Synthesis and mixed lineage kinase activity of pyrrolocarbazole and isoindolone analogs of (+)K-252a

Article abstract of DOI:10.1021/jm051074u

Structural modification of the indolecarbazole natural product (+)K-252a identified structural requirements for MLK activity and a novel series of potent fused pyrrolocarbazole MLK1/3 inhibitors. The SAR revealed that the lactam regiochemistry, the shape of the heterocycle, and aryl rings B and F are important to MLK activity. Heteroatom and alkyl replacement of the N-12 and/or N-13 indole nitrogen atoms identified the nonplanar dihydronaphthyl[3,4-a] pyrrolo[3,4-c]carbazole-7-one (8) and corresponding 5,7-dione (7) as potent cell-permeable MLK1/3 family-selective leads with in vitro activity comparable to that of (+)K-252a and determined them to be 2- to 3-fold more potent than the aglycone natural product K-252c.

Full text of DOI:10.1021/jm051074u

J. Med. Chem. 2007, 50, 433-441
433
Articles
Synthesis and Mixed Lineage Kinase Activity of Pyrrolocarbazole and Isoindolone Analogs of
(+)K-252a
Robert L. Hudkins,* Neil W. Johnson, Thelma S. Angeles, George W. Gessner, and John P. Mallamo
Cephalon, Inc., 145 Brandywine Parkway, West Chester, PennsylVania 19380
ReceiVed October 24, 2005
Structural modification of the indolecarbazole natural product (+)K-252a identified structural requirements
for MLK activity and a novel series of potent fused pyrrolocarbazole MLK1/3 inhibitors. The SAR revealed
that the lactam regiochemistry, the shape of the heterocycle, and aryl rings B and F are important to MLK
activity. Heteroatom and alkyl replacement of the N-12 and/or N-13 indole nitrogen atoms identified the
nonplanar dihydronaphthyl[3,4-a]pyrrolo[3,4-c]carbazole-7-one (8) and corresponding 5,7-dione (7) as potent
cell-permeable MLK1/3 family-selective leads with in vitro activity comparable to that of (+)K-252a and
determined them to be 2- to 3-fold more potent than the aglycone natural product K-252c.
Introduction
in several in vitro and in vivo models.7,8 1 attenuated the loss
of tyrosine hydroxylase activity and dopamine transporter
density after administration of 1-methyl-4-phenyl-tetrahydro-
pyridine (MPTP) in mice9 and reduced the development of
neurologic dysfunction in monkeys treated with MPTP.7a,9
Although 1 has no direct activity on JNK itself, the mechanism
has been shown to be a result of JNK pathway inhibition via
MLK.8 1 is a semisynthetic 3,9-bis-ethylthiomethyl derivative
of the indolocarbazole natural product (+)K-252a (2).7a,10 2 is
a nonselective ATP competitive inhibitor of numerous tyrosine
and serine/threonine kinases. One of the drawbacks of 1 is the
requirement for fermentation of the natural product starting
material (originally fermented from the culture broth of No-
cardiopsis sp.).11 An ensuing medicinal chemistry program was
initiated to identify smaller, fully synthetic MLK1/3 inhibitors
for PD. The initial objective was to define the minimum
pharmacophore and structural features of 2 required for MLK
activity in order to identify a starting core. Removal of the sugar
from glycosylated 2 to produce the aglycone K-252c (3), a
natural product produced by Nocardiopsis strain K-290,12
resulted in a molecule with MLK activity about 5-fold weaker
than that of 2, with IC50 values of 138 nM for MLK1 and 49
nM for MLK3. Because the sugar moiety is not essential, our
strategy was to conserve the lactam hydrogen bond donor/
acceptor moiety, known to be a requirement for indolocarbazoles
to bind at the conserved hinge region at the ATP site of
kinases,13 and modify the indolocarbazole ring system. We
investigated the MLK structure-activity relationships (SAR)
by making structural changes to the indolocarbazole core with
heteroatom and alkyl replacements for the indole nitrogen. An
important aspect of this work for interpreting the SAR was the
establishment of the lactam carbonyl regiochemistry necessary
for orientation of the scaffold at the hinge region for ATP
competitive inhibitors.
Several lines of evidence indicate that neuronal apoptosis may
be an important mechanism contributing to the progression of
disability in Parkinson’s disease (PD) and Alzheimer’s disease
(AD).1 Although the few available therapies afford some degree
of symptomatic relief, none prevents the progression of the
disease or delays the pathological neuronal cell death associated
with the disease. Activation of the c-jun-N-terminal kinase
(JNK)a pathway, which is critical for naturally occurring
neuronal cell death in development, has been shown to mediate
apoptotic neuronal death in response to a variety of stimuli and
may be important for the pathological cell death in neurode-
generative diseases.2
The JNKs (JNK1-3) are stress-activated protein kinases
(SAPK) that belong to the mitogen-activated protein kinase
(MAPK) superfamily and are the only kinases that phosphorylate
the transcription factor cJun on Ser63 and Ser73, an early initiating
event in the cell death process.2,3 The mixed-lineage kinases
(MLK) are an important upstream activating component of the
JNK signaling cascade and function to phosphorylate MAPK
kinases MKK4 and MKK7 of the cascade.4 The MLKs function
as serine/threonine kinases, although their catalytic domains have
features of both tyrosine and serine/threonine kinases. Overex-
pression of MLKs results in apoptotic cell death in PC12 cells
and primary sympathetic neurons. Conversely, the expression
of kinase-dead MLKs blocks apoptosis induced by trophic factor
withdrawal.4,5 Inhibitors of MLKs, and subsequently of JNK/
cJun, have the potential not only for slowing the progression
of the neurodegenerative diseases but also for improving the
function of surviving neurons.
Our research has focused on the design of potent, selective
inhibitors of MLKs for the treatment of AD and PD.6,7 1 (CEP-
1347) was the first compound from this program to advance
into clinical evaluation for Parkinson’s disease.7a Preclinical
pharmacology demonstrated that 1 promoted neuronal survival
Chemistry
Indenocarbazole imide 4 was prepared utilizing a Diels-Alder
reaction with maleimide and 2-(2-indenyl)indole as described
previously.14 Lactam isomers 5 (7-oxo) and 6 (5-oxo) were
prepared utilizing a regioselective Diels-Alder reaction with
* Correspondence author: Tel: 610-738-6283. E-mail: rhudkins@
cephalon.com.
a Abbreviations: MLK, mixed-lineage kinase; JNK, c-jun-N-terminal
kinase; MKK, MAP kinase kinase; GST, glutathione S-transferase.
10.1021/jm051074u CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/11/2007

434 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3
Hudkins et al.
essentially equivalent, whereas ethylene bridge analog 8 dis-
played a 2- to 3-fold improvement in potency (MLK1 IC50
)
46 nM, MLK3 IC50 ) 22 nM) over 3. When the N-12 nitrogen
was replaced with either sulfur (19) or oxygen (21), a 3-5-
fold loss in activity for MLK3 was observed. 19 showed a 13-
fold drop in MLK1 activity (IC50 ) 1.8 µM). The data set for
the lactam regioisomers is shown in Table 2. Methylene analog
6 was 2-fold more potent for MLK1 (IC50 138 nM) over 3 or
its regiomer 5 and was also potent for MLK3 with an IC50 value
of 40 nM. The ethylene bridge analog 9, contrary to its regiomer
8, showed a significant loss in activity with IC50 values of 1.1
and 0.7 µM for MLK1 and MLK3, respectively. One explana-
tion for the decrease in activity may be the lack of planarity
between the D-B rings. The B-D-ring dihedral angle was 30°
in C-ring expanded analog 9 due to the carbonyl-H4 crowding
effect (Figure 2). The carbonyl deshielding effect on H-4 can
Figure 1. Structures of 1 (CEP-1347), 2 ((+)-K-252a), and 3 (K-252c).
2-(2-indenyl)indole and ethyl cis-â-cyanoacrylate.14 The indi-
vidual tetrahydrocarbazole diastereomers were separated by
recrystallization, aromatized to the cyano-ester carbazoles using
DDQ, and then subjected to a reductive cyclization (RaNi, H2,
DMF, MeOH) to produce the lactams. The regiochemistry and
1
be observed in H NMR comparing 6 to 5 (δ 9.4 to δ 7.7).14
1
1
stereochemistry were assigned by H NMR and 2D H NMR
experiments and confirmed by single-crystal X-ray crystal-
lography.14 Dihydronaphthyl imide 7 (Scheme 1) and lactam
isomers 8 and 9 were produced in a similar manner starting
with 2-(3,4-dihydronaphthyl)indole 10b as the diene (Scheme
2). DDQ oxidation of 7 in dioxane produced 12. Because of
the difficulty in separating the cyano-ester tetrahydrocarbazole
isomers (13a, 13b; Scheme 2), the mixture was dehydrogenated
to a mixture of carbazoles 14a and 14b and then underwent
cyclization to produce a mixture of the lactam products.
Regiosomers 8 and 9 were separated using silica gel column
chromatography. Benzo[b]thieno- and benzo[b]furano[2,3-a]-
pyrrolo[3,4-c]carbazole imides 15 and 16 were prepared from
Michael adducts 2-(2-benzo[b]thieno)- (17) and 2-(2-benzo[b]-
furano)-3-[3-(2,5-dioxo-1H-pyrrolidinyl)]indole (18) by a novel
palladium(II)acetate/tetrachloro-1,4-benzoquinone oxidative A-E
ring closure (Scheme 3).15 Benzothiaphene and benzofuran
imides 15 and 16 were then converted to the lactam regioisomers
(19-22) using Clemmensen reduction conditions (zinc-mercury
amalgam, EtOH, HCl) and separated by reverse-phase HPLC.15
Biindene imide 23 was prepared from 2,2′-biindene16 (24) and
maleimide as shown in Scheme 4. DDQ dehydrogenation of
25 to 23 followed by a Clemmensen reduction produced lactam
26 in high yield (Scheme 4).17 2,2′-Biindene (24) was prepared
by an improved method with palladium catalyzed coupling of
2-(tributylstannyl) indene (27) with 2-bromoindene.18 Diben-
zothiaphene imide 28 was prepared by the reaction of 2,2′-bi-
benzothiaphene 29 and diethyl acetylenedicarboxylate in a sealed
tube at 190 °C (Scheme 5) to give diester 30. Intermediate
diester 30 was converted to the anhydride 31 with LiI/acetic
anhydride in pyridine (Scheme 5). Anhydride 31 was treated
with HMDS to produce imide 28, followed by Clemmensen
reduction conditions to produce lactam 32. Using a similar
procedure to prepare 28, indeno-benzothiaphene imide 33 was
prepared starting with 2-(2′-indenyl)benzothiaphene 34. The
lactam isomers 37 and 38 were separated using preparatory
HPLC.
Compounds 3, 6, and 20 were essentially planar, with the D-B-
ring dihedral angle near 0°. The sulfur analog 20 also showed
the reverse trend from its regiomer to more potent compounds,
with IC50 values of 111 and 26 nM for MLK1 and MLK3,
respectively. The oxygen replacement 22 was not tolerated for
either of the MLKs.
From this set of data, the most potent MLK profile was seen
with the 7-oxo dihydronaphthyl analog 8 and with the indeno-
carbazole (6) and benzothiaphene (20) regiomers. Shown in
Table 3 is a set of analogs prepared to evaluate the effect of
replacing both N-12 and N-13 simultaneously. In general, the
replacement of both nitrogen atoms was not favorable. CH2-S
isomers 37 and 38, although weaker than 3, were the top
compounds from this set. Bis-CH2 analog 26 showed 5-fold and
10-fold losses in activity, whereas sulfur analog 32 was inactive
(IC50 > 10 µM). All attempts to prepare the bis-benzofuran
counterpart were unsuccessful. The imide derivatives were also
tested for MLK activity with the dihydronaphthyl analog 7 (X2
) CH2CH2) displaying the most potent inhibitory profile with
IC50 values of 22 and 12 nM for MLK1 and MLK3, respectively
(Table 4). Sulfur 15 was also tolerated, whereas imides 23, 28,
and 33 lacking both N-12 and N-13 were weaker.
The emerging SAR indicated that the shape (derived from
molecular modeling) and the bridging groups were important
MLK activity. The order of activity for the pharmacophore
represented by a planar B-C-D pyrrolocarbazole ring system
was X1 ) CH2CH2 > CH2 = N > O > S when X2 ) N and X2
) CH2 = S = N > CH2CH2 >> O when X1 ) N. To
investigate this hypothesis further, we evaluated two pyrrolo-
carbazoles lacking the N-12 (39) or N-13 (40) indole nitrogen
(Figure 3).19 Analog 40 was inactive (IC50 > 10 µM) for both
MLK1 and MLK3, whereas des-N-12 carbazole 39 retained
weak activity with IC50 values of 2.9 µM for MLK1 and 1.2
µM for MLK3. 39 also retained weak survival-promoting
activity in the cholinergic spinal cord motoneuron assay, whereas
40 was inactive.19 To evaluate the contribution of aryl rings B
and F, des-aryl analogs 41 and 42 were tested and displayed
IC50 values >1 µM.20 To further evaluate whether a linear or
bent shape of the aryl rings was preferred, analogs 43 and 4421
were also tested and were inactive for MLK1 and MLK3.
Results and Discussion
The pyrrolocarbazoles and isoindolones were tested for their
MLK1 and MLK3 inhibitory activity against GST-tagged
truncated kinase-active forms of the enzymes expressed from
baculovirus constructs. The assays were established using myelin
basic protein as a substrate via a radioactive multiscreen format.
Shown in Table 1 is the MLK1/3 data for a set of lactam
regioisomers, replacing the N-12 indole nitrogen with CH2, CH2-
CH2, S, and O compared to compound 3 (K-252c) (MLK1 IC50
) 138 nM, MLK3 IC50 ) 49 nM). Methylene analog 5 was
The inhibitory activity of scaffolds 5, 6, 7, 8, and 20 were
profiled against related family members MLK2 and DLK and
for selectivity against a set of serine/threonine and tyrosine
kinases that represented targets for 2, such as protein kinase-
C10 (PKC) (mixture of Ca2+-dependent isozymes R, â, and γ),
vascular endothelial growth factor receptor-2 (VEGF-R2),22 and
trkA10,22,23 (Table 3). Interestingly, the data in Table 5 show

Pyrrolocarbazole and Isoindolone Analogs of (+)K-252a
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3 435
Scheme 1a
a Reagents and conditions: (a) n-BuLi, THF, CO2(g), -78 °C. (b) t-BuLi, THF, -78 °C, 2-tetralones. (c) NH4Cl. (d) 1 N HCl, acetone. (e) Maleimide,
190 °C. (f) DDQ, C6H5CH3, 60-70 °C. (g) DDQ, dioxane, reflux.
Scheme 2a
Scheme 4a
a Reagents and conditions: (a) 2-bromoindene, bis(triphenylphosphine)
palladium(II) chloride, ethanol. (b) Maleimide, 190 °C. (c) DDQ, C6H5CH3,
60-70 °C. (d) ZnHg, HCl, EtOH, reflux.
a Reagents and conditions: (a) cis-ethyl â-cyanoacrylate, 190 °C. (b)
DDQ, C6H5CH3, 60-70 °C. (c) RaNi, H2, DMF, MeOH (d) Silica gel
chromatography separation.
Scheme 5a
Scheme 3a
a Reagents and conditions: (a) diethyl acetylene dicarboxylate. (b) LiI,
Ac2O, pyr. (c) HMDS, MeOH, DMF. (d) ZnHg, HCl, EtOH, reflux. (e)
Reverse-phase HPLC separation.
a Reagents and conditions: (a) Pd(OAc)2, C6Cl4O2, PhCl2, reflux. (b)
ZnHg, HCl, EtOH, reflux. (c) Reverse-phase HPLC separation.
FGFR1, CDK1, CDK2, p38R, TIE2, JNK1, and âIRK and
displayed <30% inhibition at 1 µM.
The more potent compounds 5, 6, 7, 8, and 20 were evaluated
in an MLK1-cell-based assay for inhibition of MKK4 phos-
phorylation after a 1 h incubation. MLK1 was transfected into
CHO cells with dominant negative MKK4 cDNA, and phospho-
MKK4 was measured in an ELISA-based format as described
previously.8 5, 6, and 20 were weak to inactive for MLK1 cell
activity tested up to 1 µM concentration, signifying poor cell
permeability. However, dihydronaphthyl imide 7 (MLK1 en-
zyme IC50 ) 22 nM) and 7-oxo lactam 8 (MLK1 enzyme IC50
) 46 nM) displayed good MLK1 cell activity profiles with IC50
values of 90 and 274 nM, respectively (Table 6). 7 and 8 display
that the scaffolds displayed good selectivity for MLK1/3 within
the family because they only weakly inhibited DLK, and only
the 5-oxo indenocarbazole 6 inhibited MLK2 with an IC50 value
of 364 nM. The nonplanar dihydronaphthyl 8 showed activity
for PKC (IC50 ) 205 nM) but weak activity for trkA and VEGF-
R2. Planar indenocarbazoles 5 and 6 showed the opposite
selectivity profile compared to that of 8. Benzothiaphene 20
was a potent inhibitor of trkA with an IC50 value of 85 nM and
a weaker inhibitor of PKC and VEGF-R2. Compounds 5, 6, 7,
8, and 20 were screened further against PDGFRâ, EGFR,

436 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3
Hudkins et al.
Table 1. MLK1 and MLK3 Activity of N-12-Replaced (X1)
Pyrrolocarbazole Analogs
Table 3. MLK1 and MLK3 Activity of Lactam N-12 and N-13
Replacement Analogs
entry
X1
X2
MLK1a
MLK3a
3
26
32
37
38
NH
CH2
S
S
CH2
NH
CH2
S
CH2
S
138
1415
49
277
entry
X1
NH
CH2
CH2CH2
S
O
MLK1a
MLK3a
>10 000
>10 000
482
615
157
110
3
5
8
19
21
138
167
46
1795
308
49
87
22
171
270
a IC50 values are reported in nanomolar.
Table 4. MLK1 and MLK3 Activity of Imide Analogs
a IC50 values are reported in nanomolar.
Table 2. MLK1 and MLK3 Activity of N-13-Replaced (X2)
Pyrrolocarbazole Analogs
entry
X1
X2
NH
CH2
CH2CH2
CHdCH
S
O
CH2
S
MLK1a
MLK3a
3
4
7
12
15
16
23
28
33
NH
NH
NH
NH
NH
NH
CH2
S
>300
>300
22
>300
>300
12
entry
X2
NH
MLK1a
MLK3a
3
6
138
69
49
40
∼300
∼300
CH2
68
58
9
20
22
CH2CH2
S
O
1106
111
>10000
724
26
1106
>300
>300
>300
>300
>300
>300
>300
>300
S
CH2
a IC50 values are reported in nanomolar.
a IC50 values are reported in nanomolar.
Figure 2. MOPAC-minimized structure and shape of 9.
approximately a 4-fold shift from the cell-free-isolated MLK1
enzyme activity. The cell permeability of the dihydronaphthyl
core compared favorably with the cell activity of the optimized
semisynthetic 2-derived clinical compound 1 (MLK1 enzyme
IC50 ) 38 nM; MLK1 cell IC50 ) 72 nM).6 The best compounds
were further evaluated for functional survival promoting activity
as measured by the ability to enhance ChAT activity in
embryonic rat spinal cord cultures.24 ChAT catalyzes the
synthesis of the neurotransmitter acetylcholine and is considered
to be a specific biochemical marker for functional cholinergic
neurons. In the spinal cord, motor neurons are cholinergic and
express ChAT.25 ChAT activity has been used extensively to
study the effects of 2, indolocarbazole analogs, and neurotro-
phins on the survival and/or function of cholinergic neu-
rons.6,7a,10,19,24,26 In spinal cord cultures (E14-E19), a significant
number of cholinergic neurons would be expected to die in the
absence of a motor neuron survival factor, and a continual
Figure 3. Structures of pyrrolo[3,4-c]carbazole modifications.
decline in ChAT activity is observed with increasing culture
time.24a In this assay K-252a was used as an internal control
for direct comparison of the functional activity in the ChAT
assay experiments. K-252a was ineffective in the spinal ChAT
assay at concentrations of less than 100 nM and showed a
maximum enhancement of ChAT activity of 186 ( 3% above
basal levels (100%) at 300 nM. Above 300 nM, K-252a showed
a decrease in ChAT activity due to toxicity.16 The analogs were
evaluated for ChAT activity in the spinal cord cultures at 300
nM after 2 days in culture for maximum efficacy. The
experimental data shown in Table 6 represents the mean ( the
standard deviation from the three independent experiments.
Analogs 5, 6, and 20 showed a maximum enhancement of ChAT
activity at 155, 152, and 138%, respectively. The dihydronaph-

Pyrrolocarbazole and Isoindolone Analogs of (+)K-252a
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3 437
Table 5. MLK and Kinase Selectivity Profile of Selected Analogs
entry
X1
MLK1a
MLK3a
MLK2a
DLKa
PKCa
TrkAa
VEGF-R2a
2
5
6
7
8
2(+)K-252a
CH2
CH2
CH2CH2
CH2CH2
S
22
167
69
22
46
13
87
40
12
22
26
45
2664
364
339
3447
2088
360
>1000
>1000
487
>1000
>1000
250
>1000
1196
83
205
>1000
13
155
162
43
414
110
>300
>300
>300
>1000
>1000
88
20
111
a IC50 values are reported in nanomolar.
Table 6. In Vitro MLK1 Cell and Functional Activity
dropwise. The resulting yellow solution was allowed to stir for 2
h at -78 °C and then 2-tetralone (13.7 g, 12.9 mL, 93.7 mmol) in
THF (30 mL) was added dropwise, and the mixture was stirred for
1 h. The reaction was quenched by the addition of water (5 mL),
poured into a saturated NH4Cl solution (250 mL), and extracted
with ether (2 × 200 mL). The Et2O layer was washed with 100
mL of a saturated NH4Cl solution followed by drying (MgSO4)
and concentration to give an oil. Recrystallization from MeOH gave
MLK1 cell
entry
MLK1a
activityb
spinal cord ChATc
2
5
6
22
167
69
NT
22%
19%
90
181 ( 3
166 ( 8
152 ( 14
178 ( 2
171 ( 15
inactive
138 ( 1
inactive
inactive
inactive
7
22
8
46
>300
111
>10000
>300
>10000
274
NT
3%
NT
NT
NT
1
10 g (45%) of 10a as a white solid: mp 191-192 °C. H NMR
16
20
22
23
32
(DMSO-d6) δ: 2.08-2.16 (m, 2H), 2.57-2.63 (m, 1H), 2.95-
3.02 (m, 1H), 3.03 (d, J ) 16.7 Hz, 1H), 3.28 (d, J ) 16.8 Hz,
1H), 5.31 (s, 1H), 6.15 (s, 1H), 6.88 (t, J ) 7.3 Hz, 1H), 6.98 (t,
J ) 7.7 Hz, 1H), 7.04-7.08 (m, 2H), 7.31 (d, J ) 7.9 Hz, 1H),
7.38 (d, J ) 7.7 Hz, 1H), 10.98 (s, 1H). MS m/z: 264 (M + 1).
13C NMR (DMSO-d6) δ: 26.4, 34.8, 42.5, 68.5, 97.2, 111.6, 119.0,
120.1, 120.9, 125.9, 126.0, 128.1, 128.8, 129.5, 135.4, 135.9, 136.5,
146.8. Anal. Calcd for C18H17NO (C, H, N).
a IC50 values are reported in nanometers. b IC50 values are reported in
nanomolar or percent inhibition at 1 µM. NT ) not tested. c Percent of
control at 300 nM.
thyl derivatives 7 and 8 promoted the survival of spinal cord
motor neurons and enhanced ChAT activity by 178 and 171%
at 300 nM. Weak to inactive MLK inhibitors 16, 21, 22, 23,
and 32 were also inactive in this assay at 300 nM concentrations.
In conclusion, this work identified a novel series of potent
fused pyrrolocarbazole MLK1/3 inhibitors by the modification
of indolecarbazole natural product 2. The SAR revealed that
the lactam regiochemistry, the shape of the heterocycle, and
aryl rings B and F are important MLK activity. Heteroatom
and alkyl replacement of the N-12 and/or N-13 indole nitrogen
atoms identified the nonplanar dihydronaphthyl[3,4-a]pyrrolo-
[3,4-c]carbazole-7-one (8) and corresponding 5,7-dione (7) as
potent MLK1/3 family selective leads with in vitro activity
comparable to that of 2 and determined them to be 2- to 3-fold
more potent than the aglycone natural product K-252c. The
dihydronaphthyl ring system demonstrates good cell potency
and selectivity against a panel of serine/threonine and tyrosine
kinases representing a novel scaffold for further optimization.
Lead optimization and MLK1 crystallography on the dihy-
dronaphthyl scaffold leading to the progression of development
compounds will be reported in due course.
2-(3,4-Dihydronaphth-2-yl)-1H-indole (10b). A stirred solution
of 10a (5.0 g, 19.0 mmol) in acetone (150 mL) was added to 2 N
HCl (5 mL) at room temperature. The solution was stirred for 1 h,
and then water (ca. 25 mL) was added. The precipitate was collected
by filtration, washed well with water, and dried to give 4.5 g (97%)
of 10b: mp 179-180 °C. (MeOH). 1H NMR (DMSO-d6) δ: 2.71
(t, J ) 7.7 Hz, 2H), 2.89 (t, J ) 8.4 Hz, 2H), 6.67 (s, 1H), 6.98 (t,
J ) 7.1 Hz, 1H), 7.09-7.23 (m, 6H), 7.38 (d, J ) 8.0 Hz, 1H),
7.50 (d, J ) 7.8 Hz, 1H), 11.37 (s, 1H). 13C NMR (DMSO-d6) δ:
24.7, 27.8, 101.16, 111.4, 119.6, 120.6, 121.7, 122.6, 126.7, 127.2,
127.3, 127.8, 128.7, 130.7, 134.6, 135.1, 137.9, 138.6. MS(ES+)
m/z: 246 (M+). Anal. Calcd for C18H15N (C, H, N).
4c,7a,7b,12,13,13a-Hexahydro-6H,14H-naphthyl[3,4-a]pyrrolo-
[3,4-c]carbazole-5,7-(5H,7H)dione (11). A stirred mixture of 10b
(500 mg, 2.0 mmol) and maleimide (300 mg, 3.1 mmol) in a sealed
reaction vial was held at 180-190 °C in an oil bath for 0.5 h. After
cooling to ambient temperature, MeOH (5 mL) was added. The
product was precipitated, triturated, and collected to give 610 mg
(89%) of 11 as a mixture of two diastereomers. The product was
used directly in the next step: mp 256-258 °C. 1H NMR (DMSO-
d6) δ: 1.60-1.77 (m, 0.5 H) 2.09-2.13 (m, 0.5 H), 2.87-2.92
(m, 2H), 3.13-3.20 (m, 2.5 H), 3.40 (m, 1H), 3.88-3.92 (m, 1H),
4.07-4.10 (m, 0.5 H), 4.22 (d, J ) 7.7 Hz, 1H), 6.94-7.14 (m,
5H), 7.25-7.34 (m, 2H), 7.8 (m, 1H), 10.88 (s, 1H), 11.09 (s, 1H).
MS m/z: 343 (M + 1).
Experimental Section
12,13-Dihydro-6H,14H-naphthyl[3,4-a]pyrrolo[3,4-c]carbazole-
5,7(5H,7H)dione (7). Solid 2,3-dichloro-5,6-dicyano-1,4-benzo-
quinone (930 mg, 4.1 mmol) was added in one portion to a solution
of 11 (400 mg, 1.2 mmol) in toluene (50 mL). The solution was
maintained at 60-65 °C for 6 h. After cooling in an ice bath, the
solid was collected by filtration, suspended in MeOH (20 mL),
stirred for 0.5 h, and collected to give 320 mg (79%) of 7: mp
Chemistry. All reagents and anhydrous solvents were obtained
1
from commercial sources and used as received. H and 13C NMR
were obtained at 300 or 400 MHz in the solvent indicated with
tetramethylsilane as an internal standard. Coupling constants (J)
are in Hertz (Hz). Analytical HPLC was run using a Zorbax RX-
C8 5 × 150 mm2 column eluted with a mixture of acetonitrile and
water containing 0.1% trifluoroacetic acid with a gradient of 10-
100%. Column chromatography was performed on silica gel 60
(230-400 mesh).
2-2-(2-Hydroxy-1,2,3,4-tetrahydronaphthyl)indole (10a). n-
BuLi (85.3 mmol, 34 mL of 2.5 M sol in hexanes) was added
dropwise to a solution of indole (10.0 g, 85.3 mmol) in dry THF
(500 mL) at -78 °C under a nitrogen atmosphere for 15 min. The
solution was stirred for 0.5 h, after which CO2(g) was passed
through for 10 min. The solution was warmed to room temperature,
and then excess CO2(g) was removed at reduced pressure. The
solution was cooled to -78 °C, and a solution of t-BuLi (85.3
mmol, 50 mL of a 1.7 M solution in hexanes) was then added
1
258-260 °C. H NMR (DMSO-d6) δ: 2.87 (t, J ) 4.9 Hz, 2H),
3.07 (t, J ) 6.8 Hz, 2H), 7.23-7.34 (m, 4H), 7.48-7.57 (m, 2H),
8.09-8.92 (m, 1H), 8.90 (d, J ) 8.0 Hz, 1H), 11.05 (s, 1H), 11.95
(s, 1H). 13H NMR (DMSO-d6) δ: 24.6, 28.3, 112.0, 117.8, 119.7,
120.7, 121.2, 125.7, 126.2, 127.6, 127.7, 127.8, 128.6, 128.7, 129.9,
131.0, 131.2, 138.6, 142.3, 142.6, 170.4, 170.8. MS(FAB) m/z: 338
(M+). Anal. Calcd for C22H14N2O2 (C, H, N).
6H,14H-Naphthyl[3,4-a]pyrrolo[3,4-c]carbazole-5,7(5H,7H)-
dione (12). Solid 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (465
mg, 2.1 mmol) was added in one portion to a solution of 7 (200
mg, 0.59 mmol) in dry dioxane (30 mL). The mixture was stirred

438 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3
Hudkins et al.
at reflux for 12 h, and then cooled to room temperature and filtered.
The solvent was concentrated at reduced pressure. The solid residue
was held at reflux in MeOH (25 mL), cooled to room temperature,
and collected to yield 120 mg (61%) of 12. Recrystallization from
phenylphosphine) palladium(0), (775 mg, 0.7 mmol), and hexabutyl-
ditin (6.4 mL, 12.7 mmol). The reaction was held at reflux and
monitored by TLC (silica gel, 1:5 EtOAC/hexanes). After 1 h, the
starting material was consumed. The reaction was allowed to cool
to room temperature, was diluted with CH2Cl2, and was filtered
through celite. The solvent was removed at reduced pressure, and
the compound was purified through a silica gel column (5%
EtOAc-hexanes) to give 4.7 g of 2-tributylstannyl indene as a clear
oil (containing traces of hexabutyl-ditin). The compound was used
as is for the next step. 1H NMR (CDCl3) δ: 0.9 (m, 15H), 1.2 (m,
6H), 1.6 (m, 6H), 3.5(s, 2H), 7.10 (s, 1H), 7.5-7.2 (m, 4H).
2,2′-Biindene (24). To a 100 mL round-bottomed flask, fitted
with a reflux condenser, was added 2-bromoindene (1.2 g, 6.3
mmol), 27 (3.4 g, 8.4 mmol), and ethanol (70 mL). To this mixture
was added bis(triphenylphosphine) palladium(II) chloride (442 mg,
0.63 mmol). The reaction was stirred at reflux for 16 h. The reaction
was allowed to cool to room temperature, diluted with diethyl ether
(50 mL), and then filtered through a pad of alumina. The solvent
was concentrated at reduced pressure, and the product was
recrystallized from toluene to give 870 mg (60%) of 24: mp 238
1
THF-MeOH gave 12 as a brown solid: mp > 330 °C. H NMR
(DMSO-d6) δ: 7.4 (t, 1H), 7.6 (t, 1H); 7.7-7.8 (m, 3H), 8.1 (m,
1H), 8.2 (d, 1H), 8.6 (d, 1H), 9.1 (d, 1H), 10.0 (m, 1H), 11.2 (s,
1H), 12.9 (s, 1H). MS(FAB) m/z: 336 (M+). Anal. Calcd for
C22H12N2O2‚0.1 H2O (C, H, N).
3-Cyano-4-ethoxycarbonyl-1,2,3,4-tetrahydro-1,2-dihydronaph-
thyl[3,4-a]-9H-carbazole (13a) and 4-Cyano-3-ethoxycarbonyl-
1,2,3,4-tetrahydro-1,2-didronaphthyl[3,4-a]-9H-carbazole (13b).
A mixture of 10 (1.0 g, 4.1 mmol) and ethyl cis-â-cyanoacrylate
(5.0 g, 40 mmol) was heated in a sealed reaction flask and held at
180 °C with stirring for 1 h. The mixture was cooled to room
temperature and transferred to a round-bottomed flask, and excess
cyanoacrylate was removed by Kugelrohr distillation (oven tem-
perature 80-85 °C, 0.5 mm). MeOH (25 mL) was added to the
residue, and the product was triturated to give 700 mg (46%) of a
white solid. The 1H NMR showed approximately a 2:1 mixture of
13a/13b. 1H NMR (DMSO-d6) δ: 1.25 (t, 3H), 3.1-3.35 (m, 3H),
3.8 (s, m, 4H), 3.9 (m, 1H), 4.3-4.55 (m, 2H), 4.6 (d, 1H), 6.7 (d,
1H), 6.95 (s, 1H), 7.05-7.25 (m, 5H), 11.1 (s, 1H). The mixture
was used directly in the next step.
1
°C (lit. mp ) 238 °C16). H NMR (CDCl3) δ: 3.73 (s, 4H), 6.93
(s, 2H), 7.30 (m, 8H). MS m/z: 231 (M +1).
1a,3a,4,7-Tetrahydroindenyl[2,3-c]indenyl[2,3-e]isoindol-1,3-
dione (25). To a sealable borosilicate reaction tube was added 24
(98 mg, 0.4 mmol), maleimide (43 mg, 0.44 mmol), BHT (5 mg),
and CH2Cl2 (1 mL). The tube was sealed, and the reaction was
held at130 °C in an oil bath for 24 h. The reaction was allowed to
cool to room temperature, and the solvent was concentrated at
reduced pressure. The crude solid was purified via column
chromatography (silica gel, 10-75% EtOAc-hexane) to give 50
3-Cyano-4-ethoxycarbonyl-1,2-di-hydronaphthyl[3,4-a]-9H-
carbazole (14a) and 4-Cyano-3-ethoxycarbonyl-1,2-di-hydronaph-
thyl[3,4-a]-9H-carbazole (14b). 2,3-Dichloro-5,6-dicyano-1,4-
benzoquinone (900 mg, 4.0 mmol) was added in one portion to a
stirred solution of a 2:1 mixture of 13a/13b (590 mg, 1.6 mmol) in
dry toluene (50 mL). The solution was stirred at 65-70 °C for 6
h and cooled to room temperature, and the precipitate was removed
by filtration and washed with toluene (10 mL). The toluene solution
was concentrated at reduced pressure to yield a crude solid.
Purification by column chromatography (silica gel, EtOAC/hexaness
2:1) gave 510 mg (87%) of an off-white solid with a composition
1
mg (38%) of 25 as a white solid: mp 244-247 °C. H NMR
(CDCl3) δ: 3.68 (s, 4H), 3.80 (m, 2H), 4.00 (bs, 2H), 7.24 (m,
7H), 7.53 (d, J ) 7 Hz, 2H). MS m/z: 328 (M + 1).
1H-Indenyl[2,3-c]-1H-indenyl[2,3-e]isoindol-1,3-dione (23). A
mixture of 25 (50 mg, 0.15 mmol) in toluene (4 mL) was added to
solid 2,3-dichloro-5,6-dicyano-1,4-benzquinone (79 mg, 0.35 mmol)
in one portion. The reaction was heated under nitrogen at 65-70
°C for 4 h. The solution was cooled in an ice bath, and the solid
material was collected by filtration. The crude precipitate was
washed with cold methanol, leaving a pale-yellow solid (28 mg,
63%): mp 244-247 °C. 1H NMR (CDCl3) δ: 9.17 (d, J ) 4 Hz,
2H), 7.55 (m, 7H), 4.15 (s, 4H); MS m/z: 324 (M + 1). Anal.
Calc. for C22H13NO2 (C, H, N).
1
of 2:114a/14b. H NMR (DMSO-d6) δ: 1.15 and 1.4 (t, 3H), 2.9
and 3.1-3.2 (q, 2H), 4.35 and 4.6 (q, 2H), 7.2-7.7 (m, 4H), 7.9
(d, 0.5H), 8.2 (d, 0.5H), 8.4 (d,1H), 12.2 (d, 1H). The mixture was
used directly in the next step.
12,13-Dihydro-5H,6H,14H-naphthy[3,4-a]pyrrolo[3,4-c]car-
bazole-7(7H)one (8) and 12,13-dihydro-6H,7H,14H-naphthyl-
[3,4-a]pyrrolo[3,4-c]carbazole-5(5H)one (9). The mixture of
isomers 14a/14b from the preceding step (300 mg, 0.81 mmol)
was added to a Raney nickel catalyst (ca. 1 g, wet form) in 100
mL of 3:1 DMF/MeOH and hydrogenated at 35 psi on a Parr
apparatus for 16 h. The solution was diluted with DMF (50 mL),
filtered through celite, and concentrated at reduced pressure to give
210 mg (80%) of a mixture of lactam isomers. The compounds
were separated by silica gel column chromatography (2:1 EtOAc/
hexanes). The fractions containing products were pooled and
concentrated at reduced pressure. The products were recrystallized
from MeOH-ether and dried (100 °C, 0.5 mm, 12 h) to give white
1H-Indenyl[2,3-c]-1H-indenyl[2,3-e]-3H-isoindol-1-one (26).
A zinc amalgam was prepared by suspending zinc dust (122 mg,
1.9 mmol) in 1 mL of water and adding HgCl2 (35 mg, 0.08 mmol)
followed by 4 drops of concentrated HCl. This mixture was stirred
for 10 min, and the aqueous layer was decanted off. The amalgam
was washed with water and then washed repeatedly with EtOH.
The above zinc amalgam was suspended in 5 mL of EtOH, and 23
(10 mg, 0.03 mmol) was added. A few drops of concentrated HCl
were added, and the reaction was held at reflux for 3 h. The yellow
color disappeared during the first hour of heating. The reaction was
allowed to cool to room temperature, and the solution was
concentrated at reduced pressure. The residue was dissolved in 10
mL of THF-EtOAc (1:1) and washed with saturated NaHCO3 and
NaCl solutions and then dried (MgSO4). The drying agent was
removed by filtration, and the solvent was concentrated at reduced
pressure to give 8 mg (88%) of lactam 26 as a white solid: mp
1
solids. 8: Rf ) 0.3; mp > 300 °C. H NMR (DMSO-d6) δ: 2.91
(t, J ) 5.2 Hz, 2H), 3.20 (t, J ) 6 Hz, 2H), 4.87 (s, 2H), 7.19 (t,
J ) 7.3 Hz, 1H), 7.30-7.47 (m, 4H), 7.55 (d, J ) 8.1 Hz, 1H),
7.83 (d, J ) 7.7 Hz, 1H), 8.74 (s, 1H), 9.16 (d, J ) 7.9 Hz, 1H),
11.57 (s, 1H). 13H NMR (DMSO-d6) δ: 24.2, 28.6, 47.8, 111.3,
118.0, 119.3, 122.3, 124.9, 126.3, 126.5, 126.7, 126.8, 126.8, 127.5,
127.7, 128.7, 133.6, 134.1, 138.4, 138.6, 141.4, 171.8. MS(FAB)
m/z: 325 (M + 1). Anal. Calcd for C22H16N2O‚0.1 H2O (C, H, N).
9: Rf ) 0.25, mp > 300 °C. 1H NMR (DMSO-d6) δ: 2.87 (t, J )
5.6 Hz, 2H), 3.05 (t, J ) 7.2 Hz, 2H), 4.82 (s, 2H), 7.22-7.34 (m,
4H), 7.47 (t, J ) 7.8 Hz, 1H), 7.60 (d, J ) 8.1 Hz, 1H), 8.00 (d,
J ) 7.7 Hz, 1H), 8.21 (m, 1H), 8.43 (s, 1H), 11.70 (s, 1H). 13H
NMR (DMSO-d6) δ: 24.2, 28.9, 44.1, 111.9, 116.2, 119.3, 120.2,
122.2, 122.3, 125.9, 126.4, 127.3, 127.5, 130.9, 131.9, 132.3, 138.0,
140.0, 140.3, 141.1, 171.7. MS(FAB) m/z: 325 (M + 1). Anal.
Calcd for C22H16N2O‚0.25 H2O: (C, H, N).
1
256 °C. H NMR (CDCl3) δ: 9.20 (d, J ) 8 Hz, 1H), 7.50 (m,
6H), 6.24 (s, 1H), 4.83 (s, 2H), 4.05 (s, 2H), 3.95 (s, 2H); MS m/z:
310 (M + 1). Anal. Calc. for C22H13NO (C, H, N).
2,2′-Bibenzothiaphene (29). To a two-necked round-bottomed
flask fitted with a reflux condenser was added 2-bromobenzothi-
aphene (3.3 g, 15.6 mmol), 2-(tri-n-butyltin)benzothiaphene (7.3
g, 17 mmol), and toluene (40 ml). To this mixture was added
tetrakis(triphenylphosphine) palladium(0) (360 mg, 0.3 mmol) and
BHT (5 mg). The reaction was held at reflux for 16 h. After cooling
to room temperature, the solvent was removed under vacuum, and
the reaction was dissolved in DMF and filtered through celite. The
solvent was removed under vacuum, and the solid was triturated
2-(Tributylstannyl) Indene (27). To a round-bottomed flask
containing 2-bromoindene (1.64 g, 8.4 mmol) in NEt3 (75 mL) was
added palladium(II) acetate (304 mg, 11.4 mmol), tetrakis(tri-

Pyrrolocarbazole and Isoindolone Analogs of (+)K-252a
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3 439
with hexanes to give 3.58 g (13.4 mmol, 85% yield) of 2,2′biben-
zothiaphene 29 as a silver-black solid: mp 260-262 °C. 1H NMR
(DMSO-d6) δ: 7.98 (m, 2H), 7.62 (s, 1H), 7.36 (m, 2H), 7.25 (s,
1H).
g, 11.9 mmol), and 50 mL of toluene. To this mixture was added
bis(triphenylphosphine) palladium(II) dichloride (1 g, 1.5 mmol)
and BHT (5 mg). The reaction was held at reflux for 16 h. After
cooling, the solvent was removed at reduced pressure, and the
residue was dissolved in DMF/THF and filtered through celite. The
solvent was removed, and the solid was triturated with hexanes to
give 1.4 g (5.6 mmol, 47% yield) of 34 as an orange solid: mp
260-265 °C. 1H NMR (DMSO-d6) δ: 7.82 (m, 4H), 7.61 (s, 1H),
7.35 (m, 5H), 3.97 (s, 2H).
3,4-Carboethoxybenzothienyl[1,2-a]dibenzothiaphene (30). In
a sealable glass tube was placed 29 (1.02 g, 3.8 mmol), diethyl
acetylenedicarboxylate (3.1 mL, 19 mmol), and BHT (5 mg). The
reaction vessel was sealed under N2 and held at at 190 °C. The
reaction was allowed to proceed for 24 h. After allowing the reaction
vessel to cool, the contents were transferred to a round-bottomed
flask using CHCl3, and the solvent was removed under vacuum.
The solid was taken up in diethyl ether and filtered, giving 468
mg (1.07 mmol, 28%) of 3,4-carboethoxybenzothienyl[1,2-a]-
dibenzothiaphene as a pale-yellow solid. A second crop afforded
280 mg of material for a total yield of 45%: mp 206-207 °C. 1H
NMR (DMSO-d6) δ: 8.27 (d, J ) 7.4 Hz, 2H), 8.05 (d, J ) 7.9
Hz, 2H), 7.65 (m, 4H), 4.57 (q, J ) 7.1 Hz, 4H), 1.38 (t, J ) 7.1
Hz, 6H).
3,4-Carboethoxyindenyl[1,2-a]dibenzothiaphene (35). In a
sealable glass tube was placed 33 (480 mg, 3.95 mmol), diethyl
acetylenedicarboxylate (3.2 mL, 19 mmol), and BHT (10 mg). The
reaction vessel was sealed under N2 and held at 190 °C. The reaction
was allowed to proceed for 24 h. After allowing the vessel to cool,
the contents were transferred to a round-bottomed flask using
CHCl3, and solvent was removed. The crude material was passed
through a silica column, and the top fractions were collected. This
solid was taken up in diethyl ether and filtered, giving 538 mg
1
(1.29 mmol, 23%) of 35 as a pale-orange solid, mp 186 °C. H
Benzothieno[2,3-c]benzothieno[2,3-e]isoindol-1,3-dione (28).
(a) A round-bottomed flask was charged with 30 (500 mg, 1.15
mmol) and DMF (50 mL). To this mixture was added sodium
cyanide (124 mg, 2.5 mmol) and solid lithium iodide trihydrate
(476 mg, 2.5 mmol). The reaction was held at 150 °C and followed
by TLC. Additional NaCN/LiI was added as time progressed. A
total of 4 equiv each of NaCN and LiI was added over a reaction
time of 36 h, at which time the starting material was totally
consumed. The reaction mixture was cooled to room temperature
and poured over cold (0 °C) aqueous HCl. The mixture was filtered
and washed with water. The resultant solid was dried under vacuum.
The above crude solid was then placed in a round-bottomed flask,
and 50 mL of acetic anhydride was added. The reaction mixture
was then held at reflux. After 4 h at reflux, the reaction was
complete by TLC (new spot at Rf 0.65 in 1:1 EtOAc/hexanes). The
solvent was removed, and the crude oil was purified via flash
chromatography to give a bright yellow-orange solid. This solid
was triturated with diethyl ether to give 160 mg (0.44 mmol, 40%
yield) of anhydride 31 (benzothienyl[4,5-a]benzothienyl[6,7-a]-
isobenzofuran-1,3-dione) as a bright-yellow solid: mp > 300 °C.
1H NMR (DMSO-d6) δ: 9.54 (dd, J ) 5.2 Hz, J ) 2.5 Hz, 2H),
8.34 (dd, J ) 4.0 Hz, J ) 3.3 Hz, 2H), 7.73 (m, 4H). (b) 31 (75
mg, 0.2 mmol) in DMF (3 mL) was added to 1,1,1,3,3,3-
hexamethyldisilazane (4.4 mL, 20.8 mmol), followed by 30 µL (1
mmol) of methanol. The suspension became clear after ap-
proximately 15 min. TLC after 1 h showed nearly complete
consumption of the starting material. The reaction was allowed to
stir overnight for a total reaction time of 18 h. Solvent was removed
to give 65 mg (0.18 mmol, 80% yield) of 28 as a yellow solid:
NMR (DMSO-d6) δ: 8.15 (d, J ) 7.4 Hz, 1H), 7.95 (d, J ) 7.4
Hz, 1H), 7.71 (m, 2H), 7.56 (m, 2H), 7.41 (m, 2H), 4.50 (q, J )
7.0 Hz, 4H), 4.22 (s, 2H), 1.33 (t, J ) 7.0 Hz, 6H).
Indenyl[2,3-c]benzothienyl[2,3-e]isoindol-1,3-dione (33). (a) A
round-bottombottomed flask was charged with 35 (250 mg, 0.6
mmol) and pyridine (10 ml). To this mixture was added solid
lithium iodide trihydrate (677 mg, 3.6 mmol). The reaction was
held at 115 °C, followed by TLC. More LiI was added as time
progressed. A total of 5 equiv of LiI was added over a reaction
time of 36 h, at which time the starting material was totally
consumed. The reaction mixture was cooled to room temperature
and poured over cold (0 °C) aqueous HCl. The mixture was filtered
and washed with water. The resultant solid was dried under vacuum.
The above crude solid was then placed in a round-bottomed flask,
and 50 mL of acetic anhydride was added. The reaction mixture
was then held at reflux. After 4 h at reflux, the reaction was
complete by TLC (new spot at Rf 0.6 in 1:1 EtOAc/hexanes). The
solvent was removed, and the crude oil was purified via flash
chromatography to give a bright yellow-orange solid. This solid
was triturated with diethyl ether to give anhydride 36 (indenyl-
[4,5-a]benzothienyl[6,7-a]isobenzofuran-1,3-dione) (160 mg, 0.44
mmol, 40% yield) as a bright yellow solid, mp > 300 °C. 1H NMR
(DMSO d6) δ: 9.65 (d, J ) 7.2 Hz, 1H), 8.76 (d, J ) 7.6 Hz, 1H),
8.15 (s, J ) 7.4 Hz, 1H), 7.78-7.38 (m, 5H), 4.97 (s, 2H). (b)
Anhydride 36 (120 mg, 0.35 mmol) was dissolved in 3 mL of DMF.
To this mixture was added 1,1,1,3,3,3-hexamethyldisilazane (7.4
mL, 35 mmol) followed by methanol (50 µL, 1 mmol). The
suspension became clear after approximately 15 min. TLC after 1
h showed nearly complete consumption of the starting material.
The reaction was allowed to stir overnight for a total reaction time
of 18 h. Solvent was removed to give 35 mg (0.18 mmol, 30%
1
mp > 300 °C. H NMR (DMSO-d6) δ: 9.8 (dd, J ) 5.0 Hz, J )
4.1 Hz, 2H), 8.25 (dd, J ) 4.9 Hz, J ) 4.1 Hz, 2H), 7.70 (m, 4H);
MS m/z: 360 (M + 1).
1
yield) of 33 as an orange solid, mp > 280 °C. H NMR (DMSO-
Benzothieno[2,3-c]benzothieno[2,3-e]isoindol-1-one (32). To
a 10 mL ethanol suspension of Zn amalgam (3 equiv prepared as
described for 26) was added 68 mg (0.18 mmol) of 28 as a solution
in 10 mL of acetic acid. Concentrated HCl (5 mL) was added, and
the reaction was held at reflux overnight. The reaction, which
became clear and tan in color, was cooled and decanted off of the
mercury layer. After the solvent was removed, the mixture was
diluted with ethyl acetate (50 mL) and washed with saturated
NaHCO3 (2 × 25 mL). The organic layer was dried over MgSO4,
filtered, and the solvent was removed at reduced pressure. The crude
reaction mixture was purified by silica gel column chromatography
(95:5 CH2Cl2/MeOH) to give 65 mg (0.18 mmol, 100% yield) of
32 as a tan solid, mp 225-226 °C. 1H NMR (DMSO-d6) δ: 10.17
(dd, J ) 6.4 Hz, J ) 2.7 Hz, 1H), 8.22 (s, 1H), 8.21 (dd, J ) 4.1
Hz, J ) 3.7 Hz, 2H), 8.11 (dd, J ) 6.5 Hz, J ) 2.2 Hz, 1H), 7.63
(t, J ) 3.7 Hz, 2H), 7.55 (t, J ) 3.7 Hz, 2H), 5.19 (s, 2H). MS
m/z: 346 (M + 1).
d6) δ: 12.72 (s, 1H), 9.84 (d, J ) 8.3 Hz, 1H), 8.14 (d, J ) 6.4
Hz, 1H), 8.07 (d, J ) 7.5 Hz, 1H), 7.82-7.28 (m, 5H), 5.03 (s,
2H), MS m/z: 342 (M + H).
Indenyl[2,3-c]benzothienyl[2,3-e]isoindol-1 (37) and 3-one
(38). A 2 mL ethanol suspension of Zn amalgam (3 eq prepared as
described for 26) was added to 10 mg (0.3 mmol) of 30 as a solution
in 10 mL acetic acid. The reaction was held at reflux after the
addition of 1 mL of concentrated HCl. After 3h at reflux, the
reaction became clear and slightly tan. The reaction was cooled
and decanted off of the mercury layer. After most of the solvent
was removed, the mixture was diluted with ethyl acetate and washed
two times with saturated NaHCO3. The organic layer was dried
over MgSO4, was filtered, and solvent was removed to give the
compound as a mixture of isomers as a tan solid. HPLC separation
produced 37 and 38.
37: mp > 280 °C. 1H NMR (DMSO-d6) δ: 10.15 (d, J ) 5 Hz,
1H), 9.04 (s, 1H), 8.11 (d, J ) 5 Hz, 1H), 7.83 (d, J ) 7.2 Hz,
1H), 7.74 (d, J ) 7 Hz, 1H), 7.74-7.59 (m, 4H), 5.08 (s, 2H),
4.28 (s, 2H). MS m/z: 328 (M + 1).
2-(2′-Indenyl)benzothiaphene (34). To a two-necked round-
bottomed flask fitted with a reflux condenser was added 2-bro-
moindene18 (2 g, 13.3 mmol), 2-(tri-n-butyltin)benzothiaphene (5.1

440 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3
Hudkins et al.
1
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38: mp > 280 °C. H NMR (DMSO-d6) δ: 9.38 (d, J ) 7.3
Hz, 1H), 8.94 (s, 1H), 8.14-8.16 (m, 2H), 7.60-7.69 (m, 3H),
7.74-7.46 (m, 2H), 5.06 (s, 2H), 4.20 (s, 2H). MS m/z: 328 (M +
1).
Mixed-Lineage Kinase Assays. The MLK1, MLK2, and MLK3
assays were performed using the Millipore multiscreen trichloro-
acetic acid (TCA) “in-plate” format as described previously.8 Each
50 µL assay mixture contained 20 mM Hepes, pH 7.2, 5 mM
EGTA, 15 mM MgCl2, 1 mM DTT, 25 mM â-glycerophosphate,
the Km level of ATP (60 µM for MLK1 or 100 µM for MLK2 and
MLK3), 0.25 µCi [γ-32P]ATP, 0.1% BSA, 2% DMSO, and 500
µg/mL myelin basic protein. The reaction was initiated by adding
purified recombinant kinases (50, 150, and 100 ng of GST-
MLK1KD, GST-MLK2KD/LZ, and GST-MLK3KD, respectively). The
samples were incubated for 15 min at 37 °C. The reaction was
stopped by adding ice-cold 50% TCA, and the proteins were
allowed to precipitate for 30 min at 4 °C. The plates were then
washed with ice-cold 25% TCA. A supermix scintillation cocktail
was added, and the plates were allowed to equilibrate for 1 to 2 h
prior to counting using the Wallac MicroBeta 1450 PLUS scintil-
lation counter. Inhibition curves for the compounds were generated
by plotting the percent control activity versus the log of the
concentration of the compound. IC50 values were calculated by
nonlinear regression using the sigmoidal dose-response (variable
slope) equation in GraphPad Prism as follows: y ) bottom + (top
- bottom)/(1 + 10(log IC -x)*Hillslope), where y is the percent kinase
50
activity at a given concentration of compound, x is the logarithm
of the concentration of the compound, bottom is the percent of
control kinase activity at the highest compound concentration tested,
and top is the percent of control kinase activity at the lowest
compound concentration examined. The values for bottom and top
were fixed at 0 and 100, respectively. The IC50 values were reported
as the averages of duplicates that agree to within 20%.
Other Kinase Assays. Inhibition assays for recombinant receptor
tyrosine kinases (EGFR, FGFR1, âIRK, PDGFRâ, TIE2, TrkA,
and VEGFR2) and serine/threonine kinases (CDK1, CDK2, DLK,
JNK1, p38R, and PKC) were performed as described previously.27-29
Acknowledgment. We acknowledge the scientific contribu-
tions of and helpful discussions with Mark Ator, Ed Bacon,
Jim Diebold, Marcie Glicksman, Jean Husten, Sherri Meyer,
Chung Ho Park, Dave Rotella, Mary Savage, and Shi Yang.
Supporting Information Available: Elemental analysis of
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
(10) Kaneko, M.; Saito, Y.; Saito, H.; Matsumoto, T.; Matsuda, Y.;
Vaught, J. L.; Dionne, C. A.; Angeles, T. S.; Glicksman, M. A.;
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protein kinase C from microbial origin. J. Antibiot. 1986, 39, 1059-
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(14) Hudkins, R. L.; Park, C-H. Synthesis of indeno[2, 1-a]pyrrolo[3,4-
c]carbazole lactam regioisomers using ethyl cis-â-cyanoacrylate as
a dienophile and lactam precursor. J. Heterocycl. Chem. 2003, 40,
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(15) Hudkins, R. L.; Johnson, N. W. Synthesis of benzo[b]thieno- and
benzo[b]furano[2,3-a]pyrrolo[3,4-c]carbazole-5-, -7- and -5,7-dione.
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