Inhibitors of Sir2: Evaluation of Splitomicin Analogues

Article abstract of DOI:10.1021/jm030473r

Splitomicin (1) and 41 analogues were prepared and evaluated in cell-based Sir2 inhibition and toxicity assays and an in vitro Sir2 inhibition assay. Lactone ring or naphthalene (positions 7-9) substituents decrease activity, but other naphthalene substitutions (positions 5 and 6) are well-tolerated. The hydrolytically unstable aromatic lactone is important for activity. Lactone hydrolysis rates were used as a measure of reactivity; hydrolysis rates correlate with inhibitory activity. The most potent Sir2 inhibitors were structurally similar to and had hydrolysis rates similar to 1.

Full text of DOI:10.1021/jm030473r

J . Med. Chem. 2004, 47, 2635-2644
2635
In h ibitor s of Sir 2: Eva lu a tion of Sp litom icin An a logu es
J eff Posakony, Maki Hirao, Sam Stevens, J ulian A. Simon,* and Antonio Bedalov*
Clinical Research and Human Biology Divisions, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North,
Seattle, Washington 98109
Received September 19, 2003
Splitomicin (1) and 41 analogues were prepared and evaluated in cell-based Sir2 inhibition
and toxicity assays and an in vitro Sir2 inhibition assay. Lactone ring or naphthalene (positions
7-9) substituents decrease activity, but other naphthalene substitutions (positions 5 and 6)
are well-tolerated. The hydrolytically unstable aromatic lactone is important for activity. Lactone
hydrolysis rates were used as a measure of reactivity; hydrolysis rates correlate with inhibitory
activity. The most potent Sir2 inhibitors were structurally similar to and had hydrolysis rates
similar to 1.
In tr od u ction
identified splitomicin (1) as a micromolar inhibitor of
Sir2. Treatment of wild-type cells with splitomicin
created a faithful phenocopy of a Sir2 mutant. Ap-
proximately 130 analogues of splitomicin were evalu-
ated including analogues of sirtinol.8 Here, we report
the convenient syntheses for splitomicin and 41 ana-
logues and the analysis of their activity. This analysis
revealed that the hydrolytically unstable lactone ring
of splitomicin is critically important for its activity.
Epigenetic regulation of gene transcription is often
accomplished via posttranslational modification of his-
tones.1 Histone lysine methylation, acetylation and
deactylation, and serine phosphorylation are essential
to this regulation, which has been described as a
“histone code” that complements the genetic code.2
Prominent among the enzymes responsible for this
regulation are the histone deacetylases, which promote
gene silencing via cleavage of acetyl-lysine (Ac-K) on
histone tails. Two classes of histone deacetylases have
been identified.3 One class, known as the HDACs,
incorporates a divalent Zn atom in the active site that
activates a water molecule, which hydrolyzes the amide
bond. The other class of histone deacetylases is known
as Sirtuins after their founding member Sir2 in S.
cerevisiae. The Sirtuins consume NAD+ in the deacety-
lation reaction to afford free lysine, nicotinamide, and
O-acetyl ADP ribose. Both classes of histone deacety-
lases have also been shown to deacetylate and modulate
the activity of nonhistone proteins. For example, p53,
a tumor suppressor protein involved in the modulation
of a cell’s response to DNA damage, is inactivated via
deacetylation on lysine 382 by a human Sir2.4 Further-
more, the function of the transcriptional repressor
BCL6, implicated in the pathogenesis of B-cell lympho-
mas, critically depends on its deacetylation by HDAC
and Sir2-like enzymes.5 Because of their role in a variety
of cellular pathways relating to cancer, inhibitors of
histone deacetylases have been an active area of re-
search. Indeed, inhibitors of these enzymes have been
shown to induce growth arrest, differentiation, and/or
apoptotic cell death in transformed cells.6
Ch em istr y
Splitomicin and numerous derivatives were synthe-
sized by Friedel-Crafts alkylation of the appropriate
naphthol with an excess of acrylic acid and Amberlyst
15 ion exchange resin as the acid catalyst (Scheme 1).
The product was purified by first filtering off the
catalyst and then removing the solvent, followed by
column chromatography. This synthesis with its good
atom economy afforded numerous methoxy- and hy-
droxy-substituted versions of splitomicin with typical
yields of 50-85%. However, the limited solubility of 1,6-
dihydroxynaphthalene in refluxing toluene resulted in
only a 20% yield of 32. Dipyranones (e.g., 28, 36, and
39) were also isolated from reactions with dihydrox-
ynaphthalenes, although in disappointingly low yields
(4-11%). This procedure was also successful with
2-naphthalenethiol affording the thiopyranone (10) in
40% yield. The regioselectivity of alkylation followed
that expected for naphthalenes, i.e., preferred alkylation
at the 1-, 4-, 5-, and 8-positions. However, substitution
at the less-favored carbons also occurred with two
1-naphthols to afford 6 and 40, albeit in generally lower
yields (10-37%). In cases where competing alkylation
could occur (e.g., 1,3- or 1,6-dihydroxynaphthalene), only
products with the “preferred” alkylation patterns were
observed. Friedel-Crafts alkylation using propiolic acid
in place of acrylic acid was also successful. For example,
alkylation of 2-naphthol afforded the unsaturated pyra-
none 2 (58%) and alkylation of 24 afforded the partially
saturated dipyranone 29 (35%).
We used S. cerevisiae as a model organism to identify
and characterize inhibitors of NAD-dependent deacety-
lases. Sir2 is required for epigenetic silencing at three
sites in the yeast genomes: genes adjacent to telomeres,
the ribosomal DNA (r-DNA) locus, and the silent mating
loci. Using a cell-based screen for inhibitors of telomeric
silencing from a library of 6000 compounds,7 we recently
Nitrogen and oxygen nucleophiles readily reacted
with the aromatic lactone ring of 1, which facilitated
the synthesis of analogues 12-16. Deprotonation (DBU)
* To whom correspondence should be addressed. (A.B.) Tel: 206-
667-4863. E-mail: abedalov@fhcrc.org. (J .A.S.) Tel: 206-667-6241.
Fax: 206-667-4669. E-mail: jsimon@fhcrc.org.
10.1021/jm030473r CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/09/2004

2636 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Posakony et al.
Ta ble 1. Structures and Properties of Pyranone Variant and Ring-Opened Analogues
X
Y
R
compd
MGC (µM)
IC50 (µM)
SIa
75 µM inhibitionb
half-life (h)c
(CH2)2
CHdCH
CH2
(CH2)3
(CH2)3
O
O
O
O
O
1
0.49 ( 0.13
74 ( 32
86 ( 8
59 ( 20
12 ( 4
81 ( 26
65 ( 25
25 ( 8
g
97 ( 30
125 ( 43
g
125 ( 43
g
151
0.66 ( 0.16
0.73 ( 0.05
N/Ae
0.82 ( 0.03
N/A
0.87 ( 0.09
N/A
0.92 ( 0.04
0.75 ( 0.05
0.80 ( 0.05
N/A
7.5 ( 0.4
2
g
>100
3d
4
g
g
g
12.2 ( 0.3
49.5 ( 3.4
not measured
6.9 ( 0.5
13.0 ( 2.7
no hydrolysis obsd
5
6
(CH2)2
S
NH
2.4 ( 0.4
g
g
27
(CH2)2
(CH2)2
10
11f
12
13
14
15
16
OCH3
NH2
NH(Me)
NEt2
OH
5.2 ( 0.8
19
158
g26
0.79 ( 0.25
5.8 ( 2.3
g
g
0.88 ( 0.06
0.87 ( 0.08
a
b
SI ) IC50/MGC. Activity of in vitro assay at 75 µM, normalized to DMSO control. c Determined from the pseudo-first-order hydrolysis
rate constant, measured in yeast growth media, pH 4.3. Described in ref 10. e No activity (g95% of DMSO controls). f Described in ref
11. g150 µM.
d
g
Sch em e 1a
overall yield. Analogues with substituents at the 2-posi-
tion of the pyran ring (20-22) were produced by
alkylation of the anion of the â-diester of 19, followed
by ester hydrolysis and decarboxylation/ring closure
steps in poor to good (27-71%) overall yields. Relac-
tonization was achieved via acid catalysis under dehy-
drating conditions (e.g., catalytic TsOH or HCl in
refluxing benzene). Spontaneous relactonization was
observed when the ring-opened versions were stored
neat for several days (e.g., 16 and the hydroxy acid
precursors to 21 and 22).
The naphtho-oxepinone, 4, was produced by Bayer-
Villager oxidation of the tetrahydrophenanthrenone (5)
in 49% yield. Borohydride reduction of 2 in MeOH
afforded the fully reduced naphthol (18, 94%). Struc-
tures of all the compounds are given in Tables 1 and 2,
Figure 1 and Scheme 1.
Biology: Resu lts a n d Discu ssion
Sir2 inhibitory activity of splitomicin analogues was
first evaluated in vivo using a functional assay for
telomeric silencing in yeast. The test strain contains the
URA3 gene in close proximity to the left telomere of
chromosome VII, where the expression of URA3 is
repressed through Sir2-dependent silencing12 (Figure 2).
Because Ura3 is required for uracil biosynthesis, this
strain cannot grow in media lacking uracil. Compound-
mediated inhibition of Sir2 eliminates silencing and
activates the URA3 gene, which allows the cells to grow
in media lacking uracil.
In this cell-based screen, the measured output (yeast
growth) includes effects of Sir2 inhibition, compound-
mediated toxicity, and the compounds’ individual phys-
icochemical parameters, such as solubility and lipophi-
licity, which can affect drug transport. Thus, a com-
pound’s Sir2 inhibitory activity is expressed as mini-
mum growth-stimulating concentration (MGC, 10% of
maximal growth in media lacking uracil, seen with
compound 41) (Tables 1 and 3), the concentration at
which toxicity is least pronounced. A compound’s toxic-
ity (expressed as IC50) was evaluated separately by
a
Reagents and conditions: (a) Acrylic acid, Amberlyst 15,
toluene, reflux. (b) Nu. (c) DBU, RX. (d) Diethyl malonate,
pyridine, piperidine (catalytic), reflux. (e) NaBH4, pyridine. (f) 1
N LiOH, THF. (g) TsOH, benzene, reflux or room temperature,
no solvent. (h) LDA, RX.
of the hydroxyl group of 24 and subsequent alkylation
with alkyl halides or alkyl methanesulfonates were a
useful route to several analogues (25-27) obtained in
fair to excellent (37-95%) yield.
A lengthier route to analogues of 1 involved Knoev-
enagel condensation between a malonate ester and a
hydroxynaphthaldehyde, subsequent NaBH4 reduction
of the unsaturated â-diester in pyridine, ester hydroly-
sis, and decarboxylation/ring closure steps (Scheme 1).
This was the only route to compound 9, which was
obtained from 2-hydroxy-3-naphthaldehyde9 in 48%

Evaluation of Splitomicin Analogues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2637
Ta ble 2. Structures of the Substituted Naphthopyranones
position
compd
1
2
5
6
7
8
9
10
1
H
H
CO2Et
Me
CH2CHdCH2
CH2CH(Me)2
H
H
H
H
H
H
OH
OMe
OBn
H
H
H
H
H
H
H
H
H
H
†
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
†
19
20
21
22
23a
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
H
H
H
H
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O(CH2)2O(CH2)2NHBOC
OC(O)(CH2) 2-§b
OC(O)CHdCH-§
†
H
H
H
H
H
H
H
H
H
H
OH
OMe
H
H
H
H
H
H
H
OH
OMe
†
H
H
H
H
H
OH
OMe
H
(CH2)2C(O)O-§
H
H
H
H
H
OC(O)(CH2)2-§
a
b
Obtained from the NCI. §, denotes attachment at next (†) position.
lactone (2) and tetrahydrophenanthrenone (5) do not
stimulate growth. However, the five-membered lactone
(3)10 and oxepinone (4) induce slight growth at concen-
trations >10 and >20 µM, respectively, but neither
induce a sufficient growth density to determine the
MGC (see Figures 3 and 4). Oxepinone 4 is quite toxic
(IC50 ) 12 µM), which may mask its inhibition of Sir2.
Changing the lactone to a thiolactone (10) or lactam
(11)11 also eliminates growth stimulation. The orienta-
tion of the lactone ring is also important. The [1,2]-
lactone (6) and [2,3]-lactone (9) are ca. 5- and 31-fold
less active than 1 ([2,1]-lactone), respectively. Similarly,
the 6-hydroxy [1,2]-lactone (40) is also less active than
its [2,1]-lactone homologue (32, discussed below). Hy-
droxypropyl naphthol (18), which is incapable of lac-
tonization, shows no growth stimulation. That the
lactone ring orientation is important and that substit-
uents in the 7-9 positions greatly reduce the growth
stimulation (see below) suggest that a splitomicin
interacts with its target in a particular orientation,
perhaps in a binding pocket.
F igu r e 1. Miscellaneous analogues of 1.
Several ring-opened analogues (12, 14-16) have
markedly decreased activity (MGCs 5 to >150 µM).
Hydroxyamide 13 is potent, but subsequent analysis of
this compound indicated that 13 in aqueous solution is
converted to 1 over time (see Supporting Information)
and that a significant concentration of 1 accumulates
during the growth stimulation experiment. Substitution
on the lactone ring is poorly tolerated as demonstrated
by the marked increase in MGC (5.4 to >150 µM) from
methyl (20), allyl (21), isobutyl (22), carboxyethyl (19),
and phenyl (23) substitution on the lactone ring.
One interpretation of these data is that an analogue’s
growth stimulating activity is dependent upon the
F igu r e 2. Sir2 regulates telomeric silencing.
assessing the effect on growth of the same strain in
complete medium. Representative dose-response curves
can be found in Figures 3 and 4. The ratio of the MGC
to IC50 value afforded a selectivity index (SI) for each
of the compounds. Examination of splitomicin and its
analogues’ activities in vivo have helped to elucidate the
structure-activity relationships, described below.
Va r ia tion s of a n d Su bstitu tion s on th e La cton e
Rin g. The saturated six-membered lactone ring of
splitomicin is important to its activity. The unsaturated

2638 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Posakony et al.
Ta ble 3. Biological Activity of the Substituted
Naphthopyranones and Miscellaneous Analogues
amino acid residue of Sir2 itself or another component
critical to Sir2’s function. Efforts to identify covalent
splitomicin adducts have so far proven unsuccessful.
However, the fact that 13 can be converted to 1 suggests
that adduct formation may be a reversible reaction and
adducts are only present transiently.
MGC ( SD
(µM)
IC50 ( SD
(µM)
75 µM inhibitor
( SDb
no.
SIa
1
0.49 ( 0.13
f
74 ( 32
f
151
0.66 ( 0.16
0.88 ( 0.09
0.94 ( 0.13
0.86 ( 0.04
N/Ac
0.90 ( 0.15
N/A
N/A
0.17 ( 0.06
N/A
0.80 ( 0.03
0.83 ( 0.07
N/A
N/A
N/A
N/A
19
20
21
22
23d
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
5.4 ( 0.7
52 ( 17
27 ( 14
60 ( 37
43 ( 37
111 ( 48
128 ( 44
27 ( 28
86 ( 8
89 ( 34
f
131 ( 47
44 ( 26
f
39 ( 19
f
10
In the absence of a biologically relevant nucleophile,
we have used the hydrolysis rate of selected analogues
in growth media as a relative measure of lactone
reactivity (Table 1). Indeed, compounds 2-4, 10, and
11 have slower hydrolysis rates than 1 and all are less
active than 1. Compound 6 ([1,2]-lactone) is hydrolyzed
only slightly faster than 1, and its higher MGC may be
due to the orientation of the lactone ring. Compound 9
([2,3]-lactone), in contrast, has a hydrolysis rate (24.1
( 4.5 h) ca. three times that of 1, and its poor activity
may be due to its slower hydrolysis rate or the lactone
ring’s orientation. However, it is not possible to evaluate
the relative importance of these two factors from these
data. Interestingly, compound 41 also has a much longer
half-life (20.5 ( 1.6 h) than 1 but has a similar MGC to
that of 1. One explanation for this discrepancy is that
a phenyl ring in place of the naphthalene ring alters
the solubility and lipophilicity sufficiently to afford a
greater concentration of 41 at the active site in the cell.
As such, the significant structural changes from 41 and
1 may preclude their direct comparison in an SAR study.
f
f
f
0.69 ( 0.19
1.5 ( 0.5
0.97 ( 0.63
6.4 ( 0.4
4.9 ( 1.8
5.1 ( 0.2
11 ( 1
1.8 ( 0.3
16 ( 6
5.5 ( 2.0
38 ( 10
13.8 ( 4.4
f
161
85
29
13
18
g29
12
24
g107
7
g39
5
0.85 ( 0.08
0.84 ( 0.03
0.87 ( 0.09
N/A
0.84 ( 0.08
N/A
73 ( 30
70 ( 26
134 ( 14
41 ( 15
f
48 ( 5
f
f
3
miscellaneous analoguese
9
15 ( 5
f
f
f
10
0.85 ( 0.03
0.88 ( 0.04
N/A
0.15 ( 0.03
0.74 ( 0.05
0.89 ( 0.11
0.94 ( 0.02
N/A
18
40
41
42
43
44
45
f
21 ( 1
g7
144
0.85 ( 0.05
122 ( 11
f
f
f
24 ( 4
f
f
g6
8.8 ( 1.7
Su bstitu tion s on th e Na p h th a len e Rin g. The
activity of analogues with naphthalene ring substituents
depends on the position of substitution as is illustrated
by the series of hydroxy- (24, 30, 32, 34, and 37) and
methoxy-substituted (25, 31, 33, 35, and 38) analogues.
The MGC values decrease systematically in the series
of compounds with substituents at the 5-9-positions
(from 0.7 to 48 µM for hydroxy substitution and from
1.5 to >150 µM for methoxy substitution). A similar
trend is observed for the bis-lactone series (28, 36, and
39). The hydroxy- and methoxy-naphthols used to
prepare these analogues show no growth stimulating
activity. Interestingly, the 5-position tolerates even
large groups such as O-benzyl (26), an N-BOC-amino-
ethoxyethyl chain (27), and the bis-lactones (28 and 29)
without large increases in MGC values.
f
a
b
SI ) IC50/MGC. Activity of in vitro assay at 75 µM, normal-
ized to DMSO control. c No activity (g95% of DMSO controls).
Obtained from the NCI. e For structures, see Scheme 1 (com-
d
pound 9) and Figure 1 (compounds 18 and 40-45). f g150 µM.
The hydrolysis half-lives of methoxy-substituted ana-
logues (33, 35, and 38) and the 9-hydroxy analogue (37)
were determined to be 9.5 ( 0.1, 9.5 ( 0.3, 7.8 ( 0.5,
and 8.3 ( 0.3 h, respectively. These data suggest that
the increased MGC values of these analogues are due
to the substituents’ interference at the site of action and
not to a change in the hydrolysis rates, as these half-
lives are very similar to that of splitomicin.
F igu r e 3. Representative yeast proliferation assay dose-
response curves.
Miscella n eou s An a logu es. Dihydrocoumarin (41)
induces the most growth in our yeast proliferation
assays (Figure 3) and its toxicity is low (IC50 ) 122 µM,
Figure 4), which likely contributed to its potency. As
such, 41 was used as the reference for determination of
the MGC values (10% of maximal growth). Like 2,
coumarin (42) also shows no growth induction; however,
coumaranone (43) has a modest MGC (24 µM).
F igu r e 4. Representative toxicity dose-response curves.
Two other analogues (44 and 45), which, like splito-
micin, have the potential to act as acylating reagents,
show no growth stimulation. However, 44 has the
reactivity (electrophilicity) of the aromatic lactone in
forming a covalent adduct with a nucleophile (e.g.,
RNH2, RSH, and ROH). The nucleophile may be an

Evaluation of Splitomicin Analogues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2639
Con clu sion s
Forty-one analogues of splitomicin (1) were prepared
and evaluated for their relative Sir2 growth stimulating
activity in vivo and in vitro, and their toxicity profiles
were determined in vivo. The structure-activity rela-
tionships show that substituents are not well-tolerated
on the lactone nor at the 7-9-positions on the naph-
thalene ring, and the most potent analogues are struc-
turally similar to 1. Furthermore, the naphthalene ring
is not required for the activity as it can be replaced with
a benzene ring (e.g., 41). Although the aromatic lactone
is hydrolytically unstable, it is central to splitomicin’s
activity as an inhibitor of Sir2. Analogues with modified
lactone rings (e.g., contracted, oxidized, or substituted
with a heteroatom) are poor inhibitors and are less
reactive (i.e., have slower hydrolysis rates than 1),
suggesting that the reactivity of the lactone ring was
critical to the inhibition of Sir2. Our studies will serve
as a starting point toward the development of specific
inhibitors of Sir2-like deacetylases.
F igu r e 5. In vitro HDAC assay for 1 and 41.
greatest toxicity of all of the compounds evaluated in
this screen and this may have masked any growth
induction.
Toxicity a n d Selectivity In d ex (SI). Most ana-
logues with MGC values below 50 µM exhibited low
toxicity (IC50 > 60 µM), but no clear correlation of
toxicity to the MGCs was observed. This is consistent
with the observation that Sir2 activity is not required
for viability. Indeed, yeast cells lacking all five NAD+-
dependent deacetylases are viable.13 The toxicity:MGC
ratio yields a SI, a ratio that was useful in comparing
different compounds. Splitomicin (1), 13 (which is
converted to 1 in situ), 24, and 41 had the highest SI
values.
Exp er im en ta l Section
Gen er a l Meth od s. Mass spectrometry (electrospray ioniza-
tion) was performed on a Bruker Esquire ion trap mass
spectrometer or an HP Series 1100 MSD. Gas chromatogra-
phy-mass spectrometry (GC-MS) analyses (EI) were per-
formed on an HP 5890 GC with a HP 5970 detector and 30 m
DB5 column (0.25 mm i.d., 0.25 µm film) (method A), a HP
5989 detector and 60 m DB1 column (0.32 mm i.d., 0.25 µm
film) (method B), or a 15 m SPB-35 column (0.25 mm i.d., 0.25
µm film) (method C). Each GC-MS analysis was performed at
15 L/min (He) with a starting temperature of 70 °C and a
gradient of 15 °C/min from 3 to 18 min. 1H NMR spectra were
In Vitr o HDAC In h ibition . The in vitro inhibition
of Sir2 was evaluated using yeast whole cell lysates from
a strain overexpressing Sir2 as described previously.7
Inhibition of NAD+-dependent deacetylase activity was
first examined at 75 µM (Tables 1 and 3), and dose-
1
obtained on a Tecmag 300 MHz spectrometer, and H chemical
shifts are reported in ppm (δ). Flash column chromatography
was performed using silica gel (Merck, grade 9385, 230-400
mesh). Analytical thin-layer chromatography (TLC) was per-
formed using silica gel Analtech GF plates (0.25 mm), and
products were visualized using UV light. Unless otherwise
noted, reagents were purchased from Sigma-Aldrich Company
or Lancaster Synthesis. Solvents were ACS reagent grade or
better, and anhydrous solvents (Aldrich) were used as received
unless otherwise indicated. 1,2,3,4-Tetrahydro-1-phenan-
threnone (5), 1,2-dihydro coumarin (41), coumarin (42), 2-cou-
maranone (43), 1,8-dinaphthoic anhydride (44), and N-hydrox-
ysuccinimidyl (1-oxynaphthyl)acetate (45) were purchased
from Sigma-Aldrich Company and were used without further
purification. Compound 23 (1-phenyl-1,2-dihydro-3H-naphtho-
[2,1-b]pyran-3-one, NSC no. 17364) was obtained from the
National Cancer Institute. In general, the dihydronaphthopy-
ranones were susceptible to hydrolysis and were kept as solids
until preparing the DMSO stock solutions (30 mM) used in
the yeast proliferation assay, described below. The DMSO
solutions were kept tightly capped at 3-4 °C before use, and
as such, hydrolysis was less than a few percent over a period
of 2-3 weeks.
Yea st P r olifer a tion Assa y. The yeast proliferation assay
was performed as described previously.7 Briefly, DMSO stock
solutions were used to prepare stock solutions of each com-
pound in 5% DMSO in water (1.5 mM to 0.73 µM). Aliquots of
these solutions were transferred to 96 well plates containing
yeast cultures (strain UCC2210 MatR ppr1 adh4::URA3-TEL
(VII-L), ca. 13 500 cells per well) in synthetic complete media
lacking uracil to achieve 150 µM to 73 nM concentrations (150
µL total). The maximum concentration of DMSO in the yeast
cultures was 2%. Background absorbance measurements
(OD660nm) were taken at the beginning of the assay to account
for any compound precipitation at high concentrations. The
cultures were incubated at 30 °C until the OD660nm for the
culture of 41 reached ca. 0.5, which was typically reached by
40-48 h. The initial absorbance measurements were sub-
response curves were determined for 1 and 41 (IC50
)
100 and 31 µM, respectively; Figure 5). There was no
clear correlation between the activity in the yeast assay
and the in vitro HDAC assay, which is consistent with
our previous observations comparing in vivo and in vitro
inhibition of Sir2.7 Nevertheless, many of the com-
pounds that were highly active in vivo (e.g., 1, 26, and
41) inhibited Sir2 in vitro, affording 15-66% of the
activity relative to dimethyl sulfoxide (DMSO) control
at 75 µM. The concentrations sufficient for the in vivo
effect are lower than those required for the in vitro
inhibition. This might be due to higher stability of the
compounds in the acidic growth medium in the in vivo
assay. In synthetic complete medium, pH 4.3-4.5, the
half-life of 1 is 7.5 h; the pH of the in vitro reaction is
∼7.5 and 1 has a half-life of 1.5 ( 0.1 h (measured in
phosphate-buffered saline, data not shown). Alterna-
tively, the discrepancy between in vivo and in vitro
growth stimulating concentrations could be a reflection
of the differences between the histone peptide substrate
in vitro and the native nucleosomes in the context of
chromatin. A similar rationale has been proposed to
explain different in vitro vs in vivo properties of the
Sir2-R275 mutant.14 This Sir2 allele contains a point
mutation in the putative peptide binding site, where
splitomicin is proposed to act,7,15 and encodes for a fully
active histone deacetylase in vitro. Although the mutant
Sir2 protein is well-expressed and is appropriately
recruited to chromatin, it is incapable of establishing a
silenced state at the target loci.

2640 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Posakony et al.
Ta ble 4. HPLC Elution Methods
A
B
C
time
(min)
%
%
time
(min)
% (50 mM NH4OAc,
%
MeOH
time
(min)
%
%
H2Oa
CH3CNa
5 mM Et3N‚HOAc)b
H2Oa
CH3CNa
0
3
13
20
80
80
0
20
20
100
100
0
23
24
29
30
35
55
55
5
5
55
55
45
45
95
95
45
45
0
3
6
65
65
0
35
35
100
100
0
10
0
flow rate ) 0.3 mL/min
flow rate ) 0.25 mL/min
flow rate ) 0.36 mL/min
a
b
With 0.1% TFA. pH 3.5.
tracted from the final readings, and these values were plotted
vs the corresponding concentrations. MGC values were deter-
mined by assessing the concentration on the dose-response
curve at which the OD660nm was 10% of the growth observed
with 41 (typically 0.5-0.55 AU at 150 µM). Each compound
was evaluated in triplicate; uncertainties in Tables 1 and 3
and Figure 3 represent (1 SD.
Yea st Toxicity Assa y. The yeast cultures were incubated
with each compound as described above but in synthetic
complete media containing uracil. Initial OD660nm measure-
ments were taken just after preparing the plates, and the
plates were incubated at 30 °C. The final OD660nm measure-
ments were taken when the DMSO control wells had reached
0.35-0.4 AU. The final OD660nm values were subtracted from
the initial values, and the data were normalized to the DMSO
controls and plotted on a dose-response curve. The IC50 values
were determined using the software program Prism. Each
compound was evaluated in triplicate; uncertainties in Tables
1 and 3 and Figure 4 represent (1 SD.
In Vitr o HDAC Assa y. Histone deacetylase activity in the
presence or absence of splitomicin analogues was assessed by
measuring the NAD+-dependent release of free [3H]acetate
from [3H]-acetylated histone H4 peptide in the presence of
whole cell lysate prepared from an hst2∆ strain overexpressing
Sir2 as described previously.7 The hst2∆ strain contains a 2
µm plasmid with galactose inducible SIR2,16 and the extract
contained robust NAD-dependent deacetylase activity derived
exclusively from Sir2. Histone H4 peptide was acetylated
chemically using the HDAC assay kit (Upstate Biotechnology,
NY). Whole cell yeast lysate (10 µg) was incubated with [3H]-
acetylated histone H4 peptide (40 000 cpm) with or without
500 µM NAD+ in a 20 µL reaction buffered with 150 mM NaCl,
50 mM Tris-HCl, pH 8.0, and 1 mM DTT. Reactions were
incubated at 30 °C for 23 h and stopped by the addition of 5
µL of 1 N HCl and 0.15 N acetic acid. Released [3H]acetate
was extracted with 400 µL of ethyl acetate, and the activity
in 200 µL of this extract was determined using a scintillation
counter. All analogues were initially evaluated at 75 µM in
deacetylation reactions with 500 µM NAD+. Compounds 1 and
41 were also evaluated over a concentration range of 150 to
9.4 µM. Each deacetylation assay was performed in triplicate;
uncertainties in Tables 1 and 3 and Figure 5 represent (1
SD.
High -P er for m a n ce Liqu id Ch r om a togr a p h y (HP LC).
HPLC analyses were performed on HP1050 and HP1100
HPLC systems using a Supelcosil LC-18 column (25 cm × 2.1
mm, 0.3 µm; Supelco). To determine compound purity, aliquots
(1 µL) of 0.4 mM solutions (2:1 DMSO:MeOH) were analyzed
(at 230 nm) with gradient (method A) and isocratic then step
gradient (method B) elution protocols as described in Table 4.
Method C was used to monitor (280 nm) the hydrolysis rates
of selected compounds: DMSO stock solutions (30 mM) of
selected compounds were used to prepare 100 µM solutions in
complete media lacking uracil (pH 4.2-4.3, unadjusted). The
solutions were analyzed by HPLC several times over a 2-3
day period, and the peak area of unhydrolyzed product was
determined. A plot of ln(unhydrolyzed peak area) vs time
afforded a slope of the pseudo-first-order hydrolysis rate
constant. Each hydrolysis experiment was performed in trip-
licate; uncertainties in Table 1 represent (1 SD.
Gen er a l Meth od : Fr iedel-Cr a fts Alk yla tion . The naph-
thol (1 mmol), acrylic acid (2 mmol), and Amberlyst 15 ion
exchange resin (75-100 mg) were refluxed in toluene (2-3 mL)
overnight. The catalyst was filtered off and washed with hot
toluene (5 mL), and the solvent was removed under reduced
pressure. Flash chromatography of the residue afforded the
purified product.
1,2-Dih ydr o-3H-n aph th o[2,1-b]pyr an -3-on e (1). Friedel-
Crafts alkylation of 2-naphthol afforded 1 (85%) as a white
1
solid. Rf ) 0.6 (CH2Cl2). H NMR (CDCl3): [2.89 (d, J ) 7.0),
2.93 (d, J ) 6.5 Hz), 2H], 3.35 (t, J 1 ) 7.5, J 2 ) 7.3 Hz, 2H),
7.23 (d, J ) 8.8 Hz, 1H), 7.47 (overlapping dd, J 1 ) 6.7 Hz, J 2
) 1.3 Hz, 1H), 7.58 (overlapping dd, J 1 ) 6.9 Hz, J 2 ) 1.5 Hz,
1H), 7.76 (d, J ) 8.8 Hz, 1H), 7.84-7.90 (m, 2H). The HPLC,
GC-MS, and electrospray MS data for 1 and all other com-
pounds can be found in Table 5.
3H-Na p h th o[2,1-b]p yr a n -3-on e (2). Compound 2 was
prepared by Friedel-Crafts alkylation of 2-naphthol using
propiolic acid in place of acrylic acid, and the mixture was
refluxed for 4 h. Some 2-naphthol (21%) was recovered, and
the yield of 2 was 73% based on 79% conversion. Rf ) 0.45
1
(hexanes:EtOAc 3:2). H NMR (CDCl3): 6.54 (d, J ) 9.9 Hz,
1H), 7.41 (d, J ) 8.8 Hz, 1H), 7.55 (t, J ) 8.8 Hz, 1H), 7.67 (t,
J ) 8.3 Hz, 1H), 7.88 (d, J ) 7.8 Hz, 1H), 7.94 (d, J ) 8.8 Hz,
1H), 8.17 (d, J ) 8.3 Hz, 1H), 8.43 (d, J ) 9.9 Hz, 1H).
2,3-Dih yd r o-1H-n a p h th o[2,1-b]oxep in -4-on e (4). m-CP-
BA (g77% m-CPBA, remainder chlorobenzoic acid and H2O,
0.63 g, 2.5 equiv) was added in 3 aliquots over 12 h to a
solution of 1,2,3,4-tetrahydro-1-phenanthrenone (5, 0.196 g,
1 mmol) in CH2Cl2 (5 mL) while the solution was refluxed.
Then, the solvent was removed and the residue was flash-
chromatographed to afford 4 (0.104 g, 49%). Rf ) 0.7 (CH2-
Cl2). 1H NMR (CDCl3): 2.15-2.30 (m, 2H), 2.39-2.46 (m, 2H),
3.20 (t, J ) 7.0 Hz, 2H), 7.19 (d, J ) 8.8 Hz, 1H), 7.36-7.53
(m, 2H), 7.70 (d, J ) 8.8 Hz, 1H), 7.79 (dd, J 1 ) 8.2 Hz, J 2
1.3 Hz, 1H), 7.94 (d, J ) 8.4 Hz, 1H).
)
3,4-Dih ydr o-2H-n aph th o[1,2-b]pyr an -3-on e (6). Friedel-
Crafts alkylation (5 h reflux) of 1-naphthol afforded 6 (37%)
and a trace of unreacted 1-naphthol. 6: Rf ) 0.65 (EtOAc:
hexanes 1:2). 1H NMR (CDCl3): 2.88-2.90 (m, 2H), 3.13-3.17
(m, 2H), 7.27 (d, J ) 8.3 Hz, 1H), 7.47-7.58 (m, 2H), 7.60 (d,
J ) 8.3 Hz, 1H), 7.81-7.84 (m, 1H), 8.23-8.26 (m, 1H).
Compound 6 was also be prepared in 9% overall yield by
the Knoevenagel condensation, borohydride reduction, and
decarboxylation procedure, described below for 9, using 1-hy-
droxy-2-naphthaldehyde.17
1,2-Dih yd r o-3H-n a p h th o[2,3-b]p yr a n -3-on e (9). Kn o-
even a gel con d en sa tion : 3-Hydroxy-2-naphthalenecarbox-
aldehyde9 (0.11 g, 0.58 mmol), diethylmalonate (0.18 mL, 1.2
mmol), and piperidine (1 drop) were dissolved in pyridine (3
mL), and the mixture was refluxed overnight. The solvent was
removed, the residue was washed with H2O (20 mL), and the
product was extracted into EtOAc (2 × 25 mL). The organic
extract was dried (Na2SO4), and the solvent was removed. The
residue was purified by flash chromatography (hexanes:EtOAc,
2:1 to 3:2) to afford ethyl 3H-naphtho[2,3-b]pyran-3-one-2-
carboxylate (7, 0.145 g, 93%). Rf ) 0.35 (2:1 hexanes:EtOAc).
1H NMR (CDCl3): 1.44 (t, J ) 7.1 Hz, 3H), 4.44 (q, J ) 7.1
Hz, 2H), 7.49-7.57 (m, 1H), 7.60-7.68 (m, 1H), 7.73 (s, 1H),
7.87-7.97 (m, 2H), 8.17 (s, 1H), 8.65 (s, 1H).

Evaluation of Splitomicin Analogues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2641
Ta ble 5. HPLC, GC-MS, and Electrospray MS Data
Rt HPLC A
(% purity)
Rt HPLC B
(% purity)
GC-MSa Rt (method, purity),
m/z (intensity, assignment)
ESI-MS (-/+) m/z
(intensity, assignment)
1
2
14.70 (99)
16.55 (96)
12.15 min (B, >99%), 198
(100, M+), 170 (60), 128 (70).
12.4 min (A, >99%), 196
10.60 (>99)
7.97 (>99)
(90, M+), 168 (100), 139 (60)
3b
4
11.89 (>99)
16.08 (96)
8.33 (>99)
9.57 (>99)
13.1 min (A, >99%), 212
(40, M+), 184 (40), 157 (100), 129 (40)
11.9 min (B, 99%), 190 (100, M+),
170 (50), 156 (90), 128 (50)
6
13.93 (>99)
13.27 (>99)
16.22 (>99)
12.03 (>99)
15.88 (>99)
6.57 (>99)
10.33 (>99)
18.08 (98)
7
31.37 (>99)
20.87 (>99)
19.24 (>99)
21.37 (>99)
16.73 (>99)
24.62 (95)
15.4 min (B, >99%), 268 (100, M+),
223 (70), 196 (90), 139 (70)
8
(+) 563 (20, 2M + Na), 293 (80, M + Na), 242
(100, M + H-CH2dCH2)
9
12.2 min (B, >99%), 198 (100, M+),
170 (50), 128 (50)
10
11c
12
13.1 min (B, >99%), 214 (50, M+),
186 (100)
14.3 min (A, >99%), 197 (100, M+),
168 (85)
(+) 269 (20, M + K), 253 (50, M + Na), 231
(10, M + H), 199 (100, M + H-MeOH)
(-) 214 (100, M - H)
13
14
15
16
17
5.79 (>99)
13.29 (98)
12.60 (>99)
6.09 (>99)
6.18 (>99)
7.11 (>99)
6.78 (>99)
18.00 (98)
13.33 (>99)
23.76 (>99)
(-) 228 (100, M - H)
(+) 272 (100, M + H), 294 (5, M + Na)
(-) 215 (100, M - H)
(+) 307 (15, M + K), 291 (100, M + Na),
269 (5, M + H)
18
19
5.78 (98.7)
17.38 (>99)
7.36 (95)
17.42 (98)
(-) 201 (100, M - H)
(+) 309 (60, M + K), 293 (100, M + Na),
271 (30, M + H)
20
21
22
15.97 (>99)
17.38 (97)
18.06 (95)
35.04 (>99)
37.15 (>99)
36.94 (>99)
12.9 min (A, 98%), 212 (80, M+),
184 (30), 169 (100), 128 (60)
12.8 min (B, 97%), 238 (80, M+),
196 (100), 169 (50), 198 (50)
14.1 min (A, 90%), 254 (100, M+),
198 (85), 183 (80), 157 (100), 128 (100)
23d
24
25
17.96 (99)
5.93 (96.5)
13.84 (>99)
37.11 (>99)
7.48 (98)
14.74 (>99)
(-) 213 (100, M - H)
17.3 min (A, >99%), 228 (100, M+),
200 (30), 186 (45).
26e
16.17 (91)
36.18 (94)
17.9 min (A, >99%), 304 (50, M+),
91 (100)
27
28e
29
30
31
32
15.32 (95)
9.80 (94)
8.28 (>99)
6.84 (>99)
16.11 (97)
5.83 (>99)
35.8 (95)
13.86 (92)
8.87 (99)
8.31 (>99)
31.61 (>99)
6.38 (97)
(+) 424 (100, M + Na), 440 (10, M + K)
18.2 min (C, 79%), 268 (100, M+), 198 (50)
(+) 555 (100, 2M + Na), 289 (40, M + Na)
(-), 213 (100, M - H)
14.3 min (A, 99%), 228 (100, M+)
13.9 min (A, 99%), 214 (100, M+),
186 (40), 172 (75)
33
15.20 (>99)
18.50 (98)
14.3 min (A, 91%), 228 (100, M+),
200 (40), 186 (95)
34
35
36
5.42 (>99)
14.69 (96.7)
9.19 (97.7)
6.48 (>99)
16.52 (99)
13.32 (95)
(-) 213 (100, M - H)
(-) 213 (100, M - H)
13.9 min (C, 99%), 228 (100, M+), 186 (90)
17.7 min (C, >99%), 268 (100, M+),
226 (50), 198 (50)
37
38
5.42 (98)
15.46 (95)
7.66 (97)
18.57 (>99)
13.8 min (C, 90%), 228 (100, M+),
200 (70)
39
7.93 (97.4)
13.25 (>99)
9.29 (>99)
17.6 min (C, 99%), 268 (M+, 100),
240 (50), 197 (70)
40
5.95 (>99)
(-) 213 (100, M - H)
a
If GC-MS showed a purity <95%, the sample was repurified by chromatography before HPLC analysis and biological evaluation.
b Described in ref 10. c Described in ref 11. Obtained from the NCI. e Purity was 90-95% even after subsequent purification. The biological
assays were performed with compound as is.
d
Na BH4 Red u ction . Compound 7 (50 mg, 0.19 mmol) was
dissolved in dry pyridine (1 mL) at 0 °C, and NaBH4 was added
(5 mg). The solution was stirred for 1 h at 0 °C and then for
30 min at room temperature. The mixture was poured into 1
N HCl (10 mL), and the precipitated product was filtered off
and subsequently washed with H2O (50 mL). The precipitate
was dissolved in a minimum of EtOAc:hexanes (1:1) and
passed through a plug of silica. Solvent removal afforded 43
mg (86%) of ethyl 1,2-dihydro-3H-naphtho[2,3-b]pyran-3-one-
2-carboxylate (8), which was used without further purification.
Compound 8: 1H NMR (CDCl3): 1.19 (t, J ) 7.2 Hz, 3H), 3.30-
3.65 (m, 2H), 3.86 (d of d, J 1 ) 7.7 Hz, J 2 ) 5.8 Hz, 1H), 4.18
(q, J ) 7.2 Hz, 2H), [7.45-7.49 (m) overlapping with 7.51 (s),
3H], 7.70 (s, 1H), 7.77-7.82 (m, 2H).
Deca r boxyla tion . Compound 8 (20 mg, 75 µmol) was
dissolved in THF (1 mL), and 1 N LiOH (1 mL) was added.
The mixture was stirred overnight, then the solution was
acidified to pH 4 with 1 N HCl, brine (5 mL) was added, and

2642 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Posakony et al.
the product was extracted into EtOAc. The solvent was
removed, and the residue was then dissolved in benzene (10
mL) acidified with TsOH (1-2 mg). The mixture was refluxed
overnight, and then, the solvent was removed. The product
was purified by chromatography (CH2Cl2) to afford 9 (9 mg,
45 µmol, 60%). Compound 9: Rf ) 0.55 (CH2Cl2). 1H NMR
(CDCl3): 2.85-2.90 (m, 2H), 3.21 (t br, J 1 ) 7.5 Hz, J 2 ) 5.7
Hz, 2H), [7.44-7.51 (m) overlapping with 7.49 (s), 3H)], 7.69
(s, 1H), 7.78-7.81 (m, 2H).
1,2-Dih yd r o-3H -n a p h t h o[2,1-b]t h iop yr a n -3-on e (10).
Friedel-Crafts alkylation of 2-naphthalene thiol, as described
for 1 (48 h of reflux), afforded 10 in 40% yield. Rf ) 0.9
(hexanes:EtOAc 3:2). 1H NMR (CDCl3): 3.09-3.14 (m, 2H),
3.27-3.32 (m, 2H), 7.28 (d, J ) 8.6 Hz, 1H), 7.45 (t, J ) 8.0
Hz, 1H), 7.6 (t of d, J 1 ) 8.3 Hz, J 2 ) 1.3 Hz, 1H), 7.74 (d, J
) 7.8 Hz, 1H), 7.78 (d, J ) 8.8 Hz, 1H), 9.16 (d, J ) 8.8 Hz,
1H).
J 1 ) 8.6, J 2 ) 7.9 Hz, 2H), 7.15 (d, J ) 8.8 Hz, 1H), 7.26 (t of
d, J 1 ) 7.0 Hz, J 2 ) 0.9 Hz, 1H), 7.44 (t of d, J 1 ) 7.0 Hz, J 2
) 1.4 Hz, 1H), 7.63 (d, J ) 8.8 Hz, 1H), 7.75 (d, J ) 7.2 Hz,
1H), 7.87 (d, J ) 8.6 Hz), 9.6 (s, br, 1H), 12.1 (s, br, 1H).
As a neat oil, 16 slowly relactonizes to 1. Thus, preparation
of 16 for biological evaluation was more conveniently carried
out by treating a DMSO solution of 1 with 5% NaOH. After
several minutes, HPLC indicated only 16 (data not shown).
1-(3-Hydr oxypr opyl)-2-h ydr oxyn aph th alen e (18). NaBH4
(40 mg) was added to a solution of 2 (25 mg, 0.13 mmol) in
MeOH (4 mL). The mixture was stirred for 5 h and then slowly
added to 1 N HCl (10 mL). The product was extracted into
EtOAc, and the solvent was removed after drying (Na2SO4).
The residue was purified by flash chromatography (EtOAc) to
afford 18 (12 mg, 46%). Rf ) 0.35. 1H NMR (CD3CN): 1.87
(m, 2H), 3.13 (d, J ) 7.3 Hz, 2H), 3.53 (d, J ) 6.2 Hz, 2H),
7.14 (d, J ) 8.8 Hz, 1H), 7.34 (t of d, J 1 ) 8.0, J 2 ) 1.0, 1H),
7.49 (t of d, J 1 ) 8.6, J 2 ) 1.6, 1H), 7.67 (d, J ) 8.8 Hz, 1H),
7.82 (d, J ) 8.0 Hz, 1H), 7.98 (d, J ) 8.6 Hz, 1H).
Meth yl 3-(2-Hyd r oxyn a p h th -1-yl)p r op ion a te (12). Spli-
tomicin (1, 100 mg, 0.5 mmol) and TsOH (5 mg) were
disssolved in MeOH (5 mL), and the mixture was stirred
overnight. Then, the solvent was removed and the residue was
purified by flash chromatography (CH2Cl2) to yield 0.103 g
(0.45 mmol, 89%) of 12 as a white powder. Rf ) 0.55. 1H NMR
E t h yl 1,2-Dih yd r o-3H -n a p h t h o[2,1-b]p yr a n -3-on e-2-
ca r boxyla te (19). Diethylmalonate and 2-hydroxy-1-naph-
thaldehyde were subjected to conditions of the Knoevanagel
condensation described for 7 above. Purification by flash
chromatography and subsequent recrystallization from EtOH
afforded ethyl 3H-naphtho[2,1-b]pyran-3-one-2-carboxylate
(17) in 92% yield. Compound 17 was reduced using NaBH4 in
pyridine, as described for 8 above, to afford 19 in 85% yield.
Compound 17. Rf ) 0.6 (1:1 EtOAc:hexanes). MS (ESI+): m/z
307(10, M + K), 291 (M + Na), 269 (5, M + H). 1H NMR
(CDCl3): 1.33 (t, J ) 7.1 Hz, 3H), 4.40 (q, J ) 7.1 Hz, 2H),
7.44 (d, J ) 9.1 Hz, 1H), 7.58-7.63 (m, 1H), 7.71-7.77 (m,
1H), 7.92 (d, J ) 7.3 Hz, 1H), 8.08 (d, J ) 9.1 Hz, 1H), 8.30 (d,
J ) 8.3 Hz, 1H), 9.29 (s, 1H). Compound 19. Rf ) 0.75 (1:1
(CDCl3): 2.9 (dd, J 1 ) 5.7 Hz, J 2 ) 6.2 Hz, 2H), 3.3 (dd, J 1
)
5.7 Hz, J 2 ) 6.0 Hz, 2H), 3.69 (s, 3H), 7.21 (d, J ) 8.8 Hz, 1H),
7.34 (t, J ) 6.4 Hz, 1H), 7.50 (t, J ) 6.4 Hz, 1H), 7.67 (d, J )
8.8 Hz, 1H), 7.9 (overlapping d’s, J ) 8.2 Hz, 2H), 8.13 (s, 1H).
3-(2-Hyd r oxyn a p h th -1-yl)p r op ion a m id e (13). Splitomi-
cin (100 mg, 0.5 mmol) and NH4OAc (150 mg, ca. 2 mmol) were
dissolved in DMF (1 mL), and the mixture was stirred
overnight. The mixture was poured into water (25 mL), brine
(15 mL) was added, and the product was extracted into EtOAc
(2 × 25 mL). The solvent was dried (Na2SO4) and then removed
under reduced pressure. The residue was chromatographed
using EtOAc to afford 12 mg of recovered 1 and 13 (89 mg,
0.41 mmol, 94% based on 88% conversion). Rf ) 0.55 (EtOAc).
1H NMR (acetone-d6): 2.79-2.83 (m, overlapping with residual
H2O, 2H), 3.27 (dd, J 1 ) 6.0 Hz, J 2 ) 6.2 Hz, 2H), 6.6 (s br,
1H), 7.11 (d, J ) 8.8 Hz, 1H), 7.30 (t of d, J 1 ) 8.0 Hz, J 2 ) 1.0
Hz, 1H), 7.45 (t of d, J 1 ) 8.3 Hz, J 2 ) 1.0 Hz), 7.65 (d, J ) 8.6
Hz, 1H), 7.78 (d, J ) 8.3 Hz, 1H), 7.88 (d, J ) 8.6 Hz, 1H),
9.70 (s, 1H).
1
EtOAc:hexanes). H NMR (CDCl3): 1.20 (t, J ) 7.1 Hz, 3H),
3.5 (dd, J 1 ) 6.8 Hz, J 2 ) 16.0 Hz, 1H), 3.62-3.91 (m, 2H), 4.2
(overlapping q’s, each with J ) 7.1 Hz, 2H), 7.23 (d, J ) 8.2
Hz, 1H), 7.48 (ABC spin system, J AB ) 8.1 Hz, J AC ) 7.0 Hz,
J BC ) 1.0 Hz, 1H), 7.58 (ABC spin system, J AB ) 8.3 Hz, J AC
) 7.0 Hz, J BC ) 1.3 Hz, 1H), 7.78 (d, J ) 8.8 Hz, 1H), 7.84 (d,
J ) 7.5 Hz, 1H), 7.90 (d, J ) 7.5 Hz, 1H).
1,2-Dih yd r o-2-m et h yl-3H -n a p h t h o[2,1-b]p yr a n -3-on e
(20). A solution of 19 (135 mg, 0.5 mmol) in dry THF (2 mL)
was cooled to 0 °C, and LDA (0.25 mL of a 2.0 M solution in
THF/ethylbenzene) was added. The mixture was stirred for 1
h at 0 °C, and then, CH3I (0.31 mL, 5 mmol) was added. After
it was stirred for another 16 h, the solvent was removed, and
the residue was chromatographed (EtOAc:hexanes 1:1) to
afford the methylated product (Rf ) 0.85), The decarboxylation
and relactonization were carried out as described for the
synthesis of 9, except the relactonization was accomplished
in refluxing (3 h) benzene (25 mL) without adding TsOH.
Chromatographic purification (1:1 hexanes:EtOAc) of the
residue afforded 90 mg (84% overall yield) of 1,2-dihydro-2-
methyl-3H-naphtho[2,1-b]pyran-3-one-2-carboxylate (20) as a
N-Meth yl-3-(2-h yd r oxyn a p h th -1-yl)p r op ion a m id e (14).
Splitomicin (50 mg, 0.25 mmol) was dissolved in CH3CN (2
mL), and two aliquots of methylamine (2.0 M solution in
MeOH, 15 µL each) were added at 0 and 3 h. The solution
was stirred overnight, during which time a precipitate formed.
The solvent was removed, and the residue was chromato-
graphed (CH2Cl2:MeOH 25:1) to afford unreacted 1 (38 mg)
and 14 (13 mg, 0.057 mmol, 95% based on 24% conversion).
1
Rf ) 0.35. H NMR (CDCl3): 2.18 (t, J ) 7.5 Hz, 2H), 2.93 (t,
J ) 7.5 Hz, 2H), 4.34 (s, 3H), 6.71 (d, J ) 8.8 Hz, 1H), 6.87
(overlapping d of d, J 1 ) 7.0 Hz, J 2 ) 1.0 Hz, 1H), 7.05
(overlapping d of d, J 1 ) 7.0 Hz, J 2 ) 1.6 Hz, 1H), 7.20 (d, J
) 8.8 Hz, 1H), 7.33 (d, J ) 7.3 Hz, 1H), 7.50 (d, J ) 8.6 Hz,
1H), 9.90 (s, 1H).
1
white powder. Rf ) 0.55 (CH2Cl2). H NMR (CDCl3): 1.39 (d,
5.5 Hz, 3H), 2.82-2.99 (m, 2H), 3.4 (m, 1H), 7.14 (d, J ) 9.7
Hz, 1H), 7.34-7.52 (m, 2H), 7.66-7.83 (m, 3H).
N,N-Diet h yl-3-(2-h yd r oxyn a p h t h -1-yl)p r op ion a m id e
(15). Splitomicin (1, 15 mg, 76 µmol) and diethylamine (0.2
mL, 0.19 mmol) were combined and stirred for 1 h. The solvent
was removed, and the residue was chromatographed (CH2Cl2:
MeOH 100:1) to afford 15 (7.5 mg, 28 µmol) in 36% yield. Rf )
0.4. 1H NMR (acetone-d6): 1.01-1.09 (overlapping t’s, J ) 7.0
Hz, 6H), 2.93-2.97 (m, 2H), 3.30-3.40 (m, 6H), 7.09 (d, J )
8.8 Hz, 1H), 7.28 (t of d, J 1 ) 6.8, J 2 ) 1.5 Hz), 7.65 (d, J )
8.6, 1H), 7.77 (d, J ) 8.3 Hz, 1H), 7.93 (d, J ) 8.6 Hz, 1H),
10.30 (s, 1H).
3-(2-Hyd r oxyn a p h th -1-yl)p r op ion ic Acid (16). Splito-
micin (1, 0.138 g, 0.7 mmol) was dissolved in THF (5 mL), and
1 N LiOH (2.5 mL) was added. The mixture was stirred
overnight, then the pH was adjusted to ∼3 (1 N HCl), and the
product was extracted into EtOAc. The solvent was dried (Na2-
SO4) and removed under reduced pressure to afford 16 (0.145
g, 96%) as an oil. 1H NMR (DMSO-d6): 2.39-3.45 (overlapping
d’s, J 1 ) 8.6 Hz, J 2 ) 7.7 Hz, 2H), 3.15-3.23 (overlapping d’s,
1,2-Dih yd r o-2-a llyl-3H-n a p h th o[2,1-b]p yr a n -3-on e (21).
A solution of 19 (0.108 g, 0.4 mmol) in dry THF (4 mL) was
treated with LDA (2.0 M solution in THF/ethylbenzene, 0.2
mL, 0.4 mmol), and the solution was stirred for 1 h at room
temperature Allyl bromide (0.69 mL, 8 mmol) was added, and
the mixture was stirred for 48 h. The solvent was removed
under reduced pressure, and the residue was redissolved in
THF (5 mL). The decarboxylation reaction was carried out as
described for 9. As a solid residue, the hydroxy acid product
slowly relactonized (TLC), and after 3 days, the residue was
purified by chromatography (CH2Cl2) to afford 21 (68 mg, 71%).
Rf ) 0.65 (CH2Cl2). 1H NMR (CDCl3): 2.39-2.50 (m, 1H),
2.74-2.90 (m, 2H), [2.96 (d, J ) 10.6 Hz), 3.01 (d, J ) 10.9
Hz), 1H], [3.42 (d, J ) 6.2 Hz), 3.48 (d, J ) 6.0), 1H], 5.14-
5.18 (m, 1H, 5.18-5.21 (m, 1H), 5.84-5.98 (m, 1H), 7.21 (d, J
) 8.8 Hz, 1H), 7.46 (overlapping d of d, J 1 ) 6.9 Hz, J 2 ) 1.3

Evaluation of Splitomicin Analogues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2643
Hz, 1H), 7.57 (overlapping d of d, J 1 ) 6.8 Hz, J 2 ) 1.3 Hz,
1H), 7.75 (d, J ) 9.1 Hz, 1H), 7.83-7.89 (m, 2H).
2H], 3.27 (q, J ) 5.7, 2H), 3.42 (t, J 1 ) 8.1 Hz, J 2 ) 6.9 Hz,
2H), 3.62 (t, J ) 5.7 Hz, 2H), 3.90 (t, J ) 4.8 Hz, 2H), 4.30 (t,
J ) 4.8 Hz, 2H), 5.9 (s, br, 1H), 7.37 (s, 1H), 7.42-7.45 (m,
2H), 7.79-7.83 (m, 1H), 7.89-7.93 (m, 1H).
1,2-Dih yd r o-3H,6H-n a p h th o[2,1-b],[3,4-b]bisp yr a n -3,6-
d ion e (29). Treatment of 24 with propiolic acid (15 equiv)
under the Friedel-Crafts alkylation conditions (24 h reflux)
afforded 29 (35%). Rf ) 0.35 (1:1 EtOAc:hexanes). 1H NMR
(DMSO-d6): [2.99 (d, J ) 7.3 Hz), 3.01 (d, J ) 7.0 Hz), 2H],
3.50 (t, J 1 ) 7.5 Hz, J 2 ) 7.3 Hz), 6.68 (d, J ) 9.9 Hz, 1H),
7.68-7.72 (m, 2H), 8.11-8.14 (m, 1H), 8.54-8.57 (m, 1H), 8.96
(d, J ) 9.9 Hz, 1H).
1,2-Dih yd r o-3H-6-h yd r oxyn a p h th o[2,1-b]p yr a n -3-on e
(30). Friedel-Crafts alkylation of 1,3-dihydroxynaphthalene
afforded 30 (50%) after 3 h of refluxing. Rf ) 0.5 (CH2Cl2:
acetone 10:1). 1H NMR (DMSO-d6): [2.85 (d, J ) 7.0 Hz), 2.88
(d, J ) 6.5 Hz), 2H], 3.19 (t, J 1 ) 7.8 Hz, J 2 ) 7.0.0 Hz, 2H),
6.59 (s, 1H), 7.41 (m, 1H), 7.56 (m, 1H), 7.87 (d, J ) 8.3 Hz,
1H), 8.13 (d, J ) 8.3 Hz, 1H), 10.48 (s, 1H).
1,2-Dih yd r o-3H -6-m e t h oxyn a p h t h o[2,1-b]p yr a n -3-
on e (31). Compound 30 was methylated in 30% yield under
the conditions used to synthesize 25. Compound 31: Rf ) 0.85
(20:1 CH2Cl2:acetone). 1H NMR (CDCl3): [2.90 (d, J ) 7.3 Hz),
2.93 (d, J ) 6.2 Hz), 2H)] 3.27 (t, J 1 ) 7.0, J 2 ) 7.8 Hz, 2H),
3.99 (s, 3H), 6.59 (s, 1H), 7.45 (m, 1H), 7.58 (m, 1H), 7.81 (d,
J ) 7.3 Hz, 1H), 8.26 (d, J ) 7 Hz).
1,2-Dih yd r o-3H-7-h yd r oxyn a p h th o[2,1-b]p yr a n -3-on e
(32). Friedel-Crafts alkylation of 1,6-dihydroxynaphthalene
(24 h of reflux) afforded 32 (20%); although all reagents were
in solution at the beginning of the reaction, an unidentified
precipitate formed during the 24 h reflux. Rf ) 0.35 (20:1
CH2Cl2:acetone). 1H NMR (acetone-d6): 2.86-2.91 (m, 2H),
3.11-3.16 (m, 2H), 7.19-7.22 (m, 2H), 7.28 (d, J ) 8.6 Hz,
1H), 7.45 (d, J ) 8.3 Hz, 1H), 8.01 (d, J ) 9.6 Hz, 1H), 8.76 (s,
1H).
1,2-Dih yd r o-3H-7-h yd r oxym eth yln a p h th o[2,1-b]p yr a n -
3-on e (33). Compound 32 was methylated in 40% yield under
the conditions used to synthesize 25. Compound 33: Rf ) 0.85
(20:1 CH2Cl2:acetone). 1H NMR (CDCl3): [2.87 (d, J ) 7.1 Hz),
2.90 (d, J ) 5.4 Hz), 2H], 3.11 (t, J 1 ) 7.8 Hz, J 2 ) 5.5 Hz,
2H), 3.93 (s, 3H), 7.11-7.24 (m, 3H), 7.48 (d, J ) 8.1 Hz, 1H),
8.13 (d, J ) 8.5 Hz, 1H).
1,2-Dih yd r o-2-(2-m eth ylp r op yl)-3H-n a p h th o[2,1-b]p y-
r a n -3-on e (22). LDA (2.0 M in THF/ethylbenzene 0.2 mL) was
added to a solution of 19 (0.108 g, 0.2 mmol) in THF (2 mL) at
0 °C, and the solution was stirred for 1 h at room temperature.
The solvent was removed, and the residue was redissolved in
dry DMF (2 mL). To this solution was added iodo-2-methyl-
propane (0.9 mL, 8 mmol); the mixture was stirred at room
temperature for 24 h and then heated to 60-70 °C for 12 h.
The solvent was removed under reduced pressure, and the
residue was washed with H2O (10 mL, acidified to pH 4 with
1 N HCl). Brine (15 mL) was added, and the product was
extracted into EtOAc (2 × 25 mL). The EtOAc solution was
dried (Na2SO4), the solvent was removed, and the residue was
flash-chromatographed (CH2Cl2). Unreacted 19 (Rf ) 0.4, 26
mg, 25%) was recovered, and the new product (Rf ) 0.55, 62
mg as an oil), ethyl 1,2-dihydro-2-(2-methylpropyl)-3H-naph-
tho[2,1-b]pyran-3-one-2-carboxylate (22a ), was collected but
was not characterized. Compound 22a (50 mg, 0.15 mmol) was
decarboxylated using LiOH as described above for the syn-
thesis of 9. As a neat residue, the hydroxy acid product slowly
relactonized at room temperature. After 3 days, the product
was purified by filtering through a plug of silica as a solution
in CH2Cl2 to afford 22 (22 mg) in 36% overall yield from 19,
based on 75% conversion. Compound 22: Rf ) 0.75 (CH2Cl2).
1H NMR (CDCl3): 0.94 (d, J ) 6.3 Hz, 3H), 1.00 (d, J ) 6.5
Hz, 3H), 1.92-1.98 (m, 2H), 2.85-3.09 (m, 2H), 3.48 (dd, J 1
)
5.8 Hz, J 2 ) 15.4 Hz, 1H), 7.23 (d, J ) 8.9 Hz), 7.42-7.62 (m,
2H), 7.77 (d, J ) 8.9 Hz, 1H), 7.83-7.92 (m, 2H).
1,2-Dih yd r o-3H-5-h yd r oxyn a p h th o[2,1-b]p yr a n -3-on e
(24) a n d 1,2,7,8-Tetr a h yd r o-3H,6H-n a p h th o[2,1-b:3,4-b′]-
d ip yr a n -3,6-d ion e (28). Friedel-Crafts alkylation of 2,3-
dihydroxynaphthalene afforded 24 (55%) and 28 (3%) after 3
h of refluxing. Compound 24: Rf ) 0.5 (CH2Cl2:acetone 10:1).
1H NMR (DMSO-d6): [2.88 (d, J ) 7.3 Hz), 2.91 (d, J ) 6.3
Hz), 2H], 3.32 (t, under residual water peak), 7.18 (s, 1H), [7.35
(d, J ) 3.6 Hz), 7.37 (d, J ) 2.1 Hz), 2H], 7.69 (m, 1H), 7.87
(m, 1H), 10.05 (s br, 1H). Compound 28: Rf ) 0.9 (CH2Cl2:
acetone 10:1). 1H NMR (CDCl3): [2.91 (d, J ) 7.0 Hz), 2.94 (d,
J ) 6.2 Hz), 2H], 3.37 (t, J 1 ) 7.5 Hz, J 2 ) 7.3 Hz, 2H), 7.54
(d, J ) 3.4 Hz, 1H), 7.57 (d, J ) 3.4 Hz, 1H), 7.87 (d, J ) 3.4
Hz, 1H), 7.90 (d, J ) 3.4 Hz, 1H).
1,2-Dih yd r o-3H-8-h yd r oxyn a p h th o[2,1-b]p yr a n -3-on e
(34) a n d 1,2,7,8-Tetr a h yd r o-3H,9H-n a p h th o[2,1-b:6,5-b′]-
d ip yr a n -3,9-d ion e (36). Friedel-Crafts alkylation of 2,6-
dihydroxynaphthalene afforded 34 and 36 in 50 and 4% yield,
respectively, after chromatographic purification of the products
(CH2Cl2/acetone 20:1 to 10:1). Compound 34: Rf ) 0.33
1,2-Dih yd r o-3H -5-m e t h oxyn a p h t h o[2,1-b]p yr a n -3-
on e (25). Methyl iodide (25 µL, 0.4 mmol) was added to a
solution of 24 (40 mg, 0.19 mmol) and DBU (30 µL, 0.2 mmol)
in CH3CN (2 mL), and the mixture was stirred overnight.
Then, the solvent was removed and residue was flash-chro-
matographed to afford 25 (21 mg, 49%). Rf ) 0.85 (20:1 CH2-
1
(CH2Cl2/acetone 20:1). H NMR (CDCl3/DMSO-d6; referenced
1
Cl2:acetone). H NMR (CDCl3): [2.88 (d, J ) 7.0 Hz), 2.91 (d,
to DMSO-d6): [2.85 (d, J ) 7.0 Hz), 2.87 (d, J ) 7.5 Hz), 2H],
[3.28 (t, J 1 ) 7.8 Hz, J 2 ) 6.8 Hz, 2H) overlapping with water
signal], [7.09 to 7.16 (m, 3H); overlapping with residual CHCl3
signal], 7.59 (d, J ) 8.8 Hz, 1H), 7.81 (d, J ) 9.3 Hz), 9.66 (s,
J ) 6.2 Hz), 2H], 3.33 (t, J 1 ) 7.8, J 2 ) 7.0, 2H), 3.98 (s, 3H),
7.11 (s, 1H), 7.42-7.46 (m, 2H), 7.72-7.80 (m, 2H).
1,2-Dih yd r o-3H -5-b e n zyloxyn a p h t h o[2,1-b]p yr a n -3-
on e (26). DBU (47 µL, 0.32 mmol) was added to a solution of
24 (50 mg, 0.23 mmol) in CH3CN (1 mL), and after 15 min of
stirring, benzyl bromide (60 µL, 0.5 mmol) was added to the
mixture. After it was stirred overnight, the solvent was
removed and the mixture was purified by flash chromatogra-
phy to afford 26 (94% yield). Rf ) 0.5 (50:1 CH2Cl2:acetone).
1H NMR (CDCl3): [2.90 (d, J ) 7.3 Hz), 2.92 (d, J ) 6.2 Hz),
2H], 3.35 (t, J 1 ) 8.0 Hz, J 2 ) 7.0 Hz, 2H), 5.26 (s, 2H), 7.33-
7.54 (m, 8H), 7.69-7.72 (m, 1H), 7.78-7.81 (m, 1H).
1,2-Dih yd r o-3H-5-(2-(N-BOC-2-a m in oeth oxy)eth yloxy)-
n a p h th o[2,1-b]p yr a n -3-on e (27). DBU (0.39 mL, 2.6 mmol)
and 24 (0.375 g, 1.75 mmol) were combined in dry DMF (4
mL) under Ar, and the mixture was stirred for 20 min at room
temperature. Then, 2-(2-tert-butoxycarbonylaminoethoxy)-
ethanol methanesulfonic ester18 (0.75 g, 2.6 mmol) was added
and the mixture was heated to 80-85 °C for 5 h with stirring.
The solvent was removed under reduced pressure, and the
mixture was chromatographed on silica/(CH2Cl2:acetone 10:
1). Unreacted 24 (0.05 g, 13%) eluted, followed by 27 (Rf )
0.5, 0.286 g, 47% based on 87% conversion). 1H NMR (acetone-
d6): 1.39 (s, 9H), [2.92 (d, J ) 7.2 Hz), 2.95 (d, J ) 6.3 Hz),
1
1H). Compound 36. Rf ) 0.85 (CH2Cl2/acetone 20:1). H NMR
(CDCl3): [2.91 (d, J ) 7.0 Hz), 2.93 (d, J ) 7.5 Hz), 2H], 3.37
(t, J 1 ) 7.0 Hz, J 2 ) 7.8 Hz, 2H), 7.31 (d, J ) 9.3 Hz, 2H), 7.83
(d, J ) 9.2 Hz, 2H).
1,2-Dih yd r o-3H -8-m e t h oxyn a p h t h o[2,1-b]p yr a n -3-
on e (35). Friedel-Crafts alkylation using 6-methoxy-2-
naphthol yielded 35 in 63% yield. Rf ) 0.73 (CH2Cl2:acetone
1
10:1). H NMR (CDCl3): [2.97 (d, J ) 7 Hz), 2.99 (d, J ) 6.2),
2H], 3.41 (t, J 1 ) 6.7 Hz, J 2 ) 7.8 Hz, 2H), 4.00 (s, 3H), 7.23
(d, J ) 2.6 Hz, 1H), 7.27 (d, J ) 9.1 Hz, 1H), 7.31 (dd, J 1 ) 8.8
Hz, J 2 ) 2.8 Hz, 1H), 7.73 (d, J ) 8.8 Hz, 1H), 7.75 (d, J ) 9.1
Hz, 1H).
1,2-Dih yd r o-3H-9-h yd r oxyn a p h th o[2,1-b]p yr a n -3-on e
(37) a n d 1,2,11,12-Tetr a h yd r o-3H,10H-n a p h th o[2,1-b:7,8-
b′]d ip yr a n -3,10-d ion e (39). Friedel-Crafts alkylation (8 h
of reflux) using 2,7-dihydroxynaphthalene afforded 37 (69%)
and 39 (11%) after chromatographic purification (CH2Cl2/
acetone 10:1). Compound 37: Rf ) 0.4 (CH2Cl2:acetone 10:1).
1H NMR (DMSO-d6): 2.87 (d, J ) 7.2 Hz, 1H), 2.90 (d, J )
6.2 Hz, 1H), 3.19 (t, J 1 ) 7.0 Hz, J 2 ) 7.8 Hz, 2H), 6.99 (d, J
) 8.8 Hz, 1H), 7.03 (dd, J 1 ) 8.8 Hz, J 2 ) 2.3 Hz, 1H), 7.14 (d,

2644 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
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J ) 2.3 Hz, 1H), 7.71 (d, J ) 8.8 Hz, 1H), 7.76 (d, J ) 8.8 Hz,
1H), 9.89 (s, 1H). Compound 39. Rf ) 0.75 (CH2Cl2:acetone
10:1). 1H NMR (CDCl3): [2.74 (d, J ) 6.0 Hz), 2.77 (d, J ) 7.2
Hz), 2H], 3.50 (t, J 1 ) 7.0 Hz, J 2 ) 7.8 Hz, 2H), 7.22 (d, J )
8.8, 2H), 7.74 (d, J ) 8.8 Hz, 2H).
1,2-Dih yd r o-3H -9-m e t h oxyn a p h t h o[2,1-b]p yr a n -3-
on e (38). Friedel-Crafts alkylation with 7-methoxy-2-naph-
thol yielded 38 (51%); a trace of 39 (8% yield) was also isolated.
Compound 38: Rf ) 0.55 (EtOAc:hexanes 1:1). 1H NMR
(CDCl3): [3.05 (d, J ) 7.0 Hz), 3.08 (d, J ) 6.2 Hz), 2H], 3.45
(t, J 1 ) 7.0 Hz, J 2 ) 7.8 Hz, 2H), 4.08 (s, 3H), 7.23 (d, J ) 8.8
Hz, 1H), [overlapping signals 7.27 (dd, J 1 ) 9.67 Hz, J 2 ) 2.3
Hz, 1H), 7.26 (d,1H)], 7.82 (d, J ) 8.8 Hz, 1H), 7.88 (d, J ) 9.6
Hz, 1H).
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(40). Friedel-Crafts alkylation of 1,5-dihydroxy naphthalene
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2H), 3.08-3.15 (m, 2H), 6.86 (d of d, J 1 ) 7.7 Hz, J 2 ) 0.9 Hz,
1H), 7.22-7.32 (m, 2H), 7.53 (d, J ) 8.6 Hz, 1H), 7.90 (d, J )
8.6 Hz, 1H), 9.07 (s, 1H).
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Ack n ow led gm en t. We thank the laboratory of Drs.
J ohn Slattery and Danny Shen at the Fred Hutchinson
Cancer Research Center for use of their mass spectrom-
etry equipment. This work was funded in part by the
Chromosome Metabolism and Cancer Training Grant
(T32 CA09657) (J .P.), the Ellison Medical Foundation
New Scholar Award (A.B.), and RO1CA 878746 (J .A.S.).
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
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available free of charge via the Internet at http://pubs.acs.org.
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