophylline A (400 mg, 586 µmol) and methyl acrylate (126
mg, 1.46 mmol) were added, and the solution was again degassed
for 10 min. After addition of Pd(OAc)2 (13.5 mg, 58.6 µmol), the
mixture was stirred for 1.5 h at 70 °C before it was filtered over
celite. The filtrate was portioned between Et2O and H2O, the organic
phase was dried over MgSO4, and the solvent was removed in vacuo
at 20 °C. Flash chromatography eluting with petroleum ether-
EtOAc (8:2 to 1:1) gave a pale yellow solid (310 mg, 82%): mp
103 °C; [R]D25 ) -88.4 (c ) 0.05, MeOH; IR (KBr) 2932, 2838,
25
mg, 79%): mp 138 °C; [R]D ) -40.0 (c ) 0.12, MeOH); IR
(KBr) 3421, 2925, 2853, 1594, 1461, 1394, 1261, 1070, 1027 cm-1
;
1H NMR (400 MHz, methanol-d4) δ 1.33 (d, 3JH-H ) 6.20 Hz, 3H,
3
3-Me), 1.51 (d, JH-H ) 6.68 Hz, 3H, 1-Me), 1.76-1.79 (m, 2H,
2
3
CH2), 2.01 (s, 3H, 2′-Me), 2.47 (dd, JH-H ) 16.92 Hz, JH-H
)
)
2
11.08 Hz, 1H, 4-H), 2.61-2.65 (m, 2H, CH2), 2.96 (dd, JH-H
3
16.91 Hz, JH-H ) 4.28 Hz, 1H, 4-H), 3.43-3.49 (m, 1H, 3-H),
3.57-3.61 (m, 2H, CH2OH), 3.91 (s, 3H, 5′-OMe), 3.96 (s, 3H,
1
1716, 1585, 1454, 1389, 1263, 1070, 1073 cm-1; H NMR (400
3
3
MHz, methanol-d4) δ 1.30 (d, JH-H ) 6.80 Hz, 3H, 3-Me), 1.43
4′-OMe), 4.50 (q, JH-H ) 6.56 Hz, 1H, 1-H), 6.69 (s, 1H, 3′-H),
3
(d, JH-H ) 6.60 Hz, 3H, 1-Me), 2.10 (s, 3H, 2′-Me), 2.76 (dd,
6.84-6.88 (m, 2H, 6′-H, 8′-H), 6.91 (s, 1H, 6-H), 7.11-7.16 (m,
1H, 7′-H); 13C NMR (100 MHz, methanol-d4) δ 20.3 (1-Me), 20.9
(3-Me), 22.2 (2′-Me), 29.2 (CH2), 34.4 (CH2), 34.9 (3-C), 43.3 (4-
C), 57.0 (4′-OMe), 57.1 (5′-OMe), 62.7 (OCH2), 107.2, 110.6,
117.7, 120.1, 124.9, 127.1, 127.5, 131.2, 132.5, 133.1, 137.6, 138.6,
150.3, 158.0 (4′-C), 158.6 (5-C); MS (EI, 70 eV): m/z (%): 435
(24) [M]+, 434 (42) [M - H]+, 420 (100) [M - CH3 - H]+, 390
(7) [M - CH2CH2OH]+, 210 (25); HRMS (ESI): C27H34NO4
requires M + H at m/z 436.24878; found: 436.24802.
N-(Benzophenone-4-methylene)-N-dansyl-l-serine Methyl Es-
ter (28). p-Bromomethylbenzphenone (16) (39.0 mg, 142 µmol)
and NaI (42.6 mg, 284 µmol) were suspended in acetone (1.50
mL) and refluxed for 30 min, N-dansyl-L-serine methyl ester (27)
(10.0 mg, 28.4 µmol) and Cs2CO3 (185 mg, 56.8 µmol) were added,
and the solution was refluxed for 9 h. The mixture was acidified
(pH 4-5) with 0.05 N HCl and extracted with EtOAc (3 × 2 mL).
The organic phase was dried over MgSO4, and the solvent was
removed in vacuo. Flash chromatography eluting with petroleum
ether-EtOAc (1:1) gave a pale yellow solid (7.00 mg, 45%), which
2JH-H ) 17.32 Hz, 3JH-H ) 11.36 Hz, 1H, 4-H), 2.93 (dd, 2JH-H
)
17.32 Hz, 3JH-H ) 4.92 Hz, 1H, 4-H), 3.47 (d, 2JH-H ) 13.52 Hz,
1H, NCHHPh), 3.52-3.58 (m, 1H, 3-H), 3.74 (s, 3H, COOMe),
3.88-3.95 (m, 3H, NCHHPh, OCH2Ph), 3.92 (s, 3H, 5′-OMe), 3.95
(s, 3H, 4′-OMe), 4.13 (q, 3JH-H ) 6.68 Hz, 1H, 1-H), 6.27 (d, 3JH-H
) 15.8 Hz, 1H, CHCOOMe), 6.29 (d, 3JH-H ) 7.00 Hz, 2H), 6.85-
6.89 (m, 2H, 6′-H, 3′-H), 6.94-6.98 (m, 2H), 7.03-7.06 (m, 1H),
7.14-7.21 (m, 2H), 7.24-7.31 (m, 3H), 7.33-7.38 (m, 3H, 6-H),
3
8.07 (d, 1 H, JH-H ) 15.76 Hz, CHCHCOOMe); 13C NMR (100
MHz, methanol-d4) δ 19.6 (1-Me), 20.1 (3-Me), 20.9 (2′-Me), 30.9
(4-C), 47.0 (1-C), 51.2 (3-C), 52.2 (COOCH3), 52.3 (NCH2Ph),
56.9 (4′-OMe), 56.0 (5′-OMe), 75.7 (OCH2Ph), 107.0, 110.4, 117.7,
119.1, 119.6, 127.6, 127.9, 128.1, 128.7, 128.8, 129.0, 129.5, 130.0,
130.1, 130.1, 132.3, 134.5, 136.3, 137.2, 137.9, 138.0, 141.0, 142.7,
158.1 (8-C), 158.3 (4′-C), 158.8 (5-C), 169.2 (COOCH3); MS (EI,
70 eV): m/z (%): 641 (3) [M]+, 626 (100) [M - CH3]+, 542 (22)
[M - methylacrylate, C4H6O2 - Me]+, 535 (6) [M - Me - Bn]+,
520 (17), 518 (14), 434 (6) [M - 2Bn - 2Me]+, 91 (51) [CH2-
Ph]+; Anal. (C42H43NO5) C, H, N.
25
was recrystallized from petroleum ether-EtOAc: mp 53 °C; [R]D
3-(N,8-O-Dibenzyldioncophylline-A-5-yl)-2-propen-1-ol. A so-
lution of ester 24 (20.0 mg, 31.2 µmol) in dry THF (2 mL) was
cooled to -78 °C, and LiAlH4 (2.48 mg, 65.4 µmol) was added.
After 2 h stirring at that temperature, the solution was allowed to
warm to room temperature, neutralized with 0.05 n HCl, and
extracted with EtOAc (3 × 2 mL). The organic phase was washed
with water and dried over MgSO4. After removal of the solvent in
vacuo at 20 °C, the crude product was purified by flash chroma-
tography using deactivated silica gel and eluting with petroleum
) -12.7 (c ) 0.10, MeOH); IR (KBr) 3337, 2950, 2856, 1742,
1656, 1608, 1447, 1318, 1280, 1145, 1060, 941, 792, 704 cm-1
;
1H NMR (400 MHz, CDCl3) δ 2.88 (s, 6H, NMe2), 3.38 (s, 3H),
3.83-3.92 (m, 2H, â-CH2), 4.54 (d, 2JH-H ) 16.28 Hz, 1H), 4.82
3
2
(d, JH-H ) 12.24 Hz, 1H), 4.85 (d, JH-H ) 16.28 Hz, 1H, 7.19
3
3
(d, JH-H ) 7.44 Hz, 1H), 7.31 (d, JH-H ) 8.08 Hz, 1H), 7.42-
7.49 (m, 3H), 7.52-7.60 (m, 4H), 7.72 (d, 3JH-H ) 7.08 Hz, 1H),
3
3
8.21 (d, JH-H ) 7.32 Hz, 1H), 8.34 (d, JH-H ) 8.72 Hz, 1H),
8.52 (d, JH-H ) 8.48 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ
3
1
ether-EtOAc (8:2) to obtain a yellow solid (12.0 mg, 63%). H
45.6 (NMe2), 50.4 (CH2), 52.3 (OMe), 61.0 (R-C), 61.8 (â-CH2),
115.4, 119.4, 123.3, 126.5, 128.4, 128.7, 130.0, 130.1, 130.3, 130.4,
130.5, 131.0, 131.0, 132.6, 134.7, 137.0, 137.6, 141.6, 170.0, 196.2;
MS (EI, 70 eV): m/z (%): 546 (6) [M]+, 171 (100) [1-N,N-
NMR showed a 3 to 1 ratio of 3-(N,8-O-dibenzyldioncophylline-
A-5-yl)-2-propen-1-ol to 3-(N,8-O-dibenzyldioncophylline-A-5-yl)-
propan-1-ol. The main portion of the material was used as such in

Naphthylisoquinolines for Photoaffinity Labeling
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6113
dimethylnaphthalene]+,38 170 (51), 105 (44) [PhCO]+, 77 (31)
[Ph]+; HRMS (ESI): C30H30N2O6S requires M + Na at m/z
569.17223; found: 569.17091.
thalene]+,38 170 (100), 169 (26), 168 (36), 155 (21), 154 (27), 128
(23), 127 (29); HRMS (ESI): C35H32N2O6S requires for M + Na
at m/z 670.16576; found: 670.16475.
N-(Benzophenone-4-methylene)-N-dansyl-l-serine (29). A mix-
ture of N-(p-benzophenoylmethyl)-N-dansyl-L-serine methyl ester
(28) (40.0 mg, 73.2 µmol) and 2 N NaOH (146 µL, 293 µmol) in
THF-H2O (1:1, 5 mL) was refluxed for 4 h. Acidification (pH
4-5) of the solution followed by extraction with EtOAc (3 × 3
mL) and evaporation of volatile compounds yielded the crude
product. Purification by flash chromatography eluting with petro-
N-Dansyl-l-tyrosine (32). A suspension of N,O-didansyl-L-
tyrosine (31) (365 mg, 0.56 mmol) in a 1:1 mixture (40 mL) of 2
N NaOH and MeOH was refluxed for 2 h. The solution was
acidified (pH 4-5) with 0.05 N HCl and extracted with EtOAc (3
× 20 mL). The organic phase was dried over MgSO4, and the
solvent was removed in vacuo. The resulting pale yellow solid was
25
recrystallized from EtOAc (218 mg, 93%): mp 119 °C; [R]D
)
25
leum ether-EtOAc (1:1) gave a yellow oil (28.2 mg, 72%). [R]D
-60.1 (c ) 0.07, MeOH); IR (KBr) 3550, 2925, 1612, 1578, 1396,
1
) +8.8 (c ) 0.28, CHCl3); IR (KBr) 3443, 2924, 1711, 1658, 1317,
1146, 1091, 788, 591 cm-1; H NMR (400 MHz, methanol-d4) δ
1280, 1204, 1143, 703 cm-1; 1H NMR (400 MHz, CDCl3) δ 2.92
2.64 (dd, 2JH-H ) 13.88 Hz, 3JH-H ) 8.84 Hz, 1H, â-CHH), 2.83-
2
(s, 6H, NMe2), 3.88 (m, 2H), 4.59 (d, JH-H ) 16.16 Hz, 1H,
2.91 (m, 1H, â-CHH), 2.87 (s, 6H, NMe2), 3.90 (dd, 2JH-H ) 8.72
2
3
3
ArNCHH), 4.80 (m, 1H, R-H), 4.85 (d, JH-H ) 16.17 Hz, 1H,
Hz, JH-H ) 5.18 Hz, 1H, R-H), 6.34 (d, JH-H ) 8.56 Hz, 2H),
3 3
ArNCHH), 7.24 (d, 1H), 7.31 (d, 3JH-H ) 8.01 Hz, 1H), 7.47 (m,
6.69 (d, JH-H ) 8.56 Hz, 2H), 7.22 (d, JH-H ) 6.84 Hz, 1H),
7.41-7.50 (m, 2H), 7.99 (d, 3JH-H ) 6.08 Hz, 1H), 8.21 (d, 3JH-H
3
3
3H), 7.57 (m, 4H), 7.80 (d, JH-H ) 6.99 Hz, 1H), 8.21 (d, JH-H
) 7.16 Hz, 1H), 8.41 (d, 3JH-H ) 8.70 Hz, 1H), 8.55 (d, 3JH-H
)
) 8.72 Hz, 1H), 8.48 (d, JH-H ) 8.44 Hz, 1H); 13C NMR (62.5
3
8.08 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 45.8 (NMe2), 50.5
(CH2), 60.6 (R-C), 62.0 (â-CH2), 126.7, 128.3, 128.5, 128.8, 128.9,
130.2, 130.3, 130.5, 130.6, 131.0, 132.3, 132.4, 132.7, 137.1, 137.6,
141.5, 172.2, 196.4; HRMS (ESI): C29H28N2O6S requires for M
+ Na at m/z 555.15657; found: 555.15745.
MHz, methanol-d4) δ 38.8 (â-C), 46.0 (NMe2), 59.5 (R-C), 61.7
(OCH2), 115.9, 116.3, 116.4, 121.1, 124.1, 124.2, 128.3, 129.9,
130.8, 130.9, 131.1, 131.2, 137.1, 137.1, 152.8, 157.0, 175.1; MS
(EI, 70 eV): m/z (%): 428 (17), 414 (15) [M]+, 236 (28), 172
(25), 171 (100) [1-N,N-dimethylnaphthalene]+,38 170 (87), 168 (29),
155 (18), 154 (19), 121 (24), 107 (57); HRMS (ESI): C21H22N2O5S
requires for M + Na at m/z 437.11471; found: 437.11491.
N-Dansyl-2,2-difluoro-4-(4-hydroxybenzyl)-[1,3,2]oxazaboro-
lidin-5-one (33). To a solution of N-dansyl-L-tyrosine (36) (13.0
mg, 31.4 µmol) in dry THF (0.5 mL) was added NaH (0.83 mg,
34.5 µmol), and the mixture was stirred at room temperature for
30 min. After addition of BF3‚OEt2 (8.36 µL, 66.0 µmol), the
solution was refluxed for 3 h. The precipitated NaBF4 was filtered
off under nitrogen, and the resulting solution was freed from volatile
compounds in vacuo. The resulting yellow solid (14.4 mg, 99%)
could be stored under nitrogen at 4 °C for several weeks. Because
of its instability to hydrolysis it was only characterized by NMR.
4-(5-Dimethylaminonaphthalene-1-sulfonylmethyl)-benzophe-
none (30). A solution of dansyl sulfinic acid39 (30.0 mg, 0.13
mmol), 4-bromomethyl-benzophenone (16) (124 mg, 0.45 mmol),
and 2 N NaOH (65.0 µL, 0.13 mmol) in EtOH (1 mL) was stirred
over night at 80 °C. The mixture was extracted with CH2Cl2 (3 ×
2 mL), and the organic phase was washed with H2O and dried over
MgSO4. Evaporation of the solvent in vacuo yielded the crude
product, which was purified by flash chromatography eluting with
petroleum ether-EtOAc (7:3) to obtain a yellow solid (32 mg,
50%). The following analytical data completely matched that of
the observed byproduct (30): mp 61-63 °C; IR (KBr) 2940, 1658,
1608, 1574, 1448, 1412, 1314, 1278, 1141, 1124, 794, 703 cm-1
1H NMR (400 MHz, CDCl3) δ 2.90 (s, 6H, NMe2), 4.59 (s, 2H,
;
2
1H NMR (400 MHz, acetone-d6) δ 2.62 (dd, JH-H ) 13.88 Hz,
3
3
CH2), 7.10 (d, JH-H ) 8.20 Hz, 2H), 7.22 (d, JH-H ) 7.36 Hz,
1H), 7.45-7.50 (m, 3H), 7.56-7.63 (m, 4H), 7.72 (d, 3JH-H ) 7.95
Hz, 2H), 7.99 (d, 3JH-H ) 7.33 Hz, 1H), 8.46 (d, 3JH-H ) 8.59 Hz,
1H), 8.58 (d, 3JH-H ) 8.58 Hz, 1H); 13C NMR (100 MHz, CDCl3)
δ 45.6 (NMe2), 62.1 (CH2), 115.5, 118.5, 123.4, 129.1, 129.8, 130.2,
130.8, 130.9, 131.6, 131.9, 132.8, 133.3, 137.4, 137.8, 152.4, 196.2;
MS (EI, 70 eV): m/z (%): 429 (87) [M]+, 364 (19), 234 (35)
[dansyl]+, 195 (32) [p-methyl-benzophenone - H]+, 170 (100)
[1-N,N-dimethylnaphthalene - H]+,38 169 (89), 168 (68), 167 (83),
105 (55) [benzaldehyde - H]+, 77 (46) [Ph]+; HRMS (ESI):
C26H23NO3S requires for M + Na at m/z 452.12963; found:
452.12998; Anal. (C26H23NO3S): C, H, N, S.
3JH-H ) 10.72 Hz, 1H, â-CHH), 2.95 (dd, 2JH-H ) 13.88 Hz, 3JH-H
) 9.84 Hz, 1H, â-CHH), 3.75 (s, 6 H, NMe2), 4.00-4.05 (m, 1H,
3
3
R-H), 6.14 (d, JH-H ) 8.44 Hz, 2H), 6.67 (d, JH-H ) 8.32 Hz,
2H), 7.38 (d, 3JH-H ) 9.44 Hz, 1H, NH), 7.78-7.82 (m, 2H), 8.18
(d, 3JH-H ) 6.60 Hz, 1H), 8.29 (d, 3JH-H ) 7.72 Hz, 1H), 8.50 (d,
3JH-H ) 8.72 Hz, 1H), 8.76 (d, JH-H ) 8.72 Hz 1H); 19F NMR
(400 MHz, acetone-d6) δ -152.34, -150.10.
3
N-Dansyl-O-(4-benzoylbenzyloxy)-l-tyrosine (34). A mixture
of oxazaborolidinone 33, p-bromomethyl-benzophenone (16) (13.5
mg, 49 µmol), and Cs2CO3 (22.5 mg, 69 µmol) in acetone (3 mL)
was stirred at 50 °C until formation of a byproduct [Rf (EtOAc-
PE ) 1:1): 0.72] was observed (usually 6-8 h). Addition of water
(1 mL) and further stirring for 30 min at 50 °C effected hydrolysis
of the oxazaborolidinone. The solution was acidified (pH 4-5) with
0.5 N HCl and extracted with EtOAc (3 × 2 mL). The organic
phase was washed with 0.05 N HCl and dried over MgSO4.
Evaporation of the solvent in vacuo resulted in a pale yellow
solid (12.0 mg, 63%), which was recrystallized from EtOAc: mp
N,O-Didansyl-l-tyrosine (31). Dansyl chloride (7) (50.0 mg, 0.19
mmol) was suspendend in Et2O (2 mL) and added to a solution of
L-tyrosine (26.0 mg, 0.14 mmol) in 2 N NaOH, and the mixture
was stirred for 20 min at room temperature. A pale yellow gel was
formed during the reaction. The aqueous phase was washed with
Et2O, acidified (pH 4-5) with 0.05 N HCl, and extracted with
EtOAc (3 × 3 mL). The organic phase was dried over MgSO4,
and the solvent was removed in vacuo, yielding a yellow solid (32.0
mg, 69%), which was recrystallized from EtOAc: mp 122 °C;
25
83 °C; [R]D ) -10.6 (c ) 0.07, MeOH); IR (KBr) 3453, 3286,
2942, 1743, 1655, 1612, 1597, 1576, 1516, 1447, 1317, 1145, 1024,
790, 702, 627 cm-1; 1H NMR (400 MHz, CDCl3) δ 2.81 (dd, 2JH-H
25
3
[R]D ) -38.8 (c ) 0.10, MeOH); IR (KBr) 2945, 1720, 1573,
) 13.78 Hz, JH-H ) 6.56 Hz, 2H, â-CH2), 2.88 (s, 6H, NMe2),
1
1504, 1456, 1367, 1145, 866, 789, 623, 572 cm-1; H NMR (400
4.18-4.21 (m, 1H, R-H), 4.71 (d, 2JH-H ) 12.52 Hz, 1H, OCH2),
4.79 (d, 2JH-H ) 12.50 Hz, 1H, OCH2), 5.34 (d, 3JH-H ) 9.20 Hz,
MHz, CDCl3) δ 2.74 (dd, 2JH-H ) 14.04 Hz, 3JH-H ) 6.96 Hz, 1H,
â-CHH), 2.81-2.85 (m, 1H, â-CHH), 2.85 (s, 6H, NMe2), 2.90 (s,
3
3
1H, NH), 6.38 (d, JH-H ) 8.44 Hz, 2H), 6.63 (d, JH-H ) 8.56
Hz, 2H), 7.10 (d, 3JH-H ) 8.48 Hz, 2H), 7.18 (d, 3JH-H ) 7.72 Hz,
1H), 7.45-7.52 (m, 5H), 7.59-7.67 (m, 1H), 7.66 (d, 3JH-H ) 8.48
3
6H, NMe2), 4.01-4.03 (m, 1H, R-H), 5.48 (d, JH-H ) 8.32 Hz,
3
3
1H, NH), 6.50 (d, JH-H ) 8.60 Hz, 2H), 6.69 (d, JH-H ) 8.60
Hz, 2H), 7.14 (d, 3JH-H ) 7.48 Hz, 1H), 7.27 (d, 3JH-H ) 6.92 Hz,
Hz, 2H), 7.79-7.81 (m, 2H), 8.21-8.23 (m, 2H), 8.51 (d, 3JH-H
)
3
1H), 7.37-7.64 (m, 3H), 7.66 (t, JH-H ) 7.68 Hz, 1H), 8.00 (d,
8.59 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 38.3 (â-C), 45.3
(NMe2), 57.0 (R-C), 66.0 (OCH2), 123.0, 126.0, 128.0, 128.3, 129.4,
129.5, 129.8, 130.0, 130.1, 130.5, 132.8, 134.4, 136.8, 137.6, 138.9,
154.7, 170.3, 196.1; MS (EI, 70 eV): m/z (%): 608 (21) [M]+,
373 (16) [M - dansyl-H]+, 235 (17) [dansyl-H]+, 196 (41)
[p-methyl-benzophenone]+, 195 (31) [p-methyl-benzophenone -
H]+, 171 (100) [1-N,N-dimethylnaphthalene]+,38 170 (93) [1-N,N-
3JH-H ) 7.32 Hz, 1H), 8.09-8.12 (m, 2H), 8.43-8.48 (m, 2H),
8.56 (d, JH-H ) 8.75 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ
3
37.9 (â-C), 45.7 (NMe2), 57.1 (R-C), 115.8, 116.0, 119.9, 120.0,
121.8, 123.4, 123.5, 128.4, 129.0, 129.4, 129.5, 129.6, 129.6, 130.2,
130.5, 130.5, 131.1, 131.4, 131.9, 134.5, 134.5; MS (EI, 70 eV):
m/z (%): 647 (2) [M]+, 253 (18), 171 (71) [1-N,N-dimethylnaph-

6114 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Bringmann et al.
dimethylnaphthalene - H]+, 107 (36), 77 (21) [Ph]+; Anal.
(C35H32N2O6S): C, N, H calcd: 5.30; found: 5.89; S calcd: 5.27;
found: 4.77.
Supporting Information Available: Further microscopic vi-
sualizations, UV-spectroscopic investigations, mechanistic consid-
erations, and HPLC, combustion analysis, and NMR data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
3-(Dioncophylline-A-5-yl)-n-propyl N-Dansyl-O-(4-benzoyl-
benzyloxy)-l-tyrosinecarbaminate (35). The obtained N-dansyl-
O-(4-benzoylbenzyloxy)-L-tyrosine (34) (23.0 mg, 38.0 µmol) was
treated with a solution of phosgene (20%, 0.80 mL, 1.53 mmol)
and pyridine (3.00 mg, 38.0 µmol) in toluene, and the mixture was
stirred for 1.5 h at 60 °C. A white precipitate was formed. The
solvent was removed in vacuo, conveying the gases through a wash
bottle filled with 2 N NaOH. The residue was dissolved in THF (1
mL), 3-(dioncophylline-A-5-yl)-propan-1-ol (25) (14.6 mg, 33.4
µmol) was added, and the mixture was stirred overnight at room
temperature. The solvent was removed in vacuo, and the residue
was purified by preparative HPLC to give a brown solid (5.00 mg,
14%): mp 65-67 °C; [R]D25 ) +33.7 (c ) 0.08, CHCl3); 1H NMR
References
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In The Alkaloids; Cordell, G. A., Ed.; Academic Press: New York,
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Assi, L.; Schlauer, J. The Alkaloids of Triphyophyllum peltatum
(Dioncophyllaceae). Chimia 1998, 52, 18-28.
(2) (a) Ruangrungsi, N.; Wongpanich, V.; Tantivatana, P.; Cowe, H. J.;
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3
(400 MHz, methanol-d4) δ 1.56 (d, JH-H ) 6.32 Hz, 3H, 3-Me),
3
1.70 (d, JH-H ) 6.68 Hz, 3H, 1-Me), 1.97-1.99 (m, 2H, CH2),
2.13 (s, 3H, 2′-Me), 2.71-2.75 (m, 2H, CH2), 2.76-2.93 (m, 10H,
â-CH2, 4-CH2, NMe2), 3.90-3.93 (m, 4H, 3-H, 5′-OMe), 3.97 (s,
3
3
3H, 4′-OMe), 4.06 (dd, JH-H ) 8.60 Hz, JH-H ) 6.44 Hz, 1H,
R-H), 4.08-4.16 (m, 2H, CH2OR), 4.84-4.88 (m, 1H, 1-H), 6.46
3
(d, JH-H ) 8.60 Hz, 2H), 6.79-6.88 (m, 5H, 3′-H, 6′-H, 8′-H,
H-Ar), 6.90 (s, 1H, 6-H), 7.15-7.21 (m, 4H, 7′-H, H-Ar), 7.42-
7.56 (m, 5H), 7.61-7.77 (m, 4H), 8.07 (d, 3JH-H ) 6.04 Hz, 1H),
8.21 (d, 3JH-H ) 8.68 Hz, 1H), 8.42 (d, 3JH-H ) 8.60 Hz, 1H); 13
C
NMR (150 MHz, acetone-d6): δ ) 17.9 (1-Me), 19.2 (3-Me), 20.8
(2′-Me), 29.3 (CH2), 30.6 (CH2), 32.6 (4-C), 38.4 (â-C), 44.5 (3-
C), 45.6 (NMe2), 48.9 (1-C), 56.3 (4′-OMe), 56.5 (5′-OMe), 58.5
(R-C), 66.4 (ArOCH2), 68.4 (OCH2), 106.3, 109.5, 115.9, 117.3,
120.4, 121.5, 123.9, 127.5, 128.3, 129.3, 130.5, 130.7, 132.0, 132.2,
133.3, 134.5, 13.7, 137.3, 138.0, 138.1, 141.0, 150.8, 152.5 (NMe2),
152.6, 154.0, 158.0 (4′-C), 158.6 (5-C), 171.4 (COOH), 196.1;
HRMS (ESI): C63H65N3O11S requires for M + H at m/z 1070.42616;
found: 1070.42581.
Biological Materials and Methods. Cultivation and Fluores-
cence Labeling of Intraerythrocytic P. falciparum. P. falciparum
(strain FCBR) was cultured in vitro under standard conditions.
Briefly, parasites were grown in human red blood cells (blood group
A
Rh+) in RPMI 1640 medium (Sigma), supplemented with 25 mM
Hepes, 20 mM sodium bicarbonate, 10 mM D-glucose, and 0.5%
w/v AlbuMAX II (GIBCO) at 2.5% hematocrit. Cultivation was
performed at 37 °C with a gaseous phase of 90% N2, 5% O2, and
5% CO2. For fluorescence labeling a culture of P. falciparum was
incubated with 0.5 µM of the respective compound for 4 h under
the culture conditions. After the incubation period, thin blood smears
were prepared and immediately examined under a confocal-laser-
scanning microscope (Leica TCS SP) using the laser and filter
settings for DAPI fluorescence or an Axiovert S100 fluorescence
microscope (Zeiss) using an emission filter of 470 ( 40 nm. Images
were also taken in transmission mode to visualize nonfluorescent
cells. The tests for protease inhibition were performed as previously
described.37
Computational Chemistry. For the quantum chemical calcula-
tions the program package Gaussian 0340 was used together with
the Hartree-Fock method and the polarized valence double-ú basis
set 6-31G(d).34 To establish the found stationary points as local
minima (no imaginary frequencies) or transition states (exactly one
imaginary frequency), frequency calculations were performed. These
calculations were also used to get zero point corrected energies
(zero-point vibrational energies were scaled with the factor
0.913541). The population analyses were accomplished with the
NBO software42 as implemented in Gaussian 03.
Acknowledgment. This work was supported by the Fonds
der Chemischen Industrie and the Deutsche Forschungsgemein-
schaft (SFB 630 “Recognition, Preparation, and Functional
Analysis of Agents Against Infectious Diseases”). We are
grateful to Dr. W. Saeb and Dr. A. J. Price Mortimer for
previous work in the field, and Prof. Dr. F. Wu¨rthner and V.
Dehm for the fluorescence spectra.
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