Parallel synthesis and in vitro activity of novel anthranilic hydroxamate-based inhibitors of the prostaglandin H2 synthase peroxidase activity

Article abstract of DOI:10.1039/b505525c

Currently available non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin are directed at the cyclooxygenase (COX) site, but not the peroxidase (POX) activity of prostaglandin H₂ synthase (PGHS). They are thus unable to inhibit the free-radical induced tissue injury associated with PGHS peroxidase activity, which can occur independently of the COX site. A lead compound, anthranilic hydroxamic acid (AHA) was found to have significant PGHS-POX inhibitory activity (IC₅₀ = 72 μM). To define the critical parameters for PGHS-POX inhibition, we investigated 29 AHA derivatives, synthesised from their acid precursors, using solid phase synthesis. In vitro analysis demonstrated a ten-fold improvement in inhibition with 3,5-diiodoanthranilic hydroxamic acid (IC₅₀ = 7 μM). The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b505525c

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A R T I C L E
Parallel synthesis and in vitro activity of novel anthranilic
hydroxamate-based inhibitors of the prostaglandin H2 synthase
peroxidase activity†
Jean Lee,a Anthony J. Chubb,b Edelmiro Moman,a Brian M. McLoughlin,b
Caroline T. Sharkey,b John G. Kelly,c Kevin B. Nolan,a Marc Devocelle*a and
Desmond J. Fitzgerald*‡b
a Centre for Synthesis and Chemical Biology, Department of Pharmaceutical and Medicinal
Chemistry, Royal College of Surgeons in Ireland, 123 St. Stephen’s Green, Dublin 2, Ireland.
E-mail: mdevocelle@rcsi.ie
b Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, 123 St. Stephen’s
Green, Dublin 2, Ireland
c School of Pharmacy, Royal College of Surgeons in Ireland, 123 St. Stephen’s Green, Dublin 2,
Ireland
Received 20th April 2005, Accepted 2nd August 2005
First published as an Advance Article on the web 8th September 2005
Currently available non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin are directed at the
cyclooxygenase (COX) site, but not the peroxidase (POX) activity of prostaglandin H2 synthase (PGHS). They are
thus unable to inhibit the free-radical induced tissue injury associated with PGHS peroxidase activity, which can
occur independently of the COX site. A lead compound, anthranilic hydroxamic acid (AHA) was found to have
significant PGHS-POX inhibitory activity (IC50 = 72 lM). To define the critical parameters for PGHS-POX
inhibition, we investigated 29 AHA derivatives, synthesised from their acid precursors, using solid phase synthesis.
In vitro analysis demonstrated a ten-fold improvement in inhibition with 3,5-diiodoanthranilic hydroxamic acid
(IC50 = 7 lM).
POX activity.12 Furthermore, as cyclooxygenase catalysis is
Introduction
dependent on POX activity,1,13 inhibition of the peroxidase site
would also lead to inactivation of the COX site. Therefore,
there is a two-fold reason for developing PGHS peroxidase
site inhibitors. Compared to the number of available COX
inhibitors, a relatively small number of POX inhibitors have been
described. These include synthetic N-acyl-hydroxylamines and
-hydrazines,14 as well as natural peroxidase inhibitors such as
resveratrol, the cardio-protective, anti-inflammatory and cancer
preventive compound found in red wine.15
Prostaglandin H2 synthase (PGHS), or cyclooxygenase (COX),
is the enzyme catalysing the first two steps in the biotransfor-
mation of arachidonic acid to the prostaglandin hormones.1–5
There are different isozymes of PGHS encoded by distinct genes:
PGHS-1, the constitutive enzyme expressed in most tissues and
PGHS-2, which can be described in a simplified manner as the
inducible isoform, expressed at sites of inflammation and in
tumours. A third isozyme derived from the PGHS-1 gene has
recently been identified.6
We have previously reported that acetylated salicylhydrox-
amate derivatives can act as effective inhibitors of the COX
activity of PGHS by acetylating a serine residue in this active
site.16,17 Triacetylsalicylhydroxamic acid in particular can poten-
tially acetylate two molecules of PGHS, via the formation of
an intermediate acetylating species, O-acetylsalicylhydroxamic
acid, following first-step acetylation. These reactions would
finally yield one molecule of salicylhydroxamic acid (SHA, 2,
Fig. 1), a known inhibitor of myeloperoxidase.18 As numerous
peroxidases are inhibited by aromatic hydroxamic acids such
as benzohydroxamic acid (BHA, 1),19–21 we investigated if
SHA, a potential metabolite of our COX inhibitors, could in
addition interfere with the POX activity of PGHS. SHA was
not an effective inhibitor of the H2O2-dependent oxidation of
a POX co-substrate by PGHS. We thus sought more potent
peroxidase inhibitors, based on the aromatic hydroxamic acid
backbone. Such molecules substituted on the ortho position
with a group capable of forming hydrogen bonds represent the
minimal pharmacophore for the generation of COX and POX
inhibitors.14 We therefore turned our attention to anthranilic
hydroxamic acid (AHA, 3, Fig. 1) which we found inhibited
peroxidase activity of PGHS with an IC50 value of 72 lM
(vide infra) and was deemed a suitable lead compound for
optimisation by parallel solid phase synthesis.
PGHS is a bifunctional enzyme possessing two connected
active sites,7 a cyclooxygenase site (COX), which catalyses the
addition of two oxygen molecules to arachidonic acid and
a spatially and functionally distinct peroxidase site (POX),
which catalyses the reduction of a hydroperoxide group to the
corresponding alcohol. The COX active site is the target of non-
steroidal anti-inflammatory drugs (NSAIDs), such as aspirin.8
The peroxidase activity is unaffected by NSAIDs and can
function independently of COX site inhibition.9,10 Compounds
targeting the POX site are of potential therapeutic value, as
unchecked free radical generation contributing to disease pro-
gression in inflammatory conditions could occur despite NSAID
administration.11 Also, nitric oxide, which induces vasodilation
and inhibits platelet aggregation, is consumed during PGHS-
† Electronic supplementary information (ESI) available: schemes of
2-chlorotrityl chloride polystyrene resin and p-nitrophenyl carbonate
Wang resin based methods; table of conditions for modified Wang
resin method; correlation between biological activity and electronic
properties of substituted anthranilic hydroxamic acids (-AHA) MOPAC
calculations; QSAR analysis; plots of PGHS-1 peroxidase inhibi-
tion vs. hydrophobicity and molecular density; NMR spectra. See
http://dx.doi.org/10.1039/b505525c
‡ Present address: Conway Institute of Biomolecular and Biomedical
Research, University College Dublin, Belfield, Dublin 4, Ireland. E-
mail: Des.Fitzgerald@ucd.ie; Fax: +353 1 716 1286; Tel: +353 1 716
1285.
We report here the synthesis of targeted combinatorial arrays
of anthranilic hydroxamic acids and their in vitro activity
as potential inhibitors of the peroxidase catalysis of PGHS.
3 6 7 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 6 7 8 – 3 6 8 5
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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the reduced nucleophilicity of this amino group does not afford
unequivocal loading of the trityl chloride resin.
A p-nitrophenyl carbonate Wang resin has previously been
used for the successive immobilisation of anthranilic acid
through its anilino group, amidification of its carboxylic group
and cyclization-cleavage to generate quinazoline-2,4-diones.23
We followed the reported procedure23 for the modification of
the Wang resin and the loading of anthranilic acid by formation
of a carbamate linkage (ESI,† Fig. S2). The success of this latter
step was verified by sacrificing a small portion of the resin to
directly liberate anthranilic acid under acidic conditions. The
polymer-bound anthranilic acid was then used for the acylation
of hydroxylamine. However, despite several attempts, based
on different coupling chemistries (carbodiimides, phosphonium
and uronium reagents, a preformed active p-nitrophenyl ester
prepared according to Mitsunobu conditions, preformed mixed
anhydrides) and conditions using protected or free hydroxy-
lamine and on different cleavage conditions, poor yields and
purities for the anthranilic hydroxamic acid, not exceeding 50%
and 42% respectively, were obtained (see ESI,† Table S1).
The reduced nucleophilicity of the amino group in anthranilic
acid evidenced previously (vide supra) prompted us to assess the
preparation of the hydroxamates by nucleophilic displacement
of resin-bound anthranilate active esters with hydroxylamine.
This ‘catch-and-release’ strategy is based on the formation of a
polymer-bound active ester in a first step and its reaction with
hydroxylamine in a second step. It was therefore hoped that the
anilino group would not displace the intermediate resin-bound
anthranilate to form the 2-(2-amino-benzoylamino)benzoic
acid. We selected two polystyrene-based supports, the oxime
resin, a well established support for the solid phase synthesis
of hydroxamic acids24 and the hydroxybenzotriazole (HOBt)
resin 32 (Scheme 1), which was recently used by our group for
the preparation of low molecular weight hydroxamic acids.22
In our hands, the best results were obtained with the HOBt-
active esters 34. The reactivity of these intermediates afforded
shorter reaction times, for both the activation and the coupling
steps (2 h and 4 h respectively for the HOBt esters versus
17 h and 18 h respectively for the oxime esters) and a lower
number of equivalents of hydroxylamine than for their oxime
esters counterparts (0.9 equivalents for the HOBt esters versus
3 equivalents for the oxime esters). Moreover, the hydroxamic
acids 35 were obtained in quantitative yields and in high purities
Fig. 1 Anthranilic hydroxamic acid (AHA) derivatives synthesised.
The synthetic protocol used is an extension of methodology
previously reported by us in this journal for monofunctional
hydroxamic acids.22 Of the 29 anthranilic hydroxamic acids
synthesised, 22 are reported for the first time.
Results and discussion
The effect of substitution on the aromatic ring of AHA on
peroxidase activity inhibition has been evaluated in this study. A
library of 29 anthranilic hydroxamic acid derivatives containing
a wide range of substituents with different electronic and steric
properties have been prepared and tested (see Fig. 1).
Solid phase synthesis of anthranilic hydroxamic acids
The general strategy was to prepare the novel AHA compounds
by solid phase synthesis, from commercially available carboxylic
acid precursors, without the need to protect the anilino group.
An attractive solution is to use this latter function for the
attachment of anthranilic acid to the resin, and to subsequently
convert the free carboxylic group to the hydroxamate by simple
coupling chemistry with hydroxylamine.
The first resin selected was the 2-chlorotrityl chloride
polystyrene resin, which allows the anchoring of compounds
through a nucleophilic group and their later release under mild
acidic conditions (ESI,† Fig. S1). The loading of the resin with
anthranilic acid (4 equivalents relative to resin) was attempted
in the presence of diisopropylethylamine (2 equivalents relative
to resin) to generate the free anilino group. However, FTIR
analysis of the modified resin with anthranilic acid showed that
the attachment of this substrate occurred through either the
anilino or the carboxylic group (data not shown). Therefore,
Scheme
1
‘Catch-and-release’ method. Reagents and conditions:
i, PS-HOBt (1 equiv.), anthranilic acid (1.5 equiv.), diisopropylcarbodi-
imide, (4.5 equiv.), 4-(dimethylamino)pyridine, (0.6 equiv.), 1 : 1 DCM–
DMF, 2 h; ii, hydroxylamine (0.9 equiv.), 2.3 : 1 THF–MeOH, 4 h.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 6 7 8 – 3 6 8 5
3 6 7 9

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(see Experimental section). Finally, the recycling of the resin 32
was successfully undertaken to scale-up the preparation of some
hydroxamic acids to gram quantities (data not shown). The by-
products, detected by reverse-phase HPLC, were the carboxylic
acids 33, accounting for 2%, in the case of compound 10, to 23%,
in the case of compound 28, of the isolated products. Traces of
4-(dimethylamino)pyridine or N,Nꢀ-diisopropylurea were also
observed by HPLC or NMR, respectively, in some cases. We
did not investigate if 2-(2ꢀ-amino-benzoylamino)benzoic acid
derivatives were formed during the activation step, as the
final yields of hydroxamic acids were satisfactory. In addition,
this unwanted product would have been eliminated during
the washes performed after this step. In the case of N,Nꢀ-
diisopropylurea we prepared an authentic sample and tested
it in the TMPD assay (vide infra). Finally, some hydroxamic
acids from the library (17, 20, 22, 24 and 26) covering a range
of biological activities were selected and purified by reverse-
phase HPLC to a satisfactory analytical purity. The biological
activities of these purified hydroxamic acids were assessed and
found to be equivalent to those of the non-purified compounds
(see Table 1, footnote c).
Fig. 2 The PGHS-peroxidase reaction.
indicating that the hydroxamic moiety is necessary for perox-
idase inhibition. This is consistent with previous observations
on tepoxalin and naproxen hydroxamic acid, both of which are
PGHS-1 peroxidase inhibitors, whereas their carboxylic acid
derivatives are inactive.25 Both the 4-(dimethylamino)pyridine
and diisopropylurea were also shown to be inactive (IC50
1000 lM in both cases). Therefore, the inhibitory activity
observed is solely due to the hydroxamic acids.
>
Our initial observation was that the relative potency for the
inhibition of PGHS-1 peroxidase activity was AHA > BHA >
SHA (1–3, Table 1). To examine the effect of the relative position
of the amino group in AHA, we tested 3-amino BHA and 4-
amino BHA26 and found IC50 values of 146 14 lM and 373
47 lM respectively, both significantly higher than AHA which
In vitro biological activity
TMPD assays. The compounds were screened for their
ability to inhibit the PGHS-1 and -2 peroxidase activity by mea-
suring the H2O2-dependent oxidation of the chromogenic reduc-
ing co-substrate, N,N,Nꢀ,Nꢀ-tetra-p-methylphenylenediamine
(TMPD)25 (Table 1, Fig. 2). Inhibitors, at a range of concen-
trations, were pre-incubated with the enzyme before both the
co-substrate (TMPD) and substrate (H2O2) were added to start
the reaction. Initial rates of reaction were measured.
has an IC50 value of 72
26 lM. The preference shown by
the PGHS-1 peroxidase site for an amino group at the ortho
position led us to investigate the influence of substitution at
different positions on the phenyl ring of anthranilic hydroxamic
acid (4–31).
All carboxylic acid precursors of these compounds showed no
inhibition of either isozyme in this assay (i.e. IC50 > 1000 lM),
Of the 29 compounds synthesised, 3,5-diiodoanthranilic
hydroxamic acid (3,5-diiodo AHA, 24) was the most effective
Table 1 Inhibition of PGHS peroxidase activity by substituted anthranilic hydroxamic acids (-AHA)
-AHA
IC50 TMPD PGHS-1a
IC50 TMPD PGHS-2a
% PPHPb PGHS-1
16 11
3,5-I2 (24)
3,5-Br2 (23)
3,5-Cl2 (22)
5-Br (11)
3-Cl (4)
4,5-F2 (29)
5-I (15)
3-Br-5-Me (26)
4-Cl (7)
5-NO2 (18)
5-Cl (12)
4-F (8)
3,4,5,6-F4 (28)
3-CF3 (6)
5-F (13)
4-NO2 (9)
AHA (3)
3,5-(CH3)2 (25)
Nicotinic (31)
5-OH (14)
5-CH3 (17)
BHA (1)
3-OCH3 (5)
4,5-(OCH3)2 (30)
5-NHCOCH3 (10)
SHA (2)
5-OCH3 (16)
6-F (20)
6-Cl (19)
7.2 3.1c
12.2 7.1
12.9 8.3c
13.0 2.3
14 12
16 10
132 92
148 90
128 55
577 178
108 30
134 34
257 142
184 250
315 98
284 73
498 156
184 49
241 45
357 132
191 97
425 199
402 123
>1000
220 153
263 88
>1000
>1000
109 43
>1000
>1000
304 216
>1000
>1000
>1000
34
58
8
3
49 12
79 13
69 18
52 16
15
7
17 13
23 5c
27 10
28 18
29 18
77
64
79
75
2
0
4
3
35
43
9
8
78 14
80
0
49 59
68 91
69 18
72 26
84 12
84 86
110 41
115 55c
116 12
116 82
206 91
248 83
378 50
388 235
419 264c
>1000
78 20
65 20
59 20
76 13
83
63
70
67
0
6
3
8
n.d.
87 22
83
78
0
3
n.d.
85
65
1
7
3
62
6-CH3 (21)
(OCH3)3 (27)
>1000
>1000
60 12
76 19
>1000
a IC50 values for inhibition of TMPD oxidation shown in lM (200 lM H2O2 substrate; 200 lM TMPD co-substrate; n = 3, std. dev.). b Percentage
of maximal PPHP conversion using 200 lM of each compound as co-reducing agent (n = 2 to 4, std. dev.). c IC50s in lM of purified AHAs are:
5.3 1.8 for 24; 8.4 0.8 for 22; 30.3 18.3 for 26; 93.0 18.3 for 17; 921.6 66.1 for 20.
3 6 8 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 6 7 8 – 3 6 8 5

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inhibitor, with IC50 values of 7.2 3.1 lM and 16 10 lM for
PGHS-1 and PGHS-2 respectively (Table 1). This was closely
followed by the other dihalogenated compounds 3,5-dibromo
AHA (23), 3,5-dichloro AHA (22) and 4,5-difluoro AHA (29)
showing that for a meta substituent, an electron-withdrawing
group overrides the effect of an electron-releasing methyl group.
Assuming a free radical mechanism, the effects of substituents
ortho or para (positions 3 and 5) to the amino group are more
consistent with the formation of a nitrogen-centered aniline
radical32 as opposed to an oxygen centered hydroxamate radical.
This is despite the fact that semiempirical calculations show
that the enthalpy of formation of the latter is significantly lower
(see QSAR studies below). In any case, it appears that the
amino and hydroxamate groups must be in ortho positions for
activity to be observed.
An alternative explanation is that the substituents in position 5
establish favourable interactions with the active site, thus helping
the compound to adopt a different binding conformation.
Different binding modes may account for the differing levels
of inhibition of the compounds: if the free radical generated
upon reaction with compound II diffuses away, the molecule
would act as a mere reducing co-substrate, whereas if the free
radical reacts with a residue within the active site resulting in
protein damage the molecule would be an inhibitor.
The fact that only 24 (3,5-diiodo AHA) seems to be a genuine
inhibitor of PGHS-1, and possibly PGHS-2, may suggest a
different mechanism of action for this particular compound.
A two-electron oxidation of compound 24 to yield an iodine(III)
species33 could result in enzyme inactivation.
Another significant finding is that substituents at the 6
position (19, 20 and 21), ortho to the hydroxamic acid, render
the compound ineffective as an inhibitor in the TMPD assay.
This could be explained because of the steric hindrance in
the environment of the reactive centre (assuming that it is the
hydroxamate). However, ortho substituents would also force the
carbonyl group out of plane with respect to the phenyl ring, thus
destabilizing a free radical centered on the hydroxamate oxygen,
and the amino nitrogen.
with IC50 values of 12.2
7.1 lM, 12.9
8.3 lM and 15
7 lM, respectively. Monohalogenated compounds such as 5-
bromo AHA (11), 3-chloro AHA (4), and 5-iodo AHA (15) were
also found to be effective, with IC50 values of 13.0 2.3 lM, 14
12 lM and 17 13 lM, respectively for inhibition of PGHS-1
peroxidase activity.
Whereas most of the compounds showed inhibitory activity
towards the peroxidase site of PGHS-1, apart from 3,5-diiodo
AHA, all were ineffective as inhibitors towards the peroxidase
site of PGHS-2, with IC50 values greater than 100 lM. This is
not surprising since the peroxidase catalyses of PGHS-1 and -2
are markedly different.5 However, our results allow for selective
inhibition of the peroxidase site of PGHS-1.
PPHP assays. A limitation frequently overlooked in per-
oxidase inhibitor studies is that the assay is based on the
oxidation of a chromogenic reducing co-substrate (e.g. TMPD
or guaiacol) and not on the reduction of the substrate (e.g. H2O2)
itself. Therefore, apparent inhibitors may compete with TMPD
oxidation, rather than exhibit true inhibition of enzyme catalysis.
Consequently the IC50 values reflect a competition with the co-
reducing assay reagent (TMPD), and not the substrate itself
(H2O2). We have addressed this in our studies by assaying the
reduction of a lipid peroxide substrate (trans-5-phenyl-pent-
4-en-1-yl hydroperoxide, PPHP) to the corresponding alcohol
(trans-5-phenyl-pent-4-en-1-ol, PPA), using HPLC27 (Table 1).
At 200 lM, most of the AHA derivatives showed significant
reducing co-substrate activity, exceeding that of phenol (set at
approximately 40% in this assay). However, compounds 23 and
24, showed less co-reducing activity than phenol. Compound 24
(3,5-diiodo AHA) showed no activity as a reducing co-substrate
at 200 lM (i.e. well in excess of the TMPD IC50), indicating that
it is likely a true inhibitor of the PGHS-1 peroxidase site.
QSAR and modelling studies. Semiempirical calculations
were carried out for all the compounds using MOPAC at PM334
level of theory (results are given in ESI†). As already observed
in similar QSAR studies35 the correlation found between the
computed electronic properties of the compounds described
herein and their IC50 values was poor (see ESI†). Among the
properties analyzed were the heats of formation of three possible
free radicals (hydroxamate oxygen, hydroxamate nitrogen and
aniline nitrogen), ionization potentials and electron densities
and bond orders at the likely reactive centres of the inhibitors
(ESI,† Table S2).
Docking calculations were also carried out with a few
representative compounds using the program Autodock36 and a
crystal structure37 of the ovine PGHS-1 (Protein Data Bank ac-
cession number 1Q4G) and a compound II model prepared with
SYBYL (Tripos Inc.). The docking energies obtained are very
similar for all the compounds (approximately −8.0 kcal mol−1),
about one half of the value found for the natural substrate PGG2
(data not shown, manuscript in preparation).38 We conclude
that the enzyme–inhibitor interactions are weak and essentially
non-specific.
A QSAR analysis was undertaken using the QuaSAR pack-
age of the MOE39 suite of programs, using 203 2D and
3D descriptors, as reported previously.40 A weak correlation
was found between the hydrophobicity coefficient [logP(o/w),
octanol/water] and the pIC50 for PGHS-1 peroxidase inhibition
[pIC50 = − log(IC50 × 10−6), where IC50 is in lM] (ESI,†
Fig. S3). This is reasonable as the effective concentration within
an essentially hydrophobic cavity is expected to be higher for the
less water-soluble compounds.
The effects of substituents on biological activity. Aromatic
hydroxamic acids have been proposed to act as reducing co-
substrates in the peroxidase catalytic cycle of PGHS.14 Reaction
with compound II (Fig. 2) would presumably yield an oxygen-
centered free radical that could be delocalised by resonance
through the phenyl ring via a hydroxamate tautomer.28 A radical
of this type would be expected to be stabilised in ortho and para
positions, while the influence of meta substituents should be
slightly destabilising or negligible.29–31
From Table 1, it is obvious that compounds with substituents
meta to the hydroxamate, and thus ortho or para to the amino
group, are the best inhibitors. Electron-withdrawing groups (-Cl,
-Br, -F, and -CF3) as well as -I increase potency. In addition to
their inductive effect, -Br and -Cl can stabilize a free radical
by resonance.29–31 Significantly, this is also true for the more
polarisable -I. 3,5-Dihalogenated derivatives are the most active,
but the difference between their activities and those of the mono-
halogenated analogues is very small. For instance, comparing 4
(3-chloro AHA) with 22 (3,5-dichloro AHA), and 11 (5-bromo
AHA) with 23 (3,5-dibromo AHA) indicates that even one
halogen substituent at positions 3 or particularly 5 (meta to the
hydroxamate and ortho or para to the amino group, respectively)
produces an inhibitor nearly as active as the dihalogenated
compounds. The low activity of compound 10 (5-acetamido
AHA) may be due to the steric demands of the acetamido
substituent outweighing its electron withdrawing properties.
Electron-donating non-polarisable groups (-CH3, -OH, and
-OCH3), on the other hand, reduce inhibition, all compounds
containing such groups (25, 31, 14, 5, 17, 30, 16, 21 and 27)
showing less activity than AHA. Interestingly, the only excep-
tion, 3-bromo-5-methyl AHA (26), which is more effective than
AHA, has the halogen substitution meta to the hydroxamate,
Conclusion
In contrast to other peroxidases, PGHS-1 appears to favour an
amino group at the ortho position in the aromatic hydroxamic
acid peroxidase inhibitors, i.e. anthranilic hydroxamic acid.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 6 7 8 – 3 6 8 5
3 6 8 1

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Derivatisation showed that substituents ortho and para to the
amino group, i.e. meta to the hydroxamate, are preferred,
with electron-withdrawing or polarisable substituents signifi-
cantly improving inhibition. Electron-donating groups, however,
consistently decrease efficacy. All aromatic hydroxamic acid
peroxidase inhibitors tested showed significant co-substrate
activity, except for 3,5-diiodoanthanilic hydroxamic acid, which
appears to be a true PGHS-1 peroxidase inhibitor.
hep, J = 6.52 Hz, CH), 1.01–0.99 (12H, d, J = 6.52 Hz, CH3).
13C NMR (DMSO-d6, 100 MHz): d 156.76, 40.66, 23.30.
TMPD assay for PGHS peroxidase activity
Peroxidase activity was measured by monitoring the
PGHS enzyme catalysed oxidation of the reducing co-
substrate N,N,Nꢀ,Nꢀ-tetra-p-methylphenylenediamine dihy-
drochloride (TMPD), at 611 nm.25 Reactions were carried out at
37 ◦C in a 200 lL cuvette. Compounds were dissolved in DMSO
at various concentrations (0.01 lM to 1 mM) and were incubated
with 23 nM of PGHS-1 or 365 nM PGHS-2 for one minute at
37 ◦C in a 200 lL solution of 0.1 M Tris pH 8.0 and 1 lM
haematin (final DMSO concentration <1.0%). The reaction
was initiated with the addition of 200 lM TMPD followed by
200 lM H2O2. The rate of TMPD oxidation recorded in the first
4 seconds of the reaction was taken as a measure of the enzyme’s
initial velocity. Enzyme activity was normalised to the activity
of solutions containing the vehicle alone.
Each experiment was carried out in duplicate (n ≥ 3). Initial
velocities were converted to percentage of maximal activity
and plotted against the log10 of inhibitor concentrations (lM).
The titration curve was fitted to a four-parameter non-linear
regression equation (SigmaPlot 8.0, SPSS Inc.) of the form:
y = min + (max − min)/[1 + 10exp(logIC50 − x)] where max
and min are asymptotes, x is the logarithm of the inhibitor
concentration and logIC50 is the inflection point. Only data
with an R2 value greater than 0.900 were considered (n = 3).
The corresponding carboxylic acids were tested and showed no
activity (i.e. IC50 > 1000 lM).
Experimental
Materials
The 1-hydroxybenzotriazole-6-sulfonamidomethyl polystyrene
resin (PS-HOBt, 0.98 mmol g−1) was purchased from Argonaut
technologies. PGHS-1, PGHS-2 and PPHP were from Cayman
Chemicals, Michigan, USA. All other reagents and solvents,
purchased from Sigma-Aldrich and Lancaster, were of the high-
est possible quality and were used without further purification.
The elemental analyses were performed by SGS MULTILAB
(Evry, France).
Synthesis
Three arrays of 10 anthranilic hydroxamic acids were prepared
on a QUEST 210 ASW Synthesiser (Argonaut Technologies,
Cardiff, UK). The reactions were performed under an
atmosphere of nitrogen. Percentage yields were estimated from
the isolated weight of each hydroxamic acid and are based on the
amount of hydroxylamine used (0.9 equivalents of the PS-HOBt
resin initial loading). Chromatographic analysis and purification
were performed on a PerSeptive Biosystems BioCAD SPRINT
Perfusion Chromatography Workstation using POROS 20R2
reverse-phase chromatography packing (column: 4.6 mmD ×
100 mmL, 1.7 ml, self-packed; A mobile phase: 0.1% TFA in
water; B mobile phase: 0.1% TFA in acetonitrile; gradient: 2
to 60% B in 18 column volumes at 7 ml min−1 flow rate, for
analysis) (column: 10 mmD × 100 mmL, 7.9 ml, self-packed;
A mobile phase: 0.1% TFA in water; B mobile phase: 0.1%
TFA in acetonitrile; gradient: 2 to 60% B in 20 column
volumes at 12 ml min−1 flow rate, for purification). Purities
were ascertained from the percent area of the hydroxamic acid
relative to the total area of all UV absorbing components
(235 nm and 280 nm). The carboxylic acid precursors, the main
impurities in the current work, are predicted to have absorbance
maxima analogous to the corresponding hydroxamic acids.
Liquid chromatography-mass spectrometry experiments were
performed on a Quattro Micro quadrupole electrospray mass
spectrometer (Micromass, Waters Corp., USA): 10 lL of the
samples were injected in 300 lL of acetonitrile : water (60 :
40, v/v). The mass spectrometry data were acquired both in
positive and negative ion modes. Nuclear magnetic resonance
spectra were recorded on a Bruker DPX400 NMR.
PPHP assay for PGHS peroxidase activity
The PPHP assay is based on the chromatographic analysis of the
of conversion of a lipid peroxide into its corresponding alcohol
and has been adapted from a reported procedure.27 Purified
ovine PGHS-1 (4.3 nM) was pre-incubated for 3 minutes with
200 lM of each compound in 500 lL of 100 mM Tris buffer
(pH 8.0), 1 lM haematin at 37 ◦C (final DMSO concentration
<1.0%). Reactions were initiated by adding 100 lM PPHP
◦
(stock in ethanol) and incubated at 37 C for 3 minutes (final
ethanol concentration <0.5%). Reactions were terminated by
C18 reverse-phase extraction. The C18 columns (Empore C18-
HD, 3 M) were pre-equilibrated with 1 ml H2O, reaction
mixtures were then added and the columns were washed with
4 ml H2O, and eluted in 1 ml methanol. PPHP and PPA were
separated using reverse-phase HPLC on a C12 column (Synergi
4 lm MAX-RP; 150 mmL × 4.6 mmD; Phenomenex). Each
sample (200 ll) was injected using a BioCAD SPRINT HPLC
workstation (Applied Biosystems) and isocratically eluted with
65% methanol–H2O at a flow rate of 1.0 ml min−1 with detection
at 254 nm. Under these conditions all anthranilic hydroxamic
acids eluted in less then 7.0 minutes, while PPA and PPHP eluted
at 7.6 and 8.7 minutes respectively, with equal peak absorbance
integrals. Product formation was expressed as the ratio of the
integrated absorbance of PPA to total substrate and product
[i.e. PPA/(PPA + PPHP)]. Background conversion due to heme
(average = 0.38) was subtracted from each reading. Maximal
activity, found using an excess of enzyme (34 nM), was used
to convert each ratio to percentage of maximal activity. Phenol
showed 42% 12% of maximal conversion while the control (no
phenol) showed 24% 12% conversion. Assays were performed
in duplicate with n = 2 to 4.
The synthesis of the anthranilic hydroxamic acids was carried
out as previously reported22 on a 0.245 mmol scale for the resin,
except that the loading and the coupling step were performed for
2 h and 4 h respectively. 0.9 Equivalent of free hydroxylamine41
relative to the initial loading of the PS-HOBt resin was used.
The hydroxamic acids were obtained in quantitative yields and
with high purities. Compounds 4–10, 13–21, 24–26 and 28–30
are reported for the first time.
N,Nꢀ-Diisopropylurea was synthesised as follow: a solution
of 500 mg (4 mmol) of N,Nꢀ-diisopropylcarbodiimide in 2.5 ml
of THF was added to 5 ml of a 1 M NaOH solution and
the resulting mixture was vigorously stirred for 2 h at room
temperature. The organic solvent was evaporated under reduced
pressure and the aqueous solution was stored at 4 ◦C overnight.
The crystals formed were isolated by filtration, rinsed with water
Characterization of AHA derivatives
Anthranilic hydroxamic acid (3). Isolated as a light brown
solid in 94% yield, 31 mg. Found: C, 55.32; H, 5.28; N, 18.27.
1
Calc. for C7H8N2O2: C, 55.26; H, 5.30; N, 18.41%. H NMR
1
and dried under vacuum to give 57 mg (12%) of product. H
(DMSO-d6, 400 MHz): d 10.89 (1H, br s, OH), 8.85 (1H, br s,
NH), 7.31–7.29 (1H, d, J = 8 Hz, ArH6), 7.13–7.12 (1H, m,
NMR (DMSO-d6, 400 MHz): d 5.5–5.48 (2H, d, NH), 3.64 (2H,
3 6 8 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 6 7 8 – 3 6 8 5

View Article Online
J = 7 Hz, 8 Hz, ArH5), 6.69–6.67 (1H, d, J = 8 Hz, ArH3), 6.49–
116.23, 107.01, 23.31. Purity (RP-HPLC): 98% (tr = 1.05); m/z
6.47 (1H, m, J = 7 Hz, 8 Hz, ArH4), 6.23 (2H, br s, NH2). 13
C
(ES) calc. for C9H11N3O3 208.2, found 208.2.
NMR (DMSO-d6, 100 MHz): d 166.89, 149.23, 131.97, 127.93,
116.57, 115.08, 113.05. Purity (RP-HPLC): 97% (tr = 1.05); m/z
(ES) calc. for C7H8N2O2 151.2, found 151.2.
5-Bromoanthranilic hydroxamic acid (11). Isolated as a light
brown solid in 57% yield, 29 mg. 1H NMR (DMSO-d6,
400 MHz): d 11.05 (1H, s, OH), 8.96 (1H, s, NH), 7.46–7.45
(1H, d, J = 2.52 Hz, ArH6), 7.27–7.25 (1H, m, J = 8.52 Hz, J =
2.52 Hz, ArH4), 6.69–6.67 (1H, d, J = 9 Hz, ArH3), 6.40 (2H,
br s, NH2). 13C NMR (DMSO-d6, 100 MHz): d 165.54, 148.48,
134.05, 129.63, 118.26, 114.56, 104.97. Purity (RP-HPLC): 88%
(tr = 1.93); m/z (ES) calc. for C7H7N2BrO2 230.1, found 230.9.
5-Chloroanthranilic hydroxamic acid (12). Isolated as a
cream solid in 70% yield, 29 mg. 1H NMR (DMSO-d6,
400 MHz): d 11.06 (1H, s, OH), 8.98 (1H, s, NH), 7.35–7.34
(1H, d, J = 2.52 Hz, ArH6), 7.16–7.13 (1H, m, J = 9 Hz,
J = 2.48 Hz, ArH4), 6.73–6.71 (1H, m, J = 8.52 Hz, ArH3),
6.38 (2H, br s, NH2). 13C NMR (DMSO-d6, 100 MHz): d
162.31, 148.16, 138.94, 131.30, 126.82, 117.82, 113.86. Purity
(RP-HPLC): 95% (tr = 1.45); m/z (ES) calc. for C7H7N2ClO2
185.6, found 185.2.
3-Chloroanthranilic hydroxamic acid (4). Isolated as a cream
1
solid in 92% yield, 38 mg. H NMR (DMSO-d6, 400 MHz): d
11.14 (1H, s, OH), 9.05 (1H, s, NH), 7.38–7.36 (1H, dd, J =
1 Hz, J = 7.52 Hz, ArH6), 7.34–7.32 (1H, d, J = 7.52 Hz,
ArH4), 6.58–6.56 (1H, m, J = 7.52 Hz, 8 Hz, ArH5), 6.35 (2H,
br s, NH2). 13C NMR (DMSO-d6, 100 MHz): d 165.93, 157.21,
144.75, 131.60, 126.57, 118.87, 115.43. Purity (RP-HPLC): 95%
(tr = 2.52); m/z (ES) calc. for C7H7N2ClO2 185.6, found 185.1.
3-Methoxyanthranilic hydroxamic acid (5). Isolated as a
brown oil in 81% yield, 30 mg. 1H NMR (DMSO-d6, 400 MHz):
d 10.93 (1H, br s, OH), 9.21 (1H, br s, NH), 7.00–6.98 (1H,
dd, J = 1 Hz, J = 7 Hz, ArH6), 6.89–6.87 (1H, d, J = 8 Hz,
ArH4), 6.50–6.48 (1H, m, J = 7 Hz, 8 Hz, ArH5), 3.79–3.77
(3H, s, J = 5 Hz, ArOCH3), 5.94 (2H, br s, NH2). 13C NMR
(DMSO-d6, 100 MHz): d 166.75, 146.83, 139.16, 119.20, 114.24,
112.65, 111.92, 55.54. Purity (RP-HPLC): 90% (tr = 1.12); m/z
(ES) calc. for C8H10N2O3 181.2, found 181.1.
5-Fluoroanthranilic hydroxamic acid (13). Isolated as a
cream solid in 53% yield, 32 mg. 1H NMR (DMSO-d6,
400 MHz): d 10.86 (1H, br s, OH), 9.20 (1H, br s, NH), 7.17–7.14
(1H, m, J = 10 Hz, J = 3 Hz, ArH6), 7.06–7.04 (1H, m, J =
8.56 Hz, J = 3 Hz, ArH4), 6.73–6.71 (1H, m, J = 9 Hz, J = 5 Hz,
ArH3), 6.15 (2H, br s, NH2). 13C NMR (DMSO-d6, 100 MHz):
d 153.72, 145.99, 139.76, 118.84, 117.36, 113.19, 106.92. Purity
(RP-HPLC): 95% (tr = 1.05); m/z (ES) calc. for C7H7N2FO2
169.1, found 169.1.
3-Trifluoromethylanthranilic hydroxamic acid (6). Isolated
1
as a cream solid in 85% yield, 41 mg. H NMR (DMSO-d6,
400 MHz): d 11.23 (1H, br s, OH), 9.14 (1H, br s, NH), 7.58–
7.57 (1H, d, J = 7.52 Hz, ArH6), 7.51–7.49 (1H, d, J = 7.48 Hz,
ArH4), 6.68–6.66 (1H, m, J = 7.48, 7.52 Hz, ArH5), 6.51
(2H, br s, NH2). 13C NMR (DMSO-d6, 100 MHz): d 169.28,
165.82, 145.93, 132.62, 129.39, 124.90, 115.88, 114.68. Purity
(RP-HPLC): 96% (tr = 2.88); m/z (ES) calc. for C8H7N2F3O2
219.2, found 219.2.
5-Hydroxyanthranilic hydroxamic acid (14). Isolated as a
brown solid in 76% yield, 27 mg. 1H NMR (DMSO-d6,
400 MHz): d 10.19 (1H, s, OH), 8.85 (1H, s, NH), 8.71 (1H, s,
OH, ArH5), 6.74–6.73 (1H, s, J = 2.52 Hz, ArH6), 6.70–6.68
(1H, m, J = 8.52 Hz, J = 2.52 Hz, ArH4), 6.59–6.57 (1H, d,
J = 8.56 Hz, ArH3), 6.59 (2H, s, NH2). 13C NMR (DMSO-
d6, 100 MHz): d 162.32, 147.35, 139.21, 119.67, 117.65, 113.45,
106.93. Purity (RP-HPLC): 90% (tr = 1.05); m/z (ES) calc. for
C7H8N2O3 167.2, found 167.1.
4-Chloroanthranilic hydroxamic acid (7). Isolated as a cream
1
solid in 73% yield, 30 mg. H NMR (DMSO-d6, 400 MHz):
d 10.98 (1H, br s, OH), 9.39 (1H, br s, NH), 7.33–7.31 (1H,
m, J = 3.44 Hz, J = 8.20 Hz, ArH6), 6.77–6.76 (1H, m, J =
2.04 Hz, ArH3), 6.50–6.48 (1H, m, J = 6.8 Hz, J = 2.04 Hz
ArH5), 6.52 (2H, br s, NH2). 13C NMR (DMSO-d6, 100 MHz):
d 166.00, 150.60, 136.04, 129.31, 115.35, 114.80, 111.72. Purity
(RP-HPLC): 94% (tr = 1.97); m/z (ES) calc. for C7H7N2ClO2
185.6, found 185.2.
5-Iodoanthranilic hydroxamic acid (15). Isolated as a black
1
solid in 85% yield, 49 mg. H NMR (DMSO-d6, 400 MHz): d
11.02 (1H, br s, OH), 8.94 (1H, br s, NH), 7.58 (1H, s, ArH6),
7.39–7.37 (1H, d, J = 8.52 Hz, ArH4), 6.58–6.56 (1H, d, J =
8.56 Hz, ArH3), 6.39 (2H, br s, NH2). 13C NMR (DMSO-d6,
100 MHz): d 165.50, 150.91, 139.53, 135.38, 118.71, 115.50,
106.93. Purity (RP-HPLC): 93% (tr = 2.83); m/z (ES) calc. for
C7H7N2IO2 277.0, found 277.0.
4-Fluoroanthranilic hydroxamic acid (8). Isolated as a cream
1
solid in 85% yield, 32 mg. H NMR (DMSO-d6, 400 MHz): d
10.94 (1H, s, OH), 9.58 (1H, s, NH), 7.39–7.37 (1H, m, J =
6.84 Hz, J = 8.88 Hz, ArH6), 6.49–6.46 (1H, m, J = 12.28 Hz,
J = 2.72Hz, ArH3), 6.30–6.28 (1H, m, J = 8.88 Hz, J = 2.72 H
ArH5), 6.58 (2H, br s, NH2). 13C NMR (DMSO-d6, 100 MHz):
d 165.55, 163.11, 151.82, 130.04, 124.91, 109.64, 101.57. Purity
(RP-HPLC): 90% (tr = 1.27); m/z (ES) calc. for C7H7N2FO2
169.1, found 169.2.
5-Methoxyanthranilic hydroxamic acid (16). Isolated as a
brown solid in 63%, 24 mg. 1H NMR (DMSO-d6, 400 MHz): d
10.71 (1H, br s, OH), 8.89 (1H, br s, NH), 6.91–6.90 (1H, m, J =
3 Hz, ArH6), 6.84–6.82 (1H, m, J = 8.52 Hz, J = 3 Hz, ArH4),
6.67–6.65 (1H, m, J = 9 Hz, ArH3), 3.66 (3H, ArOCH3). 13C
NMR (DMSO-d6, 100 MHz): d 149.29, 143.34, 139.05, 119.31,
117.60, 111.39, 106.94, 55.41. Purity (RP-HPLC): 94% (tr =
1.05); m/z (ES) calc. for C8H10N2O3 181.2, found 181.1.
4-Nitroanthranilic hydroxamic acid (9). Isolated as a light
brown solid in 62% yield, 27 mg. 1H NMR (DMSO-d6,
400 MHz): d 11.28 (1H, br s, OH), 9.17 (1H, br s, NH), 7.58–7.58
(1H, m, J = 2 Hz, ArH6), 7.53–7.51 (1H, m, J = 8.52 Hz, ArH3),
7.28–7.25 (1H, m, J = 8.52 Hz, J = 2.52H, ArH5), 6.72 (2H,
br s, NH2). 13C NMR (DMSO-d6, 100 MHz): d 166.74, 156.90,
149.33, 133.03, 129.12, 109.81, 108.46. Purity (RP-HPLC): 90%
(tr = 1.60); m/z (ES) calc. for C7H7N2KO4 (i.e. K+ salt) 235.2,
found 235.3.
5-Methylanthranilic hydroxamic acid (17). Isolated as a
1
cream solid in quantitative yield, 36 mg. H NMR (DMSO-
d6, 400 MHz): d 10.85 (1H, br s, OH), 8.84 (1H, br s, NH),
7.14 (1H, s, ArH6), 6.97–6.95 (1H, m, J = 8.52 Hz, J =
2 Hz, ArH4), 6.63–6.60 (1H, m, J = 8.52 Hz, ArH3), 2.13
(3H, s, ArCH3), 6.01 (2H, br s, NH2). 13C NMR (DMSO-d6,
100 MHz,): d 162.33, 146.88, 132.36, 127.57, 123.04, 116.34,
113.19, 20.00. Purity (RP-HPLC): 96% (tr = 1.07); m/z (ES)
calc. for C8H10N2O2 165.2, found 165.2. Purified by RP-HPLC
(Experimental section) Found: C, 41.92; H, 3.88; N, 9.35. Calc.
for C8H10N2O2·TFA·0.5 H2O: C, 41.52; H, 4.18; N, 9.68%.
5-Acetamidoanthranilic hydroxamic acid (10). Isolated as a
1
pale cream solid in 82% yield, 37 mg. H NMR (DMSO-d6,
400 MHz): d 9.67 (1H, s, NHOH), 8.71 (1H, s, NH), 7.48–7.47
(1H, S, J = 2 Hz, ArH6), 7.30–7.28 (1H, d, J = 8.8 Hz, J =
2.76 Hz, ArH4), 6.64–6.62 (1H, d, J = 8.8 Hz, ArH3), 5.86
(2H, br s, NH2), 1.96 (3H, s, COCH3). 13C NMR (DMSO-d6,
100 MHz): d 170.85, 167.76, 147.61, 127.73, 124.39, 119.59,
5-Nitroanthranilic hydroxamic acid (18). Isolated as a yellow
1
solid in 98% yield, 40 mg. H NMR (DMSO-d6, 400 MHz):
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 6 7 8 – 3 6 8 5
3 6 8 3

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d 11.38 (1H, br s, OH), 9.15 (1H, br s, NH), 8.32–8.31 (1H,
m, J = 2.48 Hz, ArH6), 8.03–8.01 (1H, m, J = 9.04 Hz, J =
2.52 Hz, ArH4), 6.82–6.80 (1H, d, J = 9.04 Hz, ArH3), 7.61 (2H,
br s, NH2). 13C NMR (DMSO-d6, 100 MHz,): d 154.89, 139.35,
134.84, 127.36, 125.18, 115.60, 111.30. Purity (RP-HPLC): 81%
(tr = 1.62); m/z (ES) calc. for C7H7N3O4 196.2, found 196.1.
6-Chloroanthranilic hydroxamic acid (19). Isolated as a light
grey solid in 74% yield, 29 mg. 1H NMR (DMSO-d6, 400 MHz):
d 10.98 (1H, br s, OH), 9.27 (1H, br s, NH), 7.06–7.04 (1H, m, J =
8.2, J = 7.52 Hz, ArH4), 6.64–6.62 (1H, d, J = 8.2 Hz, ArH5),
6.59–6.57 (1H, d, J = 7.52Hz, ArH3), 5.20 (2H, s, NH2). 13C
NMR (DMSO-d6, 100 MHz,): d 162.00, 147.84, 130.96, 130.49,
118.89, 116.03, 113.55. Purity (RP-HPLC): 90% (tr = 1.10); m/z
(ES) calc. for C7H7N2ClO2 185.6, found 185.2.
113.31, 20.02, 17.55. Purity (RP-HPLC): 96% (tr = 1.23); m/z
(ES) calc. for C9H12N2O2 179.2, found 179.1.
3-Bromo-5-methylanthranilic hydroxamic acid (26). Isolated
1
as a cream solid in 78% yield, 40 mg. H NMR (DMSO-d6,
400 MHz): d 11.10 (1H, br s, OH), 9.04 (1H, s, NH), 7.37 (1H,
br s, ArH6), 7.20 (1H, s, ArH4), 2.15 (3H, s, ArC5, CH3), 6.04
(2H, br s, NH2). 13C NMR (DMSO-d6, 100 MHz): d 165.89,
143.25, 135.16, 127.66, 125.09, 115.43, 109.48, 19.50. Purity
(RP-HPLC): 94% (tr = 3.22); m/z (ES) calc. for C8H9N2BrO2
244.1, found 243.1.
3,4,5-Trimethoxyanthranilic hydroxamic acid (27). Isolated
as a dark brown solid in 97% yield, 49 mg. 1H NMR (DMSO-
d6, 400 MHz): d 10.95 (1H, br s, OH), 8.88 (1H, br s, NH), 6.84
(1H, s, ArH6), 3.78 (3H, s, ArC5, CH3), 3.71 (12H, br s, ArC3,
ArC4, ArC5, OCH3), 5.83 (2H, br s, NH2). 13C NMR (DMSO-
d6, 100 MHz,): d 166.45, 145.14, 142.59, 140.31, 138.97, 107.37,
106.93, 60.37, 59.98, 56.33. Purity (RP-HPLC): 87% (tr = 1.43);
m/z (ES) calc. for C10H17N2O2 241.2, found 241.0.
6-Fluoroanthranilic hydroxamic acid (20). Isolated as a
cream solid in 69% yield, 39 mg. 1H NMR (DMSO-d6,
400 MHz): d 10.90 (1H, s, OH), 9.17 (1H, br s, NH), 7.10–
7.08 (1H, q, J = 8.04, 9.52 Hz, ArH4), 6.52–6.50 (1H, d, J =
8.04 Hz, ArH5), 6.34–6.31 (1H, t, J = 9.52Hz, ArH3), 5.71 (2H,
br s, NH2). 13C NMR (DMSO-d6, 100 MHz): d 161.17, 158.90,
149.56, 131.15, 111.15, 105.78, 101.70. Purity (RP-HPLC): 90%
(tr = 1.18); m/z (ES) calc. for C7H7N2FO2 169.1, found 169.2.
3,4,5,6-Tetrafluoroanthranilic hydroxamic acid (28). Isolated
1
as a brown solid in 60% yield, 30 mg. H NMR (DMSO-d6,
400 MHz): d 11.20 (1H, br s, OH), 9.41 (1H, br s, NH), 5.84
(2H, s, NH2). 13C NMR (DMSO-d6, 100 MHz): d 158.35, 139.29,
138.99, 133.47, 124.96, 106.99, 103.15. Purity (RP-HPLC): 77%
(tr = 2.02); m/z (ES) calc. for C7H4N2F4O2 223.1, found 223.1.
4,5-Difluoroanthranilic hydroxamic acid (29). Isolated as
a cream solid in 87% yield, 46 mg. 1H NMR (DMSO-d6,
400 MHz): d 13.39 (1H, br s, OH), 11.93 (1H, br s, NH), 7.77
(1H, s, ArH6), 7.76 (1H, s, ArH2), 9.70 (2H, s, NH2). 13C NMR
(DMSO-d6, 100 MHz): d 165.94, 157.11, 132.71, 124.75, 122.37,
115.12, 105.53. Purity (RP-HPLC): 98% (tr = 1.35); m/z (ES)
calc. for C7H6N2F2O2 187.1, found 187.2.
6-Methylanthranilic hydroxamic acid (21). Isolated as a
cream solid in 74% yield, 25 mg. 1H NMR (DMSO-d6,
400 MHz): d 10.79 (1H, br s, OH), 9.23 (1H, br s, NH), 6.95–
6.94 (1H, m, J = 8 Hz, 7.52 Hz, J = 2.52 Hz, ArH4), 6.51–6.49
(1H, d, J = 8 Hz, ArH5), 6.39–6.37 (1H, d, J = 7.52 Hz, ArH3),
2.14–2.14 (3H, d, J = 2.48 Hz, ArCH3), 4.85 (2H, br s, NH2). 13
C
NMR (DMSO-d6, 100 MHz,): d 164.84, 146.06, 135.23, 129.24,
120.19, 117.68, 112.57, 19.48. Purity (RP-HPLC): 87% (tr =
1.05); m/z (ES) calc. for C8H10N2O2 165.2, found 165.1.
3,5-Dichloroanthranilic hydroxamic acid (22). Isolated as
grey crystals in 98% yield, 48 mg. 1H NMR (DMSO-d6,
400 MHz): d 11.27 (1H, s, OH), 9.16 (1H, s, NH), 7.51–
7.51 (1H, m, J = 2 Hz, ArH6), 7.51–7.40 (1H, m, J =
2 Hz, ArH4), 6.47 (2H, br s, NH2). 13C NMR (DMSO-d6,
100 MHz): d 164.72, 143.95, 130.84, 126.25, 119.98, 117.99,
115.63. Purity (RP-HPLC): 93% (tr = 3.38); m/z (ES) calc.
for C7H6N2Cl2O2 220.0, found 219.1. Purified by RP-HPLC
(Experimental section) Found: C, 38.11; H, 2,71; N, 12.53; Cl,
31.08. Calc. for C7H6Cl2N2O2: C, 38.04; H, 2.74; N, 12.67; Cl,
32.08%.
4,5-Dimethoxyanthranilic hydroxamic acid (30). Isolated as
a brown solid in 83% yield, 39 mg. 1H NMR (DMSO-d6,
400 MHz): d 10.74 (1H, br s, OH), 8.69 (1H, s, NH), 6.94 (1H, s,
ArH6), 6.31 (1H, s, ArH3), 3.70 (3H, s, ArC5, OCH3), 3.64
(3H, s, ArH4, OCH3), 6.20 (2H, br s, NH2). 13C NMR (DMSO-
d6, 100 MHz): d 167.08, 152.59, 145.86, 138.95, 111.29, 102.90,
99.86, 56.26, 55.07. Purity (RP-HPLC): 92% (tr = 1.07); m/z
(ES) calc. for C9H12N2O4 211.2, found 211.2.
Nicotinic anthranilic hydroxamic acid (31). Isolated as a
white solid in 88% yield, 28 mg. 1H NMR (DMSO-d6, 400 MHz):
d 11.20 (1H, br s, OH), 9.07 (1H, br s, NH), 8.07–8.06 (1H, m,
J = 5 Hz, J = 1.52 Hz, ArH5), 7.77–7.75 (1H, d, J = 7.52 Hz,
ArH6), 6.60–6.58 (1H, m, J = 7.56 Hz, J = 4.52 Hz, ArH3),
7.04 (2H, br s, NH2). 13C NMR (DMSO-d6, 100 MHz): d 149.96,
140.34, 136.43, 111.35, 108.55, 106.94. Purity (RP-HPLC): 98%
(tr = 1.07); m/z (ES) calc. for C6H7N3O2 152.1, found 152.1.
3,5-Dibromoanthranilic hydroxamic acid (23). Isolated as
a cream solid in 85% yield, 58 mg. 1H NMR (DMSO-d6,
400 MHz): d 11.20 (1H, br s, OH), 9.35 (1H, br s, NH), 7.72–
7.71 (1H, d, J = 2.04 Hz, ArH6), 7.72–7.53 (1H, m, J = 2.04 Hz
ArH4), 6.42 (2H, br s, NH2). 13C NMR (DMSO-d6, 100 MHz,):
d 164.36, 145.04, 136.27, 129.54, 116.26, 110.08, 105.28. Purity
(RP-HPLC): 88% (tr = 3.72); m/z (ES) calc. for C7H6N2Br2O2
309.0, found 309.0.
Acknowledgements
3,5-Diiodoanthranilic hydroxamic acid (24). Isolated as a
white solid in 79% yield, 71 mg. 1H NMR (DMSO-d6, 400 MHz):
d 11.19(1H, br s, OH), 9.11 (1H, br s, NH), 7.96 (1H, s, ArH6),
7.60 (1H, s, ArH4), 6.31 (2H, br s, NH2). 13C NMR (DMSO-
d6, 100 MHz): d 164.40, 149.34, 147.60, 140.11, 135.77, 124.91,
116.32. IR (KBr) cm−1; 3438, 3343, 3195, 1640. Purity (RP-
HPLC): 93% (tr = 4.17); m/z (ES) calc. for C7H6N2I2O2 403.9,
found 403.0. Purified by RP-HPLC (Experimental section)
Found: C, 21.38; H, 1.58; N, 6.54; I, 62.31. Calc. for C7H6I2N2O2:
C, 20.81; H, 1.50; N, 6.93; I, 62.83%.
We thank the Irish Government under its ‘Programme for
Research in Third Level Institutions’, the Research Committee
of The Royal College of Surgeons in Ireland and the Irish
Research Council for Science, Engineering and Technology
(IRCSET) postdoctoral research fellowship (AJC) for financial
support; Dr Dilip Rai, University College Dublin, for MS
analysis; Dr John O’Brien, Trinity College Dublin, for NMR
analysis.
References
3,5-Dimethylanthranilic hydroxamic acid (25). Isolated as
a cream solid in 99% yield, 37 mg. 1H NMR (DMSO-d6,
400 MHz,): d 10.91 (1H, br s, OH), 8.86 (1H, br s, NH), 7.04
(1H, s, ArH6), 6.91 (1H, s, ArH4), 2.12 (3H, s, ArC5, CH3), 2.05
(3H, s, ArC3, CH3,), 5.91(2H, br s, NH2). 13C NMR (DMSO-
d6, 100 MHz): d 167.28, 144.61, 138.94, 133.39, 125.42, 123.25,
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