Structure-activity relationships of substituted 1-pyridyl-2-phenyl-1,2- ethanediones: Potent, selective carboxylesterase inhibitors

Article abstract of DOI:10.1021/jm101101q

Inhibition of intestinal carboxylesterases may allow modification of the pharmacokinetics/pharmacodynamic profile of existing drugs by altering half-life or toxicity. Since previously identified diarylethane-1,2-dione inhibitors are decidedly hydrophobic,

Full text of DOI:10.1021/jm101101q

J. Med. Chem. 2010, 53, 8709–8715 8709
DOI: 10.1021/jm101101q
Structure-Activity Relationships of Substituted 1-Pyridyl-2-phenyl-1,2-ethanediones: Potent,
Selective Carboxylesterase Inhibitors
Brandon M. Young, Janice L. Hyatt, David C. Bouck,† Taosheng Chen, Parimala Hanumesh, Jeanine Price, Vincent A. Boyd,
Philip M. Potter, and Thomas R. Webb*
Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, Tennessee 38105,
United States. †Current address: Millennium: The Takeda Oncology Company, 75 Sidney Street, Cambridge, MA 02139.
Received August 24, 2010
Inhibition of intestinal carboxylesterases may allow modification of the pharmacokinetics/pharmacodynamic
profile of existing drugs by altering half-life or toxicity. Since previously identified diarylethane-
1,2-dione inhibitors are decidedly hydrophobic, a modified dione scaffold was designed and elaborated
into a >300 member library, which was subsequently screened to establish the SAR for esterase
inhibition. This allowed the identification of single digit nanomolar hiCE inhibitors that showed
improvement in selectivity and measured solubility.
Introduction
Benzil can inhibit hiCE intracellularly resulting in reduced
sensitivity of cells to 1.9 However, the active site of CEs is
located at the base of a deep hydrophobic pocket, and
efficacious inhibitors also tend to be highly lipophilic and
water insoluble.10 We wished to determine if the diarylethane-
1,2-dione scaffold could be modified in such a way as to
maintain or increase compound potency while simultaneously
allowing for improved solubility relative to benzil. Since the
introduction of a polar pyridine group would be expected to
enhance solubility and bioavailability, we felt the most pro-
mising strategy involved replacing one of the phenyl groups
of benzil with a pyridine group. Furthermore, we planned
to introduce a synthetically accessible functional group for
both aryl rings of the 1-phenyl-2-pyridyl-1,2-ethanedione that
would also serve as sites for diversification with the appro-
priate reagents. A survey of available synthons led to the
introduction of an amino group onto the 1-phenyl-2-pyridyl-
1,2-ethanedione scaffold in our design, which could be elabo-
rated into an amide library through parallel synthesis to
maintain potency toward CE inhibition but have improved
solubility relative to benzil, as determined by solubility mea-
surements of active compounds.
Carboxylesterases (CEsa) are widely expressed enzymes
believed to be responsible for hydrolysis of xenobiotics in the
body by catalyzing the hydrolysis of esters to the correspond-
ing alcohols and carboxylic acids.1 Since it is known that a
number of important pharmaceutical agents are substrates for
metabolism by CEs, it is apparent that compounds that
modulate the activity of these enzymes may be useful in
altering the half-life and/or toxicities that are associated with
these drugs. The conversion of antitumor prodrug 1 (CPT-11)
to 2 (SN-38), a potent topoisomerase I poison, (Figure 1) is an
example of drug activation in vivo and is likely mediated by
the human intestinal CE (hiCE, CES2), an enzyme primarily
expressed in the liver and duodenum.2,3 A second CE, hCE1
(CES1), is expressed in human liver, but this enzyme demon-
strates poor kinetic properties toward 1 and it is unclear
whether hCE1 significantly contributes to drug activation in
humans.2,3 The dose limiting toxicity for 1 is diarrhea that
likely arises in part from the direct hydrolysis of 1 in the
intestine by hiCE. This is due to the drug being excreted into
the bile and hence deposited into the duodenum. Developing
specific inhibitors of hiCE that could potentially ameliorate
toxicity of 1 would have broad clinical utility, improving
patient quality of care while potentially allowing for the
administration of higher and more effective doses.
Chemistry
Our synthetic plan is outlined in Scheme 1 and was based on
intermediate preparation step efficiency, with the intermedi-
ates subjected to diversification via amide coupling, then oxida-
tion, all in a parallel format. We began by exploring the
Sonogashira coupling conditions for 3a with 4b to give 6a. We
found that standard conditions (catalytic Pd(PPh3)2Cl2 and
CuI) did provide the desired product; however, the product
was frequently contaminated with unidentified impurities that
coeluted during chromatography. In an effort to improve the
yield and purity of 6a, we then examined conditions described
by Fu and Buchwald for the Sonogashira coupling of aryl
bromides at room temperature using Pd(PhCN)2Cl2, CuI,
We have recently identified several different chemical classes
of selective CE inhibitors, including bisbenzene sulfonamides,
isatins, trifluoroketones, and ethane-1,2-diones.4-8 In general,
these compounds are potent (Ki as low as 5 nM) and demon-
strate no cross reactivity with acetyl- or butyrylcholinesterase.
*To whom correspondence should be addressed. Phone: 901-595-3928.
Fax: 901-595-5715. E-mail: thomas.webb@stjude.org.
a Abbreviations: AChE, acetylcholinesterase; BChE, butyrylcholin-
esterase; CE, carboxylesterase; HBTU, O-(benzotriazol-1-yl)-N,N,N0,
N0-tetramethyluronium hexafluorophosphate; hCE1 (CES1), human
carboxylesterase 1; hiCE (CES2), human intestinal carboxylesterase;
MAOS, microwave assisted organic synthesis; NaHMDS, sodium bis-
(trimethylsilyl)amide; SAR, structure-activity relationship.
t
and Bu3P.11,12 These conditions worked exceptionally well,
r
2010 American Chemical Society
Published on Web 11/24/2010
pubs.acs.org/jmc

8710 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Young et al.
Figure 1. Chemical structure and hydrolysis of 1 resulting in the formation of 2.
Scheme 1. Synthesis of 1-Pyridyl-2-(aminophenyl)ethane-1,2-dionesa
a Reagents and conditions: (a) 2% CuI, 3% Pd(PhCN)2Cl2, 6% tBu3P HBF4, 1.2 equiv of iPr2NH, dioxane, 25 °C, 3 h; (b) 1.5 equiv of AcCl, 2.0 equiv
3
of Et3N, THF, 0 °C; (c) NaHMDS, Boc2O, THF, 0-25 °C; (d) KMnO4, Na2SO4, NaHCO3, aqueous acetone; (d) AcCl, MeOH, 0 °C.
proceeding rapidly (3 h at 25 °C or within 30 min at 80 °C) and
giving 6a in 86% yield. We also found that these conditions
work well on larger scales (the preparation of 6a could be
scaled up to a 60 mmol scale without compromising yield or
purity).
anhydrous HCl resulted in the production of 15c, cleanly in
60% yield.19 15c was fully characterized as derivatives, since
this aminoketone was not found to be stable to concentration
and storage (presumably because of polymerization). We
found that 15c could be used as an intermediate if the CH2Cl2
extracts were routinely rapidly concentrated following aqu-
eous workup and stored as a 1 M DMF solution.
With this general experimental protocol available, we then
began the synthesis of the remaining aminophenyl positional
isomers (14c, 16-19c) in order to broaden the scope of our
SAR studies. We did find a limitation on the scope of this
scheme in that these conditions did not afford the desired
dione intermediate in the case of 4-bromopyridine 3c. This
reaction sequence produced 12a, 12c, and 21b, but attempts to
deprotect 21b failed to yield useful amounts of 21c, presum-
ably because of accelerated polymerization. Recognizing this
scope limitation, we excluded 20-22c from our plans for
library design. We also excluded 20-amino scaffolds 14c and
17c after a screen of Boc protected compounds 14-19b
showed that compounds 14b and 17b were inactive at 30 μM
when screened against hiCE.
We then turned our attention to the scale-up of the required
1-(aminopyridyl)-2-phenylethane-1,2-dione intermediates
(Scheme 2). The details of this approach take advantage
of insights gained during the preceding synthetic scope and
limitation study. Commercially available aminobromo-
pyridines 23-27a were protected as their Boc derivatives 23-27b,
then coupled using the Buchwald-Fu Sonogashira coupling to
give 23-27c in good yield (68-85%). The Sonogashira products
were then cleanly oxidized to the give the Boc protected diketones
28-32a in 40-65% yield. In general the Boc protecting group
removal for 28-32a was slow at room temperature and required
several hours at reflux to generate the desired products 28-32b
that were then stored as 1 M solutions in DMF.
With these coupling conditions in hand, we prepared6b as a
model compound to examine the feasibility of a parallel
oxidation reaction. At the time of our investigation, several
methods had been published for the oxidation of diaryl-
alkynes into ethane-1,2-diones; however, all required harsh
oxidants and/or conditions, and most of these reports did
not elucidate the scope and limitations of functional group
compatibility with the reported reaction conditions.13-16 Of
these methods, we initially chose conditions using KMnO4
described by Walsh and Mandal to determine suitability for
adaptation of these conditions into a parallel format.17 Though
the oxidation successfully produced the desired dione 15a,
there were several concerns. The reaction was exceedingly fast
at both 0 and 25 °C with substantial loss of desired product,
due to overoxidation, if the reaction was not promptly quenched.
Additionally, the quench with sodium bisulfite solution
resulted in an exotherm and gas evolution that could create an
overpressure resulting in overflow or, in a sealed format, a
possible explosion.
At this initial stage, the parallel oxidation under these condi-
tions appeared impractical, so the synthesis was modified to
produce intermediates 14-22b that would then be diversified
in the final step. This required the use of an amino protecting
group that was stable to the KMnO4 oxidation, yet easily
removed in a subsequent step. For this purpose we chose to
introduce the Boc protecting group by treating 6a with Boc
anhydride and NaHMDS (1 M in THF) to give 6c in 78%
yield.18 The Boc derivative 6c was then oxidized to give the
desired Boc protected ethane-1,2-dione 15b in 60% yield.
Though TFA mediated deprotection (even in the presence
of triethylsilane)19 failed to deliver 15c (due to decomposition
of product under these conditions), we found that the use of
Having prepared the desired intermediates, we then
designed a library that used a selection of diverse acid chloride
building blocks for the generation of new amides to probe the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8711
Scheme 2. Synthesis of 1-(Aminopyridyl)-2-phenylethane-1,2-dionesa
t
i
a Reagents and conditions: (a) Boc2O, NaHMDS, THF 0-25 °C, 16 h; (b) 2% CuI, 3% Pd(PhCN)2Cl2, 6% Bu3P HBF4, 1.2 equiv of Pr2NH,
3
1.1 equiv of phenylacetylene, dioxane 25 °C, 3 h; (c) KMnO4, Na2SO4, NaHCO3, aqueous acetone; (d) AcCl, MeOH, 80 °C.
Scheme 3. Preparation of the CE Inhibitor Librarya
a R1 comprised a diverse selection of alkyl, aryl, and heteroaryl groups. See Supporting Information for data on individual library members.
effect of substitution on CE potency (see Scheme 3). The
parallel synthesis was undertaken in 48 position XT Mini-
block reaction synthesizer blocks. A range of primary, sec-
ondary, and tertiary aliphatic compounds and aromatic and
heteroaromatic acid chlorides were successfully utilized with
this protocol with the exception of the acid chlorides of
nicotinic and isonicotinic acid. Compounds were isolated by
preparative reverse phase HPLC purification and submitted
for screening following completion of our in-house quality
control protocols. Eighty-two percent of the targeted com-
pounds in the 308-member library were obtained with g85%
purity in g1 mg quantity.
aromatic amide analogues were identified with IC50 values
below 350 nM (Table 1) and nine additional analogues with
sub-600 nM IC50 values. Overall, aliphatic amides of 15c were
much less potent than aromatic amides, although an increase
in chain length (and therefore hydrophobicity) was associated
with increased potency, as shown in Table 1. (Assay data
for all library members can be found in the Supporting
Information.)
At this point in the project we became aware of a report of a
diarylalkyne oxidation protocol mediated by catalytic amounts
of PdBr2 and PdI2 at elevated temperature in DMSO.23 This
report was especially intriguing to us, since the reaction condi-
tions appeared compatible with several functional groups on
the diarylalkynes, including amides. The authors also noted
1-pyridyl-2-arylalkynes oxidized under these conditions but
required increased reaction time to effect conversion. We
believed this procedure would allow us to affect our original
experimental design, a parallel oxidation following intermedi-
ate diversification, so we chose to examine this potentially
efficient alternative scheme.
With 6d as a test substrate, a pilot study was performed
using 10 mol % PdBr2 at 140 °C for 9 h. Despite producing the
desired dione 15d, a thick emulsion formed during the aqu-
eous workup requiring filtration through Celite to facilitate
extraction. Though this difficulty was easily handled on a
preparative scale; the emulsion posed a serious barrier for
library purification by reverse phase HPLC. In an attempt to
circumvent the emulsion problem, we turned to microwave
assisted organic synthesis (MAOS).24,25 The application of
MAOS has steadily increased over the past decade because of
its increased heating efficiency that allows for rapid reactions
Results and Discussion
We then assessed the ability of these compounds to inhibit
hiCE and hCE1 using a series of spectrophotometric assays.20-22
In the initial screen IC50 values were measured (Table 1), and
on the basis of these results, a set of the most potent com-
pounds were then subjected to Ki determination (Table 2).
Analysis of the resulting IC50 values revealed a defined
SAR selectivity pattern. No library members utilizing the
1-(aminopyridyl)-2-phenylethanedione scaffolds 28-32 ex-
hibited an IC50 potency below 2.5 μM, with the majority of
these analogues being poorly active or inactive in our assay.
Similar results were seen from library members derived from
18c and 19c. Fortunately, better results were observed with
scaffolds 15c and 16c. Compounds based on 16c incorporat-
ing a thiophene based amide yielded several submicromolar
inhibitors. The most potent compounds that we identified at
this point were obtained from scaffold 15c. Ten substituted

8712 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Young et al.
Table 1. Assay IC50 Results for hiCE of Selected Aminopyridylphenylethane-1,2-dioneamidesa
a Data obtained from primary screen.
Table 2. Selectivity Profile of 15d-m against hiCE, hCE1, AChE, and BChE Including Measured Solubility and cLogP
Ki (nM)a
compd
hiCE
hCE1
AChE (nM)
BChE (nM)
solubility (μg/mL)b
cLogP
15d
15e
15f
15g
15h
15i
1.48 ( 0.15
4.65 ( 0.4
1.17 ( 0.12
2.86 ( 0.19
2.73 ( 0.14
3.68 ( 0.4
4.67 ( 0.39
1.55 ( 0.16
3.23 ( 0.17
3.6 ( 0.23
279 ( 45
>100000
437 ( 61
1034 ( 145
1244 ( 155
>100000
>100000
756 ( 92
>100000
381 ( 90
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
3.3
19.6
0.5
8.4
0.7
16
4.51
3.17
4.12
3.67
3.59
3.02
3.17
3.88
3.20
3.39
15j
13.7
3.1
14.1
5.1
15k
15l
15m
a Measured Ki ( standard error obtained from resynthesized, fully characterized compounds. b Solubility measured at pH 4.0.
with the potential for fewer side reactions. Since the alkyne
oxidation required elevated temperatures for good conver-
sion, we explored the use of microwave irradiation to shorten
the reaction time and suppress formation of the (unknown)
emulsifying agents. We were gratified to find that the oxida-
tion of 6d was complete within 3 min at 200 °C and that the
emulsion formation was significantly reduced during aqueous
workup. The emulsion was further minimized by switching
from ethyl acetate to chloroform for the extractive workups.
By use of this protocol, 15d was isolated in 58% yield, fol-
lowing purification on silica gel.
To examine the suitability of these new conditions for
parallel synthesis, we applied this new approach to the
resynthesis (see Scheme 4) of the 10 most potent compounds
identified in our original screen. Thus, 6a was acylated under
standard conditions with the required acid chlorides to pro-
duce 6d-m in excellent yield (g85%). In one case (6f) it was
necessary to use the corresponding carboxylic acid in the
presence of HBTU in DMF to give the desired compound,
which proceeded in excellent (92%) yield. This result is
notable in that 15c failed to acylate using this type of coupling
conditions, which necessitated the use of more reactive acid
chlorides for ourlibraryproduction. Thoughwidely available,
the number of commercially available acid chlorides is signi-
ficantly smaller than that of commercially available carboxy-
lic acids. This substantially expands the scope of this scheme.
Following purification, 6d-m were taken up in DMSO,
treated with PdBr2, and sequentially brought to 200 °C, using
microwave heating in a Biotage Initiator. Upon cooling, the
compounds werethen subjected toa parallel aqueous workup.
Following CHCl3 extraction, the crude products were con-
centrated and purified on silica to give the desired products
15d-m in 46-71% yield. Subsequent determination of Ki for
these compounds closely recapitulated the results of our
original screen and increased our confidence in the SAR
trends observed.
In the previous examination of diarylethane-1,2-diones as
CE inhibitors, it was noted that inhibitory potency increased
with cLogP measurements. After performing cLogP calcula-
tions for the library members derived from scaffold 15,26 we
measured the solubility of compounds 15d-m at pH 4.0 and
7.4 and noted some interesting correlations (Table 2 and
Supporting Information Figure 1). While the scaffold 15
library did generally follow the same trend of increased
potency with higher cLogP values that has also been reported
with other scaffolds,27 several important differences to this

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8713
Scheme 4. Parallel Synthesis of 1-Pyridyl-2-(aminophenyl)ethane-1,2-diones via Microwave Irradiationa
a Reagents and conditions: (a) 0.125 mmol of 6b, 1.3 equiv of acid chloride, 1.5 equiv of Et3N, THF; (b) 1.2 equiv of carboxylic acid, 1.3 equiv of HBTU,
1.5 equiv of Et3N, DMF, 25 °C; (c) 10 mol % PdBr2, DMSO, 200 °C, microwave heating. See Table 1 for R1 groups.
correlation were observed. Interestingly these diketones show
a pronounced difference in slope of logKi versus cLogP
between the two esterases (hiCE and hCE), whereas the
previously reported sulfonamide inhibitors show an essen-
tially identical correlation slope (see Supporting Information
Figure 1). It is also fortuitous that the slopes of logKi versus
measured solubility show a favorable difference in slope (see
Supporting Information Figure 1). The “outliers” were also of
interest, such as 15e, which has a cLogP of 3.17 and 6 times the
measured solubility (19.6 μg/mL vs 3.3 μg/mL) of our most
potent compound 15d which hasa cLogPof 4.51. Compounds
15i and 15l were also identified as potent analogues with
cLogP values of 3 with good measured solubilities of 16.0 and
14.1 μg/mL, respectively. Thus, we established that it is pos-
sible to maintain potent activity and high selectivity while
improving solubility with this particular scaffold.
We further determined the selectivity of 15d-m versus
hCE1, acetylcholinesterase (AChE), and butyrylcholinesterase
(BChE). As shown in Table 2, all 10 compounds were inactive
against AChE and BChE. Compounds 15d and 15f were
shown to be the most potent inhibitors of hCE1 among our
10 most active analogues and showed the desired selectivity
(100-fold or more) in favor of hiCE inhibition. It is also of
interest to note that all four analogues with measured solubi-
lities greater than 10 μg/mL were inactive against hCE1.
Graphical analysis of the Ki for the 1,2-diones vs either their
cLogP or aqueous solubilities indicated good correlation
coefficients (see Supporting Information Figure 1). Interest-
ingly, better correlations were observed with the former
parameter with hiCE, and the latter with hCE1. We were
surprised by the r2 values that were obtained by these analyses
(as high as 0.83), considering the relatively small data sets that
were analyzed. While it is apparent that the active sites for
CEs are highly hydrophobic, the reasons for the differences
observed between the two human enzymes are unclear. More
importantly, however, these analyses could provide param-
eters that will guide the design and synthesis of novel inhibitors
with additional improvements in selectivity and potency.
In summary, we have prepared a number of amino-
substituted 1-phenyl-2-pyridylethane-1,2-dione scaffolds, which
were readily diversified using commercially available acid
chlorides. This library produced a clear SAR pointing to a
single scaffold (15) that when properly modified gave potent
(single digit nanomolar), selective, and relatively soluble hiCE
inhibitors. The top 10 active analogues identified in the
primary screen were synthesized on a preparative scale using
an efficient microwave assisted protocol that reduces the
number of required intermediate preparatory steps and allows
the introduction of carboxylic acids as a point of diversity for
future libraries. The measured Ki for these compounds con-
firmed our initial screening results, and further profiling
showed that they have significant selectivity for hiCE versus
hCE1, AChE, and BChE. Several of these compounds also
gave notable improvements in measured solubility relative to
benzil, confirming that the diarylethane-1,2-dione scaffold
can be elaborated into a highly potent and selective leadlike
compound class. Additionally we have uncovered a trend in
the SAR that allows us to couple improvements in solubility
and selectivity over hCE1. With aromatic amides of scaffold
15 as a starting point, we plan to use these observations to
prepare compounds for eventual in vivo experiments and will
report these results in due course.
Experimental Section
General Methods. All reagents and anhydrous solvents were
purchased from Sigma-Aldrich or Acros Organics and used as
received. Reactions were set up in air and carried out under
nitrogen atmosphere. Parallel synthesis was accomplished using
MiniBlock XT synthesizers (purchased from Mettler-Toledo
AutoChem) placed on a stirring hot plate. Intermediates were
prepared using standard glassware purchased from Chemglass
or in glass microwave vials with inert septa-aluminum crimp
caps purchased from Biotage or Chemglass. Flash chromatog-
raphy was carried out on prepacked silica cartridges using a
Biotage SP4 or Biotage Isolera chromatography system. 1H and
13C NMR spectra were recorded on a Bruker-400 MHz spectro-
meter at 400 and 101 MHz respectively. Prepurification and QC
analysis was performed on a Waters Acquity UPLC/PDA/
ELSD/MS system using a BEH C18 2.1 mm ꢀ 50 mm column
with a 90:10 to 5:95 0.1% aqueous formic acid/acetonitrile 2 min
gradient elution method. Library purification was performed
using a Dionex mass directed HPLC purification system using a
Phenomenex Gemini Aixia packed C18 30 mm ꢀ 50 mm, 5 μm
column using either 0.1% aqueous formic acid/acetonitrile or
0.1% aqueous ammonium bicarbonate/acetonitrile gradient
adjusted based on prepurification results. Fully characterized
compounds have purities of g95%; purity values for library
members are contained in the Supporting Information.
3-(Pyridine-2-ylethynyl)aniline (6a). In a 20 mL glass micro-
wave vial, CuI (0.019 g, 0.1 mmol, 0.02 equiv), Pd(PhCN)2Cl2
(0.057 g, 0.15 mmol, 0.03 equiv), and Bu3PHBF4 (0.087 g,
t
0.3 mmol, 0.06 equiv) were combined; the vial was sealed with an
aluminum crimp cap, evacuated, and placed under a nitrogen
atmosphere. The mixture was diluted with 15 mL of anhydrous
1,4-dioxane followed by 2-bromopyridine (0.5 mL, 5.00 mmol,
1 equiv), 3-ethynylaniline (0.70 g, 6.00 mmol, 1.2 equiv), and
iPr2NH (1.4 mL, 10 mmol, 2 equiv). The mixture was then
stirred at 25 °C until the starting materials were consumed as
observed by TLC. This was visually indicated by the precipita-
tion of Pr2NH-HBr. The crimp cap was removed and the thick
slurry diluted with EtOAc and transferred to a fritted funnel
containing Celite and filtered. The filtrate was concentrated and
the residue purified by chromatography on silica gel using a
0-60% EtOAc/hexane gradient to give 0.72 g of 6a in 74% yield
as an off white solid.
tert-Butyl (3-(Pyridin-2-ylethynyl)phenyl)carbamate (6c). In a
100 mL round-bottom flask under nitrogen, 6a (0.971 g, 5.0 mmol,
1 equiv) and di-tert-butyl dicarbonate (1.20 g, 5.5 mmol, 1.1 equiv)
were combined and dissolved in 10 mL of anhydrous THF. The
mixture was cooled to 0 °C and treated with NaHMDS (1 M in
THF, 10.5 mmol, 2.1 equiv) dropwise over 20 min. The mixture
was allowed to slowly warm to 25 °C overnight, then treated
with 30 mL of saturated NH4Cl. The mixture was extracted with

8714 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Young et al.
EtOAc (3 ꢀ 20 mL), and the combined organics were washed
with saturated NaCl, dried with MgSO4, filtered, and concen-
trated. The residue was purified by chromatography on silica gel
using a 0-40% EtOAc/hexane gradient to give 1.14 g of 6c in
78% yield as an off white solid.
logistics and screening analysis: Anang Shelat, Ph.D., Peter
Shu, and Hong Ruan. We thank Jimmy Cui from the High
Throughput Screening Center for setting up the automation
protocol. B.M.Y. thanks John Reichwein, Ph.D., for helpful
discussions. This work was supported in part by NIH Grant
CA108775, National Cancer Institute Grant P30CA027165,
and the American Lebanese Syrian Associated Charities
(ALSAC), St. Jude Children’s Research Hospital.
tert-Butyl (3-(2-Oxo-2-(pyridin-2-yl)acetyl)phenyl)carbamate
(15b). In a 250 mL round-bottom, 6c (1.47 g, 5 mmol, 1 equiv)
was dissolved in acetone (60 mL) at 25 °C and treated with a
0.22% NaHCO3/2.2% MgSO4 aqueous solution (30 mL).
KMnO4 (1.97 g, 12.5 mmol, 2.5 equiv) was added portionwise
over 5 min to ensure dissolution in the vigorously stirred
solution. After the indicated period, the reaction was quenched
by dropwise addition of 50% aqueous NaHSO3 (30 mL) fol-
lowed by stirring for 1 h. The milky suspension was filtered
through a fritted filter containing Celite. The filtrate was
extracted with EtOAc (3 ꢀ 25 mL), and the combined organics
were washed with saturated NaCl, dried with MgSO4, filtered,
and concentrated. The residue was purified by chromatography
on silica gel using a 0-40% EtOAc/hexane gradient to give 0.98 g
of 15b in 60% yield as a yellow solid.
1-(3-Aminophenyl)-2-(pyridin-2-yl)ethane-1,2-dione (15c). In
a 100 mL round-bottom flask under nitrogen, anhydrous MeOH
(20 mL) was cooled to 0 °C and treated with acetyl chloride (7.96 mL,
112 mmol, 10 equiv) over 30 min. After 15 min, the resulting
solution was transferred (via cannula or syringe) to a separate
250 mL round-bottom flask that contained a solution of 15b
(3.63 g, 11.15 mmol, 1 equiv) in anhydrous MeOH under nitro-
gen at 25 °C. The mixture was stirred until the starting material
was consumed as judged by TLC or LCMS. The reaction was
quenched by the slow addition of saturated NaHCO3 (100 mL).
The mixture was extracted with CH2Cl2 (3 ꢀ 30 mL), and the
combined extracts were washed with saturated NaCl, dried with
MgSO4, filtered, and concentrated to give 1.51 g of 15c as an
orange oil in 60% yield. The oil was immediately taken up in
6.7 mL of DMF to prepare a 1 M stock solution.
Procedure for the Acylation of Amino(phenyl, pyridyl)-1,2-
ethane Dione Library. A 48 position Mettler-Toldeo Miniblock
XT containing 11.5 mm ꢀ 110 mm reaction tubes with stir bars
was charged with 75 μL of the desired amino(phenyl, pyridyl)-
1,2-ethanedione (0.075 mmol), 1 M DMF stock solution, fol-
lowed by Et3N (16 μL, 0.113 mmol, 1.5 equiv). The reaction
block was topped with an inert atmosphere manifold and purged
with nitrogen. The vessels were then treated with 100 μL of the
appropriate acid chloride stock solution (1 M in THF) and
allowed to stir overnight at 25 °C. 2.5 mL of saturated NH4Cl
solution was added to each vessel and mixture extracted with
EtOAc (2.5 mL). The organic extracts were transferred to a
Genevac 16 mm ꢀ 100 mm test tube rack and concentrated. The
residue was dissolved in DMSO (1.75 mL) and transferred to a
2 mL Thomson filter plate (25 μm), prepacked with Celite, con-
nected to a Waters 96-well 2 mL collection plate. Upon filtra-
tion, the collection plate was analyzed by UPLC and product
bearing wells purified via reverse phase chromatography. Final
purity was measured by the total wavelength current (TWC)
from λ = 210-400 nm on UPLC.
Supporting Information Available: General synthetic methods,
characterization data for all intermediates and potent ana-
logues, analytical data for amide library, and assay description.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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