Nucleoside analogues with a 1,3-diene-Fe(CO)3 substructure: Stereoselective synthesis, configurational assignment, and apoptosis-inducing activity

Article abstract of DOI:10.1002/chem.201301672

The synthesis and stereochemical assignment of two classes of iron-containing nucleoside analogues, both of which contain a butadiene-Fe(CO)₃ substructure, is described. The first type of compounds are Fe(CO)₃-complexed 3'-alkenyl-2′,3′-dideoxy- 2′,3′-dehydro nucleosides (2,5-dihydrofuran derivatives), from which the second class of compounds is derived by formal replacement of the ring oxygen atom by a CH₂ group (carbocyclic nucleoside analogues). These compounds were prepared in a stereoselective manner through the metal-assisted introduction of the nucleobase. Whilst the furanoid intermediates were prepared from carbohydrates (such as methyl-glucopyranoside), the carbocyclic compounds were obtained by using an intramolecular Pauson-Khand reaction. Stereochemical assignments based on NMR and CD spectroscopy were confirmed by X-ray structural analysis. Biological investigations revealed that several of the complexes exhibited pronounced apoptosis-inducing properties (through an unusual caspase 3-independent but ROS-dependent pathway). Furthermore, some structure-activity relationships were identified, also as a precondition for the design and synthesis of fluorescent and biotin-labeled conjugates. I gotta Fe-ling: Iron-containing nucleoside analogues, which were first synthesized during an exercise in stereoselective π-complex chemistry, exhibited pronounced cytotoxic and apoptosis-inducing activities, even against resistant cancer cell lines. Both hetero- (X=O) and carbocyclic (X=CH₂) compounds were studied, and a synthetic route to R′-labeled derivatives was developed as a precondition for future biological experiments. TDS=thexyldimethylsilyl. Copyright

Full text of DOI:10.1002/chem.201301672

FULL PAPER
DOI: 10.1002/chem.201301672
ꢀ
Nucleoside Analogues with a 1,3-Diene Fe(CO)3 Substructure:
Stereoselective Synthesis, Configurational Assignment,
and Apoptosis-Inducing Activity
Christoph Hirschhꢀuser,[a] Juraj Velcicky,[a] Daniel Schlawe,[a] Erik Hessler,[a]
Andrꢁ Majdalani,[a] Jçrg-Martin Neudçrfl,[a] Aram Prokop,*[b] Thomas Wieder,[c] and
Hans-Gꢂnther Schmalz*[a]
Dedicated to Professor Stefan Toma on the occasion of his 76th birthday
Abstract: The synthesis and stereo-
chemical assignment of two classes of
iron-containing nucleoside analogues,
tive manner through the metal-assisted
introduction of the nucleobase. Whilst
the furanoid intermediates were pre-
pared from carbohydrates (such as
methyl-glucopyranoside), the carbocy-
clic compounds were obtained by using
an intramolecular Pauson–Khand reac-
tion. Stereochemical assignments based
on NMR and CD spectroscopy were
confirmed by X-ray structural analysis.
Biological investigations revealed that
several of the complexes exhibited pro-
nounced apoptosis-inducing properties
(through an unusual caspase 3-inde-
pendent but ROS-dependent pathway).
Furthermore, some structure–activity
relationships were identified, also as a
precondition for the design and synthe-
sis of fluorescent and biotin-labeled
conjugates.
ꢀ
both of which contain a butadiene
Fe(CO)3 substructure, is described. The
first type of compounds are Fe(CO)3-
complexed
3’-alkenyl-2’,3’-dideoxy-
2’,3’-dehydro nucleosides (2,5-dihydro-
furan derivatives), from which the
second class of compounds is derived
by formal replacement of the ring
oxygen atom by a CH2 group (carbocy-
clic nucleoside analogues). These com-
pounds were prepared in a stereoselec-
Keywords: antitumor
agents
·
apoptosis · carbonyl ligands · iron ·
nucleosides
Introduction
anti-malaria drug chloroquine). These and many other[4] ex-
amples demonstrate the still-underestimated potential of
metal-containing compounds for future pharmaceutical ap-
plications.
In recent years, the field of bio-organometallic chemistry
has enjoyed increasing attention because several transition-
metal–organometallic compounds have been shown to ex-
hibit significant biological activities.[1] Among the most-
prominent examples are ferrocene analogues of well-estab-
lished drugs, such as ferrocifene[2] (derived from the breast-
cancer agent tamoxifen) and ferroquine[3] (related to the
In 2004, we introduced a new class of iron-containing nu-
ꢀ
cleoside analogues with a butadiene Fe(CO)3 substruc-
ture.[5] Some of these agents were found to induce apoptosis
in BJAB tumor cells at low micromolar concentrations. Fur-
thermore, the cytosine derivative N69 (Figure 1), a particu-
larly active compound, was found to target new caspase-in-
dependent but ROS-dependent apoptosis pathways in mela-
noma cells.[6] The surprising biological properties and the
fact that the metal-free congener of N69 were found to be
more or less inactive prompted us to further explore this
class of iron-containing nucleoside analogues.[7] Herein, we
[a] Dr. C. Hirschhꢀuser, Dr. J. Velcicky, Dr. D. Schlawe, Dr. E. Hessler,
Dr. A. Majdalani, Dr. J.-M. Neudçrfl, Prof. Dr. H.-G. Schmalz
Department of Chemistry, University of Cologne
Greinstrasse 4, 50939 Kçln (Germany)
Fax : (+49)221-470-3064
E-mail: schmalz@uni-koeln.de
[b] Dr. Dr. A. Prokop
Children’s Hospital Cologne
Department of Paediatric Oncology
Amsterdamer Strasse 59, 50735 Kçln (Germany)
E-mail: prokopa@kliniken-koeln.de
[c] Dr. T. Wieder
Department of Dermatology
University Medical Center Tꢁbingen
Liebermeisterstrasse 25, 72076 Tꢁbingen (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201301672.
Figure 1. Structures of ferrocifen, ferroquine, and N69.
Chem. Eur. J. 2013, 19, 13017 – 13029
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13017

report the stereoselective synthesis and configurational as-
signment of a variety of Fe(CO)3-containing nucleoside ana-
logues of type 1 and their carbocyclic relatives of type 2
(Figure 2). Moreover, we report the results of biological in-
vestigations, which have led to the identification of struc-
ture–activity relationships concerning the apoptosis-inducing
activities of these compounds.
diene precursors of type 4 were considered to be prepared
by a Wittig reaction from aldehydes of type 5, which were
accessible from suitable carbohydrate building blocks, as de-
scribed by Rehnberg and Magnusson.[9]
We started our investigation with the synthesis of simpli-
fied dienes 10 and 11, which lacked the 5’-substitutent.
As we had previously communicated,[10] the synthesis
(Scheme 2) started with the conversion of (+)-l-arabinose
ꢀ
Figure 2. Two types of nucleoside analogues with a 1,3-diene Fe(CO)3
substructure as target structures. NB=nucleobase.
Results and Discussion
ꢀ
Scheme 2. Synthesis of vinyl dihydrofurans 10 and 11: a) BnOH, HCl(g)
,
ꢀ
Synthesis of vinyl dihydrofuran-derived Fe-containing nu-
cleosides of type 1: Our strategy for the synthesis of nucleo-
side analogues of type 1 is outlined in Scheme 1. We intend-
08C, 5 h; b) (CH3)2C
A
dine, p-TsCl, RT, 48 h; d) 1m HCl, THF, 508C, 12 h; e) NaOMe, MeOH,
RT, 16 h; f) LiBr, TMU, toluene, reflux, 1.5 h; g) for compound 10:
Ph3P=CHCO2Et, THF, 508C, 5 h; h) for compound 11: [Ph3PCH3]+Brꢀ,
nBuLi, ꢀ908C to ꢀ58C, 4 h. Bn=benzyl, p-Ts=para-toluenesulfonyl.
(6) into the benzyl glycoside under acidic conditions to ex-
clusively afford the thermodynamically favored a-product,
which was subsequently transformed into cis-acetonide 7.
Tosylation of the remaining OH group, acetal cleavage, and
subsequent base-induced epoxide formation delivered com-
pound 8. Upon treatment with LiCl and tetramethylurea
(TMU) in toluene, the epoxide underwent a ring-contractive
rearrangement (and subsequent water elimination) to yield
unsaturated aldehyde 9. Wittig olefination with the appro-
priate reagents finally afforded dienes 10 and 11, respective-
ly.
Scheme 1. Planned synthesis of iron-containing nucleoside analogues of
type 1.
The synthesis of the corresponding diene ligands with an
additional (silyl-protected) CH2OH substituent (16–20;
Scheme 3) started from commercially available methyl-(d)-
glucopyranosides, that is, compounds 12a and 12b, respec-
tively.
ed to introduce the nucleobase (NB) in a diastereoselective
fashion through Lewis acid-mediated SN reactions, such as
by applying the Vorbrꢁggen variation[8a] of the Hilbert–
Johnson method.[8b–d] As a key feature, we expected the
Fe(CO)3 fragment to shield the complexed face (“lower
hemisphere”) of the p ligand, thus forcing the nucleobase to
preferentially approach from the less-shielded “upper hemi-
sphere” (Scheme 1). Therefore, the stereoselective genera-
In both the a- and b-series, the selective silylation of the
primary alcohol function[11] and subsequent Mitsunobu reac-
tion yielded mixtures of diastereomeric epoxides 13 and
14.[12] Without separation, the diastereomer mixtures (13/14)
were subjected to the ring-contractive rearrangement/con-
densation conditions (LiCl, TMU, toluene, reflux, slow addi-
tion of the substrate) to yield the expected aldehydes of
type 15. The yield of this transformation was found to be
highly dependent on the quality of the reagents that were
used (in particular TMU).[13] Finally, Wittig olefination reac-
tions to afford compounds 16a, 16b, 17a, and 19a were
performed by directly adding the appropriate Wittig re-
agents (ylenes) to the crude reaction mixture of aldehyde
15a or 15b. The corresponding olefinations that yielded
ꢀ
tion of the “planar chiral” diene Fe(CO)3 substructure
through diastereoselective complexation (3!4) and a relia-
ble assignment of the configuration of the resulting com-
plexes were pivotal. Two factors were proposed to control
the diastereoselective complexation of compound 4:
1) Steric shielding of the “upper hemisphere” by a bulky
substituent (R1) and 2) pre-coordination of the Fe(CO)x re-
agent by the alkoxy group (OR3), thus directing it towards
the “lower hemisphere” of the diene ligand. The required
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ꢀ
Nucleoside Analogues with a 1,3-Diene Fe(CO)3 Substructure
FULL PAPER
type 21 was observed for the R1-unsubstituted substrates.
Thus, the complexation of compound 10 afforded com-
pounds 21a/22a in a diastereomeric ratio (d.r.) of 3.0:1,
whilst compound 11 gave rise to compounds 21b/22b with a
d.r. of 2.1:1 (Scheme 4). The (a-configured) substrates that
Scheme 4. Complexation of dienes 10, 11, and 16–20. Yields and selectivi-
ties are listed in Table 1.
contained an additional R1 substituent (16a, 17a, 18a, and
20a) reacted with increased diastereoselectivity, again in
favor of the desired complexes of type 21. Amide 19a dis-
played lower diastereoselectivity; reversed diastereoselectiv-
ity was only observed in the case of substrate 16b and com-
plexes 21d and 22d were formed in a ratio of 1:2.7.
Scheme 3. Synthesis of 4-substituted 3-alkenyl-2,5-dihydrofuran ligands:
a) TDSCl, pyridine, RT, 18 h to 60 h; b) for compounds 13a/14a: DIAD,
PPh3, toluene, RT, 1 h, then reflux, 2 h; for compounds 13b/14b: DEAD,
PPh3, benzene, RT, 0.5 h, then reflux, 4 h; c) LiBr, TMU, toluene, reflux,
3 h; d) for compounds 16a, 16b, 17a, and 19a: starting from an appropri-
ate mixture of compounds 13/14: LiBr, TMU, toluene, reflux, 3 h, then
From these results, we concluded that two factors (as
mentioned above) contributed to the observed diastereose-
lectivities: 1) Pre-coordination of the incoming Fe(CO)x re-
agent to the alkoxy (OR3) functionality directed its delivery
to the diene from the same hemisphere,[16] as evidenced by
the complexation of compounds 10 and 11. 2) The steric
shielding of the “upper hemisphere” by the R1 substituent
directed the incoming iron fragment towards the opposite
face of the ligand, as confirmed by the increased diastereo-
selectivity in the complexation of compounds 16a, 17a,
18a, and 20a. The outcome of the complexation of com-
pound 16b, in which the repulsive effect of the R1 and the
attractive (pre-coordination) effect of the R3 substituent are
opposed to each other, indicates that the pre-coordination is
the dominant effect (Figure 3).
Ph3P=CH
ꢀ908C to ꢀ58C, 4 h; f) NaH, H
CH2=CH(CH)3O(C=O)CH2Cl, RT, 15 min, then compound 15a, RT,
ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
0.5 h. TDS=thexyldimethylsilyl.
compounds 18a and 20a were carried out by employing iso-
lated aldehyde 15a. In the latter case, a Wittig–Horner–
Emmons reagent that was prepared from 4-pentenyl chlor-
oacetate was used.[14]
With a range of dienes of type 4 in hand, we next investi-
gated the complexation of these ligands to Fe(CO)3 by using
Fe2(CO)9 as the reagent of choice.[15] Of particular interest
was the evaluation of the factors that were responsible for
the diastereoselectivity. Diastereomeric ratios (d.r.) were de-
termined by 1H NMR spectroscopy of the mixtures after
column chromatography on silica gel (see below for assign-
ment of the diastereomers). As shown in Table 1, significant
diastereoselectivity in favor of the desired complexes of
It must be mentioned that separation of the diastereomer-
ic complexation products by column chromatography on
silica gel was often difficult, in particular in the case of
ester-substituted compounds, such as 21c/22c, which were
only separable by HPLC. However, for the larger-scale syn-
theses of such complexes, the separation was ach-
ieved by temporary desilylation (TBAF, THF/
water), flash column chromatography on silica gel
Table 1. Complexation of different dienes according to Scheme 4.
Diene Products R1
R2
R3(a) R3(b) 21/22
Yield [%]
of the resulting alcohols, and subsequent re-protec-
tion.[5] More conveniently, separation of the dia-
stereomers was achieved by column chromatogra-
phy on silica gel at a later stage, that is, after the
introduction of the nucleobase.
The main remaining goal was the introduction of
the nucleobase. As our initial studies had shown,
the Fe-assisted nucleophilic substitution reactions
at the acetal center of complexes of type 3 occur-
red with significant levels of diastereoselectivity.[17]
In particular, nucleoside formation by applying the
10
11
16a
16b
17a
18a
21a/22a
21b/22b
21c/22c
H
H
COOEt
H
OBn
OBn
OMe
H
OMe
OMe
H
H
H
3.0:1.0[a] 79
2.1:1.0[a] 77
4.3:1.0[a] 77
CH2OTDS COOEt
21d/22d CH2OTDS COOEt
21e/22e CH2OTDS COOMe
21 f/22 f
OMe 1.0:2.7[a] 65
H
H
3.8:1.0[a] 86
3.3:1.0[a] 72
CH2OTDS
H
19a
20a
21g/22g CH2OTDS
21h/22h CH2OTDS
OMe
OMe
H
H
2.1:1.0[b] 74
4.0:1.0[a] 52
[a] Ratio determined by 1H NMR spectroscopy. [b] Ratio determined after isolation.
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A. Prokop, H.-G. Schmalz et al.
Figure 3. The pre-coordination of the incoming Fe(CO)x reagent to the
alkoxy substituent mainly determines the diastereoselectivity of the com-
plexation step.
Vorbrꢁggen variation of the Hilbert–Johnson method[8] af-
forded mixtures of diastereomeric nucleoside analogues
with a significant preference for the b product. This reaction
can be best rationalized in terms of a SN1-type mechanism,
as shown in Scheme 5. We suggest that the cationic inter-
mediate that is generated from compound 3 is not only sta-
bilized by the anomeric oxygen atom, but also by the buta-
Scheme 5. Reaction of complexes of type 3 with silylated nucleobases
that were derived from cytosine (C*; X=NH, R4 =H), uracil (U*; X=O,
R4 =H), thymine (T*; X=O, R4 =CH3), or 5-bromo-uracil (BrU*; X=O,
R4 =Br) under Lewis acidic conditions (LA, Table 2).
diene Fe(CO)3 fragment.[18] Then, the steric influence of
ꢀ
the iron fragment directs the nucleophilic attack to the
“upper hemisphere”, thereby resulting in the predominant
formation of the desired b diastereomer. The antagonizing
steric effect of the R1 substituent becomes evident by com-
paring the reactions of compounds 21b and 21 f. Whilst
compound 21b reacted under the conditions given in
Table 2 (bis-TMS-uracile, SnCl4, TMS= trimethylsilyl) to
yield compounds 23a and 23b in a 19:1 ratio, the corre-
sponding reaction of compound 21 f (Table 2, entry 2) pro-
duced compounds 24a and 24b in only a 5.6:1 ratio. This
decrease in stereoselectivity for the desired b product can
clearly be attributed to the steric demand of the R1-
CH2OTDS substituent of compound 21 f, which partially im-
pedes the nucleophilic attack from the “upper hemisphere”.
As the various reactions summarized in Table 2 demon-
strate, the b products were generally formed with significant
levels of diastereoselectivity, independent of the nucleophile
and the Lewis acid that were employed. This result indicates
the dominance of the directing effect of the Fe(CO)3 frag-
ment.
The arguments discussed above are supported by the out-
come of reactions that were performed with complex 22c, in
which the Fe(CO)3 fragment and the sterically demanding
silyl group were located in the same (“upper”) hemisphere.
In this case, the Lewis-acid-mediated introduction of uracil
Table 2. Introduction of the nucleobase (NB) according to Scheme 5.
Entry
SM
R1
R2
OR3
NB[a]
Lewis acid (equiv)
T [8C]
Products
b/a
Yield [%]
1
2
3
4
5
6
7
8
9
21b
21 f
21 f
21 f
21c
21c
21c
21c
21c
21e
H
H
H
H
H
OBn
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
U
U
T
C
U
T
BrU
C
C
SnCl4 (2.9)[c]
SnCl4 (2.4)[c]
SnCl4 (3.0)[c]
TMSOTF (6)
SnCl4 (3.4)[c]
SnCl4 (4.5)[c]
SnCl4 (5.6)[c]
SnCl4 (4.5)[c]
TMSOTF (6)
TMSOTF (6)
40
40
40
RT
40
40
40
40
RT
RT
23b/23a
24b/24a
25b/25a
26b/26a
27b/27a
28b/28a
29b/29a
30
19:1[d]
5.6:1[d]
1.6:1[d]
2.3:1[d]
6.6:1[d]
2.4:1[d]
4.5:1[d]
n.d.
99
79
82
96
84
81
72
18
77
67
CH2OTDS
CH2OTDS
CH2OTDS
CH2OTDS
CH2OTDS
CH2OTDS
CH2OTDS
CH2OTDS
CH2OTDS
COOEt
COOEt
COOEt
COOEt
COOEt
COOMe
N69/31a
32b/32a
3.4:1[d]
4.5:1[d]
10
C
11
12
21g
CH2OTDS
CH2OTDS
OMe
OMe
C
C
TMSOTF (6)
TMSOTF (6)
RT
RT
33b/33a
34b/34a
2.3:1[e]
96
57
21h
n.d.[f]
13
14
15
16
17
21i[b]
21j[b]
21k[b]
21l[b]
21m[b]
CH2OAc
CH2OAc
CH2OTBS
CH2OTBS
CH2OTBDPS
COOEt
COOEt
COOEt
COOEt
COOEt
OMe
OMe
OMe
OMe
OMe
C
BrU
C
BrU
C
TMSOTF (6)
SnCl4 (5.5)[c]
TMSOTF (6)
SnCl4 (5.5)[c]
TMSOTF (6)
RT
RT
RT
RT
RT
35b/35a
36b/36a
37b/37a
38b/38a
39b/39a
11.5:1[d]
3.3:1[d]
6.7:1[d]
4.7:1[d]
8.2:1[d]
75
69
77
51
55
18
19
21n[b]
COOEt
COOEt
OMe
OMe
C
C
TMSOTF (6)
TMSOTF (6)
RT
RT
40b/40a
41b/41a
3.5:1[d]
58
85
21o[b]
5.1:1[d]
Reaction conditions: (TMS)2NB, Lewis acid, CH2Cl2, 2 h to 20 h. [a] C=cytosine, U=uracil, T=thymine, BrU=5-bromo-uracil. [b] Complexes 21i–21o
were prepared by the desilylation of compound 21c and subsequent re-protection (see the Supporting Information). [c] SnCl4 was added to the refluxing
reaction mixture over 2 h. [d] d.r. was calculated from the yields of the isolated diastereomers. [e] d.r. determined by 1H NMR spectroscopy. [f] com-
pound 34a was not isolated. n.d.=not determined.
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ꢀ
Nucleoside Analogues with a 1,3-Diene Fe(CO)3 Substructure
FULL PAPER
and thymine proceeded with the complete retention of con-
figuration to give the products 42a and 43a, respectively, in
high yields (Scheme 6).
Scheme 6. Fully diastereoselective introduction of the nucleobase by
using complex 22c.
Scheme 7. Further derivatization of compounds N69 and 32b: a) TBAF,
As Table 2 shows, this method (which was originally de-
veloped during the course of a mechanistic investigation)[10]
was applicable for the efficient introduction of different nu-
cleobases. Thus, a reliable synthetic access to various nucleo-
side analogues of type 1 was established, as a precondition
for investigating the key structural features that are respon-
sible for the biological activity of these compounds. For the
introduction of uracil, 5-bromo-uracil, or thymine, substrates
of type 21 were reacted with the silylated nucleobases by
using SnCl4 as a Lewis acid in refluxing CH2Cl2 to yield the
corresponding nucleosides (as an a/b mixture) in which the
pyrimidine unit was bound through the N-1 atom. Interest-
ingly, attempts to introduce cytosine under these conditions
(Table 1, entry 3) primarily afforded the N-4’-bound cytosine
derivative (30), which was an isomer of N69 (Figure 4).
THF (containing 0.1–0.5% water), RT, 3 h, 37% yield; b) TMANO, tol-
uene, 08C, 1.5 h, 74% yield; c) R2O
ACHTUNGRTNE(NUG C=O)Cl, Et3N, DMAP, CH2Cl2, 08C
to RT, 16 h. TBAF=tetra-n-butylammonium fluoride, TMANO=trime-
thylamine N-oxide, DMAP=4-dimethylaminopyridine.
rivatives of compound 45 required O-5’ functionalization
prior to the introduction of the nucleobase. Thus, starting
from compound 21c, the TDS group was first cleaved off
and the resulting alcohol was reacted with an appropriate
chlorosilane or activated carboxylic acid derivative before
the nucleobase was introduced under the optimized condi-
tions (Table 2, entries 13–19; also see the Supporting Infor-
mation). Remarkably, all of our attempts to cleave the ester
function in compounds N69 or 32b (to give acid 44) com-
pletely failed under a variety of conditions (see the Support-
ing Information). Therefore, the preparation of different
carboxylic acid derivatives (such as amide 33b and ester
34b) required the introduction of these moieties earlier in
the synthesis, that is, at the stage of the Wittig reaction
(Scheme 3). “Metal-free” N69 derivative 46 was prepared
by oxidative decomplexation with TMANO. Finally, inspired
by carbamate-protected nucleoside prodrugs, such as capeci-
tabine,[20] we also prepared some carbamate derivatives of
N69, that is, compounds 47a–47d, by reacting compound
32b with commercially available chloroformates under
standard conditions (Scheme 7).
Figure 4. Structures of the desired cytosine derivative N69 (Table 2,
entry 9) and its N-4’-linked isomer (30; Table 2, entry 8).
ꢀ
However, this undesired reaction pathway could be sup-
pressed by using an excess of TMSOTf instead of SnCl4.
Whilst SnCl4 facilitates desilylation at the N-4’ position, the
beneficial effect of TMSOTf might be associated to its abili-
ty to keep the NH2 group TMS-protected during the reac-
tion, thus guaranteeing the N-1-selective formation of cyto-
sine nucleosides (Table 2, entries 4, 9–13, 15, and 17–19).[19]
For the biological investigations, additional analogues of
lead compound N69 and its methyl ester congener (32b)
were prepared (Scheme 7). Removal of the TDS group pro-
ceeded smoothly with TBAF under standard conditions
(THF, water) to give the C-5’-unprotected nucleoside (45).
It should be mentioned that the synthesis of various O-5’-de-
Configurational assignment of substituted vinyl dihydrofur-
ꢀ
an Fe(CO)3 complexes: The configurational assignment of
ꢀ
various diene Fe(CO)3 complexes, that is, the position of
the Fe(CO)3 fragment with respect to the plane of chirality
(Scheme 1), was not trivial at all and deserves some detailed
discussion. The relative configuration could not be deter-
mined by NMR spectroscopy, although characteristic NMR
patterns were observed for stereochemically related com-
plexes. Therefore, additional analytical methods had to be
employed. We considered circular dichroism (CD) as a suit-
able method, which allows for a credible assignment of the
absolute configuration, at least where reference spectra of
comparable complexes with confirmed configuration are
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A. Prokop, H.-G. Schmalz et al.
available. Martelli, Bolard, and co-workers[21] found a strong
correlation between the absolute configuration of acyclic
(for which no CD-based assignment was possible) was ac-
complished by comparison of the 1H NMR spectra. As
shown in Figure 6, the compounds showed distinct signal
patterns, in particular within the range d=4.5–6.0 ppm.
Thus, the configuration of all of the compounds that were
investigated could be unambiguously assigned.
ꢀ
diene Fe(CO)3 complexes with a terminal electron-with-
drawing substituent (ester, aldehyde, or ketone) and the
sign of the Cotton effects at longer wavelengths (about
400 nm). This relationship allowed us to tentatively assign
the configurations of various complexes of type 21 and 22
with an ester group at the R2 position (Table 3). Whilst all
of the complexes of type 21 showed a positive Cotton effect
at about 400 nm (Table 3, entries 1–13), their diastereomeric
Table 3. Cotton effects (q) of various complexes at about 400 nm.
Entry Complex Type R1
R3(a) R3(b) q (max) l [nm]
1
2
3
4
5
6
7
8
21a
21c
21d
27a
27b
28a
28b
29a
29b
30
31a
N69
21c-1
22a
22c
22d
42a
43a
22c-1
21
21
21
21
21
21
21
21
21
21
21
21
21
22
22
22
22
22
22
H
OBn
H
H
+2793
+1566
395
402
390
398
397
394
397
398
397
384
389
391
394
390
387
395
388
387
384
CH2OTDS OMe
1
Figure 6. Section of the H NMR spectra of complexes of type 21a/b and
CH2OTDS
CH2OTDS
CH2OTDS
CH2OTDS
CH2OTDS
H
U
H
T
OMe +2690
H
U
H
T
H
BrU
C[a]
H
C
H
H
H
22a/b, which show the characteristic patterns of both stereochemical
series.
+1937
+3878
+2000
+3990
+1891
+3140
+1220
+1020
+920
H
CH2OTDS BrU
Synthesis of carbocyclic nucleosides of type 2: Although the
dihydrofuran-derived nucleoside analogues of type 1 were
found to be reasonably stable against air and moisture,[22] we
envisioned that carbocyclic analogues of type 2 might exhib-
it even higher stability. Moreover, carbocyclic nucleosides
often display different biological properties, such as in-
creased resistance towards enzymatic degradation, as well as
decreased toxicity.[23] For the synthesis of carbocyclic nucleo-
sides of type 2, we decided to introduce the Fe(CO)3 frag-
ment in the final step through the diastereoselective com-
plexation of diene precursors of type 54 because these
enoates should be readily prepared through Wittig olefina-
tion reactions.
9
CH2OTDS
CH2OTDS
CH2OTDS
CH2OTDS
CH2OH
H
H
C
H
OMe
OBn
10
11
12
13
14
15
16
17
18
19
+770
H
ꢀ2938
CH2OTDS OMe
ꢀ3689
CH2OTDS
CH2OTDS
CH2OTDS
CH2OH
H
U
T
OMe
OMe ꢀ620
H
H
H
ꢀ4267
ꢀ4520
ꢀ1740
[a] The cytosine was bound at the N-4 position.
counterparts (22) exhibited a negative Cotton effect within
the same range (Table 3, entries 14–19). Conclusive evidence
was obtained from the crystal structure of desilylated com-
plex 22c-1 (Figure 5).
The stereostructural assignment of complexes that lacked
an electron-withdrawing ester substituent on the diene unit
A synthesis of the required aldehyde precursors (53) has
previously been developed in our laboratories and shall only
be discussed briefly here (Scheme 8).[24] This synthesis start-
ed with the PCC oxidation of C-silylated propargyl alcohol
ꢀ
48 and the subsequent formation of diallyl acetal 49, which
was further transformed into furan derivative 50 through a
Pauson–Khand reaction[25] and subsequent kinetic resolution
by means of CBS reduction.[26] After the Luche reduction of
compound 50,[27] alkoxide-induced desilylation, and acetyla-
tion (to afford compound 51), the diastereoselective intro-
Figure 5. Crystal structure of compound 22c-1, which was obtained after
the desilylation of complex 22c.
Scheme 8. Projected synthesis of carbocyclic nucleoside analogues of
type 2 through diastereoselective complexation.
13022
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Chem. Eur. J. 2013, 19, 13017 – 13029

ꢀ
Nucleoside Analogues with a 1,3-Diene Fe(CO)3 Substructure
FULL PAPER
duction of the nucleobase was achieved through a Pd-cata-
lyzed allylic substitution reaction. Finally, the resulting com-
pounds of type 52 were converted into aldehydes 53 through
acid acetal hydrolysis and subsequent silylation of the OH
function (Scheme 9).
Scheme 11. Synthesis of nucleosides of type 2 continued: a) Ph3P=
CHCOOEt
(1.2 equiv),
THF,
508C,
5 h,
to
RT,
16 h;
b) (EtO)2OPCH2COOEt (1.75 equiv), nBuLi (1.55 equiv in hexanes),
THF, 08C, then compound 53, 08C to RT, 16 h; c) Fe2(CO)9, Et2O, reflux,
24 h.
Scheme 9. Synthesis of the aldehyde precursors of carbocyclic nucleosides
of type 2: a) PCC (1.5 equiv), CH2Cl2, RT, 3 h; b) allyl-OH (excess), p-
TsOH (cat.), benzene, reflux, 15 h, 88% yield from compound 48;
c) [Co2(CO)8] (1.1 equiv), CH2Cl2, 4 ꢃ molecular sieves, RT, 2 h, then
TMANO (8.8 equiv), air, 08C to RT, 15 h, 76% yield; d) kinetic resolu-
tion through CBS reduction (34% yield from compound 50); e) NaBH4,
CeCl3, MeOH, 08C, 0.5 h, quantitative yield; f) tBuOK, DMSO/water
(19:1), RT, 1 h, 87% yield; g) Ac2O, Et3N, DMAP (cat.), CH2Cl2, RT, 1 h,
99% yield; h) NB or derivative, Pd0 (cat.); i) p-TsOH, acetone/water,
reflux, 3 h; j) TDSCl, (1.5 equiv), pyridine, RT, 16 h. PCC=pyridinium
chlorochromate.
Table 4. Compounds that were synthesized according to Scheme 10.
Series
R
X
Wittig
Yield of
Yield of Yield of
d.r.
(2/55)
method[a] 54 [%]
2 [%]
55 [%]
a
b
c
d
e-1
e
H
F
OH
OH
A
A
A
A
B
–
99
95
99
99
47
57
73
57
55
42
17
27
20
n.d.
n.d.
n.d.
2.8:1
2.2:1
3.8:1
n.d.
n.d.
n.d.
Br OH
Me OH
H
H
NHBz
NH2
81
98[b]
[a] Method A:
Ph3P=CHCOOEt,
THF,
508C;
method B:
(EtO)2OPCH2COOEt, nBuLi in hexanes, THF, 08C to RT. [b] Com-
pound 54e was prepared from compound 54e-1 (2m NH3 in water,
40 equiv, RT, 16 h).
The Wittig olefination of aldehydes 53 to afford their cor-
responding enoates (54) proceeded smoothly. The final com-
plexation was performed with Fe2(CO)9 to give the desired
nucleosides of type 2 with a decent degree of diastereoselec-
tivity (Scheme 10, Scheme 11, Table 4, and Table 5). Howev-
er, the diastereomeric by-products (55) could be separated
off by means of flash column chromatography on silica gel
in all cases. Because the kinetic resolution of compound rac-
50 gave rise to both enantiomers, we also conducted the syn-
thesis of a set of “unnatural”, that is enantiomeric target nu-
cleosides (ent-2).[28] In most cases, Wittig olefination of the
aldehydes (53) was efficiently achieved by using the com-
mercial reagent Ph3P=CH2COOEt (method A).
However, in the case of (protected) cytosine- and ade-
nine-derivatives 53e and 53g (Scheme 10, Scheme 11,
Table 4, and Table 5), a Wittig–Horner–Emmons procedure
(method B) was applied to circumvent the difficult removal
of triphenylphosphine-oxide contaminants from the products
(54e-1 and 54g-1, respectively). Not unexpectedly, the com-
plexation of dienes of type 54 occurred preferentially from
the less-hindered hemisphere (d.r. between 2.2:1 and 3.8:1).
Table 5. Compounds that were synthesized according to Scheme 11.
Series R2
R2
Wittig
Yield of Yield of Yield of d.r.
method[a] 54 [%]
2 [%]
55 [%]
ACHTUNGTRENNUNG(2/55)
f (rac) NH2
g-1
g
H
B
A
–
99
45
28
25
11
n.d.
11
4.1:1
n.d.
2.3:1
R2[b] NHAc
OH NH2
95
99[c]
Scheme 10. Synthesis of carbocyclic nucleosides of type 2: a) Ph3P=
[a] Method A:
Ph3P=CHCOOEt,
THF;
method B:
CHCOOEt
(1.2 equiv),
THF,
508C,
5 h,
to
RT,
16 h;
(EtO)2OPCH2COOEt, nBuLi in hexanes, THF. [b] For compounds 53g-1
and 54g-1, R1 =OCbz; for compounds 2g-1 and 55g-1, R1 =OH.
[c] Compound 54g was prepared from compound 54g-1 (2m NH3 in
water, 40 equiv, RT, 16 h). Cbz=carboxybenzyl.
b) (EtO)2OPCH2COOEt (1.75 equiv), nBuLi (1.55 equiv in hexanes),
THF, 08C, then compound 53, 08C to RT, 16 h; c) Fe2(CO)9, Et2O, reflux,
24 h.
Chem. Eur. J. 2013, 19, 13017 – 13029
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13023

A. Prokop, H.-G. Schmalz et al.
Uracil-nucleoside analogues 2a–2d were obtained in 40–
70% yield after purification; minor diastereomers 55a–55d
were also isolated in most cases. For the synthesis of cyto-
sine-derivative 2e, we employed benzoyl-protected deriva-
tive 53e,[29] which was olefinated to give compound 54e-1.
However, after complexation to give compound 2e-1, nucle-
ophilic deprotection with ethoxide[30] was unsuccessful. Nev-
ertheless, diene 54e-1 could be deprotected in quantitative
yield by using ammonia (2m in MeOH, 16 h, RT), thus de-
livering deprotected compound 54e, which, on complexa-
tion, afforded the desired nucleoside analogue (2e) in 42%
yield.
A related strategy was employed in the synthesis of gua-
nine derivative 2g. In this case, the deprotection of diene
precursor 54g-1 was again achieved with ammonia. Then,
subsequent complexation afforded the desired product (2g)
in only 25% yield. The synthesis of the corresponding ade-
nine derivative was conducted both in the racemic (rac-2 f)
and the enantiomerically pure series (ent-2 f).
associated to Cotton effects at about 390 nm confirmed the
configurational assignments.
NMR spectroscopy was used as a second method to probe
the configurational assignments. As observed in the furanose
series, diastereomeric complexes of type 2 and type 55
1
showed characteristic differences in their H NMR spectra
(Figure 8). In compounds of type 2, the Fe(CO)3 fragment
distorts the diene ligand in such a fashion that the olefinic
H-2’ proton moves out of the plane, thereby increasing the
ꢀ
ꢀ
dihedral angle between the C H-2 and C H-3’ bonds to ap-
proximately 908. Consequently, no coupling between these
two protons is observed, as shown for compound 2c
(Figure 8, spectrum 1). Owing to the lack of other coupling
partners, the H-2’ atom (d=1.85 ppm) is observed as a sin-
glet and the H-3’ atom (d=5.09 ppm) as a doublet of dou-
blets (dd). In the case of the corresponding diastereomer
(55c), the angle between the H-2’ and H-3’ atoms is smaller
than 908 (Figure 8, Spectrum 2), which results in significant
coupling (4.2 Hz) between the H-2’ and H-3’ signals. Ac-
cordingly, the H-2’ signal appears as a doublet and the H-3’
signal appears as a doublet of doublet of doublets (ddd).[31]
Configurational assignment of nucleoside analogues of
Type 2: The assigned relative configuration of these com-
plexes was confirmed by CD spectroscopy, in comparison
with the data for the dihydrofuran series (see above). Be-
cause the methylene group in the carbocyclic compounds
was isosteric with the oxygen atom in the corresponding fur-
anoids, related chiroptical properties were expected. Indeed,
the recorded CD spectra of compounds 2a and 2c–2e were
similar to those of their corresponding dihydrofuran deriva-
tives, as shown in Figure 7. Moreover, in the cases of fluo-
rouracil derivative 2b and purines ent-2 f and 2g (for which
no reference data were available), the characteristic curves
Cytotoxicity data and structure–activity relationships: The
cytotoxic properties of the various iron-containing nucleo-
side analogues were tested in vitro by using an established
cell assay. For this purpose, Burkitt-like lymphoma (BJAB)
cells were grown in a culture medium that contained fetal
calf serum. After incubation with the potential cytotoxic
agents for 48 h, the release of lactate dehydrogenase (LDH)
was measured as a quantitative indicator of deceased
cells.[32]
A comparison of the LD50 values of various nucleoside
analogues of type 1 (Table 6) indicated certain structural
features that were connected to potent cytotoxicity: Firstly,
a lipophilic R1 substituent seemed to be necessary. Whereas
other silyl ether congeners of N69, such as compounds 37b
and 39b, still showed significant cytotoxic activity, depro-
tected derivative 45, as well as ester-substituted analogues
35b, 40b, and 41b, were practically inactive. Secondly, the
R2 substituent appeared to be a variable position tolerant of
various substituents, because all of the tested N69 deriva-
tives that were modified at this position (26b, 32b, 33b,
34b) exhibited cytotoxicity, although morpholine derivative
33b was less active. Thirdly, the use of cytosine as a nucleo-
base gave rise to the highest cytotoxicities (which was why
most of the compounds that were prepared were cytosine
derivatives). However, the functionalization of the cytosine
amino group as a carbamate resulted in a loss of activity. As
we had reported previously,[5] decomplexation (removal of
the Fe(CO)3 fragment) also led to a greatly diminished ac-
tivity. Closer investigations were undertaken for N69, as the
most potent compound of type 1, and for morpholine deriv-
ative 33b, as a compound with improved water solubility.
The cytostatic activity of these compounds, as determined
by counting the living cells (again by using BJAB cells), was
significant in both cases, with N69 several times more active
than compound 33b (Figure 9). In addition, the apoptosis-
Figure 7. CD spectra of compounds 29b (spectrum 1) and 2c (spectrum
2). The positive Cotton effect at about 390 nm is indicative of the abso-
lute configuration shown (cf. Table 3).
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ꢀ
Nucleoside Analogues with a 1,3-Diene Fe(CO)3 Substructure
FULL PAPER
enantiomers (ent-2) in compar-
ison to their “natural” counter-
parts. This difference was the
most pronounced in the case of
fluorouracil
derivative
2b
(Table 7, entries 3 and 4). Both
purine derivatives, that is, com-
pounds 2 f and ent-2g, also
showed significant cytotoxic
activity (Table 7, entries 12
and 13).[33] DNA-fragmentation
was quantified for compounds
2a, ent-2a, 2c, 2d, and ent-2d
at selected concentrations after
72 h. The values that were ob-
tained corresponded to their
respective cytotoxicity levels,
thereby confirming that cell
death was due to apoptosis in
these cases as well.
Synthesis of N69 conjugates:
The data presented above
demonstrate the biological po-
tential of N69 and its related
Fe-containing nucleoside ana-
logues. Most interestingly, the
clear structure–activity rela-
tionships (including stereo-
chemical aspects) suggest that
these compounds do not act as
Figure 8. 1H NMR spectra of compounds 2c (spectrum 1) and 55c (spectrum 2) and conformational analysis.
“normal”
nucleoside
ana-
logues. Therefore, it is likely
inducing activity of these two compounds was demonstrated
(and quantified) by measuring the DNA fragmentation after
72 h. These results were in accordance with characteristic
morphological changes (blebbing) that were observed by mi-
croscopy. In the case of N69, Western Blot analysis also
showed that apoptosis induction was connected with proteo-
lytic processing and activation of caspase-3. Furthermore,
we clearly demonstrated that N69 neither decreased the via-
bility of healthy, primary leukocytes nor induced apoptosis,
as determined by DNA fragmentation in NIH 3T3 fibro-
blasts (data not shown). Thus, nucleoside analogues that
that the compounds act through an (unknown) protein-
based mechanism, which constitutes a challenge to future bi-
ological and biochemical investigations to elucidate the pro-
tein targets that are involved. Against this background, suit-
able procedures for the synthesis of labeled derivatives have
been developed. Based on the structure–activity relation-
ships discussed above, N69 conjugates of type 56 with biotin,
fluorescein, or 7-dimethylaminocoumarin-4-acetic acid
(DMAC)[34] have emerged as desirable targets, in which the
label is attached through a flexible alkyl linker onto the
ester unit of the N69 core. As indicated in Figure 10, three
synthetic approaches were evaluated. Initially, it seemed fea-
sible to synthesize compounds of type 56 directly from N69
or its methyl-ester analog (32b) by cleavage of the ester and
subsequent coupling with a labeled linker. However, we did
not succeed in obtaining the free acid (44, Scheme 7; for the
tested conditions, see the Supporting Information). Also, all
attempts to synthesize conjugates of type 56 by cross-meta-
thesis (by employing pentenyl ester 34b) and subsequent hy-
drogenation failed (see the Supporting Information). How-
ever, the synthesis of the labeled derivatives (56, n=0) was
achieved by introducing a masked 1,6-aminohexanol linker
early in the synthesis (Scheme 12).
ꢀ
possess a characteristic butadiene Fe(CO)3 substructure are
promising candidates for the development of iron-containing
anti-cancer drugs with pro-apoptotic activity.
The cytotoxic properties of a series of carbocyclic com-
pounds of type 2 (and their enantiomers ent-2) were also de-
termined. The results (Table 7) reveal that the “natural”
enantiomers (2) showed similar cytotoxic properties to their
oxacyclic congeners. Again, cytosine derivative 2e (Table 7,
entry 9) was more potent than the thymine-, uracil-, and
halo-uracil-substituted compounds, although it was less
potent than N69. Again, functionalization of the cytosine
amine (2e-1; Table 7, entry 11) quenched its activity. A no-
table result was the increased activity of the “unnatural”
Chem. Eur. J. 2013, 19, 13017 – 13029
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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13025

A. Prokop, H.-G. Schmalz et al.
Table 6. Cytotoxicity of nucleoside analogues of type 1 against BJAB cells.
Phthalimide- and tetrachlorophthalimide (TCP)-
protected linkers 57a and 57b were transformed
into the corresponding Wittig salts and coupled
with aldehyde 15b to yield dienes 58a and 58b, re-
spectively. Following the established route, these
dienes were reacted with Fe2(CO)9 to yield com-
plexes of type 59, which were subsequently trans-
formed into the corresponding nucleoside ana-
logues of type 60. Although the deprotection of
the TCP derivative 60b with 1,2-diaminoethane
did deliver the desired amine (61), contamination
of the product with excess 1,2-diaminoethane pre-
sented a problem. However, the facile deprotection
of phthalimide derivative 60a with hydrazine in
EtOH at 608C yielded compound 61 in high purity
(75% yield after aqueous workup). Labeling with
biotin (56a) and DMAC (56c) was achieved under
standard peptide-coupling conditions. The reaction
of compound 61 with fluorescein isothiocyanate
(FITC) in DMF directly afforded compound 56b.
The regioselectivity of these reactions was con-
firmed by heteronuclear multiple-bond correlation
(HMBC) spectroscopy, which indicated that no by-
products had been formed that would result from
electrophile attack at the (less nucleophilic) cyto-
sine-NH2 function. Thus, reliable procedures for
the synthesis of labeled N69 derivatives have been
established, which constitute a valid basis for
future biological studies. An initial cytotoxicity
assay revealed pleasing activity for biotin conju-
gate 56a (LD50 =17 mm), whilst fluorescein deriva-
tive 56b and the (much-smaller and less-polar)
DMAC conjugate 56c were inactive.[35]
[c]
Entry Compound R1
R2
NB
LD50[b] [mm] LD50 [mm]
1
2
3
N69
32b
26b
CH2OTDS
CH2OTDS
CH2OTDS
COOEt
COOMe
H
C
C
C
10
–
18
18
24
–
4
5
33b
34b
CH2OTDS
C
C
88
–
91
23
CH2OTDS
CH2OTBS
CH2OTBDPS COOEt
CH2OAc
6
7
8
37b
39b
35b
COOEt
C
C
C
30
14
>100
–
–
–
COOEt
9
40b
41b
COOEt
C
C
>100
>100
–
–
10
COOEt
11
12
13
14
15
16
17
18
45
CH2OH
COOEt
COOEt
COOEt
COOEt
COOMe
COOMe
COOMe
COOMe
C
>100
>100
72
–
–
–
–
–
–
–
–
29b
38b
28b
47a
47b
47c
47d
CH2OTDS
CH2OTBS
CH2OTDS
CH2OTBS
CH2OTBS
CH2OTBS
CH2OTBS
BrU
BrU
T
>100
C-a[a] >100
C-b[a] >100
C-c[a] >100
C-d[a] >100
[a] Carbamoyl-protected cytosine (c.f. Scheme 7). [b] based on LDH concentration
after 48 h. [c] based on DNA fragmentation after 72 h. TBS=tert-butyldimethylsilyl.
Conclusion
As a contribution to the field
of bio-organometallic chemis-
try, we have developed reliable
schemes for the synthesis of
enantiomerically pure nucleo-
side analogues that possess a
ꢀ
characteristic
butadiene
Fe(CO)3 substructure. The
stereoselective generation of
ꢀ
the planar chiral butadiene
Fe(CO)3 unit was achieved by
the diastereoselective com-
plexation of appropriate chiral
diene ligands, governed both
by (repulsive) steric factors
and by attractive pre-coordina-
tion effects. The introduction
of the nucleobase also pro-
ceeded with good-to-excellent
levels of diastereoselectivity
Figure 9. Cytostatic activity of compounds N69 and 33b, as well as N69-induced cleavage of caspase-3.
13026
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Chem. Eur. J. 2013, 19, 13017 – 13029

ꢀ
Nucleoside Analogues with a 1,3-Diene Fe(CO)3 Substructure
FULL PAPER
Table 7. Cytotoxicity of carbocyclic Fe-containing nucleoside analogues
of type 2.
Entry
Compound
Type
NB
LD50[a] [mm]
1
2
3
4
5
6
7
2a
ent-2a
2b
ent-2b
2c
2
ent-2
2
ent-2
2
U
U
54
23
>100
28
>100
81
FU
FU
BrU
BrU
T
ent-2c
2d
ent-2
2
79
8
9
ent-2d
2e
ent-2
2
T
C
33
40
10
11
12
13
ent-2e
2e-1
2 f
ent-2
2
C
Bz-C
G
39
>100
28
2
ent-2g
ent-2
A
32
[a] Based on LDH release after 48 h. FU = 5-fluoro-1H,3H-pyrimidine-
2,4-dione BrU = 5-bromo-1H,3H-pyrimidine-2,4-dione.
Scheme 12. Synthesis of conjugates of N69 with different molecular
labels: a) PPh3 (1.1–1.4 equiv), toluene, RT, 0.5–4 days, for R=H: 66%
yield, for R=Cl: 95% yield; b) (i) Wittig salt (1.8 equiv–2.0 equiv),
LiHMDS (1.3 equiv–1.7 equiv), THF, RT, 0.5 h, (ii) compound 15a
(1 equiv), reflux, 2 h, for R=H: 95% yield, for R=H: 85% yield;
c) (i) Fe2(CO)9 (1.3 equiv), Et2O, RT 2 h, reflux, 2 h, for R=H: 77%
yield, d.r. 4.0:1, for R=Cl: 71% yield, d.r. 3.1:1 (diastereomers not sepa-
rated); (ii) isolation of compound 59a by temporary desilylation: TBAF
(1.2 equiv), THF (containing 0.25% water), ꢀ158C to 108C, 3 h, 92%
yield; (iii) TDSCl, pyridine, 08C to RT, 12 h, 89% yield; d) (TMS)2Cyt
(4 equiv), TMSOTf (6 equiv), CH2Cl2, RT, 5–24 h, for R=H: 60% yield,
ꢀ
for R=Cl: 27% yield; e) for R=H: H2N NH2, EtOH, 608C, 2 h, 75%
yield; f) for R=Cl: (H2NCH2)2 (4 equiv), EtOH/THF (1.3:1), 608C;
g) biotin, EDCI·HCl (2 equiv), HOBT, DiPEA (2 equiv), DMF, 08 to RT
overnight, 63% yield; h) FITC, DMF, ꢀ108C to 88C overnight, 84%
yield; i) DMAC-carboxylic acid, HBTU (2 equiv), DiPEA (2 equiv),
DMF, ꢀ108C to RT overnight, 36% yield. LiHMDS=lithium bis(trime-
thylsilyl)amide,
TMSOTf=trimethylsilyl
trifluoromethanesulfonate,
HOBT=hy-
Figure 10. N69 conjugates with biological labels and our attempted syn-
thetic approaches (DMAC=7-dimethylaminocoumarine).
EDCI=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,
droxybenzotriazole, DiPEA=N,N-diisopropylethylamine, HBTU=2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate.
from the face opposite the bulky Fe(CO)3 fragment. The
relative configuration of the iron-complexes was assigned by
means of NMR and CD spectroscopy and confirmed
through X-ray crystallography in one case.
ing was exploited in the synthesis of labeled N69 derivatives
in which a fluorescent or a biotin unit was connected to the
ether function through a 6-aminohexyl linker. Therefore, we
have paved the way for further biological investigations of
N69 and related iron-containing nucleoside analogues, with
the aim of identifying molecular targets and other aspects of
their clearly unusual mechanism of action.
Whilst the lead compound, that is, N69, had previously
been shown to exhibit pronounced apoptosis-inducing prop-
erties (even against highly resistant tumor cells), cytotoxicity
studies of the various complexes that were prepared during
the course of this study revealed that compounds of both
series (i.e., compounds of type 1 and their carbocyclic ana-
logues of type 2) were able to induce apoptosis in BJAB
tumor cells, in particular if cytosine was present as a nucleo-
base. Also, a lipophilic substituent at the R1 position was
shown to be essential for activity. In contrast, variation of
the substituent at the R2 position was possible and this find-
Acknowledgements
This work was financially supported by the Deutsche Forschungsgemein-
schaft (DFG, FOR-630).
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A. Prokop, H.-G. Schmalz et al.
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ꢀ
Nucleoside Analogues with a 1,3-Diene Fe(CO)3 Substructure
FULL PAPER
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be significantly active. The synthesis (through cross-metathesis) af-
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Supporting Information).
[33] Another noteworthy observation connected to the absolute stereo-
chemistry of these compounds is the sometimes significantly lower
Received: May 1, 2013
Published online: August 9, 2013
Chem. Eur. J. 2013, 19, 13017 – 13029
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
13029