Iron-containing nucleoside analogues with pronounced apoptosis-inducing activity

Article abstract of DOI:10.1002/anie.200353132

Bioactive organometallic compounds: Iron-containing nucleoside analogues, which can be stereoselectively synthesized in a few steps starting from simple carbohydrates, cause tumor cells to undergo apoptosis (programmed cell death; see picture for the typi

Full text of DOI:10.1002/anie.200353132

Angewandte
Chemie
Antitumor Agents
Iron-Containing Nucleoside Analogues with
Pronounced Apoptosis-Inducing Activity**
Daniel Schlawe, AndrØ Majdalani, Juraj Velcicky,
Erik Heßler, Thomas Wieder,* Aram Prokop,* and
Hans-Günther Schmalz*
Scheme 1. Diastereoselective nucleoside formation via an iron-stabi-
lized cationic intermediate.
We have previously demonstrated that h4-butadiene-Fe(CO)3
complexes of type 1 undergo highly selective substitution
reactions at the acetal center via Fe(CO)3-stabilized cationic
intermediates of type 2.[1] As an example, the SnCl4-catalyzed
reaction of complex 1 (R = H) with a silylated uracil
derivative[2] afforded the nucleoside analog 3 with greater
95% diastereoselectivity in high yield (Scheme 1).
Encouraged by the high efficiency of this transformation
and the growing importance of bio-organometallic chemis-
try,[3] we felt challenged to apply this methodology to the
stereoselective synthesis of iron-containing nucleoside ana-
logs of type 4 or 5 (NB = nucleobase) also possessing a 5’-
CH2OR’ substituent.Such compounds could exhibit useful
biological activities, since nucleoside analogs in general have
a great pharmacological potential (e.g. as anti-viral and anti-
tumoral drugs)[4] and transition-metal carbonyl complexes
offer some unique new opportunities for drug development.[5]
Herein, we disclose the discovery of new iron-containing
nucleoside analogs, which surprisingly exhibit strong apopto-
sis-inducing properties.[6] The synthesis of complexes 10 and
12 (Scheme 2) as precursors for nucleoside analogs of type 4
[*] Dr. D. Schlawe, A. Majdalani, Dr. J. Velcicky, E. Heßler,
Prof. Dr. H.-G. Schmalz
Institut für Organische Chemie
Universität zu Köln
Greinstrasse 4, 50939 Köln (Germany)
Fax: (+49)221-470-3064
E-mail: schmalz@uni-koeln.de
Dr. Dr. A. Prokop
Department of Pediatric Oncology/Hematology
University Medical Center CharitØ, Campus Virchow, Humboldt
University of Berlin
13353 Berlin (Germany)
Fax: (+49)30-450-559999
E-mail: aram.prokop@charite.de
Scheme 2. a) TDSCl, pyridine, RT, 16 h (98%); b) DEAD, PPh3, ben-
zene, 808C, 4 h (81%); c) LiBr, tetramethylurea, toluene, reflux, 2.5 h
Priv.-Doz. Dr. T. Wieder
Department of Physiology I
Eberhard-Karls University Tübingen
72076 Tübingen (Germany)
Fax: (+49)7071-293-073
=
=
(68%) d) H2C PPh3, THF, ꢀ788C !RT (86%); or Ph3P C(H)CO2Et,
THF, reflux, 1 h (95%); e) [Fe2(CO)9] Et2O, reflux, 18 h (for R=H,
72%, 10:11=3.3:1) or 3 h (for R=CO2Et, 77%, 12:13=4.3:1);
DEAD=diethylazodicarboxylate.
E-mail: thomas.wieder@uni-tuebingen.de
[**] This work was supported by grants from the Deutsche For-
schungsgemeinschaft (Schm 875/1 to H.G.S.), Deutsche JosØ
Carreras Leukämie-Stiftung e.V. (DJCLS-R01/02; to T.W. and A.P.),
the Verein zur Förderung der Tagesklinik e.V. (to A.P. and T.W.), the
Berliner Krebsgesellschaft e.V. (to T.W.), and the Fonds der
Chemischen Industrie (to H.G.S.). The authors would like to thank
Dr. P. T. Daniel for kindly providing BJAB cells and laboratory
equipment. Technical assistance by A. Richter in the performance of
cell assays is gratefully acknowledged. The authors would also thank
Dr. J. Lex, Dr. H. Schmickler, and Dr. M. Schäfer for advanced X-ray
crystallographic, NMR spectroscopic and mass spectrometric
measurements.
started with commercial a-methylglucopyranoside (6), which
was first converted into the aldehyde 7 by silylation of the
primary hydroxyl group (TDS = thexyldimethylsilyl), Mitsu-
nobu epoxide formation, and subsequent LiBr-induced rear-
rangement/ring contraction.[7] Complexation of the dienes 8
and 9, obtained from 7 by Wittig olefination, with [Fe2(CO)9]
in refluxing Et2O proceeded with significant diastereoselec-
tivity[8] to preferentially give the endo-complexes 10 and 12,
respectively.While chromatographic separation of the mix-
tures (10/11 and 12/13) was possible only by preparative
HPLC, the corresponding primary alcohols, obtained by
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2004, 43, 1731 –1734
DOI: 10.1002/anie.200353132
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1731

Communications
tetrabutylammonium fluoride (TBAF) induced desilylation,
were readily separable by simple flash chromatography and
could be resilylated in high yield.[9]
For the synthesis of nucleoside analogs of type 5, complex
19 was synthesized starting from commercially available
ribonolactone 14 (Scheme 3).Selective trityl protection of the
Figure 1. Silylated nucleobases (NB*) used for the synthesis of iron-
containing nucleoside analogs (see Figure 2 and Table 1); TMS=tri-
methyl silane.
Table 1: Synthesis of iron-containing nucleoside analogs and some
cytotoxicity data.
Starting NB* Conditions[a] Product Yield [%][b] d.r.[c]
material
LD90 [mM][d]
10
10
10
12
12
12
12
19
19
19
19
19
19
5e
5 f
4g
4g
U*
T*
C*
U*
T*
BrU*
C*
U*
T*
BrU*
C*
ClP*
BzA*
–
A
A
B
A
A
A
B
B
B
B
B
B
B
C
D
E
F
4a
4b
4c
4d
4e
4 f
4g
5a
5b
5c
5d
5e
5 f
5g
5g
4h
21
79
82
96
84
81
72
83
76
75
60
84
79
23
56
78
37
74
5.6:1
1.6:1
2.3:1
6.6:1
2.4:1
4.5:1
3.6:1
>50:1
>50:1
>50:1
>50:1
>50:1
>50:1
–
n.d.
n.d.
30
n.d.
>100
>100
20
>100
ꢁ100
ꢁ100
ꢁ30
n.d.
n.d.
n.d.
n.d.
>100
>100
Scheme 3. a) TrCl, pyridine, 558C, 16 h (67%); b) Tf2O, pyridine,
=
CH2Cl2, ꢀ788C!ꢀ158C, 2 h (85%); c) Bu3SnCH CH2, 3.5 mol%
Ph3As, 0.5 mol% [Pd2(dba)3], LiCl, THF, RT, 2 h (87%); d) [Fe2(CO)9]
EtOAc, RT!reflux over 4 h (69%, 17:18=1.4:1); then chromatogra-
phy; e) DIBAH, toluene, ꢀ788C, 2 h (92%); f) HC(OMe)3, cat. TsOH,
MeOH, RT, 5 h (99%); g) TDSCl, imidazol, CH2Cl2, RT, 16 h (96%);
TrCl=tritylchloride, Tf2O=trifluoro methanesulfonic anhydride,
dba=1,5-diphenyl-1,4-pentadiene-3-one, DIBAH=didsobutylalumi-
numhydride, TsOH=4-methyl benzenesulfonic acid
–
–
–
–
–
–
primary hydroxy group, formation of the enol triflate 15, and
subsequent Stille coupling with tributylvinylstannane
afforded diene 16 in 50% total yield.[10] Complexation of 16
proved to be more difficult than that of dienes 8 or 9.Best
results were obtained on treatment of 16 with four equiva-
lents of [Fe2(CO)9] in ethyl acetate, slowly heating the mixture
from ambient to reflux temperature.The resulting 14.:1
mixture of complexes 17 and 18 was easily separated by flash
chromatography.[9] Conversion of 17 into 19 proceeded
smoothly through DIBAH reduction, acidic methanolysis,
and silylation.
[a] Reaction conditions: A) 3.0–5.0 equivalents NB* (see Figure 2) in
refluxing CH2Cl2, 3.0–5.5 equivalent SnCl4 as 1m solution in heptane
added over 2 h by perfusion, then H2O; B) 4 equivalents NB*, 6 equiv-
alents TMSOTf, CH2Cl2, RT, 2–8 h, then H2O; C) 1 atm. NH3, DMAP,
dioxane/MeOH (20:1), RT, 3 weeks; D) 7m NH3 in MeOH, RT, 20 h;
E) 1.5 equivalents TBAF, H2O, THF, RT, 3 h; F) 6 equivalents TMANO,
toluene, RT, 1.5 h. [b] Yields of isolated separated epimers. [c] Diastereo-
meric ratio (4/epi-4 or 5/epi-5, respectively), [d] Cytotoxicity of the
products as measured by LDH release of cultivated BJAB mock cells
48 h after treatment (n.d.=not determined). Owing to the steep increase
of the biological activity beyond a certain threshold concentration, LD90
values are given instead of LD50.
The synthesis of nucleoside analogs of type 4 and 5 was
accomplished (in analogy to Scheme 1) by treating complexes
10, 12, and 19 with silylated nucleobases (Figure 1) under
Vorbrüggen-type conditions (Table 1).[2,11] In the case of
uracile (U), 5-bromouracile (BrU) and thymine (T), an
excess of SnCl4 was used as a Lewis acid to give the desired
N1-substituted products.In contrast, TMSOTf (instead of
SnCl4) had to be used for cytosine (C) to avoid formation of
the regioisomeric N4-substituted products.[12]
high yield (Figure 2).The introduction of adenine as a
nucleobase was achieved in a two step sequence starting
from 19 using the silylated reagents ClP* or BzA* (see
Figure 1).From the resulting products ( 5e and 5 f, respec-
tively) the adenosine derivative 5g was then obtained in good
yield by ammonolysis (Table 1).
Starting from complexes 10 and 12 gave the naturally
configured nucleosides 4 together with varying amounts of
their epimeric counterparts (epi-4).[13] Remarkably, complex
19 reacted with complete diastereoselectivity under the same
conditions to yield exclusively nucleoside analogs of type 5 in
For comparison purposes in the biological assays (see
below), the iron-free nucleoside 20 was prepared by oxidative
decomplexation of 4g with trimethylamine-N-oxide
(TMANO).Also, the desilylated cytosine derivative 4h was
obtained from 4g by reaction with TBAF (Scheme 4).
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Angewandte
Chemie
Figure 2. Various iron-containing nucleoside analogues prepared (see Table 1).
role of the metal–carbonyl fragment for the biological
function.However, any general (unspecific) activity of the
diene–Fe(CO)3 unit could be excluded because the close
analogs 4e, 4 f, and 5a, as well as the iron-containing
precursor compounds of type 10 and 12 did not exhibit any
activity.Also the desilylated product 4h (derived from 4g)
was found to be inactive.
In further experiments we were able to show that the
cytotoxic effect of the active compounds results from the
induction of apoptosis.This process, the programmed cell
death, which is one the most fundamental processes in cell
biology,[15] has become a key concept in modern cancer
therapy.[16]
The first evidence that iron-containing nucleosides can
induce apoptosis came from the observation of typical
morphological changes[15] (blebbing) after treatment of cul-
tured tumor cells (BJAB cells)[17] with compound 4g
(Figure 3).
Scheme 4. Reagents and conditions: a) TMANO, toluene, 08C!RT
(74%); b) TBAF, H20, THF, RT, 3 h (37%).
While preliminary tests for anti-HIV activity gave only
disappointing results,[14] some of the iron-containing nucleo-
side analogs exhibit pronounced cytotoxic activities against
tumor cells (Table 1).While both regioisomer series ( 4 and 5)
show activity, a significant influence of the nucleobase was
observed, the cytosine derivatives 4c, 4g, and 5d (Figure 2)
being the most potent compounds.Decomplexation of 4g to
20 led to a sharp drop of activity, which indicates the essential
Figure 3. Treatment of cultured BJAB tumor cells (left) with 4g
(20 mmolLꢀ1) leads to the induction of apoptosis after 24 h (right) as
indicated by typical morphological changes (blebbing).
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Communications
[3] Reviews: a) W.Beck, K.Severin, Chem. Unserer Zeit 2002, 36,
356; b) “The Bio Side of Organometallics”: R.Dagani, Chem.
Eng. News 2002, 80(37), 23; c) R.H.Fish, G.Jaouen, Organo-
metallics 2003, 22, 2166.
As well as inducing apoptosis, many cytostatic substances
cause (undesired) concentration-dependent membrane-
damage leading to cell death by necrosis.By determination
of lactate dehydrogenase (LDH) release into the culture
medium we could show that 4g does not significantly reduce
the viability of BJAB cells after 4 h of incubation at
concentrations ꢂ 100 mm (Figure 4a).This result indicates
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[7] a) G.Magnusson, N.Rehnberg, J. Org. Chem. 1990, 55, 5467;
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[9] The stereochemical assignments of the complexation products
were based on NMR and CD spectral correlations and X-ray
crystal-structure analysis of 18 and the desilylation product
obtained from 13.
Figure 4. Influence of compound 4g on a) the viability after 4 h and
b) the apoptosis of BJAB cells after 48 h. Viability was determined by
the LDH release assay[18] and is expressed as percentage of the value
measured in the control experiment ꢃSD (n=3). Apoptosis was
measured by flow cytometric analysis and is expressed as percentage
of cells with hypodiploid DNA ꢃSD (n=3).[19] See the Supporting
Information for experimental details.
[10] Methodology based on: a) I.Kalwinsh, K-H. .Metten, R.
Brückner, Heterocycles 1995, 40, 939; b) F.C. Gꢀrth, A.
Umland, R.Brückner, Eur. J. Org. Chem. 1998, 1055.
[11] a) H.Vorbrüggen, K.Krolikiewicz, B.Bennua, Chem. Ber. 1981,
114, 1234; b) H.Vorbrüggen, G.Hꢀfle, Chem. Ber. 1981, 114,
1256; c) TL. .Su, B.Bennua, H.Vorbrüggen, HJ..Lindner,
Chem. Ber. 1981, 114, 1269; d) H.Vorbrüggen, B.Bennua, Chem.
Ber. 1981, 114, 1279.
that such a necrosis-like, lytic mechanism is not responsible
for the cytotoxicity of compound 4g.In contrast, 4g very
potently induces DNA fragmentation after 48 h of treatment
in up to 80% of the cells.Apoptosis induction was concen-
tration-dependent (EC50 of 30 mm; Figure 4b).
[12] The assignments of N1 versus N4 substitution at the heterocycle
are based on NMR spectroscopy (NOE and long-range cou-
pling).
[13] The configurational assignments were demonstrated by NMR
spectroscopy (coupling and NOE between the hydrogen atoms
at the substituted dihydrofuran centers).
[14] We thank Dr.J.Balzarini, Rega Institute, Leuven, for the
performance of anti-viral tests.
[15] See: M.D.Jacobson, N.McCarthy, Apoptosis, Oxford University
Press, Oxford, 2002, and also: S.Grimm, Chem. Unserer Zeit
2003, 37, 172; F.Hꢀffeler, Biol. Unserer Zeit 2004, 34, 16.
[16] D.Hanahan, R.A.Weinberg, Cell 2000, 100, 57.
[17] BJAB: Burkitt-like lymphoma cell line as characterized before:
T.Wieder, F.Essmann, A.Prokop, K.Schmelz, K.Schulze-
We also tested the antileukemic potency of the new
nucleosides.Compound 4g very efficiently induced apoptosis
in an ex vivo DNA-fragmentation assay using primary,
leukemic lymphoblasts of patients suffering from childhood
acute lymphoblastic leukemia (ALL).[20]
In conclusion, we have shown that iron-containing nucleo-
side analogs of type 4 and 5 are easily prepared and represent
a new class of cytostatic, apoptosis-inducing agents.Current
investigations are focusing on the mechanism of action
(including the specific role of the metal carbonyl group) and
the development of compounds with improved pharmaco-
logical properties.
Osthoff, R.Beyaert, B.Dꢀrken, P.T.Daniel,
1378.
Blood 2001, 97,
[18] T.Wieder, C.E.Orfanos, C.C.Geilen,
11025.
J. Biol. Chem. 1998, 273,
Received: October 22, 2003 [Z53132]
[19] T.Wieder, A.Prokop, B.Bagci, F.Essmann, D.Bernicke, K.
Schulze-Osthoff, B.Dꢀrken, H-.G.Schmalz, PT. .Daniel, G.
Henze, Leukemia 2001, 15, 1735.
[20] The ability of 4g to induce apoptosis in BJAB cells was
additionally shown by Western blot analysis of caspase-3
processing, which was already observed after 18 h of incubation
(20 mm); for details of the method, see: A.Prokop, T.Wieder, I.
Sturm, F.Essmann, K.Seeger, C.Wuchter, W-.D.Ludwig, G.
Henze, B.Dꢀrken, P.T.Daniel, Leukemia 2000, 14, 1606.
Keywords: antitumor agents · carbonyl complexes · iron ·
.
nucleosides · synthetic methods
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1734
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