Design and evaluation of hydroxamate derivatives as metal-mediated inhibitors of a protein tyrosine kinase

Article abstract of DOI:10.1021/jm061058c

Protein tyrosine kinases use two Mg²⁺ ions as cofactors in catalysis, one as the ATP-Mg complex (M1) and the other as an essential activator (M2), The M2-binding site has high affinity for transition metal cations such as cobalt and zinc. Takin

Full text of DOI:10.1021/jm061058c

7532
J. Med. Chem. 2006, 49, 7532-7539
Design and Evaluation of Hydroxamate Derivatives as Metal-Mediated Inhibitors of a Protein
Tyrosine Kinase
Xianfeng Gu,†,# Yuehao Wang,# Anil Kumar,† Guofeng Ye,† Keykavous Parang,*,† and Gongqin Sun*,#
Department of Biomedical and Pharmaceutical Sciences and Department of Cell and Molecular Biology, UniVersity of Rhode Island,
Kingston, Rhode Island 02881
ReceiVed September 5, 2006
Protein tyrosine kinases use two Mg2+ ions as cofactors in catalysis, one as the ATP-Mg complex (M1)
and the other as an essential activator (M2). The M2-binding site has high affinity for transition metal
cations such as cobalt and zinc. Taking advantage of this high affinity, we examined hydroxamates as metal-
mediated inhibitors against C-terminal Src kinase (Csk), a protein tyrosine kinase. Of a small group of
amino acid hydroxamates, tyrosine and phenylalanine hydroxamates inhibited Csk activity only in the presence
of Co2+. Four classes of phenylalanine and tyrosine hydroxamate derivatives were synthesized and evaluated
as metal-mediated inhibitors of Csk, leading to improved inhibition and a better understanding of the
structure-activity relationships. This study suggests that hydroxamates may serve as a general scaffold for
developing metal-mediated inhibitors against protein tyrosine kinases. To the best of our knowledge, this is
the first report of designing metal-mediated inhibitors against a protein tyrosine kinase by targeting a metal
binding site.
Introduction
much higher affinity for the M2 binding site than Mg2+ does
(Kd ) 2.3 mM for Mg2+, 12 µM for Mn2+, 12 µM for Co2+
,
Protein tyrosine kinases (PTK) phosphorylate tyrosine hy-
droxyl groups in specific proteins.1 They are essential compo-
nents of signal transduction pathways that transmit extracellular
signals to intracellular targets. PTKs can be transiently activated
following signals for cell growth or differentiation.1 Aberrant
activation of specific PTKs is a key step in the initial
transformation, angiogenesis, and metastasis for many types of
cancers.2,3 PTK inhibitor development has become a major
approach for drug discovery.4 Most current PTK inhibitors target
the ATP-binding site. Although selective inhibitors competitive
with ATP have been identified for specific protein kinases,5,6
the discovery process is still labor-intensive because of the high
structural homology between the ATP-binding site of protein
kinases.7 Therefore, identification of functionally critical binding
sites and development of novel design strategies to target these
sites are important for the long-term success of PTK inhibitors
as successful drugs.
In addition to the Mg2+ (M1) bound to ATP to form the
ATP-Mg complex as the phosphate donating substrate, a
second Mg2+ (M2) is essential for PTK catalysis.8-10 All other
PTKs so far examined, including Src,8,11 FGFR,8 Yes,9 v-Fps
(a member of the Fps/Fes family),12 IRK,13 and EGFR,14 also
require M2 for activity. Crystal structure of IRK with catalytic
ligands confirmed the presence of two Mg2+ ions in the active
site.15 The cAMP-dependent protein kinase (PKA), a Ser/Thr
kinase, also binds to two Mg2+ in the active site, but the second
Mg2+ is inhibitory by reducing its kcat.16 It appears likely that
protein kinases require two Mg2+ ions for catalyzing the
phosphorylation reaction.
and 20 µM for Ni2+). Although Zn2+ could not serve as a Csk
activator, it binds to the M2-binding site with a Kd of 0.65 µM,
thus serving as a potent inhibitor competitive against M2.10 The
high affinity of Zn2+ for the Csk M2-binding site is exploited
for a single-step affinity purification of GST-Csk from total
bacterial extract on a Zn2+-iminodiacetic acid agarose column.17
The experiment demonstrates that Zn2+ could bind to the M2-
binding site of Csk and iminodiacetic acid simultaneously. This
result suggests that appropriate compounds can bind tightly to
Csk by targeting M2.
Metal chelation and coordination are much stronger interac-
tions than most of the weak interactions in biochemistry.
Incorporating such interactions to inhibitor design is a well-
established strategy.18-23 Cisplatin, the most effective drug
available today against several types of human cancers,19,20 and
hydroxamate-containing inhibitors against proteases21-23 all take
advantage of this tight binding. To the best of our knowledge,
no metal-mediated inhibitor against PTKs has been reported so
far.
We describe the evaluation of hydroxamate compounds as
metal-mediated PTK inhibitors. We first identified tyrosine
hydroxamate (TyrH) and phenylalanine hydroxamate (PheH)
as potent metal-mediated inhibitors from screening a group of
simple amino acid hydroxamates. A systematic effort was taken
to determine the structure-activity relationships and optimize
the inhibition. Four classes of PheH and TyrH derivatives were
synthesized: (1) analogues with modified hydroxamate func-
tional group (5b-f); (2) phenyl-substituted hydroxamate ana-
logues (17a-r); (3) analogues with modified linker between
hydroxamate and phenyl functional groups (18u,v); (4) TyrH
analogues with modified R-amino group (-NH2) (23a-d).
These studies established a new family of PTK inhibitors.
Although Mg2+ is the physiological cofactor, Mn2+, Co2+
,
and Ni2+ can also substitute Mg2+ for binding to M2 to support
PTK activity.10 Furthermore, Mn2+, Co2+, and Ni2+ all have
Results and Discussion
* To whom correspondence should be addressed. For K.P.: phone, 401-
874-4471; fax, 401-874-5787; e-mail, kparang@uri.edu. For G.S.: phone,
401-874-5937; fax, 401-874-2202; e-mail, gsun@uri.edu, kparang@uri.edu.
† Department of Biomedical and Pharmaceutical Sciences.
Chemistry. Scheme 1 shows the solid-phase synthesis of
tyrosine hydroxamate (5a) and thiohydroxamate (5b) in class
I. Hydroxylamine Wang resin (1) was reacted with Fmoc-Tyr-
# Department of Cell and Molecular Biology.
10.1021/jm061058c CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/08/2006

Metal-Mediated Inhibitors against Csk
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7533
Csk in the absence of Co2+ but inhibited Csk with an IC50 of
9.5 µM in the presence of 0.2 mM Co2+ (Figure 1A). PheH
exhibited similar Co2+-dependent inhibition patterns with an IC50
of 15.5 µM (data not shown). As controls, the amino acid Tyr
or Phe up to 1 mM did not inhibit Csk activity in the presence
or absence of 0.2 mM CoCl2. The requirement of both Co2+
and the hydroxamate group indicated that the inhibition was
dependent on Co2+ binding to the hydroxamate group.
Scheme 1. Solid-Phase Synthesis of Tyrosine Hydroxamate
(5a) and Thiohydroxamate (5b)a
Two mechanisms could explain the Co2+ dependence of the
inhibition. The first is based on Co2+ directly binding to Csk at
the M2-binding site and on the inhibitors binding to Co2+. An
alternative explanation would be that Co2+ and the inhibitors
formed some kind of complex(es), which inhibited Csk. This
second mechanism would not depend on Co2+ directly binding
to Csk. The results from two experiments strongly favored the
first possibility. In the first experiment, the inhibition of Csk
by 25 µM TyrH in the presence of 12 mM MgCl2 and various
concentrations of CoCl2 was determined (Figure 1B). TyrH
exhibited no inhibition in the absence of Co2+. As the
concentration of Co2+ increased, the level of inhibition by TyrH
a Reagents: (i) HBTU, NMM/DMF (0.4 M); (ii) Lawesson’s reagent,
THF; (iii) piperidine in DMF (20%); (iv) TFA/CH2Cl2 (70%).
(tBu)-OH (2) in the presence of HBTU and N-methylmorpholine
(NMM) to yield polymer-bound hydroxamate derivative (3a).
The reaction between 3a and 2,4-bis(4-methoxyphenyl)-[1,3,2,4]-
dithiadiphosphetane 2,4-disulfide (Lawesson’s reagent) in THF
for 3 days afforded polymer-bound thiohydroxamate derivative
3b. The deprotection of the Fmoc group in 3a and 3b with
piperidine followed by treatment with TFA afforded 5a and 5b,
respectively.
Scheme 2 displays the synthesis of N-methyl- and N-
benzyltyrosine hydroxamates (5c and 5d) in class I. Polymer-
bound Fmoc protected methyl ester tyrosine (8) was synthesized
from Wang resin (6) and Fmoc-Tyr(OH)-OMe using the
Mitsunobu reaction in the presence of triphenylphosphine (PPh3)
and diisopropylazodicarboxylate (DIAD). The deprotection of
Fmoc and methyl ester groups of 8 with NaOH (0.4 M) afforded
polymer-bound tyrosine 9. The subsequent reaction of 9 with
the N-methyl- or N-benzylhydroxylamine hydrochloride in the
presence of diisopropylcarbodiimide (DIC) and triethylamine
(Et3N) followed by cleavage using TFA afforded 5c and 5d,
respectively.
Scheme 3 displays the syntheses of compounds 17a-r and
18u,v in classes II and III, respectively. The syntheses of
polymer-bound compounds 13a-r and 14s,t were accomplished
by the reaction between hydroxylamine Wang resin (1) and
substituted Fmoc-Phe-OH derivatives (11a-r and 12s,t) in the
presence of HBTU. Deprotection of Fmoc group with piperidine
followed by cleavage with TFA gave 17a-r and 18u,v,
respectively.
Scheme 4 shows the procedure for the preparation of
compounds 23a-d in class IV. The hydroxyl groups in (S)-3-
(4-hydroxylphenyl)propionic acid derivatives (19a-d) were
protected by the acetyl group in the presence of acetic anhydride.
Tyrosine acetylated compounds 20a-d were reacted with
hydroxylamine Wang resin (1) in the presence of HBTU to give
21a-d. After removal of the Fmoc group in 21a with piperidine
(20%), the free amino group was reacted with benzyl bromide
(BnBr) in pyridine to afford 21a′. The deprotection of acetyl
group in 21a′ and 21b-d with sodium methoxide followed by
cleavage with TFA/DCM (70%) afforded 23a-d.
also increased, reaching about 80% at 0.2 mM Co2+. The Co2+
-
dependent inhibition by TyrH correlated to the binding of Co2+
to the M2 binding site predicted by the Kd (12 µM).
The second experiment compared the inhibition of Csk by
25 µM TyrH in the presence of different combinations of
divalent metal cations (Figure 1C). On the basis of the affinities
of metal cations for the M2 binding site, Co2+ occupies 90%
of the M2-binding site in the presence of 12 mM Mg2+ and 0.2
mM Co2+ but only 20% of the M2-binding site in the presence
of 2 mM Mn2+ and 0.2 mM Co2+. If the inhibition of Csk by
TyrH is dependent on Co2+ occupying the M2-binding site,
much higher inhibition would be expected in the Co2+-Mg2+
combination. On the other hand, if Co2+ and TyrH formed
inhibitory complex(es), the level of inhibition would be
independent of which divalent metal cation occupies the M2-
binding site. TyrH (25 µM) inhibited Csk about 80% in 12 mM
Mg2+ and 0.2 mM Co2+ (condition b of Figure 1C) but only
13% in the presence of 2 mM Mn2+ and 0.2 mM Co2+
(condition e). In the presence of 12 mM MgCl2 (condition a),
12 mM MgCl2 + 2 mM MnCl2 (condition c), and 2 mM MnCl2
(condition d), 25 mM TyrH did not inhibit Csk. These results
indicated that TyrH inhibition of Csk required Co2+ occupying
the M2-binding site and argued for the interpretation that TyrH
inhibited Csk as a metal-mediated inhibitor.
The inhibition of Csk by TyrH was noncompetitive against
ATP (data not shown), suggesting that TyrH did not interfere
with the binding of ATP-M1. When poly-E4Y was the variable
peptide substrate, the inhibition was mixed but leaning toward
competitive with a Ki of 2 µM, determined by the Dixon plot
(1/V versus [I]). The near competitiveness with poly-E4Y
suggested that TyrH binding site was close to or partially
overlapped with the peptide-binding site. TyrH also inhibited
Src and FGFR in a Co2+-dependent manner (Ki ) 18 µM for
Src; Ki ) 23 µM for FGFR in the presence of 12 mM MgCl2
and 0.2 mM CoCl2). These results suggested that hydroxamate
compounds could potentially be used as a general scaffold for
developing metal-mediated inhibitors against various PTKs.
TyrH inhibits Csk Activity in a Co2+-Dependent Manner.
To explore the possibility of developing metal-mediated PTK
inhibitors targeting the M2-binding site of Csk, a group of
commercially available amino acid hydroxamates were evalu-
ated. In the presence of 12 mM MgCl2, none of the hydrox-
amates at 25 µM inhibited Csk more than 5% (Table 1). When
0.2 mM CoCl2 in addition to 12 mM MgCl2 was present,
however, both TyrH and PheH inhibited Csk about 80%, while
ArgH, LysH, LeuH, NorvalH, and GlyH also inhibited Csk,
although to lesser extents. The addition of 0.2 mM Mn2+ or
Ni2+ did not lead to hydroxamate inhibition of Csk.
In order to further explore the structural requirements for
designing metal-mediated inhibitors against PTKs, four classes
of tyrosine and phenylalanine hydroxamate derivatives were
synthesized and evaluated for their potency to inhibit Csk in
the presence of cobalt:
The concentration-dependent inhibition of Csk by TyrH and
PheH was then determined. TyrH up to 1 mM did not inhibit
I. Analogues with Modified Hydroxamate Functional
Group. To understand the mechanisms of this metal-mediated

7534 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Gu et al.
Scheme 2. Synthesis of N-Methyl- and N-Benzyltyrosine Hydroxamates (5c and 5d)a
a Reagents: (i) DIAD, Ph3P, CH2Cl2; (ii) NaOH (0.4 M); (iii) N-methylhydroxylamine hydrochloride or N-benzylhydroxylamine hydrochloride, DIC,
Et3N, CH3CN; (iv) TFA/CH2Cl2 (70%).
Scheme 3. Synthesis of Phenylalanine and Tyrosine Hydroxamate Derivatives (17a-r and 18u,v)a
a Reagents: (i) HBTU, NMM/DMF (0.4 M); (ii) piperidine/DMF (20%); (iii) TFA/CH2Cl2 (70%).
Scheme 4. Synthesis of Tyrosine Hydroxamate Analogues with Modified R-Amino Group (-NH2) (23a-d)a
a Reagents: (i) Ac2O, pyridine; (ii) 1, HBTU, NMM/DMF (0.4 M); (iii) piperidine/DMF (20%); (iv) BnBr, pyridine; (v) NaOMe, MeOH; (vi) TFA/
CH2Cl2 (70%).
inhibition, the role of the hydroxamate group was first examined.
To further verify the importance of the hydroxamate functional
group and its role as the metal binding motif in M2 mediated
inhibition, the -CO, -NH, and -OH moieties of the hydrox-
amate group (-CO-NH-OH) were modified (Table 2).
When the hydroxamate group in 5a was replaced with
thiohydroxamate group in 5b, the inhibitory potency was
enhanced by approximately 4-fold, possibly because of the
stronger binding affinity of sulfur to metals. N-Alkylation or
N-arylation of the -NH group significantly reduced the
inhibitory potency toward Csk as shown in N-methyl- and
N-benzyltyrosine hydroxamate analogues (5c and 5d).
L-Tyrosine (5e), which contains an -OH group instead of a
-NHOH, did not inhibit Csk in the presence or the absence of
Co2+ even at the high concentration of 1 mM. Additionally,
replacement of the -OH group in the hydroxamate group by
-NH2 in L-tyrosine hydrazide (5f) reduced the inhibitory
potency by approximately 8-fold.
Structure-activity relationships by these analogues revealed
that moieties (-OH, -CO, or -NH) of the hydroxamate group
are required for binding to Co2+. These results further confirm
that the -NHOH group plays key roles in the inhibition process
involving the chelation with metal and/or interaction with the
target binding site.

Metal-Mediated Inhibitors against Csk
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7535
Table 1. Inhibition of Csk by Hydroxamates
Table 2. Inhibitory Potency of Class I Analogues Containing Modified
Hydroxamate Functional Groups in the Presence of Co2+ (0.2 mM)
inhibition of Csk, %
25 µM compd
12 mM MgCl2
12 mM MgCl2 + 0.2 mM CoCl2
none
L-HisH
L-ArgH
L-LysH
L-TyrH
L-LeuH
â-AlaH
DL-NorvalH
DL-Asp γ-H
L-Glu γ-monoH
DL-PheH
GlyH
0
0
0
3
0
4
0
0
0
0
0
0
0
2
36
26
83
62
7
40
0
0
77
45
a IC50 is the concentration required to produce 50% inhibition in
phosphorylation of poly-E4Y by Csk (average of triplicate experiments).
The upper limit of the standard error of the mean (SEM) was (10%. b No
inhibition up to 1000 µM.
Table 3. Inhibitory Potency of Phenyl-Substituted Analogues (Class II)
in the Presence of Co2+ (0.2 mM)
compd
R1
R2
R3
NH2
CN
N3
B(OH)2
NO2
H
CH3
CH(CH3)2
Ph
4-NO2Ph
3-NO2Ph
F
F
Cl
Br
I
R4
n
IC50 (µM)a
17a
17b
17c
17d
17e
17f
17g
17h
17i
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
I
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
I
H
H
H
H
H
H
H
H
H
H
H
H
F
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
2
47
28
21
34
7
15.5
9.5
3.3
12
4.9
6.1
11
17j
17k
17l
17m
17n
17o
17p
17q
17r
18u
18v
18
Figure 1. Characterization of Co2+-dependent inhibition of Csk by
L-TyrH. The data are the average of triplicate experiments. The upper
limit of the standard error of the mean (SEM) was (10%: (A) inhibition
pattern of Csk in the presence of variable concentrations of L-TyrH
and the presence or absence of Co2+; (B) Co2+ concentration-dependent
inhibition of Csk (l-TyrH ) 25 µM, MgCl2 ) 12 mM); (C) Csk
inhibition in the presence of different metals combinations, (a) 12 mM
MgCl2, (b) 12 mM Mg2+ and 0.2 mM Co2+, (c) 12 mM MgCl2 + 2
H
H
H
H
I
6.3
5.6
3.2
3.4
3.4
57.0
18.0
H
H
H
H
OH
OH
OH
H
H
H
H
a Average of triplicate experiments. The upper limit of the standard error
of the mean (SEM) was (10%.
mM MnCl2, (d) 2 mM MnCl2, (e) 2 mM Mn2+ and 0.2 mM Co2+
.
II. Phenyl-Substituted Hydroxamate Analogues. The in-
hibition of Csk by TyrH, PheH, and LeuH suggests that a
hydrophobic pocket on Csk near the M2 binding site is involved
in the binding to these inhibitors. To optimize this interaction,
different functional groups were introduced to the aryl ring of
PheH to probe the interactions of the aryl ring with such a
hydrophobic pocket. These substitution groups had different size,
polarity, and charge and allowed us to systematically probe the
binding environment of the aryl ring (Table 3).
Introducing polar groups (-NH2, -CN, -N3, -B(OH)2) at
the para position of the phenyl ring in 17a-d in phenylalanine
hydroxamates reduced the inhibitory potency (IC50 ) 21-47
µM). On the other hand, introducing alkyl groups (CH3, CH-
(CH3)2) at the para position of the phenyl ring in 17g and 17h
gradually enhanced the inhibitory potency compared to 17f
according to the volume of the alkyl group.
Similar to the alkyl-substituted compounds, introducing
bulkier halogens on the phenyl ring in 17n-p enhanced the

7536 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Gu et al.
Table 4. Inhibitory Potency of Tyrosine Hydroxamate Analogues
Containing a Modified R-Amino Group (Class IV) in the Presence of
Co2+ (0.2 mM)
Furthermore, the replacement of R-amino group in TyrH with
-OH and -H completely eliminated the inhibitory potency
(Table 4), indicating the importance of this group. The exact
nature of complexation between these groups and Co2+ or
interaction with M2 remains unknown. It appears that a number
of other factors in addition to complexation contribute to
inhibitory potency. For example, by introduction of a bulkier
group on the phenyl ring, the inhibitory potency was enhanced
significantly (Table 3). Structure-activity relationships provide
insights about the contribution of different functional groups
in hydroxamate derivatives in metal-mediated inhibition. This
led to a much better understanding of the inhibitory mechanism
and improved inhibition. Structural studies of the enzyme-
inhibitor complex is needed to determine the exact nature of
compd
R
IC50 (µM)a
23a
23b
23c
23d
NHBn
OH
Ph
29
no inhibitionb
290
H
no inhibitionb
a Average of triplicate experiments. The upper limit of the standard error
of the mean (SEM) was (10%. b No inhibition up to 1000 µM.
the interactions of these functional groups with Csk and Co2+
.
inhibitory potency. For example, 4-iodo-substituted analogue
(17p) showed approximately 3-fold higher inhibition compared
to TyrH. These results indicate that by introduction of a bulkier
group on the phenyl ring, the interaction between the hydrox-
amate analogues and a hydrophobic pocket of Csk enhances
the inhibitory potency.
Compound 17i (IC50 ) 12 µM), which has an unsubstituted
phenyl group perpendicular to the phenyl of phenylalanine
hydroxamate, did not exhibit improved inhibitory potency
compared to 17h. Substitution of the phenyl group with NO2 at
the para and meta positions in compounds 17j (IC50 ) 4.9 µM)
and 17k (IC50 ) 6.1 µM), respectively, showed enhanced
inhibitory potency, possibly by changing the conformation of
the substituted phenyl group to a more favorable position for
interaction with the hydrophobic pocket.
III. Analogues with Modified Linker between Hydrox-
amate and Phenyl Functional Groups. Two TyrH derivatives
were synthesized that had different -(CH2)n- linker size
between the hydroxamate group and the phenyl, where n is
changed from 1 in TyrH (5a) to 0 and 2 in 18u and 18v,
respectively (Table 3). Shortening or lengthening the distance
between the hydroxamate group and the phenyl in 18u and 18v
significantly reduced the inhibitory potency. These results
suggest that an optimal distance is required between the
hydrophobic side chain and hydroxamate group for a favorable
interaction with the M2-binding site.
IV. TyrH Analogues with Modified r-Amino Group
(-NH2). To explore the role of R-amino group of TyrH in
inhibition of Csk, compounds 23a-d with modified R-amino
group were synthesized (Table 4). The substitution on the
R-amino group of TyrH with the benzyl group in 23a led to
3-fold reduced inhibitory potency compared to 5a. The replace-
ment of R-amino group with -OH and -H in 23b and 23d,
respectively, completely eliminated the inhibitory potency.
Additionally, the replacement of an R-amino group with a
phenyl group in 23c reduced the inhibitory potency by 31-fold
compared to 5a. These results suggest that the presence of the
R-amino group of TyrH is critical in generating inhibitory
potency possibly through a favorable interaction with residues
located in the kinase domain.
Conclusions
This study established hydroxamate, represented by TyrH,
as a scaffold for metal-mediated inhibitors against a protein
tyrosine kinase and revealed a number of important structural
features for the inhibition. To the best of our knowledge, this
is the first reported design of metal-mediated inhibitors against
protein tyrosine kinases by targeting a metal-binding site.
Tyrosine thiohydroxamate (5b) and compounds containing
hydrophobic residues on the phenyl ring (17h and 17p-r)
showed improved inhibitory potency compared to TyrH. Taken
together, these results suggest that exploring further structural
diversity of functional groups of hydroxamate analogues may
lead to more potent Csk inhibitors. Additionally, this design
strategy lays the foundation for developing metal-mediated
inhibitors against other PTKs.
Experimental Section
Chemistry. All solid-phase reactions were carried out in Bio-
Rad polypropylene columns by shaking and mixing at room
temperature using a Glass-Col small tube rotator under dry
conditions unless otherwise stated. Real-time monitoring of loading
of compounds on resin beads was carried out with a Thermo-Nicolet
550 FT-IR spectrophotometer coupled with a Nic-Plan microscope
using OMNIC software. The chemical structures of final products
were characterized by nuclear magnetic resonance spectra (1H
NMR, 13C NMR) determined on a Bruker NMR spectrometer (400
MHz). 13C NMR spectra are fully decoupled. Chemical shifts were
reported in parts per millions (ppm). The chemical structures of
final products were confirmed by a high-resolution PE Biosystems
Mariner API time-of-flight electrospray mass spectrometer. Final
compounds were purified (>99%) on a Phenomenex Prodigy 10
µm ODS reversed-phase column (2.1 cm × 25 cm) with a Hitachi
HPLC system using a gradient system of acetonitrile and water
(CH3CN/H2O, 0-40%, pH 7.0, 30 min). The purity of final products
(>99%) was confirmed by analytical HPLC. The analytical HPLC
was performed on the Hitachi analytical HPLC system on a C18
Shimadzu Premier 3 µm column (150 cm × 4.6 mm) using two
different isocratic systems and a flow rate of 1 mL/min with UV
detection at 260 nm. L-Tyrosine (5e), L.-HisH, L-ArgH, l-LysH,
L-LeuH, â-AlaH, DL-NorvalH, DL-Asp γ-H, L-Glu γ-monoH, DL-
PheH, and GlyH were purchased from Sigma-Aldrich Co. L-
Tyrosine hydrazide (5f) was purchased from MP Biomedicals Co.
General Procedure for the Synthesis of 5a,b, 17a-r, and 18u,v.
Hydroxylamine Wang resin (1, 50 mg, 2 mmol/g) was swollen in
DMF (5 mL) for 15 min. N-(9-Fluorenylmethoxycarbonyl)-L-
phenylalanine analogues (2, 11a-r, or 12s,t; 0.4 mmol) and HBTU
(170 mg, 0.5 mmol) were dissolved in NMM/DMF (5 mL, 0.4 M)
and added to the swollen resin. The reaction mixture was shaken
for 30 min. The resin was collected by filtration and washed
successively with DMF (3 × 5 mL), dichloromethane (3 × 5 mL),
and methanol (2 × 5 mL) to afford 3a, 13a-r, and 14s,t,
Hydroxamates and thiohydroxamates have been previously
reported to create complexes with Co2+ and other transition
metals.24-30 On the basis of the structure-activity relationship
data, it appears that the -CO, -NH, and -OH moieties in the
hydroxamate group (-CONHOH) and the R-amino group are
binding to Co2+ or M2 site. Structure-activity relationships by
analogues in Table 2 revealed that replacement or elimination
of -OH, -CO, or -NH moieties of the hydroxamate group
significantly modified the inhibitory potencies against Csk.

Metal-Mediated Inhibitors against Csk
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7537
respectively. The resin 3a (50 mg, 0.1 mmol) was swollen in THF
(15 mL) for 10 min. Lawesson’s reagent (121 mg, 0.3 mmol) was
added to the swollen resin. The reaction mixture was shaken for 3
days. The resin was collected by filtration and washed successively
with DMF (3 × 5 mL) and methanol (2 × 5 mL) to afford 3b.
Piperidine in DMF (5 mL, 20%) was added to 3a, 3b, 13a-r, and
14s,t, and the reaction mixture was shaken for 10 min. The resins
were collected by filtration, washed successively with DMF (3 ×
5 mL), dichloromethane (3 × 5 mL), and methanol (2 × 5 mL) to
give 4a,b, 15a-r, and 16s,t. The cleavage was carried out by
addition of TFA in dichloromethane (70%, 5 mL). After 30 min of
shaking of the mixtures at room temperature, the resins were
collected by filtration. The solvents of filtrate solutions were
evaporated, and the residues were dried under vacuum. The crude
mixtures were purified by HPLC as described above to yield 5a,b,
17a-r, and 18u,v, respectively (overall yield, 30-60%).
General Procedure for the Synthesis of 5c,d. Wang resin (6,
100 mg; 1.1 mmol/g) was swollen in dichloromethane (5 mL) for
15 min. N-(9-Fluorenylmethoxycarbonyl)-L-tyrosine methyl ester
(210 mg, 0.5 mmol) and triphenylphosphine (144 mg, 0.54 mmol)
were dissolved in dichloromethane (2 mL) and added to the swollen
resin. Diispropylazodicarboxylate (DIAD, 90 µL, 0.47 mmol) was
diluted to 400 µL with dichloromethane and added dropwise to
the mixture at room temperature. The reaction mixture was shaken
overnight. The resin was collected by filtration, washed successively
with dichloromethane (5 mL), DMF (3 × 5 mL), and methanol (2
× 5 mL), and dried under vacuum to afford 8. The solution of
sodium hydroxide in methanol (6 mL, 0.4 M) was added to the
dried resin, and the reaction mixture was shaken for 6 days. The
resin was collected by filtration, washed successively with DMF
(3 × 5 mL), dichloromethane (3 × 5 mL), and methanol (2 × 5
mL), and dried under vacuum to give 9. The resin was swollen in
THF (5 mL) for 15 min. N-Methylhydroxylamine hydrochloride
or N-benzylhydroxylamine hydrochloride (RNHOH‚HCl, 0.6 mmol)
and diisopropylcarbodiimide (93 µL, 0.6 mmol) were added to the
swollen resin and mixed. Triethylamine (Et3N, 18 µL, 1.3 mmol)
was added to the mixture. The reaction mixture was shaken
overnight. The resin was collected by filtration and washed
successively with DMF (3 × 5 mL), dichloromethane (3 × 5 mL),
and methanol (2 × 5 mL) to afford 10a or 10b. The cleavage was
carried out by addition of TFA in dichloromethane (70%, 5 mL)
to 10a or 10b. After 30 min of shaking of the mixtures at room
temperature, the resins were collected by filtration and washed with
dichloromethane (2 mL). The solvents of filtrate solutions were
evaporated, and the residues were dried under vacuum. The crude
mixtures were purified by HPLC as described above to yield 5c or
5d (overall yield, ∼18-20%).
22a-d. The cleavage was carried out by addition of TFA in
dichloromethane (70%, 5 mL). After 30 min of shaking of the
mixtures at room temperature, the resins were collected by filtration
and washed with dichloromethane (2 mL). The solvents of filtrate
solutions were evaporated, and the residues were dried under
vacuum. The crude mixtures were purified by HPLC as described
above to yield 23a-d (overall yield, 25-30%).
(S)-2-Amino-N-hydroxy-3-(4-hydroxyphenyl)propanethioam-
ide (5b). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.75-2.85 (m,
1H, CH2), 3.12-3.25 (m, 1H, CH2), 3.60-3.75 (m, 1H, CH), 6.63
(d, J ) 8.00 Hz, 2H, aromatic H), 6.99 (d, J ) 8.00 Hz, 2H,
aromatic H), 7.85 (br s, 2H, NH2), 9.10 (s, 1H, OH), 9.22 (s, 1H,
OH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 38.07, 59.31,
115.80, 127.44, 131.17, 156.84, 166.40. HR-MS (ESI-TOF) (m/z):
C9H12N2O2S calcd, 212.0619; found, 213.2830 [M + 1]+, 214.2910
[M + 2]+.
(S)-2-Amino-N-hydroxy-3-(4-hydroxyphenyl)-N-methylpro-
1
panamide (5c). H NMR (DMSO-d6, 400 MHz, δ ppm): 2.88-
2.94 (m, 1H, CH2), 3.09-3.13 (m, 1H, CH2), 3.15 (s, 3H, CH3),
4.34-4.38 (m, 1H, CH), 6.71 (d, J ) 8.40 Hz, 2H, aromatic H),
7.01 (d, J ) 8.40 Hz, 2H, aromatic H), 8.13 (s, 2H, NH2), 9.42 (s,
1H, OH), 10.84 (s, 1H, OH). 13C NMR (DMSO-d6, 100 MHz, δ
ppm): 35.23, 36.87, 52.41, 116.23, 125.76, 131.39, 157.43, 168.28.
HR-MS (ESI-TOF) (m/z): C10H14N2O3 calcd, 210.1004; found,
211.3252 [M + 1]+, 212.355 [M + 2]+.
(S)-2-Amino-N-benzyl-N-hydroxy-3-(4-hydroxyphenyl)pro-
1
panamide (5d). H NMR (DMSO-d6, 400 MHz, δ ppm): 2.83-
2.87 (m, 1H, CH2), 3.04-3.08 (m, 1H, CH2), 4.36-4.40 (m, 1H,
CH), 4.75 (s, 2H, CH2), 6.69 (d, J ) 7.60 Hz, 2H, aromatic H),
6.97 (d, J ) 7.60 Hz, 2H, aromatic H), 7.30-7.36 (m, 5H, aromatic
H), 7.97 (br s, 2H, NH2), 9.38 (s, 1H, OH), 10.57 (s, 1H, OH). 13
C
NMR (DMSO-d6, 100 MHz, δ ppm): 35.48, 52.59, 116.26, 125.63,
128.40, 129.03, 129.26, 131.43, 138.80, 157.47, 168.85. HR-MS
(ESI-TOF) (m/z): C16H18N2O3 calcd, 286.1317; found, 287.2736
[M + 1]+, 288.3632 [M + 2]+.
(S)-2-Amino-3-(4-aminophenyl)-N-hydroxypropanamide (17a).
1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.83-2.87 (m, 2H, CH2),
3.62 (t, J ) 7.00 Hz, 1H, CH), 5.63 (br s, 2H, NH2), 6.55 (d, J )
8.00 Hz, 2H, aromatic H), 6.87 (d, J ) 8.00 Hz, 2H, aromatic H),
8.23 (s, 2H, NH2), 9.26 (s, 1H, OH), 10.94 (s, 1H, NH). 13C NMR
(DMSO-d6, 100 MHz, δ ppm): 37.22, 52.89, 115.61, 123.27,
130.75, 147.25, 165.23. HR-MS (ESI-TOF) (m/z): C9H13N3O2
calcd, 195.1008; found, 196.3336 [M + 1]+, 244.3152 [M + 2]+.
(S)-2-Amino-3-(4-cyanophenyl)-N-hydroxypropanamide (17b).
1H NMR (DMSO-d6, 400 MHz, δ ppm): 3.05-3.10 (m, 2H, CH2),
3.81-3.84 (m, 1H, CH), 7.41 (d, J ) 8.00 Hz, 2H, aromatic H),
7.82 (d, J ) 8.00 Hz, 2H, aromatic H), 8.40 (br s, 2H, NH2), 9.31
(br s, 1H, OH), 11.01 (s, 1H, NH). HR-MS (ESI-TOF) (m/z):
C10H11N3O2 calcd, 205.0851; found, 206.2166 [M + 1]+.
(S)-2-Amino-3-(4-azidophenyl)-N-hydroxypropanamide (17c).
1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.94-2.99 (m, 2H, CH2),
3.69-3.74 (m, 1H, CH), 7.10 (d, J ) 8.40 Hz, 2H, aromatic H),
7.24 (d, J ) 8.40 Hz, 2H, aromatic H), 8.28 (br s, 2H, NH2), 9.29
(s, 1H, OH), 10.95 (s, 1H, NH). HR-MS (ESI-TOF) (m/z):
C9H11N5O2 calcd, 221.0913; found, 222.2411 [M + 1]+, 223.2430
[M + 2]+.
General Procedure for the Synthesis of 23a-d. To (S)-3-(4-
hydroxylphenyl)propionic acid derivatives (19a-d) (0.5 mmol) in
dry pyridine (4 mL) was added acetic anhydride (Ac2O, 2 mL).
The reaction mixtures were stirred at room temperature overnight.
The solvent was removed under vacuum, and the mixtures were
purified by silica gel column chromatography using hexane and
ethyl acetate as eluents to yield (S)-3-(4-acetoxylphenyl)propionic
acid derivatives (20a-d). HBTU (170 mg, 0.5 mmol) and 20a-d
were dissolved in the NMM/DMF (5 mL, 0.4 M) and added to the
swollen hydroxylamine Wang resin (50 mg; loading, 2.0 mmol/g).
The reaction mixtures were shaken for 30 min. The resins were
collected by filtration and washed successively with DMF (3 × 5
mL), dichloromethane (3 × 5 mL), and methanol (2 × 5 mL) to
give 21a-d. The Fmoc group in 21a was removed by piperidine
in DMF (20%). To the resulting polymer-bound unprotected amine
was added benzyl bromide (0.5 mL, 4 mmol) in dry pyridine (5
mL). The mixture was shaken overnight. The resin was collected
by filtration and washed successively with DMF (3 × 5 mL),
dichloromethane (3 × 5 mL), and methanol (2 × 5 mL) to afford
21a′. The acetyl groups in 21a′, 21b, 21c, and 21d were removed
by addition of sodium methoxide (20 mg) in methanol (5 mL).
The mixtures were shaken for 2 h. The resins were collected by
filtration and washed successively with DMF (3 × 5 mL),
dichloromethane (3 × 5 mL), and methanol (2 × 5 mL) to give
4-((S)-2-(Hydroxycarbamoyl)-2-aminoethyl)phenylboronic Acid
1
(17d). H NMR (DMSO-d6, 400 MHz, δ ppm): 2.80-3.20 (m,
2H, CH2), 3.68-3.72 (m, 1H, CH), 7.12-7.22 (m, 2H, aromatic
H), 7.72-7.78 (m, 2H, aromatic H), 8.0 (s, 2H, BOH), 8.28 (br s,
2H, NH2), 9.28 (s, 1H, OH), 10.95 (s, 1H, NH). HR-MS (ESI-
TOF) (m/z): C9H13BN2O4 calcd, 224.0968; found, 224.2403 [M]+,
225.2274 [M + 1]+.
(S)-2-Amino-N-hydroxy-3-(4-nitrophenyl)propanamide (17e).
1H NMR (DMSO-d6, 400 MHz, δ ppm): 3.00-3.10 (m, 2H, CH2),
3.73-3.77 (m, 1H, CH), 7.50 (d, J ) 8.80 Hz, 2H, aromatic H),
7.81 (br s, 2H, NH2), 8.22 (d, J ) 8.80 Hz, 2H, aromatic H), 9.25
(br s, 1H, OH), 10.90 (br s, 1H, NH). 13C NMR (DMSO-d6, 100
MHz, δ ppm): 37.80, 52.32, 124.46, 131.74, 144.14, 147.63,
165.12. HR-MS (ESI-TOF) (m/z): C9H11N3O4 calcd, 225.0749;
found, 226.2678 [M + 1]+, 227.2962 [M + 2]+.

7538 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Gu et al.
(S)-2-Amino-N-hydroxy-3-phenylpropanamide (17f). 1H NMR
(DMSO-d6, 400 MHz, δ ppm): 2.87-3.10 (m, 2H, CH2), 3.75 (t,
J ) 7.00 Hz, 1H, CH), 7.20-7.25 (m, 2H, aromatic H), 7.26-
7.37 (m, 3H, aromatic H), 8.30 (br s, 2H, NH2), 9.30 (s, 1H, OH),
10.98 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 37.84,
52.57, 128.05, 129.45, 130.23, 135.73, 165.00. HR-MS (ESI-TOF)
(m/z): C9H12N2O2 calcd, 180.0899; found, 181.3126 [M + 1]+,
182.3207 [M + 2]+.
3.72 (t, J ) 7.20 Hz, 1H, CH), 7.16 (d, J ) 8.00 Hz, 2H, aromatic
H), 7.54 (d, J ) 8.00 Hz, 2H, aromatic H), 8.11 (br s, 2H, NH2),
9.22 (s, 1H, OH), 10.94 (s, 1H, NH). 13C NMR (DMSO-d6, 100
MHz, δ ppm): 37.31, 52.41, 121.35, 132.43, 132.50, 135.25,
165.08. HR-MS (ESI-TOF) (m/z): C9H11BrN2O2 calcd, 258.0004;
found, 259.1935 [M + 1]+, 261.1879 [M + 2]+.
(S)-2-Amino-N-hydroxy-3-(4-iodophenyl)propanamide (17p).
1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.90-3.10 (m, 2H, CH2),
3.70-3.77 (m, 1H, CH), 7.02 (d, J ) 8.00 Hz, 2H, aromatic H),
7.69 (d, J ) 8.00 Hz, 2H, aromatic H), 8.28 (br s, 2H, NH2), 9.31
(s, 1H, OH), 10.99 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz,
δ ppm): 37.33, 52.37, 94.29, 132.65, 135.51, 138.15, 164.90. HR-
MS (ESI-TOF) (m/z): C9H11IN2O2 calcd, 305.9865; found, 307.1113
[M + 2]+.
(S)-2-Amino-N-hydroxy-3-(2-iodophenyl)propanamide (17q).
1H NMR (DMSO-d6, 400 MHz, δ ppm): 3.08-3.20 (m, 2H, CH2),
3.70-3.80 (m, 1H, CH), 7.01-7.09 (m, 1H, aromatic H), 7.20-
7.25 (m, 1H, aromatic H), 7.35-7.40 (m, 1H, aromatic H), 7.85-
7.92 (m, 1H, aromatic H), 8.48 (br s, 2H, NH2), 9.27 (s, 1H, OH),
10.94 (s, 1H, NH). HR-MS (ESI-TOF) (m/z): C9H11IN2O2 calcd,
305.9865; found, 307.1118 [M + 2]+.
(S)-2-Amino-N-hydroxy-3-p-tolylpropionamide (17g). 1H NMR
(DMSO-d6, 400 MHz, δ ppm): 2.29 (s, 3H, CH3), 2.86-2.99 (m,
2H, CH2), 3.72 (t, J ) 7.00 Hz, 1H, CH), 7.05-7.17 (m, 4H,
aromatic H), 8.26 (br s, 2H, NH2), 9.29 (s, 1H, OH), 10.96 (s, 1H,
NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 21.53, 37.44, 52.64,
130.00, 130.10, 132.64, 137.09, 165.05. HR-MS (ESI-TOF) (m/z):
C10H14N2O2 calcd, 194.1055; found, 195.3455 [M + 1]+, 196.3175
[M + 2]+.
(S)-2-Amino-N-hydroxy-3-(4-isopropylphenyl)propanamide
(17h). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 1.19 (d, 6H, CH3),
2.83-2.90 (m, 2H, CH2), 2.90-3.03 (m, 1H, CH), 3.69-3.74 (m,
1H, CH), 7.13 (d, 2H, J ) 8.00 Hz, aromatic H), 7.21 (d, J ) 8.00
Hz, 2H, aromatic H), 8.17 (br s, 2H, NH2), 9.29 (s, 1H, OH), 10.96
(s, 1H, NH). HR-MS (ESI-TOF) (m/z): C12H18N2O2 calcd,
222.1368; found, 223.1797 [M + 1]+, 224.1997 [M + 2]+.
(S)-2-Amino-N-hydroxy-3-[4-(phenyl)phenyl]propanamide (17i).
1H NMR (DMSO-d6, 400 MHz, δ ppm): 3.01-3.08 (m, 2H, CH2),
3.73-3.81 (m, 1H, CH), 7.25-7.40 (m, 2H, aromatic H), 7.42-
7.50 (m, 2H, aromatic H), 7.60-7.70 (m, 5H, aromatic H), 8.24
(br s, 2H, NH2), 9.31 (s, 1H, OH), 10.98 (s, 1H, NH). HR-MS
(ESI-TOF) (m/z): C15H17N2O2 calcd, 256.1212; found, 257.2748
[M + 1]+.
(S)-2-Amino-N-hydroxy-3-(4-hydroxy-3,5-diiodophenyl)pro-
panamide (17r). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.72-
2.92 (m, 2H, CH2), 3.68-3.75 (m, 1H, CH), 7.58 (br s, 2H, aromatic
H), 8.22 (br s, 2H, NH2), 9.34 (s, 1H, OH), 10.95 (s, 1H, NH). 13
C
NMR (DMSO-d6, 100 MHz, δ ppm): 35.78, 52.55, 88.09, 131.76,
140.69, 155.45, 165.03. HR-MS (ESI-TOF) (m/z): C9H10I2N2O3
calcd, 447.8781; found, 448.7776 [M + 1]+.
(S)-2-Amino-N-hydroxy-2-(4-hydroxyphenyl)acetamide (18u).
1H NMR (DMSO-d6, 400 MHz, δ ppm): 4.57-4.64 (m, 1H, CH),
6.75-6.85 (m, 2H, aromatic H), 7.25-7.35 (d, 2H, aromatic H),
8.52 (s, 2H, NH2), 9.26 (s, 1H, OH), 9.77 (s, 1H, OH), 11.08 (s,
1H, NH). HR-MS (ESI-TOF) (m/z): C8H10N2O3 calcd, 182.0691;
found, 183.6436 [M + 1]+, 205.5888 [M + Na]+, 221.5359 [M +
K]+.
(S)-2-Amino-N-hydroxy-3-[4-(nitrophenyl)phenyl]propana-
1
mide (17j). H NMR (DMSO-d6, 400 MHz, δ ppm): 3.01-3.12
(m, 2H, CH2), 3.79-3.83 (m, 1H, CH), 7.34-7.43 (m, 2H, aromatic
H), 7.76-7.83 (m, 2H, aromatic H), 7.91-8.00 (m, 2H, aromatic
H), 8.27-8.38 (m, 4H, aromatic H and NH2), 9.33 (s, 1H, OH),
11.01 (s, 1H, NH). HR-MS (ESI-TOF) (m/z): C15H15N3O4 calcd,
301.1063; found, 302.4769 [M + 1]+.
(S)-2-Amino-N-hydroxy-4-(4-hydroxyphenyl)butanamide (18v).
1H NMR (DMSO-d6, 400 MHz, δ ppm): 1.89-2.09 (m, 2H, CH2),
2.10-2.16 (m, 2H, CH2), 3.45-3.60 (m, 1H, CH), 6.60 (d, J )
7.60 Hz, 2H, aromatic H), 6.90 (d, 2H, aromatic H), 8.30 (br s,
2H, NH2), 9.29 (s, 1H, OH), 9.38 (s, 1H, OH), 11.10 (s, 1H, NH).
HR-MS (ESI-TOF) (m/z): C10H14N2O3 calcd, 210.1004; found,
211.0421 [M + 1]+.
(S)-2-Amino-N-hydroxy-3-[3-(nitrophenyl)phenyl]propana-
1
mide (17k). H NMR (DMSO-d6, 400 MHz, δ ppm): 3.00-3.13
(m, 2H, CH2), 3.76-3.86 (m, 1H, CH), 7.35-7.42 (m, 2H, aromatic
H), 7.72-7.82 (m, 3H, aromatic H), 8.11-8.20 (m, 1H, aromatic
H), 8.22-8.26 (m, 1H, aromatic H), 8.34 (br s, 2H, NH2), 8.40-
8.45 (m, 1H, aromatic H), 9.34 (s, 1H, OH), 11.02 (s, 1H, NH).
HR-MS (ESI-TOF) (m/z): C15H15N3O4 calcd, 301.1063; found,
302.4702 [M + 1]+.
(S)-2-Benzylamino-N-hydroxy-3-(4-hydroxyphenyl)propana-
1
mide (23a). H NMR (DMSO-d6, 400 MHz, δ ppm): 2.60-2.78
(m, 2H, CH2), 3.00-3.11 (m, 1H, CH2), 3.47-3.52 (m, 1H, CH2),
3.67-3.74 (m, 1H, CH), 6.64 (d, J ) 8.40 Hz, 2H, aromatic H),
6.97 (d, J ) 8.40 Hz, 2H, aromatic H), 7.17-7.30 (m, 5H, aromatic
H), 8.81 (s, 1H, NH), 9.14 (s, 1H, OH), 10.45 (s, 1H, NH). HR-
MS (ESI-TOF) (m/z): C16H18N2O3 calcd, 286.1317; found, 287.1699
[M + 1]+, 288.2465 [M + 2]+.
(S)-2-Amino-3-(4-fluorophenyl)-N-hydroxypropanamide (17l).
1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.90-3.05 (m, 2H, CH2),
3.75 (t, J ) 7.20 Hz, 1H, CH), 7.10-7.30 (m, 4H, aromatic H),
8.30 (br s, 2H, NH2), 9.31 (s, 1H, OH), 10.99 (s, 1H, NH). 13C
NMR (DMSO-d6, 100 MHz, δ ppm): 36.97, 52.58, 116.30, 132.16,
132.24, 163.55, 164.96. HR-MS (ESI-TOF) (m/z): C9H11FN2O2
calcd, 198.0805; found, 199.2988 [M + 1]+.
(S)-2-Amino-3-(3,4-difluorophenyl)-N-hydroxypropana-
mide (17m). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.85-3.10
(m, 2H, CH2), 3.74 (t, J ) 7.0 Hz, 1H, CH), 7.00-7.10 (m, 1H,
aromatic H), 7.20-7.30 (m, 1H, aromatic H), 7.42-7.46 (m, 1H,
aromatic H), 7.92 (br s, 2H, NH2), 9.26 (s, 1H, OH), 10.88 (br s,
1H, NH). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 37.24, 52.58,
118.28, 118.45, 119.17, 119.34, 127.20, 127.27, 165.44. HR-MS
(ESI-TOF) (m/z): C9H10F2N2O2 calcd, 216.0710; found, 217.2563
[M + 1]+.
(S)-N,2-Dihydroxy-3-(4-hydroxyphenyl)propanamide (23b).
1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.50-2.63 (m, 1H, CH2),
2.70-2.85 (m, 1H, CH2), 3.80-3.95 (m, 1H, CH), 5.20-5.30 (d,
J ) 6.40 Hz, 1H, OH), 6.65 (d, J ) 8.00 Hz, 2H, aromatic H),
6.99 (d, J ) 8.00 Hz, 2H, aromatic H), 8.70 (s, 1H, OH), 9.10 (s,
1H, OH), 10.40 (s, 1H, NH). HR-MS (ESI-TOF) (m/z): C9H11-
NO4: calcd, 197.0688; found, 198.2101 [M + 1]+.
(S)-N-Hydroxy-3-(4-hydroxyphenyl)-2-phenylpropanamide
(23c). 1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.76 (dd, J ) 6.00
Hz, J ) 13.60 Hz, 1H, CH2), 3.19 (dd, J ) 9.20 Hz, J ) 13.60
Hz, 1H, CH2), 3.49 (dd, J ) 6.00 Hz, J ) 9.20 Hz, 1H, CH), 6.61
(d, J ) 8.40 Hz, 2H, aromatic H), 6.95 (d, J ) 8.40 Hz, 2H,
aromatic H), 7.20-7.40 (m, 5H, aromatic H), 8.74 (br s, 1H, OH),
9.14 (s, 1H, OH), 10.55 (s, 1H, NH). 13C NMR (DMSO-d6, 100
MHz, δ ppm): 38.40, 51.30, 115.74, 127.53, 128.60, 128.98,
130.50, 130.57, 141.42, 156.40. HR-MS (ESI-TOF) (m/z): C15H15-
NO3 calcd, 257.1052; found, 258.2814 [M + 1]+, 259.2742 [M +
2]+.
(S)-2-Amino-3-(4-chlorophenyl)-N-hydroxypropanamide (17n).
1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.90-3.05 (m, 2H, CH2),
3.75 (t, J ) 7.00 Hz, 1H, CH), 7.23 (d, J ) 8.40 Hz, 2H, aromatic
H), 7.40 (d, 2H, J ) 8.40 Hz, aromatic H), 8.30 (br s, 2H, NH2),
9.31 (s, 1H, OH), 10.99 (s, 1H, NH). 13C NMR (DMSO-d6, 100
MHz, δ ppm): 37.11, 52.43, 129.38, 132.16, 132.80, 134.76,
164.88. HR-MS (ESI-TOF) (m/z): C9H11ClN2O2 calcd, 214.0509;
found, 215.2769 [M + 1]+, 217.2745 [M + 2]+.
N-Hydroxy-3-(4-hydroxyphenyl)propanamide (23d). 1H NMR
(DMSO-d6, 400 MHz, δ ppm): 2.16 (t, J ) 7.20 Hz, 2H, CH2),
2.69 (t, J ) 7.20 Hz, 2H, CH2), 6.63 (d, J ) 8.40 Hz, 2H, aromatic
(S)-2-Amino-3-(4-bromophenyl)-N-hydroxypropionamide (17o).
1H NMR (DMSO-d6, 400 MHz, δ ppm): 2.88-3.01 (m, 2H, CH2),

Metal-Mediated Inhibitors against Csk
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7539
(9) Sun, G.; Budde, R. J. Expression, purification and initial characteriza-
tion of human Yes protein tyrosine kinase from a bacterial expression
system. Arch. Biochem. Biophys. 1997, 345, 135-142.
(10) Sun, G.; Budde, R. J. Substitution studies of the second divalent metal
cation requirement of protein tyrosine kinase CSK. Biochemistry
1999, 38, 5659-5665.
(11) Budde, R. J. A.; Ramdas, L.; Sun, G. Cloning, expression, purification
and characterization of the alternate splice Src variants for drug
discovery. J. Mol. Catal. 2001, 11, 805-809.
(12) Saylor, P.; Wang, C.; Hirai, T. J.; Adams, J. A. A second magnesium
ion is critical for ATP binding in the kinase domain of the oncoprotein
v-Fps. Biochemistry 1998, 37, 12624-12630.
(13) Vicario, P. P.; Saperstein, R.; Bennun, A. Role of divalent metals in
the kinetic mechanism of insulin receptor tyrosine kinase. Arch.
Biochem. Biophys. 1988, 261, 336-345.
(14) Koland, J. G.; Cerione, R. A. Activation of the EGF receptor tyrosine
kinase by divalent metal ions: comparison of holoreceptor and
isolated kinase domain properties. Biochim. Biophys. Acta. 1990,
1052, 489-498.
(15) Hubbard, S. R. Crystal structure of the activated insulin receptor
tyrosine kinase in complex with peptide substrate and ATP analog.
EMBO J. 1997, 16, 5572-5581.
(16) Adams, J. A.; Taylor, S. S. Divalent metal ions influence catalysis
and active-site accessibility in the cAMP-dependent protein kinase.
Protein Sci. 1993, 2, 2177-2186.
(17) Sun, G.; Budde, R. J. Affinity purification of Csk protein tyrosine
kinase based on its catalytic requirement for divalent metal cations.
Protein Expression Purif. 2001, 21, 8-12.
(18) Kim, D. H.; Mobashery, S. Mechanism-based inhibition of zinc
proteases. Curr. Med. Chem. 2001, 8, 959-865.
(19) Cohen, S. M.; Lippard, S. J. Cisplatin: from DNA damage to cancer
chemotherapy. Prog. Nucleic Acid Res. Mol. Biol. 2001, 67, 93-
130.
(20) Farrell, N. Current status of structure-activity relationships of
platinum anticancer drugs: activation of the trans geometry. Met.
Ions Biol. Syst. 1996, 32, 603-639.
(21) Janc, J. W.; Clark, J. M.; Warne, R. L.; Elrod, K. C.; Katz, B. A.;
Moore, W. R. A novel approach to serine protease inhibition: kinetic
characterization of inhibitors whose potencies and selectivities are
dramatically enhanced by zinc(II). Biochemistry 2000, 39, 4792-
4800.
H), 6.95 (d, J ) 8.40 Hz, 2H, aromatic H), 8.68 (s, 1H, OH), 9.14
(s, 1H, OH), 10.33 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz,
δ ppm): 30.96, 35.23, 115.87, 129.92, 131.95, 156.33, 169.23. HR-
MS (ESI-TOF) (m/z): C9H11NO3 calcd, 181.0739; found, 182.2973
[M + 1]+, 183.3021 [M + 2]+, 204.2666 [M + Na]+, 220.2325
[M + K]+.
Kinase Activity Assays. PTK activities were determined by
measuring the phosphorylation of poly-E4Y by the kinase using an
acid precipitation assay as previously described.8,10 The standard
reaction contained the appropriate amount of the kinase, 1 mg mL-1
poly-E4Y and 0.2 mM [32P]-ATP (1,000 dpm pmol-1) in the kinase
assay buffer (75 mM EPPS, pH 8.0, 12 mM MgCl2, 5% glycerol,
0.005% Triton X-100). The appropriate amount of kinase was
predetermined to ensure that the kinase activity was linear with
the amount of enzyme added. The reaction volume was 50 µL,
and the reaction was allowed to proceed for 30 min at 30 °C. At
the end of the reaction, 35 µL of the reaction mixture was spotted
into a strip of filter paper (1 cm × 2 cm), and the reaction was
stopped by immersing the filter paper in warm 5% TCA. The filter
paper was washed three times in 5% TCA for 10 min each.
Phosphorylated and unphosphorylated poly-E4Y were both pre-
cipitated into the filter paper, and the amount of phosphate
incorporated onto poly-E4Y was determined by liquid scintillation
counting.
To determine the inhibition of Csk by a hydroxamate compound,
the reaction mixture also contained 0.2 mM CoCl2 and various
concentrations of the compound. To determine the effect of other
metal cations on the inhibition, other metal salts were used in the
place of CoCl2. When the Km and Kcat were determined with regard
to one substrate, the kinase activity was determined at various
concentrations of that substrate in the range of 20-200 µM for
ATP or 20-200 µg mL-1 for poly-E4Y. The Kcat and Km values
were determined by Lineweaver-Burk plots with linear regression
using the Microsoft Excel program.
Acknowledgment. We acknowledge the financial support
from American Cancer Society (Grant RSG CDD-106966).
(22) Katz, B. A.; Clark, J. M.; Finer-Moore, J. S.; Jenkins, T. E.; Johnson,
C. R.; Ross, M. J.; Luong, C.; Moore, W. R.; Stroud, R. M. Design
of potent selective zinc-mediated serine protease inhibitors. Nature
1998, 391, 608-612.
Supporting Information Available: 1H NMR and 13C NMR
spectra for representative compounds in different classes and their
analytical HPLC profiles in two diverse systems. This material is
available free of charge via the Internet at http://pubs.acs.org.
(23) Katz, B. A.; Luong, C. Recruiting Zn2+ to mediate potent, specific
inhibition of serine proteases. J. Mol. Biol. 1999, 292, 669-684.
(24) Rao, M. J.; Sethuram, B.; Rao, T. N. Synthesis and spectroscopic
and fungicidal characterization of hydroxamic acids and their metal
chelates. J. Inorg. Biochem. 1985, 24, 155-160.
References
(1) Hubbard, S. R.; Till, J. H. Protein tyrosine kinase structure and
function. Annu. ReV. Biochem. 2000, 69, 373-398.
(25) Farkas, E.; Kovacs, A. Hydroxamic acid derivatives of phenylalanine,
tyrosine and 3,4-dihydroxyphenylalanine (DOPA) as ligands. J.
Coord. Chem. 1991, 24, 325-332.
(26) Chang, C. A.; Sekhar, V. C.; Garg, B. S.; Guziec, F. S., Jr.; Carrera
Russo, T. Equilibria and kinetics of some metal complexes of peptide
hydroxamic acids. Inorg. Chim. Acta 1987, 135, 11-18.
(27) Li, J.-Z.; Zhang, C.-C.; Zhou, B.; Qin, S.-Y. Synthesis of cobalt(II)
complexes with p-tert-butylcalix[4]arene-supported dihydroxamic
acids. J. Chem. Res. 2004, 9, 624-625.
(28) Abu-Dari, K.; Raymond, K. N. Coordination isomers of biological
iron transport compounds. 8. The resolution of tris(hydroxamato) and
tris(thiohydroxamato) complexes of high-spin iron(III). J. Am. Chem.
Soc. 1977, 99, 2003-2005.
(29) Nagata, K.; Mizukami, S. Studies on thiohydroxamic acids and their
metal chelates. IV. Reaction of thiohydroxamic acids with metal ions.
Chem. Pharm. Bull. 1967, 15, 61-69.
(2) Hunter, T. Protein kinases and phosphatases: the yin and yang of
protein phosphorylation and signaling. Cell 1995, 27, 225-236.
(3) Biscardi, J. S.; Tice, D. A.; Parsons, S. J. c-Src, receptor tyrosine
kinases, and human cancer. AdV Cancer Res. 1999, 76, 61-119.
(4) Uckun, F. M.; Mao, C. Tyrosine kinases as new molecular targets in
treatment of inflammatory disorders and leukemia. Curr. Pharm. Des.
2004, 10, 1083-1091.
(5) Thiesing, J. T.; Ohno-Jones, S.; Kolibaba, K. S.; Druker, B. J. Efficacy
of STI571, an abl tyrosine kinase inhibitor, in conjunction with other
antileukemic agents against bcr-abl-positive cells. Blood 2000, 96,
3195-3199.
(6) Wakeling, A. E.; Guy, S. P.; Woodburn, J. R.; Ashton, S. E.; Curry,
B. J.; Barker, A. J.; Gibson, K. H. ZD1839 (Iressa): an orally active
inhibitor of epidermal growth factor signaling with potential for
cancer therapy. Cancer Res. 2002, 62, 5749-5754.
(7) Stout, T. J.; Foster, P. G.; Matthews, D. J. High-throughput structural
biology in drug discovery: protein kinases. Curr. Pharm. Des. 2004,
10, 1069-1082.
(8) Sun, G.; Budde, R. J. Requirement for an additional divalent metal
cation to activate protein tyrosine kinases. Biochemistry 1997, 36,
2139-2146.
(30) Abu-Dari, K.; Cooper, S. R.; Raymond, K. N. Coordination chemistry
of microbial iron transport compounds. 15. Electrochemistry and
magnetic susceptibility of iron(III)-hydroxamate and -thiohydrox-
amate complexes. Inorg. Chem. 1978, 17, 3394-3397.
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