Synthesis and structure-activity relationships of novel 5-(hydroxamic acid)methyl oxazolidinone derivatives as 5-lipoxygenase inhibitors

Article abstract of DOI:10.1080/14756366.2020.1786082

Oxazolidinone hydroxamic acid derivatives were synthesised and evaluated for inhibitory activity against leukotriene (LT) biosynthesis in three in vitro cell-based test systems and on direct inhibition of recombinant human 5-lipoxygenase (5-LO). Thirteen

Full text of DOI:10.1080/14756366.2020.1786082

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and structure-activity relationships of
novel 5-(hydroxamic acid)methyl oxazolidinone
derivatives as 5-lipoxygenase inhibitors
Oludotun A. Phillips , Mira A. Bosso & Charles I. Ezeamuzie
To cite this article: Oludotun A. Phillips , Mira A. Bosso & Charles I. Ezeamuzie (2020) Synthesis
and structure-activity relationships of novel 5-(hydroxamic acid)methyl oxazolidinone derivatives as
5-lipoxygenase inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 1471-1482,
DOI: 10.1080/14756366.2020.1786082
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1471–1482
https://doi.org/10.1080/14756366.2020.1786082
ARTICLE
Synthesis and structure-activity relationships of novel 5-(hydroxamic acid)methyl
oxazolidinone derivatives as 5-lipoxygenase inhibitors
Oludotun A. Phillipsa , Mira A. Bossob and Charles I. Ezeamuzieb
b
aFaculty of Pharmacy, Department of Pharmaceutical Chemistry, Kuwait University, Safat, Kuwait; Faculty of Medicine, Department of
Pharmacology & Toxicology, Kuwait University, Safat, Kuwait
ABSTRACT
ARTICLE HISTORY
Received 10 April 2020
Revised 21 May 2020
Accepted 16 June 2020
Oxazolidinone hydroxamic acid derivatives were synthesised and evaluated for inhibitory activity against
leukotriene (LT) biosynthesis in three in vitro cell-based test systems and on direct inhibition of recombin-
ant human 5-lipoxygenase (5-LO). Thirteen of the 19 compounds synthesised were considered active
((50% inhibitory concentration (IC50) ꢀ 10 mM in two or more test systems)). Increasing alkyl chain length
on the hydroxamic acid moiety enhanced activity and morpholinyl-containing derivatives were more
active than N-acetyl-piperizinyl derivatives. The IC50 values in cell-based assay systems were comparable
to those obtained by direct inhibition of 5-LO activity, confirming that the compounds are direct inhibitors
of 5-LO. Particularly, compounds PH-249 and PH-251 had outstanding potencies (IC50 < 1 mM), compar-
able to that of the prototype 5-LO inhibitor, zileuton. Pronounced in vivo activity was demonstrated in
zymosan-induced peritonitis in mice. These novel oxazolidinone hydroxamic acid derivatives are, therefore,
potent 5-LO inhibitors with potential application as anti-allergic and anti-inflammatory agents.
KEYWORDS
Hydroxamic acid derivatives;
oxazolidinone-hydroxa-
mates; 5-lipoxygenase
inhibitors; leukotrienes
1. Introduction
hydroxyureas as 5-LO inhibitors with therapeutic potentials. The
N-hydroxy-arachidonamides (1a–c, Figure 1) and the arylhydroxa-
mic acids (2a, Figure 1) containing a terminal N-hydroxyl group
(designated as “type A hydroxamic acids”) were the most potent
inhibitors of 5-LO in vitro in their series but suffered rapid meta-
bolic hydrolysis in vivo13,14. However, further studies identified the
arylhydroxamic acids of structure (2b, Figure 1) having terminal
small N-acyl groups like acetyl, which are designated as “type B
hydroxamic acids” containing reversed substitution pattern. These
“type B hydroxamic acids” are potent 5-LO inhibitors that are
orally active and less prone to rapid in vivo metabolic hydrolysis.
In addition, they showed higher plasma bioavailability, longer dur-
ation, and are more potent orally active inhibitors of leukotriene
biosynthesis compared with the type A hydroxamic acids15.
Further intensive investigations led to the discovery of Zileuton,
( )1–(1-(benzo[b] thiophen-2-yl)-ethyl)-1-hydroxyurea (3a, Figure
1), a hydroxyurea derivative of the Fe3þ-chelating type inhibitor as
the only 5-LO inhibitor currently in clinical use. Zileuton is com-
mercially available as a racemate (R and S enantiomers), with both
enantiomers exhibiting in vitro 5-LO inhibitory activity. However, it
is plagued with significant drawbacks such as liver toxicity, weak
potency and short half-life7, thus requiring higher frequency of
administration (an extended-release dosage form has been intro-
duced) accompanied by liver enzyme test. Based on these disad-
vantages, the need for intensive research to discover newer and
potentially more effective 5-LO inhibitors with favourable pharma-
cokinetic, pharmacodynamic and safety profiles for treating
related human diseases is highly imperative. In this light, the N-
The biosynthesis of bioactive leukotrienes (LTs) from arachidonic
acid (AA) is catalysed by the crucial enzyme 5-Lipoxygenase (5-LO)
with the help of 5-LO-activating protein (FLAP), the accessory pro-
tein that presents AA to 5-LO1–4. LTs are pro-inflammatory media-
tors that are implicated in a variety of human inflammatory and
allergic diseases, including, asthma, allergic rhinitis, cardiovascular
diseases (e.g. atherosclerosis and myocardial infarction), arthritis,
inflammatory bowel diseases and certain forms of cancer3,5–8
.
Since 5-LO and its isoforms have been implicated in the patho-
physiology and progression of several human diseases, it is there-
fore identified as a viable therapeutic target. Therefore, discovery
and development of selective inhibitors of 5-LO for therapeutic
intervention have been subjects of active research, which are pre-
sented in patents and scientific publications3,7,9,10
.
Generally, 5-LO inhibitors can be classified into 4 main types
based on their mechanism of action: (i) substrate or competitive
analogues, (ii) inhibitors of 5-LO activating protein (FLAP), (iii)
redox-active inhibitors that could interfere with the free radical
chemistry, (iv) iron-chelating (Fe3þ ion) inhibitors – bind the puta-
tive iron at the active site11. Several compounds based on these
four different types have been synthesised, isolated as natural
products and investigated as potent selective inhibitors of 5-LO
with promising therapeutic usefulness3,7. However, only few 5-LO
inhibitors progress to clinical trials due to insufficient bioavailabil-
ity, pharmacokinetics and/or toxicity related problems12.
The scientific literature contains several reports on the discov-
ery and synthesis of iron-complexing hydroxamates and hydroxy urea atreleuton (VA-2291, 3b, Figure 1) is currently in
CONTACT Oludotun A. Phillips
Charles I. Ezeamuzie ezeamuzie@hsc.edu.kw
dphillips@hsc.edu.kw
Department of Pharmaceutical Chemistry, Kuwait University, P. O. Box 24923, Safat 13110, Kuwait;
Department of Pharmacology & Toxicology, Kuwait University, P. O. Box 24923, Safat 13110, Kuwait
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1472
O. A. PHILLIPS
Figure 1. Chemical structures of 5-lipoxygenase inhibitors and antibacterial agents.
clinical trials for the treatment of cardiovascular diseases and vas-
cular inflammation7, while the orally active hydroxamate contain-
ing a sulphonamide linker (4, Figure 1) has been reported as a 5-
LO inhibitor with potential use in 5-LO mediated cancers3.
2. Results and discussion
2.1. Chemistry
The oxazolidinone hydroxamic acid derivatives (PH-series intermedi-
ates and PH-series, Table 1) evaluated in this study were prepared
as outlined in the chemical reaction sequence in Scheme 1 according
to previously reported experimental methods with some modifica-
tions24,25. Starting from the commercially available starting materials
morpholine 6, piperazine 7 and 3,4-difluoronitrobenzene 8, the key
intermediate hydroxylamine derivatives 15 and 16 (TFA salt) were
prepared in seven and ten chemical reaction steps, respectively. The
nucleophilic acylation reactions of the hydroxylamine derivatives 15
and 16 (TFA salt) with different acyl anhydrides or acid chlorides
yielded the respective 5-(N-alkanoxy-N-alkanamide) methyl oxazolidi-
none intermediate derivatives, PH-series intermediates. The final
hydroxamic acid derivatives PH-series were obtained from base-pro-
moted hydrolysis of the 5-N-alkanoxy-N-alkanamide oxazolidinones,
PH-series intermediates. Ten of the compounds reported in this
study were previously reported by our laboratory and were shown to
be devoid of significant antibacterial or monoamine oxidases
The oxazolidinone scaffold is an important pharmacophore in
some clinically used antibacterial, psychoactive and anticoagulant
agents16–20. In addition, structural modifications around the oxazo-
lidinone 5-membered ring have resulted in novel compounds with
antiepileptic, anticancer and antithyroid activities19,21–23. Based on
the clinical success of drug molecules containing oxazolidinone
scaffold, namely the antibacterial drugs linezolid (5a, Figure 1)
and tedizolid phosphate (5b, Figure 1) and the well-documented
pharmacological properties of the hydroxamic acid derivatives we
decided to investigate a series of novel oxazolidinones containing
type B hydroxamic acid functionality as inhibitors of 5-LO.
We hereby report the synthesis and structure-activity relation-
ships of novel oxazolidinone hydroxamic acid (PH-series inter-
mediates and PH-series, Figure 1) derivatives with potent in vitro
5-LO inhibitory activity based on the structural modifications
around the oxazolidinone heterocycle as
drug discovery.
a
scaffold for

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1473
Table 1. Clog P values and in vitro inhibitory activity of oxazolidinone hydroxamates.
Compd.
code
Clog
P values
Human whole blood LTB4
Human monocyte LTC4
Mouse mast cell LTC4
Direct inhibition of 5-LO
activity. IC50 (CI) lM
ꢁ
ꢁ
ꢁ
ꢁ
Structure of compounds
IC50 (CI) lM
IC50 (CI) lM
IC50 (CI) lM
PH-23
0.7148
3.3 (1.2–9.2)
18.7 (8.9–39.4)
20.7 (13.7–31.2)
>30
PH-199
1.2438
1.4 (0.9–2.3)
8.2 (5.4–12.6)
6.3 (3.1–12.6)
11.8 (0.8–37.8)
PH-204
PH-205
PH-206
2.9859
1.5528
2.4779
10.7 (5.3–21.8)
2.3 (1.2–4.4)
7.9 (3.5–17.8)
>50
21.8 (10.9–43.5)
13.8 (8.5–22.6)
37.3 (16.8–82.9)
>30
14.1 (10.0–19.8)
>50
3.9 (1.4–11.8)
>30
PH-211
PH-237
PH-239
PH-241
PH-245
PH-244
1.2988
4.2239
2.1718
4.3018
1.6278
2.1868
1.3 (0.8–1.9)
>50
7.5 (5.7–9.8)
>50
8.7 (6.7–11.3)
>50
3.5 (0.6–12.3)
>30
3.5 (1.9–6.3)
2.2 (1.4–3.5)
3.4 (1.9–6.1)
5.5 (3.5–8.5)
8.0 (5.8–11.0)
3.2 (2.0–5.1)
ND
3.1 (1.9–5.2)
2.8 (2.3–3.4)
ND
4.7 (1.6–14.2)
3.3 (1.8–6.3)
ND
ND
ND
ND
PH-246
PH-247
PH-249
PH-251
PH-201
5.5419
2.8308
3.3598
3.8888
0.2758
1.5 (1.1–2.2)
2.3 (1.6–3.4)
0.7 (0.3–1.6)
1.9 (1.2–3.0)
38.0 (13.8–70.5)
7.1 (4.1–12.4)
2.5 (1.8–3.4)
0.9 (0.7–1.2)
2.4 (1.8–3.4)
>50
3.3 (2.5–4.3)
2.9 (2.1–4.0)
1.3 (1.0–1.8)
0.2 (0.1–0.5)
>50
21.6 (6.9–62.5)
3.4 (1.4–5.7)
1.9 (1.3–2.9)
1.6 (0.6–2.8)
>30
PH-212
PH-208
1.1338
2.0589
>50
32.7 (16.8–63.4)
>50
>50
>50
14.8 (3.2–38.5)
>30
38.2 (7.5–84.5)
PH-213
0.8739
2.4830
22.5 (3.2–77.4)
0.7 (0.5–0.9)
23.3 (11.3–48.0)
0.5 (0.3–0.6)
>50
7.8 (1.0–38.7)
0.8 (0.2–3.2)
Zileuton
0.4 (0.2–0.6)
ꢁ
CI ¼ 95% Confidence Interval; ND ¼ not determined.

1474
O. A. PHILLIPS
Scheme 1. Synthetic route for the oxazolidinone hydroxamic acid derivatives. (i) DCM/TFA/0ꢃC r.t.; (ii) DCM/TEA/acetic anhydride/0ꢃC r.t.; (iii) DMF/NaH/tert-Butyl-N-
(tertbutoxycarbonyl)carbamate/0 ꢃC r.t. or 0–60 ꢃC.; (iv) DCM/TFA/0 ꢃC r.t.; (v) DCM/TEA/RCOCl or (RCO)2O/0ꢃC r.t.; (vi) MeOH/THF/NaOH/0ꢃC.
inhibitory activities24,25. Six of the previously reported compounds are mouse mast cells (BMMC). Furthermore, in order to confirm that
morpholinyl derivatives (PH-23, PH-199, PH-204, PH-205, PH-206, the in vitro inhibitory activity of the compounds was not due to
and PH-211), and others are N-acetyl piperazinyl derivatives (PH-201,
PH-212, PH-208, and PH-213). While the novel oxazolidinone
hydroxamic acid derivatives, namely PH-237, PH-239, PH-241, PH-
244, PH-245, PH-246, PH-247, PH-249, and PH-251) and their PH-
series intermediates were prepared and reported for the first time in
the present study. These novel compounds were fully characterised
by appropriate spectroscopic and analytical methods as described in
the experimental section. The characteristic signals for the N-hydroxa-
mic acid N–OH appeared between 9.90 and 10.20 ppm, which is
the direct toxic effects on the cells, the effect of the compounds
on the viability of isolated human monocytes after 3 h and 24 h
treatment was performed. Finally, in vivo anti-inflammatory studies
were performed using the zymosan-induced peritoneal inflamma-
tion model in mice, which is a well-known model in which the LTs
are known to play a critical role26.
The inhibitory activity data for the tested compounds obtained
in three cell-based in vitro test systems - LTB4 release from human
whole blood, LTC4 release from isolated human monocytes and
LTC4 release from IgE/antigen-activated mouse mast cells, are
shown in Table 1. Similar data on the direct inhibitory effect on
the enzymatic activity of recombinant human 5-LO, together with
the calculated Clog P (log of partition coefficient) values, which is
an indication of the lipophilicity of each compound, are also
shown in Table 1. In each case, the 50% inhibitory concentration
(IC50) (95% confidence interval) values of the compounds were
compared with those of the reference drug, and the only clinically
available 5-LO inhibitor, zileuton.
exchangeable with D2O and as broadband around 3171–3467 cmꢂ1
,
in the nuclear magnetic resonance and infra-red spectra, respectively.
The calculated log of the partition coefficient (Clog P), which is an
indication of the lipophilicity of the compounds was estimated using
the PerkinElmer ChemBioDraw Ultra 19.0 Computer Software and
was reported as Clog P values in Table 1.
2.2. Pharmacology
Of the 19 compounds tested, 13 were found to have good
(IC50 < 10 mM) to excellent (IC50 < 1 mM) inhibitory activity in at
least two of the test systems, while six compounds (PH-211, PH-
239, PH-241, PH-247, PH-249, and PH-251) were active in all
four test systems. On human whole blood and isolated human
monocytes, compound PH-249 (IC50 ¼ 0.7 mM and 0.9 mM, respect-
ively), containing the heptanoyl moiety on the hydroxamate nitro-
Nineteen synthesised oxazolidinones containing the hydroxamic
acid functionality were evaluated in three different cell-based
in vitro assay systems for inhibition against the release of LTs, and
by direct inhibition of 5-LO activity in a cell-free assay, using zileu-
ton as a reference drug. The cell-based test systems included
inhibition of 5-LO-dependent generation of LTB4 from activated
human whole blood, inhibition of 5-LO product LTC4 from isolated
human monocytes and inhibition of 5-LO product LTC4 from aller- gen, had potencies that were similar to those of zileuton (IC50
¼
gen/Immunoglobulin (IgE)-activated bone marrow-derived 0.7 mM and 0.5 mM, respectively, Table 1), whereas on mast cells
E

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1475
that were activated by an allergic mechanism, compound PH-251, activity in the cell-free system were also the most active in inhibit-
containing the octanoyl moiety on the hydroxamate nitrogen, was ing LT biosynthesis in cell-based systems, whereas the inactive
the most active (IC50 ¼ 0.2 mM). In this later test system, com- analogue, PH-237 (containing the isovaleryloxy and isovaleryl moi-
pound PH-251 was slightly more potent than zileuton (IC50
¼
eties on the hydroxamate nitrogen), was inactive in both systems.
These results indicate that the mechanism of inhibition of LT bio-
synthesis by the active compounds is by direct inhibition of 5-LO,
and that their potencies are comparable to that of zileuton. In
addition, one of the most active compounds, PH-251 demon-
strated an outstanding potency in inhibiting LT biosynthesis in
mouse mast cells (IC50 ¼ 0.2 mM), which was slightly better than
that of zileuton (IC50 ¼ 0.4 mM, Table 1). Since mast cell activation
through IgE/allergen interaction is the basis of all allergic dis-
eases27, compounds like PH-251 have a particularly high potential
of being developed into useful drugs for the treatment of allergic
diseases, including asthma.
0.4 mM). In the cell-free system, both PH-249 and PH-251 were
also the most active (IC50 ¼ 1.9 mM and 1.6 mM, respectively,
Table 1).
Structure-activity relationship revealed that the inhibitory activ-
ities of the compounds were highly dependent on the substitu-
tion pattern around the phenyloxazolidinone moiety, whereby the
morpholinyl-containing derivatives were more active compared
with the N-acetyl-piperizinyl-containing derivatives. Also, the
N–OH hydroxamate-containing oxazolidinones were significantly
more active than their respective precursors, with the exception
of the N-(hexanoyloxy)hexanamide derivative PH-246, which was
also quite active in cell-based systems (IC50 values of 1.5 mM,
7.1 mM and 3.3 mM, for whole blood, isolated monocytes and mast
cells, respectively, Table 1). The reason for this discrepancy is not
immediately clear given that the compound contains an N-hexa-
noyloxy functionality which might make it liable to hydrolysis by
plasma esterases. Moreover, it is possible that the observed activ-
ity in these systems may reflect that of its hydrolytic metabolite,
PH-247. However, since PH-246 is the only 5-N-alkanyloxy-N-alka-
namide derivative with reasonable activity compared to that of
PH-247, this may suggest that the N-hexanoyloxy moiety is more
readily hydrolysed by hydrolases than the other N-alkanoyloxy
groups. Hence, PH-246 may be serve as a pro-drug for the release
of PH-247 in vivo. In addition, the absence of cellular esterase
activity may explain why PH-246 is very weak in inhibiting the
activity of isolated 5-LO enzyme in cell-free experiments. Further
studies are planned to investigate the stability of these com-
pounds in vitro at different pH conditions and in the presence of
hydrolases in plasma.
The finding that most of the active compounds were generally
more active in human whole blood than on isolated human
monocytes is very interesting, given that in drug discovery studies,
compound instability in plasma is a well-recognised problem. One
possible explanation could be that the compounds are more
active on granulocytes than monocytes or that a plasma factor
enhances their entry into cells. These possibilities are currently
being investigated.
It was further observed that all the N-acetyl-piperizinyl contain-
ing oxazolidinone derivatives tested showed either weak activity
(IC50 values in the ranges of 10.0–30 mM) or were essentially
inactive (IC50 values > 30 mM, Table 1). This demonstrates that the
presence of the morpholine heterocycle is essential for activity in
these compounds.
From these results, it can be concluded that these novel oxazo-
lidinones, especially the morpholinyl-containing derivatives, are
potent inhibitors of 5-LO that affect cells from both humans and
mice. Their action is also independent of the mode of cell activa-
tion, as they equally affect cells activated by lipopolysaccharide
(LPS)/N-formyl methyl-leucyl-phenylalanine (FMLP), calcium iono-
phore and antigen-antibody immune complex. These findings are
very important in that they show that the active compounds have
the potential of being useful, not only in allergic diseases, such as
asthma, allergic rhinitis, atopic dermatitis, but also in many other
inflammatory diseases in which LTs, induced by a variety of stim-
uli, are known to be involved6,7
.
In order to exclude the possibility that the in vitro inhibitory
activity of the compounds, was a result of direct toxicity of the
compound on the cells, we studied the effect of the most active
compound (PH-249) on the viability of isolated human monocytes
after 3 h and 24 h treatment. As shown in Figure 2, at concentra-
tions up to 50 lM (a concentration far beyond that required for
100% inhibition of LT release), no significant effect on cell viability
was detected, whether cells were cultured with the drug for 3 h or
24 h. This shows that the compounds are not cytotoxic.
To determine if the compounds were active in vivo, the repre-
sentative compound PH-249 was also tested in the zymosan-
induced peritoneal inflammation, a well-known model in which LT
is known to play a critical role26. As shown in Figure 3, intra-peri-
toneal administration of zymosan (plus vehicle) resulted in a
highly significant increase in the cellular influx and LTC4 content
of the recovered peritoneal lavage fluid after 3 h. Pre-treatment
with 10 or 30 mg/kg of PH-249 resulted in highly significant inhib-
ition of total LTC4 content of the lavage fluid (p < 0.001), Figure
125
Further analysis also revealed that among the hydroxamates,
activity generally increased with the length of the straight-chain
hydrocarbon moiety on the hydroxamate nitrogen, until the hep-
tanoyl group. The increase in chain length to the octanoyl group
resulted in a slight decrease in potency. On the other hand, cyclic
hydrocarbon-containing analogues (cyclobutyl and cyclopentyl),
were generally less potent, with the exception of the cyclopropyl
derivative, PH-211. In the morpholine containing hydroxamic
acids, IC50 values seem to correlate with the Clog P values (indica-
tive of the lipophilicity of the compounds), thus suggesting that
the optimal Clog P values are in the range of 3.3598 for PH-249
and 3.8888 for PH-251, (Table 1).
3h
24h
100
75
50
25
0
0.05%
Triton-X
Veh
1
10
30
M)
50
Data from studies with an isolated 5-LO enzyme (Table 1)
showed that the inhibitory effect of the compounds paralleled
their effects in cell-based assays. For example, compounds PH-249
PH-249 (
m
Figure 2. Effect of compound PH-249 on the viability of isolated human mono-
cytes. Cells were exposed to the test compound or triton-X, as positive control,
and PH-251, which were the most active in inhibiting 5-LO for 3 h or 24h, and viability was assessed by the MTT method, n ¼ 3.

1476
O. A. PHILLIPS
activities that were comparable to those of zileuton. One com-
pound, PH-251, was particularly effective on mast cell LT release
induced by allergen/IgE interaction (IC50 ¼ 0.2 mM). Structure-activ-
ity relationship studies revealed that oxazolidinone derivatives
containing the (N-(OH)COR) hydroxamate functionality with rela-
tively longer straight-chain alkyl groups (-R) along with the mor-
pholine heterocycle demonstrated the best activity. The optimal
alkyl chain length appeared to be C ¼ 6 (PH-249, R ¼ hexyl), as
extending the length to C ¼ 7 (PH-251, R ¼ heptyl) resulted in
either a slight loss or no change in activity, except for mast cells
in which a further increase in activity was observed. Results also
suggest that the active compounds are non-toxic and possess
strong in vivo anti-inflammatory activity. Hence, they have the
potential for development as drugs for the treatment of allergic
and inflammatory diseases.
(A)
400
300
200
100
0
###
***
***
***
)
)
h
h
0
0
1
30)
(
(3
(
Ve
9
n
to
+
+ Ve
4
49
o
S
B
2
2
-
-
m
u
P
y
H
e
Z
PH
Zil
+
+ P
+
4. Experimental
50
40
30
20
10
0
4.1. Synthesis
(B)
###
4.1.1. Materials and methods
The starting materials 3,4-difluoronitrobenzene, morpholine,
piperazine, n-butyllithium, sodium hydride, and other common
reagents and solvents used for the synthesis of the oxazolidi-
nones, including, dichloromethane (DCM), diethyl ether, dimethyl-
formamide (DMF), ethyl acetate, methanol, tetrahydrofuran (THF)
were purchased from Merck (formerly Sigma-Aldrich) Germany.
Among the previously reported compounds, six are morpholinyl
derivatives (PH-23, PH-199, PH-204, PH-205, PH-206, and PH-
211), and the others are N-acetyl piperazinyl derivatives (PH-201,
PH-212, PH-208 and PH-213). These were previously reported
from our laboratory and synthesised according to literature meth-
ods24,25. The remaining nine oxazolidinone hydroxamic acid deriv-
atives, namely PH-237, PH-239, PH-241, PH-244, PH-245, PH-
246, PH-247, PH-249 and PH-251 and their respective PH-series
intermediates are reported for the first time (Table 1). Purification
of compounds was performed on silica gel column chromatog-
raphy using silica gel (Kieselgel 60, 70–230 mesh; Aldrich-Sigma,
**
**
)
h
e
V
0)
(1
0)
(3
0
3
(
Veh
+
+
49
+
49
o
S
ton
-2
-2
u
m
H
P
H
PB
ile
Z
Zy
P
+
+
Drug Treatment (mg/Kg)
Figure 3. In vivo inhibitory effect of compound PH-249 on zymosan-induced Germany) and thin-layer chromatography (TLC) was conducted on
peritoneal inflammation in mice. Animals were pre-treated subcutaneously with
PH-249, zileuton or vehicle, 30min before induction of peritoneal inflammation
Germany). Solid products were recrystallised from a suitable solv-
with intraperitoneal injection of 0.2ml activated zymosan (2 mg/ml). Peritoneal
0.25 mm pre-coated silica gel plates (60F254, Merck, Darmstadt,
ent and or solvent mixtures. Melting points were determined on a
lavage fluids obtained after 2 h were analysed for total LTC4 content (A) and total
cellular content (B). Values are means sem, n ¼ 6–7. ###p < 0.001 with respect Stuart Scientific melting point apparatus (SMP1, Stuart, Stone, UK)
ꢁꢁꢁ
p < 0.001;
ꢁꢁ
p < 0.01, with respect to zymosan
to PBS plus vehicle;
plus vehicle.
and were uncorrected. Further structural elucidation was per-
formed using 1H- and 13 C-NMR (decoupled experiments) spectra
in DMSO-d6 using solvent peaks as reference signals and were
recorded on Bruker DPX 400 MHz and Bruker Avance II 600 NMR
spectrometers. Two-dimensional NMR spectra experiments, namely
2 D COSY (COrrelated SpectroscopY) and 2 D HSQC (Heteronuclear
Signal Quantum Coherence) experiments were also performed on
representative compounds (PH-242 (containing the cyclobutylcar-
bonyloxy and the cyclobutylcarbonyl moieties on the hydroxa-
mate nitrogen), PH-245 (containing the cyclobutylcarbonyl group
on the hydroxamate nitrogen), PH-246 (containing hexanoyloxy
and the hexanoyl moieties on the hydroxamate nitrogen), PH-247
(containing the hexanoyl moiety on the hydroxamate nitrogen) to
assist in proton and carbon assignments. Chemical shifts and cou-
3(A), but for cellular infiltration, only the inhibition by the higher
dose reached statistical significance, p < 0.01, (Figure 3(B)). At
30 mg/kg, zileuton achieved a similar effect with respect to both
parameters. These results show that the representative compound
PH-249 containing the heptanoyl moiety has in vivo biological
activity that is comparable to that of the reference drug, zileuton
and thus suggests that these novel oxazolidinone hydroxamic acid
derivatives have potential anti-inflammatory actions in vivo.
3. Conclusion
In conclusion, the synthesised novel oxazolidinone hydroxamic pling constants (J, Hz) of protons and carbons are reported in
acid derivatives were screened in four in vitro test systems for parts per million (ppm) downfield and up-field from solvent
inhibitory activity against the release of LTs or direct inhibition of DMSO-d6 (d ¼ 2.5; 39.7) peak as reference. Mass spectra data were
5-LO enzyme activity. Thirteen of the compounds had good or recorded on a Thermo Scientific DFS Gas Chromatography/Mass
excellent activity in at least two test systems, while six compounds Spectrometer (DFS GC-MS, Thermo Fisher Scientific, Bremen,
were active in all four systems. The most active compounds had Germany) and Waters QToF/Mass Spectrometer (LC-MS/MS ESI,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1477
Waters Corporation, Milford, MA). Infra-red (IR) spectra were 135.37, 133.64, 133.57, 119.19, 119.17, 114.04, 114.02, 106.69,
recorded on JASCO FT-IR-6300 (JASCO, Tokyo, Japan) 106.52, 70.66, 70.04, 66.11, 56.31, 50.68, 48.12. MS 311.1 (Mþ)24,25
Spectrometer. Elemental analyses were performed on an
.
Elementar Vario Micro Cube CHN Analyser apparatus (Elementar,
4.1.4. General procedure for the synthesis of the (R)-N-((3-(3-flu-
oro-4-morpholinophenyl)-2-oxoxazolidin-5-yl)methyl)-N-hydroxyal-
kanamide (PH-series)
Langenselbold, Germany), and analyses indicated by the symbols
of the elements (CNH) were within 0.4% of the theoretical val-
ues. Elemental analyses (CHN) were used to confirm the purity of
all newly synthesised compounds (>95%), Analyses were per-
formed by the General Facility-Science (GF-S), Faculty of Science,
Kuwait University, Kuwait. The structures of the oxazolidinones
and their Clog P values were sketched and estimated, respectively
using the PerkinElmer ChemBioDraw Ultra 19.0 Software.
A solution of the N-hydroxylamine derivative, compound 15 (1.0
eq.) in anhydrous DCM or CH3CN (30–50 ml) was treated with trie-
thylamine (6.0 eq.) and dropwise addition of the respective acid
anhydride or acid chloride (3.0 eq.) at 0 ꢃC and stirred to room
temperature overnight. The reaction mixture was diluted with a
10% solution of potassium carbonate (20 ml) and the DCM layer
was separated and washed with water, brine, dried (Na2SO4), fil-
tered and concentrated to give the crude product. Purification by
normal phase silica gel column chromatography and/or recrystal-
lised using suitable organic solvent or mixtures to give the inter-
mediate products N-alkanoxy-N-((3-3-fluoro-4-morpholinophenyl)-
2-oxooxazolidin-5-yl) methyl)alkanamide PH-series intermediates
(PH-237, PH-240, PH-242, PH-243, PH-246, PH-248 and PH-250).
A solution of the PH-series intermediates (1 eq.) in methanol:THF
(4:1, v/v) was stirred at 0 ꢃC and treated with a 1.0 eq. NaOH solu-
tion in water (ꢅ20 ml). The reaction mixture was stirred for 1 h
10 min and neutralised to pH of ꢅ7 by addition of a solution of
1.0 eq. HCl in water (30 ml). The reaction mixture was concen-
trated to remove THF and methanol, and the aqueous residue
was saturated with NaCl and extracted with DCM (2 ꢄ 30 ml) and
the organic layer was separated, dried (Na2SO4), filtered and con-
centrated to give the crude product. Purification was performed
either by silica gel column chromatography and/or recrystallisation
using suitable organic solvent or mixtures to give the (R)-N-((3-(3-
fluoro-4-morpholinophenyl)-2-oxoxazolidin-5-yl)methyl)-N-hydroxy-
alkanamide derivatives (PH-239, PH-241, PH-244, PH-245, PH-
247, PH-249, and PH-251) as the final products.
4.1.2. (R)-tert-butyl (tert-butoxycarbonyl)oxy ((3-3-fluoro-4-mor-
pholinophenyl)-2-oxooxazolidin-5-yl) methyl)carbamate (13)
An ice-cooled (0 ꢃC) solution of tert-butyl N-(tert-butoxycarbony-
loxy) carbamate (4.97 g, 21.31 mmol) in anhydrous DMF (30 ml)
under nitrogen was treated portion-wise with 60% sodium hydride
in mineral oil (770 mg, 22.75 mmol) and stirred for 30 min. The
reaction mixture was treated with drop-wise addition of a solution
of [N-3-(3-fluoro-4-morpholinylphenyl)-2-oxo-5-oxazolidinyl] methyl
methanesulfonate (6.00 g, 16.02 mmol) in DMF (70 ml). The reac-
tion mixture was stirred to room temperature for 60 h, quenched
by addition of water (200 ml) and extracted with ethyl acetate
(3 ꢄ 100 ml). The ethyl acetate (EtOAc) extract was diluted with
hexane (60 ml) and washed with water, brine, dried (Na2SO4), fil-
tered and concentrated to give the crude as a brownish oil. Silica
gel column chromatography eluted with EtOAc-Hexane 2:1 gave
the title compound 1324,25 as a pale-yellow viscous oil (7.0 g, yield
85%), solidifies upon cooling in the fridge to a yellow solid after a
long period of time. 1H-NMR (DMSO-d6, 600 MHZ): d 7.51 (dd, 1H,
J ¼ 15.0 Hz, 2.5 Hz, phenyl H), 7.19 (dd, 1H, J ¼ 8.8 Hz, 2.5 Hz, phe-
nyl H), 7.09 (t, 1H J ¼ 9.6 Hz, phenyl H), 4.87 (m, 1H, oxazolidinone
H), 4.15 (t, 1H, J ¼ 4.6 Hz, oxazolidinone H), 3.97 (m, 1H, oxazolidi-
none H), 3.82 (m, 1H, methylene H), 3.72–3.76 (br. t, 5H, morpho-
line H and methylene H), 2.97 (br. t, 4H, morpholine H), 1.41–1.48
(br,18H). MS 511.2 (Mþ)24,25
4.1.4.1. (R)-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-
yl)methyl)-N-hydroxy-3-methylbutanamide (PH-239). The inter-
mediate compound PH-237, was prepared via the general proced-
ure from compound 15 (1.50 g, 4.82 mmol), isovaleric anhydride
(2.83 ml, 14.46 mmol), triethylamine (4.05 ml; 28.41 mmol) in
anhydrous DCM (30 ml) to give crude product. Purification by sil-
ica gel column chromatography (EtOAc-Hexane, 1:2 to 1:1) gave
the intermediate (R)-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxa-
zolidin-5-yl)methyl)-3-methyl-N-((3-methylbutanoyl)oxy)butanamide
PH-237, as a white solid 0.815 g, yield 35%, mp 89–90 ꢃC. IR (KBr,
cmꢂ1): ꢀ 2960, 2871, 2822, 1797, 1759, 1672, 1520, 1471, 1445,
1404, 1329, 1289, 1169, 1136, 1119, 1065. 1H-NMR (CDCl3,
600 MHZ): d 7.46 (dd, 1H, J ¼ 2.6 Hz, 14.3 Hz, phenyl H), 7.11 (m,
1H, phenyl H), 6.94 (t, 1H, J ¼ 9.1 Hz, phenyl H), 4.80–4.84 (br. m,
1H, oxazolidinone H), 4.10 (d, 2H, J ¼ 4.6 Hz, methylene CH2), 4.05
(t, 1H, J ¼ 8.9 Hz, oxazolidinone H), 3.88–3.91 (m, 5H, morpholine H
and oxazolidinone H), 3.07 (t, 4H, J ¼ 4.7 Hz, morpholine H),
2.39 (d, 2H, J ¼ 7.1 Hz, NOCOCH2CH(CH3)2), 2.19–2.51 (m, 1H,
NOCOCH2CH(CH3)2), 2.06–2.12 (m, 3H, NCOCH2CH (CH3)2 and
4.1.3. (R)-3-(3-fluoro-4-morpholinophenyl)-5-((hydroxyamino)me-
thyl)oxazolidin-2-one (15)
Compound 13 (14.69 g, 28.71 mmol) was dissolved in anhydrous
DCM (25 ml) and cooled to 0 ꢃC in an ice-bath. This solution was
treated with rapid dropwise addition of trifluoroacetic acid (20 ml)
and the reaction mixture was stirred overnight. The reaction mix-
ture was concentrated to give a gummy residue, which was
treated with
a 10% potassium carbonate solution in water
(100 ml) to give a basic solution. The resulting gelatinous precipi-
tate was collected by filtration to give an off-white solid (8.40 g),
the filtrate was extracted with DCM and the DCM layer was dried
(Na2SO4) and concentrated to give a second crop (0.240 g). The
titled compound 15 was obtained as an off-white solid (8.64 g,
yield 94%)24,25, mp 134–137 ꢃC. This product was used for further
reactions without further purification. 1H-NMR (DMSO-d6,
400 MHZ): d 7.50 (dd, 1H, J ¼ 15.1 Hz, 2.5 Hz, phenyl H), 7.45 (s,
N–OH, 1H), 7.20 (dd, 1H, J ¼ 2.1 Hz, 8.7 Hz, phenyl H), 7.06 (t, 1H,
J ¼ 9.6 Hz, phenyl H), 6.02 (br. s, 1H, N–H), 4.70–4.83 (m, 1H, oxazo-
lidinone H), 4.09 (t, 1H, J ¼ 8.9 Hz, oxazolidinone H), 3.82 (dd, 1H,
J ¼ 6.8 Hz, 8.9 Hz, oxazolidinone H), 3.73 (br. t, 4H, J ¼ 4.6 Hz, mor-
pholine H), 2.97–3.10 (br. m, 6H, CH2N(OH)H, partially overlaps
NCOCH2CH
NOCOCH2CH(CH3)2),
(CH3)2),
1.05
0.93
(dd,
(dd,
6H,
6H,
J ¼ 1.4 Hz,
6.6 Hz,
13.0 Hz,
J ¼ 6.1 Hz,
NCOCH2CH(CH3)2). 13 C-NMR (DMSO-d6, 600 MHZ): d 155.33 (d,
J ¼ 243.20 Hz), 153.70, 135.55 (d, J ¼ 8.76 Hz), 133.32 (d,
J ¼ 10.80 Hz), 119.22 (d, J ¼ 4.16 Hz), 114.13, (d, J ¼ 2.80 Hz), 106.67
(d, J ¼ 25.88 Hz), 70.10, 66.10, 50.66, 50.30, 47.15, 24.95, 22.11. MS
479.4 (Mþ). Anal. Calcd. for C24H34FN3O6: C: 60.11, H: 7.15, N: 8.76;
with the morpholine H triplet signal at 2.96, J ¼ 4.6 Hz), 13 C-NMR found C: 59.71, H: 7.41, N: 8.55. A solution of the intermediate PH-
(DMSO-d6, 600 MHZ): d 165.28, 155.35, 154.21, 153.74, 135.42, 237 (0.80 g, 1.67 mmol) in MeOH:THF (28:7 ml) was treated with

1478
O. A. PHILLIPS
NaOH solution (133 mg in 20 ml water). Purification by recrystal- H), 2.36–2.39 (m, 2H, NCOCH2CH2CH2CH3), 1.44–1.48 (m, 2H,
lisation (EtOAc-hexane 2:1) gave the titled compound PH-239 as a NCOCH2CH2CH2CH3), 1.24–1.30 (m, 2H, NCOCH2CH2CH2CH3), 0.85
white solid (328 mg, yield, 55%), mp 122–124.5 ꢃC. IR (KBr, cmꢂ1): (t, 2H, J ¼ 7.4 Hz, NCOCH2CH2CH2CH3). 13 C-NMR (DMSO-d6,
600 MHZ): d 173.93, 154.53 (d, J ¼ 244.06 Hz), 153.90, 135.49 (d,
ꢀ 3384, 3196, 2955, 2869, 1752, 1729, 1633, 1610, 1521, 1447,
1429, 1333, 1272, 1233, 1173, 1119, 1068. 1H-NMR (DMSO-d6,
(d, J ¼ 2.84 Hz), 106.64 (d, J ¼ 26.00 Hz), 69.87, 66.09, 50.66, 50.64,
600 MHZ): d 9.93 (s, 1H, N–OH, exchangeable with D2O), 7.48 (dd,
J ¼ 8.78 Hz), 133.40 (d, J ¼ 10.79 Hz), 119.20 (d, J ¼ 4.06 Hz), 114.10
50.30, 47.45, 31.22, 26.21, 21.81, 13.68. MS ESþ (m/z): 396.2037
(Mþ þ H), MS (m/z): 395.2 (Mþ). Anal. Calcd. for C19H26FN3O5: C:
57.71, H: 6.63, N: 10.63; found C: 58.10, H: 6.61, N: 10.39.
1H, J ¼ 2.5 Hz, 14.9 Hz, phenyl H), 7.17 (dd, 1H, J ¼ 2.3 Hz, 8.7 Hz,
phenyl H), 7.06 (t, 1H, J ¼ 8.4 Hz, phenyl H), 4.85–4.90 (br. m, 1H,
oxazolidinone H), 4.13 (t, 1H, J ¼ 8.9 Hz, oxazolidinone H), 4.05 (dd,
1H, J ¼ 6.5 Hz, 13.7 Hz, oxazolidinone H), 3.72–3.76 (m, 5H, mor-
pholine H and methylene H), 3.67 (dd, 1H, J ¼ 4.5 Hz, 14.6 Hz,
methylene H), 2.96 (t, 4H, J ¼ 4.6 Hz, morpholine H), 2.25–2.30 (m,
2H, NCOCH2CH(CH3)2), 1.99–2.04 (m, 1H, NCOCH2CH(CH3)2), 0.88 (t,
6H, J ¼ 5.9 Hz, NCOCH2CH(CH3)2). 13 C-NMR (DMSO-d6, 600 MHZ): d
173.22, 154.55 (d, J ¼ 243.45 Hz), 153.95, 135.53 (d, J ¼ 8.77 Hz),
133.43 (d, J ¼ 10.83 Hz), 119.24 (d, J ¼ 4.11 Hz), 114.11 (d,
J ¼ 2.79 Hz), 106.66 (d, J ¼ 26.24 Hz), 69.87, 66.13, 50.69, 50.27,
47.48, 24.42, 22.48. MS ESþ (m/z): 396.1874 (Mþ þ H), MS (m/z):
395.2 (Mþ). Anal. Calcd. for C19H26FN3O5: C: 57.71, H: 6.63, N:
10.63; found C: 57.28, H: 6.94, N: 10.32.
4.1.4.3. (R)-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-
yl)methyl)-N-hydroxycyclobutanecarboxamide (PH-245). The inter-
mediate compound PH-242 was prepared via the general proced-
ure
from
compound
15
(2.20 g,
7.07 mmol),
cyclobutanecarbonylchloride (2.42 ml, 21.20 mmol), triethylamine
(5.94 ml; 42.40 mmol) in anhydrous DCM (30 ml) to give a brown-
yellowish gummy crude product. Purification by silica gel column
chromatography (EtOAc-Hexane, 1:2 to 1:1) gave the intermediate
(R)-N-((cyclobutanecarbonyl)oxy)-N-((3-(3-fluoro-4-morpholino phe-
nyl) ꢂ2-oxooxazolidin-5-yl)methyl)cyclobutanecarboxamide PH-
242 as a white solid 0.487 g, yield 15%, mp 122–124 ꢃC. IR (KBr,
cmꢂ1): ꢀ 2963, 2857, 1788, 1741, 1678, 1517, 1445, 1410, 1329,
1261, 1096, 1021. 1H-NMR (DMSO-d6, 600 MHZ): d 7.48 (dd, 1H,
J ¼ 2.4 Hz, 14.9 Hz, phenyl H), 7.17 (dd, 1H, J ¼ 2.3 Hz, 8.8 Hz, phe-
nyl H), 7.07 (t, 1H, J ¼ 9.3 Hz, phenyl H), 4.83–4.87 (br. m, 1H, oxa-
zolidinone H), 4.09–4.15 (br. t, 2H, oxazolidinone H, overlaps with
oxazolidinone H, triplet, at 4.10 ppm, J ¼ 9.0 Hz), 3.87–3.90 (br., 1H,
methylene H), 3.73–3.75 (m, 5H, morpholine H and methylene H),
3.32–3.35 (br., 1H, cyclobutane H), 3.09–3.19 (br., 1H, cyclobutane
H), 2.96 (t, 4H, J ¼ 4.7 Hz, morpholine H), 1.17–2.34 (m, 12H, cyclo-
4.1.4.2. (R)-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-
yl)methyl)-N-hydroxypentanamide (PH-241). The intermediate
compound 0PH-240, was prepared via the general procedure
from compound 15 (2.20 g, 7.07 mmol), valeric anhydride (4.18 ml,
21.20 mmol), triethylamine (5.94 ml; 42.40 mmol) in anhydrous
DCM (30 ml) to give crude product. Purification by silica gel col-
umn chromatography (EtOAc-Hexane, 1:2 to 1:1) gave the inter-
mediate (R)-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-
yl)methyl)-N-(pentanoyloxy)pentamide PH-240 as a white solid
1.10 g, yield 33%, mp 75–77.5 ꢃC. IR (KBr, cmꢂ1): ꢀ 2963, 2930,
2857, 1797, 1739, 1683, 1627, 1572, 1518, 1447, 1409, 1327, 1236,
1214, 1138, 1120, 1057. 1H-NMR (DMSO-d6, 600 MHZ): d 7.48 (dd,
1H, J ¼ 2.5 Hz, 14.9 Hz, phenyl H), 7.18 (dd, 1H, J ¼ 2.2 Hz, 8.8 Hz,
phenyl H), 7.07 (t, 1H, J ¼ 9.4 Hz, phenyl H), 4.84–4.88 (br. m, 1H,
oxazolidinone H), 4.10–4.19 (br. t, 2H, oxazolidinone H, overlaps
with oxazolidinone H triplet, at 4.11 ppm, J ¼ 9.0 Hz), 3.84–3.92
(br., 1H, methylene H), 3.74 (t, 5H, J ¼ 4.6 Hz, morpholine H and
methylene H), 2.96 (t, 4H, J ¼ 4.6 Hz, morpholine H), 2.50 (br., 2H,
methylene –CH2– overlapping with DMSO signal), 2.11–2.27 (br.,
2H, methylene H), 1.56–1.61 (m, 2H, methylene H), 1.40–1.50 (br.,
2H, methylene –CH2–), 1.31–1.37 (m, 2H, methylene H) 1.20–1.29
(br., 2H, methylene H), 0.88 (t, 3H, J ¼ 7.4 Hz, methyl H), 0.82 (br., t,
6H, J ¼ 6.5 Hz, methyl H). 13 C-NMR (DMSO-d6, 600 MHZ): d 154.56
(d, J ¼ 243.26 Hz), 153.75, 135.56 (d, J ¼ 8.72 Hz), 133.36 (d,
J ¼ 10.62 Hz), 119.24 (d, J ¼ 4.16 Hz), 114.13 (d, J ¼ 2.78 Hz), 106.66
(d, J ¼ 25.88 Hz), 70.16, 66.13, 50.69, 50.67, 47.15, 30.85, 30.75,
butane H). 13 C-NMR (DMSO-d6, 600 MHZ):
d
154.54 (d,
J ¼ 243.26 Hz), 153.75, 135.57 (d, J ¼ 8.72 Hz), 133.34 (d,
J ¼ 10.75 Hz), 119.23 (d, J ¼ 4.13 Hz), 114.16, 106.69 (d,
J ¼ 26.26 Hz), 70.20, 66.12, 50.68, 50.67, 47.15, 34.94, 24.43, 24.32,
24.21, 24.11, 17.99, 17.44. MS ESþ (m/z): 498.2177 (Mþ þ Na), MS
(m/z): 475.2 (Mþ). A solution of the intermediate PH-242 (0.487 g,
1.02 mmol) in MeOH:THF (28:7 ml) was treated with NaOH solution
(84 mg in 20 ml water). Purification by recrystallisation (EtOAc-hex-
ane 2:1) gave the titled compound PH-245 as off-white solid
248 mg, yield 92%, mp 132–133.5 ꢃC. IR (KBr, cmꢂ1): ꢀ 3224, 2953,
2858, 1740, 1722, 1629, 1523, 1476, 1425, 1331, 1233, 1198, 1114,
1
1081. H-NMR (DMSO-d6, 600 MHZ): d 9.80 (s, 1H, N–OH, exchange-
able with D2O), 7.49 (dd, 1H, J ¼ 2.5 Hz, 14.9 Hz, phenyl H), 7.18
(dd, 1H, J ¼ 2.3 Hz, 8.9 Hz, phenyl H), 7.06 (t, 1H, J ¼ 9.4 Hz, phenyl
H), 4.84–4.89 (br. m, 1H, oxazolidinone H), 4.12 (t, 1H, J ¼ 8.9 Hz,
oxazolidinone H), 4.02 (dd, 1H, J ¼ 6.8 Hz, 14.6 Hz, oxazolidinone
H), 3.73–3.76 (m, 5H, morpholine H and methylene H), 3.66 (dd,
1H, J ¼ 4.5 Hz, 14.6 Hz, methylene H), 3.46–3.52 (m, 1H, cyclobu-
tane H), 2.96 (t, 4H, J ¼ 4.7 Hz, morpholine H), 1.75–1.93 (m, 6H,
cyclobutane H). 13 C-NMR (DMSO-d6, 600 MHZ): d 175.29, 155.55 (d,
J ¼ 243.92 Hz), 153.95, 135.52 (d, J ¼ 8.77 Hz), 133.42 (d,
J ¼ 10.69 Hz), 119.23 (d, J ¼ 3.77 Hz), 114.11 (d, J ¼ 2.89 Hz), 106.66
(d, J ¼ 26.03 Hz), 69.80, 66.13, 50.69, 50.67, 50.56, 47.50, 35.72,
24.39, 17.73. MS ESþ (m/z): 394.1899 (Mþ þ H), MS (m/z): 393.2
(Mþ). Anal. Calcd. for C19H24FN3O5: C: 58.01, H: 6.15, N: 10.68;
found C: 57.61, H: 6.02, N: 10.42.
25.97, 21.55, 21.49, 13.62, 13.49. MS ESþ (m/z): 480.2300 (Mþ
þ
H), MS (m/z): 479.3 (Mþ). Anal. Calcd. for C24H34FN3O6: C: 60.11; H:
7.15; N: 8.76; found C: 60.06; H, 6.87; N, 8.74. A solution of the
intermediate PH-240 (0.900 g, 1.88 mmol) in MeOH:THF (28:7 ml)
was treated with NaOH solution (150 mg in 20 ml water).
Purification by recrystallisation (EtOAc-hexane 2:1) gave the titled
compound PH-241 as an off-white solid 700 mg, yield 94%, mp
121–122.5 ꢃC. IR (KBr, cmꢂ1): ꢀ 3187, 2959, 2931, 2860, 1743, 1720,
1626, 1522, 1447, 1425, 1331, 1271, 1234, 1196, 1115, 1071. 1H-
NMR (DMSO-d6, 600 MHZ): d 9.94 (s, 1H, N–OH, exchangeable with
D2O), 7.48 (dd, 1H, J ¼ 2.5 Hz, 15.0 Hz, phenyl H), 7.17 (dd, 1H,
J ¼ 2.3 Hz, 8.9 Hz, phenyl H), 7.06 (t, 1H, J ¼ 9.4 Hz, phenyl H),
4.85–4.89 (br. m, 1H, oxazolidinone H), 4.12 (t, 1H, J ¼ 8.9 Hz, oxa-
4.1.4.4. (R)-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-
yl)methyl)-N-hydroxycyclopentanecarboxamide
(PH-244).
The
intermediate compound PH-243 was prepared via the general
zolidinone H), 4.04 (dd, 1H, J ¼ 6.6 Hz, 15.6 Hz, oxazolidinone H), procedure from compound 15 (2.20 g, 7.07 mmol), cyclopentane-
3.72–3.76 (m, 5H, morpholine H and methylene H), 3.67 (dd, 1H, carbonylchloride (2.58 ml, 21.20 mmol), triethylamine (5.94 ml;
J ¼ 4.6 Hz, 14.9 Hz, methylene H), 2.96 (t, 4H, J ¼ 4.7 Hz, morpholine 42.40 mmol) in anhydrous DCM (30 ml) to give a brown-yellowish

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1479
gummy crude product. Purification by silica gel column chroma- 600 MHZ):
d
154.49 (d, J ¼ 246.37 Hz), 153.67, 135.50 (d,
tography (EtOAc-Hexane, 1:2 to 1:1) gave the intermediate (R)-N- J ¼ 8.78 Hz), 133.27 (d, J ¼ 10.43 Hz), 119.17 (d, J ¼ 4.13 Hz), 114.10
((cyclopentanecarbonyl)oxy)-N-((3-(3-fluoro-4-morpholino phenyl)- (d, J ¼ 2.71 Hz), 106.64 (d, J ¼ 26.06 Hz), 70.09, 66.06, 50.62, 50.61,
2-oxooxazolidin-5-yl)methyl)cyclopentanecarboxamide PH-243 as 47.11, 31.07, 30.94, 30.53, 30.40, 23.50, 21.68, 21.57, 13.64, 13.60.
a white solid 1.20 g, yield 34%, recrystallised from EtOAc-Et2O, mp MS ESþ (m/z): 508.3000 (Mþ þ H), MS (m/z): 507.3 (Mþ). Anal.
114.5–116 ꢃC. IR (KBr, cmꢂ1): ꢀ 2960, 2853, 1779, 1754, 1671, 1515, Calcd. for C26H38FN3O6: C: 61.52; H: 7.55; N: 8.28; found C: 61.44;
1449, 1415, 1327, 1225, 1193, 1117, 1087. 1H-NMR (DMSO-d6, H, 7.53; N, 7.95. A solution of the intermediate compound PH-246
600 MHZ): d 7.48 (dd, 1H, J ¼ 2.3 Hz, 14.9 Hz, phenyl H), 7.17 (dd, (4.50 g, 8.87 mmol) in MeOH:THF (84:21 ml) was treated with NaOH
1H, J ¼ 2.3 Hz, 8.9 Hz, phenyl H), 7.07 (t, 1H, J ¼ 9.4 Hz, phenyl H), solution (709 mg in 20 ml water). Purification by recrystallisation
4.83–4.88 (br. m, 1H, oxazolidinone H), 4.09–4.16 (br. t, 2H, oxazoli- (EtOAc-hexane 2:1) gave the titled compound PH-247 as an off-
dinone H, overlaps with oxazolidinone H triplet, at 4.12 ppm, white solid 3.23g, yield 89%, mp 118.5–120.5 ꢃC. IR (KBr, cmꢂ1): ꢀ
J ¼ 9.0 Hz), 3.84–3.95 (br., 1H, methylene H), 3.74 (t, 5H, J ¼ 4.6 Hz, 3187, 2957, 2930, 2858, 1745, 1719, 1626, 1524, 1475, 1426, 1332,
1
morpholine H and methylene H), 2.96 (t, 5H, J ¼ 4.6 Hz, morpho- 1270, 1258, 1234, 1196, 1114, 1073. H-NMR (DMSO-d6, 600 MHZ): d
line H and cyclopentane H), 2.64–2.79 (br., 1H, cyclopentane H), 9.92 (s, 1H, N–OH, exchangeable with D2O), 7.48 (dd, 1H, J ¼ 2.5 Hz,
1.44–1.96 (m, 16H, cyclopentane H). 13 C-NMR (DMSO-d6, 600 MHZ): 15.0Hz, phenyl H), 7.17 (dd, 1H, J ¼ 2.2 Hz, 8.8Hz, phenyl H), 7.06 (t,
d 154.52 (d, J ¼ 243.39 Hz), 153.71, 135.36 (d, J ¼ 8.85 Hz), 133.33 1H, J ¼ 9.4Hz, phenyl H), 4.85–4.89 (br. m, 1H, oxazolidinone H),
(d, J ¼ 10.77 Hz), 119.22 (d, J ¼ 4.16 Hz), 114.10 (d, J ¼ 2.37 Hz), 4.12 (t, 1H, J ¼ 8.9 Hz, oxazolidinone H), 4.04 (dd, 1H, J ¼ 6.7 Hz,
106.64 (d, J ¼ 26.22 Hz), 70.24, 66.10, 50.66, 50.65, 47.15, 29.49, 14.7Hz, oxazolidinone H), 3.73–3.76 (m, 5H, morpholine
H
29.22, 29.08, 29.05, 25.53, 25.26. MS ESþ (m/z): 504.2700 (Mþ
þ
and methylene H), 3.67 (dd, 1H, J ¼ 4.3Hz, 14.8Hz, methylene H),
H), MS (m/z): 503.2 (Mþ). Anal. Calcd. for C26H34FN3O6: C: 62.01; H: 2.96 (t, 4H, J ¼ 4.7Hz, morpholine H), 2.35–2.38 (m, 2H,
6.81; N: 8.34; found C: 62.25; H: 6.64; N: 8.08. A solution of the NCOCH2CH2CH2CH2CH3), 1.45–1.49 (m, 2H, NCOCH2CH2CH2CH2CH3),
intermediate PH-243 (0.900 g, 1.79 mmol) in MeOH:THF (28:7 ml) 1.22–1.30 (m, 4H, NCOCH2CH2CH2CH2CH3), 0.85 (t, 3H, J ¼ 7.0 Hz,
was treated with NaOH solution (143 mg in 20 ml water). NCOCH2CH2CH2CH2CH3). 13 C-NMR (DMSO-d6, 600 MHZ): d 173.90,
Purification by recrystallisation (EtOAc-hexane 2:1) gave the titled 154.51 (d, J ¼ 244.10 Hz), 153.88, 135.46 (d, J ¼ 8.79Hz), 133.38 (d,
compound PH-244 as off-white solid 578 mg, yield 76%, recrystal- J ¼ 10.70 Hz), 119.17 (d, J ¼ 4.18 Hz), 114.07 (d, J ¼ 2.64 Hz), 106.62
lised from EtOAc-Et2O; mp 164.5–166.5 ꢃC. IR (KBr, cmꢂ1): ꢀ 3301, (d, J ¼ 26.24 Hz), 69.86, 66.07, 50.64, 50.62, 50.26, 47.42, 31.46, 30.88,
2964, 2869, 1752, 1655, 1640, 1520, 1477, 1444, 1398, 1328, 1239, 23.70, 21.80, 13.71. MS ESþ (m/z): 410.2000 (Mþ þ H), MS (m/z):
1132, 1107. 1H-NMR (DMSO-d6, 600 MHZ): d 9.90 (s, 1H, N–OH, 409.2 (Mþ). Anal. Calcd. for C20H28FN3O5: C: 58.67; H: 6.89; N: 10.26;
exchangeable with D2O), 7.49 (dd, 1H, J ¼ 2.5 Hz, 15.00 Hz, phenyl found C: 58.32, H: 6.87, N: 10.61.
H), 7.18 (dd, 1H, J ¼ 2.3 Hz, 8.8 Hz, phenyl H), 7.06 (t, 1H, J ¼ 9.4 Hz,
phenyl H), 4.84–4.89 (br. m, 1H, oxazolidinone H), 4.13 (t, 1H,
4.1.4.6. (R)-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-
yl) methyl)-N-hydroxyheptanamide (PH-249). The intermediate
J ¼ 8.9 Hz, oxazolidinone H), 4.02 (dd, 1H, J ¼ 6.5 Hz, 14.2 Hz, oxazo-
lidinone H), 3.73–3.76 (m, 5H, morpholine H and methylene H),
compound PH-248 was prepared via the general procedure from
3.68 (dd, 1H, J ¼ 4.2 Hz, 14.6 Hz, methylene H), 3.10–3.20 (m, 1H,
compound 15 (6.00 g, 19.27 mmol), heptanoyl chloride (8.95 ml,
57.81 mmol), triethylamine (16.21 ml; 115.62 mmol) in anhydrous
DCM (30 ml) to give crude product. Purification by silica gel col-
umn chromatography (EtOAc-Hexane, 3:4) gave the intermediate
cyclopentane H), 2.96 (t, 4H, J ¼ 4.6 Hz, morpholine H), 1.45–1.80
(m, 8H, cyclopentane H). 13 C-NMR (DMSO-d6, 600 MHZ): d 176.77,
154.55 (d, J ¼ 243.43 Hz), 153.96, 135.51 (d, J ¼ 8.75 Hz), 133.43 (d,
J ¼ 10.72 Hz), 119.23 (d, J ¼ 4.18 Hz), 114.10 (d, J ¼ 2.87 Hz), 106.64
(R)-N-(3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)
(d, J ¼ 26.14 Hz), 69.90, 66.13, 50.69, 50.68, 50.58, 47.48, 29.27,
methyl)-N-(heptanoyloxy) heptanamide PH-248 as a white solid
29.20, 25.58, 25.56. MS ESþ (m/z): 408.2039 (Mþ þ H), MS (m/z):
6.82 g, yield 66%, mp 68–70.5 ꢃC. IR (KBr, cmꢂ1): ꢀ 2960, 2929,
407.2 (Mþ). Anal. Calcd. for C20H26FN3O5: C, 58.96; H, 6.43; N,
2855, 1794, 1740, 1685, 1519, 1446, 1410, 1329, 1236, 1217, 1140,
1119, 1066. 1H-NMR (DMSO-d6, 600 MHZ):
d 7.48 (dd, 1H,
10.31; found C, 58.57; H, 6.31; N, 10.01.
J ¼ 2.5 Hz, 14.7 Hz, phenyl H), 7.17 (dd, 1H, J ¼ 2.2 Hz, 8.8 Hz, phe-
4.1.4.5. (R)-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-
nyl H), 7.06 (t, 1H, J ¼ 9.3 Hz, phenyl H), 4.84–4.88 (br. m, 1H, oxa-
yl) methyl)-N-hydroxyhexanamide (PH-247). The intermediate zolidinone H), 4.10–4.18 (br. t, 2H, oxazolidinone H, overlaps with
compound PH-246 was prepared via the general procedure from oxazolidinone H triplet at 4.11, J ¼ 9.0 Hz), 3.84–3.94 (br., 1H,
compound 15 (6.0 g, 19.27 mmol), hexanoyl chloride (8.08 ml, methylene H), 3.73 (t, 5H, J ¼ 4.6 Hz, morpholine H and methylene
57.82 mmol), triethylamine (16.20 ml; 115.64 mmol) in anhydrous H), 2.96 (t, 4H, J ¼ 4.6 Hz, morpholine H), 2.50 (br., 2H, methylene
DCM (90 ml) to give crude product. Purification by silica gel col- –CH2– overlapping with DMSO signal), 2.10–2.26 (br., 2H, methy-
umn chromatography (EtOAc-Hexane, 1.3:2) gave the intermediate lene H), 1.15–1.64 (m, 16H, methylene H), 0.86–0.87 (m, 6H, two
(R)-N-((3-(3-flouro-4-morpholino phenyl)methyl)-N-(hexanoyloxy)- methyl H). 13 C-NMR (DMSO-d6, 600 MHZ):
d
154.53 (d,
hexanamide PH-246 as a white solid 4.50 g, yield 46%, mp J ¼ 244.26 Hz), 153.72, 135.54 (d, J ¼ 8.77 Hz), 133.31 (d,
80–82.8 ꢃC. IR (KBr, cmꢂ1): ꢀ 2958, 2930, 2856, 1794, 1740, 1685, J ¼ 10.73 Hz), 119.19 (d, J ¼ 4.01 Hz), 114.11 (d, J ¼ 2.43 Hz), 106.66
1517, 1446, 1408, 1329, 1237, 1216, 1140, 1119, 1063. 1H-NMR (d, J ¼ 26.01 Hz), 70.13, 66.10, 50.66, 50.65, 47.13, 31.03, 30.87,
(DMSO-d6, 600 MHZ): d 7.48 (dd, 1H, J ¼ 2.5 Hz, 14.9 Hz, phenyl H), 30.74, 28.04, 27.94, 23.83, 21.87, 21.83, 13.79. MS ESþ (m/z):
7.18 (dd, 1H, J ¼ 2.3 Hz, 8.8 Hz, phenyl H), 7.06 (t, 1H, J ¼ 9.3 Hz, 535.3498 (Mþ þ H), 538.3384 (Mþ þ Na), MS (m/z): 535.4 (Mþ).
phenyl H), 4.84–4.88 (br. m, 1H, oxazolidinone H), 4.10–4.18 (br. t, Anal. Calcd. for C28H42FN3O6: C: 62.78; H: 7.90; N: 7.84; found C:
2H, oxazolidinone H, overlaps with oxazolidinone H triplet at 4.11, 62.72; H, 7.90; N, 7.51. A solution of the intermediate PH-248
J ¼ 9.0 Hz), 3.84–3.94 (br., 1H, methylene H), 3.74 (t, 5H, J ¼ 4.6 Hz, (6.28 g, 11.72 mmol) in MeOH:THF (84:21 ml) was treated with
morpholine H and methylene H), 2.96 (t, 4H, J ¼ 4.6 Hz, morpho- NaOH solution (928 mg in 20 ml water). Purification by recrystal-
line H), 2.50 (br., 2H, methylene –CH2– overlapping with DMSO lisation (EtOAc-hexane 2:1) gave the titled compound PH-249 as
signal), 2.10–2.28 (br., 2H, methylene H), 1.16–1.64 (m, 12H, methy- an off-white solid 4.30 g, yield 87%, mp 123–125 ꢃC. IR (KBr, cmꢂ1):
lene H), 0.82–0.88 (m, 6H, two methyl H). 13 C-NMR (DMSO-d6, ꢀ 3188, 2957, 2923, 2855, 1743, 1719, 1626, 1525, 1473, 1426,

1480
O. A. PHILLIPS
1332, 1271, 1235, 1196, 1115, 1073. 1H-NMR (DMSO-d6, 600 MHZ): 173.96, 154.56 (d, J ¼ 244.06Hz), 153.95, 135.51 (d, J ¼ 8.78Hz),
d 9.92 (s, 1H, N–OH, exchangeable with D2O), 7.48 (dd, 1H, 133.43 (d, J ¼ 10.79 Hz), 119.21 (d, J ¼ 4.06 Hz), 114.08 (d,
J ¼ 2.5 Hz, 14.9 Hz, phenyl H), 7.18 (dd, 1H, J ¼ 2.2 Hz, 8.8 Hz, phe- J ¼ 2.84 Hz), 106.64 (d, J ¼ 26.00 Hz), 69.94, 66.13, 50.69, 50.68, 50.26,
nyl H), 7.06 (t, 1H, J ¼ 9.3 Hz, phenyl H), 4.85–4.89 (br. m, 1H, oxa- 47.44, 31.57, 31.14, 28.70, 28.49, 24.10, 22.04, 13.93.
zolidinone H), 4.12 (t, 1H, J ¼ 8.9 Hz, oxazolidinone H), 4.04 (dd,
MS ESþ (m/z): Calcd for C22H32FN3O5 þ H: 438.2404, found:
1H, J ¼ 6.7 Hz, 14.7 Hz, oxazolidinone H), 3.73–3.75 (m, 5H, mor- 438.2402 (Mþ þ H), Calcd. for C22H32FN3O5 þ Na: 460.2224, found:
pholine H and methylene H), 3.67 (dd, 1H, J ¼ 4.2 Hz, 14.9 Hz, 460.2214 (Mþ þ Na), MS (m/z): 437.2 (Mþ). Anal. Calcd. for
methylene H), 2.96 (t, 4H, J ¼ 4.7 Hz, morpholine H), 2.35–2.38 (m, C22H32FN3O5: C: 60.40; H: 7.37; N: 9.60; found C: 60.54; H, 7.10;
2H,
NCOCH2CH2CH2CH2CH2CH3),
1.44–1.49
1.21–1.27
0.85 (t,
(m,
(m,
2H,
6H,
N, 9.43.
NCOCH2CH2CH2CH2CH2CH3),
NCOCH2CH2CH2CH2CH2CH3),
3H,
J ¼ 7.0 Hz,
NCOCH2CH2CH2CH2CH2CH3). 13 C-NMR (DMSO-d6, 600 MHZ):
d
4.2. Biological activity
173.93, 154.53 (d, J ¼ 244.01 Hz), 153.92, 135.49 (d, J ¼ 8.73 Hz),
133.41 (d, J ¼ 10.27 Hz), 119.19 (d, J ¼ 4.15 Hz), 114.07 (d,
J ¼ 2.80 Hz), 106.63 (d, J ¼ 26.27 Hz), 69.90, 66.10, 50.67, 50.65,
50.27, 47.43, 31.54, 31.02, 28.36, 24.02, 21.91, 13.85. MS ESþ (m/z):
424.2521 (Mþ þ H), MS (m/z): 423.3 (Mþ). Anal. Calcd. for
C21H30FN3O5: C: 59.56; H: 7.14; N: 9.92; found C: 59.58, H: 7.52,
N: 10.05.
4.2.1. General
The solvents, drugs and other reagents, including, polyethylene
glycol (PEG), dimethylsulphoxide (DMSO), N-formyl methyl-leucyl-
phenylalanine (FMLP), zileuton, zymosan, mouse anti-dinitrophenyl
(DNP) IgE, dinitrophenyl-conjugated bovine serum albumin (DNP-
BSA), foetal bovine serum (FBS), heparin, calcium ionophore
A23187, LPS, DTT and glutathione peroxidase (GPx) were obtained
from Sigma-Aldrich (St. Louis, MO). All compounds for in vitro
experiments were solubilised in DMSO and diluted down in PBS.
The final concentration of the solvent did not exceed 0.05%. For
in vivo experiments, the compounds were made up in drug
vehicle (4% DMSO/67.2% PEG/28.8% PBS).
4.1.4.7. (R)-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-
yl) methyl)-N-hydroxyoctanamide (PH-251). The intermediate com-
pound PH-250 was prepared via the general procedure from com-
pound 15 (2.43 g, 7.80 mmol), octanoyl chloride (3.27 ml,
23.40 mmol), triethylamine (6.60ml; 46.80mmol) in anhydrous DCM
(35ml) to give crude product. Purification by silica gel column chro-
matography (EtOAc-hexane, 1:2; 3:4) gave the intermediate(R)-N-((3-
(-Fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)-N-(octa-
noyloxy)octanamide PH-250 as a white solid 2.20 g, yield 50%, mp
49–53 ꢃC. IR (KBr, cmꢂ1): ꢀ 2954, 2925, 2853, 1790, 1750, 1673,
4.2.2. In vitro assay for the inhibition of 5-LO product LTB4 from
human whole blood
All compounds were evaluated for inhibitory activity against the
5-LO-dependent generation of LTB4 from activated human whole
blood. With ethical approval from the Health Sciences Centre
Ethical Committee of Kuwait University, heparinised fresh human
1
1520, 1442, 1417, 1377, 1328, 1272, 1225, 1193, 1120, 1075. H-NMR
(DMSO-d6, 600 MHZ): d 7.48 (dd, 1H, J ¼ 2.5Hz, 14.9Hz, phenyl H),
7.17 (dd, 1H, J ¼ 2.3Hz, 8.8Hz, phenyl H), 7.06 (t, 1H, J ¼ 9.3 Hz, phe- blood samples from apparently healthy individuals of both sexes
nyl H), 4.84–4.88 (br. m, 1H, oxazolidinone H), 4.09–4.18 (br. t, 2H, were obtained from the Kuwait Central Blood Bank. All donors
oxazolidinone H, overlaps with oxazolidinone H triplet at 4.11, gave informed consent and the work was carried out in accord-
J ¼ 9.1 Hz), 3.84–3.94 (br., 1H, methylene H), 3.73 (t, 5H, J ¼ 4.6 Hz, ance with the “Declaration of Helsinki” for experiments involving
morpholine H and methylene H), 2.96 (t, 4H, J ¼ 4.6 Hz, morpholine human subjects. Aliquots of 185 ml of whole blood were dispensed
H), 2.50 (br., 2H, methylene –CH2– overlapping with DMSO signal), into each well of a 96-well culture plate containing 5 ml of the pri-
2.10–2.24 (br., 2H, methylene H), 1.20–1.64 (m, 20H, methylene H), ming agent, LPS (1 mg/ml final concentration). After a 15 min incu-
0.83–0.87 (m, 6H, two methyl H). 13 C-NMR (DMSO-d6, 600 MHZ): d bation at 37 ꢃC, 5 ml of the test compounds (0.001–30 mM) or the
154.53 (d, J ¼ 244.26 Hz), 153.72, 135.56 (d, J ¼ 8.77 Hz), 133.31 (d,
reference drug, zileuton (as positive control) or the vehicle (0.05%
DMSO), was added. After further incubation for 15 min at 37 ꢃC,
J ¼ 10.72 Hz), 119.18 (d, J ¼ 4.01 Hz), 114.08 (d, J ¼ 2.43Hz), 106.66
(d, J ¼ 26.01Hz), 70.14, 66.10, 50.66, 50.65, 47.11, 31.07, 31.03, 28.34,
5 ml of FMLP (1 mM final concentration) was added to stimulate LT
28.33, 28.25, 28.23, 23.89, 21.97, 13.86. MS ESþ (m/z): 564.3386 (Mþ
production. The reaction was stopped 15 min later, and the super-
þ H), MS (m/z): 563.3 (Mþ). Anal. Calcd. for C30H46FN3O6: C: 63.92;
H: 8.23; N: 7.45; found C: 64.01; H, 8.26; N, 7.43. A solution of the
intermediate PH-250 (2.15g, 3.81mmol) in MeOH:THF (56:14 ml)
was treated with NaOH solution (305mg in 20 ml water).
Purification by recrystallisation (EtOAc-hexane 2:1) gave the titled
compound PH-251 as an off-white solid 1.30 g, yield 78%, mp
119–122 ꢃC. IR (KBr, cmꢂ1): ꢀ 3184, 2956, 2923, 2854, 1743, 1720,
1626, 1524, 1471, 1446, 1427, 1332, 1272, 1235, 1197, 1115, 1075.
1H-NMR (DMSO-d6, 600 MHZ): d 9.93 (s, 1H, N–OH, exchangeable
with D2O), 7.48 (dd, 1H, J ¼ 2.5 Hz, 14.9 Hz, phenyl H), 7.17 (dd, 1H,
J ¼ 2.2 Hz, 8.8 Hz, phenyl H), 7.06 (t, 1H, J ¼ 9.4 Hz, phenyl H),
4.85–4.89 (br. m, 1H, oxazolidinone H), 4.12 (t, 1H, J ¼ 8.9Hz, oxazo-
lidinone H), 4.04 (dd, 1H, J ¼ 6.7 Hz, 14.7Hz, oxazolidinone H),
3.73–3.75 (m, 5H, morpholine H and methylene H), 3.67 (dd, 1H,
natants were recovered by centrifugation and stored at ꢂ40 ꢃC
pending analysis of LTB4 content.
4.2.3. In vitro assay for the inhibition of 5-LO product LTC4 from
isolated human monocytes
Mononuclear cells were first isolated from heparinised blood using
the Ficoll-Hypaque gradient centrifugation method. Monocytes
were subsequently purified by adherence to plastic according to
standard protocols. The purity of the adherent monocytes
(CD14þ) was routinely confirmed by flow cytometry to be >95%
and viability (by trypan blue exclusion method) was routinely
>97%. Adherent monocytes were then washed and incubated in
190 ml culture medium RPMI-1640 supplemented with 100 U/ml
J ¼ 4.2 Hz, 14.9 Hz, methylene H), 2.96 (t, 4H, J ¼ 4.6Hz, morpholine penicillin, 100 mg/ml streptomycin and 10% heat-inactivated foetal
H), 2.34–2.38 (m, 2H, NCOCH2CH2CH2CH2CH2CH2CH3), 1.44–1.50 bovine serum (Sigma-Aldrich, St Louis, MO). Cells were then incu-
(m, 2H, NCOCH2CH2CH2CH2CH2CH2CH3), 1.18–1.30 (m, 8H, bated with the various test compounds (0.001–30 lM) or vehicle
NCOCH2CH2CH2CH2CH2CH2CH3),
0.85
(t,
3H,
J ¼ 7.0 Hz, (0.03% DMSO) or zileuton (positive control) for 15 min before
NCOCH2CH2CH2CH2CH2CH2CH3). 13 C-NMR (DMSO-d6, 600 MHZ): d being stimulated with 5 ml of the calcium ionophore A23187 at a

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1481
final concentration of 2.5 mM. After further incubation for 15 min, 4.2.7. In vitro toxicity testing
the culture supernatants were recovered by centrifugation and Adherent human monocytes, prepared as described above were
stored at ꢂ40 ꢃC pending LTC4 determination.
cultured with various concentrations of the test compounds or
vehicle, or with 0.05% Triton-X as a positive control for 3 h or
24 h. At the end of the culture, cell viability was determined using
4.2.4. In vitro assay for the inhibition of 5-LO product LTC4 from
allergen/IgE-activated bone marrow-derived mouse mast (5 mg/ml) was added to each well and incubated at 37 ꢃC for 4 h.
the standard MTT assay method. Briefly, 15 lL of the MTT solution
After removing the supernatant, 200 lL of DMSO was added to
dissolve the crystals. Absorbance at 570 nm was then measured in
a microplate reader. Viability was expressed as a percentage of
the vehicle-treated cells.
cells (BMMC)
Bone marrow-derived mast cells (BMMC) were generated from
pathogen-free 5 to 7-weeks-old male Balb/c mice according to the
method of Davis et al.28. Essentially, bone marrow cells were
obtained by flushing the femoral bone marrow and the recovered
cells cultured in RPMI 1640 medium supplemented with 10% FBS,
100 U/ml penicillin, and 100 mg/ml streptomycin, 25 mM HEPES,
1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids,
0.0035% 2-mercaptoethanol, and 30 ng/ml mouse recombinant IL-
3, with culture medium, replaced every 2 days. Cells were used
after 4–8 weeks of culture, by which time at least 97% of the cells
would have differentiated into mast cells, as routinely obtained in
our laboratory.
4.2.8. Evaluation of in vivo activity in zymosan-induced peritoneal
inflammation model in mice
Female Balb/c mice 6 to 8-weeks-old obtained from the Animal
Resources Centre of the Health Sciences Centre, Kuwait University,
were used. They were maintained under temperature-controlled
conditions with an artificial 12-h light/dark cycle and allowed
standard chow and water ad libitum. The study was carried out in
compliance with the Regulations for the Use of Laboratory
Animals in the Health Sciences Centre, Kuwait University, and
complied with the National Institute of Health guide for the care
The generated BMMCs were seeded at 5 ꢄ 104 cells/well in a
96-well flat-bottom culture plate and passively sensitised over-
night with 0.5 mg/ml anti-DNP monoclonal IgE antibody. The cells
were then washed twice to remove any unbound antibody and
subsequently resuspended in reaction buffer (135 mM NaCl, 5 mM
KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 0.05% BSA and
20 mM HEPES, pH 7.4). They were then pre-incubated with the
test compounds (0.001 ꢂ 30 lM) or the solvent (0.05% DMSO) for
15 min before being stimulated with the specific antigen, DNP-
BSA (10–30 ng/ml). After 30 min incubation at 37 ꢃC, the amount
of LTC4 released into the supernatant was determined by ELISA as
described below.
and use of laboratory animals (NIH Publication
revised 1978).
# 8023,
The zymosan-induced peritonitis model - a recognised LT-
mediated inflammatory reaction26, was used. Five groups of mice,
7 per group, were treated subcutaneously with either PH-249 (the
most active compound on human cells) at doses of 10 or 30 mg/
kg or drug vehicle alone or zileuton for comparison. After 30 min,
all groups were injected intraperitoneally with 0.2 ml of activated
zymosan (2 mg/ml) except the control group that received 0.2 ml
PBS. After 2 h, all animals were killed, and the peritoneal exudate
collected by washing the cavity with 3 ml of heparinised (10 IU/ml)
PBS. Cells in the exudate were recovered by centrifugation and
counted in a haemocytometer while the supernatant volume was
recorded and then stored frozen at ꢂ78 ꢃC until used for the
determination of LTC4 by ELISA as detailed above.
4.2.5. Assay of released leukotrienes
Appropriately diluted supernatants were assayed for the released
products – LTs (LTB4 and LTC4), by the enzyme immunoassay (EIA)
method using assay kits supplied by R&D Systems (Minneapolis,
MN) and following the manufacturer’s instructions.
4.2.9. Statistical analysis
All data were analysed using GraphPad Prism software (GraphPad
Software, San Diego, CA). The 50% inhibition concentration (IC50)
values were determined from the concentration-response curves
by non-linear regression analysis (normalised variable). Differences
between experimental groups were first analysed by one-way
ANOVA, followed by Bonferroni’s post-hoc test. A p-value of less
than 0.05 was taken as statistically significant.
4.2.6. Evaluation of the direct inhibition of 5-LO activity in a cell-
free assay
The assay was based on the oxidation of the dye 20,70-dichlorodi-
hydrofluorescein diacetate (H2DCF-DA) to a highly fluorescent
product by 5-LO enzymatic products29. H2DCF-DA (60 mM) was
first pre-cleaved by incubating with 450 mU/ml recombinant
human 5-LO enzyme in Tris buffer (containing 50 mM Tris, pH ¼
7.5, 2 mM EDTA, 2 mM CaCl2, 1 mM DTT and 0.6 U/ml glutathione
peroxidase) for 10 min at room temperature. Then, to each well of
a black 96-well plate was added 25 ml of the above enzyme/dye
solution, followed by 25 ml of the test compounds or zileuton
(0.001–30 mM) or drug vehicle, in duplicates. After 10 min incuba-
tion at room temperature, the reaction was started with the add-
ition of 50 ml of substrate solution (Tris buffer containing 20 mM
ATP and 20 mM arachidonic acid (AA). After a further 20 min, the
reaction was terminated with 100 ml acetonitrile. Fluorescence was
read at 500 nm excitation and 520 nm emission, with NovostarR
microplate reader (BMG Labtech, Offenburg, Germany).
Appropriate controls, including 100 mM nordihydroguaiaretic acid
(NDGA), were included to isolate only the 5-LO-attributable,
NDGA-inhibitable RFU values.
Acknowledgements
The authors acknowledge the HSC Animal Resources Centre for
assistance with research equipment and animals. The authors are
also grateful to Ms. Elizabeth Philip, for her excellent tech-
nical assistance.
Ethical approval statement
Experiments involving living animals and their care were per-
formed in strict compliance with the Regulations for the Use of
Laboratory Animals in the Health Sciences Centre, Kuwait
University, and complied with the National Institute of Health
guide for the care and use of laboratory animals (NIH Publication

1482
O. A. PHILLIPS
# 8023, revised 1978). The protocol was approved by the Health
Sciences Centre Animal Experimentation Ethical Committee of
Kuwait University. All efforts were made to minimise the animal’s
suffering and to reduce the number of animals used.
All experiments involving human blood were conducted with
ethical approval from the Health Sciences Centre Ethical
Committee of Kuwait University and carried out in accordance
with the “Declaration of Helsinki” for experiments involving
human subjects. All donors gave informed consent.
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Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This study was supported by Kuwait University (KU) Research
Sector Grant [MR01/14]; KU, HSC OMICS Research Unit Grant
[GM01/15]; KU General Facilities Science (GF-S), Faculty of Science
Grants [GS01/03, GS01/05, and GS02/10].
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