Hydroxamates: Relationships between structure and plasma stability

Article abstract of DOI:10.1021/jm900648x

Hydroxamates are valuable tools for chemical biology as well as interesting leads for medicinal chemistry. Although many hydroxamates display nanomolar activities against metalloproteases, only three hydroxamates have reached the market, among which is the HDAC inhibitor vorinostat. Failures in development are generally attributed to lack of selectivity, toxicity, or poor stability. To help medicinal chemists with respect to plasma stability, we have performed the first and preliminary study on structure-plasma stability for hydroxamates. We define some structural rules to predict or improve the plasma stability in the preclinical stage.

Full text of DOI:10.1021/jm900648x

6790 J. Med. Chem. 2009, 52, 6790–6802
DOI: 10.1021/jm900648x
Hydroxamates: Relationships between Structure and Plasma Stability
Marion Flipo,†,‡,§, Julie Charton,†,‡,§, Akila Hocine,†,‡,§, Sandrine Dassonneville,†,‡,§, Benoit Deprez,*,†,‡,§, and
Rebecca Deprez-Poulain*,†,‡,§,
†INSERM U761 Biostructures and Drug Discovery and ‡Faculteꢀ de Pharmacie, Univ Lille Nord de France, 3 rue du Pr Laguesse,
Lille F-59006, France, §Institut Pasteur de Lille, IFR 142, Lille F-59021, France, and PRIM, Lille F-59006, France
Received May 16, 2009
Hydroxamates are valuable tools for chemical biology as well as interesting leads for medicinal
chemistry. Although many hydroxamates display nanomolar activities against metalloproteases, only
three hydroxamates have reached the market, among which is the HDAC inhibitor vorinostat. Failures
in development are generally attributed to lack of selectivity, toxicity, or poor stability. To help
medicinal chemists with respect to plasma stability, we have performed the first and preliminary study
on structure-plasma stability for hydroxamates. We define some structural rules to predict or improve
the plasma stability in the preclinical stage.
Introduction
(ZBG).15 For example, some teams have shifted back to carbo-
xylic acids or tetrazoles.16,17 Other series include o-aminobenza-
mides or retrohydroxamates.18,19 Nevertheless, after 25 years,
the first hydroxamate was approved in 2006 for marketing:
vorinostat (SAHA, Merck & Co.), a histone deacetylase
(HDAC) inhibitor for the treatment of cancer.20
Hydroxamic acids are often potent bioactive molecules.1,2
Because of their chelating group, they target metalloproteases
and can serve as both biological probes3 and leads. They can
also be used as bioisosters of carboxylic acid, being weak
acids.4 Recently reported biological activities of these com-
pounds include: inhibition ofpeptide deformylase (PDF) or of
botulinum neurotoxin A protease.5,6 Also, inhibition of the
protease responsible for the shedding of the extracelullar
domain of HER-2 by hydroxamates has been described.7
Furthermore, inhibition of aggrecanases by hydroxamates
has been reported in the literature, following the initial
work on matrix metalloproteases (MMP).8 The related tumor
necrosis factor converting enzyme (TACE, ADAM17) is
also inhibited by hydroxamates.9 Other hydroxamates are
active on Plasmodium falciparum, either by inhibiting metal-
loproteases or zinc hydrolases like histone deacetylases
(HDAC).10,11 Finally, the field of human histone deacetylase
hydroxamate inhibitors has been extensively studied and led
to the successful development of vorinostat (SAHA).12
Although hydroxamates are often very potent enzyme inhi-
bitors, several challenges need to be addressed in the context of
drug discovery. First, a relatively low selectivity (due to a
significant contribution of Zn binding to their affinity to the
target) may lead to several adverse effects. For example, broad
MMP inhibition in patients gives rise to “stiffening” of the joints
referred to as musculoskeletal syndrome (MSS).13 Although this
lack of selectivity hampered clinical development of the first
generation of inhibitors, the discovery of more selective hydro-
xamates has been possible thanks to chemical modulation.14
Second, pharmacokinetics and toxicological issues are not easily
solved. These challenges have forced medicinal chemists to
search for surrogates for this highly efficient zinc binding group
Hydroxamic acids may be hydrolyzed to the corresponding
carboxylic acid under physiological conditions (Scheme 1).21
Although involvement of the liver aldehyde oxidase was
proposed, the hydrolytic activity of the plasma is the most
widely accepted explanation.22-24 Some hydroxamates are
prone to hydrolysis in plasma. This is deleterious to their
distribution and efficiency because the carboxylic acid is
generally much less active. Hydrolysis may also contribute
to toxicity because of the mutagenicity of the byproduct
hydroxylamine.25 Other metabolites of the hydroxamic
function include glucuronides and sulfonates.26,27 Recently,
glycosylhydroxamates have been proposed as pro-drugs.28
Although many hydroxamates are disclosed in the literature,
as well as their pharmacokinetics, no consistent information
is yet available on structure-plasma stability relation-
ships (SPSR). We and other groups have tried to improve the
pharmacokinetics properties of hydroxamates by chemical
modulation.10,29 Unstable molecules exhibit a rapid clearance
and short half-life, resulting in poor in vivo exposure of the
organism and thus poor bioactivity. Determining plasma
stability is critical for prioritizing compounds before in vivo
experiments. This is therefore an important driver for the
medicinal chemist. We report here the first and preliminary
in vitro structure-plasma stability relationships (SPSR) of
hydroxamates in rat plasma that could be useful for drug
design. We discuss the structural features that potentially affect
in vitro stability and relate our findings to those reported in the
literature on hydroxamates.
*To whom correspondence should be addressed. For B.D. and
R.D-P.: phone, þ33 (0)320 964 709; fax, (þ33) 320964709. E-mail,
rebecca.deprez@univ-lille2.fr (R.D-P.); benoit.deprez@univ-lille2.fr
(B.D.). Home pages: U761, http ://www.u761.lille.inserm.fr; PRIM,
http://www.drugdiscoverylille.org.
Chemistry
Compounds 1-3 (Figure 1) were synthesized as previously
described.10 Compound 1 and (Z)-2 were respectively a hit
r
pubs.acs.org/jmc
Published on Web 10/12/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6791
Scheme 4. Synthesis of Compounds 6-10a
Figure 1. Structures of compounds 1-3.
Scheme 1. Metabolic Hydrolysis of Hydroxamic Acids in Plasma
a Reagents and conditions: (a) (1) EtONa/EtOH, 1 h, 50 °C; (2)
C6H5CH2Br, 2 h, 50 °C, 83%. (b) KOH, abs EtOH, 4 h, room temp,
80%. (c) p-fluoro-benzylamine, EDCI/HOBt, DMF, DIEA, room
temp, 12 h. (d) KOH, abs EtOH, 12 h, room temp. (e) (1) Ethyl
chloroformate, TEA, CH2Cl2, 40 min, 0 °C; (2) H2NO-Trt, 1 h, room
temp; (3) TFA 2%/CH2Cl2, triisopropylsilane, 5 min room temp.
(f) H2NOTrt, DIEA, CH2Cl2, 3 h, room temp, 79%. (g) TFA 2%/
CH2Cl2, triisopropylsilane, 5 min room temp.
Scheme 2. Synthesis of Compound 4a
a Reagents and conditions: (a) (1) CDI, THF, DIEA, DMF, 1.5 h,
room temp; (2) p-F-C6H4-CH2-NH2, room temp, 3 h, 81%. (b) KOH,
abs EtOH, 12 h, room temp, 80%. (c) PyBrop, CH3-NH-OH, CH2Cl2,
DIEA/DMF, 12 h, room temp, 40%.
byproduct.31 Compounds 6 and 8-10 were obtained from the
corresponding substituted diethylmalonate, using N-tritylhy-
droxylamine (Scheme 4). Compound 6 required the synthesis
of the 2-benzyl-2-methylmalonic acid diethyl ester precursor.
Hydroxamate 7 was obtained from the chlorocarbonylacetic
acid ethyl ester (Scheme 4). Compound 11 differs from 1 by
the inversion of amide function and was obtained from
phenylalanine using a solid support strategy that allowed
both anchoring and protection of the hydroxamate moiety
(Scheme 5).
Scheme 3. Synthesis of Prodrug 5a
A secondseries of compounds wasinvestigated withtheaim
of varying the structure and length of the chain between the
terminal hydroxamic moiety and an aryl group (compounds
12-22).
a Reagents and conditions: (a) camphor sulfonic acid, CH2Cl2, 2 h,
room temp, (b) NaOH 0.1 M, dioxane, 3.5 h, room temp, 50%.
These compounds were obtained from the corresponding
carboxylic acid and O-trityl-hydroxylamine by activation
using either oxalylchloride or EDCI/HOBt (Scheme 6). For
compound 19, the acid precursor was synthesized by a
Sonogashira reaction from the corresponding acetylenic deri-
vative and 4-iodobenzoic ethyl ester (Scheme 6). Compound
22 derived from N-Boc-L-phenylalanine (Scheme 6). Finally,
SAHAand compounds 20-21were synthesized asreportedin
the literature (Scheme 7).32,33
and a lead, which were identified in our optimization program
aiming at the development of inhibitors of the plasmodial zinc
metalloprotease PfAM1.10 In the course of this program,
several analogues were developed for SAR purposes and also
for structure-plasma stability relationships (SPSR). Synth-
eses of compounds 4-11 are described in Schemes 2-5.
Compound 4 was obtained in three steps from 2-benzylma-
lonic acid monoethyl ester (Scheme 2). Coupling of N-methyl-
hydroxylamine required some optimization because the
classical protocol used for unsubstituted hydroxylamine
(e.g., activation by oxalyl chloride) was unsuccessful
(Table 1). The best activator was PyBropa (Table 1). Com-
pound 5 was designed as a prodrug of 1.30 Its synthesis
proceeds as described in Scheme 3, giving 5 with an overall
yield of 50%. Indeed, the ethyl ester derivative is a major
Plasma Stabilities
Rat plasma stabilities were evaluated for hydroxamates
1-22 and expressed as their corresponding half-lives. The
stability of 1 was also measured in the presence of phenyl-
methylsulfonyl fluoride (PMSF), an esterase inhibitor. Stabi-
lity of compounds 1, (Z)-2, (E)-2, 14, and 15 was further
evaluated in human plasma. In all cases, quantification was
performed in duplicate using LC-MSMS (MRM or SIM
detection modes) in the presence of an internal standard.
a Abbreviations: AcOEt, ethyl acetate; AcOH, acetic acid; CDI: N,N0-
carbonyldiimidazole; CH3CN, acetonitrile; DCM, dichloromethane;
DIEA, diisopropylethylamine; DMF, dimethylformamide; DMSO, di-
methylsulfoxide; EDCI, N-ethyl-3-(3-dimethyaminopropyl)-carbodii-
mide; Et2O, diethyl ether; EtOH, ethanol; EWG, electron-
withdrawing group; HOBT, N-hydroxybenzotriazole; MeOH, metha-
nol; PyBrop, bromo-tris-pyrrolidinophosphonium hexafluoropho-
sphate; TEA, triethylamine; TFA, trifluoroacetic acid; THF,
tetrahydrofuran; Trt, trityl.
Results and Discussion
Evidencing Esterase Implication. All compounds are stable
when incubated in potassium phosphate buffer (pH 7.4),

6792 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Scheme 5. Solid-Phase Synthesis of 11a
Flipo et al.
a Reagents and conditions: (a) piperidine 20%/DMF 80%, 30 min; (b) D,L-Fmoc-Phe-OH, HATU, DIEA, DMF, 12 h (twice); (c) piperidine
20%/DMF 80%, 30 min; (d) RCOOH, HOBt, TBTU, DIEA, DMF 3 h; (e) TFA 2%/CH2Cl2, triisopropylsilane, 2 min.
Table 1. Reaction Conditions for the Coupling of N-Methyl Hydroxylamine with 4b
reagent
solvent
temp (°C)
time (h)
conversiona (%)
27
comments
oxalyl chloride44
ethyl chloroformate
TBTU/HOBt
EDCI/HOBt45
PyBrop
DCM
DCM
DMF
DMF
DCM
0 then rt
0 then rt
4
1.5
complex mixture
obtention of diethyl ester
complex mixture
complex mixture
rt
rt
rt
overnight
overnight
overnight
60
25
70
a 4b conversion to final product determined by HPLC (215 nm).
Scheme 6. Synthesis of Compounds 19a, 12-22a
of ibuprofen that an important structural feature for resis-
tance to metabolism is the spacer unit between the hydro-
xamate group and the phenyl ring.35 They concluded that
introduction of a larger spacer enhances metabolism to the
corresponding carboxylic acid. However, we believe that the
dramatic drop in stability observed in our series between 13
and 14 cannot be explained solely by the increase of acces-
sibility of the carbonyl center. In fact, SAHA which has the
longest alkylidene spacer has an intermediate half-life of
9.7 h (Table 4). Rather, recognition of the phenyl group by
esterases is likely to be a key component of hydrolysis.
Interestingly, replacing a methylene moiety by an oxygen
atom does not alter the half-life (15 vs 18) or increases the
half-life by 100% (14 vs 17). Decreasing flexibility (due to the
introduction of a trans double bond) better protects from
hydrolysis (16 vs 14). Interestingly, hydroxamate 19 is less
stable than its analogue 12. An explanation could be that 19
has a greater lipophilicity (AlogP = 2.2 and 0.8 for 19 and 12,
respectively)36 and that this longer substituent enhances the
hydrophobic contact with the esterases.37 Along with the
possible hydrogen bond with NH in 22, this explanation
could also be valid for the difference between 14 and 22, the
latter bearing an additional hydrophobic tBoc-amino group
(AlogP = 1.3 and 1.8 for 14 and 22, respectively).
a Reagents and conditions: (a) cyclopropylacetylene, PdCl2(PPh3)2,
CuI, NEt3, DMF, 70 °C, 90%; (b) NaOH, EtOH, H2O, room temp,
98%; (c) (i) oxalylchloride (1.2 equiv), DCM, cat. DMF, 45 min, 0 °C; (ii)
DIEA (3 equiv), O-tritylhydroxylamine (0.85 equiv), DCM, 0 °C then
room temp, 3 h; or carboxylic acid 0.1 M/DMF (1 equiv), DIEA (2.4
equiv), EDCI (1.1 equiv), HOBt (1.1 equiv), room temp, 5 min then O-
tritylhydroxylamine (0.85 equiv) 0.1 M/DMF, DIEA (2 equiv), room
temp, 5 h; (d) TFA 2%/DCM, triisopropylsilane, 5 min, room temp.
suggesting that degradation occurring in rat plasma was
most likely enzymatic.34 To demonstrate the esterasic acti-
vity of plasma, we preincubated rat plasma with 2 mM
PMSF (phenylmethylsulfonyl fluoride), a known broad
spectrum serine-hydrolase inhibitor, in experiments aiming
at the measurement of the half-life of 1 (Table 2). The
increase in half-life in the presence of PMSF is similar to
that of enalapril, an ester prodrug known to be hydrolyzed
by plasma esterases, showing that hydrolysis is enzyme-
dependent.
Rat Plasma Stabilities. In vitro half-lives of compounds in
rat plasma are presented in Tables 3-6. The following
paragraphs are based largely on pairwise comparisons of
plasma stabilities. These results are consolidated and put
into perspective in the Summary.
Influence of Substituent and Spacer in Arylalkanoic Hydro-
xamate Derivatives. In the series designed to explore the
influence of the length and nature of the chain between the
aryl ring and the hydroxamate function, large variations of
half-lives are observed (11-22, Table 4). While benzohy-
droxamic and phenylacetohydroxamic acids 12 and 13 are
very stable, the homologous compound 14 is much less
stable. Summers et al. showed for hydroxamate derivatives
Phenylalanine derivatives 11 and 22 that bear a benzyl
group in alpha to the hydroxamate are rapidly hydrolyzed.
In contrast, compounds 20 and 21 are more stable because
they lack a benzyl substituent on the CR.
Influence of the Nature of the Substituent in Malonic
Hydroxamic series. Plasma stabilities for malonic compound
1 and analogues 2-4 and 6-10 are presented in Table 3. In
the malonic series, again, the nature of the substituant on the
malonic carbon (e.g., alpha to the hydroxamate function)
has a great impact on stability. Indeed, the half-lives range
from 0.8 to 33 h. For example, 7, which bears no substituent,
displays an intermediate stability of 10.5 h, comparable to
that of the glycine derivatives 20 and 21 in the previous series.
The shortest half-lives were observed for hydroxamic acids 1,
4, and 6. Again, as for the previous series, the benzyl
substituent appears to be deleterious for the stability as
it can be seen when comparing 1 and 7, as well as 6 and 8.
In contrast, introduction of a methyl group on the same
malonic carbon decreases the susceptibility to hydrolysis
(6 vs 1 and 8-10 vs 7). Interestingly, in the benzyl series,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6793
Scheme 7. Synthesis of Derivatives 20-21a
a Reagents and conditions: (a) DIEA, DMF, THF, room temp, 1 h; (b) NaH, ethylbromoacetate, THF, room temp, 12 h; (c) NH2OH HCl, NaOMe,
MeOH, room temp, 12 h.
3
Table 2. Influence of PMSF on Plasma Stability of 1 and Enalapril
(t1/2 in h)
Table 3. Rat Plasma Stabilities of Analogues of Malonyl Hydroxamic
Acid 1
compd
w/o PMSF
2 mM PMSF
1
enalapril
0.8
0.05
15
>24
methylation on the nitrogen of the hydroxamate did not
increase stability (4 vs 1 and 6).
Electronic and Geometric Effects. Three R,β-unsaturated
derivatives were prepared and tested ((E)-2, (Z)-2, and 3).
The three compounds are more stable than the saturated
analogue 1. This improved stability could be attributed to the
dispersion of the electrophilic character on two centers.
Another explanation could be the influence of steric
constraints caused by the insaturation. Indeed, for 2, the
Z configuration is much more stable than the E configura-
tion. This could be due to a better recognition by hydrolases
of the extended configuration of (E)-2 or a steric protection
of the electrophilic carbonyl by the aromatic ring in (Z)-2.
The high stability of 3 is probably mainly due to its cyclic
nature.
Summary
Comparison of half-lives of all direct analogues of phenyl-
propionohydroxamic acid 14 provides useful information
(Table 5). Conformationnally flexible analogues 14, 22, 1,
and 11 are globally highly unstable. Compounds (E)-2 and 16
that unveil the hydroxamate moiety are more stable than the
previous flexible compounds, probably due to the less electro-
philic carbonyl group vicinal to a double bond. (Z)-2 presents
two stabilizing features that are the steric hindrance of the
hydroxamate and the less electrophilic carbonyl group.
Finally, it is possible that an extended conformation ((E)-2,
1, and 14) of the arylated chain is favorable to the recognition
by esterases. A phenylbutanoic ester chain is also found in
enalapril which is very rapidly hydrolyzed.
We have shown that methylation of the R position to the
electophilic carbonyl increases in each case the stability. This
effect is consistent with the frequent occurrence of neopentyl
centers in R position to hydroxamic acids developed as lead
compounds. However, a highly substituted center is not
always required to obtain stable compounds, as we have
demonstrated here (SAHA, 7 vs 1). Gilmore et al. were
surprised that a compound lacking a substituent at the
R position is as stable as its neopentyl analogue.38 In our
opinion, this stability reflects more the fact that both com-
pounds are devoid of a correctly placed aryl substituent
favoring the hydrolysis.
Inconclusion, plasmastabilityofhydroxamates seemstobe
the result of two opposing factors (Figure 3). Stabilizing
factors are the steric hindrance around the hydroxamate

6794 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Table 4. Rat Plasma Stabilities of Hydroxamic Acids 11-22
Flipo et al.
Table 6. Enhancement of Apparent Half-Life of 1 Using Prodrug 5
Figure 2. Structures of NVP-LAQ824, panobinostat (NVP-
LBH589), and belinostat (PXD101).
Figure 3. Hydrolysis promoting or protecting factors. EWG: elec-
tron-withdrawing group.
Table 5. Comparing Stabilities of Analogues of 14
presence of an extended phenylpropiono- or phenylbutyro-
hydroxamic motif. These results allow us to hypothesize a
preliminary pharmacophore for plasma hydrolysis or stability
of hydroxamic acids (Figure 3).
In case of significant hydrolysis, it may be interesting to
design a prodrug. Only a few prodrugs of hydroxamates are
described.28,30 Our attempts to protect 1 as prodrug 5 did not
significantly improve the half-life (Table 6).39 Prodrug 5 is
hydrolyzed into 1 with a half-life of 0.3 h, and then compound
1 ishydrolyzed into the corresponding carboxylicacidin0.8 h,
resulting in an almost unchanged half-life of 1.
The result obtained with trans-cinnamic compound (Z)-2 is
of high interest in the light of the current development of
cinnamic inhibitors of botulinum neurotoxin A or histone
deacetylases (Figure 2).40,41 For example, the direct analogue
of cinnamic acid, belinostat PXD101, is currently evaluated in
18 clinical trials in cancer therapy.42
compd
R-
t1/2 (h)
14
H-
1.5
<1
0.8
22a
1
Boc-NH-
F-C6H4-CH2-NHCO-
F-C6H4-CH2-CONH-
H-
F-C6H4-CH2-NHCO-
F-C6H4-CH2-NHCO-
11
16
1.0
6.2
(E)-2
(Z)-2
4.1
22.0
a Derived from D-Phe.
group and the mesomeric effects that reduce the electro-
philic nature of the carbonyl. Among potential hydrolysis-
promoting factors, we have identified hydrophobicity and the
Several papers report species differences for the plasma
stability of amides or hydroxamates.43-45 It is known that
generally speaking, rat plasma is “more aggressive” than

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6795
Table 7. Comparison of Stability (t1/2 in h) in Various Media
2H), 7.08-7.37 (m, 10H), 8.86 (t, J = 6.0 Hz, NHCO), 9.08 (br s,
OH), 10.70 (br s, CONHO). trLCMS 4.41 min. MS [M þ H]þ
m/z 315.
compd
rat plasma
human plasma
buffer (PBS, pH = 7.4)
1
0.8
4.1
22
>24
>24
>24
>24
>24
>24
>24
>24
>24
>24
1-Hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylic Acid 4-
Fluoro-benzylamide (3). See Supporting Information; beige
powder (70%); purity 99%. 1H NMR (DMSO-d6) δ ppm:
4.58 (d, J = 6.0 Hz, 2H), 7.14-7.20 (m, 2H), 7.38-7.41 (m,
3H), 7.76-7.81 (m, 2H), 8.05 (d, J = 7.5 Hz, 1H), 8.85 (s, 1H),
10.06 (t, J = 6.0 Hz, NH), 11.82 (s, 1H, OH). 13C NMR
(DMSO-d6) δ ppm: 42.5, 113.2, 115.8 (d, JCF = 21 Hz), 118.4,
121.9, 123.9, 130.0 (d, JCF = 8 Hz), 130.8, 134.0, 135.9, 139.7,
141.4, 158.1, 161.9 (d, JCF = 240 Hz), 163.1. trLCMS 5.10 min.
MS [M þ H]þ m/z 313.
(E)-2
(Z)-2
14
1.5
2.0
15
human plasma.23 Not surprisingly, independently of their
half-lives in rat plasma, all our compounds are very stable in
human plasma (t1/2 > 24 h) (Table 7). The difference in
stability in human and rodent plasma remains a specific
hurdle in the development of hydroxamates. In fact, hydro-
xamates must be stable in preclinical in vivo models (often
rodents) for proof of concept. In this context, our results help
to rationalize structure-stability relationships. These should
help medicinal chemists to reconcile the pharmacophore of
their target and the structural requirements for rodent plasma
stability.
2-Benzyl-N-(4-fluoro-benzyl)-N0-hydroxy-N0-methyl-malona-
mide (4). 2-Benzyl-malonic acid diethyl ester (7.5 g, 30 mmol)
was added to a solution of KOH (1.68 g, 30 mmol) in EtOH
(45 mL). The solution was stirred at room temperature for 6 h
and evaporated. The residue was dissolved in NaHCO3 5%
(20 mL) and extracted with ethyl acetate. The aqueous layer was
acidified and extracted three times with ethyl acetate. The
combined organic layers were dried over MgSO4, filtered, and
evaporated to give (4a) as a colorless oil (86%). Purity 99%.
1H NMR (DMSO-d6) δ 1.23 (t, J = 7.2 Hz, 3H), 3.26 (d, J =
7.8 Hz, 2H), 3.73 (t, J = 7.8 Hz, 1H), 4.19 (q, J = 7.2 Hz, 2H),
7.23-7.34 (m, 5H), 10.11 (s, 1H, COOH). MS [M þ H]þ
m/z 223. 2-Benzyl-malonic acid monoethyl ester (4a) (7.556 g,
34 mmol) was dissolved in DMF (25 mL) and DIEA (7.1 mL,
41 mmol). CDI (6.06 g, 37.4 mmol) was dissolved in THF
(50 mL) and added to the carboxylic acid solution. The reaction
mixture was stirred at room temperature for 1.5 h and
4-fluorobenzylamine (3.872 mL, 34 mmol) dissolved in DMF
(40 mL) and DIEA (11.8 mL, 68 mmol) was added. The solution
was stirred at room temperature for 3 h and evaporated. The
crude product was dissolved in ethyl acetate, washed 10 times
with H2O, three times with KHSO4 solution (pH = 3), and with
aq NaCl, dried over MgSO4, filtered, and evaporated to give the
ester as a beige powder (81%). Purity 95%. 1H NMR (DMSO-
d6) δ 1.14 (t, J = 7.2 Hz, 3H), 3.04-3.10 (m, 2H), 3.69 (dd, J =
6.6 Hz, J = 9.0 Hz, 1H), 4.08 (q, J = 7.2 Hz, 2H), 4.19-4.23 (m,
2H), 7.05-7.09 (m, 4H), 7.19-7.28 (m, 5H), 8.60 (t, J = 5.4 Hz,
NH). trLCMS 6.02 min. MS [M þ H]þ m/z 330. The ester (8.88 g,
27 mmol) was added to a solution of KOH (4.54 g, 81 mmol) in
EtOH (50 mL). The solution was stirred at room temperature
overnight and evaporated. The residue was dissolved in H2O
(20 mL) and extracted with ethyl acetate. The aqueous layer was
acidified and extracted three times with ethyl acetate. The
combined organic layers were dried over MgSO4, filtered, and
evaporated to give (4b) as a white powder (80%). Purity 99%. 1H
NMR (DMSO-d6) δ 2.96-3.09 (m, 2H), 3.60 (dd, J = 6.3 Hz, J =
9.3 Hz, 1H), 4.11 (dd, J = 5.4 Hz, J = 15.3 Hz, 1H), 4.11 (dd, J =
6.3 Hz, J = 15.3 Hz, 1H), 6.97-7.06 (m, 4H), 7.16-7.29 (m, 5H),
8.53 (t, J = 6.0 Hz, NHCO), 12.61 (s, 1H, COOH). trLCMS
4.93 min. MS [M þ H]þ m/z 302. 2-Benzyl-N-(4-fluoro-benzyl)-
malonamic acid (4b) was dissolved in DMF (4 mL), and DIEA
(1.2 equiv, 0.96 mmol) and PyBrop (0,96 mmol, 124 mg) were
added. The solution was stirred at room temperature for 1 min and
Experimental Section
Chemistry. General Information. 2-Chlorotrityl N-Fmoc-
hydroxylamine, polymer-bound, 100-200 mesh, was purchased
from Sigma-Aldrich Inc. NMR spectra were recorded on a
Bruker DRX-300 spectrometer. Chemical shifts are in parts
per million (ppm). The assignments were made using one-
dimensional (1D) 1H and 13C spectra and two-dimensional
(2D) HSQC and COSY spectra. Mass spectra were recorded
on a MALDI-TOF Voyager-DE-STR spectrometer or with a
LCMS-MS triple-quadrupole system (Varian 1200ws). The
purities of the desired compounds were confirmed by reversed
phase HPLC or LCMS, using UV detection (215 nM): HPLC
analyses were performed using a C18 TSK-GEL Super ODS
2 μm column (dimensions 50 mm ꢀ 4.6 mm). A gradient starting
from 100% H2O/0.05% TFA and reaching 20% H2O/80%
CH3CN /0.05% TFA within 10 min at a flow rate of 1 mL/
min was used. LCMS gradient starting from 100% H2O/0.1%
formic acid and reaching 20% H2O /80% CH3CN/0.08%
formic acid within 10 min at a flow rate of 1 mL/min was used.
€
Melting points were measured on a Buchi B-540 apparatus and
are uncorrected. All commercial reagents and solvents were used
without further purification. Organic layers obtained after
extraction of aqueous solutions were dried over MgSO4 and
filtered before evaporation in vacuo. Thick layer chromatogra-
phy was performed with Silica Gel 60 (Merck, 40-63 μm).
Purification yields were not optimized.
2-Benzyl-N-(4-fluoro-benzyl)-N0-hydroxy-malonamide (1). See
Supporting Information; white powder; yield 88%; purity 100%.
1H NMR (DMSO-d6) δ ppm: 2.96-3.10 (m, 2H), 3.35-3.40 (m,
1H), 4.16 (dd, J = 15, J = 5.7 Hz, 1H), 4.27 (dd, J = 15, J = 6 Hz,
1H), 7.04-7.27 (m, 9H), 8.40 (t, J= 5.6 Hz, NHCO), 8.93 (s, OH),
10.58 (s, CONHO). 13C NMR (DMSO-d6) δ ppm: 35.0, 42.1,
52.5, 115.5 (d, JCF = 21.1 Hz), 126.8, 128.8, 129.4, 129.5 (d, JCF
=
8.4 Hz), 136.0 (d, JCF = 2.3 Hz), 139.6, 161.7 (d, JCF = 240.4 Hz),
166.3, 168.9. trLCMS 4.31 min. MS [M þ H]þ m/z 317. mp 193-
194 °C.
N-methylhydroxylamine HCl in DMF (4 mL), and DIEA
3
(2.4 equiv, 332 μL) was added. The solution was stirred at room
temperature overnight and evaporated. The residue was dissolved
in ethyl acetate and washed three times with aq NaHCO3 5% and
once with aq NaCl, dried over MgSO4, filtered, and evaporated.
The residue was purified by TLC (DCM/MeOH 96/4) to give (4) as
a white powder (40%). Purity 99%. 1H NMR (DMSO-d6) δ 2.95
(dd, J=7.1Hz, J= 13.6 Hz, 1H), 3.07 (s, 3H), 3.04-3.11 (m, 1H),
4.03 (t, J = 7.0 Hz, 1H), 4.16 (dd, J = 5.7 Hz, J = 15.3 Hz, 1H),
4.27 (dd, J = 6.1 Hz, J = 15.1 Hz, 1H), 7.05-7.27 (m, 9H), 8.32 (s,
NH), 9.93 (s, OH). 13C NMR (DMSO-d6) δ ppm: 35.1, 36.5, 41.9,
51.1, 115.3 (d, JCF = 21.1 Hz), 126.5, 128.5, 129.3, 129.4, 136.0,
140.1, 161.5 (d, JCF = 241.7 Hz), 169.2, 169.4. tR LCMS 4.69 min.
MS [M þ H]þ m/z 331.
N-(4-Fluoro-benzyl)-N0-hydroxy-2-[1-phenyl-meth-(Z)-ylidene]-
malonamide ((Z)-2). See Supporting Information; white powder;
1
yield 96%; purity 99%. H NMR (DMSO-d6) δ 4.37 (d, J =
6.0 Hz, 2H), 7.12-7.18 (m, 2H), 7.32-7.40 (m, 5H), 7.43 (s, 1H),
7.51-7.54 (m, 2H), 8.26 (t, J = 6.0 Hz, NHCO), 9.13 (s, OH),
11.01 (s, CONHO). 13C NMR (DMSO-d6) δ ppm: 42.6, 115.6
(d, JCF = 21 Hz), 129.3, 129.5, 129.9, 130.1, 130.6, 134.2, 136.2,
137.3, 161.8 (d, JCF = 246 Hz), 163.5, 164.6. trLCMS 4.23 min. MS
[M þ H]þ m/z 315.
2-[1-Phenyl-meth-(Z)-ylidene]-malonic Acid Monoethyl Ester
((E)-2). See Supporting Information; white powder; yield 70%;
purity 95%. 1H NMR (DMSO-d6) δ ppm: 4.30 (d, J = 6.0 Hz,

6796 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Flipo et al.
2-(5,5-Dimethyl-[1,4,2]dioxazol-3-yl)-N-(4-fluoro-benzyl)-3-
phenyl-propionamide (5). 2-Benzyl-N-(4-fluoro-benzyl)-N0-hy-
droxy-malonamide (1) (1.00 g, 3.16 mmol) was dissolved
in DCM (100 mL) and 2,2-diethoxypropane (1.528 mL,
9.48 mmol), and camphorsulfonic acid (734 mg, 3.16 mmol)
were added. The reaction mixture was stirred at room tem-
perature for 2 h, and aq Na2CO3 solution (20 mL) was added.
The aqueous layer was extracted four times with diethyl ether.
The combined organic layers were dried over MgSO4, filtered,
and evaporated. The residue was dissolved in dioxane (8 mL),
and 0.1 N NaOH solution (8 mL) was added. The reaction
mixture was stirred at room temperature for 3.5 h, water was
added, and reaction mixture was extracted three times with
ethyl acetate. The combined organic layers were dried over
MgSO4, filtered, and evaporated. The residue was purified by
TLC (DCM/MeOH 96/4 to give (5) as a white powder (50%)
purity 96%. 1H NMR (CDCl3) δ 1.44 (s, 3H), 1.46 (s, 3H), 3.11
(dd, J = 8.1, J = 13.8 Hz, 1H), 3.30 (dd, J = 7.2, J = 14.1 Hz,
1H), 3.60 (t, J = 7.8 Hz, 1H), 4.32-4.38 (m, 2H), 6.78 (br s,
NH), 6.90-6.96 (m, 2H), 7.06-7.10 (m, 2H), 7.17-7.26 (m,
5H). trLCMS 6.20 min. MS [M þ H]þ m/z 357.
7.07-7.26 (m, 9H), 8.30 (t, J = 6.0 Hz, NH). tR LCMS 5.40 min.
MS [M þ H]þ m/z 316. Acid (317 mg, 1 mmol) was dissolved in
DCM (8 mL) and TEA (155 μL, 1.1 mmol). Ethyl chloroformate
(105 μL, 1.1 mmol) was added dropwise at 0 °C, the solution was
stirred for 40 min at 0 °C, and O-tritylhydroxylamine (206 mg,
0.75 mmol) in DCM (2 mL) was added. The solution was stirred
at room temperature for 1 h and evaporated. The residue was
dissolved in ethyl acetate and washed four times with aq
NaHCO3 5% and once with 1N HCl solution and once with
aq NaCl, dried over MgSO4, filtered, and evaporated. The crude
product was purified by TLC (DCM/MeOH 98/2) to give
2-benzyl-N-(4-fluoro-benzyl)-2-methyl-N0-trityloxy-malonamide
as a white powder (60%). Purity 98%. 1H NMR (DMSO-d6) δ
0.95 (s, 3H), 2.65 (d, J = 13.5 Hz, 1H), 3.03 (d, J = 13.5 Hz, 1H),
4.14 (d, J = 5.7 Hz, 2H), 6.83-6.85 (m, 2H), 7.04-7.15 (m, 7H),
7.33 (s, 15H), 8.21 (t, J = 5.7 Hz, NH), 10.13 (s, CONHO).
trLCMS 8.59 min. MS [M - H]- m/z 571. The protected hydro-
xamic acid (239 mg, 0.4 mmol) was dissolved in TFA 2%/DCM
(0.03 M), and triisopropylsilane was added dropwise until the
yellow color disappeared. The reaction mixture was stirred
5 min at room temperature, solvents were removed under reduced
pressure, and the residue was washed with petroleum ether and
purified by TLC (DCM/MeOH/TEA 6/1.5/2.5) to give (6) as a
beige powder (60%). Purity 99%. 1H NMR (CD2Cl2) δ: 1.24 (s,
3H), 2.99 (s, 2H), 4.34 (s, 2H), 7.00-7.23 (m, 9H). 13C NMR
(CDCl3) δ ppm: 17.8, 43.3, 44.8, 53.1, 115.5 (d, JCF = 21.3 Hz),
127.2, 128.4, 129.5 (d, JCF = 7.5 Hz), 130.0, 133.2, 135.4,
162.2 (d, JCF = 244.4 Hz), 170.1, 172.1. trLCMS 4.66 min. MS
[M þ H]þ m/z 331.
2-Benzyl-N-(4-fluoro-benzyl)-N0-hydroxy-2-methyl-malonamide
(6). Sodium (633 mg, 27.5 mmol) was added slowly to absolute
EtOH (30 mL) at 0 °C. The solution was stirred at room
temperature for 30 min, and diethylmethylmalonate (4.30 mL,
25 mmol) was added. The reaction mixture was stirred at 50 °C for
1 h, and benzylbromide (2.392 mL, 20 mmol) was added. The
reaction mixture was stirred at 50 °C for 2 h and evaporated. The
residue was dissolved in DCM and washed three times with aq
NaHCO3 5%, once with NaOH solution (1N) and with H2O,
dried over MgSO4, filtered, and evaporated to give 2-benzyl-2-
methyl-malonic acid diethyl ester (6a) as a colorless oil (83%).
Purity 95%. 1H NMR (CD2Cl2) δ 1.27 (t, J = 7.2 Hz, 6H), 1.32 (s,
3H), 3.22 (s, 2H), 4.20 (q, J = 7.2 Hz, 4H), 7.13-7.16 (m, 2H),
7.23-7.33 (m, 3H). trLCMS 6.93 min. MS [M þ Na]þ m/z 287.
Diester (6a) (3.854 g, 14.6 mmol) was added to a solution of KOH
(819 mg, 14.6 mmol) in EtOH (50 mL). The solution was stirred at
room temperature for 4 h and evaporated. The residue was
dissolved in NaHCO3 5% solution (20 mL) and washed with
DCM. The aqueous layer was acidified and extracted three times
with DCM. The combined organic layers were dried over MgSO4,
filtered, and evaporated to give 2-benzyl-2-methyl-malonic acid
monoethyl ester as a white powder (80%). Purity 97%. 1H NMR
(CD2Cl2) δ: 1.30 (t, J = 7.2 Hz, 3H), 1.43 (s, 3H), 3.21 (d, J = 13.5
Hz, 1H), 3.32 (d, J = 13.5 Hz, 1H), 4.23 (q, J = 7.2 Hz, 2H),
7.17-7.20 (m, 2H), 7.28-7.35 (m, 3H). MS [M - H]- m/z 235.
2-Benzyl-2-methyl-malonic acid monoethyl ester (8.2 mmol)
was dissolved in DMF (20 mL) and DIEA (4.966 mL,
28.7 mmol). EDCI (1.895 g, 9.8 mmol) and HOBt (1.505 g,
9.8 mmol) were added, the solution was stirred for 5 min, and
4-fluorobenzylamine (942 μL, 8.2 mmol) was added. The solu-
tion was stirred at room temperature overnight and evaporated.
The residue was dissolved in ethyl acetate and washed four times
with aq NaHCO3 5% and once with 1N HCl solution and once
with aq NaCl, dried over MgSO4, filtered, and evaporated to
give 2-benzyl-N-(4-fluoro-benzyl)-2-methyl-malonamic acid
ethyl ester as a white powder (66%). Purity 98%. 1H NMR
(DMSO-d6) δ 1.14 (t, J = 7.2 Hz, 3H), 1.21 (s, 3H), 3.10 (d, J =
13.2 Hz, 1H), 3.17 (d, J = 13.2 Hz, 1H), 4.08 (q, J = 7.2 Hz, 2H),
4.18-4.32 (m, 2H), 7.06-7.28 (m, 9H), 8.33 (t, J = 6.0 Hz, NH).
trLCMS 6.58 min. MS [M þ H]þ m/z 344. Ester (3.8 mmol) was
added to a solution of KOH (6.405 g, 11.4 mmol) in EtOH
(9 mL). The solution was stirred at room temperature overnight
and evaporated. The residue was dissolved in H2O and washed
with DCM. The aqueous layer was acidified and extracted three
times with DCM. The combined organic layers were dried over
MgSO4, filtered, and evaporated to give 2-benzyl-N-(4-fluoro-
benzyl)-2-methyl-malonamic acid as a beige powder (84%).
N-(4-Fluoro-benzyl)-N0-hydroxy-malonamide (7). O-Tritylhy-
droxylamine (468 mg, 1.7 mmol) was dissolved in DCM
(10 mL), and DIEA (346 μL, 2 mmol) was added. The flask
was put in an ice bath, and chlorocarbonylacetic acid ethyl ester
(2 mmol, 253 μL) was added dropwise. Reaction mixture was
stirred at room temperature for 3 h and washed with aq
NaHCO3 5% (six times) and once with aq NaCl, dried over
MgSO4, filtered, and evaporated. The residue was washed with
petroleum ether to give N-trityloxy-malonamic acid ethyl ester
1
as a white powder (79%). Purity 99%. H NMR (DMSO-d6)
δ ppm: 1.10 (t, J = 7.2 Hz, 3H), 2.94 (s, 2H), 3.97 (q, J =
7.2 Hz, 2H), 7.32 (s, 15H), 10.47 (s, NH). trLCMS 5.10 min. MS
(M - H)- m/z 388. N-Trityloxy-malonamic acid ethyl ester
(468 mg, 1.2 mmol) was dissolved in DCM (7 mL), and KOH
(202 mg, 3.6 mmol) was added as a solution in EtOH (10 mL).
The reaction mixture was stirred at room temperature overnight
and evaporated. The residue was dissolved in H2O and washed
with DCM (three times). The aqueous layer was acidified
by KHSO4 (pH = 6) and extracted with DCM (four times).
The combined organic layers were dried over MgSO4, filtered,
and evaporated to give N-trityloxy-malonamic acid as a white
powder (90%). Purity 98%. 1H NMR (DMSO-d6) δ ppm: 2.84
(s, 2H), 7.32 (s, 15H). trLCMS 5.68 min. MS [M - H]- m/z 360.
Acid (360 mg, 1 mmol) was dissolved in DMF (10 mL), and
DIEA (346 μL, 2 mmol), EDCI (210 mg, 1.1 mmol), and HOBt
(168 mg, 1.1 mmol) were added. The reaction mixture was
stirred at room temperature for 5 min, and 4-fluorobenzylamine
(115 μL, 1 mmol) was added. The reaction mixture was stirred at
room temperature overnight and evaporated. The crude pro-
duct was dissolved in DCM, washed three times with 5% aq
NaHCO3 and once with aq NaCl, dried over MgSO4, filtered,
and evaporated. The residue was washed with petroleum ether
to give N-(4-Fluoro-benzyl)-N0-trityloxy-malonamide as a
white powder (85%). Purity 95%. 1H NMR (DMSO-d6)
δ ppm: 2.72 (s, 2H), 4.15 (d, J = 6.0 Hz, 2H), 7.07-7.31 (m,
19H), 8.68 (br s, 1H, NHCO), 10.43 (br s, 1H, CONHO). trLCMS
6.85 min. MS [M - H]- m/z 467. The protected hydroxamic acid
(190 mg, 0.4 mmol) was dissolved in TFA 2%/DCM (0.03 M),
and triisopropylsilane was added dropwise until the yellow color
disappeared. The reaction mixture was stirred 5 min at room
temperature, solvents were removed under reduced pressure,
1
Purity 95%. H NMR (CD2Cl2) δ 1.86 (s, 3H), 3.06 (d, J =
13.2 Hz, 1H), 3.16 (d, J = 13.2 Hz, 1H), 4.25 (d, J = 6.0 Hz, 2H),

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6797
and the residue was washed with petroleum ether to give (7) as a
beige powder (90%). Purity 97%. 1H NMR (DMSO-d6) δ 2.95
(s, 2H), 4.26 (d, J = 5.4 Hz, 2H), 7.10-7.16 (m, 2H), 7.29-7.33
(m, 2H), 8.51 (br s, NHCO), 8.93 (br s, OH), 10.56 (s, CONHO).
(q, J = 7.2 Hz, 1H), 4.37 (s, 2H), 7.02-7.07 (m, 2H), 7.29-7.34
(m, 2H). 13C NMR (CD3OD) δ ppm: 14.2, 42.0, 45.0, 114.7 (d,
JCF = 22.0 Hz), 128.9 (d, JCF = 8.2 Hz), 134.4, 162.1 (d,
J
m/z 241.
CF = 244 Hz), 169.1, 171.1. trLCMS 2.98 min. MS [M þ H]þ
13C NMR (DMSO-d6) δ ppm: 41.3, 41.9, 115.5 (d, JCF
=
21.7 Hz), 129.5 (d, JCF = 7.8 Hz), 135.9, 138.1, 161.6 (d,
N-(4-Fluoro-benzyl)-N0-hydroxy-2,2-dimethyl-malonamide (9).
J
m/z 227.
CF = 239.2 Hz), 164.2, 166.9. trLCMS 2.81 min. MS [M þ H]þ
2,2-Dimethylmalonic acid diethyl ester (950 μL, 5 mmol) was
dissolved in EtOH (20 mL), and KOH (280 mg, 5 mmol) was
added. The solution was stirred at room temperature for 4 h and
evaporated. The residue was dissolved in a 5% NaHCO3 solution
and washed with DCM. The aqueous layer was acidified (pH =
1) and extracted three times with DCM. The combined organic
layers were dried over MgSO4, filtered, and evaporated to give
2,2-dimethylmalonic acid diethyl ester as a colorless oil (79%).
1H NMR (CDCl3) δ 1.27 (t, J = 7.2 Hz, 3H), 1.46 (s, 6H), 4.20 (q,
J = 7.2 Hz, 2H). 2,2-Dimethylmalonic acid diethyl ester (575 mg,
3.5 mmol) was dissolved in DMF (10 mL) and DIEA (2.12 mL,
12.2 mmol). EDCI (805 mg, 4.2 mmol) and HOBt (643 mg,
4.2 mmol) were added, and the reaction mixture was stirred at
room temperature for 5 min. 4-Fluorobenzylamine (402 μL,
3.5 mmol) was added, and the solution was stirred overnight
and evaporated. The residue was dissolved in DCM and washed
three times with aq NaHCO3 5% and three times with 1N HCl
solution and once with brine, dried over MgSO4, filtered, and
evaporated to give N-(4-fluoro-benzyl)-2,2-dimethyl-malonamic
acid ethyl ester as a white powder (85%). Purity 95%. 1H NMR
(CD2Cl2) δ: 1.26 (t, J = 7.2 Hz, 3H), 1.47 (s, 6H), 4.18 (q, J =
7.2 Hz, 2H), 4.42 (d, J = 5.7 Hz, 2H), 6.67 (br s, NH), 7.01-7.09
(m, 2H), 7.24-7.30 (m, 2H). trLCMS 5.01 min. MS [M þ H]þ
m/z 268. Ester (535 mg, 2 mmol) was added to a solution of KOH
(337 mg, 6 mmol) in EtOH (7 mL). The solution was stirred at
room temperature overnight and evaporated. The residue was
dissolved in H2O and washed with DCM. The aqueous layer was
acidified and extracted three times with DCM. The combined
organiclayersweredried over MgSO4, filtered, andevaporated to
give N-(4-fluoro-benzyl)-2,2-dimethyl-malonamic acid as a white
powder (80%). Purity 99%. 1H NMR (DMSO-d6) δ 1.31 (s, 6H),
4.24 (d, J = 6 Hz, 2H), 7.09-7.15 (m, 2H), 7.27-7.28 (m, 2H),
8.25 (t, J = 6 Hz, NH), 12.53 (br s, COOH). trLCMS 3.78 min. MS
[M - H]- m/z 238. Acid (48mg, 0.2mmol) was dissolvedinDCM
(1.5 mL) with catalytic DMF (10 μL). The mixture was cooled at
0 °C (ice bath), and oxalyl chloride (20.6 μL, 0.24 mmol) was
added dropwise. The reaction mixture was stirred 45 min at
0 °C and then evaporated under reduced pressure. The residue
was dissolved in DCM (1.5 mL) and cooled at 0 °C. DIEA
(123 μL, 0.48 mmol) was added, and then O-trityl-hydroxylamine
(41 mg, 0.15 mmol) was added. The reaction mixture was stirred
2 h at room temperature. The residue was dissolved in ethyl
acetate and washed with NaHCO3 5% and brine. The organic
phase was washed dried over MgSO4 and evaporated under
reduced pressure. The crude product was purified by TLC
(DCM/MeOH 95/5) to give N-(4-fluoro-benzyl)-2,2-dimethyl-
N0-trityloxy-malonamide as a white powder (63%). Purity 99%.
trLCMS 7.60 min. MS [M - H]- m/z 495. The protected hydro-
xamic acid (31 mg, 0.062 mmol) was dissolved in TFA 2%/DCM
(0.03 M), and triisopropylsilane was added drop by drop until the
yellow color disappeared. The reaction mixture was stirred 5 min
at room temperature, solvents were removed under reduced
pressure, and the residue was washed with petroleum ether and
purified by TLC (DCM/MeOH/TEA 6/1.5/2.5) to give (9) as a
beige powder (75%). Purity 99%. 1H NMR (CD3OD) cis/trans
isomer mixture (55/45) δ: 1.31 (s, 1.35H), 1.41 (s, 1.35H), 1.44
(s, 3.3H), 4.12 (s, 1.1 H), 4.37 (s, 0.9H), 7.04 (t, J = 8.7 Hz, 0.9H),
7.20 (t, J = 8.7 Hz, 1.1H), 7.28-7.31 (m, 0.9H), 7.44-7.48 (m,
1.1H). trLCMS 3.18 min. MS [M þ H]þ m/z 255.
N-(4-Fluoro-benzyl)-N0-hydroxy-2-methyl-malonamide (8).
2-Methyl-malonic acid diethyl ester (871 mg, 5 mmol) was
dissolved in EtOH (20 mL), and KOH (280 mg, 5 mmol) was
added. The reaction mixture was stirred at room temperature
for 4 h and evaporated. The residue was dissolved in aq 5%
NaHCO3 solution and washed with DCM (five times). The
aqueous layer was acidified (pH = 1) and extracted with DCM
(4 times). The combined organic layers were dried over
MgSO4, filtered, and evaporated to give 2-methyl-malonic
acid monoethyl ester as a white powder (71%). Purity 98%.
1H NMR (CDCl3) δ ppm: 1.29 (t, J = 7.2 Hz, 3H), 1.46 (d, J =
7.5 Hz, 3H), 3.47 (q, J = 7.5 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H),
8.88 (br s, COOH). Acid (518 mg, 3.5 mmol) was dissolved
in DMF (10 mL), and DIEA (2.12 mL, 12.2 mmol), EDCI
(805 mg, 4.2 mmol), and HOBt (643 mg, 4.2 mmol) were
added. The reaction mixture was stirred at room temperature
for 5 min, and 4-fluorobenzylamine (402 μL, 3.5 mmol) was
added. The reaction mixture was stirred at room temperature
overnight and evaporated. The crude product was dissolved in
DCM, washed three times with aq NaHCO3 5% and once with
aq NaCl, dried over MgSO4, filtered, and evaporated. The
residue was washed with petroleum ether to give N-(4-fluoro-
benzyl)-2-methyl-malonamic acid ethyl ester as a white pow-
der (75%). Purity 95%. 1H NMR (CD2Cl2) δ ppm: 1.27 (t, J =
7.2 Hz, 3H), 1.43 (d, J = 7.2 Hz, 3H), 3.33 (q, J = 7.2 Hz, 1H),
4.19 (q, J = 7.2 Hz, 2H), 4.38 (dd, J = 6.0 Hz, J = 15.0 Hz,
1H), 4.47 (dd, J = 6.0 Hz, 15.0 Hz, 1H), 6.73 (br s, NH),
7.02-7.10 (m, 2H), 7.25-7.31 (m, 2H). trLCMS 4.52 min. MS
[M þ Na]þ m/z 276. Ester (659 mg, 2.6 mmol) was dissolved in
EtOH (10 mL), and KOH (440 mg, 7.8 mmol) was added. The
reaction mixture was stirred at room temperature overnight
and evaporated. The residue was dissolved in H2O and washed
with DCM (three times). The aqueous layer was acidified
(pH = 1) and extracted with DCM (four times). The combined
organic layers were dried over MgSO4, filtered, and evapo-
rated to give N-(4-Fluoro-benzyl)-2-methyl-malonamic acid
as a white powder (45%). Purity 99%. 1H NMR (DMSO-d6)
δ ppm: 1.19 (d, J = 7.2 Hz, 3H), 3.32 (q, J = 7.2 Hz, 1H), 4.22
(dd, J = 6.0 Hz, 15.3 Hz, 1H), 4.30 (dd, J = 6.0 Hz, 15.3 Hz,
1H), 7.10-7.18 (m, 2H), 7.27-7.31 (m, 2H), 8.58 (t, J =
6.0 Hz, NH), 12.49 (br s, COOH). trLCMS 3.47 min. MS
[M - H]- m/z 224. The previous carboxylic acid (115 mg,
0.5 mmol) was dissolved in DMF (5 mL), and DIEA (260 μL,
1.5 mmol), EDCI (108 mg, 0.55 mmol), and HOBt (86 mg,
0.55 mmol) were added. The reaction mixture was stirred
at room temperature for 5 min, and O-tritylhydroxylamine
(103 mg, 0.38 mmol) was added. The reaction mixture was
stirred at room temperature overnight and evaporated. The
crude product was dissolved in ethyl acetate, washed three
times with aq 1 N NaOH solution and once with aq NaCl,
dried over MgSO4, filtered, and evaporated. The residue was
purified by TLC (DCM/MeOH 97/3) and precipitated in
petroleum ether to give N-(4-fluoro-benzyl)-2-methyl-N0-tri-
tyloxy-malonamide as a white powder (19%). Purity 99%.
trLCMS 7.05 min. MS [M - H]- m/z 481. The protected hydro-
xamic acid (34 mg; 0.07 mmol) was dissolved in TFA 2%/
DCM (0.03 M), and triisopropylsilane was added drop by
drop until the yellow color disappeared. The reaction mixture
was stirred 5 min at room temperature, solvents were removed
under reduced pressure, and the residue was washed with
petroleum ether to give (8) as a white powder (89%). Purity
Cyclopropane-1,1-dicarboxylic Acid 4-Fluoro-benzylamide
Hydroxyamide (10). Cyclopropane-1,1-dicarboxylic acid dieth-
yl ester (887 μL, 5 mmol) was dissolved in EtOH (20 mL), and
KOH (280 mg, 5 mmol) was added. The solution was stirred at
room temperature for 4 h and evaporated. The residue was
1
97%. H NMR (CD3OD) δ 1.41 (d, J = 7.2 Hz, 3H), 3.17

6798 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Flipo et al.
dissolved in a 5% NaHCO3 solution and washed with DCM.
The aqueous layer was acidified (pH = 1) and extracted three
times with DCM. The combined organic layers were dried over
MgSO4, filtered, and evaporated to give cyclopropane-1,
1-dicarboxylic acid ethyl ester (79%). 1H NMR (CDCl3)
δ 1.28 (t, J = 7.2 Hz, 3H), 1.73-1.78 (m, 2H), 1.82-1.86 (m,
2H), 4.25 (q, J = 7.2 Hz, 2H). The previous carboxylic acid
(568 mg, 3.5 mmol) was dissolved in DMF (10 mL) and DIEA
(2.12 mL, 12.2 mmol). EDCI (805 mg, 4.2 mmol) and HOBt
(643 mg, 4.2 mmol) were added, and the reaction mixture was
stirred at room temperature for 5 min, 4-fluorobenzylamine
(402 μL, 3.5 mmol) was added, and the solution was stirred
overnight and evaporated. The residue was dissolved in DCM
and washed three times with aq NaHCO3 5% and three times
with 1N HCl solution and once with brine, dried over MgSO4,
filtered, and evaporated to give 1-(4-fluoro-benzylcarbamoyl)-
cyclopropanecarboxylic acid ethyl ester (59%). Purity 95%. 1H
NMR (CD2Cl2) δ 1.24 (t, J = 7.2 Hz, 3H), 1.57-1.69 (m, 4H),
4.13 (q, J = 7.2 Hz, 2H), 4.47 (d, J = 5.7 Hz, 2H), 7.02-7.10 (m,
2H), 7.29-7.35 (m, 2H), 9.10 (br s, NH). trLCMS 5.52 min. MS
[M þ H]þ m/z 266. Ester (530 mg, 2 mmol) was added to a
solution of KOH (337 mg, 6 mmol) in EtOH (7 mL). The
solution was stirred at room temperature overnight and evapo-
rated. The residue was dissolved in H2O and washed with DCM.
The aqueous layer was acidified and extracted three times with
DCM. The combined organic layers were dried over MgSO4,
filtered, and evaporated to give 1-(4-fluoro-benzylcarbamoyl)-
cyclopropanecarboxylic acid as a white powder (87%). Purity
98%, 1H NMR (DMSO-d6) δ 1.34-1.42 (m, 4H), 4.33 (d, J =
5.7 Hz, 2H), 7.10-7.18 (m, 2H), 7.28-7.34 (m, 2H), 9.06 (t, J =
5.7 Hz, NH), 13.07 (br s, COOH). trLCMS 3.96 min. MS [M -
H]- m/z 236. Acid (47 mg, 0.2 mmol) was dissolved in DCM
(1.5 mL) with catalytic DMF (10 μL). The mixture was cooled at
0 °C (ice bath), and oxalyl chloride (20.6 μL, 0.24 mmol) was
added dropwise. The reaction mixture was stirred 45 min at 0 °C
and then evaporated under reduced pressure. The residue was
dissolved in DCM (1.5 mL) and cooled at 0 °C. DIEA (123 μL,
0.48 mmol) was added, and then O-trityl-hydroxylamine (41 mg,
0.15 mmol) was added. The reaction mixture was stirred 2 h
at room temperature. The residue was dissolved in ethyl
acetate and washed with NaHCO3 5% and brine. The organic
phase was washed, dried over MgSO4, and evaporated under
reduced pressure. The crude product was purified by TLC
(DCM/MeOH 95/5) to give cyclopropane-1,1-dicarboxylic acid
4-fluoro-benzylamide trityloxy-amide as a yellow powder
(49%). Purity 99%. trLCMS 7.52 min. MS [M - H]- m/z 493.
The protected hydroxamic acid (32 mg, 0.065 mmol) was
dissolved in TFA 2%/DCM (0.03 M), and triisopropylsilane
was added dropwise until the yellow color disappeared. The
reaction mixture was stirred 5 min at room temperature, sol-
vents were removed under reduced pressure, and the residue was
washed with petroleum ether and purified by TLC (DCM/
MeOH/TEA 6/1.5/2.5) to give (10) as a beige powder (75%).
Purity 99%. 1H NMR (CD3OD) cis/trans isomer mixture (65/
35) δ 1.29-133 (m, 2H), 1.37-1.41 (m, 2H), 4.12 (s, 0.7 H), 4.39
(s, 1.3 H), 7.05 (t, J = 8.7 Hz, 1.3H), 7.20 (t, J = 8.7 Hz, 0.7H),
7.29-7.35 (m, 1.3H), 7.46-7.51 (m, 0.7H). 13C NMR (CD3OD)
DMF and DIEA (895 μL, 5.42 mmol, 8 equiv). The mixture was
then added to the resin. The resin was shaken overnight at room
temperature and then washed with DMF and DCM. The
coupling was performed twice. To remove the Fmoc protecting
group, the resin was treated with a piperidine/DMF 20/80
cocktail for 30 min. Half of the resin was used for the next step.
The resin was washed with DMF. 4-Fluorophenylacetic acid
(210 mg, 1.35 mmol, 4 equiv) was activated with HOBt
(210 mg, 1.35 mmol, 4 equiv) and TBTU (435 mg, 1.35 mmol,
4 equiv) in 7 mL of DMF and DIEA (450 μL, 2.71 mmol,
8 equiv). The mixture was then added to the resin. The resin was
shaken 3 h at room temperature then washed with DMF and
DCM. The coupling was performed twice, and the resin was
washed with DCM. Cleavage from the resin was accomplished
by treatment with a mixture of TFA (100 μL), TIS (50 μL) in
DCM (5 mL) for 2 min. The resin was filtered, and the cleavage
was performed five times. The filtrate was neutralized with a
piperidine/methanol/water 10/45/45 mixture to avoid the con-
version of hydroxamic acid into carboxylic acid. The organic
solvents were evaporated under reduced pressure, and the aqu-
eous phase was extracted four times with ethyl acetate. The
combined organic layers were washed with water and brine and
then dried over MgSO4 and evaporated. The residue was washed
with petroleum ether to obtain (11) as a beige powder (81%).
Purity 98%. 1H NMR (DMSO-d6) δ: 2.73-2.80 (m, 1H),
2.90-2.95 (m, 1H), 3.35 (s, 2H), 4.36-4.40 (m, 1H), 7.03-7.21
(m, 9H), 8.45 (d, J = 5.7 Hz, CONH), 8.90 (s, OH), 10.74 (s,
CONHO). 13C NMR (DMSO-d6) δ ppm: 38.5, 41.5, 52.2, 115.2
(d, JCF = 21.1 Hz), 126.7, 128.5, 129.6, 131.2 (d, JCF = 7.6 Hz),
132.9, 138.1, 161.4 (d, JCF = 238.2 Hz), 168.2, 170.1. tR LCMS
4.29 min. MS [M - H]- m/z 315.
N-Hydroxy-benzamide (12). Benzoic acid (12a) (354 mg,
2.9 mmol) was dissolved in DCM (0.4M) with catalytic DMF.
To this solution cooled to 0 °C (ice bath) was added dropwise
oxalyl chloride (299 μL, 3.48 mmol). The mixture was stirred at
room temperature for 1 h and evaporated under reduced
pressure (temp max 25 °C). The residue was dissolved in
DCM (0.4M). To this solution cooled to 0 °C (ice bath) was
added dropwise DIEA (1.4 mL, 1.2 g, 7.8 mmol.). Then
O-tritylhydroxylamine (878 mg, 3.19 mmol) was added and
the mixture was stirred at room temperature for 4 h. Control
of reaction was performed by TLC (DCM/MeOH 95/5, UV and
H2SO4 visualization). The mixture was washed once with aq
NaHCO3 5% and three times with water, and the combined
organic layers were dried over MgSO4 and evaporated. O-Trityl
hydroxamate intermediate was dissolved in TFA 2%/DCM
(0.03 M), and triisopropylsilane was added dropwise until the
yellow color disappeared. Solvents were removed under reduced
pressure, and the residue was washed with petroleum ether to
1
give compound (12) (47%). White powder; purity 100%. H
NMR 300 MHz (DMSO-d6) δ ppm: 7.45 (m, 3H), 7.74 (d, J =
9 Hz, 2H), 9.00 (s, 1H), 11.19 (s, 1H). 13C NMR (MeOD) δ ppm:
166.8, 132.2, 131.4, 128.3, 126.7. mp: 129.6-131 °C. trLCMS
2.9 min. MS [M þ H]þ m/z 138.
N-Hydroxy-2-phenyl-acetamide (13). Phenylacetic acid (13a)
(500 mg, 3.67 mmol) was dissolved in DCM (42 mL) with DIEA
(2.43 mL, 1.9 g, 14.69 mmol). EDCI (774 mg, 4.04 mmol) and
HOBt (618 mg, 4.04 mmol) were added. The mixture was stirred
at room temperature for 5 min, and the O-tritylhydroxylamine
(1.01 g, 3.33 mmol) was added. The mixture was stirred at room
temperature for 5 h. The reaction mixture was evaporated under
reduced pressure, and the residue was dissolved in DCM
and washed three times with aq NaHCO3 solution (5%) and
once with water and the organic layer was dried over MgSO4
and evaporated. O-Trityl hydroxamate intermediate was dis-
solved in TFA 5%/DCM (0.03 M), and triisopropylsilane
was added dropwise until the yellow color disappeared. Sol-
vents were removed under reduced pressure, and the residue was
washed with petroleum ether to give compound (13). Yield 30%.
δ ppm: 14.1, 27.1, 42.2 (A form), 42.3 (B form), 114.7 (d, JCF
22.3 Hz) (A form), 115.6 (d, JCF = 22.3 Hz) (B form), 128.9 (d,
CF = 8.0 Hz) (A form), 130.9 (d, JCF = 8.5 Hz) (B form), 134.6
=
J
(A form), 137.6 (B form), 162.0 (d, JCF = 243 Hz) (A form),
163.2 (d, JCF = 247 Hz) (B form), 169.0, 170.7. trLCMS 3.18 min.
MS [M þ H]þ m/z 253.
2-[2-(4-Fluoro-phenyl)-acetylamino]-N-hydroxy-3-phenyl-pro-
pionamide (11). 2-Chlorotrityl N-Fmoc-hydroxylamine, poly-
mer-bound, 100-200 mesh (1.504 g, 0.68 mmol), was treated
with a piperidine/DMF 20/80 cocktail for 30 min to remove the
Fmoc-protecting group. The resin was washed with DMF and
DCM. N-Fmoc-phenylalanine (1.050 g, 2.71 mmol, 4 equiv) was
activated with HATU (1.030 g, 2.71 mmol, 4 equiv) in 15 mL of
1
White powder, purity 100%. H NMR 300 MHz (DMSO-d6)

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6799
δ ppm: 3.27 (s, 2H), 726 (m, 5H), 8.75 (br s, 1H), 10.64 (s, 1H).
13C NMR (MeOD) δ ppm: 169.4, 135.1, 128.6, 128.1, 126.6,
39.2. trLCMS 2.4 min. MS [M þ H]þ m/z 152.
stirred at room temperature for 16 h.The reaction mixture was
evaporated under reduced pressure, the residue was dissolved in
DCM and washed three times with aq NaHCO3 solution (5%)
and once with water, and the organic layer was dried over
MgSO4 and evaporated. O-Trityl hydroxamate intermediate
was dissolved in TFA 5%/DCM (0.03 M), and triisopropylsi-
lane was added dropwise until the yellow color disappeared.
Solvents were removed under reduced pressure, and the residue
was washed with petroleum ether to give compound (17) (7%).
N-Hydroxy-3-phenyl-propionamide (14). 3-Phenyl-propionic
acid (14a) (500 mg, 3.33 mmol) was dissolved in DCM (40 mL).
EDCI (702 mg, 3.66 mmol), HOBt (561 mg, 3.66 mmol), and
DIEA (2206 μL, 13.3 mmol) were added. The mixture was
stirred at room temperature for 5 min, and O-tritylhydroxyl-
amine (917 mg, 3.33 mmol) was added. The mixture was stirred
at room temperature for 5 h. The solvent was evaporated under
reduced pressure, and the residue was dissolved in DCM and
washed three times with aq NaHCO3 solution (5%) and once
with water and the organic layer was dried over MgSO4 and
evaporated. O-Trityl hydroxamate intermediate was dissolved
in TFA 5%/DCM (0.03 M) and triisopropylsilane was added
dropwise until the yellow color disappeared. Solvents were
removed under reduced pressure, and the residue was washed
with petroleum ether to give compound (14) (10%). White
1
White powder, purity 98%. H NMR 300 MHz (DMSO-d6)
δ ppm: 4,62 (s, 2H), 6.94 (d, J = 8,1 Hz, 2H), 7.05 (t, J = 7.2 Hz,
1H), 7.34 (t, J = 7.8 Hz, 2H), 9.24 (br s, 1H). trLCMS 2.85 min.
MS [M þ H]þ m/z 168.
N-Hydroxy-3-phenoxy-propionamide (18). 3-Phenoxy-propionic
acid (18a) (300 mg, 2.80 mmol) was dissolved in DMF (20 mL).
EDCI (415 mg, 2.16 mmol), HOBt (415 mg, 2.70 mmol), and
NMM (793 μL, 7.2 mmol) were added. The mixture was stirred at
room temperature for 5 min, and O-tritylhydroxylamine (497 mg,
2.16 mmol) was added. The mixture was stirred at room tempera-
ture overnight. The solvent was evaporated under reduced pressure
and the residue was dissolved in DCM and washed three times with
aq NaHCO3 (5%) and once with water and the organic layer was
dried over MgSO4 and evaporated. O-Trityl hydroxamate inter-
mediate was dissolved in TFA 5%/DCM (14 mL), and triisopro-
pylsilane was added dropwise until the yellow color disappeared.
Solvents are removed under reduced pressure, and the residue was
washed with diethyl ether/pentane and petroleum ether to give
compound (18) (37%). White powder, purity 98%. 1H NMR
300 MHz (MeOD) δ ppm: 2.53 (t, J = 6.0 Hz, 1H), 4.22 (t,
J = 6.0 Hz, 1H), 6.91 (m, 3H) 7.24 (m, 2H). trLCMS 3.18 min. MS
[M þ H]þ m/z 182.
1
powder, purity 98%. H NMR 300 MHz (DMSO-d6) δ ppm:
2.24 (t, J = 7.2 Hz, 2H), 2.79 (t, J = 7.0 Hz, 2H) 7.29-7.16 (m,
5H), 8.68 (s, 1H), 10.35 (s, 1H). 13C NMR (MeOD) δ ppm:
171.9, 142.2, 129.5, 129.4, 127.2, 35.7, 32.6. trLCMS 3.1 min. MS
[M þ H]þ m/z 166.
N-Hydroxy-4-phenyl-butyramide (15). 4-Phenyl-butyric acid
(15a) (1 g, 6.09 mmol) was dissolved in DCM (15 mL) and DMF
(30 μL) was added. The reaction mixture was cooled to 0 °C (ice
bath) and oxalyl chloride (627 μL, 7.3 mmol) was added drop-
wise. Reaction mixture was stirred at room temperature for 1 h.
The solvent was evaporated under reduced pressure, the residue
was dissolved in DCM (10 mL), and O-tritylhydroxylamine
(1425 mg, 5.17 mmol) in solution in DCM (5 mL) with DIEA
(3 mL, 18.27 mmol) was added dropwise. The mixture was
stirred at room temperature for 3 h and washed three times with
aq NaHCO3 solution (5%) and once with water, and the organic
layer was dried over MgSO4 and evaporated. The residue was
precipitated in diethyl ether and filtrated. O-Trityl hydroxamate
intermediate was dissolved in TFA 5%/DCM (0.03 M), and
triisopropylsilane was added dropwise until the yellow color
disappeared. Solvents were removed under reduced pressure,
and the residue was washed with diethyl ether/pentane 50/50
and 25/75 mixture to give compound (15) (68%). White powder,
4-Cyclopropylethynyl-N-hydroxy-benzamide (19). Ethyl-4-
iodobenzoate (1.2 mL, 7.64 mmol), triethylamine (10.2 mL,
72.4 mmol), CuI (276 mg, 1.45 mmol), PdCl2(PPh3), and
ethynylcyclopropane (622 mg, 9.41 mmol) were dissolved in
DMF (28 mL). The mixture was stirred at 70 °C overnight. The
solvent was evaporated under reduced pressure, and the residue
was dissolved in DCM and filtrated on celite. The DCM
solution was washed with brine, and the organic layer was dried
over MgSO4 and evaporated. The residue was purified by flash
chromatography (cyclohexane/ethyl acetate) to give ethyl-
4-cyclopropylethynyl-benzoate (yellow oil, 90%). Purity 98%.
1H NMR 300 MHz (DMSO) δ ppm: 0.85 (m, 2H), 0.92 (m, 2H),
1.31 (t, J = 6.9 Hz, 3H) 1.58 (m, 1H), 4.3 (q, J = 7.2 Hz, 2H),
7.74 (d, J = 8.7 Hz, 2H), 7.89 (d, J = 8.7 Hz, 2H). trLCMS
7.5 min. MS [M þ H]þ m/z 215. Ester (1.3 g, 6.07 mmol) and
NaOH (486 mg, 12.15 mmol) were dissolved in EtOH (25 mL)
and H2O (500 μL). The mixture was stirred at room temperature
overnight. The solvent was evaporated under reduced pressure.
The residue was dissolved in DCM and washed with HCl (1N)
solution. The organic layer was dried over MgSO4 and evapo-
rated to give 4-cyclopropylethynylbenzoic acid (19a) (white
solid, 98%). Purity 98%. 1H NMR 300 MHz (DMSO) δ ppm:
0.76 (m, 2H), 0.91 (m, 2H), 1.57 (m, 1H), 7.44 (d, J = 8.4 Hz,
2H), 7.87 (d, J = 8.4 Hz, 2H). trLCMS 5.54 min. MS (ESIþ):
m/z = 187 (M þ H)þ. mp = 220-222 °C. 4-Cyclopropylethy-
nylbenzoic acid (1.0 g, 5.38 mmol), was dissolved in DCM
(20 mL) with catalytic DMF and oxalyl chloride (577 mL,
6.72 mmol) was added dropwise at 0 °C (ice bath). The reaction
mixture was stirred at 0 °C for 45 min and evaporated under
reduced pressure. The residue was dissolved in DCM (20 mL)
with catalytic DMF and DIEA (2.67 mL, 16.14 mmol) and
O-tritylhydroxylamine (1.259 g, 4.573 mmol) were added. The
reaction mixture was stirred at room temperature for 5 h, and
the solvent was removed under reduced pressure. The residue
was dissolved in DCM and washed three times with aq NaHCO3
solution (5%) and once with water and the organic layer was
dried over MgSO4 and evaporated. The residue was purified
by flash chromatography (cyclohexane/ethyl acetate) to give
4-cyclopropylethynyl-N-trityloxy-benzamide (white solid, 46%).
1
purity 98%. H NMR 300 MHz (DMSO-d6) δ ppm: 1.78 (m,
2H), 1.96 (t, J = 9.0 Hz, 2H), 2.54 (t, J = 9.0 Hz, 2H), 7.33-7.10
(m, 5H), 8.66 (s, 1H), 10.35 (s, 1H). 13C NMR (MeOD) δ ppm:
172.7, 142.8, 129.4, 129.3, 126.9, 36.2, 33.2, 28.6. trLCMS 4.5 min.
MS [M þ H]þ m/z 180.
(E)-N-Hydroxy-3-phenyl-acrylamide (16). (E)-3-Phenyl-acrylic
acid (16a) (300 mg, 2.02 mmol) was dissolved in DMF (20 mL).
EDCI (465 mg, 2.42 mmol), HOBt (465 mg, 3.03 mmol), and
NMM (890 μL, 13.3 mmol) were added. The mixture was stirred at
room temperature for 5 min, and O-tritylhydroxylamine (669 mg,
2.42 mmol) was added. The mixture was stirred at room tempera-
ture overnight. The solvent was evaporated under reduced pres-
sure, the residue was dissolved in DCM and washed three times
with aq NaHCO3 (5%) and once with water and the organic layer
was dried over MgSO4 and evaporated. O-Trityl hydroxamate
intermediate was dissolved in TFA 5%/DCM (19 mL) and
triisopropylsilane was added dropwise until the yellow color
disappeared. Solvents were removed under reduced pressure, and
the residue was washed with petroleum ether to give compound
(16) (87%). White powder, purity 98%. 1H NMR 300 MHz
(DMSO-d6) δ ppm: 6.45 (d, J = 15.6 Hz, 1H), 7.38-7.56 (m,
5H þ 1H), 9.03 (s, 1H), 10.09 (s, 1H). 13C NMR (DMSO-d6)
δ ppm: 164.9, 140.3, 134.7, 129.5, 128.6, 127.4, 116.9. trLCMS
3.30 min. MS [M þ H]þ m/z 164.
N-Hydroxy-2-phenoxy-acetamide (17). Phenoxyacetic acid
(17a) (300 mg, 1.97 mmol) was dissolved in DMF (20 mL).
O-Tritylhydroxylamine (649.8 mg, 2.36 mmol), EDCI (452.43
mg, 2.36 mmol), HOBt (451.76 mg, 2.95 mmol), and NMM
(866 μL, 797.0 mg, 7.88 mmol) were added. The mixture was

6800 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Flipo et al.
Purity 98%. 1H NMR 300 MHz (DMSO) δ ppm: 0.72 (m, 2H),
0.88 (m, 2H), 1.53 (m, 1H), 7.34 (m, 19H), 10.94 (s, 1H). trLCMS
8.31 min. MS [M þ H]þ m/z 442. mp = 155-156 °C. O-Trityl
hydroxamate intermediate (500 mg, 1.12 mmol) was dissolved in
TFA 2%/DCM (20 mL), and triisopropylsilane was added
dropwise until the yellow color disappeared. Solvents were
removed under reduced pressure, and the residue was washed
with petroleum ether to give compound (19) (93%). White
was dried over MgSO4 and evaporated. The residue was purified
by TLC (cyclohexane/ethyl acetate 80/20) to give intermediate
(21b) (25%). Purity 95%. H NMR 300 MHz (CDCl3) δ ppm:
1
0.90 (d, J = 6.7 Hz, 6H), 1.16 (t, J = 7.1 Hz, 3H), 1.81-1.86 (m,
1H), 3.06 (d, J = 6.8 Hz, 2H), 4.00-4.13 (m, 4H), 7.46-7.60 (m,
3H), 7.81-7.85 (m, 2H). To intermediate (21b) (3 mmol) in MeOH
(10 mL) were added hydroxylamine hydrochloride (229 mg,
3.3 mmol) and sodium methylate (12.6 mL, 6.3 mmol). The
reaction mixture was stirred at room temperature overnight
and solvents evaporated. HCl solution (pH = 3) was added,
and the reaction mixture was extracted with ethyl acetate
(5 times). The combined organic layers were dried over MgSO4
and evaporated. The residue was purified by TLC (DCM/MeOH
95/5) to give (21) as a solid (29%). Purity 95%. 1H NMR
300 MHz (CDCl3) δ ppm: 0.92 (d, J = 6.7 Hz, 6H), 1.76-1.88
(m, 1H), 2.98 (d, J = 7.2 Hz, 2H), 3.72 (s, 2H), 7.54-7.67 (m,
3H), 7.81 (d, J = 8.7 Hz, 2H). 13C NMR (DMSO-d6) δ ppm:
164.7, 139.7, 133.2, 129.6, 127.7, 56.3, 48.1, 26.4, 20.3. mp =
122-123 °C.
((R)-1-Hydroxycarbamoyl-2-phenyl-ethyl)-carbamic Acid tert-
Butyl Ester (22). Boc-(D)-phenylalanine (500 mg, 1.88 mmol) was
dissolved in DMF (20 mL). EDCI (433 mg, 2.26 mmol), HOBt
(541 mg, 2.82 mmol), and NMM (828 μL, 7.5 mmol) were added.
The mixture was stirred at room temperature for 5 min, and
O-tritylhydroxylamine (519 mg, 1.88 mmol) was added. The
mixture was stirred at room temperature for 72 h. The solvent
was evaporated under reduced pressure and the residue was
dissolved in DCM and washed three times with aq NaHCO3
solution (5%) and once with water and the organic layer was
dried over MgSO4 and evaporated. O-Trityl hydroxamate inter-
mediate was dissolved in TFA 5%/DCM (2 mL) and triisopro-
pylsilane (19 μL) was added. Solvents were removed under
reduced pressure, and the residue was washed with diethyl
ether/pentane and petroleum ether to give compound (22)
(20%). White powder, purity 98%. 1H NMR 300 MHz
(MeOD) δ ppm: 1.36 (s, 9H), 2.84 (dd, J = 9.0 and 15.0 Hz,
1H), 3.04 (dd, J = 6.0 and 12.0 Hz, 1H), 4.18 (t, J = 6.0 Hz, 1H),
7.21-7.27 (m, 5H). 13C NMR (MeOD) δ ppm: 169.5, 156.0,
136.9, 128.9, 128.0, 126.3, 79.2, 53.8, 38.0, 27.2. trLCMS 4.36 min.
MS [M - H]- m/z 279.
Plasma Stabilities. Lithium-heparin plasma from Sprague-
Dawley rats (mixed gender pool) were from Sera Laboratories
International Ltd. Human plasma was a mixed gender pool
from donors. PMSF (phenylmethylsulfonyl fluoride) and
enalapril maleate were purchased from Sigma-Aldrich Inc. Then
40 μL of a 5 mM solution in DMSO of the sample were added to
1.960 mL of plasma, previously incubated or not with PMSF at
the desired concentration, to obtain a 100 μM final solution. The
mixture was gently stirred 96 h at 37 °C. Aliquots of 200 μL were
taken at various times (from 0 to 96 h) and diluted with 200 μL of
acetonitrile. Then 10 μL of the 2 mM solution in methanol of the
internal standard were added. The mixture was centrifuged and
supernatant was extracted three times with 2 mL of AcOEt.
The combined organic layers were evaporated and diluted with
200 μL of methanol.
For experiments with PMSF, incubations were performed in
duplicate in microtiterplates from Matrix with 80 μL of rat
plasma (Sprague-Dawley pooled mixed gender from Sera
Laboratories International Ltd.) for each time point. Plasma
was preincubated at 37 °C 5 min and then incubated 30 min with
PMSF at a final concentration of 2 mM when needed, before
compound addition to a final concentration of 10 μM 1%
DMSO. The reaction was terminated at 0, 1, 2, 4, 8, 24, and
48 h by the addition of acetonitrile containing the internal
standard (IS). After centrifugation, supernatant was analyzed.
Analysis and quantification used a LC-MS/MS triple-quadrupole
system (Varian 1200ws) under MRM or SIM detection using
adequate parameters (see Supporting Information for mode
of ionization, declustering potential, collision-activated dissocia-
tion, and collision energy for each compound). A calibration curve
1
powder, purity 98%. H NMR 300 MHz (DMSO-d6) δ ppm:
0.75 (m, 2H), 0.92 (m, 2H), 1.56 (m, 1H), 7.41 (d, J = 8.5 Hz, 2H),
7.69 (d, J = 8.5 Hz, 2H), 11.24(s, 1H). 13C NMR(MeOD) δppm:
167.5, 132.6, 132.2, 128.9, 128.1, 97.2, 75.8, 9.1, 0.8. trLCMS
4.30 min. MS [M þ H]þ m/z 200. mp = 171-172 °C.
N-Hydroxy-2-[isobutyl-(4-methoxy-benzenesulfonyl)-amino]-
acetamide (20). To a solution of isobutylamine (3.5 mmol) in
DMF (7 mL, 0.5 M) was added DIEA (608 μL, 3.5 mmol) and
4-methoxybenzenesulfonylchloride (3.5 mmol) in solution in
THF (7 mL, 0.5 M). The reaction mixture was stirred at room
temperature for 1 h, and the solvents were evaporated under
reduced pressure. The residue was dissolved in DCM and
washed with HCl 1N solution and H2O. The organic layer
was dried over MgSO4 and evaporated to give intermediate
1
(20a) (88%). Purity 95%. H NMR 300 MHz (CDCl3) δ ppm
0.85 (d, J = 6.7 Hz, 6H), 1.65-1.74 (m, 1H), 2.73 (t, J = 6.7 Hz,
2H), 3.86 (s, 3H), 6.94-6.99 (m, 2H), 7.76-7.78 (m, 2H). To a
suspension of NaH (3 mmol) in THF (8 mL) was added (20a)
sulfonamide (3 mmol) in THF (8 mL). The reaction mixture was
stirred at room temperature for 30 min and ethylbromoacetate
(3.3 mmol) was added. The reaction mixture was stirred at room
temperature overnight and stopped with H2O. THF was eva-
porated under reduced pressure, and the reaction mixture was
extracted with ethyl acetate. The combined organic layers were
dried over MgSO4 and evaporated. The residue was purified by
TLC (cyclohexane/ethyl acetate 80/20) to give intermediate
(20b) (72%). Purity 98%. 1H NMR 300 MHz (CDCl3) δ ppm:
0.89 (d, J = 6.7 Hz, 6H), 1.20 (t, J = 7.1 Hz, 3H), 1.83-1.88 (m,
1H), 3.02 (d, J = 7.5 Hz, 2H), 3.86 (s, 3H), 4.01-4.13 (m, 4H),
6.94-6.99 (m, 2H), 7.75-7.81 (m, 2H). To intermediate (20b)
(3 mmol) in MeOH (10 mL) was added hydroxylamine hydro-
chloride (229 mg, 3.3 mmol) and sodium methylate (12.6 mL,
6.3 mmol). The reaction mixture was stirred at room temperature
overnight and solvents were evaporated. HCl solution (pH =3)
was added, and the reaction mixture was extracted with ethyl
acetate (five times). The combined organic layers were dried
over MgSO4 and evaporated. The residue was purified by flash
chromatography (DCM/MeOH) to give (20) as a solid (53%).
Purity 98%. 1H NMR 300 MHz (CDCl3) δ ppm: 0.91 (d, J =
6.7 Hz, 6H), 1.78-1.84 (m, 1H), 2.94 (d, J = 7.3 Hz, 2H),
3.69 (s, 2H), 3.89 (s, 3H), 7.01 (d, J = 8.7 Hz, 2H), 7.75 (d, J =
8.8 Hz, 2H), 9.4 (s, 1H). 13C NMR (DMSO-d6) δ ppm: 164.9,
162.8, 131.5, 129.9, 114.6, 56.3, 56.1, 48.3, 26.7, 20.5. mp =
125-126 °C.
2-(Benzenesulfonyl-isobutyl-amino)-N-hydroxy-acetamide(21).
To a solution of isobutylamine (3.5 mmol) in DMF (7 mL,
0.5 M) was added DIEA (608 μL, 3.5 mmol) and benzenesulfo-
nylchloride (3.5 mmol) in solution in THF (7 mL, 0.5 M). The
reaction mixture was stirred at room temperature for 1 h, and the
solvents were evaporated under reduced pressure. The residue
was dissolved in DCM and washed with HCl 1N solution and
H2O. The organic layer was dried over MgSO4 and evaporated to
give (21a) (80%). Purity 95%. 1H NMR 300 MHz (CDCl3)
δ ppm 0.86 (d, J = 6.7 Hz, 6H), 1.67-1.76 (m, 1H), 2.75 (d,
J = 6.7 Hz, 2H), 7.48-7.60 (m, 3H), 7.87-7.90 (m, 2H). To a
suspension of NaH (3 mmol) in THF (8 mL) was added (21a)
sulfonamide (3 mmol) in THF (8 mL). The reaction mixture
was stirred at room temperature for 30 min and ethyl-
bromoacetate (3.3 mmol) was added. The reaction mixture was
stirred at room temperature overnight and stopped with H2O. THF
was evaporated under reduced pressure and the reaction mixture
was extracted with ethyl acetate. The combined organic layers

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6801
for each compound allowed the linear relationship between
concentration and signal intensity (given as peak area ratio
analyte/IS). Acquisition and analysis of data were performed with
MS Workstation software (version 6.3.0 or higher). The degrada-
tion half-life (t1/2) values were calculated using the following
equation: t1/2 = 0.693/k where k is the first-order degradation rate
constant. The degradation rate constant (k) was estimated by one-
phase exponential decay nonlinear regression analysis of the
degradation time course data using Xlfit software (version 2.1.2
or higher).
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Dow, G. S.; Kozikowski, A. P. A series of potent and selective,
triazolylphenyl-based histone deacetylase inhibitors [HDACIs]
with activity against pancreatic cancer cells and Plasmodium
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(12) (a) Charrier, C.; Clarhaut, J.; Gesson, J.-P.; Estiu, G.; Wiest, O.;
Roche, J.; Bertrand, P. Synthesis and Modeling of New Benzo-
furanone Histone Deacetylase Inhibitors that Stimulate Tumor
Suppressor Gene Expression. J. Med. Chem. 2009, 52 (9), 3112–
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Yan, T. P.; Fournel, M.; Maroun, C.; Li, Z.; Lemieux, A.-M.; Nicolescu,
Acknowledgment. We thank Virginie Leroux for technical
ꢀ
assistance and Pr. Andre Tartar for scientific discussion. We
are grateful to the institutions that support our laboratory
ꢀ
A.; Rahil, J.; Lefebvre, S.; Panetta, A.; Besterman, J. M.; Deziel,
R. Novel HDAC6 isoform selective chiral small molecule histone
deacetylase inhibitors. Bioorg. Med. Chem. Lett. 2009, 19 (3), 688–
692.
ꢀ
(Inserm, Universite Lille Nord de France, and Institut Pasteur
de Lille) and PRIM: Pole de Recherche Interdisciplinaire
^
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T. L.; DeCrescenzo, G. A.; Freskos, J. N.; Getman, D. P.;
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ꢀ
du Medicament. Data management was performed using
Pipeline Pilot from Accelrys. We also thank the following
institutions or companies: CAMPLP and VARIAN Inc. This
project was supported by the Fondation pour la Recherche
Medicale, Nord-Pas-de-Calais (RAD07001EEA).
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Supporting Information Available: Mass spectrometry para-
meters for each compound, example of plasma stability curve of
1 and enalapril, with or without PMSF, compound 9, prodrug 5,
structures and half-lives of hydroxamates found in the literature
(iv or po conditions), and experimental conditions for com-
pounds 1-3. This material is available free of charge via the
Internet at http://pubs.acs.org.
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