Synthesis, modeling, and in vitro activity of (3′S)-epi-K-252a analogues. Elucidating the stereochemical requirements of the 3′-sugar alcohol on trkA tyrosine kinase activity

Article abstract of DOI:10.1021/jm040178m

Utilizing our recently published semisynthetic approach to the (3′S)-K-252a diastereomer, we report the first synthesis of the (3′R)-10 diastereomer and a set of related epimers, with the goal of defining the Stereochemical role of the 3′-sugar hydroxyl g

Full text of DOI:10.1021/jm040178m

3776
J. Med. Chem. 2005, 48, 3776-3783
Synthesis, Modeling, and In Vitro Activity of (3′S)-epi-K-252a Analogues.
Elucidating the Stereochemical Requirements of the 3′-Sugar Alcohol on trkA
Tyrosine Kinase Activity
Diane E. Gingrich,† Shi X. Yang,‡ George W. Gessner,‡ Thelma S. Angeles,‡ and Robert L. Hudkins*,†
Departments of Medicinal Chemistry and Biochemistry, Cephalon, Inc., 145 Brandywine Parkway,
West Chester, Pennsylvania 19380
Received October 4, 2004
Utilizing our recently published semisynthetic approach to the (3′S)-K-252a diastereomer, we
report the first synthesis of the (3′R)-10 diastereomer and a set of related epimers, with the
goal of defining the stereochemical role of the 3′-sugar hydroxyl group on trkA tyrosine kinase
activity and selectivity. (3′R)-10 displayed potent trkA inhibitory activity with an IC50 value of
4 nM. The corresponding deshydroxy epimer (3′S)-14 was 7-fold more potent than its 3′R
counterpart (natural stereochemistry) with a trkA IC50 value of 3 nM and demonstrated >280-
fold selectivity over PKC (IC50 ) 850 nM). In cells, (3′S)-14 displayed potent inhibition of trkA
autophosphorylation with an IC50 < 10 nM. Molecular modeling studies revealed that the
3′-OH, due to the inverted geometry, forms significant H-bonding interactions with Glu27 and
Arg195, an interaction that is not attainable with the natural isomers.
Introduction
Functionally, K-252a specifically inhibits NGF-induced
autophosphorylation of trkA in cells without inhibiting
âFGF, EGF, PDGF, or insulin-induced phosphorylation
of their receptor kinases.10 (+)K-252a also inhibits other
NGF-induced events such as differentiation and sur-
vival of PC12 cells, neurite formation, and induction of
c-fos.11
trkA is the high-affinity receptor-linked tyrosine
kinase for the neurotrophin, nerve growth factor (NGF).1
The receptor is composed of an extracellular NGF-
binding domain, a single transmembrane sequence, and
the cytoplasmic tyrosine kinase domain. NGF binding
and receptor activation leads to receptor oligomerization
and tyrosine phosphorylation of specific intracellular
substrates such as PLCγ, PI3 kinase, ras, and raf/MEK/
Erk1.2 Tyrosine kinase activity is an absolute require-
ment for signal transduction through this class of
receptor.
The role of the polypeptide growth factor NGF in the
growth, differentiation, and survival of central and
peripheral neurons is well established.3 Whereas effec-
tive NGF/trkA signaling is important for neuronal
systems, in the oncology field aberrant expression of
NGF and trkA receptor kinase are implicated in the
development and progression of human prostatic car-
cinoma and pancreatic ductal adrenocarcinoma.4 NGF
may also play a role in the development of breast
cancer.5 Accordingly, small molecule inhibitors of trkA
tyrosine kinase have been proposed as a therapeutic
approach for the treatment of prostate and pancreatic
cancers, based on the premise that prostatic and pan-
creatic carcinoma cell growth utilize an NGF/trkA
autocrine mechanism for continued proliferation.4a,6
This hypothesis is currently being tested in the clinic
with trkA inhibitors derived from the indolocarbazole
(+)K-252a class.
While (+)K-252a is a potent trkA inhibitor, its lack
of selectivity and overt toxicity limit its usefulness as a
drug. An ensuing medicinal chemistry program that was
focused on optimizing the trkA activity on the (+)K-252a
scaffold produced two compounds advancing into clinical
evaluation: 2b (CEP-2563), a water soluble Lys-âAla
prodrug form for 2c (CEP-751),12 and 2a (CEP-701)6e,13
(Figure 1), a compound with improved oral bioavailabil-
ity. 2a and 2c are potent trkA inhibitors with IC50
values of 4 nM and 3 nM, respectively. 2a displays
>100-fold selectivity against FGF-R1 and PDGF-Râ,
with modest selectivity against PKC (55-fold). In both
rat and human prostate cancer models, 2a significantly
inhibited metastasis and growth of primary and meta-
static tumors and showed significant induction of apo-
ptotic death in tumor cells without toxicity to normal
tissues. In 10 different prostate cancer animal models,
2a significantly inhibited tumor growth independent of
growth rate, hormone sensitivity, metastatic capability,
or state of tumor differentiation.4a,13 In the slow-growing
androgen-sensitive Dunning H rat prostate tumor model,
2a induced significant incidences of tumor regression
independent of effects on cell cycle and in the absence
of pronounced morbidity or toxicity following oral ad-
ministration.14 The aggressive behavior and poor prog-
nosis of pancreatic ductal adrenocarcinoma contributes
to the survival rate being one of the poorest for all
cancers. The NGF-trk receptor axis has been implicated
as the critical factor involved in the growth and pro-
gression of this disease.4d Preclinical studies with orally
administered 2a showed significant and selective inhi-
The glycosylated indolocarbazole natural product
(+)K-252a (1), isolated from actinomadura7a and no-
cardiopsis species K-252,7b,c is a potent ATP competitive
inhibitor of trkA with an IC50 value of 13 nM.8,9
* To whom correspondence should be addressed. Phone 610-738-
6283, e-mail rhudkins@cephalon.com.
† Department of Medicinal Chemistry.
‡ Department of Biochemistry.
10.1021/jm040178m CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/03/2005

(3′S)-epi-K-252a Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3777
the exocyclic olefin intermediate 8.19 Osmylation of
olefin 8 followed by deprotection of the TBS group using
methanolic HCl produced (3′R)-10. The enantioselective
approach to each methyl isomer (3′S)-14 and (3′R)-17
was through reductive ring opening of the pure chiral
epoxides. Epoxide (3′R)-12 was prepared from tosylate
(3′R)-11 using sodium hydride in THF. Treatment of
(3′R)-12 with lithium triethylborohydride followed by
TBS deprotection generated (3′S)-14, the deshydroxyl
methyl analogue of (3′R)-10. Scheme 3 outlines the
synthetic route to the deshydroxyl methyl isomer
(3′R)-17 with the natural configuration. (3′S)-2 was
converted to the tosylate (3′S)-15 (tosyl chloride, DMAP,
CH2Cl2) then treated with sodium hydride to give the
epoxide (3′S)-16. Lithium triethylborohydride reduction
of epoxide 16 produced the chiral tertiary methyl alcohol
(3′R)-17.
Results and Discussion
The kinase data for the K-252a analogues is shown
in Table 1. The compounds were evaluated in a trkA
enzyme-based assay utilizing an ELISA-based format9
with time-resolved fluorescence readout and recom-
binant human phospholipase C-gamma/glutathione
S-transferase fusion protein as a substrate. The primary
objective was to establish the stereochemical preference
of the 3′-OH on trkA activity and PKC selectivity by
evaluating diastereomeric pairs of inhibitors. Since
protein kinase C is involved in the transduction of a
number of essential regulatory signals in the heart and
has been linked to toxicity, the goal was to improve the
PKC selectivity greater than 100-fold.20 Compounds
were also profiled against the angiogenesis target
VEGF-R2, since 2a displayed only about 16-fold separa-
tion and (+)K-252a is a potent inhibitor. As previously
reported, the natural isomer (3′R)(+)K-252a 1 displays
IC50 values of 13 nM for trkA, 250 nM for PKC, and 43
nM for VEGF-R2.16 The K-252a 3′-epimer, (3′S)-5, was
11-fold more potent for trkA with an IC50 value of 1.2
nM and showed a 2- to 3-fold increase in potency for
both PKC and VEGF-R2. The 3′-epi amide (3′S)-6 was
24-fold weaker for trkA than the 3′-epi methyl ester 5
with an IC50 value of 29 nM. Compound 2a, which
contains the natural 3′S stereochemistry, is a 4 nM
inhibitor of trkA with 55-fold selectivity over PKC.
Inverting the 3′-chirality to (3′R)-10 resulted in equiva-
lent trkA activity. However, in a manner analogous
to epi-5, inverting the 3′-OH increased the potency
(decreased selectivity) for both PKC and VEGF-R2.
(3′R)-10 proved to be an exceptionally potent inhibitor
of the angiogenesis target VEGF-R2 with an IC50 value
of 4 nM.
Figure 1. Structures of (+)-K-252a and advanced K-252a-
derived analogues.
bition of tumor growth in six pancreatic xenograft
models in the absence of toxicity.4a,13
Previous structure-activity relationship (SAR) stud-
ies from our labs involved elaborating the natural
product (+)K-252a (2′S,3′R,5′R enantiomer).15 The re-
sults from these studies revealed that substitution on
positions 3 or 9 of aryl rings B and/or F typically reduced
trkA activity, whereas the sugar 3′-position was impor-
tant for modulating potency. In a preceding paper we
reported an efficient semisynthetic approach to the
(3′S)-K-252a diastereomer and its kinase profile.16. In
this paper we describe SAR studies designed to further
define the stereochemical role of the 3′-hydroxyl group
on trkA activity and selectivity, including the first
synthesis of the (3′R)-10 diastereomer and a set of
related 3′-epimers.
Synthetic Methods
Outlined in Schemes 1-3 are the routes to prepare
the 3′-epimers. (3′S)-epi-K-252a was prepared as shown
in Scheme 1.16,17 Ketone 3 was prepared in 83% yield
in two steps from (+)K-252a by lithium borohydride
reduction to diol (3′S)-2, followed by periodic acid
oxidative cleavage to 3.18 Inversion of the natural C-3
stereocenter was accomplished via cyanide attack to
ketone 3 producing cyanohydrins (3′S)-4a and (3′R)-4b.
Attempted isolation of 4a and 4b resulted in retrograde
conversion back to the ketone. To circumvent this
dilemma, the cyanohydrin intermediates were imme-
diately subjected to a Pinner reaction, which produced
(3′S)-5 and a small amount of amide (3′S)-6 isolated due
to incomplete hydrolysis.
Shown in Scheme 2 are the synthetic routes to
prepare (3′R)-10 and its deshydroxy derivative (3′S)-14.
Protection of (+)K-252a using TBSCl and DMAP in
DMF produced the TBS-lactam. Lithium borohydride
reduction to the protected diol was followed consecu-
tively by thiocarbonate formation to intermediate 7 and
Corey-Winter olefination using trimethyl phosphite to
While the K-252a epimers showed a clear preference
for the 3′-OH stereochemistry for trkA, a similar com-
parison between diastereomers (3′S)-2a and (3′R)-10 did
not distinguish the isomers. The 3′-hydroxymethyl of
2a may be more important and involved in a significant
hydrogen bond analogous to the 3′-OH on (3′R)-10. The
lactam N or O distance to the hydroxyl is < 0.5Å in both
cases. 2c, which differs from 2a only by a 3′-methoxy
vs 3′-OH group, displays equivalent trkA activity as 2a,
indicating the hydroxylmethyl may be the more critical
functionality for trkA activity. To help clarify the
contribution of the individual hydroxyl groups on diols

3778 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Gingrich et al.
Scheme 1a
a Reagents and conditions: (a) LiBH4, THF, rt, 81%; (b) H5IO6, THF, rt, 83%; (c) nBu4NCN, CH2Cl2, 0 °C-rt, 60%. (d) HClg, CH3OH-
dioxane; (e) 6 N HCl.
Scheme 2a
a Reagents and conditions: (a) TBSCl, DMAP, DMF (b) LiBH4, THF, rt, 81% (c) NaH, CCl2S, THF; (d) P(EtO)3 (e) OsO4 (f) Et3N, pTsCl,
(g) NaH, THF (h) lithium triethylborohydride (i) methanol, HCl.
(3′S)-2 and (3′R)-10 the deshydroxy isomers (3′R)-17 and
(3′S)-14 were prepared. The (3′R)-17 methyl com-
pound with the natural 3′-configuration showed a 5-fold
loss in trkA activity (IC50) 20 nM) compared to diol
(3′S)-2a. Conversely, methyl compound (3′S)-14 with the
unnatural configuration of the OH displayed equivalent
activity compared to diol (3′R)-10, with a trkA IC50 of
3 nM. In addition, by removing the hydroxyl group
(3′S)-14 showed a 10-fold loss in PKC activity (IC50
)
850 nM), resulting in >280-fold selectivity against trkA.

(3′S)-epi-K-252a Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3779
Scheme 3a
a Reagents and conditions: (a) lithium borohydride, THF; (b)
p-toluenesulfonyl chloride, DMAP, CH2Cl2; (c) NaH, THF; (d)
lithium triethylborohydride.
Table 1. Kinase Activity of (3′R)-1 and (3′S)-5 Analogues
Figure 2. Inhibition of NGF-stimulated trkA phosphorylation
in 3T3 cells.
to be = 5 nM with complete inhibition at 10 nM,
indicating that the compound is highly cell permeable
and comparable to the IC50 of 1.2 nM in the isolated
trkA kinase assay (Figure 2A). Alternatively, K-252a
1, which displays an enzyme IC50 of 13 nM, does not
show complete inhibition at 100 nM (Figure 2A). The
methyl compound (3′S)-14 demonstrated very high cell
permeability with complete inhibition at 10 nM and
<3-fold shift from the isolate enzyme IC50 (Figure 2B).
The natural diol (3′R)-2a (IC50 ) 4 nM) shows complete
inhibition at 100 nM, while (3′S)-10 (IC50 ) 4 nM) shows
complete inhibition at 50 nM (Figure 2B).
entry
R1
OH
CO2Me OH
CH2OH
R2
trkAa
PKCa VEGF-R2a PKC/trkA
(3′-R)-1
(3′-S)-5
(3′-S)-2a OH
(3′-R)-10 CH2OH OH
(3′-R)-17 OH
(3′-S)-14 Me
CO2Me 13 ( 4
250
43 ( 16
19 ( 2
65 ( 12
4 ( 1
76 ( 20
11 ( 3
90 ( 24
-
19
95
1.2 ( 0.2 114
4 ( 1
4 ( 2
20 ( 2
3 ( 1
29 ( 2
218
79
310
850
663
55
20
15
Me
OH
283
23
(3′-S)-6
(3′-R)-2c OMe
CONH2 OH
CH2OH 2.9 ( 0.3 114
39
a IC50 values are reported in nM. See Experimental Section for
assay details. The trkA and VEGF-R2 assays were run in
triplicate, the PKC value is the average of two determinations.
The significant conclusion from this study is the
3′-epi configuration of isomers (3′S)-5 and (3′S)-14 en-
hance potency at the trkA receptor, suggesting an addi-
tional receptor interaction due to the hydroxyl group
geometry. Figure 3 depicts the geometry differences
with energy minimized models of (+)K-252a 1 and epi-
5. Structural insight into the trkA receptor binding
interactions of the isomers is extremely valuable for the
design of future potent and selective inhibitors. To gain
some understanding of the binding differences between
the natural and epi isomers, a homology model of trkA
was constructed using its known sequence alignment
with the average coordinates of three FGF-R1 inhibitor
complexes (PDB codes 1FGI, 2FGI, 1AGW) as tem-
plates. FGF-R1 was identified from a BLAST search and
displays 39% sequence identity to trkA. The model was
built using the Composer module within Biopolymer in
Sybyl 6.92 and renumbered from Thr1-Leu304. An
initial 3D structure was obtained using the structurally
Analogous to isomers 5 and 10, compound (3′S)-14
showed a 7-fold increase in activity toward VEGF-R2.
The key results generated from the methyl compounds
support the conclusion that the geometry of the 3′-OH
group is important for trkA activity and that the trans
3′-OH group below the sugar may possibly be involved
in a significant H-bonding interaction.
Since trkA is an intracellular kinase it was important
to establish the cell permeability of the epi-isomers. The
compounds were evaluated in cells for dose-related
inhibition of trkA autophosphorylation utilizing serum-
starved NIH3T3 cells with varying concentrations of
compound for 1 h at 37 °C.21 The cells were stimulated
with NGF followed by immunoprecipitation with an
anti-trk antibody, probed with an antiphosphotyrosine
antibody, and then detected by enhanced chemilumi-
nescence. The results from the immunoblots are shown
in Figure 2. The cellular IC50 for (3′S)-5 was estimated

3780 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Gingrich et al.
Figure 3. Energy-minimized models of (3′S)-5 and
(+)K-252a 1.
conserved backbone regions. The structurally variable
loop regions were then constructed from database
searching of known kinase crystal structures. The
refinement of the homology model was obtained through
energy minimizations starting with a steepest descent
(SD), then a conjugate gradient (CD) calculation using
an MMFF94s force field to 0.05 kcal mol-1 Å-1. The
model was optimized further using a cycle of molecular
dynamics calculations followed by an MMFF94s force
field minimization. Within the trkA model, the coordi-
nates of the ATP cavity were identified for docking
studies by superimposing 1FGI and transferring the
coordinates of the ATP-competitive oxindole inhibitor
Su-5402. The cavity for docking studies was defined as
6.5 Å around SU-5402. The ATP-competitive docking
mode of the indolocarbazole staurosporine natural
product has been clearly established, with nine X-ray
crystal structure complexes being solved. More recently,
the crystal structure has been solved for (+)K-252a
complexed with an unphosphorylated form of mutated
c-Met kinase domain at 1.8 Å resolution (PDB code
1ROP).22
Figure 4. (3′S)-5 docked in the trkA model showing important
interactions.
Figure 5. Hydrophobic surface map of the (3′S)-5 trkA model.
(3′S)-5 was docked using the FlexX module within
Sybyl 6.92 and minimized in the model. The docking
mode including the significant H-bonding interactions
with the trkA receptor is shown in Figure 4. This
proposed model is consistent with the lactam moiety
mimicking ATP with two key hydrogen bonds (donor
and acceptor) anchoring the lactam to the hinge region
as was reported with the K-252a/c-Met structure and
with the staurosporine crystal structures. The lactam
N-H shares a hydrogen with the Met101 backbone
carbonyl (1.84 Å), while the lactam CdO hydrogen
bonds with the backbone amide of Glu99 (1.65 Å). The
sugar 3′-OH is involved in two hydrogen bonding
interactions. An important feature of this trkA model,
similar to that reported in FGF-R1 complexes,23 is the
glycine-rich nucleotide-binding loop adopts various con-
formations for ligand interactions. In this model the
3′-alcohol hydrogen shares a H-bond with the Glu27
carbonyl in the glycine rich loop (3.0 Å), while the
alcohol oxygen forms a H-bond with Arg195 side chain
from the C-terminal domain of the activation loop.
Potential electrostatic contacts are feasible between the
furan oxygen and Leu25 (3.22 Å), and the Arg163
backbone is in close proximity (3.24 Å) to the 3′-ester.
Hydrogen bonding interactions are essential for ATP
competitive kinase inhibitor binding, especially to the
linker region, and contribute to additional potency as
shown with the epi-K-252a series. However, a signifi-
cant amount of the binding energy in the indolocarba-
zole class is due to hydrophobic interactions. For
example, the K-252a sugar moiety contributes signifi-
cantly to the trkA activity through electrostatic interac-
tions, but the aglycon K-252c retains good trkA potency
with an IC50 value of 84 nM. In the trkA model the
inhibitor is located in the hydrophobic ATP cavity with
Phe98 and Tyr100 forming the base of the pocket. The
F-ring phenyl group forms a favorable aromatic π-stack-
ing interaction with the phenyl side chain of Phe34. The
indolocarbazole makes significant van der Waals con-
tacts with the surface of the pocket lined with residues
Leu25, Ala29, Val50, Gly104, Cys165, Leu166, and
Gly176. Figure 5 depicts a 6 Å hydrophobic surface map
applied around (3′S)-5. An important feature is the
binding of ring F of the indolocarbazole in a deep
hydrophobic pocket formed by Phe34, with Asp177,
Ile175, and Gly176 juxtaposed to the F-ring, while the
B-ring is positioned on the border of the accessible
solvent channel. Polar residues Arg102, Asp105, Asn107,
and Arg108 line the solvent channel neighboring the
3-position.
In conclusion, we report the first synthesis of the
(3′R)-10, and a set of related 3′-epimers, with the goal
of defining the role of stereochemistry at the 3′-sugar

(3′S)-epi-K-252a Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3781
Compound 8. Compound 718 was dissolved in trimethyl
phosphite (2 mL) and heated to reflux for 3 h. The reaction
mixture was cooled to room temperature and flushed through
a flash silica gel column using chloroform-methanol (20:1) to
remove trimethyl phosphite. The product was purified by flash
chromatography (silica gel; ethyl acetate:hexane; 1:1) to give
compound 8 as a pale yellow solid (95% yield). MS (ESI+): m/e
position on trkA tyrosine kinase activity and selectivity.
The key results generated support the conclusion that
the geometry of the 3′-OH group influences the trkA
activity and selectivity. Whereas the diastereomer
(3′R)-10 showed potent trkA activity equivalent to the
natural isomer with an IC50 of 4 nM, epi-isomers (3′S)-5
and (3′S)-14 were 7- and 13-fold more potent for trkA
than the 3′R counterparts. In each case, the epimers
were more potent for VEGF-R2. Methyl compound
(3′S)-14 was identified as a potent inhibitor of trkA with
an IC50 of 3 nM and demonstrated >280-fold selectivity
over PKC, a significant improvement over 2a. In cells,
(3′S)-14 displayed potent inhibition of trkA autophos-
phorylation (IC50 < 10 nM), showing less than a 3-fold
shift from the cell-free isolated enzyme assay. To aid in
further understanding the molecular basis for the
increased potency of the epimers, a homology model of
trkA tyrosine kinase was constructed. Docking studies
reveal that the 3′-OH, due to its geometry, is involved
in significant H-bonding interactions with Glu27 and
Arg195, not attainable with the 3′ natural stereochem-
istry enantiomers. Compound (3′S)-14 showed good rat
pharmacokinetic properties for further evaluation in
trkA relevant models, which will be reported separately.
1
406 (M + H)+, H NMR (CDCl3) δ 2.62 (s, 3H), 2.85 (d, 1H),
3.37-3.45 (m, 1H), 4.95 (s, 1H), 5.00 (s, 2H), 5.09 (s, 1H), 6.29
(s, 1H), 6.90 (d, 1H), 7.33-7.53 (m, 5H), 7.91 (d, 1H), 9.41 (d,
1H).
Compound (3′R)-10. To a stirred solution of 8 (350 mg,
0.67 mmol) in THF (10 mL) at room temperature under
nitrogen was sequentially added pyridine (0.44 mL, 5.39 mmol)
and osmium tetroxide (6.73 mL, 0.67 mmol, 0.1 M soln. in
CCl4). The yellow mixture was stirred at room-temperature
overnight. After 36 h, aqueous sodium bisulfite solution
(concentrated 30 mL) was added and the contents were stirred
for 30 min, then extracted with EtOAc (2 × 20 mL), dried over
sodium sulfate, filtered and concentrated in vacuo to a light
brown film. The product was purified by flash chromatography
on silica gel using ethyl acetate to yield a yellow solid (280
mg, 76%). MS (ESI) m/e 544 (M + H), 1H NMR (CDCl3) δ 0.56
(d, 6H), 1.079 (s, 9H), 2.04 (dd, 1H), 2.12 (broad s, 1H), 2.40
(s, 3H), 2.86 (dd, 1H), 3.52 (broad s, 3H), 4.99 (s, 2H), 6.98
(dd, 1H), 7.32 (t, 1H), 7.39-7.46 (m, 4H), 7.97 (dd, 2H), 9.35
(d, 1H).
To a flask containing methanol (2 mL) at 0 °C under
nitrogen was added acetyl chloride (4 drops). Compound 9 (40
mg, 0.072 mmol) in methanol (1 mL) was added dropwise to
the solution of methanol-HCl. This mixture was stirred at 0
°C for 1 h and then warmed to room-temperature overnight.
The solvent was removed in vacuo leaving 10 as a tan solid
Experimental Section
Chemistry. All reagents and solvents were obtained from
commercial sources and used as received. 1H 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 mm column eluting with a mixture of
acetonitrile and water containing 0.1% trifluoroacetic acid with
a gradient of 10-100%. Column chromatography was per-
formed on silica gel 60 (230-400 mesh). M-Scan Inc., West
Chester, PA, performed high-resolution mass spectra (FAB).
1
(21 mg, 66%). MS (ESI) m/e 440 (M + H); H NMR (CDCl3) δ
2.05 (dd, 1H), 2.43 (s, 3H), 2.90 (dd, 1H), 3.57 (s, 1H), 3.61 (s,
2H), 5.04 (s, 2H), 6.28 (s, 1H), 7.02 (dd, 1H), 7.33-7.54 (m,
6H), 7.95 (d, 1 H), 8.02 (d, 1H), 9.32 (d, 1H); HRFAB-MS calcd
for C26H21N3O4, 440.1610; found 440.1622.
Compound 11. To a stirred solution of 9 (0.23 g, 0.42 mmol)
in THF (10 mL) at 0 °C under nitrogen were added triethyl-
amine (57.9 µl, 0.42 mmol), DMAP (25.4 mg, 0.208 mmol), and
p-toluenesulfonyl chloride (79.1 mg, 0.42 mmol). The reaction
mixture was stirred at 0 °C for 1 h and then slowly warmed
to room-temperature overnight. Thin-layer chromatography
analysis (hexanes-ethyl acetate, 2:1) showed the presence of
starting material so the reaction mixture was warmed for an
additional 1 h. The reaction was then diluted with ethyl ace-
tate (30 mL) and washed with water (3 × 15 mL). The organic
phase was dried over sodium sulfate, filtered, and concentrated
in vacuo to a yellow film. The product was purified by flash
chromatography on silica gel using hexanes-ethyl acetate
(2:1) to give 11 as a light yellow film (0.16 g, 55%) and starting
Compound (3′S)-5. To a stirred solution of 3 (451 mg, 1.11
mmol) in CH2Cl2 (5 mL)-dioxane (1 mL) under nitrogen was
added tetrabutylammonium cyanide (740 mg, 2.77 mmol) and
acetic acid (95 µL, 1.66 mmol) at room temperature. The
reaction mixture was stirred for 24 h, then concentrated in
vacuo. The dark oil was dissolved in ethyl acetate (20 mL) and
dioxane (2 mL) and washed with water (3 × 10 mL) and brine
(1 × 10 mL). The organic phase was dried over magnesium
sulfate, filtered, and concentrated in vacuo to a brown solid.
This product was used immediately in the next step without
further purification. HCl(g) was bubbled into methanol (4 mL)
for 10 min. Then a solution of crude cyanohydrin (4a/4b) (450
mg, 1.04 mmol) in methanol-dioxane (2:1, 3 mL) was added
to the HCl-methanol solution at 0 °C. The reaction mixture
was sealed, stirred at 0 °C for 2 h, and then placed in a
refrigerator for 48 h. The mixture was warmed to room
temperature, and then 6 N HCl was added carefully. The
mixture was stirred for 30 min and then concentrated to
dryness. The resulting residue was dissolved in a 50% metha-
nol-water solution, stirred overnight at room temperature,
and then concentrated to dryness. The product was purified
by flash chromatography on silica gel using hexanes-ethyl
acetate (1:1) to yield epi-K252a 5 as an off-white solid. MS
1
material (0.8 g, 35% yield). MS (APCI) m/e 708 (M + H); H
NMR (CDCl3) δ 0.57 (d, 6 H), 1.08 (s, 9H), 2.01 (dd, 1H), 2.33
(s, 3H), 2.42 (s, 3H), 3.88 (dd, 1H), 3.86 (dd, 2H), 4.98 (s, 2H),
6.97 (dd, 1H), 7.14 (d, 2H), 7.24-7.49 (m, 7H), 7.75 (d, 1H),
7.91 (d, 1H), 9.35 (d, 1H).
Compound (3′R)-12. To a stirred solution of 11 (0.14 g,
0.20 mmol) in THF (5 mL) at 0 °C under nitrogen was added
sodium hydride (15.8 mg, 0.40 mmol). After vigorous evolution
of hydrogen the reaction mixture became cloudy. Thin-layer
chromatography analysis showed product and starting mate-
rial. Additional sodium hydride (2 equiv) was then added, and
the contents of the flask were stirred for an additional 2 h
and then warmed gently for 4 h. The reaction mixture was
then cooled to 0 °C, quenched with water, diluted with ethyl
acetate, and washed with water and brine. The organic phase
was dried over sodium sulfate, filtered, and concentrated in
vacuo to a yellow film (100 mg, 95%). MS (APCI) m/e 536 (M
1
(ESI) m/e 468 (M + H); H NMR (CDCl3) δ 2.42 (s, 3H, 2.77
(dd, 1H), 2.91 (s, 3H), 2.95 (dd, 1H), 4.99 (s, 2H), 7.13 (dd, 1H),
7.33 (t, 1H), 7.44 (dd, 2H), 7.64 (t, 2H), 7.98 (d, 1H), 9.16 (d,
1H); HRFAB-MS calcd for C27H21N3O5, 468.1559; found
468.1558. Anal. C27H21N3O5‚0.66H2O, C, H, N.
Amide (3′S)-6 was obtained by eluting the column with ethyl
1
+ H); H NMR (CDCl3) δ 0.56 (d, 6H), 1.08 s, 9H), 2.32-2.38
acetate to give a light orange solid. MS (ESI) m/e 453 (M +
1
(m, 4H), 2.57 (d, 2 H), 3.01 (dd, 1H), 4.99 (s, 2H), 7.01 (d, 1H),
H); H NMR (DMSO-d6) δ 2.33 (s, 3H), 2.87 (m, 1H), 4.94 (s,
7.33-7.56 (m, 5H), 7.73 (d, 1H), 7.94 (d, 1H), 9.46 (d, 1H).
2H), 6.58 (s, 1H), 7.19-7.64 (m, 6H), 7.81 (m, 3H), 7.96 (d,
1H), 8.59 (s, 1H), 9.17 (d, 1H); HRFAB-MS calcd for C26H20N4O4,
453.1563; found 453.1583.
Compound (3′S)-14. To a stirred solution of 12 (100 mg,
0.19 mmol) in THF (5 mL) at 0 °C under nitrogen was added

3782 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Gingrich et al.
2 equiv of lithium triethylborohydride (0.37 mL of a 1 M
solution in THF, 0.37 mmol) dropwise with evolution of gas.
An additional 2 equiv of lithium triethylborohydride was
added, and the reaction was stirred at 0 °C for 30 min and
then warmed to room temperature. The reaction mixture was
cooled to 0 °C and quenched with water, diluted with ethyl
acetate, and washed with water and brine. The organic phase
was dried over magnesium sulfate, filtered, and concentrated
in vacuo. The product was purified by flash chromatography
on silica gel using hexanes-ethyl acetate (1:1) to give 13 as a
pale yellow film (90.8 mg, 91%). A methanol-HCl solution,
prepared by adding acetyl chloride (5 drops) to methanol (2
mL), was added to a stirred solution of 13 in methanol at 0 °C
under nitrogen. The reaction mixture was stirred at 0 °C for
30 min and then allowed to warm to room-temperature
overnight. The solvent was removed in vacuo leaving a yellow
solid, which was purified by silica gel chromatography using
hexanes-ethyl acetate (1:1) to give 14 (30 mg, 42%). MS (ESI)
concentrations of inhibitor. The reaction is initiated by addition
of trkA kinase and allowed to proceed for 15 min at 37 °C. An
antibody to phosphotyrosine (UBI) is then added, followed by
a secondary enzyme-conjugated antibody, alkaline phosphatase-
labeled goat antimouse IgG (Bio-Rad). The activity of the
bound enzyme is measured via an amplified detection system
(Gibco-BRL). Inhibition data are analyzed using the sigmoidal
dose-response (variable slope) equation in GraphPad Prism.
VEGF-R2 Receptor-Linked Tyrosine Kinase Assay.
Enzyme inhibition studies were performed as described previ-
ously26 using a modification of the ELISA described for trkA
kinase.9 The 96-well microtiter plate (FluoroNUNC or Costar
High Binding) was coated with 10 µg/mL recombinant human
PLC-γ/GST. Kinase assays were performed in 100 µL reaction
mixtures containing 50 mM HEPES (pH 7.4), Km level of ATP,
10 mM MnCl2, 0.1% BSA, 2% DMSO, and various concentra-
tions of drug. The reaction was initiated by adding baculoviral
recombinant human enzyme VEGF-R2 and allowed to pro-
ceed for 15 min at 37 °C. The detection antibody, Eu-N1 anti-
phosphotyrosine (PT66) antibody, was added, and the plate
was gently agitated. After 5 min, the fluorescence of the
resulting solution was measured using the Victor Multilabel
Counter.
1
m/e 424 (M + H)+, H NMR (CDCl3) δ 1.39 (s, 3H), 2.29 (dd,
1H), 2.37 (s, 3H), 2.91 (dd, 1H), 5.05 (s, 2H), 6.19 (s, 1H), 6.97
(t, 1H), 7.32-7.50 (m, 5H), 7.78 (d, 1H), 7.95 (d,1H), 9.33 (d,
1H); HRFAB-MS calcd for C26H21N3O3, 424.1661; found
424.1656. Anal. C26H23N3O3, C, H, N.
trkA Cell-Based Autophosphorylation Assay. The in-
hibition of NGF-stimulated phosphorylation of trk was per-
formed, essentially as described previously.21a NIH3T3 cells
transfected with trkA (NIH3T3-trkA) were plated in 100-mm
tissue culture dishes. Subconfluent cells were then serum-
starved by replacing media with serum-free DMEM-containing
0.05% BSA. At this point, test compounds were added to the
NIH3T3-trkA cells and incubated for 1 h at 37 °C. Except for
the negative control, NGF (10 ng/mL) was added to the positive
control and compound-treated cells. After 5 min of ligand
stimulation, the cells were rinsed with ice-cold PBS and then
lysed with RIPA buffer containing 10 mM Tris(HCl), pH 7.2,
150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1%
sodium dodecyl sulfate (SDS), protease inhibitors, and 1 mM
sodium vanadate. Immunoprecipitation and immunoblotting
were then performed as previously described.21b Clarified cell
lysates corresponding to equal amounts of protein were im-
munoprecipitated with anti-trk antibody for 1 h, followed by
incubation with Protein A-Sepharose for another hour at 4 °C.
Immunoprecipitates were washed with lysis buffer, analyzed
on a 7.5% SDS-polyacrylamide gel, and then transferred onto
Millipore PVDF membrane. The membrane was immunoblot-
ted with 4G10 anti-phosphotyrosine antibody, followed by
incubation with horseradish peroxidase coupled goat anti-
mouse IgG. Phosphorylated bands were visualized by en-
hanced chemiluminescence.
Compound (3′R)-17. To a stirred solution of 16 (90.1 mg,
0.15 mmol) in THF (4 mL) at 0 °C under nitrogen was added
lithium triethylborohydride (0.45 mL of a 1 M soln in THF,
0.45 mmol) slowly dropwise. The reaction mixture was stirred
at 0 °C for 1 h and then slowly warmed to room-temperature
overnight. The mixture was cooled to 0 °C, quenched with
methanol, stirred at 0 °C for 15 min, and then warmed to room
temperature. The solvent was removed in vacuo, leaving a
yellow oil. The product was purified by flash chromatography
on silica gel using ethyl acetate-hexane (1:1) to give 17 as a
white solid (75 mg, 83%). MS (ESI) m/e 424 (M + H); 1H NMR
(CDCl3) δ 1.69 (s, 3H), 1.99 (s, 3H), 2.86 (dd, 1H), 3.03 (dd,
1H), 4.37 (m, 3H), 4.93 (s, 2H), 6.43 (t, 1H), 6.95 (d, 1H), 7.03
(t, 1H), 7.18 (t, 1H), 7.44 (t, 1H), 7.79 (d, 1H), 7.99 (d, 1H),
8.69 (d, 1H); HRFAB-MS calcd for C26H21N3O3, 424.1661; found
424.1663.
Protein Kinase C Multiscreen “In-Plate” Assay. Protein
kinase C activity was assessed using the Millipore Multiscreen
TCA “in-plate” assay as previously described.24,15a,b,16 Assays
were performed in 96-well Multiscreen-DP plates (Millipore
Cat. # MADPNOB50). Each 40-µL assay mixture contained
20 mM HEPES, pH 7.4, 10 mM MgCl2, 2.5 mM EGTA, 2.5
mM CaCl2, 80 µg/mL phosphatidylserine, 3.2 µg/mL diolein,
200 µg/mL histone H-1 (Fluka Cat. # 53412), 5 µM [γ-32P]ATP
(Km level), 1.5 ng of PKC (UBI Cat. # 14508; mixed isozymes
of R, â, γ), 0.1% BSA, 2% DMSO, and various concentrations
of test compound. The reaction was allowed to proceed for 10
min at 37 °C and then quenched by adding 25 µL of ice-cold
50% trichloroacetic acid. The plates were allowed to equilibrate
for 30 min at 4 °C and then washed 4× with 200 µL of ice cold
25% TCA. After being washed, the plates were removed from
the vacuum manifold and the bottom of each plate was blotted.
The flexible plate underdrain was removed, and the bottom
was again blotted to remove any excess liquid. The plates were
then placed in Wallac cassette holders with the bottom sealed
with adhesive tape (Wallac Cat. # 1450-462). SuperMix (Cat.
# 1200-439) scintillation cocktail (25 µL/well) was added, and
the top of the plate was sealed with adhesive tape. The samples
were equilibrated for 1 h prior to counting. The radioactivity
was determined in the Wallac MicroBeta 1450 PLUS scintil-
lation counter by using a protocol that has been previously
normalized for counting 32P in Millipore opaque plates.
Supporting Information Available: HPLC and C, H, N
analysis. This material is available free of charge via the
Internet at http://pubs.acs.org.
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JM040178M