From imide to lactam metallo-pyridocarbazoles: Distinct scaffolds for the design of selective protein kinase inhibitors

Article abstract of DOI:10.1021/jo901641k

(Chemical Equation Presented) Organometallic pyridocarbazole scaffolds are investigated as protein kinase inhibitors.Whereas our previous designs employed solely a maleimide pharmacophore for achieving the two crucial canonical hydrogen bonds to the hinge region of the ATP binding site, we have now extended our investigations to include the related lactam metallo-pyridocarbazoles. The synthetic access of the two regioisomeric lactam pyridocarbazoles is described, and the distinct biological properties of the two lactam scaffolds are revealed by employing a ruthenium half sandwich complex as a model system, resulting in organometallic lead structures for the inhibition of the protein kinases TrkA and CLK2. These new lactam metallo-pyridocarbazoles expand our existing molecular toolbox and assist toward the generation of metal complex scaffolds as lead structures for the design of selective inhibitors for numerous kinases of the human kinome.

Full text of DOI:10.1021/jo901641k

pubs.acs.org/joc
From Imide to Lactam Metallo-pyridocarbazoles: Distinct Scaffolds for
the Design of Selective Protein Kinase Inhibitors
Nicholas Pagano,†,§ Eric Y. Wong,‡ Tom Breiding,† Haidong Liu,†, Alexander Wilbuer,†
Howard Bregman,§ Qi Shen, Scott L. Diamond,‡ and Eric Meggers*,†
†
€
Fachbereich Chemie, Philipps-Universitat Marburg, Hans-Meerwein-Strasse, 35032 Marburg, Germany,
‡Institute of Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104,
§Department of Chemistry, University of Pennsylvania, 231 S. 34th Street, Philadelphia, Pennsylvania 19104,
and Key Laboratory of Organic Synthesis of Jiangsu Province, Department of Chemistry and Chemical
Engineering, Dushu Lake Campus, Suzhou University, Suzhou 215123, P. R. China
meggers@chemie.uni-marburg.de
Received July 30, 2009
Organometallic pyridocarbazole scaffolds are investigated as protein kinase inhibitors. Whereas our
previous designs employed solely a maleimide pharmacophore for achieving the two crucial
canonical hydrogen bonds to the hinge region of the ATP binding site, we have now extended our
investigations to include the related lactam metallo-pyridocarbazoles. The synthetic access of the two
regioisomeric lactam pyridocarbazoles is described, and the distinct biological properties of the two
lactam scaffolds are revealed by employing a ruthenium half sandwich complex as a model system,
resulting in organometallic lead structures for the inhibition of the protein kinases TrkA and CLK2.
These new lactam metallo-pyridocarbazoles expand our existing molecular toolbox and assist toward
the generation of metal complex scaffolds as lead structures for the design of selective inhibitors for
numerous kinases of the human kinome.
Introduction
envisioned that substitutionally inert transition metals could
act as a scaffold to organize organic ligands in three-dimen-
sional space. As a proof-of-principle, we chose kinases as our
main targets and started by morphing the indolocarbazole
natural product class and synthetic derivatives thereof into
Recently, our group has initiated a research program that
aims at exploring the versatility of organometallic complexes
as structural scaffolds for the design of enzyme inhibitors.
Unlike traditional applications for metals in medicine, we
DOI: 10.1021/jo901641k
r
Published on Web 11/03/2009
J. Org. Chem. 2009, 74, 8997–9009 8997
2009 American Chemical Society

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FIGURE 2. Ruthenium half sandwich complexes as a model
system to compare the protein kinase inhibition properties of
imide and lactam metallo-pyridocarbazoles. Compounds 1-3 are
racemic, but only one enantiomer is shown for clarity.
the combination of the globular shape and rigidity of these
scaffolds.
FIGURE 1. Indolocarbazole natural products and derivatives
as an inspiration for metallo-pyridocarbazoles as protein kinase
inhibitors.
To date, all published MPC complexes contain the mal-
eimide pharmacophore MPC-a, whereas the indolocarba-
zole alkaloids staurosporine and K252a, among others,
possess a lactam moiety instead (Figure 1).12 The lactam or
imide is essential for the binding to most kinases, as they both
can undergo two key hydrogen bonds with the hinge region
of the ATP binding site. However, at the same time it is
known that certain kinases prefer the one pharmacophore
over the other. Thus, in order to expand the versatility and
generality of our MPC scaffold for the design of selective
kinase inhibitors, we were seeking synthetic access of the two
regioisomeric lactam pyridocarbazole complexes MPC-b
and MPC-c (Figure 1). We here now report our progress
into this direction and disclose the synthesis of lactam
ruthenium complexes 1 and 2 (Figure 2). In fact, it turns
out that lactam complexes 1 and 2 and the previously
reported maleimide complex 3 differ significantly in their
kinase selectivity profiles. For example, whereas maleimide 3
is a promising lead structure for developing selective inhibi-
tors for GSK3 and Pim1,3,5-8 lactam 1 constitutes a lead
structure for TrkA, and lactam 2 is a potential starting point
for CLK2 inhibitor design.
chemically inert metallo-pyridocarbazoles (MPC, Figure 1).1
On the basis of this scaffold, we have reported over the past
5 years organometallic inhibitors for the kinases GSK3,2-7
Pim1,4,6,8 MSK1,4 PAK1,9 and PI3K.10 Multiple co-crystal
structures of metal complexes bound to the ATP binding sites
of kinases are deposited in the Protein Databank (e.g., 2BZH,
2BZI, 2OI4, 2BZJ, 2IWI, 3CST, 3BWF, 3FXZ), all of them
confirming that the metal exerts a purely structural role and is
not in direct contact with any residue in the active site.7-11
Some of the published inhibitors belong to the most potent
and selective compounds known for their respective kinases,
and we hypothesize that this is caused at least in part by
(1) Meggers, E.; Atilla-Gokcumen, G. E.; Bregman, H.; Maksimoska, J.;
Mulcahy, S. P.; Pagano, N.; Williams, D. S. Synlett 2007, 8, 1177–1189.
(2) Bregman, H.; Williams, D. S.; Atilla, G. E.; Carroll, P. J.; Meggers, E.
J. Am. Chem. Soc. 2004, 126, 13594–13595.
(3) Williams, D. S.; Atilla, G. E.; Bregman, H.; Arzoumanian, A.; Klein,
P. S.; Meggers, E. Angew. Chem., Int. Ed. 2005, 44, 1984–1987.
(4) Bregman, H.; Carroll, P. J.; Meggers, E. J. Am. Chem. Soc. 2006, 128,
877–884.
(5) Atilla-Gokcumen, G. E.; Williams, D. S.; Bregman, H.; Pagano, N.;
Meggers, E. ChemBioChem 2006, 7, 1443–1450.
Results and Discussion
(6) Bregman, H.; Meggers, E. Org. Lett. 2006, 8, 5465–5468.
(7) Atilla-Gokcumen, G. E.; Pagano, N.; Streu, C.; Maksimoska, J.;
Filippakopoulos, P.; Knapp, S.; Meggers, E. ChemBioChem 2008, 9, 2933–
2936.
(8) Debreczeni, J. E.; Bullock, A. N.; Atilla, G. E.; Williams, D. S.;
Bregman, H.; Knapp, S.; Meggers, E. Angew. Chem., Int. Ed. 2006, 45, 1580–
1585.
(9) Maksimoska, J.; Feng, L.; Harms, K.; Yi, C.; Kissil, J.; Marmorstein,
R.; Meggers, E. J. Am. Chem. Soc. 2008, 130, 15764–15765.
(10) Xie, P.; Williams, D. S.; Atilla-Gokcumen, G. E.; Milk, L.; Xiao, M.;
Smalley, K. S. M.; Herlyn, M.; Meggers, E.; Marmorstein, R. ACS Chem.
Biol. 2008, 3, 305–316.
Synthesis. A retrosynthetic analysis of the ruthenium
complexes 1 and 2 is shown in Scheme 1. The complexes 1
and 2 are accessed from the ruthenium half sandwich com-
plex 4 and the triisopropylsilyl (TIPS)-protected lactam
pyridocarbazoles 5 and 6, respectively. Both lactams 5 and
6 can be synthesized in two consecutive reduction steps
starting with the imide pyridocarbazole 7. Heterocycle 7
ꢀ
(11) Maksimoska, J.; Williams, D. S.; Atilla-Gokcumen, G. E.; Smalley,
K. S. M.; Carroll, P. J.; Webster, R. D.; Filippakopoulos, P.; Knapp, S.;
Herlyn, M.; Meggers, E. Chem.;Eur. J. 2008, 14, 4816–4822.
ꢀ
(12) Sanchez, C.; Mendez, C.; Salas, J. A. Nat. Prod. Rep. 2006, 23, 1007–
1045.
ꢀ
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SCHEME 1. Retrosynthetic Analysis of Lactam Pyridocarbazole
Complexes 1 and 2
and heated under high vacuum to completely remove the Boc
group to afford the 2-pyridin-2-yl-1H-indole 11 in 83% over
two steps. Alternatively, the potassium trifluoroborate
analogue of indole 8 could also be used for this transfor-
mation.13,15,16 Before conducting the next two C-C bond
formation reactions, the benzyl-protection group was first
exchanged for a TIPS group in order to render the scaffold
more soluble in organic solvents. This was accomplished by
hydogenolysis with H2, Pd/C furnishing the phenol deriva-
tive 12 (71%), followed by treatment with TIPS triflate in the
presence of the base di(isopropyl)ethylamine to afford the
TIPS-protected pyridoindole 13 (88%). 2-Pyridin-2-yl-1H-
indole 13 was deprotonated with LiHMDS and reacted with
dibromomaleimide 10 to afford monobromide 14 in 82%
yield. The final C-C bond could then be installed by a
photocyclization of 14 in toluene with a medium pressure
Hg lamp using an uranium filter (50% transmission at
350 nm) to provide pyridocarbozole 7 (83%). In summary,
the bis-silyl protected ligand 7 was prepared in a total of six
steps in an overall yield of 35%.
The first of two reduction steps conducted on the imide
pyridocarbazole 7 is shown in Scheme 3. Two equivalents of
NaBH4 were added at 0 °C each hour for a total of eight
hours, followed by warming to room temperature to furnish
regioisomeric products 15 and 16 in a ratio of 3:1 in a
combined yield of 84%. Slow addition of the reducing agent
at low temperature is critical, preventing the loss of the tert-
butyldimethylsilyl (TBS) protection group. Migration of the
TBS group to the newly formed sp3 oxygen atom retains the
TBS group required for the solubility of the compounds in
organic solvents and the successful separation of the two
regioisomers using column chromatography.
SCHEME 2. Synthesis of Maleimide Pyridocarbazole 7a
The constitutions of the two regioisomeric lactams 15 and
16 were assigned by rotational frame nuclear Overhauser
effect spectroscopy (ROESY) analysis of both regioisomers
15 and 16 (Figure 3). We were initially surprised by the
observed regioselectivity because we expected the reaction to
occur primarily at the more electrophilic carbonyl group a of
heterocycle 7 (Scheme 3).17,18 This carbonyl group is directly
conjugated to the electron poor pyridine, unlike the carbonyl
b, which is directly conjugated to the electron rich indole.
Additionally, we suspected that nucleophilic attack of car-
bon center b would be more hindered as a result of the
hydride approaching from the same side as the bulky TIPS
group.19-21 However, a likely explanation for this observed
result could be the difference in the transition state energies,
with migration of the TBS group preferring the more elec-
tron rich oxygen atom conjugated to the indole.
aReaction conditions: first, 1.1 equiv of 8, 1.0 equiv of 9, 0.1 equiv of
Pd(0) catalyst, 2.75 equiv of Na2CO3, DME/H2O, reflux, overnight;
then 10:1 mass ratio silica gel/indole, high vacuum, 80 °C, overnight.
Transformation of heterocycles 15 and 16 to their corre-
sponding lactams is outlined in Scheme 3. First, the TBS
group of 15 was replaced by an ethyl group through treat-
ment with 0.1% ethanol in the presence of 10% TFA to
itself can be assembled from three easily accessible building
blocks (8-10) in a series of six linear steps that include three
C-C bond formation reactions.13,14
The synthesis of imide pyridocarbazole 7 is outlined in
Scheme 2. Suzuki-Miyaura cross-coupling of boronic acid 8
with R-halogenated pyridine 9 furnished the desired 2-pyr-
idin-2-yl-1H-indole with the Boc protection group partially
removed. Unable to completely separate the two com-
pounds, the purified mixture was adsorbed onto silica gel
(15) Molander, G. A.; Canturk, B.; Kennedy, L. E. J. Org. Chem. 2009,
74, 973–980.
(16) Molander, G. A.; Ellis, N. Acc. Chem. Res. 2007, 40, 275–286.
(17) Link, J. T.; Danishefsky, S. J. Tetrahedron Lett. 1994, 49, 9135–9138.
(18) Issa, F.; Fischer, J.; Turner, P.; Coster, M. J. J. Org. Chem. 2006, 71,
4703–4705.
(19) Mase, N.; Nishi, T.; Takamori, Y.; Yoda, H.; Takabe, K. Tetra-
hedron Asymm. 1999, 10, 4469–4471.
(20) Mase, N.; Nishi, T.; Hiyoshi, M.; Ichihara, K.; Bessho, J.; Yoda, H.;
Takabe, K. J. Chem. Soc., Perkin Trans. 1 2002, 707–709.
(21) Mangaleswaran, S.; Argade, N. P. Synthesis 2004, 10, 1560–1562.
(13) Pagano, N.; Maksimoska, J.; Bregman, H.; Williams, D. S.; Webster,
R. D.; Xue, F.; Meggers, E. Org. Biomol. Chem. 2007, 5, 1218–1227.
(14) Bregman, H.; Williams, D. S.; Meggers, E. Synthesis 2005,
1521–1527.
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FIGURE 3. 1H-1H-ROESY spectra (DMSO-d6, 600 MHz) of compounds 15 and 16. (A) Compound 15: through-space correlation between
H2 and H6. (B) Compound 16: through-space correlation between H2 and H3.
afford heterocycle 17 in near quantitative yield. This ether
derivative could then smoothly be reduced to lactam 5 in
77% yield using phenylselenol and a slight excess of TFA in
refluxing methylene chloride.22,23 It is noteworthy, that
initial attempts to directly convert TBS-protected ligand 15
to lactam 5 failed and we attributed this to the size of the
TBS ether, precluding the necessary nucleophilic attack of
phenylselenol. In analogy, treatment of TBS-protected ligand
16 with 0.1% ethanol in now 20% TFA afforded intermediate
18, which due to its poor solubility was immediately reacted
with phenylselenol in refluxing methylene chloride to furnish
lactam 6 in excellent yield (94%) over two steps.
Introduction of the ruthenium half sandwich fragment
using both lactam ligands 5 and 6 is shown in Scheme 4.
Unable to optimize the coordination step using regular
heating with an oil bath, we were pleased to discover that
the complexation proceeds very smoothly under micro-
wave irradiation. Lactams 5 and 6 were each reacted with
(22) Link, J. T.; Raghavan, S.; Danishefsky, S. J. J. Am. Chem. Soc. 1995,
117, 552–553.
(23) Link, J. T.; Raghavan, S.; Gallant, M.; Danishefsky, S. J.; Chou,
T. C.; Ballas, L. M. J. Am. Chem. Soc. 1996, 118, 2825–2842.
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SCHEME 3. Synthesis of Lactam Pyridocarbazoles 5 and 6
from Maleimide 7
Kinase Inhibition. To identify similarities and differences
between the new lactam half sandwich complexes 1 and 2
compared to the imide compound 3, racemic mixtures of
complexes 1-3 were screened at one concentration against a
panel of 253 human kinases (Millipore KinaseProfiler).24 As
expected, the racemic mixture of maleimide 3 displayed
high potencies for GSK3 (R-isoform: 0% activity at 30 nM,
10 μM ATP) and Pim1 and Pim2 (1% and -1% activity at
30 nM, respectively, 10 μM ATP), whereas most other pro-
tein kinases were not inhibited to a significant degree
(see Supporting Information). In contrast, lactams 1 and 2
displayed only very moderate inhibition of GSK3 with acti-
vities above 40% at concentrations of 100 nM (10 μM ATP).
Furthermore, under the conditions of the screening (100 nM
compound, 10 μM ATP), lactam 1 demonstrated a surpri-
sing selectivity for just very few kinases (Flt3(D835Y),
MLCK, Pim1, and TrkA), whereas lactam 2 showed highest
inhibitory activity against the protein kinases CLK2,
PKG1β, and Pim1 (1%, 2%, and 2% remaining activities
at 100 nM of 2, 10 μM ATP, respectively; see Supporting
Information).
To investigate the inhibition of TrkA and CLK2 in more
detail, the racemic half sandwich complexes 1-3 were first
resolved into their individual enantiomers. The chiral se-
paration of imide 3 has been reported recently,8 and the
chiral separation of lactam ruthenium complexes 1 and 2 was
accomplished by resolving the individual enantiomers of
their TIPS-protected half sandwich analogues 19 and 20,
with the TIPS group serving as a handle to increase solubility
in organic solvents. The resolution of 10 mg of each en-
antiomer of TIPS-protected complexes 19 and 20 was
achieved using an analytical HPLC chiral column, and
subsequently all four stereoisomers were treated separately
with TBAF and purified over column chromatography.
Assignment of the absolute configuration for each enantio-
mer for both complexes was achieved by comparing in-
dividual CD spectra with known reference compounds
(see Supporting Information).
Table 1 displays the IC50 values of the individual enantio-
mers of 1-3 against Pim1, GSK3β, TrkA, and CLK2. The
data demonstrate that by simply removing one carbonyl
group of imide 3 a profound influence on the potency and
selectivity of the half sandwich scaffold is achieved. As
reported recently, (RRu)-3 and some derivatives show a high
selectivity for GSK3 and Pim1, whereas the enantiomer
(SRu)-3 is a very selective inhibitor of Pim1.5-8 However,
replacing one carbonyl group by a methylene group of
the Pim1 inhibitor lead structure (SRu)-3 (IC50 = 0.2 nM)
reduces the binding affinities for Pim1 by the factors of
200 and 30 for the lactam complexes (SRu)-1 and (SRu)-2,
respectively. Even more pronounced, replacing the imide
group for a lactam in the GSK3 inhibitor lead structure
(RRu)-3 (IC50 = 1.0 nM) reduces the binding affinities for
GSK3β by factors of 330 and 1000, for the complexes (RRu)-
1 and (RRu)-2, respectively.
ruthenium precursor 4 in the presence of K2CO3 in an-
hydrous DMF at 80 °C. After only 10 min, both reactions
were complete as judged by thin layer chromatography.
After column chromatography, TIPS-protected ruthenium
complexes 19 and 20 were isolated in high yields of 79% and
87%, respectively. Lactam inhibitor complexes 1 and 2 were
then prepared by careful treatment with tetrabutylammo-
nium fluoride (TBAF) (76% and 71%, respectively). It
should be noted that lactam ruthenium complexes 20 and
2, in contrast to 19 and 1, are slightly air-sensitive, slowly
oxidizing to their corresponding imide analogues if left in
solution for long periods of time. Also, this process is
accelerated in the presence of base. Therefore, it is critical
to thoroughly purge both reactions 6 f 20 and 20 f 2 with
inert gas. Furthermore, as a precaution, DMSO stock solu-
tions of ruthenium complex 2 were purged with nitrogen and
stored frozen at -20 °C until assayed for activity.
This dramatic loss in binding affinities of (RRu)-3 for
GSK3 by removing a single carbonyl group can be rationa-
lized with the help of a recently solved crystal structure of a
related ruthenium half sandwich complex with GSK3β.7
(24) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam,
S. Science 2002, 298, 1912–1934.
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SCHEME 4. Synthesis of Lactam Complexes 1 and 2a
aFormed as racemates; only one enantiomer is shown for clarity.
TABLE 1. IC50 Values of Imide and Lactam Half Sandwich Complexes
Thus, having eliminated the intrinsic preference of the
half sandwich scaffold for GSK3 and Pim1 by trans-
forming the imide group of 3 into lactam moieties, the
individual enantiomers of complexes 1 and 2 can now be
used as lead structures for other kinases. In fact, Table 1
demonstrates that the enantiomer (RRu)-1 displays a sig-
nificant selectivity for TrkA over Pim1 (4-fold) and
GSK3β (16.5-fold) and is therefore a promising lead
structure for the development of further improved inhi-
bitors of protein kinase TrkA, whereas (SRu)-2 displays an
IC50 of just 5 nM (100 μM ATP) against CLK2, thus
serving as a lead structure for the development of CLK2
inhibitors.
1-3 and (RRu)-21 against Pim1, GSK3β, TrkA, and CLK2a
Pim1
GSK3β
TrkA
CLK2
b
b
c
c
(RRu)-3
(SRu)-3
(RRu)-1
(SRu)-1
(RRu)-2
(SRu)-2
(RRu)-21
0.7
0.2
80
40
25
1.0
15
330
1000
1000
370
65
40
20
50
75
15
6
30
5
6
390
d
1200
aIC50 data were obtained by phosphorylation of substrate peptides
with [γ-32P]ATP or [γ-33P]ATP and 100 μM ATP in the presence
different concentrations of organometallic kinase inhibitors. Each
IC50 value was determined from at least two independent measurements
and the average was taken. See Experimental Section for more details.
c
bRacemic 3: IC50=35 nM at 100 μM ATP. Racemic 1: 26% CLK2
Improved Inhibitor for Protein Kinase TrkA. To verify that
the obtained lactam complexes are indeed promising
lead structures for the design of organometallic complexes
as inhibitors for protein kinases different from Pim1
or GSK3, we decided to use (RRu)-1 as a starting point
for the proof-of-principle design of a more potent and
selective inhibitor for TrkA. From previous structure
activity relationships with organoruthenium half sand-
wich complexes,5,13,14 we envisioned that the complex
(RRu)-21, differing from complex (RRu)-1 by a fused
benzene ring at the pyridine moiety and a methylester
at the cyclopentadienyl ring, might be an improved
inhibitor for TrkA with a diminished affinity for Pim1.
The synthesis of racemic 21 is summarized in Scheme 5.
Starting with 5-(triisopropylsilyloxy)indole 22, Boc-
protection afforded 23 (98%), which was subsequently
converted to the stable trifluoroborate 24 (55%). Suzuki-
Miyaura cross-coupling of 24 with triflate 25 in reflux-
ing anhydrous THF furnished 2-isoquinolin-3-yl-1H-in-
dole 26 (78%) with complete loss of the Boc group. Next,
isoquinolinyl-indole 26 was deprotonated with LiHMDS
and condensed with dibromomaleimide 10 to afford the
rather unstable monobromide 27 (79%). The final C-C
bond could then be installed by a photocyclization in
toluene with a medium pressure Hg lamp using a uranium
filter to provide heterocycle 28 in a yield of 86%. Next, 28
was converted to the lactam 29 in a yield of 47% by first
treatment with NaBH4, followed by EtOH with 0.1% TFA,
and subsequently PhSeH in the presence of 2 equiv of TFA.
The lactam ligand 29 was reacted with ruthenium half
sandwich complex 30 in the presence of K2CO3 in an-
hydrous DMF at 80 °C under microwave irradiation. After
10 min and column chromatography, TIPS-protected
activity at 100 nM 1 and 10 μM ATP. d50% CLK2 activity at 100 nM of
(RRu)-21 at 10 μM ATP.
FIGURE 4. Crystal structure of a maleimide-containing ruthenium
pyridocarbazole half sandwich complex with GSK3β (PDB code
2JLD) showing the hydrogen-bonding interactions of the male-
imide carbonyl groups in the ATP binding site of GSK3β.
Figure 4 shows the hydrogen bond interactions of the
maleimide pyridocarbazole in the ATP binding site of
GSK3β and reveals that both carbonyl groups are involved
in hydrogen bonds, one with the hinge residue valine 135 and
a second water-mediated contact with aspartate 200. This
explains the preference of GSK3 for the maleimide over the
lactam, which can provide only one of the two hydrogen-
bonding contacts.25
(25) For the binding modes of small molecules to the ATP binding sites of
protein kinases, see for example: Ghose, A. K.; Herbertz, T.; Pippin, D. A.;
Salvino, J. M.; Mallamo, J. P. J. Med. Chem. 2008, 51, 5149–5171.
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SCHEME 5. Synthesis of Lactam Complex 21a
aThe half sandwich complexes were formed as racemates, but only the (RRu)-enantiomer is shown for clarity.
ruthenium complex 31 was isolated in a yield of 54%.
Finally, treatment of 31 with TBAF at 0 °C afforded the
ruthenium complex 21 (66%) and the racemic mixture of 21
was resolved into its enantiomers at this final stage using an
analytical chiral HPLC column. The absolute configura-
tion for both enantiomers was assigned by comparison of
CD spectra with known reference compounds (see Sup-
porting Information).
IC50 values of (RRu)-21 at 100 μM ATP against TrkA,
GSK3β, and Pim1 are presented in Table 1. Gratifyingly,
(RRu)-21 shows a significantly improved potency and selec-
tivity compared to the initial lead structure (RRu)-1. With an
IC50 value of 6 nM for TrkA, (RRu)-21 prefers binding to
TrkA over Pim1 by a factor of 65 (IC50 = 390 nM) and even a
factor of 200 over GSK3β (IC50 = 1200 nM). Thus, (RRu)-21
displays high selectivity over Pim1 and GSK3 and probably
most other protein kinases. In fact, a screening of complex
(RRu)-21 against a panel of human wild-type protein kinases
demonstrates the excellent selectivity profile of (RRu)-21
(Figure 5). From 229 tested protein kinases, 200 remained
more than 50% active at a concentration of 100 nM of (RRu)-
21 (10 μM ATP), and only three kinases, including TrkA
and TrkB, displayed activities below 10% under these con-
ditions. Thus, it can be concluded that (RRu)-21 con-
stitutes a highly selective molecular probe for the protein
kinase TrkA.
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in vacuo. The crude material was adsorbed onto silica gel and
chromatographed using hexanes/EtOAc (8:1 to 1:1). The
combined product eluents were isolated as a mixture of a
Boc-protected 2-pyridin-2-yl-1H-indole and compound 11.
The resulting orange solid (2.82 g) was dissolved into EtOAc
and adsorbed onto silica gel (28.2 g) using rotary evaporation.
The powder was heated to 80 °C overnight under high vacuum.
The silica gel was cooled to room temperature, filtered through
cotton with CH2Cl2/MeOH (15:1) and the filtrate dried in vacuo
to provide 2-pyridin-2-yl-1H-indole 11 (2.27 g, 83% over 2
steps) as a yellow solid. 1H NMR (300 MHz, CDCl3): δ (ppm)
9.54 (s, 1H), 8.57 (ddd, J = 4.9, 1.7, 1.0 Hz, 1H), 7.79-7.67 (m,
2H), 7.51-7.48 (m, 2H), 7.43-7.29 (m, 4H), 7.18-7.14 (m, 2H),
6.98 (dd, J = 8.8, 2.4 Hz, 1H), 6.94 (dd, J = 2.1, 0.8 Hz, 1H),
5.12 (s, 2H). 13C NMR (75 MHz, CDCl3): δ (ppm) 153.9, 150.6,
149.4, 137.9, 137.6, 136.8, 132.2, 129.7, 128.7, 128.0, 127.8,
122.1, 119.9, 114.8, 112.3, 104.3, 100.5, 71.1. IR v (cm-1) 3192,
1622, 1594, 1562, 1543, 1469, 1443, 1299, 1257, 1214, 1150, 1119,
785, 775, 741. HRMS calcd for C20H16N2ONa (M þ Na)þ
323.1155, found (M þ Na)þ 323.1152.
FIGURE 5. Selectivity profile of (RRu)-21 tested against a panel of
229 human wild-type protein kinases at a single concentration of
100 nM and an ATP concentration of 10 μM. Duplicate measure-
ments were performed and the average was taken.
Compound 12. A suspension of 2-pyridin-2-yl-1H-indole 11
(2.14 g, 7.13 mmol) in ethanol (98 mL) was purged with nitrogen
for 15 min. Following thorough nitrogen purge, palladium
(10 wt % on activated carbon, dry) (2.14 g) was added, and
the reaction mixture was carefully purged with hydrogen gas for
approximately 10 min. The reaction mixture was then stirred at
room temperature under an atmosphere of hydrogen, monitor-
ing by thin layer chromatography. After 2 h, the resulting dark
reaction mixture was filtered through Celite with CH2Cl2/
MeOH (10:1), and the filtrate was concentrated to dryness in
vacuo. The crude material was adsorbed onto silica gel and
subjected to silica gel chromatography with hexanes/EtOAc
(1:1). The combined product eluents were dried in vacuo to
provide the 2-pyridin-2-yl-1H-indole 12 (1.07 g, 71%) as an
off-white solid. 1H NMR consistent with literature reported
spectrum.3
Compound 13. A solution of 2-pyridin-2-yl-1H-indole 12 (1.07
g, 5.10 mmol) in DMF (18.3 mL) was cooled to 0 °C, and N,N-
diisopropylethylamine (4.50 mL, 25.5 mmol) was added drop-
wise. After stirring for 10 min at 0 °C, triisopropylsilyl trifluoro-
methanesulfonate (1.44 mL, 5.36 mmol) was added dropwise
over 15 min, and the reaction mixture was stirred at room
temperature for an additional 1 h. The resulting yellow solution
was cooled to 0 °C, and excess 1 M NH4OAc was carefully
added. The mixture was then further diluted with water and
extracted with CH2Cl2. The combined organic layers were
washed with brine, dried using Na2SO4, filtered, and concen-
trated to dryness in vacuo. The crude material was adsorbed
onto silica gel and subjected to silica gel chromatography with
CH2Cl2/MeOH (100:1). The combined product eluents were
dried in vacuo to provide the 2-pyridin-2-yl-1H-indole 13
(1.65 g, 88%) as a white solid. 1H NMR (300 MHz, CDCl3): δ
(ppm) 10.30 (s, 1H), 8.59 (ddd, J = 4.9, 1.7, 1.0 Hz, 1H), 7.80 (td,
J = 8.0, 1.1 Hz, 1H), 7.70 (ddd, J = 8.0, 7.4, 1.7 Hz, 1H),
7.17-7.13 (m, 3H), 6.95 (dd, J = 2.1, 0.8 Hz, 1H), 6.86 (dd, J =
8.8, 2.3 Hz, 1H), 1.39-1.27 (m, 3H), 1.17 (d, J = 6.9 Hz, 18H).
13C NMR (75 MHz, CDCl3): δ (ppm) 150.7, 150.1, 149.1, 137.5,
136.7, 132.5, 129.8, 121.9, 119.9, 117.8, 111.8, 109.9, 100.4, 18.1,
12.8. IR v (cm-1) 3150, 2940, 2861, 1595, 1545, 1442, 1292, 1280,
1222, 1150, 963, 879, 804, 776, 679. HRMS calcd for
C22H31N2OSi (M þ H)þ 367.2200, found (M þ H)þ 367.2198.
Compound 14. A solution of high-vacuum-dried 2-pyridin-
2-yl-1H-indole 13 (1.55 g, 4.23 mmol) in freshly distilled THF
(11 mL) was cooled to -15 °C. Lithium bis(trimethylsilyl)amide
(1 M solution in hexanes) (12.7 mL, 12.7 mmol) was added
dropwise over 15 min, and the resulting orange reaction mixture
was stirred at -15 °C for an additional 45 min. A solution of
vacuum-dried 10 (1.64 g, 4.44 mmol) in freshly distilled THF
Conclusions
We have here reported synthetic access to the class of
lactam metallo-pyridocarbazoles MPC-b and MPC-c, thus
complementing the previously developed imide metallo-
pyridocarbazole scaffold MPC-a and therefore expanding
our toolbox of using metallo-pyridocarbazoles as general
scaffolds for the design of selective kinase inhibitors. The
distinct properties of the lactam scaffolds have been demon-
strated by using the half sandwich complex 3as a model system.
By replacing the imide pharmacophore of 3 for the lactam
moieties in 1 and 2, we have changed the selectivity profile from
GSK3/Pim1 selectivity of (RRu)-3 and Pim1 selectivity of
(SRu)-3 to TrkA selectivity of (RRu)-1 and CLK2 selectivity
of (SRu)-2. These lactam metallo-pyridocarbazoles will serve as
scaffolds for the design of protein kinase inhibitors with new
selectivity profiles as has been demonstrated with the TrkA-
selective organoruthenium complex (RRu)-21.
Experimental Section
All reactions were carried out using oven-dried glassware and
conducted under a positive pressure of nitrogen unless other-
wise specified. NMR spectra were recorded on an Avance 300
(300 MHz), DRX-400 (400 MHz), or DRX-500 (500 MHz)
spectrometer. ROESY experiments were recorded on a Bruker
Avance 600 MHz spectrometer. Infrared spectra were recorded
on either a Bruker Alpha FTIR or Nicolet 510 FTIR spectro-
meter. High resolution mass spectra were obtained with a
Finnigan LTQ-FT instrument using either APCI or ESI. Re-
agents and solvents were used as received from standard sup-
pliers unless otherwise specified. CD spectra were recorded on a
JASCO J-810 spectropolarimeter. Ruthenium complexes 4 and
30 were prepared as previously reported.5,26
Compound 11. A biphasic suspension of pyridine 9 (0.87 mL,
9.1 mmol), indole 8 (3.67 g, 10.0 mmol), tetrakis(triphenyl-
phosphine)palladium(0) (1.05 g, 0.91 mmol), and Na2CO3
(2.65 g, 25.0 mmol) in 1,2-dimethoxyethane (39 mL) and water
(11 mL) was purged with nitrogen for 15 min and then refluxed
overnight. The resulting orange reaction mixture was cooled
to room temperature, diluted with water, and extracted with
EtOAc. The combined organic layers were washed with
brine, dried using Na2SO4, filtered, and concentrated to dryness
(26) Gill, T. P.; Mann, K. R. Organometallics 1982, 1, 485–488.
9004 J. Org. Chem. Vol. 74, No. 23, 2009

Pagano et al.
JOCArticle
(11 mL) cooled to 0 °C was added, and the reaction mixture was
stirred at -15 °C for 20 min and room temperature overnight.
The resulting dark purple reaction mixture was carefully poured
into stirring ice cold water (300 mL) and extracted with EtOAc.
The combined organic layers were washed with brine, dried
using Na2SO4, filtered, and concentrated to dryness in vacuo.
The crude material was adsorbed onto silica gel and subjected to
silica gel chromatography with hexanes/EtOAc (6:1 to 3:1). The
combined product eluents were dried in vacuo to provide
1216, 1079, 1039, 981, 883, 832, 789, 668, 484. HRMS calcd
for C32H45N3O3Si2Na (M þ Na)þ 598.2892, found (M þ Na)þ
598.2897.
Compound 16. 1H NMR (300 MHz, DMSO-d6): δ (ppm)
12.76 (s, 1H), 9.41 (dd, J = 8.4, 1.7 Hz, 1H), 9.03 (dd, J = 4.3,
1.7 Hz, 1H), 9.00 (d, J = 2.0 Hz, 1H), 7.76 (d, J = 2.4 Hz, 1H),
7.72 (dd, J = 8.4, 4.3 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.09 (dd,
J = 8.7, 2.5 Hz, 1H), 6.63 (d, J = 2.0 Hz, 1H), 1.36-1.26 (m,
3H), 1.10 (d, J = 6.7 Hz, 18H), 0.73 (s, 9H), 0.03 (s, 3H), -0.50
(s, 3H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 170.8, 149.4,
149.0, 143.6, 138.9, 137.2, 134.7, 131.9, 122.3, 122.1, 122.0,
119.0, 115.4, 114.6, 112.7, 111.9, 78.7, 25.6, 17.8, 17.6, 12.1,
-3.4, -3.9. IR v (cm-1) 3156, 3071, 2945, 2927, 2865, 1686,
1670, 1530, 1471, 1280, 1251, 1213, 1094, 984, 957, 878, 831, 792,
780, 682, 665. HRMS calcd for C32H46N3O3Si2 (M þ H)þ
576.3072, found (M þ H)þ 576.3080.
1
monobromide 14 (2.25 g, 82%) as an orange solid. H NMR
(300 MHz, CDCl3): δ (ppm) 10.22 (s, 1H), 8.58 (d, J = 4.2 Hz,
1H), 7.61 (ddd, J = 7.8, 7.8, 1.8 Hz, 1H), 7.31 (d, J = 8.1 Hz,
1H), 7.25 (d, J = 8.7 Hz, 1H), 7.18 (ddd, J = 7.5, 4.9, 1.2 Hz,
1H), 6.99 (d, J = 2.3 Hz, 1H), 6.88 (dd, J = 8.7, 2.3 Hz, 1H),
1.34-1.23 (m, 3H), 1.12 (d, J = 7.0 Hz, 18H), 1.00 (s, 9H), 0.49
(s, 6H). 13C NMR (75 MHz, CDCl3): δ (ppm) 173.4, 171.1,
151.1, 149.8, 149.4, 141.7, 137.2, 136.8, 131.5, 128.2, 125.6,
122.8, 122.7, 118.9, 112.5, 110.4, 101.7, 26.5, 19.1, 18.3, 12.9,
-4.2. IR v (cm-1) 3337, 2947, 2865, 1703, 1635, 1473, 1313,
1269, 1213, 1186, 1086, 971, 892, 826, 735. HRMS calcd
for C32H45BrN3O3Si2 (M þ H)þ 654.2177, found (M þ H)þ
654.2182.
Compound 7. An orange suspension of monobromide 14 (1.10 g,
1.68 mmol) in toluene (250 mL) was irradiated with a medium
pressure Hg lamp using a uranium filter (50% transmission at
350 nm) for 6 h with constant nitrogen flow through the reaction
mixture. The resulting red solution was concentrated, and the
reaction was repeated using an additional 1.10 g (1.68 mmol) of
14. Once combined, the red solution was concentrated to dryness in
vacuo, adsorbed onto silica gel, and subjected to silica gel chromato-
graphy with hexanes/EtOAc (6:1 to 1:1). The combined product
eluents were dried in vacuo to provide pyridocarbazole 7 (1.60 g,
83%) as a yellow solid. 1H NMR (300 MHz, CDCl3):δ(ppm) 10.48
(s, 1H), 9.42 (dd, J = 8.5, 1.7 Hz, 1H), 8.99 (dd, J = 4.3, 1.7 Hz,
1H), 8.69 (d, J= 2.5 Hz, 1H), 7.63 (dd, J=8.5,4.3Hz,1H),7.41(d,
J = 8.7 Hz, 1H), 7.18 (dd, J = 8.7, 2.5 Hz, 1H), 1.46-1.34 (m, 3H),
1.18 (d, J = 7.3 Hz, 18H), 1.07 (s, 9H), 0.63 (s, 6H). 13C NMR
(75 MHz, CDCl3): δ (ppm) 175.7, 174.1, 151.5, 150.4, 140.8, 138.7,
134.9, 134.6, 131.0, 123.3, 122.9, 122.1, 121.5, 120.4, 115.4, 114.6,
111.9, 26.7, 19.3, 18.3, 12.9, -3.9. IR v (cm-1) 3456, 2946, 2866,
1751, 1695, 1527, 1473, 1338, 1314, 1281, 1216, 1182, 905, 827,
795. HRMS calcd for C32H44N3O3Si2 (M þ H)þ 574.2916, found
(M þ H)þ 574.2926.
Compound 17. A yellow suspension of 15 (260 mg, 0.452
mmol) in CH2Cl2 (23.4 mL) containing 0.1% ethanol was cooled
to 0 °C. Trifluoroacetic acid (2.6 mL) was then carefully added,
and the resulting orange solution was stirred at 0 °C for 10 min.
The reaction mixture was then carefully quenched with addition
of saturated aqueous NaHCO3 and extracted with CH2Cl2. The
combined organic layers were washed with brine, dried using
Na2SO4, filtered, and concentrated to dryness in vacuo to
provide 17 (220 mg, 99%) as a light yellow solid. See Supporting
Information for a ROESY analysis. 1H NMR (300 MHz,
DMSO-d6): δ (ppm) 12.57 (s, 1H), 9.28 (d, J = 1.3 Hz, 1H),
9.07 (dd, J = 4.3, 1.7 Hz, 1H), 8.65 (d, J = 2.6 Hz, 1H), 8.63 (dd,
J = 8.3, 1.7 Hz, 1H), 7.73 (dd, J = 8.4, 4.3 Hz, 1H), 7.55 (d, J =
8.7 Hz, 1H), 7.07 (dd, J = 8.7, 2.6 Hz, 1H), 6.43 (d, J = 1.4 Hz,
1H), 3.67 (m, 1H), 3.39 (m, 1H), 1.40-1.27 (m, 3H), 1.15 (t, J =
7.0 Hz, 3H), 1.11 (d, J = 7.3 Hz, 18H). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) 170.3, 150.1, 149.0, 138.3, 137.5, 134.7,
132.9, 132.7, 126.6, 122.4, 121.5, 121.1, 119.5, 114.1, 113.4,
112.4, 83.5, 60.7, 17.9, 15.3, 12.1. IR v (cm-1) 3284, 3172,
3072, 2942, 2864, 1689, 1471, 1338, 1280, 1260, 1214, 1069,
1049, 881, 814, 792, 726, 682, 667, 621, 483. HRMS calcd
for C28H35N3O3SiNa (M þ Na)þ 512.2340, found (M þ Na)þ
512.2338.
Compound 5. A suspension of 17 (220 mg, 0.450 mmol) in
CH2Cl2 was purged with nitrogen for approximately 15 min.
Phenylselenol (191 μL, 1.80 mmol) followed by trifluoroacetic
acid (52 μL, 0.675 mmol) were then added, and the resulting
dark orange solution was heated to reflux for 5 h. The reaction
mixture was then cooled to room temperature and carefully
quenched with addition of saturated aqueous NaHCO3. The
CH2Cl2 was then removed in vacuo, and the resulting yellow
precipitate was collected, washed with twice with water, fol-
lowed by diethyl ether (2ꢀ) to provide lactam 5 (153 mg, 77%)
as a light yellow solid. 1H NMR (400 MHz, 100 °C, DMSO-d6):
δ (ppm) 12.06 (s, 1H), 9.05 (dd, J = 4.0, 1.3 Hz, 1H), 8.77 (s, 1H),
8.53 (dd, J = 8.3, 1.1 Hz, 1H), 8.34 (s, 1H), 7.67 (dd, J = 8.1, 4.1
Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 4.85
(s, 2H), 1.38 (m, 3H), 1.16 (d, J = 7.3 Hz, 18H). 13C NMR (100
MHz, 100 °C, DMSO-d6): δ (ppm) 171.1, 149.3, 148.4, 138.1,
135.9, 134.3, 134.1, 131.8, 126.2, 122.4, 120.7, 120.5, 118.6,
114.5, 113.4, 111.6, 44.2, 17.4, 11.7. IR v (cm-1) 3283, 3163,
3060, 2940, 2865, 1680, 1661, 1469, 1261, 1214, 1171, 1058, 978,
914, 879, 671, 658, 623, 553, 457. HRMS calcd for C26H32N3O2-
Si (M þ H)þ 446.2258, found (M þ H)þ 446.2258.
Compounds 15 and 16. A solution of pyridocarbazole 7
(510 mg, 0.890 mmol) in THF-EtOH (1:1) (51 mL) was cooled to
0 °C. Sodium borohydride (68 mg, 1.79 mmol) was added every
hour for a total of 8 h, and the stirred reaction mixture was then
allowed to naturally warm to room temperature overnight. The
resulting yellow reaction mixture was again cooled to 0 °C,
carefully quenched with addition of saturated aqueous NH4Cl,
and extracted with EtOAc. The combined organic layers were
washed with brine, dried using Na2SO4, filtered, and concen-
trated to dryness in vacuo. The crude material was adsorbed
onto silica gel and subjected to silica gel chromatography with
hexanes/EtOAc (6:1 to 3:1). The combined product eluents were
dried in vacuo to individually provide pure 15 (320 mg, 63%)
and 16 (110 mg, 21%). The constitutions of 15 and 16 were
assigned by ROESY analysis (see Figure 3).
Compound 15. 1H NMR (300 MHz, DMSO-d6): δ (ppm)
12.55 (s, 1H), 9.21 (d, J = 1.7 Hz, 1H), 9.07 (dd, J = 4.3, 1.7 Hz,
1H), 8.66 (d, J = 2.6 Hz, 1H), 8.59 (dd, J = 8.4, 1.7 Hz, 1H), 7.73
(dd, J = 8.4, 4.3 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 7.07 (dd, J =
8.7, 2.6 Hz, 1H), 6.67 (d, J = 1.8 Hz, 1H), 1.39-1.27 (m, 3H),
1.11 (d, J = 7.4 Hz, 18H), 0.81 (s, 9H), 0.21 (s, 3H), -0.21 (s,
3H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 170.3, 150.1,
149.0, 138.6, 137.3, 135.6, 134.7, 133.2, 125.6, 122.4, 121.1,
120.9, 119.5, 114.0, 113.4, 112.4, 79.1, 25.6, 17.9, 17.6, 12.1,
-4.0. IR v (cm-1) 3293, 3075, 2928, 2862, 1695, 1472, 1272,
Compound 6. A suspension of 16 (45 mg, 0.078 mmol) in
CH2Cl2 (3.6 mL) containing 0.1% ethanol was cooled to 0 °C.
Trifluoroacetic acid (0.9 mL) was then carefully added, and the
resulting orange solution was allowed to naturally warm to
room temperature, stirring for a total of 3 h. Following thin
layer chromatography analysis, the reaction mixture was purged
with nitrogen for approximately 15 min. Phenylselenol (34 μL,
0.312 mmol) was then added, and the reaction was heated to
J. Org. Chem. Vol. 74, No. 23, 2009 9005

Pagano et al.
JOCArticle
reflux overnight. The reaction mixture was then cooled to room
temperature and carefully quenched with addition of saturated
aqueous NaHCO3. The CH2Cl2 was then removed in vacuo, and
the resulting yellow precipitate was collected and washed twice
with water, followed by diethyl ether (2ꢀ) to provide lactam 6
(32 mg, 94% over 2 steps) as a white solid. 1H NMR (500 MHz,
100 °C, DMSO-d6): δ (ppm) 12.35 (s, 1H), 9.53 (d, J = 8.4 Hz,
1H), 8.99 (dd, J = 4.2, 1.5 Hz, 1H), 8.13 (s, 1H), 7.69-7.64 (m,
2H), 7.46 (d, J = 2.0 Hz, 1H), 7.10 (dd, J = 8.7, 2.2 Hz, 1H), 4.89
(s, 2H), 1.35 (septet, J = 7.4, 3H), 1.15 (d, J = 7.4 Hz, 18H). IR v
(cm-1) 3156, 3063, 2943, 2865, 1678, 1652, 1533, 1463, 1279,
1244, 1209, 1174, 964, 868, 809, 787, 672, 568, 434. HRMS calcd
for C26H31N3O2SiNa (M þ Na)þ 468.2078, found (M þ Na)þ
468.2083.
Compound 19. To a vacuum-dried solid mixture of lactam 5
(20 mg, 0.045 mmol), ruthenium precursor 426 (21 mg, 0.050
mmol), and K2CO3 (7.0 mg, 0.050 mmol) was added freshly
distilled DMF (2.0 mL), and the resulting suspension was
purged with nitrogen for approximately 10 min. The reaction
mixture was then heated to 80 °C for 10 min in a microwave
reactor (CEM Discover). The resulting dark red solution was
concentrated using high vacuum and coevaporated once with
EtOAc. The crude material was subjected to silica gel chroma-
tography with toluene:acetone (3:1). The combined product
eluents were dried in vacuo to provide complex 19 (23 mg,
79%) as an orange film. 1H NMR (300 MHz, CDCl3): δ (ppm)
8.86 (dd, J = 5.1, 1.1 Hz, 1H), 8.56 (d, J = 2.4 Hz, 1H), 8.17 (dd,
J = 8.2, 1.1 Hz, 1H), 7.32-7.28 (m, 2H), 7.15 (dd, J = 8.8, 2.5
Hz, 1H), 6.92 (s, 1H), 5.20 (s, 5H), 4.76 (s, 2H), 1.48-1.38 (m,
3H), 1.21 (d, J = 7.2 Hz, 18H). 13C NMR (100 MHz, CDCl3): δ
(ppm) 200.6, 173.0, 154.1, 151.1, 148.7, 147.9, 146.1, 131.7,
129.3, 128.0, 124.5, 121.0, 119.6, 119.4, 115.8, 114.7, 114.0,
80.9, 45.1, 18.4, 13.0. IR v (cm-1) 3183, 3077, 2942, 2925,
2865, 1951, 1731, 1686, 1607, 1544, 1462, 1405, 1267, 1244,
1210, 1189, 1127, 1056, 997, 977, 922, 896, 884, 835, 805, 791,
688. HRMS calcd for C32H36N3O3RuSi (M þ H)þ 640.1564,
found (M þ H)þ 640.1561.
with EtOAc. The combined organic layers were dried using
Na2SO4, filtered, and concentrated to dryness in vacuo. The
crude material was then subjected to silica gel chromatography
with CH2Cl2/MeOH (20:1). The combined product eluents were
dried in vacuo to provide complex 1 (13 mg, 76%) as a light red
solid. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 9.19 (dd, J =
5.1, 1.1 Hz, 1H), 8.79 (s, 1H), 8.72 (s, 1H), 8.56 (dd, J = 8.2, 1.1
Hz, 1H), 8.20 (d, J = 2.2 Hz, 1H), 7.57 (dd, J = 8.3, 5.2 Hz, 1H),
7.31 (d, J = 8.7 Hz, 1H), 6.93 (dd, J = 8.8, 2.6 Hz, 1H), 5.48 (s,
5H), 4.80 (s, 2H). 13C NMR (125 MHz, DMSO-d6): δ (ppm)
201.7, 171.6, 155.3, 150.0, 149.4, 146.2, 144.9, 132.7, 128.9,
128.7, 124.0, 120.6, 120.2, 115.0, 114.9, 114.2, 108.4, 81.6,
44.6. IR v (cm-1) 3263, 3108, 2930, 2856, 1940, 1680, 1608,
1473, 1406, 1223, 1186, 1048, 1024, 1004, 808, 793, 698. HRMS
calcd for C23H15N3O3RuNa (M þ Na)þ 506.0049, found (M þ
Na)þ 506.0055.
Compound 2. To a vacuum-dried sample of complex 20
(11 mg, 0.017 mmol) was added freshly distilled THF
(1.1 mL). and the resulting solution was purged with nitrogen
while cooling to -40 °C. Ice-cold nitrogen-purged tetrabutyl-
˚
ammonium fluoride (1 M solution in THF, dried over 4 A
molecular sieves) (19 μL, 0.019 mmol) was then added, and the
reaction mixture immediately became a dark suspension. Within
1 min, the reaction was quenched with excess glacial acetic acid,
and the resulting yellow solution was concentrated to dryness in
vacuo. The crude material was then subjected to silica gel
chromatography with CH2Cl2/MeOH (20:1). The combined
product eluents were dried in vacuo to provide complex 2 (5.8
mg, 71%) as a yellow orange solid. 1H NMR (300 MHz, DMSO-
d6): δ (ppm) 9.25 (dd, J = 8.3, 1.2 Hz, 1H), 9.11 (dd, J = 5.2, 1.2
Hz, 1H), 8.95 (s, 1H), 8.31 (s, 1H), 7.57 (dd, J = 8.3, 5.2 Hz, 1H),
7.39-7.36 (m, 2H), 6.99 (dd, J = 8.8, 2.3 Hz, 1H), 5.46 (s, 5H),
4.84 (s, 2H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 201.6,
172.0, 154.0, 151.5, 150.5, 146.1, 144.7, 143.8, 131.7, 124.3,
122.1, 121.1, 115.8, 114.6, 114.2, 111.7, 105.6, 81.6, 44.3. IR v
(cm-1) 3232, 3108, 2928, 2854, 1938, 1651, 1606, 1583, 1520,
1452, 1401, 1334, 1216, 1184, 1024, 1004, 814, 789, 715, 693, 559.
HRMS calcd for C23H16N3O3Ru (M þ H)þ 484.0230, found
(M þ H)þ 484.0233.
Compound 20. To a vacuum-dried solid mixture of lactam
6 (10 mg, 0.023 mmol), ruthenium precursor 4 (11 mg, 0.025
mmol) and K2CO3 (3.5 mg, 0.025 mmol) was added freshly
distilled DMF (1.0 mL) and the resulting suspension was purged
with nitrogen for approximately 10 min. The reaction mixture
was then heated to 80 °C for 10 min in a microwave reactor
(CEM Discover). The resulting yellow-orange solution was
concentrated using high vacuum and coevaporated once with
EtOAc. The crude material was subjected to silica gel chroma-
tography with toluene/acetone (3:1). The combined product
eluents were dried in vacuo to provide complex 20 (13 mg,
87%) as an yellow solid. 1H NMR (300 MHz, CDCl3): δ (ppm)
9.41 (dd, J = 8.3, 1.2 Hz, 1H), 8.81 (dd, J = 5.2, 1.2 Hz, 1H),
7.48 (d, J = 2.4 Hz, 1H), 7.40 (dd, J = 8.3, 5.2 Hz, 1H), 7.33 (d,
J = 8.8 Hz, 1H), 7.13 (dd, J = 8.8, 2.4 Hz, 1H), 6.26 (s, 1H), 5.19
(s, 5H), 4.95 (s, 2H), 1.40-1.28 (m, 3H), 1.17 (d, J = 7.2 Hz,
18H). 13C NMR (125 MHz, CDCl3): δ (ppm) 200.4, 173.7,
153.2, 153.1, 149.6, 147.8, 145.2, 145.0, 133.2, 124.6, 123.3,
121.0, 118.6, 115.7, 115.2, 111.9, 110.7, 80.8, 45.1, 18.3, 13.0.
IR v (cm-1) 2943, 2865, 1951, 1676, 1608, 1583, 1520, 1456,
1432, 1421, 1397, 1334, 1275, 1247, 1207, 1189, 923, 882, 685.
HRMS calcd for C32H36N3O3RuSi (M þ H)þ 640.1564, found
(M þ H)þ 640.1537.
Resolution of (RRu)-1 and (SRu)-1. Baseline separation of the
enantiomers of half sandwich complex 19 was achieved using the
Chiral Pak 1B analytical HPLC column (Daicel/Chiral
Technologies). First, 19 was dissolved in a minimal amount of
methylene chloride and carefully filtered. Then, each injection
was run using an ethanol gradient of 20% to 80% over 20 min in
hexanes. A flow rate of 0.7 mL/min was used, and injection
volumes of approximately 50 μL could be reached without
compromising baseline separation. Each enantiomer was then
treated separately with TBAF to provide both (RRu)-1 and
(SRu)-1. Retention of enantiomeric purity was confirmed using
the same Chiral Pak 1B analytical HPLC column. For this,
10 mM stock solutions (RRu)-1 and (SRu)-1 in DMSO were
diluted with an equal volume of ethanol and carefully filtered.
Then, analytical injections (5 μL) were run using an ethanol
gradient of 50%to95% over 20 min inhexanes with a flow rate of
0.7 mL/min. (RRu)-1 and (SRu)-1 were determined to be 99% and
98% enantiopure, respectively. Absolute configurations were
assigned by CD-spectroscopy (see Supporting Information).
Resolution of (RRu)-2 and (SRu)-2. Baseline separation of the
enantiomers of half sandwich complex 20 was achieved using the
Chiral Pak 1B analytical HPLC column (Daicel/Chiral
Technologies). First, 20 was dissolved in a minimal amount of
methylene chloride and carefully filtered. Then, each injection
was run using an acetone gradient of 25% to 75% over 20 min in
hexanes. A flow rate of 0.7 mL/min was used and injection
volumes of approximately 50 μL could be reached without
compromising baseline separation. Additionally, a detection
wavelength of 470 nm was used to avoid acetone absorption.
Compound 1. To a vacuum-dried sample of complex 19 (23
mg, 0.036 mmol) was added freshly distilled THF (2.3 mL), and
the resulting solution was purged with nitrogen while cooling to
0 °C. Tetrabutylammonium fluoride (1 M solution in THF,
˚
dried over 4 A molecular sieves) (47 μL, 0.047 mmol) was then
added, and the reaction mixture immediately became a dark red
suspension. Within 1 min, the reaction was quenched with excess
saturated aqueous NH4Cl (orange color returns) and extracted
9006 J. Org. Chem. Vol. 74, No. 23, 2009

Pagano et al.
JOCArticle
Each enantiomer was then treated separately with TBAF to
provide both (RRu)-2 and (SRu)-2. Retention of enantiomeric
purity was confirmed using the same Chiral Pak 1B analytical
HPLC column. First, 10 mM stock solutions (RRu)-2 and (SRu)-
2 in DMSO were diluted with an equal volume of ethanol and
carefully filtered. Then, analytical injections (5 μL) were run
using an acetone gradient of 40% to 75% over 2 h in hexanes
with a flow rate of 0.7 mL/min and a detection wavelength of
470 nm. (RRu)-2 and (SRu)-2 were determined to be 99% and
98% enantiopure, respectively. Absolute configurations were
assigned by CD-spectroscopy (see Supporting Information).
5-(Triisopropylsilyloxy)indole (22).27 A solution of 5-benzy-
loxyindole (2.50 g, 11.2 mmol) in EtOAc (100 mL) was purged
with nitrogen for 15 min. Following thorough nitrogen purge,
palladium (10 wt % on activated carbon, dry) (2.50 g) was
added, and the reaction mixture was carefully purged with
hydrogen gas for approximately 10 min. The reaction mixture
was then stirred at room temperature under an atmosphere
of hydrogen, monitoring by thin layer chromatography. After
45 min, the resulting dark reaction mixture was filtered through
Celite with EtOAc, and the filtrate concentrated to dryness in
vacuo. The resulting light yellow solid was then redissolved in
DMF (40 mL) and purged with nitrogen while cooling to 0 °C.
Then, N,N-diisopropylethylamine (9.70 mL, 56.0 mmol) was
added dropwise. After stirring for 10 min at 0 °C, triisopropyl-
silyl trifluoromethanesulfonate (3.0 mL, 11.2 mmol) was added
dropwise over 15 min, and the reaction mixture was stirred at
room temperature for an additional 1 h. The reaction mixture
was cooled to 0 °C, and excess 1 M NH4OAc was carefully
added. The mixture was then further diluted with water and
extracted with EtOAc. The combined organic layers were
washed with brine, dried using Na2SO4, filtered, and concen-
trated to dryness in vacuo. The crude material was subjected to a
short flash column using only CH2Cl2. The combined product
eluents were dried in vacuo to provide indole 22 (2.85 g, 87%
(10.2 mL). Following the addition of triisopropyl borate
(2.73 mL, 11.8 mmol), the reaction mixture was cooled to 0 °C,
and lithium diisopropylamide (2.0 M solution in heptane/tetra-
hydrofuran/ethylbenzene) (5.9 mL, 11.8 mmol) was added drop-
wise over 1 h. After stirring for an additional 30 min at 0 °C, 2 M
HCl (20 mL) was carefully added and allowed to stir at room
temperature for 10 min. The resulting layers were separated, and
the aqueous layer was extracted using EtOAc. The combined
organic layers were dried using Na2SO4, filtered, and concentrated
to dryness in vacuo to provide 1-(tert-butoxycarbonyl)-5-(triiso-
propylsilyloxy)indol-2-yl-boronic acid as an orange foam, which
due to instability was converted to the trifluoroborate 24 by
reacting with KHF2 (5.7 mL, 4.5 M in water) in MeOH. After
30 min, the solution was evaporated to dryness, and the crude solid
was dissolved in acetone, sonicated, and then separated from the
remaining insoluble material by filtration. The combined filtrates
were concentrated in vacuo, the crude product was redissolved in a
minimal amount of hot acetone, and the subsequent addition of
hexane led to the precipitation of the product, which was filtered,
collected, and dried in vacuo to provide compound 24 as a white
solid (2.11 g, 55%). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 7.85
(d, J = 8.8 Hz, 1H), 6.83 (d, J = 2.4 Hz, 1H), 6.64 (dd, J = 8.8, 2.4
Hz, 1H), 6.30 (s, 1H), 1.55 (s, 9H), 1.27-1.19 (m, 3H), 1.06 (d, J =
7.1 Hz, 18H). 13C NMR (75 MHz, DMSO-d6): δ (ppm) 151.2,
150.1, 132.6, 131.7, 114.9, 114.4, 111.6, 108.8, 81.1, 27.7, 17.8, 12.0
(C-B not observed). IR v (cm-1), 2968, 2943, 2867, 1699, 1458,
1366, 1303, 1256, 1188, 1127, 984, 969, 884, 873, 851, 689, 626, 478.
HRMS calcd for C22H34BF3NO3Si (M - K)- 456.2359, found
(M - K)- 456.2348.
Compound 26. To a vacuum-dried solid mixture of trifluoro-
borate 24 (2.23 g, 4.51 mmol), triflate 25 (1.00 g, 3.61 mmol),
tetrakis(triphenylphosphine)palladium(0) (416 mg, 0.36 mmol),
and Na2CO3 (1.05 g, 9.93 mmol) was added freshly distilled
anhydrous THF (20 mL). After purging with nitrogen for
15 min, the reaction mixture was heated to reflux overnight.
The resulting orange reaction mixture was cooled to room
temperature, diluted with water, and extracted with EtOAc.
The combined organic layers were washed with brine, dried
using Na2SO4, filtered, and concentrated to dryness in vacuo.
The crude material was adsorbed onto silica gel and purified by
chromatography using hexanes/EtOAc (10:1). The combined
product eluents were dried in vacuo to provide compound 26
(1.17 g, 78%) as a white solid. 1H NMR (300 MHz, CDCl3): 9.71
(s, 1H), 9.22 (s, 1H), 8.10 (s, 1H), 7.94 (d, J = 8.1 Hz, 1H), 7.84
(d, J = 8.3 Hz, 1H), 7.68 (ddd, J = 8.2, 6.9, 1.3 Hz, 1H), 7.54
(ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.25 (d, J = 8.6 Hz, 1H), 7.14 (d,
J = 2.3 Hz, 1H), 7.00 (dd, J = 2.1, 0.9 Hz, 1H), 6.84 (dd, J =
8.7, 2.3 Hz, 1H), 1.37-1.25 (m, 3H), 1.15 (d, J = 6.9 Hz, 18H).
13C NMR (75 MHz, CDCl3): δ (ppm) 152.3, 150.3, 144.7, 138.0,
136.8, 132.3, 131.0, 130.3, 128.0, 128.0, 127.1, 126.9, 117.4,
115.5, 111.8, 109.9, 99.3, 18.3, 13.0. IR v (cm-1) 3409, 2944,
2866, 1621, 1575, 1470, 1447, 1407, 1258, 1211, 1182, 1143, 1123,
991, 960, 880, 853, 789, 752, 684, 641, 623, 575, 548, 502, 471.
HRMS calcd for C26H33N2OSi (M þ H)þ 417.2357, found
(M þ H)þ 417.2355.
1
over 2 steps) as a clear oil. H NMR consistent with literature
reported spectrum.27
1-(tert-Butoxycarbonyl)-5-(triisopropylsilyloxy)indole (23). A
solution of indole 22 (2.85 g, 9.86 mmol) in THF (7.3 mL) was
purged with nitrogen while cooling to 0 °C. To the solution was
added di-tert-butyl dicarbonate (2.39 mL, 10.4 mmol) followed
by the careful addition of solid (dimethylamino)pyridine (1.44 g,
10.4 mmol). The resulting yellow suspension was stirred over-
night, naturally warming to room temperature. The reaction
mixture was cooled to 0 °C, and 1 M HCl (7 mL) was carefully
added. After stirring at room temperature for 5 min, the organic
layer was separated, and the aqueous layer was extracted with
EtOAc. The combined organic layers were washed with brine,
dried using Na2SO4, filtered, and concentrated to dryness in
vacuo. The crude material was adsorbed onto silica gel and
subjected to silica gel chromatography with hexanes/EtOAc
(20:1). The combined product eluents were dried in vacuo to
provide 23 (3.76 g, 98%). 1H NMR (300 MHz, CDCl3): δ (ppm)
7.95 (d, J = 8.5 Hz, 1H), 7.54 (d, J = 3.6 Hz, 1H), 7.02 (d, J =
2.4 Hz, 1H), 6.88 (dd, J = 8.9, 2.5 Hz, 1H), 6.45 (d, J = 3.7 Hz,
1H), 1.66 (s, 9H), 1.33-1.21 (m, 3H), 1.11 (d, J = 6.9 Hz, 18H).
13C NMR (75 MHz, CDCl3): δ (ppm) 152.0, 150.0, 131.7, 130.4,
126.6, 117.7, 115.7, 110.8, 107.3, 83.6, 28.4, 18.2, 12.9. IR ν
(cm-1) 2941, 2866, 1723, 1458, 1376, 1327, 1280, 1257, 1217,
1183, 1150, 1122, 1078, 962, 877, 857, 811, 764, 725, 685, 637,
500, 478. HRMS calcd for C22H36NO3Si (M þ H)þ 390.2459,
found (M þ H)þ 390.2458.
Compound 27. A solution of isoquinolinyl-indole 26 (750 mg,
1.80 mmol) in freshly distilled THF (4.7 mL) was prepared after
drying 26 under high vacuum. After cooling the solution to
-15 °C, lithium bis(trimethylsilyl)amide (1 M solution in
hexanes) (5.4 mL, 5.4 mmol) was added dropwise over 15 min,
and the resulting orange solution was stirred at -15 °C for an
additional 45 min. A solution of vacuum-dried maleimide 10
(697 mg, 1.89 mmol) in freshly distilled THF (4.7 mL) cooled to
0 °C was added, and the reaction mixture was stirred at -15 °C
for 20 min and room temperature overnight. The resulting dark
purple reaction mixture was carefully poured into stirring ice-
cold water (200 mL) and extracted with EtOAc. The combined
organic layers were washed with brine, dried using Na2SO4,
Potassium-1-(tert-butoxycarbonyl)-5-(triisopropylsilyloxy)indol-
2-yl-trifluoroborate (24). Indole 23 (3.04 g, 7.81 mmol) was dried
under high vacuum and dissolved in freshly distilled THF
(27) Kondo, Y.; Kojima, S.; Sakamoto, T. J. Org. Chem. 1997, 62, 6507–
6511.
J. Org. Chem. Vol. 74, No. 23, 2009 9007

Pagano et al.
JOCArticle
filtered, and concentrated to dryness in vacuo. The crude
material was adsorbed onto silica gel and subjected to silica
gel chromatography with hexanes/EtOAc (10:1 to 8:1). The
combined product eluents were dried in vacuo to provide the
dried in vacuo to provide lactam 29 (51 mg, 47% over 3 steps)
as a light yellow solid. The constitution of 29 was confirmed
by ROESY analysis. 1H NMR (300 MHz, DMSO-d6): δ (ppm)
12.27 (s, 1H), 9.60 (s, 1H), 8.94 (s, 1H), 8.90 (d, J = 2.5 Hz,
1H), 8.53 (d, J = 8.5 Hz, 1H), 8.42 (d, J = 7.9 Hz, 1H), 8.09 (t,
J = 7.4 Hz, 1H), 7.90 (t, J = 7.4 Hz, 1H), 7.59 (d, J = 8.7 Hz,
1H), 7.09 (dd, J = 8.7, 2.6 Hz, 1H), 5.25 (s, 2H), 1.40-1.34 (m,
3H), 1.14 (d, J = 7.3 Hz, 18H). 13C NMR (125 MHz, DMSO-
d6): δ (ppm) 171.0, 168.3, 153.5, 148.5, 137.9, 135.3, 132.6,
132.2, 132.1, 129.6, 127.3, 127.0, 126.7, 124.9, 122.3,
119.5, 116.5, 115.8, 114.5, 111.9, 48.6, 17.9, 12.1. IR v
(cm-1) 3187, 3069, 2941, 2893, 2863, 1686, 1665, 1623, 1572,
1461, 1369, 1258, 1209, 1185, 1159, 1077, 1040, 1015, 998, 974,
881, 801, 745, 710, 679, 627, 600, 559, 503, 449. HRMS calcd
for C30H34N3O2Si (M þ H)þ 496.2415, found (M þ H)þ
496.2427.
1
unstable monobromide 27 (1.0 g, 79%) as an orange solid. H
NMR (300 MHz, CDCl3): δ (ppm) 10.00 (s, 1H), 9.21 (s, 1H),
7.94 (d, J = 8.1 Hz, 1H), 7.71-7.66 (m, 2H), 7.61-7.57 (m, 2H),
7.26 (d, J = 8.6 Hz, 1H), 7.00 (d, J = 2.0 Hz, 1H), 6.86 (d, J =
8.3 Hz, 1H), 1.30 (septet, J = 7.4 Hz, 3H), 1.14 (d, J = 7.4 Hz,
18H), 0.98 (s, 9H), 0.47 (s, 6H). IR v (cm-1) 3335, 2942, 2892,
2863, 1697, 1648, 1625, 1580, 1466, 1431, 1312, 1266, 1217, 1182,
1076, 977, 897, 879, 844, 824, 795, 742, 709, 680, 619, 582,
556, 467, 405. HRMS calcd for C36H47BrN3O3Si2 (M þ H)þ
706.2313, found (M þ H)þ 706.2313.
Compound 28. An orange suspension of monobromide 27
(800 mg, 1.14 mmol) in toluene (250 mL) was irradiated with a
medium pressure Hg lamp using an uranium filter (50% trans-
mission at 350 nm) for 4 h with constant nitrogen flow through
the reaction mixture. The resulting bright orange suspension
was concentrated to dryness in vacuo, adsorbed onto silica gel,
and subjected to silica gel chromatography with hexanes/EtOAc
(6:1 to 3:1). The combined product eluents were dried in vacuo
to provide heterocycle 28 (610 mg, 86%) as a yellow solid.
1H NMR (300 MHz, CDCl3): δ (ppm) 10.23 (d, J = 8.6 Hz, 1H),
9.95 (s, 1H), 9.31 (s, 1H), 8.87 (d, J = 2.5 Hz, 1H), 8.09 (d,
J = 8.0 Hz, 1H), 8.00 (ddd, J = 8.6, 7.1, 1.5 Hz, 1H), 7.98 (ddd,
J = 8.0, 7.0, 0.9 Hz, 1H), 7.46 (d, J = 8.7 Hz, 1H), 7.21 (dd,
J = 8.7, 2.5 Hz, 1H), 1.49-1.39 (m, 3H), 1.20 (d, J = 7.3 Hz,
18H), 1.09 (s, 9H), 0.68 (s, 6H). 13C NMR (100 MHz, CDCl3):
qδ (ppm) 175.4, 173.9, 154.3, 151.2, 142.6, 135.5, 135.2,
132.2, 131.7, 129.9, 128.8, 128.5, 127.8, 123.1, 121.6, 120.4,
116.1, 115.6, 111.6, 26.8, 19.5, 18.3, 12.9, -3.5 (two hidden
carbons). IR v (cm-1) 2939, 2862, 1686, 1482, 1463, 1362,
1333, 1301, 1276, 1248, 1212, 1178, 1156, 1080, 976, 884,
866, 846, 821, 787, 753, 682, 665, 607, 583, 478, 407. HRMS
calcd for C36H46N3O3Si2 (M þ H)þ 624.3072, found (M þ H)þ
624.3072.
Ruthenium Complex 31. To a vacuum-dried solid mixture of
lactam 29 (10 mg, 0.020 mmol), ruthenium precursor 305 (11 mg,
0.022 mmol), and K2CO3 (3.0 mg, 0.022 mmol) was added
freshly distilled DMF (1.0 mL) and the resulting suspension
was purged with nitrogen for approximately 10 min. The reac-
tion mixture was then heated to 80 °C for 10 min in a microwave
reactor (CEM Discover). The resulting dark red solution was
concentrated using high vacuum and coevaporated once with
EtOAc. The crude material was subjected to silica gel chromato-
graphy with toluene:acetone (3:1). The combined product elu-
ents were dried in vacuo to provide ruthenium complex 31
1
(8.3 mg, 54%) as a red film. H NMR (300 MHz, CDCl3): δ
(ppm) 9.34 (s, 1H), 8.72 (d, J = 2.5 Hz, 1H), 8.31 (d, J = 8.1 Hz,
1H), 8.12 (d, J = 8.1 Hz, 1H), 7.96 (t, J = 7.4 Hz, 1H), 7.76 (t,
J = 7.4 Hz, 1H), 7.30 (d, J = 8.7 Hz, 1H), 7.20 (dd, J = 8.7, 2.5
Hz, 1H), 6.77 (s, 1H), 6.02 (m, 1H), 5.89 (m, 1H), 5.46 (m, 1H),
5.34 (m, 1H), 5.19 (d, J = 5.4 Hz, 2H), 3.75 (s, 3H), 1.48-1.38
(m, 3H), 1.22 (d, J = 7.2 Hz, 18H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 199.1, 172.7, 167.2, 157.9, 151.7, 148.3, 147.8,
140.3, 133.3, 132.9, 129.4, 128.5, 127.3, 127.0, 126.6, 124.3,
124.1, 119.7, 117.3, 116.2, 114.6, 113.7, 93.8, 86.0, 85.3, 77.7,
75.3, 52.2, 48.5, 18.2, 12.8. IR v (cm-1) 2923, 2854, 1962, 1714,
1684, 1601, 1457, 1440, 1380, 1289, 1271, 1251, 1209, 1144, 884,
755. HRMS calcd for C37H40N3O4RuSi (M - CO þ H)þ
720.1826, found (M - CO þ H)þ 720.1823.
Compound 29. A solution of imide 28 (135 mg, 0.217 mmol) in
THF-MeOH (1:1) (14 mL) was cooled to 0 °C. Sodium boro-
hydride (16 mg, 0.421 mmol) was added every hour for a total of
5 h, and the stirred reaction mixture was then allowed to
naturally warm to room temperature overnight. The reaction
mixture was again cooled to 0 °C, carefully quenched with
addition of saturated aqueous NH4Cl, and further diluted with
water and extracted with CH2Cl2. The combined organic layers
were washed with brine, dried using Na2SO4, filtered, and
concentrated to dryness. The crude material was adsorbed onto
silica gel and subjected to silica gel chromatography with
hexanes/EtOAc (6:1 to 3:1) to provide a yellow solid. A yellow
suspension of this solid (100 mg) in CH2Cl2 (9.0 mL) containing
0.1% ethanol was cooled to 0 °C, trifluoroacetic acid (1.0 mL)
was then carefully added, and the resulting orange solution was
stirred at 0 °C for 15 min. The reaction mixture was then
carefully quenched with saturated aqueous NaHCO3 and ex-
tracted with CH2Cl2. The combined organic layers were washed
with brine, dried using Na2SO4, filtered, and concentrated to
dryness in vacuo. A suspension of the crude material in CH2Cl2
(10 mL) was purged with nitrogen for 15 min. Phenylselenol
(68 μL, 0.640 mmol) followed by trifluoroacetic acid (18.4 μL,
0.240 mmol) were added, and the resulting dark orange solution
was heated to reflux for 5 h. The reaction mixture was cooled to
room temperature, carefully quenched with saturated aqueous
NaHCO3, and extracted with CH2Cl2. The combined organic
layers were washed with brine, dried using Na2SO4, filtered, and
concentrated to dryness in vacuo. The crude material was
adsorbed onto silica gel and subjected to silica gel chromatog-
raphy first with hexanes/EtOAc (3.5:1) and followed using
CH2Cl2/MeOH (80:1). The combined product eluents were
Ruthenium Complex 21. To a vacuum-dried sample of com-
plex 31 (14.8 mg, 0.020 mmol) was added freshly distilled THF
(2.1 mL), and the resulting solution was purged with nitrogen
while cooling to 0 °C. Tetrabutylammonium fluoride (1 M
˚
solution in THF, dried over 4 A molecular sieve) (25 μL, 0.025
mmol) was then added, and the reaction mixture immediately
became a dark red-brown suspension. Within 1 min, the reaction
was quenched with excess saturated aqueous NH4Cl and ex-
tracted with EtOAc. The combined organic layers were dried
using Na2SO4, filtered, and concentrated to dryness in vacuo.
The crude material was then subjected to silica gel chromato-
graphy first with CH2Cl2/hexanes (5:1) and followed by using
CH2Cl2/MeOH (30:1). The combined product eluents were
dried in vacuo to provide ruthenium complex 21 (7.8 mg,
1
66%) as a dark red solid. H NMR (300 MHz, DMSO-d6): δ
(ppm) 9.80 (s, 1H), 9.00 (s, 1H), 8.79 (s, 1H), 8.51 (d, J = 8.4 Hz,
1H), 8.39-8.36 (m, 2H), 8.13 (ddd, J = 8.4, 7.2, 1.2 Hz, 1H),
7.92 (t, J = 7.5 Hz, 1H), 7.29 (d, J = 8.7 Hz, 1H), 6.99 (dd, J =
8.7, 2.6 Hz, 1H), 6.20 (m, 2H), 5.67 (m, 2H), 5.20 (m, 2H), 3.48 (s,
3H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 200.8, 171.0,
166.0, 159.5, 150.9, 149.3, 146.4, 139.2, 133.4, 132.2, 129.7,
128.8, 127.8, 127.1, 126.8, 124.8, 123.7, 115.8, 115.7, 115.4,
114.2, 109.4, 89.9, 89.6, 81.4, 81.0, 79.2, 51.8, 47.9. IR v
(cm-1) 2918, 2850, 2957, 1953, 1668, 1601, 1464, 1440, 1379,
1286, 1259, 1160, 1022, 798, 755, 718, 680, 559, 438, 402. HRMS
calcd for C28H20N3O4Ru (M - CO þ H)þ 564.0492, found
(M - CO þ H)þ 564.0491.
9008 J. Org. Chem. Vol. 74, No. 23, 2009

Pagano et al.
JOCArticle
Resolution of (RRu)-21 and (SRu)-21. Baseline separation of
the enantiomers of racemic half sandwich complex 21 was
achieved using a Chiral Pak 1B analytical HPLC column
(Daicel/Chiral Technologies). First, 21 was dissolved in a mini-
mal amount of CH2Cl2 and carefully filtered. Then, each injec-
tion was run using an EtOH gradient of 50% to 90% over 20 min
in hexanes. A flow rate of 0.7 mL/min was used, and injection
volumes of approximately 25 μL could be reached without
compromising baseline separation. Amounts of 2.8 mg of both
(RRu)-21 and (SRu)-21 were isolated, and the absolute config-
uration was assigned by CD-spectroscopy (see Supporting
Information).
Protein Kinase Assays Using [γ-33P]ATP. Protein kinases
(human) and substrates were purchased from Upstate Biotech-
nology USA, Millipore. Various concentrations of (RRu)-3,
(SRu)-3, or (RRu)-21 were incubated at room temperature in
20 mM MOPS, 30 mM Mg(OAc)2, 0.8 μg/μL BSA, 5% DMSO
(resulting from the inhibitor stock solution), pH 7.0, in the
presence of substrate (GSK3β: 20 μM phosphoglycogen
synthase peptide-2; Pim1: 50 μM S6 kinase/Rsk2 substrate
peptide 2; TrkA: 150 μM Trk peptide KKKSPGEYVNIEFG)
and kinase (0.4-2.0 nM GSK3β, 0.3-1.6 nM Pim1, 4.9-9.8 nM
TrkA). After 15 min, the reaction was initiated by adding ATP
to a final concentration of 100 μM, including approximately
0.05 μCi/μL [γ-33P]ATP (GSK3β and Pim1) or 0.1 μCi/μL
[γ-33P]ATP (TrkA). Reactions were performed in a total volume
of 25 μL. After 30 min (GSK3β and Pim1) or 2 h (TrkA), the
reaction was terminated by spotting 15 μL (TrkA) or 17.5 μL
(GSK3β, Pim1) on a circular P81 phosphocellulose paper
(2.1 cm diameter, Whatman), followed by washing four times
(5 min each wash) with 0.75% phosphoric acid and once with
acetone. The dried P81 papers were transferred to a scintillation
vial, 5 mL of scintillation cocktail was added, and the counts per
minute (CPM) were determined with a Beckmann 6000 scintilla-
tion counter. IC50 values were defined to be the concentration of
inhibitor at which the CPM was 50% of the control sample,
corrected by the background.
Protein Kinase Assays Using [γ-32P]ATP. Protein kinases
(human) and substrates were purchased from Upstate Biotech-
nology USA, Millipore. Various concentrations of (RRu)-1,
(SRu)-1, (RRu)-2, and (SRu)-2 were incubated at room tempera-
ture in 20 mM MOPS, 30 mM MgCl2, 0.8 μg/μL BSA, 5%
DMSO (resulting from the inhibitor stock solution), pH 7.0, in
the presence of substrate (GSK3β and CLK2: 20 μM and
100 μM phosphoglycogen synthase peptide-2, respectively;
Pim1: 100 μM S6 kinase/Rsk2 substrate peptide 2; TrkA: 250
μM Trk peptide KKKSPGEYVNIEFG) and kinase (2.4 nM
GSK3β, 0.8 nM Pim1, 8.0 nM TrkA, 0.6 nM CLK2). After
15 min, the reaction was initiated by adding ATP to a final
concentration of 100 μM, including approximately 0.2 μCi/μL
[γ-32P]ATP. Reactions were performed in a total volume of
25 μL and incubated at 30 °C. After 30 min (Pim1) or 45 min
(GSK3β, TrkA, and CLK2), the reaction was terminated by
spotting 17.5 μL on a circular P81 phosphocellulose paper
(2.1 cm diameter, Whatman), followed by washing four times
(5 min each wash) with 0.75% phosphoric acid and once with
acetone. The dried P81 papers were transferred to a scintillation
vial, 5 mL of scintillation cocktail was added, and the counts per
minute (CPM) were determined with a Beckmann 6000 scintilla-
tion counter. IC50 values were defined to be the concentration of
inhibitor at which the CPM was 50% of the control sample,
corrected by the background.
Acknowledgment. We thank the U.S. National Institutes
of Health for financial support (GM071695 and U54-
HG003915).
Supporting Information Available: Screening results of
compounds 1-3 and (RRu)-21 against a panel of human kinases;
assignment of the absolute configurations of (RRu)-1, (SRu)-1,
(RRu)-2, (SRu)-2, and (RRu)-21; 1H-1H-ROESY analysis of com-
pound 17; and NMR spectra for all new compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 74, No. 23, 2009 9009