Biotinylated biphenyl ketone-containing 2,4-dioxobutanoic acids designed as HIV-1 integrase photoaffinity ligands

Article abstract of DOI:10.1016/j.bmc.2006.07.064

The diketo acid (DKA) class of HIV-1 integrase inhibitors are thought to function by chelating divalent metal ions within the enzyme catalytic center. However, differences in mutations conferring resistance among sub-families of DKA inhibitors suggest tha

Full text of DOI:10.1016/j.bmc.2006.07.064

Bioorganic & Medicinal Chemistry 14 (2006) 7816–7825
Biotinylated biphenyl ketone-containing 2,4-dioxobutanoic
acids designed as HIV-1 integrase photoaffinity ligands
Xue Zhi Zhao,a Elena A. Semenova,b Chenzhong Liao,a Marc Nicklaus,a
Yves Pommierb and Terrence R. Burke, Jr.a,*
aLaboratory of Medicinal Chemistry, CCR, NCI, NIH, Frederick, MD 21702, USA
bLaboratory of Molecular Pharmacology, CCR, NCI, NIH, Bethesda, MD 20892, USA
Received 16 June 2006; revised 28 July 2006; accepted 29 July 2006
Available online 26 September 2006
Abstract—The diketo acid (DKA) class of HIV-1 integrase inhibitors are thought to function by chelating divalent metal ions within
the enzyme catalytic center. However, differences in mutations conferring resistance among sub-families of DKA inhibitors suggest
that multiple binding orientations may exist. In order to facilitate identification of DKA-binding sites, biotin-tagged biphenyl
ketone-containing 2,4-dioxobutanoic acids were prepared as DKA photoaffinity probes. Introduction of biotin was obtained by
means of Huisgen [3+2] cycloaddition ‘click chemistry.’ Two photoprobes, 5a and 5b, were prepared bearing short and long linker
segments, respectively, between the biotin and DKA nucleus. The greatest inhibitory potency was shown by 5b, which inhibited 30-
processing and strand transfer reactions with IC50 values of >333 lM and 12.4 lM, respectively. In cross-linking assays designed to
measure disruption of substrate DNA binding, the photoprobes behaved similarly to a reference DKA inhibitor. Analogues 5a and
5b represent novel photoaffinity ligands, which may be useful in clarifying the HIV-1 binding interactions of DKA inhibitors.
Published by Elsevier Ltd.
1. Introduction
18 and 2,9 and non-carboxylic acid-containing analogues
such as 5-CITEP (3)10 as well as a variety of other ana-
logues that contain imbedded heteroatoms, which re-
place aspects of the dioxobutanoic acid functionality
(4,11 Fig. 1). Evidence suggests that these inhibitors che-
late divalent Mg2+ ions that are held in the IN active site
by a conserved triad of acidic residues Asp64, Asp116,
and Glu152, termed the ‘DDE’ motif.2,12 In spite of
their shared structural features and proposed common
mechanism of action, data from resistant mutants13 as
Integrase (IN) is an essential enzyme in the viral life cy-
cle of HIV-1, the causative agent of acquired immuno-
deficiency syndrome (AIDS).1 IN functions by
catalyzing the insertion of virally transcribed DNA into
the host genome in a two-stage process. In the first step,
termed ‘30-processing’ (30-P), a GT dinucleotide pair is
cleaved from the viral cDNA 30-terminus. Placement
of this processed cDNA into the host genome occurs
in a second step referred to as ‘strand transfer’ (ST).2
Inhibitors of IN have been the object of intense develop-
ment due to their potential utility as new therapeutics
for the treatment of AIDS.2–5 Although a large number
of structurally diverse IN inhibitors have been reported,
in cellular HIV infection assays many of these fail to
provide cytoprotection or target processes other than
integration.6 An exception is provided by diketo acid
inhibitors (DKA), which can afford good antiviral effica-
cy and exhibit potent inhibition of IN in whole cells as
well as in extracellular assays.7 DKA inhibitors are
exemplified by 4-aryl-2,4-dioxobutanoic acids such as
O
O
O
O
OH
OH
N3
O
O
1
2
Cl
O
OH
O
O
H
N
N
N
H
N
N
N
F
N
N
O
H
N
3
4
S
O
Keywords: HIV-1 integrase; Inhibitor; Biotin; Photoaffinity.
*
Figure 1. DKA class HIV-1 IN inhibitors with functionally equivalent
diketo acid-derived substructures in bold.
Corresponding author. Tel.: +1 301 846 5906; fax: +1 301 846
6033; e-mail: tburke@helix.nih.gov
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2006.07.064

X. Z. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 7816–7825
7817
well as computational studies14 indicate that distinct
binding sites may be involved with different subclasses
of DKA inhibitors. Accordingly, direct determination
of DKA-binding sites under physiologically relevant
conditions is needed.
at some distance from the photophore functionality.23,24
However, DKA photoprobes are structurally simple and
can consist of nothing more than a biphenyl ketone moi-
ety and a diketo acid side chain.19 For such compounds,
introduction of biotin through a linker onto the 4-posi-
tion of the BP ring system results in trifunctional ana-
logues of type 5 (Fig. 2). We envisioned construction
of the necessary ‘linker’ segments using Huisgen [3+2]
azide–alkyne cycloaddition-mediated ‘click chemis-
try.’25,26 Similar methodology has proven useful in the
biotin tagging of other photophore-containing ligands.27
Based on this approach, targets 5a and 5b were de-
signed, which differ in the length of their cycloaddi-
tion-derived triazole-containing linker segments (Fig. 2).
Photoaffinity labeling represents a highly useful means of
identifying sites of molecular interactions.15 This tech-
nique relies on incorporation into the ligand of interest
of chemical functionality that is inert until specific light
activation. Among several potential photo-reactive
groups that include diazirines and aryl azides, the benzo-
phenone moiety (BP) has proven to be advantageous16 be-
cause of its chemical stability and unreactivity toward
ambient light (activation is achieved at wavelengths in
the range 350–360 nm). Additionally, once photo-activat-
ed BP groups react preferentially with C–H bonds, even
under aqueous conditions.
Preparation of 5a and 5b required the synthesis of the
common intermediate azide 13 (Scheme 1). The route
began by coupling commercially available 4-methyl-
benzaldehyde (6) with 1,3-dibromobenzene (7) in the
presence of n-BuLi to yield the biphenylmethyl alcohol
8 (Scheme 1).8 Further reaction with n-BuLi followed
by addition of N-methoxy-N-methylacetamide provided
the methyl ketone 9. This was subjected to Collins oxi-
dation to give the diketone 10 (59% overall yield from
6).28,29 Heating 10 with N-bromosuccinimide (NBS) in
the presence of dibenzoyl peroxide30 resulted in selective
benzylic bromination to provide intermediate 11. This
was reacted with sodium azide to yield 12 (60% yield
from 10). Condensation of the aryl methyl ketone 12
with diethyl oxalate using either lithium bis(trimethylsi-
lyl)amide (LHMDS) in THF at ꢀ20 ꢀC or sodium hy-
dride in toluene at reflux31 gave the diketo ester 13 in
32% yield following HPLC purification. The photo-
affinity probe 14 was prepared by deprotecting ester 13
using lithium hydroxide in THF and H2O. The interme-
diate 10 was converted to the diketo acid ester 15 and
hydrolyzed to yield the photoprobe 16 (Scheme 2).
We have previously reported the incorporation of BP
groups into coumarin-based IN inhibitors17 and these
agents have recently been used to identify a putative cou-
marin binding site on the IN protein.18 Using a similar ap-
proach we set out to identify binding sites associated with
the DKA inhibitors. For this purpose, we designed
photoprobes that utilized a BP moiety both as part of
the photophore and simultaneously as a critical compo-
nent of the DKA pharmacophore.19 However, the use
of these inhibitors as photoaffinity ligands is potentially
limited by low cross-linking efficiency that results in high
background noise. Such drawbacks could be overcome by
including biotin as part of the photoaffinity ligand to facil-
itate selective purification of covalent adducts.15 Biotin
tagging of phenylazide,20 diazirine,21 and BP-containing
photoprobes22–24 has proven to be useful in other systems.
In the current report, we detail the application of biotin
tagging to the design and synthesis of BP-containing
DKA photoprobes as potential tools for identifying bind-
ing sites of DKA-based IN inhibitors.
The commercially available N-hydroxysuccinimide ac-
tive ester of biotin (17a)32 was dissolved in DMF and
to this was added neat propargylamine followed by an
aqueous solution of sodium bicarbonate to afford the
alkyne 18a (Scheme 3). Similar reaction using the
chain-extended analogue 17b32 gave the corresponding
propargylamide 18b. Initial attempts at Huisgen Cu(I)-
catalyzed [3+2] cycloaddition of 18a with the azide-con-
taining b-diketoacid ethyl ester 13 in the absence of a
2. Results and discussion
2.1. Design and synthesis of biotin-tagged photoprobes
Recent examples of large biotin-tagged affinity ligands
have appeared in which the biotin residue is situated
Benzophenone Photophore
Biotin-Tag
O
O
OH
H
OH
O
O
N
H
Linker
5
O
HN
S
H
Diketo Acid
5a Linker =
5b Linker =
O
H
N
N
NH
N
N
N
NH
N
N
N
H
O
Figure 2. Structures of biotin-tagged BP-containing DKA photoprobes.

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X. Z. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 7816–7825
OH
Br
CHO
OH
Br
Br
b
a
+
8
6
7
O
O
O
d
c
9
10
O
O
O
O
f
e
Br
N3
11
12
O
O
OH
O
O
OH
OH
g
N3
OEt
O
N3
O
14
13
Scheme 1. Reagents and conditions: (a) 7, n-BuLi, THF, ꢀ78 ꢀC, then 6, ꢀ78 ꢀC to rt (79% yield); (b) n-BuLi, THF, ꢀ78 ꢀC, then N-methoxy-N-
methylacetamide, ꢀ78 ꢀC to rt (79% yield); (c) CrO3, pyridine, CH2Cl2, 0 ꢀC (94% yield); (d) NBS, benzoyl peroxide, CCl4, reflux (70% yield); (e)
NaN3, acetone/H2O (5:1), reflux (85% yield); (f) LHMDS, THF, ꢀ20 ꢀC, then (CO2Et)2 (32% yield); (g) LiOHÆH2O, THF/H2O, rt (85% yield).
O
O
OH
O
O
OH
OEt
OH
b
a
10
O
O
15
16
Scheme 2. Reagents and conditions: (a) LHMDS, THF, ꢀ78 ꢀC, then (CO2Et)2, (78% yield); (b) LiOHÆH2O, THF/H2O (82% yield).
H
N
O
O
O
H
O
a
N
N
n
O
HN
H
S
H
O
17a: n=0
17b: n=2
H
N
O
b
O
H
N
H
N
H
n
O
HN
S
H
18a: n=0 (64%)
18b: n=2 (67%)
O
O
OH
H
N
OR
O
N
O
N
N
H
N
H
O
N
H
n
O
HN
S
H
c
5a: (n=0); R = H (83%)
5b: (n=2); R = H (84%)
19a: n=0; R = Et (18%)
19b: n=2; R = Et (25%)
Scheme 3. Reagents and conditions: (a) propargylamine, NaHCO3 (aq), DMF; (b) 13, (+)-sodium L-ascorbate, CuSO4Æ5H2O, DIPEA, tBuOH/H2O,
rt; HPLC purification; (c) LiOHÆH2O THF/H2O; HPLC purification.
nitrogen base failed to give good results, even after
extended reaction times.26 However, inclusion of diiso-
propylethylamine (DIPEA) in the reaction mixture im-
proved the reaction33 and the 1,4-substituted triazole-
containing adduct 19a was obtained in 18% yield after
HPLC purification. Hydrolysis of the ethyl ester fol-
lowed by HPLC purification yielded the final biotin-
tagged DKA photoprobe 5a in 83% yield. A similar
series of reactions applied to the linker-elongated
propargylamide 18b gave the desired photoprobe 5b.

X. Z. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 7816–7825
7819
2.2. Evaluation of inhibitory potencies against HIV-1
integrase
to examine whether the biotin and linker appendages
unduly perturbed DKA binding. One characteristic of
many DKA inhibitors is the selective inhibition of ST
reactions relative to 30-P reactions. For compound 2,9
which represents a reference DKA lacking any BP func-
tionality or biotin-linker appendages, ST selectivity was
approximately 50-fold. High ST selectivity was also ob-
served for BP-containing DKA inhibitors 14 and 16,
which lack biotin and linker moieties. Biotinylated
photoprobe 5a bearing a relatively short linker segment
exhibited significantly reduced ST selectivity (approxi-
mately 3-fold selectivity). However, by increasing the
length of the linker segment, photoprobe 5b regained
the lost ST selectivity and underwent an increase in
inhibitory potency.
Synthetic compounds were examined for their ability to
inhibit HIV-1 IN using in vitro assays in the presence of
Mg2+ as the metal cofactor.34 [c-32P]-Labeled 21 base
pair (bp) oligonucleotide duplexes corresponding to
the terminal U5 sequence of the HIV-1 LTR were used
as substrate. Cleavage of two bases during 30-P resulted
in 19-bp products, while strand transfer products (STP)
were formed by covalent joining of the 19-bp sequences
into identical duplexes that further served as target
DNA. Reactions were run at 37 ꢀC in the presence of
various concentrations of inhibitors. Analysis was done
by gel electrophoresis and PhosphorImager visualiza-
tion. Bands corresponding to 19-bp 30-P products as
well as STP bands were quantitated and plotted to pro-
vide inhibition constants for both the 30-P and ST reac-
tions. A tabulation of IC50 values obtained in this
fashion is shown in Table 1.
Compound 5b exhibited ST inhibitory potency and
selectivity over 30P very similar to compound 16, which
was unencumbered by any biotin-linker appendage. This
potentially indicates that the biotin-linker functionality
in 5b does not unduly perturb binding. Additionally,
although the inhibitory potency of 5b (ST
IC50 = 12.4 lM) is reduced relative to the parent DKA
inhibitor 2 (ST IC50 = 0.76 lM), it is similar to the
Because the biotin-labeled photoprobes 5a and 5b were
designed to tag relevant DKA sites, it was of interest
potency
of
a
BP-containing
inhibitor
(ST
Table 1. Inhibitory potencies of synthetic analogues calculated from
in vitro HIV-1 integrase assays
IC50 = 10 lM)17 that was recently used to identify a
novel IN binding site.18
Compound
IC50 SEa (lM)
30-P
ST
2.3. Inhibitor effects on binding of DNA to integrase
2
43.6 27.3
>333
>333
0.76 0.24
14.0 9.4
33.7 18.9
40.7 23.3
12.4 5.7
16
14
5a
5b
In addition to changing ST selectivity, alterations in the
binding of the biotin-tagged photoprobes to IN may dif-
ferentially affect binding of DNA. To examine effects of
inhibitors on the binding of DNA within the catalytic
site, a disulfide cross-linking assay was run as depicted
in Figure 3. This assay utilized DNA substrate having
136.7 9.8
>333
a Values represent the averages of three independent experiments
conducted as described in Ref. 34.
Figure 3. Results of IN–DNA disulfide cross-linking assay performed as reported in Ref. 34. (Top left) Schematic representation showing HIV-1 IN
cysteine (Cys) residues. (Top right) Mutant IN along with cross-linking strategy. Asterisk represents the 50 32P-label. (Bottom) SDS–PAGE analysis
showing the effects of increasing concentrations of inhibitors (shown above the gel) on formation of cross-linked IN–DNA.

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X. Z. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 7816–7825
a 50-AC cystosine residue that was modified to include
thiol-reactive functionality appended to its N4 position.
Mutant IN was used, which contained a Q148C muta-
tion in the flexible loop proximal to the catalytic site.
Other reactive Cys residues within the IN protein were
converted to non-reactive Ser residues so that disulfide
cross-linking of the DNA only occurred with the
Cys148 residue.35 As shown in Figure 3, parent DKA
inhibitor 2 as well as 5a and 5b inhibit DNA–IN
cross-linking only at concentrations significantly higher
than their ST IC50 values.
to catalytic residues Asp64, Asp116, and Glu152.7 Con-
sistent with this, in the current study automated docking
of inhibitors preferentially situated them close to the ac-
tive site, even through a broad search region was em-
ployed and differences did exist between the highest
scored poses of inhibitors. It is widely assumed that
DKA inhibitors function by chelating one or two
Mg2+ ions required for productive catalysis. Figure 5
overlays the best scoring pose for 5a and 5b in which
the diketo acid groups chelate a Mg2+ ion. Figure 6 pro-
vides an overall visual inspection of 5a and 5b bound to
the full-length IN–DNA complex. We chose to present
these highest-scored poses even though they are quite
different looking and might suggest different binding
models for 5a and 5b at first glance. However, as is well
known, scores calculated by docking programs are at
best semi-quantitative and do not usually permit exact
reproduction of ranking of assayed compounds. There-
fore, we could have ‘hand-picked’ poses for 5a and 5b
that look much more similar to each other, albeit at
the cost of introducing our bias into the reported results.
A second experiment was run to measure effects of
inhibitors on overall binding of DNA to IN. The assay
employed DNA that mimicked the viral U5 LTR and
contained an abasic site corresponding to the adenine
in the conserved terminal CA-dinucleotide (Fig. 4).
Nitrogen nucleophiles within the IN protein can form
Schiff-bases with the abasic C10-hemiacetal. Subsequent
in situ reduction using NaBH4 results in irreversible
cross-linking of the DNA to the IN.36 Results of this as-
say indicated that the parent DKA inhibitor 2 as well as
the biotin-tagged photoprobes 5a and 5b behaved simi-
larly in not affecting the overall binding of DNA at con-
centrations within the range needed to inhibit ST
catalysis (Fig. 4). Significant inhibition of DNA binding
was observed for photoprobe 5a at 333 lM, however
this was well above its ST inhibitory concentration of
40.7 lM.
2.4. Molecular modeling studies
To examine whether reasonable binding modes to IN
were possible for 5a and 5b, docking calculations were
carried out using a structural model of the full-length
HIV-1 IN dimer complexed with donor DNA.37 As
pointed out above, mutations conferring resistance to
DKA inhibitors map to the enzyme active site proximal
Figure 5. Overlapped binding modes of 5a (khaki) and 5b (cyan).
Figure 4. Results of IN–DNA Schiff-base cross-linking assay as performed in reference 36. (Top) Assay schematic showing imine formation between
an abasic site in the DNA substrate and an amine nucleophile from the IN protein (left), with subsequent NaBH4 reduction to the amine cross-linked
product (right). Asterisk represents the 50 32P-label. (Bottom) SDS–PAGE showing effects of increasing inhibitor concentrations on formation of IN–
DNA cross-linked product.

X. Z. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 7816–7825
7821
3.2. Docking experiments
Docking studies of compounds listed in Table 1 were
performed on a structural model of the full-length
HIV-1 IN dimer complexed with an oligonucleotide cor-
responding to the U5 LTR.37 Experiments were per-
formed using the program Glide 4.0, which is part of
the First Discovery suite (Schro¨dinger Inc.).38 Coordi-
nates of the full-length substrate-complexed dimer were
prepared for Glide 4.0 calculations by running the First
Discovery suite scripts pprep and impref. The pprep
script produces a new receptor file in which all residues
are neutralized except those that are relatively close to
the ligand (if the protein is complexed with a ligand)
or form salt bridges. The impref script runs a series of
restrained impact energy minimizations using the Im-
pact utility. Minimizations were run until the average
root mean square deviation (rmsd) of the non-hydrogen
˚
atoms reached 0.3 A. In order to study the binding
modes of inhibitors in the presence of metal ions, grid
files were generated representing the shape and proper-
ties of the receptor including Mg2+ in the active site.
Glide uses two boxes that share a common center to
organize its calculations: a larger enclosing box and a
smaller binding box. The grids themselves are calculated
within the space defined by the enclosing box. The bind-
ing box defines the space through which the center of the
defined ligand will be allowed to move during docking
calculations. It provides a measure of the effective size
of the search space. The only requirement on the enclos-
ing box is that it be large enough to contain all ligand
atoms, even when the ligand center is placed at an edge
or vertex of the binding box. Grid files were generated
with catalytic residues Asp64, Asp116, and Glu152 at
the center of the two boxes. The size of the binding
Figure 6. Hypothetical binding of 5a (A) and 5b (B) to a model of full-
length IN complexed with donor DNA. Protein coloring is rendered in
gradations from blue to red proceeding from the N-terminus to the
C-terminus, respectively.
˚
box was set at 20 A in order to explore a large region
The key conclusion of these molecular modeling studies
is that inhibitors 5a and 5b can be reasonably accommo-
dated in the IN–DNA complex.
of the protein. The three-dimensional structures of the
compounds 2, 5a, 5b, 14, and 16 were constructed using
ISIS/Draw and imported into the Maestro interface. The
initial geometry of the structures was optimized using
the OPLS-2005 force field39 performing 1000 steps of
conjugate gradient minimization. The compounds were
subjected to flexible docking using the pre-computed
grid files. For each compound the 100 top-scored poses
were saved and analyzed.
In conclusion, the current work has resulted in biotin-
tagged BP-containing DKAs that were prepared using
Huisgen [3+2] cycloaddition ‘click chemistry.’ Two
photoprobes were obtained that differed in the length of
the segment joining the biotin to the BP ring system. Ana-
logue 5a, which contained a short linker segment, exhibit-
ed reduced selectivity toward ST inhibition relative to 30-P
and at higher doses it inhibited total DNA binding. Ana-
logue 5b contained a longer linker segment and it showed
selective inhibition of ST reactions that was comparable
to an analogue (16), which lacked any biotin or linker
functionality. Analogues 5a and 5b represent novel
photoaffinity probes that may be useful in studying the
binding of DKA inhibitors to IN. Such work is currently
in progress and will be reported elsewhere.
3.3. Chemistry
High-resolution mass spectra (HRMS) were obtained
from UCR Mass Spectrometry Facility, University of
California at Riverside and fast-atom bombardment
mass spectra (FABMS) were acquired with a VG Ana-
lytical 7070E mass spectrometer under the control of a
VG 2035 data system. FABMS matrixes used were glyc-
1
erol or nitrobenzoic acid. Both H and 13CNMR data
were obtained on a Varian 400 MHz instrument and
are reported in ppm relative to TMS and referenced to
the solvent in which they were run. IR spectra were
run neat using a Jasco FT/IR-615 instrument. Solvent
was removed by rotary evaporation under reduced pres-
sure and anhydrous solvents were obtained commercial-
ly and used without further drying. Preparative high-
pressure liquid chromatogaphy (HPLC) was conducted
3. Experimental
3.1. Biological evaluation
Experimental conditions for all biological procedures
have been previously reported.34

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X. Z. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 7816–7825
using a Waters Prep LC4000 system having photodiode
array detection and using a YMC J’sphere ODS-H80
column (250 · 20 mm ID, S-4 lm, 80 A) at a flow rate
of 10 mL/min. with binary solvent system consisting of
A = 0.1% aqueous TFA and B = 0.1% TFA in
acetonitrile.
though a column of neutral aluminum oxide and the fil-
trate was evaporated to dryness. Purification by silica
gel flash chromatography (hexanes/ethyl acetate) affor-
ded the desired diketone product 10 as a colorless crys-
˚
1
talline solid (545 mg, 94% yield): Mp 77.8 – 78.0 ꢀC. H
NMR (CDCl3) d 8.30 (m, 1H), 8.13–8.11 (m, 1H), 7.93–
7.91 (m, 1H), 7.69–7.66 (m, 2H), 7.56–7.52 (m, 1H),
7.27–7.24 (m, 2H), 2.60 (s, 3H), 2.41 (s, 3H). 13C
NMR (CDCl3) d 197.3, 195.5, 143.8, 138.4, 137.1,
134.3, 134.1, 131.5, 130.2, 129.6, 129.2, 128.6, 26.7,
21.6. FABMS (+ve) m/z 239 [MH+]. IR t (cmꢀ1) 1680,
1651, 1602, 1300, 923.
3.3.1. 3-Bromo-a-(4-methylphenyl)-benzenemethanol (8).
To an oven-dried 100 mL round bottom flask under ar-
gon was added a solution of n-butyl lithium 1.6 M in hex-
anes (5.53 mL, 8.85 mmol), and the solution was cooled
(ꢀ78 ꢀC) and diluted with anhydrous THF (20 mL). To
this was added dropwise over 5 min 1,3-dibromobenzene
(1.0 mL, 8.04 mmol) and the reaction mixture was stirred
(2.5 h at ꢀ78 ꢀC) then 4-methylbenzaldehyde (1.05 mL,
8.84 mmol) was added over 15 min and the reaction mix-
ture was slowly warmed to room temperature (1.5 h). The
reaction mixture was quenched by the addition of ice-cold
H2O (20 mL) and extracted with ether (3· 100 mL). The
combined organic extract was washed with saturated
aqueous NaHCO3 and brine then dried over anhydrous
Na2SO4 and evaporated to yield a colorless oil. Purifica-
tion by silica gel flash chromatography (hexanes/ethyl
acetate) afforded bromide 8 as a colorless oil (1.86 g,
79% yield). 1H NMR (CDCl3) d 7.53 (t, 1H, J = 1.4 Hz),
7.37 (dt, 1H, J = 1.2 Hz, 6.8 Hz), 7.25 (dd, 1H,
J = 8.0 Hz), 7.20–7.12 (m, 5H), 5.67 (s, 1H), 2.51 (br s,
1H), 2.33 (s, 3H). 13C NMR (CDCl3) d 146.1, 140.3,
137.6, 130.3, 129.9, 129.3 (3C), 126.5 (2C), 125.0, 122.5,
75.3, 21.1. FABMS (+ve) m/z 275, 277 [MH+ꢀH2].
3.3.4.
1-[3-[4-(Bromomethyl)benzoyl]phenyl]-ethanone
(11). A suspension of 10 (1.38 g, 5.80 mmol), N-bromo-
succinimide (NBS) (1.24 g, 6.97 mmol), and benzoyl
peroxide (42 mg, 0.17 mmol) in anhydrous carbon tet-
rachloride (150 mL) was refluxed under argon (24 h).
The reaction mixture was cooled to room temperature
and filtered, the filter pad was washed with carbon tet-
rachloride (2· 30 mL), and the combined filtrate was
evaporated. The resulting residue was purified on silica
gel flash chromatography to provide 11 as a colorless
1
oil (1.29 g, 70% yield). H NMR (CDCl3) d 8.27 (m,
1H), 8.11–8.08 (m, 1H), 7.89–7.86 (m, 1H), 7.72–7.68
(m, 2H), 7.53–7.45 (m, 1H), 7.44–7.43 (m, 2H), 4.45
(s, 2H), 2.56 (s, 3H). 13C NMR (CDCl3) d: 197.0,
194.8, 142.4, 137.6, 137.0, 136.6, 133.9, 131.7, 130.3
(2C), 130.1, 129.3, 129.0 (2C), 32.0, 26.6. FABMS
(+ve) m/z 317, 319 [MH+]. IR t (cmꢀ1) 1730, 1685,
1656, 1603, 1236.
3.3.2. 1-[3-[Hydroxy(4-methylphenyl)methyl]phenyl]-eth-
anone (9). To a solution of n-butyl lithium 1.6 M in hex-
anes (0.59 mL, 0.94 mmol) at ꢀ78 ꢀC under argon was
added a solution of 8 (125 mg, 0.42 mmol) in anhydrous
THF (4 mL). The reaction mixture was stirred at ꢀ78 ꢀC
for 1 h, then N-methoxy-N-methylacetamide (59 lL,
0.56 mmol) was added and the reaction mixture was
slowly warmed to room temperature over 1 h. The reac-
tion mixture was quenched by the addition of ice-cold
H2O (10 mL) then extracted with ether (3· 30 mL).
The combined organic extract was washed with saturat-
ed aqueous NaHCO3and brine then dried over anhy-
drous Na2SO4 and evaporated to yield a colorless oil.
Purification by silica gel flash chromatography (hex-
anes/ethyl acetate) afforded 9 as a colorless oil (80 mg,
3.3.5. 1-[3-[4-(Azidomethyl)benzoyl]phenyl]-ethanone (12).
Sodium azide (391 mg, 6.02 mmol) was added to a solu-
tion of 11 (954 mg, 3.01 mmol) in acetone (15 mL) with
H2O (3 mL) and the mixture was stirred at reflux (over-
night). The mixture was cooled to room temperature,
concentrated, and the residue was purified by silica gel
flash chromatography to provide 12 as a colorless oil
(711 mg, 85% yield). 1H NMR (CDCl3) d 8.31 (m,
1H), 8.16–8.13 (m, 1H), 7.95–7.92 (m, 1H), 7.79–7.77
(m, 2H), 7.58–7.53 (m, 1H), 7.43–7.41 (m, 2H), 4.45 (s,
2H), 2.60 (s, 3H). 13C NMR (CDCl3) d 197.1, 195.2,
140.4, 137.8, 137.2, 136.7, 134.1, 131.8, 130.5 (2C),
129.5, 128.8, 128.0 (2C), 54.2, 26.7. FABMS (+ve) m/z
280 [MH+]. IR t (cmꢀ1) 2097 (N3), 1685 (C@O), 1657,
1595, 1237.
1
79% yield). H NMR (CDCl3) d 7.97 (m, 1H), 7.81–
7.78 (m, 1H), 7.55–7.52 (m, 1H), 7.40–7.32 (m, 1H),
7.23–7.20 (m, 2H), 7.12–7.10 (m, 2H), 5.81 (s, 1H),
2.54 (s, 3H), 2.29 (s, 3H). 13C NMR (CDCl3) d 198.2,
144.5, 140.5, 137.6, 137.2, 131.1, 129.3 (2C), 128.6,
127.3, 126.5 (2C), 126.1, 75.6, 26.6, 21.1. FABMS
(+ve) m/z 241 [MH+].
3.3.6. 4-[3-[4-(Azidomethyl)benzoyl]phenyl]-2-hydroxy-4-
oxo-2-butenoic acid ethyl ester (13). To a stirred solution
of lithium bis(trimethylsilyl)amide (LHMDS), 1.0 M in
THF (2.48 mL, 2.48 mmol) at ꢀ20 ꢀC was added drop-
wise a solution of 12 (230 mg, 0.82 mmol) in anhydrous
THF and the reaction mixture was stirred (1 h at 20 ꢀC),
then diethyl oxalate (224 lL, 1.65 mmol) was added.
The reaction mixture was slowly warmed to room tem-
perature (3 h) then quenched with ice-cold H2O
(20 mL), extracted with chloroform (3· 100 mL), and
the combined organic extract was washed with dilute
aqueous HCl then brine, dried over anhydrous Na2SO4.
Filtration followed by evaporation gave a yellow oil,
which was subjected to HPLC purification (linear gradi-
ent of 40% B to 100% B over 35 min; retention
3.3.3.
1-[3-(4-Methylbenzoyl]phenyl)-ethanone
(10).
Chromium(III) oxide (1.47 g, 14.7 mmol) was added in
several portions to a solution of pyridine (2.38 mL,
29.5 mmol) in anhydrous dichloromethane (40 mL) at
0 ꢀC and the mixture was stirred at 0 ꢀC (20 min). To
this was added quickly a solution of 9 (588 mg,
2.45 mmol) in anhydrous dichloromethane (10 mL)
and the mixture was stirred at room temperature
(10 min). The resulting black product was filtered

X. Z. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 7816–7825
7823
time = 30.6 min) to provide 13 as a white solid following
lyophilization (100 mg, 32% yield). H NMR (CDCl3) d
d6) d 8.29 (dd, 1H, J = 1.2, 8.0 Hz), 8.20 (d, 1H,
J = 1.2 Hz), 7.94 (d, 1H,J = 7.6 Hz), 7.70 (t, 1H,
J = 7.6 Hz), 7.64 (d, 2H, J = 8.4 Hz), 7.35 (d, 2H,
J = 8.4 Hz), 7.05 (s, 1H), 2.38 (s, 3H). FABMS (ꢀve)
m/z 309 [(MꢀH)ꢀ]. HRMS calcd for C18H14O5[Mꢀ]:
310.0836. Found: 310.0849.
1
8.36 (m, 1H), 8.20–8.18 (m, 1H), 8.00–7.97 (m, 1H),
7.82–7.79 (m, 2H), 7.63 (t, 1H, J = 7.6 Hz), 7.45 (d,
2H, J = 8.0 Hz), 7.07 (s, 1H), 4.45 (s, 2H), 4.38 (qt,
2H, J = 7.2 Hz), 1.38 (t, 3H, J = 7.2 Hz). 13C NMR
(CDCl3) d 194.9, 189.5, 170.4, 161.9, 140.6, 138.1,
136.6, 135.2, 134.6, 131.4, 130.5 (2C), 129.1, 129.0,
128.0 (2C), 98.0, 62.7, 54.2, 14.0. FABMS (ꢀve) m/z
378 [(MꢀH)ꢀ]. HRMS calcd for C20H18N3O5 [MH+]:
380.1246. Found: 380.1234. IR t (cmꢀ1) 2100 (N3),
1732 (C@O), 1703, 1665, 1595.
3.3.10. Biotin propargylamide (18a). To a solution of (+)-
biotin N-hydroxysuccinimide active ester (17a)31 (78 mg,
0.23 mmol) in DMF (1.0 mL) was added propargyl-
amine (31 lL, 0.45 mmol) and the solution was stirred
at room temperature (24 h). The solution was concen-
trated and subjected directly to preparative HPLC puri-
fication (linear gradient of 5% B to 40% B over 25 min;
retention time = 29.2 min) to afford 18a as a white solid
3.3.7. 4-[3-[4-(Azidomethyl)benzoyl]phenyl]-2-hydroxy-4-
oxo-2-butenoic acid (14). To a solution of ethyl ester 13
(25 mg, 0.07 mmol) in THF (800 lL) with H2O (400 lL)
was added lithium hydroxide monohydrate (5 mg,
0.16 mmol). The reaction mixture was stirred at room
temperature (1 h) then concentrated under reduced pres-
sure and subjected directly to HPLC purification (linear
gradient of 40% B to 75% B over 30 min; retention
time = 22.3 min) to provide 14 as a white solid following
lyophilization (15 mg, 65% yield). 1H NMR (DMSO-d6)
d 8.30 (d, 1H, J = 8.0 Hz), 8.24 (s, 1H), 7.96 (d,
1H,J = 8.8 Hz), 7.76 (d, 2H, J = 8.0 Hz), 7.71 (t, 1H,
J = 7.6 Hz), 7.53 (d, 2H, J = 8.0 Hz), 7.01 (s, 1H), 4.58
(s, 2H). FABMS (ꢀve) m/z 350 [(MꢀH)ꢀ].
1
following lyophilization (42 mg, 64% yield). H NMR
(DMSO-d6) d 8.18 (t, 1H, J = 5.4 Hz), 6.37 (s, 1H),
6.30 (s, 1H), 4.25 (dd, 1H, J = 5.2 Hz, 7.6 Hz), 4.09–
4.05 (m, 1H), 3.77 (dd, 2H, J = 2.4 Hz, 5.2 Hz), 3.07–
3.04 (m, 1H), 3.03 (t, 1H, J = 2.4 Hz), 2.77 (dd, 1H,
J = 5.2 Hz, 12.4 Hz), 2.52 (d, 1H, J = 12.4 Hz), 2.03 (t,
2H, J = 7.2 Hz), 1.56–1.53 (m, 1H), 1.47–1.42 (m, 3H),
1.28–1.24 (m, 2H). FABMS (+ve) m/z 282 [MH+].
HRMS calcd for C13H20N3O2S [MH+]: 282.1276.
Found: 282.1275.
3.3.11. Biotinamidohexanoyl-6-amino-hexanoyl propa-
rgylamide (18b). To biotinamidohexanoyl-6-amino-hex-
anoic acid N-hydroxysuccinimide active ester (17b)32
(50 mg, 0.088 mmol) in DMF (2.0 mL) were added
propargylamine (112 lL, 1.64 mmol) and saturated
aqueous sodium bicarbonate solution (0.5 mL), and
the reaction mixture was stirred at room temperature
(2 days). The mixture was concentrated and directly
purified by preparative HPLC (isocratic 5% B over
10 min then a linear gradient from 5% B to 40% B over
25 min; retention time = 32.4 min) to give 18b as a white
solid following lyophilization (30 mg, 67% yield). 1H
NMR (DMSO-d6) d 8.16 (s, 1H), 7.65 (br s, 2H), 6.36
(s, 1H), 6.30 (s, 1H), 4.25 (br s, 1H), 4.08 (br s, 1H),
3.78 (dd, 2H, J = 2.0 Hz, 5.2 Hz), 3.05–3.03 (m, 1H),
3.02 (t, 1H, J = 2.4 Hz), 2.95–2.94 (m, 4H), 2.77 (dd,
1H, J = 4.8 Hz, 12.4 Hz), 2.53 (d, 1H, J = 12.4 Hz),
2.03–1.95 (m, 6H), 1.60–1.52 (m, 1H), 1.48–1.38 (m,
7H), 1.31–1.25 (m, 6H), 1.17–1.16 (m, 4H). FABMS
(+ve) m/z 508 [MH+]. HRMS calcd for C25H42N5O4S
[MH+]: 508.2958. Found: 508.2977.
3.3.8. 2-Hydroxy-4-[3-(4-methylbenzoyl)phenyl]-4-oxo-2-
butenoic acid ethyl ester (15). To a stirred solution of
LHMDS, 1.0 M in THF (3.0 mL, 3.0 mmol) at ꢀ78 ꢀC
was added dropwise a solution of ketone 10 (240 mg,
1.0 mmol) in anhydrous THF. The resulting mixture
was stirred (1 h at ꢀ78 ꢀC) then diethyl oxalate
(271 lL, 2.0 mmol) was added and the reaction mixture
was slowly warmed to room temperature (3 h). The reac-
tion mixture was quenched with ice-cold H2O (20 mL)
and extracted with chloroform (3· 80 mL). The com-
bined organic extract was washed with dilute aqueous
HCl then brine, dried over anhydrous Na2SO4, and
evaporated. Purification by silica gel flash chromatogra-
phy using chloroform/methanol afforded product 15 as a
1
colorless oil (266 mg, 78% yield). H NMR (CDCl3) d
8.33 (t, 1H, J = 1.6 Hz), 8.16 (m, 1H), 7.96 (m, 2H),
7.68 (d, 2H, J = 8.0 Hz), 7.59 (t, 1H, J = 8.0 Hz), 7.27
(d, 2H, J = 8.0 Hz), 7.05 (s, 1H), 4.36 (qt, 2H,
J = 7.2 Hz), 2.41 (s, 3H), 1.37 (t, 3H, J = 7.2 Hz). 13C
NMR (CDCl3) d 195.2, 189.6, 170.3, 161.9, 143.9,
138.7, 135.0, 134.6, 134.1, 131.0, 130.2 (2C), 129.2
(2C), 129.0, 128.9, 98.0, 62.7, 21.7, 14.0. FABMS
(+ve) m/z 339 [MH+]. IR t (cmꢀ1) 3033, 2862, 1731,
1655, 1604.
3.3.12. Biotinylated photoprobe ethyl ester (19a). To
azide-containing DKA13 (16 mg, 0.042 mmol) and bio-
tin-containing 18a (16 mg, 0.057) in tert-butanol
(0.1 mL) and N,N-diisopropylethylamine (DIPEA)
(0.1 mL) were added freshly prepared ascorbate, 1.0 M
in H2O, (0.8 mL, 0.8 mmol) and copper sulfate pentahy-
drate, 0.3 M in H2O (0.3 mL, 0.09 mmol). The heteroge-
neous mixture was stirred vigorously at room
temperature (24 h), then concentrated under reduced
pressure and subjected to preparative HPLC purifica-
tion (linear gradient of 5% B to 100% B over 35 min;
retention time = 25.2 min) to provide 19a as a white sol-
3.3.9. 2-Hydroxy-4-[3-(4-methylbenzoyl)phenyl]-4-oxo-2-
butenoic acid (16). To a solution of ethyl ester 15 (59 mg,
0.17 mmol) in THF (800 lL) with H2O (400 lL) was
added lithium hydroxide monohydrate (15 mg,
0.47 mmol). The reaction mixture was stirred at room
temperature (1 h), then concentrated under reduced
pressure and purified directly by preparative HPLC (lin-
ear gradient of 40% B to 70% B over 30 min; retention
time = 24.6 min) to afford 16 as a white solid following
1
id following lyophilization (5 mg, 18% yield). H NMR
(CDCl3) d 8.34 (t, 1H, J = 1.6 Hz), 8.18 (dt, 1H,
J = 1.6 Hz, 8.4 Hz), 7.96 (dt, 1H, J = 1.6 Hz, 6.0 Hz),
1
lyophilization (42 mg, 82% yield). H NMR (DMSO-

7824
X. Z. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 7816–7825
7.76 (d, 2H, J = 8.4 Hz), 7.72 (s, 1H), 7.62 (t, 1H,
J = 7.8 Hz), 7.38 (d, 2H, J = 8.4 Hz), 7.06 (s, 1H), 6.96
(t, 1H, J = 5.6 Hz), 5.58 (d, 2H, J = 3.6 Hz), 4.58–4.51
(m, 2 H), 4.39 (t, 2H, J = 8.0 Hz), 4.38 (dd, 2H,
J = 7.2 Hz, 14.4 Hz), 3.13 (dd, 1H, J = 4.8 Hz, 7.6 Hz),
2.91 (dd, 1H, J = 4.8 Hz, 13.4 Hz), 2.73 (d, 1H,
J = 13.4 Hz), 2.28–2.17 (m, 2H), 1.68–1.55 (m, 2H),
1.44–1.40 (m, 2H), 1.38 (t, 3H, J = 7.2 Hz), 0.84–0.81
(m, 2H). FABMS (+ve) m/z 661 [MH+]. FABMS
(ꢀve) m/z 659 [(MꢀH)ꢀ]. HRMS calcd for
C33H37N6O7S [MH+]: 661.2439. Found: 661.2415.
4.09–4.05 (m, 1H), 3.06–3.01 (m, 1H), 2.94–2.93 (m,
4H), 2.76 (dd, 1H, J = 5.2 Hz, 12.8 Hz), 2.52 (d, 1H,
J = 12.8 Hz), 2.04–1.94 (m, 6H), 1.55–1.53 (m, 1H),
1.43–1.38 (m, 7H), 1.34–1.23 (m, 6H), 1.19–1.14 (m,
4H). FABMS (ꢀve) m/z 857 [(MꢀH)ꢀ]. HRMS calcd
for C43H54N8O9NaS [M+Na+]: 881.3627. Found:
881.3628.
Acknowledgments
The authors thank Drs. James A. Kelley and Christo-
pher Lai of the Laboratory of Medicinal Chemistry,
NCI, for mass spectral data. This research was support-
ed in part by the Intramural Research Program of the
NIH, Center for Cancer Research, National Cancer
Institute.
3.3.13. Biotinylated photoprobe ethyl ester (19b). Reac-
tion of biotin-containing 18b (19 mg, 0.037 mmol) and
azide-containing DKA 13 in a fashion similar to that
reported for the synthesis of 19a followed by preparative
HPLC purification (linear gradient of 5% B to 65% B
over 35 min; retention time = 31.4 min) gave 19b as a
white solid following lyophilization (8 mg, 25% yield).
1H NMR (DMSO-d6) d 8.32 (d, 1H, J = 8.0 Hz), 8.23
(m, 1H), 7.98 (s, 1H), 7.96 (d, 1H, J = 8.0 Hz), 7.72–
7.66 (m, 3H), 7.42 (d, 2H, J = 8.4 Hz), 7.10 (s, 1H),
6.36 (br s, 2H), 5.67 (s, 2H), 4.30–4.24 (m, 5H), 4.09–
4.05 (m, 1H), 3.04–3.03 (m, 1H), 2.94–2.93 (m, 4H),
2.76 (dd, 1H, J = 5.2 Hz, 12.8 Hz), 2.52 (d, 1H,
J = 12.8 Hz), 2.04–2.01 (m, 2H), 2.00–1.94 (m, 4H),
1.58–1.53 (m, 1H), 1.45–1.39 (m, 7H), 1.34–1.21 (m,
6H), 1.26 (t, 3H, J = 6.8 Hz), 1.18–1.10 (m, 4H). FAB-
MS (+ve) m/z 887 [MH+]. HRMS calcd for
C45H58N8O9NaS [M+Na+]: 909.3940. Found: 909.3927.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2006.07.064.
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d 8.28 (d, 1H, J = 7.6 Hz), 8.19 (d, 1H, J = 7.6 Hz), 8.09
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