Synthesis and preliminary cytotoxicity studies of achiral indolyl-substituted titanocenes

Article abstract of DOI:10.1021/om901031m

From the reaction of various 6-indolylfulvenes (la-f) with Super Hydride (LiBEt₃H), followed by transmetalation with titanium tetrachloride (TiCl₄), six indolyl-substituted titanocenes, bis[( 1 -methylindol-2-yl)cyclopentadienyl]titanium(IV) dichloride (3a), bis[( 1 -methyl-5-methoxyindol-2-yl)cyclopentadienyl]titanium(IV) dichloride (3b), a dihydrochloride derivative of bis[(l-methyl3-dimethylaminomethylindol-2-yl) cyclopentadienyl]titanium(IV) dichloride (3c), bis[(l-methylindol-3yl) cyclopentadienyl]titanium(IV) dichloride (3d), bis[(l-methyl-5-methoxyindol-3- yl)cyclopentadienyl]titanium(IV) dichloride (3e), and bis[(l-methylmethoxyindol- 3-yl)cyclopentadienyl]titanium(IV) dichloride (3f), were obtained. The six titanocenes 3a-f were tested for their cytotoxicity through MTT-based in vitro tests on CAKI-I cell lines in order to determine their IC₅₀ values. Titanocenes 3a-f were found to have IC₅₀ values of 47 (±9), 15 (±2), 8.2 (±1.9), 21 (±5), 11 (±1), and 170 (±40) μM, respectively.

Full text of DOI:10.1021/om901031m

1
032 Organometallics 2010, 29, 1032–1040
DOI: 10.1021/om901031m
Synthesis and Preliminary Cytotoxicity Studies of Achiral
Indolyl-Substituted Titanocenes
Megan Hogan, Brendan Gleeson, and Matthias Tacke*
UCD School of Chemistry and Chemical Biology, Conway Institute of Biomolecular and
Biomedical Research, Centre for Synthesis and Chemical Biology (CSCB),
University College Dublin, Belfield, Dublin 4, Ireland
Received November 30, 2009
From the reaction of various 6-indolylfulvenes (1a-f) with Super Hydride (LiBEt H), follo-
3
wed by transmetalation with titanium tetrachloride (TiCl ), six indolyl-substituted titanocenes,
bis[(1-methylindol-2-yl)cyclopentadienyl]titanium(IV) dichloride (3a), bis[(1-methyl-5-methoxy-
indol-2-yl)cyclopentadienyl]titanium(IV) dichloride (3b), a dihydrochloride derivative of bis[(1-methyl-
4
3-dimethylaminomethylindol-2-yl)cyclopentadienyl]titanium(IV) dichloride (3c), bis[(1-methylindol-3-
yl)cyclopentadienyl]titanium(IV) dichloride (3d), bis[(1-methyl-5-methoxyindol-3-yl)cyclopentadienyl]-
titanium(IV) dichloride (3e), and bis[(1-methylmethoxyindol-3-yl)cyclopentadienyl]titanium(IV)
dichloride (3f), were obtained. The six titanocenes 3a-f were tested for their cytotoxicity through
MTT-based in vitro tests on CAKI-1 cell lines in order to determine their IC50 values. Titano-
cenes 3a-f were found to have IC50 values of 47 ((9), 15 ((2), 8.2 ((1.9), 21 ((5), 11 ((1), and
1
70 ((40) μM, respectively.
7
1. Introduction
clinical trials in patients with metastatic renal cell carcinoma
8
or metastatic breast cancer and so was not pursued.
Although the idea of metal-based drugs is by no means
1
novel, there is still a certain prejudice against their use in the
€
€
Decades after Kopf and Kopf-Maier discovered the chemo-
therapeutic potential of these metallocene dihalides, interest
in the field was renewed with McGowan’s elegant synthesis
of ring-substituted cationic titanocene dichloride deriva-
tives, which are water-soluble and show significant activity
2
clinic. Compounds such as ferroquin and NAMI-A
3
ImH[trans-ImDMSORuCl ]) have received some recent
(
4
attention; however the field of medicinal chemistry is still
dominated by organic molecules. While cisplatin and deri-
vatives thereof remain one of the few great success stories in
the field of metal-based cancer chemotherapy, there is still
considerable opportunity for the advancement of organo-
metallic drugs targeting cancer. For instance, titanium-based
reagents have significant potential against solid tumors. In
fact, budotitane ([cis-diethoxybis(1-phenylbutane-1,3-dionato)-
titanium(IV)]) looked very promising during its preclinical
evaluation but did not go beyond phase I clinical trials,
although a Cremophor EL-based formulation was found for
9
against ovarian cancer. More recently, novel methods start-
1
0-17
18-20
ing from fulvenes
and other precursors
allow direct
access to substituted titanocenes via reductive dimeriza-
tion with titanium dichloride or else carbolithiation or
hydridolithiation of the fulvene followed by transmetalation
with titanium tetrachloride.
(8) Kr o€ ger, N.; Kleeberg, U. R.; Mross, K. B.; Edler, L.; Sass, G.;
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Mendoza, O.; M u€ ller-Bunz, H. J. Organomet. Chem. 2004, 689, 2242.
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(
4
this rapidly hydrolyzing molecule. Much more robust in this
aspect of hydrolysis is titanocene dichloride (Cp TiCl ),
(
2
2
which showed medium antiproliferative activity in vitro,
5
oꢀ n, C.;
,6
but promising results in vivo. Titanocene dichloride
reached clinical trials, but showed low efficacy in phase II
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(
14) Strohfeldt, K.; M u€ ller-Bunz, H.; Pampill oꢀ n, C.; Sweeney, N. J.;
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pubs.acs.org/Organometallics Published on Web 01/28/2010
r 2010 American Chemical Society

Article
Titanocene Y (bis[(p-methoxybenzyl)cyclopentadienyl]tita-
Organometallics, Vol. 29, No. 4, 2010 1033
nium(IV) dichloride), which has an IC50 value of 21 μM
when tested on the LLC-PK cell line (long lasting cell-pig
1
2
kidney epithelial “carcinoma”) and 30 μM against the
human renal cell carcinoma CAKI-1 cell line, was synthe-
sized through hydridolithiation of p-anisylfulvene and Super
Hydride (LiBEt H) followed by transmetalation with tita-
3
nium tetrachloride (TiCl ). The structure of titanocene Y can
4
Figure 1. Structure of titanocene Y.
be seen in Figure 1. The antiproliferative activity of titano-
2
cene Y has been studied in 36 human tumor cell lines and in
1
numerous potential pharmaceutical applications have been
investigated for this class of heterocyclic compounds in recent
years.
2
2
explanted human tumors. These in vitro and ex vivo
experiments showed that prostate, cervix, and renal cell
cancer are prime targets for these novel classes of titano-
cenes, and the IC50 values for the breast-cancer cell lines were
very promising. These results were underlined by first mecha-
nistic studies concerning the effect of these titanocenes on
apoptosis and the apoptotic pathway in prostate cancer
3
2
The main idea behind the research presented in this work
was to evaluate the cytotoxic effects of indolyl-substituted
achiral titanocenes with respect to lead compound titano-
cene Y, as well as to investigate the differences in cytotoxi-
cities when slight structural modifications are made to the
indolyl moiety. In order to avoid ambiguities in the cytotoxic
profiles of the compounds in vitro, the titanocene derivatives
presented are achiral, and thus issues regarding isomeric
mixtures are not of concern.
2
3
cells. Furthermore, first animal studies have reported
that xenografted Ehrlich’s ascites tumors in mice could
be successfully treated upon administration of an ansa-
2
4
25
26
titanocene, and likewise, xenografted CAKI-1, A431,
2
7
and MCF-7 tumors in mice were successfully treated upon
administration of titanocene Y. In fact, the treatment of
titanocene Y against xenografted CAKI-1 tumors in mice
was shown to be more effective than that of cisplatin, and
what’s more, in the MCF-7 experiment, the tumor size was
reduced following treatment with titanocene Y.
2
. Results and Discussions
2.1. Synthesis. The synthesis of methoxy-substituted in-
doles was achieved by the N-alkylation with the corresponding
alkyl halide, and 1-methyl-3-dimethylaminomethylindole
was synthesized by a Mannich reaction with formaldehyde
3
3
In an alternative synthesis, by means of the carbolithiation
of 6-(dimethylamino)fulvene with lithiated aryls or hetero-
3
4
and dimethylamine.
2
8
aryls, titanocene C and others
29-31
The synthesis of indole 2-carboxaldehydes was achieved
by ortho-lithiation of the heterocyclic precursor compound
with n-BuLi followed by formylation with dimethylforma-
mide (DMF), while the synthesis of indole 3-carboxalde-
hydes was accomplished by the Vilsmeier-Haack reaction
with phosphorus oxychloride and DMF.
The synthesis of fulvenes 1a-f is based on a modified
version of the Stone and Little method and described in
ref 11; the corresponding aldehyde was reacted with cyclo-
pentadiene in the presence of pyrrolidine as a base catalyst to
yield the desired product in yields of 47-100%. Their struc-
tures are shown in Figure 2.
were obtained and
showed IC50 values as low as 5.4 μM when tested on the
LLC-PK cell line. This was significant progress, since
Cp TiCl exhibits an IC value of only 2000 μM against
3
5
2
2
50
1
1
LLC-PK, which explains partly the failed phase II clinical
trials against renal cell carcinoma. While the mode of action
of these compounds is still unclear, it is evident (by their IC50
values) that the substitution of the cyclopentadienyl (Cp)
ring with the aryl or heteroaryl moiety affects their cytotoxic
activity. By substituting the Cp ring with a molecule that
already has proven biological activity, it is hoped to syner-
gistically enhance the activity of the titanocene derivative,
and even create a “biomolecule mimic”. Titanocenes that
include an indolyl moiety may be of particular interest, as
3
6
37
Using the method described in ref 12 and depicted in
Schemes 1 and 2 these fulvenes were reacted with LiBEt H
3
to obtain the correspondingly functionalized lithium cyclo-
pentadienide intermediates 2a-f. The exocyclic double
bonds in the fulvenes have increased polarity due to the
inductive effects of their respective indolyl groups. This
increased polarity allows for selective nucleophilic attack at
this double bond and not at the diene component of the
fulvenes. Two equivalents of the lithium cyclopentadienide
intermediate were then transmetalated with one equivalent
(
21) Kelter, G.; Sweeney, N.; Strohfeldt, K.; Fiebig, H. H.; Tacke, M.
Anti-Cancer Drugs 2005, 16, 1091–1098.
22) Oberschmidt, O.; Hanauske, A. R.; Rehmann, F. J. K.; Strohfeldt,
K.; Sweeney, N.; Tacke, M. Anti-Cancer Drugs 2005, 16, 1071–1073.
23) O’Connnor, K.; Gill, C.; Tacke, M.; Rehmann, F. J. K.; Strohfeldt,
K.; Sweeney, N.; Fitzpatrick, J. M.; Watson, R. W. G. Apoptosis 2006, 11,
205.
24) Valaderes, M. C.; Ramos, A. L.; Rehmann, F. J. K.; Sweeney,
N.; Strohfeldt, K.; Tacke, M. Eur. J. Pharmacol. 2006, 534, 26.
25) Fichtner, I.; Pampill oꢀ n, C.; Sweeney, N. J.; Strohfeldt, K.;
Tacke, M. Anti-Cancer Drugs 2006, 17, 333–336.
26) Fichtner, I.; Bannon, J. H.; O’Neill, A.; Pampill oꢀ n, C.; Sweeney,
(
(
1
(
of TiCl , resulting in the formation of one equivalent of the
4
(
appropriately indolyl-substituted titanocene 3a-f (Figure 3)
in yields of 22-91% and the byproduct lithium chloride
following a 16 h reflux.
(
N. J.; Strohfeldt, K.; Watson, R. W. G.; Tacke, M.; McGee, M. M. Br. J.
Cancer 2007, 97, 1234.
(
27) Beckhove, P.; Hanauske, A. R.; Oberschmidt, O.; Pampill oꢀ n, C.;
Schirrmacher, V.; Sweeney, N. J.; Strohfeldt, K.; Tacke, M. Anti-Cancer
Drugs 2007, 18, 311–315.
(32) Aggarwal, B.; Ichikawa, H. Cell Cycle 2005, 4 (9), 1201–1215.
(33) Chakrabarty, M.; Basak, R.; Hargaya, Y.; Takayanagi, H.
Tetrahedron 2005, 61 (7), 1793–1801.
(34) Snyder, H. R.; Eliel, E. L. J. Am. Chem. Soc. 1948, 70 (5), 1703–
1705.
(35) Comins, D. L.; Killpack, M. O. J. Org. Chem. 1987, 52, 104–109.
(36) Everett, S. A.; Naylor, M. A.; Stratford, M. R. L.; Patel, K. B.;
Ford, E.; Mortensen, A.; Ferguson, A. C.; Vojnovic, B. J. Chem. Soc.,
Perkin Trans. 2 2001, 1989–1997.
(
28) Pampill oꢀ n, C.; Sweeney, N.; Strohfeldt, K.; Tacke, M. J. Orga-
nomet. Chem. 2007, 692, 2153.
29) Pampill oꢀ n, C.; Claffey, J.; Hogan, M.; Strohfeldt, K.; Tacke, M.
Trans. Met. Chem. 2007, 32, 434–441.
30) Pampill oꢀ n, C.; Claffey, J.; Strohfeldt, K.; Tacke, M. Eur. J. Med.
Chem. 2008, 43, 122.
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O’Shea, D.; Tacke, M. Helv. Chim. Acta 2009, 91, 1787–1797.
(
(
(
(37) Stone, K. J.; Little, R. D. J. Org. Chem. 1984, 49, 1849–1853.

1
034 Organometallics, Vol. 29, No. 4, 2010
Hogan et al.
Figure 2. Structures of 6-indolylfulvenes 1a-f.
Scheme 1. Synthesis of Titanocenes 3a-c.
For 2c and 2f, the presence of dimethylamino and methyl-
methoxy substituents, respectively, leads to an increased
solubility in ethereal solution; therefore the intermediate
could not be isolated on a frit, and instead the solvent was
simply removed. This modification resulted in the increase of
impurities in the titanocene product, and therefore further
purification of 3c and 3f was necessary.
2.2. Structural Discussion. A single crystal of titanocene 3e
suitable for X-ray diffraction experiments was obtained by
the slow diffusion of pentane into saturated solutions of the

Article
Organometallics, Vol. 29, No. 4, 2010 1035
Figure 3. Structures of indolyl-substituted titanocenes 3a-f.
Scheme 2. Synthesis of Titanocenes 3d-f

1
036 Organometallics, Vol. 29, No. 4, 2010
Hogan et al.
Table 1. Selected Bond Lengths and Angles for Crystal Structures
of Titanocene Y and Compound 3e
Table 2. Crystal Data and Structure Refinement for 3e
empirical formula H Cl Ti
C
32
32
N
2
O
2
2
titanocene Y
3e
fw
temp
wavelength
cryst syst
595.4
100(2) K
0.71073 A
monoclinic
Bond Lengths (pm)
˚
Ti-C(1)
Ti-C(2)
Ti-C(3)
Ti-C(4)
Ti-C(5)
238.5(3)
240.9(3)
240.3(3)
234.4(3)
237.2(3)
242.8(3)
241.3(3)
238.6(3)
233.6(3)
235.2(3)
141.2(4)
139.3(4)
140.5(4)
140.9(4)
140.5(4)
141.7(4)
140.1(4)
138.1(4)
141.6(4)
139.6(4)
236.8(1)
236.6(1)
150.9(4)
151.7(4)
150.2(4)
151.9(4)
2.06(1)
244.6(2)
239.6(2)
233.6(2)
233.9(2)
241.1(2)
240.6(2)
241.0(2)
239.8(2)
233.7(2)
236.9(2)
141.7(2)
140.7(2)
142.5(2)
140.4(2)
140.9(2)
141.9(2)
140.5(2)
140.0(3)
140.7(3)
142.5(3)
237.7(1)
237.2(1)
150.6(2)
150.0(2)
149.8(3)
150.6(3)
2.06(1)
space group
unit cell dimens
1
P2 /c (no. 14)
˚
˚
a=18.193(2) A, R=90°
b=22.650(3) A, β=91.509(2)°
˚
c=6.7549(7) A, γ=90°
2782.5(5) A
3
˚
volume
Z
density (calcd)
absorp coeff
F(000)
0
Ti-C(1 )
4
1.421 Mg/m
0.533 mm
1240
0
Ti-C(2 )
3
0
Ti-C(3 )
-1
0
Ti-C(4 )
0
Ti-C(5 )
3
cryst size
0.40 ꢀ 0.10 ꢀ 0.05 mm
C(1)-C(2)
C(2)-C(3)
C(3)-C(4)
C(4)-C(5)
C(5)-C(1)
C(17)-C(18)
C(18)-C(19)
C(19)-C(20)
C(20)-C(21)
C(21)-C(17)
Ti-Cl(1)
θ range for data collection 1.80 to 30.56°
index ranges
reflns collected
indep reflns
completeness to θ=30.56° 99.10%
absorp corr
max. and min. transmn
refinement method
data/restraints/params
-25 e h e 25, -32 e k e 32, -9 e l e 9
32 410
8462 [R(int)=0.0292]
semiempirical from equivalents
0.9738 and 0.7425
full-matrix least-squares on F
8462/0/356
1.046
R1=0.0471, wR2=0.1161
R1=0.0571, wR2=0.1223
1.262 and -0.340 e A
2
2
goodness-of-fit on F
final R indices [I>2σ(I)]
R indices (all data)
largest diff peak and hole
Ti-Cl(2)
C(1)-C(6)
C(6)-C(7)
C(17)-C(22)
C(22)-C(23)
Ti-Cent1
-3
˚
Ti-Cent2
2.06(1)
2.06(1)
Bond Angles (deg)
Cent1-Ti-Cent2
Cl1-Ti-Cent1
Cl2-Ti-Cent1
Cl(2)-Ti-Cl(1)
130.83(2)
106.03(2)
106.07(2)
92.343(18)
compound in chloroform. Selected bond lengths and angles
are displayed in Table 1, while the crystal data and refine-
ment details for 3e are found in Table 2.
Figure 4. Molecular structure of titanocene 3e. Thermal ellip-
soids are drawn at the 50% probability level.
The lengths of bonds between the metal center and the
carbon atoms of the cyclopentadienyl rings bound to the
metal ion are similar for both titanocene Y and 3e (Figures 1
and 4). They vary between 233.6 and 242.8 pm for titanocene
Y and between 233.6 and 244.6 pm for 3e. The same applies
for the carbon-carbon bonds of the cyclopentadienyl rings
with bond lengths between 138.1 and 141.7 pm for titanocene
Y and for 3e between 140.0 and 142.5 pm. The dihedral angle
created by C(1)-C(6)-C(7)-C(8) is 101.4(3)° for titanocene
Y and 123.3(2)° for 3e, whereas the dihedral angle C(17)-
C(22)-C(23)-C(24) is 88.7(3)° for titanocene Y and 119.2°
for 3e. These values show that for titanocene Y the benzyl
ring substituents are not coplanar with the cyclopentadienyl
rings, but approximately perpendicular in their arrange-
ment, whereas for 3e the indolyl ring substituents are orien-
ted at a more obtuse angle. The titanium-chlorine bond
lengths are almost identical for both structures, with values
between 236.3 and 237.3 pm. The Cl(1)-Ti-Cl(2) angle for
titanocene Y was measured as 95.94(3)° and for 3e as
to be seen between the substituted indolyl groups, but some
soft van der Waals interactions are present. The absence of
solvent molecules in the crystal unit cells is a particular
advantage when it comes to biological testing.
2.3. Cytotoxicity Studies. As seen in Figure 5, compound
3c, which contains dimethylamino groups, has an IC50 value
of 8.2 μM and is the most cytotoxic compound of this paper.
This protonated compound exhibits an increase in solubility
due to the positive charges at the nitrogen centers. However,
the use of phosphate buffer solution throughout the cell
testing may result in deprotonation of 3c during the cell
testing. It is interesting to note that when DMSO is omitted
from the cell testing, titanocene 3c is able to be solubilized in
the aqueous serum-media itself; however the IC50 value then
decreases to 13 μM. This may be a result of the aforemen-
tioned deprotonation in aqueous media.
The second most cytotoxic compound of this study, 3e,
demonstrates an IC50 value of 11 μM and is very similar, in
both structure and cytotoxic potential, to titanocene 3b,
which has an IC50 value of 15 μM. These are the most
comparable to the lead compound, titanocene Y, as all three
incorporate a methoxy substitution on the fused ring moiety.
As aforesaid, titanocene Y displays an IC50 activity of 30 μM,
which means that compounds 3c, 3e, and 3b are all at least
twice as cytotoxic as the lead compound against this cell line.
Interestingly, when the methoxy moiety is repositioned from
9
2.34(2)°. The two structures show similar conformations.
The benzyl substituents, in the case of titanocene Y, and the
indolyl substituents in the case of 3e are oriented away from
each other, so that steric hindrances are minimized. The data
collection for the structures was done at 100 K, so rotational
isomers do not exist in the unit cell, leading to an efficient
crystal lattice packing system. There are no π-π interactions

Article
Organometallics, Vol. 29, No. 4, 2010 1037
into the synthesis and cytotoxicity of these indolyl-substi-
tuted titanocenes, we have been able to establish that these
titanocene Y derivatives have further cytotoxic potential to
be achieved through the incorporation of various functional
groups in the correct positions of the indolyl ring. The
optimum positioning of the substitution pattern on the
indolyl ring can lead to nearly a 4-fold decrease in cytotoxi-
city in comparison to titanocene Y and may speak for a
potential evaluation against renal-cell cancer.
4. Experimental Section
4.1. General Conditions. Titanium tetrachloride (1.0 M solu-
tion in toluene), Super Hydride (LiBEt H, 1.0 M solution in
3
THF), 1-methylindole, n-BuLi (2.0 in cyclohexane), and all
other chemicals, with the exception of 5-methoxyindole, were
obtained commercially from Aldrich Chemical Co. and used
without any further purification. 5-Methoxyindole was obtai-
ned from Apollo Scientific Ltd. and used without any further
purification. Solvents were distilled in a Grubbs system and
collected under an atmosphere of nitrogen prior to use. Manipu-
lations of air- and moisture-sensitive compounds were done
using standard Schlenk techniques, under a nitrogen atmo-
sphere. NMR spectra were measured on a Varian 300, 400, or
Figure 5. Cytotoxicity studies of titanocenes 3a-cagainst CAKI-1
cells.
5
00 MHz spectrometer. Chemical shifts are reported in ppm and
are referenced to TMS. IR spectra were recorded on a Varian
100 FT-IR Excalibur Series employing a KBr disk. UV-vis
spectra were recorded on a Cary 50 Scan UV-visible spectro-
3
3
-1
-1
photometer (λ in nm, ε in dm mol cm ). A single crystal of
titanocene 3e suitable for X-ray diffraction experiments was
grown by the diffusion of pentane into a saturated solution of 3e
in chloroform at room temperature. X-ray diffraction data for
the compound were collected on a Bruker Smart Apex diffract-
ometer at 100 K. A semiempirical absorption correction on the
3
8
raw data was performed using the program SADABS. The
crystal structure was then solved by direct methods (SHELXS-
NT97) and refined by full matrix least-squares methods
3
9
2
against F . Further details about the data collection are listed
in Table 2, as well as reliability factors. Additional details are
available free of charge from the Cambridge Structural Data-
base under CCDC No. 755706. MS data were collected on a
quadrupole tandem mass spectrometer (Quattro Micro, Micro-
mass/Water’s Corp., USA) and prepared as solutions of tetra-
hydrofuran (THF) or water. C,H,N analysis was carried out
with an Exeter-CE-440 elemental analyzer, and Cl was determi-
ned by mercurimetric titrations. CC=column chromatography.
Figure 6. Cytotoxicity studies of titanocenes 3d-fagainst CAKI-1
cells.
the ring system to an alkyl group off of the nitrogen, the
cytotoxicity of the compound is decreased dramatically,
which is seen pictorially in Figure 6, where titanocene 3f
displays an IC50 value of 170 μM. Compounds 3a and 3d are
very similar structurally, but differ in their cytotoxicities by
4.2. Synthesis. (1-Methylindol-2-yl)carboxaldehyde. C H ON.
1-Methylindol-2-yl)carboxaldehyde was prepared as per the lite-
rature in 76% yield and verified via HNMR. H NMR (300 MHz,
CDCl
7.19 (dd, J=9.9, 4.0 Hz, 1H), 4.11 (s, 3H).
10 9
(
35
1
1
3
): δ 9.89 (s, 1H), 7.74 (d, J=8.1 Hz, 1H), 7.46-7.37 (m, 2H),
2
-fold. Whereas titanocene 3a has an IC50 of 21 μM, 3d has
an IC50 of 47 μM. As pertains to this study, the difference in
cytotoxicities is consistent with the analogous pair 3e and 3b,
in which the more cytotoxic of the two is the one synthesized
from the Vilsmeier-Haack precursor (in which the cyclopenta-
dienyl is bridged to an indole derivative at the 3-position) as
opposed to the lithiated precursor (in which the cyclopenta-
dienyl is bridged to an indole derivative at the 2-position).
As mentioned previously, no solvent is contained within
the unit cell of the crystal of 3e. This is obviously advanta-
geous for any potential biological application.
6-(1-Methylindol-2-yl)fulvene. C H -CH-C H N-CH (1a).
3
To a Schlenk flask with (1-methylindol-2-yl)carboxaldehyde
(2.41 g, 15.1 mmol) dissolved in 38 mL of methanol were added
.5 mL (37.8 mmol) of freshly cracked cyclopentadiene (Cp) and
.86 mL (22.7 mmol) of pyrrolidine at room temperature under
5
4
8
5
2
1
a nitrogen atmosphere. The solution was stirred at ambient
temperature for 20 h to give a dark red solution. The reaction
was quenched with 1.5 mL (24.2 mmol) of acetic acid and 30 mL
of water. The product was extracted into chloroform (3 ꢀ20 mL),
dried over magnesium sulfate, and filtered, and the solvent was
removed under reduced pressure to give a dark red solid.
Purified by CC (alumina gel, pentane/dichloromethane, 1:1).
1
3
. Conclusions and Outlook
Red solid. Yield: 1.48 g, 7.1 mmol, 47%. H NMR (400 MHz,
The hydridolithiation of 6-substituted fulvenes has been
found to be a very effective and reproducible way to medium
to highly cytotoxic aryl- and heteroaryl-substituted titano-
cenes of high purity. Following these investigative studies
(
38) Sheldrick, G. M. SADABS Version 2.03; University of Goettin-
gen: Germany, 2002.
39) Sheldrick, G. M. SHELXS97 and SHELXL97; University of
Goettingen: Germany, 1997.
(

1
038 Organometallics, Vol. 29, No. 4, 2010
Hogan et al.
CDCl ): δ 7.21 (d, J=9.0 Hz, 1H), 7.14 (s, 1H), 7.06 (d, J=2.4
3
cyclopentadienide intermediate was prepared as described for
2a, with1b (3.0 g, 12.6 mmol), Super Hydride (13.8 mL, 13.8 mmol),
and diethyl ether (80 mL). Yield: 1.32 g (5.4 mmol, 43%), pale
yellow powder. Procedure followed as per 3a with 2.7 mL (2.7
Hz, 1H), 6.97-6.91 (m, 2H), 6.81 (d, J = 5.2 Hz, 1H), 6.65 (d,
J = 5.2 Hz, 1H), 6.51 (d, J = 4.5 Hz, 1H), 6.33 (d, J = 5.1 Hz,
1
3
1
H), 3.86 (s, J=3.7 Hz, 3H), 3.80-3.78 (s, 3H). C NMR (101
MHz, CDCl ): δ 154.34, 144.79, 136.41, 135.07, 130.91, 128.24,
26.42, 124.96, 120.30, 114.79, 110.38, 110.06, 107.08, 101.90
3
4
mmol) of TiCl and 40 mL of THF. The final dark brown solid was
1
washed with pentane and then dried in vacuo: 3b (0.86 g, 1.44 mmol,
1
(
(
C H -CH-C H N-CH ), 30.05 (NCH ). HRMS: 208.1119
5
53%). H NMR (300 MHz, CDCl ): δ7.16 (d, J=8.8, 2H), 6.98 (d,
4
8
5
3
3
3
þ
þ
[M þH] , C15
H
14N ; calcd 208.1123).
J=2.2, 2H), 6.84 (dd, J=8.8, 2.3, 2H), 6.41 (s, 8H), 6.07 (s, 2H),
1
3
5Bis[(1-methylindol-2-yl)cyclopentadienyl]titanium(IV) Dichloride,
3
4.27 (s, 4H), 3.82 (s, 6H), 3.66(s, 6H). C NMR (126 MHz, CDCl )
(
Hydride (7.8 mL of a 1 M solution in THF) in a Schlenk flask
η -C H -CH -C H N-CH ) TiCl (3a). A solution of Super
δ 154.16, 132.87, 130.88, 123.73, 117.32, 114.94, 111.11, 109.63,
102.21, 100.30 (C H and (C H (OCH )(NCH ))), 55.98(-OCH ),
5
4
2
8
5
3 2
2.
5
4
8
4
3
3
3
was concentrated by removal of the solvent at 80 °C under
29.98 (-NCH
3
),28.69(C
5
H
4
-CH
2
-C10
2 2
H10ON). UV-vis (CH Cl ):
-
2
vacuum of 10 mbar for 40 min and subsequently returned to
room temperature. The concentrated reagent was dissolved in dry
diethyl ether (10 mL), and to this solution was added 1a (1.48 g,
λ 207 nm (ε 12 333), λ 217 nm (ε 17 700), λ 227 nm (ε 36 900), λ 278
nm (ε 20 633), λ 306 nm (ε 17 166), λ 400 nm (weak). IR: 3102, 2933,
2830, 1839, 1703, 1618, 1482, 1350, 1288, 1213, 1161, 1093, 1032,
-
þ
2
O] ).Anal.
7
.1 mmol) in 50 mL of dry diethyl ether. The solution was stirred
940, 796. ES-MS (pos.) in THF: 542.57 ([M - 2Cl þH
Calcd for C32 Ti (595.38): C 64.55, H 5.42, Cl 11.91, N
4.71. Found: C 64.95, H 5.56, Cl 11.41, N 4.43
for 20 h, during which time the lithium cyclopentadienide
intermediate 2a precipitated from the solution and the color of
the solution changed from orange to yellow. The yellow lithium
cyclopentadienide intermediate 2a was collected on a frit, dried
in vacuo, and then transferred to a preweighed Schlenk flask
under nitrogen (yield 0.70 g, 3.25 mmol, 42%).
2 2 2
H32Cl N O
34
1-Methyl-3-dimethylaminomethylindole.
12 16 2
C H N . A 2.6 mL
amount of dimethylamine solution (40% in water) and 4 mL of
glacial acetic acid were added together at 0 °C. Formaldehyde
(37%, 1.5 mL) and 1-methylindole (2.2 mL, 18 mmol) were added
in that order while stirring in the ice bath. The reaction was stirred at
50 °C for 1 h and then allowed to stand at room temperature for
24 h. The mixture was poured into 4 g of sodium hydroxide in
40 mL of water, and the oil was extracted into diethyl ether (3 ꢀ
15 mL). The ether extract was washed with water, dried over mag-
4
2a was dissolved in 35 mL of THF, and TiCl (1.6 mL, 1.6
mmol) was added in situ at room temperature. The resultant
dark red solution was refluxed for 16 h. The solution was then
cooled, and the THF was removed under reduced pressure. The
remaining residue was dissolved in dichloromethane and filtered
through Celite. The purple filtrate was filtered twice more by
gravity filtration, and the solvent was removed under reduced
pressure, resulting in a dark red-black solid, 3a, which was
nesium sulfate, filtered, and concentrated to yield 3.38 g (18 mmol,
1
99%) of a honey-colored oil. H NMR (400 MHz, CDCl
3
): δ 7.72
(d, J= 7.9 Hz, 1H), 7.31 (d, J= 8.1 Hz, 1H), 7.24 (t, J=7.5 Hz,
1H), 7.14 (t, J=7.4 Hz, 1H), 7.01 (s, 1H), 3.77 (s, 3H), 3.64 (s, 2H),
washed with pentane (2 ꢀ 50 mL) and dried in vacuo. Yield:
1
13
0
J=7.7, 2H), 7.28 (d, J=9.0, 2H), 7.18 (t, J=7.4, 2H), 7.08 (d,
.53 g, 0.99 mmol, 61%. H NMR (300 MHz, CDCl ): δ 7.51 (d,
3
2.30 (s, 6H). C NMR (101 MHz, CDCl ): δ 136.93, 128.38,
3
128.32, 121.49, 119.35, 119.02, 111.77, 109.10, 54.40, 45.32, 32.63.
(1-Methyl-3-dimethylaminomethylindol-2-yl)carboxaldehyde.
J=7.4, 2H), 6.42 (s, 8H), 6.14 (s, 2H), 4.31 (s, 4H), 3.70 (s, 6H).
1
3
C NMR (101 MHz, CDCl
28.20, 123.88, 121.13, 120.05, 119.53, 114.82, 108.96, 100.58
and C N-CH ), 34.17 (C N-CH ), 22.60 (-C
-). UV-vis (CH Cl ): λ 204 nm (ε 11 726), λ 215 nm
3
): δ 154.02, 137.51, 131.96, 129.57,
13 16 2
C H N O. (1-Methyl-3-dimethylaminomethylindol-2-yl)car-
3
5
1
boxaldehyde was prepared based on the published procedure
in 94% yield and confirmed by H NMR.
1
(
C
5
H
4
8
H
5
3
8
H
5
3
5
H
4
-
CH
2
2
2
A solution of n-BuLi (9.4 mL, 18.8 mmol) was added to a
Schlenk flask containing 1-methyl-3-dimethylaminomethylin-
dole (3.23 g, 17.1 mmol) dissolved in 30 mL of THF at -78 °C.
The solution was warmed to 30 °C and stirred at this tempera-
ture for 1 h, followed by an additional hour at room tempera-
ture. Then DMF (1.3 mL, 17.1 mmol) was added at -30 °C, and
the reaction was allowed to come to room temperature over 24 h.
The reaction was quenched by pouring onto 30 mL of ice cold
2 N HCl. The mixture was washed with ether, and the aqueous
layer was made distinctly basic with a 40% solution of KOH.
The basic phase was extracted with diethyl ether (3 ꢀ 20 mL).
The ether was collected, dried over magnesium sulfate, filtered,
(
(
ε 16 964), λ 229 nm (ε 45 089), λ 266 nm (ε 17 946), λ 286 nm
ε 17 172), λ 321 nm (ε 13 779), λ 400 nm (weak). IR: 3048, 2927,
1
911, 1611, 1465, 1319, 1234, 1096, 802, 738. ES-MS (pos.) in
-
þ
THF: 464.56 ([M - 2Cl ] ). Anal. Calcd for C30
535.32): C 67.3, H 5.27, Cl 13.2, N 5.23. Found: C 66.8, H 5.35,
Cl 12.9, N 5.35.
-Methyl-5-methoxyindole. C H ON. Prepared as per the
2 2
H28Cl N Ti
(
1
1
0
11
3
3
1
literature procedure in 62% yield and confirmed via H NMR.
1
3
H NMR (400 MHz, CDCl ): δ 7.21 (d, J=8.9 Hz, 1H), 7.09 (d,
J=2.4 Hz, 1H), 7.02 (d, J=3.0 Hz, 1H), 6.89 (dd, J=8.8, 2.4
Hz, 1H), 6.40 (dd, J=3.0, 0.7 Hz, 1H), 3.85 (s, 3H), 3.77 (s, 3H).
(
1-Methyl-5-methoxyindol-2-yl)carboxaldehyde. C11
Prepared as per the published procedure in 55% yield and
H
11
O
2
N.
and concentrated to yield 3.48 g (16.1 mmol, 94%) of a dark
): δ 10.25 (s, 1H),
7.84 (d, J=8.2 Hz, 1H), 7.47-7.31 (m, 1H), 7.17 (ddd, J=7.9,
6.6, 1.1 Hz, 1H), 4.08 (s, 1H), 3.87 (s, 1H), 2.28 (s, 3H).
3
5
1
yellow-brown oil. H NMR (300 MHz, CDCl
3
1
1
verified by H NMR. H NMR (300 MHz, CDCl ): δ 9.84 (s,
H), 7.27 (dd, J=9.7, 6.7 Hz, 1H), 7.14 (s, 1H), 7.12-7.07 (m,
H), 4.06 (s, 3H), 3.85 (s, 3H).
3
1
2
6-((1-Methyl-3-dimethylaminoindol-2-yl)fulvene. C
H N (1c). As described for 1a, with ((1-methyl-3-dimethyl-
15 2
H -CH-C12-
5 4
6
-(1-Methyl-5-methoxyindol-2-yl)fulvene. C
5
H
4
-CH-C10
H10ON
(
1b). As described for 1a, with ((1-methyl-5-methoxyindolyl)2-)
aminomethylindole)2-)carboxaldehyde (3.47 g, 16 mmol), Cp
(2.56 mL, 32 mmol), pyrrolidine (1.97 mL, 24 mmol), methanol
(40 mL), and acetic acid (1.8 mL, 32 mmol). Red oil. Yield: 4.33 g,
carboxaldehyde (3 g, 15.8 mmol), Cp (2.5 mL, 31.6 mmol),
pyrrolidine (2 mL, 23.7 mmol), methanol (30 mL), and acetic
acid (1.8 mL, 31.6 mmol). Red solid. Yield: 3.3 g, 13.9 mmol,
8
7
1
16 mmol, 100%. H NMR (400 MHz, CDCl
3
): δ 7.78 (d, J=8.0,
1H), 7.33 (d, J=8.1, 1H), 7.30 (d, J=6.9, 1H), 7.26 (s, 1H), 7.16
1
8%. H NMR (400 MHz, CDCl ): δ 7.21 (d, J=9.0 Hz, 1H),
3
.14 (s, 1H), 7.06 (d, J=2.4 Hz, 1H), 6.97 - 6.91 (m, 2H), 6.81
(t, J=6.9, 1H), 6.61 (dd, J=3.6, 1.7, 1H), 6.54 (d, J=5.2, 1H),
6.38 (d, J=5.2, 1H), 6.25 (d, J=5.2, 1H), 3.79 (s, 2H), 3.72 (s,
(
1
d, J=5.2 Hz, 1H), 6.65 (d, J=5.2 Hz, 1H), 6.51 (d, J=4.5 Hz,
C
1
3
13
H), 6.33 (d, J = 5.1 Hz, 1H), 3.86 (s, 3H), 3.78 (s, 3H).
3
3H), 2.24 (s, 6H). C NMR (101 MHz, CDCl ): δ 148.08,
NMR (101 MHz, CDCl ): δ 154.34, 144.79, 136.41, 135.07,
138.80, 135.81, 135.15, 132.12, 128.35, 125.64, 125.37, 123.28,
122.02, 120.19, 120.16, 113.31, 109.60, (C -CH-C12 ),
53.08 (-CH -N-(CH ), 44.63 (-CH -N-(CH ), 31.88 (N-
CH ; calcd 265.1700).
3
1
1
30.91, 128.24, 126.42, 124.96, 120.30, 114.79, 110.38, 110.06,
N), 55.84 (-OCH
5
H
4
H N
15 2
07.08, 101.90 (C
H
5 4
-CH-C
H
8 4
3
), 30.05
2
3
)
2
2
)
3 2
þ
þ
þ
þ
N
2
(
2
-NCH ). HRMS: 238.1224 ([M þ H] , C H NO ; calcd
3
). HRMS: 265.1695 ([MþH] , C18
H
21
3
16 16
38.1228).
Bis[(1-methyl-5-methoxyindol-2-yl)cyclopentadienyl]titanium(IV)
Dihydrochloride Derivative of Bis[(1-methyl-3-dimethylamino-
5
methylindol-2-yl)cyclopentadienyl]titanium(IV) Dichloride, (η -
5
Dichloride, (η -C
5
H
4
-CH
2
-C10H10ON)
2
TiCl
2
. (3b). The lithium
C
5
4
H -CH
2
-C12
H
15
N
2
)
2
TiCl
2
3 2HCl. (3c). A solution LiBEt3
H

Article
6.2 mL of a 1 M solution in THF) in a Schlenk flask was
Organometallics, Vol. 29, No. 4, 2010 1039
(
2H), 7.32-7.16 (m, 4H), 7.09 (t, J=7.2, 2H), 6.83 (s, 2H), 6.35
(s, 4H), 6.26 (s, 4H), 4.18 (s, 4H), 3.72 (s, 6H). C NMR (101
1
3
concentrated by removal of the solvent at 80 °C under vacuum
-
2
of 10 mbar for 40 min and subsequently returned to room
temperature. In another Schlenk flask, 1c (1.48 g, 5.6 mmol) was
dissolved in 30 mL of dry diethyl ether and added via cannula to
MHz, CDCl ): δ 138.06, 136.99, 127.63, 127.52, 122.00, 121.70,
3
119.06, 119.00, 116.33, 112.39, 109.30 (C
32.71 (C N-CH ), 26.62 (C -CH -C
(CH Cl ): λ 203 nm (ε 12 844), λ 217 nm (ε 19 288), λ 227 nm
(ε 51 955), λ 257 nm (ε 20 888), λ 295 nm (ε 16 000), λ 400 nm
(weak). Anal. Calcd for C30 Ti (535.38): C 67.3, H 5.27,
5
H , C
4 8
5
H N-CH
3
),
8
H
5
3
5
H
4
2
8
5
H N-CH
3
). UV-vis
the concentrated LiBEt
3
H at room temperature. The solution
2
2
was stirred for 20 h, during which time the lithium cyclopenta-
dienide intermediate 2c precipitated from the solution and the
color of the solution became paler. As the intermediate was too
fine to be collected on a frit, the solvent was removed from the
Schlenk flask and the remaining brownish residue was dissolved
2 2
H28Cl N
Cl 13.2, N 5.23. Found: C 67.8, H 5.42, Cl 12.7, N 4.98. IR: 3103,
2925, 1609, 1468, 1332, 1236, 1054, 830, 740. ES-MS (pos.) in
-
þ
THF: 464.51 ([M - 2Cl ] ).
in THF (30 mL). To this was added 2.8 mL (2.8 mmol) of TiCl
4
11 2
(1-Methyl-5-methoxyindol-3-yl)carboxaldehyde. C11H O N.
3
Prepared as per the published procedure in 69% yield and
6
at room temperature, and the color of the solution turned dark
red. The solution was refluxed for 16 h, then cooled to room
temperature, and the solvent was removed under reduced
pressure. The remaining residue was dissolved in chloroform
and filtered through Celite to remove the lithium chloride. The
red filtrate was filtered twice more by gravity filtration. The
solvent was removed under reduced pressure to yield a red solid,
which was washed with pentane (30 mL) and dried in vacuo
1
1
verified by H NMR. H NMR (300 MHz, CDCl ): δ 9.95 (s,
3
1H), 7.79 (d, J=2.4 Hz, 1H), 7.63 (s, 1H), 7.26 (s, 2H), 6.99 (dd,
J = 8.9, 2.5 Hz, 1H), 3.90 (s, 3H), 3.85 (s, 3H).
6-(1-Methyl-5-methoxyindol-3-yl)fulvene. C H -CH-C10H -
5 4 10
ON (1e). As described for 1a, with 2.2 g (11.6 mmol) of (1-
methyl-5-methoxyindol-3-yl)carboxaldehyde dissolved in methanol
(20 mL) and THF (20 mL), 1.8 mL (23.2 mmol) of Cp, 1.4 mL
(
crude yield 2.0 g, 3.1 mmol, 110%).
The crude titanocene was purified by making the dihydro-
(17.4 mmol) of pyrrolidine, and 1.3 mL of acetic acid. Yield: 2.75 g
1
(11.6 mmol, 100%), red solid. H NMR (300 MHz, CDCl ): δ 7.45
3
chloride derivative. The crude titanocene (0.58 g, 0.9 mmol) was
dissolved in dichloromethane, and to the solution was added an
excess ethereal solution of hydrogen chloride (2.5 mL of a 2 M
solution in ether). A precipitate was immediately formed.
Diethyl ether (30 mL) was then added. After 15 min of stirring,
the yellow supernatant liquid was removed via syringe and the
(s, 1H), 7.34 (s, 1H), 7.15 (dd, J=9.7, 5.6, 2H), 6.86 (dd, J=8.9,
2.3, 1H), 6.69 (d, J=5.1, 1H), 6.58-6.50 (m, 1H), 6.35 (dt, J=5.0,
1
3
4.0, 2H), 3.81 (s, 3H), 3.72 (s, 3H). C NMR (101 MHz, CDCl ): δ
3
154.37, 138.73, 131.93, 131.16, 131.07, 129.40, 127.83, 127.07,
125.81, 118.21, 112.01, 111.92, 109.61, 99.65 (C -CH-C N),
54.82 (-OCH ), 32.51 (-NCH
). HRMS: 238.1225 ([M þ H] ,
16NO ; calcd 238.1228).
Bis[(1-methyl-5-methoxyindol-3-yl)cyclopentadienyl]titanium(IV)
5
H
4
8 4
H
þ
3
3
þ
remaining brown powder 3c was dried in vacuo (0.5 g, 0.69
O): δ 7.58 (d, J=7.7, 2H),
C H
16
1
mmol, 76%). H NMR (300 MHz, D
2
5
7
6
2
1
1
.40 (d, J = 8.1, 2H), 7.29-7.20 (m, 2H), 7.19-7.12 (m, 2H),
Dichloride, (η -C
cyclopentadienide intermediate was prepared as described for
2a, with 1e (2.5 g, 10.5 mmol), LiBEt H (11.6 mL, 11.6 mmol),
5 4 2 2 2
H -CH -C10H10ON) TiCl (3e). The lithium
.32 (s, 4H), 6.08 (s, 4H), 4.34 (s, 4H), 4.06 (s, 4H), 3.46 (s, 6H),
13
.70 (s, 12H). C NMR (101 MHz, DMSO): δ 140.94, 136.98,
33.40, 127.80, 124.62, 122.02, 120.45, 119.12, 116.73, 110.40,
3
and diethyl ether (80 mL). Yield: 0.53 g (2.16 mmol, 20.6%),
pale yellow powder.
Procedure followed as per 3a with 1.1 mL (1.1 mmol) of TiCl
4
01.37 (C
H
5 4
and C
H
8 4
2 3 2
N-), 65.39 (N-CH -N(CH ) ), 50.94
(
N-CH -N(CH ) ), 30.76 (-NCH ), 26.54 (-C H -CH -).
2
3 2
3
5
4
2
Anal. Calcd for C36
9.6, N 7.76. Found: C 59.21, H 5.84, Cl 19.17, N 6.43. UV-vis
CH Cl ): λ 219 nm (ε 80 240), λ 282 nm (ε 26 626), λ 292 nm
ε 24 819), λ 400 nm (weak). IR: 2958, 2712, 1621, 1470, 1248,
H44Cl
4
4
N Ti (722.43): C 59.8, H 6.14, Cl
and 40 mL of THF. The final dark brown solid was washed with
pentane and then dried in vacuo: 3e (0.60 g, 1.0 mmol, 91%). H
1
1
(
(
NMR (300 MHz, CDCl
3
): δ 7.17 (d, J = 8.8, 2H), 7.01 (d, J =
.1, 2H), 6.88 (dd, J = 8.8, 2.2, 2H), 6.78 (s, 2H), 6.37 (s, 4H),
2
2
2
6
1
3
.27 (s, 4H), 4.15 (s, 4H), 3.83 (s, 6H), 3.69 (s, 6H). C NMR
): δ 153.60, 137.94, 132.41, 128.03, 127.96,
22.21, 116.16, 111.93, 111.89, 110.05, 109.99, 101.06 (C and
1
2
159, 1027, 922, 829, 747. ES-MS (pos.) in water: 615.5 ([M -
-
þ þ
(
126 MHz, CDCl
3
HCl - 2Cl þ2H
2
OþH ] ).
1
5 4
H
(
cording to the literature procedure in 95% yield and confirmed
9
1-Methylindol-3-yl)carboxaldehyde. C10H ON. Prepared ac-
3
6
(
2
C H (OCH )(NCH ))), 56.04 (-OCH ), 32.83 (-NCH ),
8
4
3
3
3
3
1
1
6.61 (C
5
H
4
-CH
2
-C10
H
10ON). UV-vis (CH
2
Cl
2
): λ 205 nm
by H NMR. H NMR (300 MHz, CDCl
3
): δ 9.93 (s, 1H),
.31-8.25 (m, 1H), 7.61 (s, 1H), 7.33 (dd, J=3.4, 1.6 Hz, 2H),
.31-7.27 (m, 1H), 3.80 (s, 3H).
-(1-Methylindol-3-yl)fulvene. C H -CH-C H N-CH (1d).
(
(
(
ε 16 080), λ 215 nm (ε 27 440), λ 227 nm (ε 57 600), λ 250 nm
ε 31 320), λ 278 nm (ε 28 000), λ 300 nm (ε 22 000), λ 400 nm
weak). IR: 3108, 2926, 1618, 1488, 1220, 1135, 1034, 794. ES-
8
7
6
5
4
8
5
3
-
þ
2
O] ). Anal. Calcd for
MS (pos.) in THF: 542.53 ([M - 2Cl þH
Ti (595.38): C 64.55, H 5.42, Cl 11.91, N 4.71.
Found: C 64.32, H 5.51, Cl 11.43, N 4.22.
-Methylmethoxyindole. C N-CH -OCH
the literature procedure in 92% yield and confirmed via H
As described for 1a, with (1-methylindol-3-yl)carboxaldehyde
2.3 g, 14.4 mmol), methanol (40 mL), Cp (2.8 mL, 36 mmol),
32 2 2 2
C H32Cl N O
(
pyrrolidine (1.8 mL, 21.6 mmol), and acetic acid (2.5 mL, 43.2
mmol). Crude yield 3.44 g (16.6 mmol) of dark red oil, purified
by CC (alumina gel, dichloromethane): 1.57 g, 7.6 mmol, 67%
1
H
8 6
2
3
. Prepared as per
4
0
1
1
3
1
1
NMR and C NMR. H NMR (300 MHz, CDCl
3
): δ 7.63 (d,
red solid. H NMR (300 MHz, CDCl
3
): δ 7.83 (d, J = 7.5 Hz,
J=7.8 Hz, 1H), 7.50-7.44 (m, 1H), 7.27-7.18 (m, 1H), 7.18-
1
5
2
1
1
3
H), 7.59 (s, 1H), 7.48 (s, 1H), 7.38-7.20 (m, 3H), 6.78 (d, J =
1
3
7
.10 (m, 2H), 6.56-6.50 (m, 1H), 5.41 (s, 2H), 3.21 (s, 3H).
C
.1 Hz, 1H), 6.64 (d, J=3.4 Hz, 1H), 6.43 (dd, J=9.0, 7.4 Hz,
1
3
NMR (101 MHz, CDCl ): δ 136.38, 129.20, 128.12, 122.19,
H), 3.88 (s, 3H). C NMR (101 MHz, CDCl ): δ 140.38,
3
3
1
20.98, 120.32, 111.23, 109.54, 102.63, 77.41, 55.86.
1-Methylmethoxyindol-3-yl)carboxaldehyde. C H O N.
Prepared as per the literature procedure in 87% yield and
37.12, 133.28, 131.92, 130.53, 128.46, 128.29, 127.13, 122.99,
21.20, 119.61, 119.05, 113.33, 109.98 (C H -CH-C H N-CH ),
(
11 11
2
5
4
8
5
3
3
6
þ
þ
3.32 (C
8
H
5
N-CH
3
). HRMS: 208.1118 ([M þ H] , C15
H14N ;
1
13
1
verified by H and C NMR. H NMR (300 MHz, CDCl
0.05 (s, 1H), 8.35-8.26 (m, 1H), 7.80 (s, 1H), 7.53 (dd, J=5.8,
2.9 Hz, 1H), 7.36 (dd, J=6.5, 2.8 Hz, 2H), 5.51 (s, 2H), 3.31 (s,
3
): δ
calcd 208.1123).
Bis[(1-methylindol-3-yl)cyclopentadienyl]titanium(IV) Dichloride,
1
5
(
η -C
tadienide intermediate was prepared as described for 2a, with
fulvene 1d (1.78 g, 8.6 mmol), LiBEt H (9.46 mL, 9.46 mmol),
5 4 2 8 5 3 2
H -CH -C H N-CH ) TiCl2. (3d). The lithium cyclopen-
1
3
3
H). C NMR (101 MHz, CDCl
3
): δ 184.91, 138.14, 137.17,
1
25.60, 124.53, 123.44, 122.17, 119.07, 110.57, 78.29, 56.37.
3
and diethyl ether (80 mL). Yield: 0.72 g (3.35 mmol, 39%),
yellow powder. Procedure followed as per 3a with 1.7 mL (1.7
mmol) of TiCl and 40 mL of THF. The final dark brown solid
4
(
40) Adams, D. R.; Abraham, A.; Asano, J.; Breslin, C.; Dick, C.;
Ixkes, U.; Johnston, B. F.; Johnston, D.; Kewnay, J.; Mackay, S.;
MacKenzie, S. J.; McFarlane, M.; Mitchell, L.; Spinks, D.; Takano,
Y. Bioorg. Med. Chem. Lett. 2007, 17, 6579–6583.
was washed with pentane and then dried in vacuo: 3d (0.55 g, 1.02
): δ 7.52 (d, J = 7.8,
1
mmol, 60%). H NMR (300 MHz, CDCl
3

1
040 Organometallics, Vol. 29, No. 4, 2010
Hogan et al.
6
-(1-Methoxymethylindol-3-yl)fulvene. C
5
H
4
-CH-C
8
H
5
N-CH
2
-
C 64.55, H 5.42, Cl 11.91, N 4.71. Found: C 64.72, H 5.61, Cl 11.52,
N 4.36.
OCH (1f). As described for 1a, with (1-methylmethoxy-
3
indol-3-yl)carboxaldeyde (3.0 g, 15.8 mmol), Cp (2.5 mL, 31.6
mmol), pyrrolidine (2.0 mL, 23.7 mmol), acetic acid (1.8 mL,
4.3. MTT-Based Assay (MTT=3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-2H-tetrazolium bromide). Preliminary in vitro cell
tests were performed on CAKI-1 cells to compare the cytotoxi-
city of the compounds presented in this work. The cell line was
obtained from the ATCC (American Type Culture Collection)
(HTB-47) in 2001 and maintained in Dulbecco’s modified Eagle
medium containing 10% (v/v) FBS (fetal bovine serum), 1% (v/v)
penicillin streptomycin, and 1% (v/v) L-glutamine. The cyto-
toxicities of titanocenes 3a-f were determined by an MTT-
3
3
7
7
1
1.6 mmol), methanol (30 mL), and THF (20 mL). Dark red oil:
.5 g, 15.0 mmol, 95% yield. H NMR (300 MHz, CDCl ): δ
3
1
.83 (d, J =7.1 Hz, 1H), 7.65 (s, 1H), 7.52 (d, J= 7.8 Hz, 1H),
.46 (s, 1H), 7.31 (dd, J=16.6, 8.3 Hz, 2H), 6.78 (d, J=5.7 Hz,
H), 6.65 (d, J=5.1 Hz, 1H), 6.49 (d, J=5.3 Hz, 1H), 6.42 (d,
1
3
J=4.9 Hz, 1H), 5.52 (s, 2H), 3.30 (s, 3H). C NMR (75 MHz,
CDCl ): δ 141.70, 135.95, 133.71, 130.39, 129.61, 129.15, 126.84,
3
4
1
1
23.43, 121.66, 119.53, 119.13, 110.46 (C
OCH ), 77.93 (-CH -OCH ), 28.12 (-OCH
[M þH] , C H NO ; calcd 238.1228).
5
H
4
, C
). HRMS: 238.1218
8
H
5
N-CH
2
-
based assay.
3
2
3
3
Specifically, cells were seeded in 96-well plates containing
200 μL of medium and were incubated at 37 °C and 5% carbon
dioxide for 24 h to allow for exponential growth. Then the
compounds used for the testing were dissolved in the most
minimal amount of dimethyl sulfoxide (DMSO) possible and
þ
þ
(
16
16
Bis[(1-methylmethoxyindol-3-yl)cyclopentadienyl]titanium(IV)
5
Dichloride, (η -C
solution of LiBEt
5
H
4 2 8 5 2 3 2
-CH -C H N-CH -OCH ) TiCl2. (3f). A
3
H (4.2 mL of a 1 M solution in THF) in a
-
4
Schlenk flask was concentrated by removal of the solvent at 80 °C
diluted with medium to obtain stock solutions of 5ꢀ10 M in
concentration and less than 0.7% of DMSO. The cells were then
treated with varying concentrations of the compounds and
incubated for 48 h at 37 °C. At that time, the solutions were
removed from the wells, the cells were washed with PBS (phos-
phate buffer solution), and fresh medium was added to the wells.
Following a recovery period of 24 h incubation at 37 °C, indivi-
dual wells were treated with 200 μL of a solution of MTT in
medium (5 mg of MTT per 11 mL of medium). The cells were
incubated for 3 h at 37 °C. The medium was then removed, and
the purple formazan crystals were dissolved in 200 μL of DMSO
per well. Absorbance was then measured at 540 nm by a Wallac-
Victor (Multilabel HTS Counter) plate reader or a SpectraMax
190 microplate reader. Cell viability was expressed as a percen-
tage of the absorbance recorded for control wells. The values
used for the dose-response curves of Figure 5 represent the
values obtained from four consistent MTT-based assays for
each compound tested.
-
2
under vacuum of 10 mbar for 40 min and subsequently retur-
ned to room temperature. The concentrated reagent was dis-
solved in dry diethyl ether (20 mL), and to this solution 1f (0.9 g,
3.8 mmol) was added in 50 mL of dry diethyl ether. The solution
was stirred for 16 h, and the solvent was removed under reduced
pressure. The brownish residue 2f was dissolved in THF (30 mL),
and to this was added 2.1 mL (2.1 mmol) of TiCl at room tempe-
4
rature. The solution was refluxed for 16 h. The solution was then
cooled and the solvent was removed under reduced pressure. The
remaining residue was dissolved in chloroform and filtered through
Celite to remove the lithium chloride. The brown filtrate was filtered
twice more by gravity filtration. The solvent was removed under
reduced pressure to yield a brown solid. The brown solid was
purified by redissolving in chloroform (40 mL) and adding pentane
(20 mL) until a precipitate formed. The suspension solution was
filtered by gravity filtration. The reddish-brown filtrate was col-
lected, and the solvent was removed under reduced pressure. The
resultant brown solid was washed with pentane (30 mL) and dried
1
in vacuo. Yield 3f: 0.50 g, 0.84 mmol, 22%. H NMR (400 MHz,
Acknowledgment. The authors thank the Higher Edu-
cation Authority (HEA), the Irish Research Centre for
Science, the Centre for Synthesis and Chemical Biology
(CSCB), the University College Dublin (UCD), and
COST D39 for funding. The authors also thank Dr. Helge
M u€ ller-Bunz of UCD for the X-ray crystal data.
CDCl
.27-7.22 (m, 2H), 7.16-7.11 (m, 4H), 6.97 (s, 2H), 6.35 (t, J =
.6 Hz, 4H), 6.28 (t, J=2.6 Hz, 4H), 5.38 (s, 4H), 4.21 (s, 4H), 3.22
3
): δ 7.53 (d, J = 7.9 Hz, 2H), 7.46 (d, J = 8.2 Hz, 2H),
7
2
(
13
s, 6H). C NMR (101 MHz, CDCl
27.45, 125.67, 121.41, 121.22, 119.03, 118.19, 114.89, 112.98,
08.96 (C and C N-CH -OCH ) 76.25 (-N-CH -OCH ),
4.93 (-N-CH -OCH ), 25.53 (-C H -CH -). UV-vis (CH -
3
): δ 136.50, 136.50, 135.77,
1
1
5
H
4
8
H
5
2
3
2
3
5
2
3
5
4
2
2
2
Cl ): λ 204 nm (ε 12 500), λ 215 nm (ε 17 250), λ 227 nm (ε 44 500),
Supporting Information Available: Crystallographic data for
3e given as a CIF file are available free of charge via the Internet
at http://pubs.acs.org.
λ 265 nm (ε 18 375), λ 281 nm (ε 16 625), λ 293 nm (ε 13 750),
λ 400 nm (weak). IR: 3108, 3048, 2924, 1927, 1703, 1610, 1461,
1
348, 1235, 1181, 1084, 909, 813, 741. ES-MS (pos.) in THF: 542.57
-
þ
(
[M - 2Cl þH
2
O] ). Anal. Calcd for C32
H
32Cl
2
N
2
O
2
Ti (595.38):
(41) Mossman, T. J. Immunol. Methods 1983, 65.