A convenient procedure for the solid-phase synthesis of hydroxamic acids on PEGA resins

Article abstract of DOI:10.1016/j.tetlet.2011.10.103

An efficient method for the solid-phase synthesis of hydroxamic acids is described. The method comprises the nucleophilic displacement of esters immobilized on PEGA resins with hydroxylamine/sodium hydroxide in isopropanol. The hydroxyaminolysis protocol

Full text of DOI:10.1016/j.tetlet.2011.10.103

Tetrahedron Letters 52 (2011) 7121–7124
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A convenient procedure for the solid-phase synthesis of hydroxamic
acids on PEGA resins
Nitin S. Nandurkar a, Rico Petersen a, Katrine Qvortrup a, Vitaly V. Komnatnyy a, Kennedy M. Taveras a,
Sebastian T. Le Quement a, Robin Frauenlob a, Michael Givskov b, Thomas E. Nielsen a,
⇑
a Department of Chemistry, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
b Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient method for the solid-phase synthesis of hydroxamic acids is described. The method com-
prises the nucleophilic displacement of esters immobilized on PEGA resins with hydroxylamine/sodium
hydroxide in isopropanol. The hydroxyaminolysis protocol is compatible with a broad range of PEGA-
supported peptide and peptidomimetic esters. The methodology was found to be compatible with two
new strategies for the synthesis of solid-supported lactams and diketopiperazines, respectively, both
relying on the high inter- and intramolecular reactivity of cyclic N-acyliminium ions with electron-rich
aromatics and heteroaromatics, ultimately affording hydroxamic acid derivatives in high purities.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 17 September 2011
Revised 7 October 2011
Accepted 18 October 2011
Available online 31 October 2011
Keywords:
Hydroxamic acids
Solid-phase synthesis
Hydroxylamine
HDAC inhibitor
PEGA resin
Hydroxamic acids represent an important class of compounds
with a wide spectrum of biological properties, such as antibacte-
rial, antifungal and anticancer (Fig. 1).1,2 The ability of hydroxa-
mates to chelate metal ions, such as Fe3+ and Zn2+, has been
widely explored in biology and medicine, as exemplified by
hydroxamic acid based siderophores produced by microorganisms
for the harvesting of iron from iron-deficient environments. The
siderophore desferrioxamine B (Desferal, 1) is used medically to
treat iron poisoning, which can arise following blood transfusion
to patients with genetic blood diseases.3
plored. There have been several reports describing the solid-phase
synthesis of hydroxamic acids.6 For example, hydroxylamine deriv-
atives, being either N-tethered to MBHA,7 and Tentagel resins,8 or O-
tethered to Wang,9 Sasrin,10 and trityl resins,11 have served as the
starting point for the synthesis of a range of hydroxamic acids. An-
other approach has been a stepwise method, where esters were
cleaved from the resin to give the corresponding carboxylic acids
that were subsequently reattached to a hydroxylamine resin using
A large number of recent studies have dealt with hydroxamic
acids as potent inhibitors of Zn2+-containing enzymes, such as ma-
trix metallo-proteinases (MMPs), and histone deacetylases (HDACs)
in particular. Although hydroxamic acids have been widely
explored in the pharmaceutical industry for decades, the number
of clinical failures associated with this compound class is substan-
tial (e.g., for 3 and 4). However, the launch of the HDAC inhibitor,
vorinostat (SAHA, suberoyl anilide hydroxamic acid, 2) in 2006 by
Merck for the treatment of cutaneous T-cell lymphoma,4 has drawn
renewed attention to the chemistry and biomedical properties of
hydroxamic acids.
O
O
H2N
N
HO
O
HN
H
N
O
OH
N
OH
N
H
N
H
O
O
SAHA, 2
O
(HDAC inhibitor)
1
Desferal,
N
OH
(chelating agent)
H
O
N
O
O
OH
O
O
H
N
OH
S
N
N
H
N
N
H
Given the growing number of potent hydroxamic acids identified
in drug and probe discovery efforts,5 methods for the parallel and
combinatorial synthesis of hydroxamic acids have been widely ex-
O
OH
S
O
4
Marimastat,
3
Prinomastat,
(MMP inhibitor)
(MMP inhibitor)
⇑
Corresponding author. Tel.: +45 4525 2134; fax: +45 4593 3968.
E-mail address: ten@kemi.dtu.dk (T.E. Nielsen).
Figure 1. Clinically tested hydroxamic acids.
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.10.103

7122
N. S. Nandurkar et al. / Tetrahedron Letters 52 (2011) 7121–7124
Table 1
a peptide coupling agent, followed by cleavage to provide the
hydroxamic acids.12
Optimization study for the conversion of resin-bound ester 8 into the corresponding
hydroxamic acid 9
The solid-phase synthesis of hydroxamic acids via direct
hydroxyaminolysis of an ester-linked substrate has also been re-
ported. These methods generally require treatment of the esteri-
fied resin with excess amounts of NH2OH for prolonged periods
of time to generate effectively the corresponding hydroxamic
acids.13 In a few reports, activated resins have been employed to
facilitate a more rapid cleavage.14 In one highly interesting report,
the hydroxyaminolysis of an ester-linked substrate using aqueous
hydroxylamine and KCN as the catalyst was also reported.15 As
part of ongoing efforts to synthesize hydroxamic acids, we herein
communicate our results on the hydroxyaminolysis of ester-linked
substrates on PEGA resins, thus providing a convenient procedure
for the release of hydroxamic acids from the solid support.
Initially, a solid-supported substrate (8, Scheme 1), to be used
for rapid optimization of the reaction conditions necessary for
hydroxamic acid release, was constructed.
50% aq. NH2OH
(13 equiv)
O
O
H
N
H
N
OH
O
N
H
base (1.2 equiv).
solvent, 1 h
8
9
O
O
Entry
Base
Solvent
Purity (%)a
1
2
3
4
5
None
NaOH
NaOH
NaOH
KOH
MeOH
t-BuOH
i-PrOH
MeOH
MeOH
84
88
>95
>95
92
a
Determined by RP-HPLC/MS of the crude products (254 nm).
Table 2
Conversion of ester-linked substrates into the corresponding hydroxamic acids 10–27
via hydroxyaminolysisa
Starting with amino-functionalized PEGA800 resin (0.4 mmol/
g),16 the HMBA (hydroxymethylbenzoic acid) linker was attached
using a N,N,N0,N0-tetramethyl-O-(benzotriazol-1-yl)uronium tetra-
fluoroborate (TBTU) mediated amide coupling procedure.17 The
HMBA linker was then esterified with Fmoc-Gly-OH using the
1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT) proto-
col.18 Fmoc deprotection with 20% piperidine in DMF and final
TBTU-coupling of benzoic acid then yielded solid-supported sub-
strate 8. The purity of 8 (>95%) was tested via release from the
HMBA linker, as the corresponding carboxylic acid, with aqueous
NaOH (0.1 M).19
50% aq. NH2OH
(13 equiv)
O
O
OH
R
S
O
R
N
H
-
NaOH (1.2 equiv),
i-PrOH, 1 h
10 27
Entry
1
R
Product
Purity (%)b
92
H
N
10
O
H
N
2
3
11
12
91
69
MeO
O2N
With solid-supported ester-linked substrate 8 in hand several
cleavage reaction conditions were tested. Using 50% aqueous
NH2OH (13 equiv), a range of protic solvents and basic additives
were first examined (see Table 1 for selected results).
O
H
N
O
The results showed how the combination of NH2OH with NaOH
in i-PrOH or MeOH was extremely efficient for the formation of the
desired hydroxamic acid 9 in excellent purity (>95%). A range of
bases (KOH, NaOH, KOt-Bu) and solvents (toluene, 1,4-dioxane,
DMF, and THF) was examined (results not shown), but none proved
nearly as efficient as those noted in Table 1. Critical for a successful
hydroxylaminolysis reaction is good solubility of the base in the
solvent, which must still be able to swell effectively the resin,
while not transforming the formed hydroxamic acid further. Inter-
estingly, when the cleavage was performed in the absence of base,
hydroxamic acid 9 could be obtained in good purity (84%). A rapid
examination of the reagent stoichiometries showed the impor-
tance of maintaining a low amount of base, as decomposition of
N
F
H
4
5
6
13
14
15
>95
82
N
O
H
N
O
H
N
>95
O
H
N
the hydroxamic acid
conditions.
9 was observed under strongly basic
7
8
9
16
17
18
84
86
55
O
O
Having developed an efficient protocol for the release of
hydroxamic acid 9 from the solid support, we next investigated
the scope of the methodology for a range of solid-supported esters
(Table 2).19 All the ester-linked substrates were synthesized
according to the synthetic strategy presented in Scheme 1.
H
N
8
N
H
O
HN
O
HO
b
a
H
N
H2N PEGA800
(0.4 mmol/g)
FmocHN
10
11
19
20
74
94
PEGA800
H
O
N
5
O
6
O
O
O
O
c
d
H
= HMBA-PEGA800
H2N
N
O
H
N
7
8
O
O
Scheme 1. Solid-phase synthesis of ester-linked substrate 8. Reagents and condi-
tions: (a) HMBA, TBTU, NEM, DMF; (b) FmocGlyOH, MSNT, MeIm, CH2Cl2; (c) 20%
piperidine (DMF), (d) PhCO2H, TBTU, N-ethylmorpholine (NEM), DMF.

N. S. Nandurkar et al. / Tetrahedron Letters 52 (2011) 7121–7124
7123
O
Table 2 (continued)
H
N
H
Standard
SPPS
O
HO
H
O
O
5
Entry
R
Product
Purity (%)b
89
N
PEGA800
N
H
5
32
O
HN
H
N
= HMBA-PEGA800
CO2H
12
21
Standard
SPPS
O
OMe
O
36
NBoc
OMe
O
O
H
N
e
H2N
N
H
O
O
N
H
O
Et
13
22
87
H
N
H
H
N
O
O
N
O
5
O
28
O
N
H
O
a
33
H
N
O
N
14
15
23
24
93
91
N
H
NBoc
O
H
N
O
O
OMe
O
N
O
O
H
N
OMe
H
H
N
f, b, g
O
O
29
b, c
N
H
O
O
O
H
N
H
O
O
O
5
O
H
N
O
N
16
17
25
26
>95
>95
N
34
NH
N
H
O
N
H
O
N
H
O
O
OMe
HN
O
30
O
OMe
83%
H
N
d
d
86%
N
H
O
O
H
N
H
O
O
O
OH
O
N
H
5
N
N
H
O
O
N
H
N
H
N
OH
35
N
18
27
>95
N
H
N
H
31
H
O
HN
O
O
N
OMe
OMe
a
A reaction time of 1 h gave the crude product in >80% yield from 50 mg of resin.
As determined by RP-HPLC/MS of the crude products (254 nm).
b
Scheme 2. Solid-phase synthesis of compounds 31 and 35. Reagents and condi-
tions: (a) 36, TBTU, NEM, DMF; (b) 10% TFA (aq); (c) indole, 50% TFA (CH2Cl2);
(d) 50% NH2OH, NaOH, i-PrOH; (e) CH2CHCOCl, CH2Cl2; (f) OsO4, NaIO4, DBU,
H2O:THF (1:1); (g) 50% TFA (CH2Cl2).
A series of unhindered esters incorporating substituted aro-
matic amide moieties was examined (entries 1–8). Gratifyingly,
the protocol proved well compatible with these substrates, gener-
ally providing the desired hydroxamic acids in reasonable to excel-
lent purities (69?95%). In further experiments, the compatibility
with sterically more congested substrates was also demonstrated
(entries 10–18), revealing a synthetically useful protocol giving
hydroxamic acids in excellent purities (74?95%). Some limitation
was observed for the more hindered proline derivative 18 (entry 9),
that could only be formed in moderate purity (55%).
in high purity (83%). In both cases, only minor amounts of the cor-
responding carboxylic acids were observed (<10%).
In summary, an efficient protocol for the solid-phase synthesis
of hydroxamic acids has been developed. The protocol relies on
readily available HMBA esters, which are easily accessible on PEGA
supports. Hydroxamic acids were then efficiently formed following
hydroxyaminolysis with NH2OH/NaOH in isopropanol. The meth-
odology is useful for the synthesis of peptidic and peptidomimetic
hydroxamic acids in good to excellent purities. In addition, two
new N-acyliminium reactions for the synthesis of substituted lac-
tams and diketopiperazines, respectively, were also developed.
Both heterocyclic scaffolds were synthesized effectively, embed-
ded in highly pure products released from the solid support, thus
paving the way for the synthesis of larger and structurally more
complex libraries of hydroxamic acids. Biological evaluation of
the synthesized hydroxamic acids as HDAC inhibitors is ongoing,
and the results will be reported in due course.
Efforts in our laboratories have dealt with the use of solid-sup-
ported N-acyliminium intermediates,20 conveniently formed by
intramolecular aldehyde-amide condensation reactions, for the
synthesis of a wide range of constrained peptidomimetic scaf-
folds.21 In this context, we have been particularly interested in
the solid-phase synthesis of tetrahydro-b-carbolines and tetrahy-
droisoquinolines (THIQs), which are known to exhibit a plethora
of biological activities.22 To advance the scope of this methodology,
we sought to demonstrate that the protocol shown in Table 2 could
also be applied in this context. Previous efforts have focused on
intramolecular reactions of cyclic N-acyliminium intermediates.
Below, we present two new variants thereof, one useful for the
synthesis of substituted lactams, the other applicable to the syn-
thesis of polycyclic diketopiperazines. In this context, compounds
28 and 32 (Scheme 2) were constructed on a solid support using
standard solid-phase synthesis protocols. Compound 28 was acyl-
ated with masked aldehyde building block 36,23 prior to reaction
with indole as an external nucleophile under acidic reaction condi-
tions.19 The desired lactam hydroxamic acid 31 was subsequently
released in 86% purity from the solid support. Similarly, acryloylat-
ed peptide 33 was subjected to OsO4/NaIO4-mediated oxidative
cleavage,24 followed by treatment with 50% TFA (CH2Cl2).19 The
resulting diketopiperazine hydroxamic acid 35 was finally isolated
Acknowledgments
The Hans Christian Oersted Postdoc Program, Technical Univer-
sity of Denmark (DTU), Carlsberg Foundation, Lundbeck Founda-
tion, Danish Councils for Strategic and Independent Research,
and Holm Foundation are gratefully acknowledged for the financial
support.
Supplementary data
Supplementary data (experimental synthetic procedures and
data for selected compounds) associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.2011.10.103.

7124
N. S. Nandurkar et al. / Tetrahedron Letters 52 (2011) 7121–7124
first for 2 min, followed by washing with DMF (ꢀ2), and then for 18 min. The
References and notes
deprotected resin was then washed with DMF (ꢀ6), CH2Cl2 (ꢀ6) and then DMF
(ꢀ6) again. Subsequent attachment of amino acids and/or capping units was
accomplished using appropriate cycles of TBTU-mediated coupling and Fmoc
deprotection reactions as described above.
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General procedure for the synthesis of hydroxamic acids by hydroxyaminolysis of
ester-linked substrates: NaOH (1.2 equiv) was weighed and dissolved in
a
minimum amount of i-PrOH. A solution of 50% NH2OH (aq) (13 equiv) was then
added. The solution was transferred to the resin and the swollen resin was
gently stirred with a spatula. After cleavage for 15 h, the cleavage solution was
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filtered, washed with H2O (ꢀ6), DMF (ꢀ6), MeOH (ꢀ6) and CH2Cl2 (ꢀ6), and
lyophilized for 20 h. The resin was then treated with indole (3 equiv) in 50%
TFA (CH2Cl2). The reaction was allowed to proceed for 5 min. After removal of
the reaction mixture by suction, the remaining resin was washed with H2O
(ꢀ6), 20% piperidine in DMF (ꢀ2), H2O (ꢀ6), DMF (ꢀ6), MeOH (ꢀ6) and CH2Cl2
(ꢀ6), dried under vacuum and lyophilized.
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Procedure for the synthesis of substrate 34 from 32: Acryloyl chloride (10 equiv)
was dissolved in dry CH2Cl2 and transferred to solid-supported peptide 32. The
swollen resin was stirred gently with a spatula and allowed to react for 1 h. The
resin was filtered and washed with H2O (ꢀ6), DMF (ꢀ6) and CH2Cl2 (ꢀ6), then
dried using suction for 30 min. The resin was transferred to a capped reaction
vial. The oxidative cleavage was carried out by addition of DBU (5 equiv) and
NaIO4 (10 equiv) to the resin pre-swollen in H2O:THF (1:1). After 10 min of
shaking, a solution of OsO4 in t-BuOH (2.5% w/w, 0.05 equiv) was added and
the mixture was left under shaking for 20 h. The mixture was transferred to a
syringe (5 ml) equipped with a polypropylene filter. After removal of the
reaction mixture by suction, the resin was washed with H2O (ꢀ6), 10% TFA (aq)
(ꢀ4), H2O (ꢀ6), DMF (ꢀ6) and CH2Cl2 (ꢀ6) and dried under vacuum. The resin
was then subjected to treatment with 50% TFA (CH2Cl2), stirred gently with a
spatula and allowed to react, in a sealed syringe, for 25 h. After removal of the
reaction mixture by suction, the remaining resin was washed with H2O (ꢀ6),
20% piperidine in DMF (ꢀ2), H2O (ꢀ6), DMF (ꢀ6), MeOH (ꢀ6) and CH2Cl2 (ꢀ6),
dried under vacuum and lyophilized.
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Compound 35: 1H NMR (500 MHz, DMSO-d6) d 10.31 (br s, 1H), 8.14 (m, 1H),
6.94 (s, 1H), 6.73 (s, 1H), 5.10 (s, 1H), 4.56 (dd, J = 8.5, 7.5 Hz, 1H), 4.09 (q,
J = 6.8 Hz, 1H), 3.75 (s, 3H), 3.68 (s, 3H), 3.14 (dd, J = 15.5, 7.2 Hz, 1H), 3.05 (m,
2H), 3.01 (s, 3H), 2.90 (dd, J = 15.0, 9.1 Hz, 1H), 1.92 (m, 2H), 1.46 (m, 2H), 1.38
(m, 2H), 1.19 (m, 2H), 1.11 (d, J = 6.9 Hz, 3H); 13C NMR (125 MHz, DMSO-d6) d
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55.2, 53.0, 38.1, 31.8, 30.9, 29.1. 28.4, 25.4, 24.5, 17.7; MS (ESI) calcd for
C
23H33N4O7 [M+H]+ 477.2 found 477.3. Isolated yield after prep. RP-HPLC: 15%.
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amino-functionalized PEGA800 resin was carried out by pre-mixing HMBA
(3.0 equiv), NEM (4.0 equiv) and TBTU (2.88 equiv) in DMF for 5 min. The
volume of DMF was kept to a minimum amount sufficient for full coverage and
swelling of the beads. The resulting solution was added to the resin, and
allowed to react for 2 h. After removal of the reaction mixture by suction, the
remaining resin was washed with DMF (ꢀ6), CH2Cl2 (ꢀ6), and then dried under
vacuum. Coupling of the first amino acid to the HMBA-derivatized resin was
accomplished by treating the freshly lyophilized resin with a mixture of the
Fmoc-protected amino acid (3.0 equiv), MeIm (5.0 equiv) and MSNT (3.0 equiv)
in CH2Cl2. The MSNT-mediated coupling was repeated once. Removal of the
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