Synthesis of glutamic acid analogs as potent inhibitors of leukotriene A4 hydrolase

Article abstract of DOI:10.1016/j.bmc.2008.03.042

Leukotriene B₄ (LTB₄) is a potent pro-inflammatory mediator that has been implicated in the pathogenesis of multiple diseases, including psoriasis, inflammatory bowel disease, multiple sclerosis and asthma. As a method to decrease the level of LTB₄ and possibly identify novel treatments, inhibitors of the LTB₄ biosynthetic enzyme, leukotriene A₄ hydrolase (LTA₄-h), have been explored. Here we describe the discovery of a potent inhibitor of LTA₄-h, arylamide of glutamic acid 4f, starting from the corresponding glycinamide 2. Analogs of 4f are then described, focusing on compounds that are both active and stable in whole blood. This effort culminated in the identification of amino alcohol 12a and amino ester 6b which meet these criteria.

Full text of DOI:10.1016/j.bmc.2008.03.042

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4963–4983
Synthesis of glutamic acid analogs as potent inhibitors
of leukotriene A4 hydrolaseq
Thomas A. Kirkland,a,* Marc Adler,a John G. Bauman,a Ming Chen,a
Jesper Z. Haeggstro¨m,d Beverly King,c Monica J. Kochanny,a Amy M. Liang,b
Lisa Mendoza,c Gary B. Phillips,a Marjolein Thunnissen,e Lan Trinh,b Marc Whitlow,a
Bin Ye,a Hong Ye,a John Parkinsonc and William J. Guilforda
aDepartment of Medicinal Chemistry, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, United States
bDepartment of Molecular Pharmacology, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, United States
cDepartment of Immunology, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, United States
dDepartment of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden
eMolecular Biophysics, Lund University, Getingeva¨gen 60, SE-221 00 Lund, Sweden
Received 8 January 2008; revised 13 March 2008; accepted 14 March 2008
Available online 20 March 2008
Abstract—Leukotriene B4 (LTB4) is a potent pro-inflammatory mediator that has been implicated in the pathogenesis of multiple
diseases, including psoriasis, inflammatory bowel disease, multiple sclerosis and asthma. As a method to decrease the level of LTB4
and possibly identify novel treatments, inhibitors of the LTB4 biosynthetic enzyme, leukotriene A4 hydrolase (LTA4-h), have been
explored. Here we describe the discovery of a potent inhibitor of LTA4-h, arylamide of glutamic acid 4f, starting from the corre-
sponding glycinamide 2. Analogs of 4f are then described, focusing on compounds that are both active and stable in whole blood.
This effort culminated in the identification of amino alcohol 12a and amino ester 6b which meet these criteria.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
the biosynthesis of LTB4 may be useful for the treatment
of these inflammatory conditions.
Leukotriene B4 (LTB4) is a potent pro-inflammatory
activator of inflammatory cells, including neutrophils,
eosinophils, monocytes, macrophages, T cells and B
cells that is a functional link between early innate and
late adaptive immune responses.1 There is substantial
evidence that LTB4 plays a significant role in the ampli-
fication of many inflammatory disease states2 including
asthma,3 inflammatory bowel disease (IBD),4 chronic
obstructive pulmonary disease (COPD),5 arthritis,6 pso-
riasis,7 and atherosclerosis.8 LTB4 also stimulates the
production of various cytokines and may play a role
in immunoregulation.9 Therefore, a therapeutic agent
that inhibits the response of cells to LTB4 or inhibits
Two pharmacologically distinct human LTB4 receptors,
BLT110 and BLT2,11 have recently been described.
BLT1 is a high affinity LTB4 receptor (Kd = 0.1–
0.7 nM) with a restricted expression in inflammatory
cells, for example, neutrophils, monocytes, thymus,
and spleen. BLT2 has a wider tissue expression profile
but a 20-fold lower affinity for LTB4 (Kd = 23 nM) than
BLT1. Although the presence of two receptors can pro-
vide for a graded immune response, it makes antago-
nism of a LTB4 receptor a more difficult therapeutic
target. Therefore, we focused on the reduction of
LTB4 production as a therapeutic approach.
The biosynthesis of LTB4 from arachidonic acid (AA)
involves the action of three enzymes: phospholipase A2
(PLA2), to release AA from the membrane lipids; 5-
lipoxygenase (5-LO), to form the unstable epoxide
leukotriene A4 (LTA4); and leukotriene A4 hydrolase
(LTA4-h), to form LTB4.12 In addition to being the
Keywords: Leukotriene A4; Inhibitor; Crystal structure.
q
Note to the editor: The atomic coordinates described in this paper
have been deposited with the Protein Data Bank.
Corresponding author. Tel.: +1 805 544 8524x692; fax: +1 805 543
1531; e-mail: Thomas.kirkland@promega.com
*
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.042

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T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
precursor to LTB4, LTA4 is a precursor of the
pro-inflammatory cysteinyl leukotrienes LTC4, LTD4,
and LTE413 via LTC4 synthase and a precursor of lipoxy-
genases to give the anti-inflammatory mediators, lipoxins
A4 (LXA4) and B4 (LXB4).14 Thus the targeting of LTA4-
h would leave other immune response pathways intact.
Since LTA4-h is considered a drugg able target with
available functional and PD assays, and plays a role in
inflammatory diseases,20 several companies have re-
ported programs designed to discover a potent and
selective LTA4-h inhibitor.20b,21 Our goal at the start
of the Berlex program was to identify a non-hydroxamic
acid containing inhibitor using the peptidase assay. The
peptidase assay is more suitable for high-throughput
screening than the hydrolase assay and was used during
the early stage of the project described here. Compound
2 was identified through high-throughput screening as
an inhibitor of the peptidase activity of recombinant hu-
man LTA4-h (peptidase assay)22 with an IC50 of
280 nM. The secondary assay for this project was inhibi-
tion of LTB4 synthesis in human whole blood (the whole
blood assay),23 and compound 2 was weakly active in
this assay with a potency of 7 lM. The whole blood as-
say gave insights into cell permeability and protein bind-
ing, and high potency in this assay was the ultimate goal
for inhibitors.
LTA4-h is a monomeric, soluble 69 kD enzyme.15
LTA4-h is a bifunctional zinc-dependent metalloenzyme
of the M1 class of metallohydrolases which catalyzes
two reactions at the same active site: a stereospecific
epoxide hydrolase reaction and a non-specific peptidase
reaction. Based on biochemical studies, mutational
analysis, and X-ray crystallography,16 the two en-
zyme-catalyzed reactions are exerted via distinct and
yet overlapping active sites.17 The bifunctional nature
of LTA4-h allows for the determination of inhibition con-
stants using either the hydrolase or peptidase activity
using either LTA4 or L-alanine-p-nitroanilide as sub-
strates, respectively. The differences in the binding and ki-
netic constants for the two substrates18 make direct
comparison of inhibitor data from the hydrolase and pep-
tidase assays problematic, but comparison of inhibition
data within either assay can be used to rank compounds.
H
N
H2N
O
O
High resolution crystal structures of recombinant hu-
man LTA4-h with bound inhibitors have been reported
in the literature.19 The hydroxamic acid 1 (Ki = 1.6 nM)
bound in a LTA4-h crystal structure is believed to
make many of the same interactions as LTA4
(Fig. 1). As can be seen in Figure 1, the interaction be-
tween LTA4-h and molecule 1 can be divided into four
distinct regions. A hydrophobic pocket containing the
two aromatic rings of inhibitor 1 in Figure 1 is the pro-
posed binding site of the long alkyl tail of LTA4. Just
outside the hydrophobic pocket are Glu271 and
Gln136 which form a salt bridge with the free amine
on 1. The zinc atom which chelates to the hydroxamic
acid would bind to the epoxide of LTA4. Basic residue
Arg563, which forms a salt bridge with the terminal
carboxylic acid of 1, could make a similar interaction
with LTA4.
2
Compound 2 was chemically simple and easy to derivatize,
and thus was an attractive lead compound. Following is a
description of the efforts to optimize this lead into novel,
potent and cell-permeable inhibitors of LTA4-h.
2. Chemistry
The synthesis of compounds 2–12 was carried out in the
following manner. Compound 2 was synthesized through
carbonyl diimidazole (CDI) mediated coupling of 13 and
Boc-glycine followed by acidic deprotection (Scheme 1).
Coupling of other simple amino acids to benzyloxyaniline
was done in a similar manner.
Figure 1. A hydroxamic based inhibitor bond to rhLTA4-h (PDB entry 2VJB). Green is used for the inhibitor’s carbon atoms. H-bonds are depicted
as green lines. The zinc is displayed as a peach colored sphere that is chelated to His295, His299, and Glu318 (gold atoms). The hydroxamic acid also
˚
chelates the zinc and the carboxylic acid forms H-bond with Arg563 (2.6 A) and Gly268 (2.4 A).
˚

T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
4965
H
N
H2N
H2N
a, b
O
O
O
2
13
Scheme 1. General synthesis of amino acid analogs. Reagents and condition: (a) CDI, Boc-Gly, CH2Cl2, reflux; (b) TFA, CH2Cl2.
Synthesis of aspartic acid and glutamic acid derivatives
was accomplished using isobutyl chloroformate (IBC)
coupling conditions followed by sequential deprotection
as shown for the synthesis of compound 4f (Scheme 2).
The same conditions were used successfully to couple ani-
lines with both the alpha carboxylic acid and the side-
chain carboxylic acid. During the process of varying the
aniline portion of the lead series, it was found that stan-
dard peptide coupling agents such as HATU or EDC/
HOBt were also effective in coupling to glutamic acid
derivatives.
Initial modification of the glutamic acid portion of 4f fo-
cused on ester and amide derivatives. Some of these deriv-
atives could be synthesized simply by beginning with the
appropriate glutamic esters and glutamines (Scheme 2).
Amides 11a–e were synthesized through coupling the
appropriate amine with intermediate 14 using EDC/
HOBt conditions, followed by deprotection if necessary.
Replacement of the carboxylic acid of 4f with a hetero-
cycle was accomplished through the following sequence
(Scheme 3). Aminonitrile 15 was synthesized through
OBn
O
O
H2N
O
O
a, b
+
OH
O
HO
N
H
OBn
NHBoc
NHBoc
14
13
c
d, c
OBn
O
O
OBn
O
O
HO
N
H
NH2
NHR
N
H
4f
NH2
11a-e
Scheme 2. Synthesis of c-glutamic acid analogs. Reagents and condition: (a) IBC, N-methylmorpholine, THF, À20 ꢁC; (b) NaOH, MeOH; (c) TFA,
CH2Cl2; (d) NH2R, EDC, HOBt, DMF.
OPh
O
O
H2N
O
N
a, b
N
H
+
OH
H2N
OPh
NHBoc 15
NHBoc
c, d
H
OPh
N
O
N
N
N
H
NH2
12e
Scheme 3. Synthesis of acid replacement analogs of 4f. Reagents and condition: (a) IBC, N-methylmorpholine, THF, À20 ꢁC; (b) cyanuric chloride,
DMF; (c) formic hydrazide, K2CO3, EtOH; (d) TFA, CH2Cl2.

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T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
dehydration of the glutamide formed through coupling
4-phenoxyaniline and Boc-Gln, and then conversion to
the triazole and deprotection led to compound 12e.
amino acids having hydrophobic, basic, and acidic side
chains with the ultimate goal of making a positive inter-
action with the zinc ion by displacing the water
molecules.
Other intermediates were used to synthesize extended
inhibitors. Reduction of the protected amino ester
and sulfonylation provided the versatile mesylate 16
(Scheme 4). This mesylate could be displaced by a vari-
ety of heterocycles using similar conditions to those
shown for the phthalimide for compound 12h. Depro-
tection of the amine provided the final class of inhibi-
tors in this series.
Substitution on the glycine portion of 2 with hydropho-
bic groups was explored first (Table 1). The decrease in
the inhibitory activity of analogs with an increase in the
size of the hydrophobic group, analogs 3a–c, confirmed
the prediction based on the analysis of Figure 2 that the
binding pocket is compact and disfavors non-polar
groups. The lack of inhibitory activity with the proline
analog 3d can be explained by both the increase in
non-polar groups and the loss of a hydrogen bond be-
tween LTA4-h and the secondary amine. Next we ex-
plored the glycine analogs with polar side chains with
the anticipation of filling the polar pocket and having
a positive interaction with the zinc. However, substitu-
tion of glycine with an amino acid having a polar side
chain, glutamine (Gln, 3e), or a basic side chain, lysine
and histamine (Lys and His, 3f–g), or acidic side chain,
glutamic acid or aspartic acid (Glu or Asp, 4a–b) all
showed reduced activity.
3. Results and discussion
The strategy for altering 2 was based on interactions ob-
served in the crystal structure (Fig. 2). The interaction of
this compound with the enzyme appears to be mostly
hydrophobic, with the two phenyl rings extending into
the presumed binding site for the aliphatic chain of
LTA4.16 The amine of 2 forms the only direct hydrogen
bond (H-bond) to the protein through the carbonyl of
˚
Gly269 (2.9 A). The amine is also the closest inhibitor
˚
atom to the active site zinc (5.5 A) and there are several
A crystal structure of 4b bound to the enzyme (Fig. 3)
shows the side-chain carboxylic acid interacting with
˚
the zinc (2.8 A), although not within the chelation
waters that fill the gap between the two atoms. The ini-
tial plan was to probe the binding site through the use of
OPh
O
O
O
H2N
a, b, c
OPh
+
N
OH
O
OMs
H
16
NHBoc
NHBoc
d, e
OPh
O
O
N
N
H
NH2
12h
O
Scheme 4. Synthesis of extended analogs of 4f. Reagents and condition: (a) IBC, N-methylmorpholine, THF, À20 ꢁC; (b) NaBH4, MeOH/THF; (c)
MsCl, pyridine; (d) potassium phthalimide, DMF; (e) TFA, CH2Cl2.
Figure 2. Compound 2 bound to rhLTA4 (PDB entry 3CHO).

T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
4967
Table 1. Amino acid analogs
improvement in potency (Table 2). Shifting the position
of the amino substitution on the chain by coupling to
the aniline through the side-chain carboxylic acid pro-
vided a breakthrough. Compound 4f was 40 times more
potent than 4a, and over 10 times more potent than our
lead compound 2. The specificity of the binding was
shown with the decrease in potency by shortening the
carbon chain, 4e, or inverting the configuration at the
amino group, 4g.
H
N
R
O
Number
R
Peptidase IC50 WBA IC50
NH2
2
280
7000
Examining the crystal structure of 4f gives some ratio-
nale for this potency increase (Fig. 4). The carboxylic
O
˚
acid binds to the zinc (2.1 A) and becomes the fourth
NH2
Me
member of the chelation sphere. The carboxylic acid
also forms H-bonds to the side chains of Glu296
3a
3b
3c
3d
3e
3f
150
>10,000
O
O
˚
˚
(2.6 A) and Tyr383 (2.8 A), two residues that have been
shown to be catalytically active.16 In addition, the amine
NH2
˚
˚
forms H-bonds to Gln136 (2.7 A) and Glu271 (2.9 A). A
similar interaction was observed with the free amine in
the hydroxamic acid based inhibitor of LTA4-h
(Fig. 1). Based on the proposed model of the enzyme’s
peptidase activity,17 the glutamic acid portion of 4f fills
the same pocket as the N-terminal residue of the sub-
strate peptide.
>2000
>2000
>2000
>2000
10,000
5800
ND
Me
Me
NH2
ND
O
O
H
N
However, compound 4f was 400-fold less active in the
whole blood assay than in the peptidase assay. There
are many possible reasons for this significant drop in
activity, but lack of cell permeability caused by the zwit-
terionic nature of the alpha amino acid was considered
the most likely. With the interaction between the zinc
and carboxylic acid observed in the crystal structure,
we felt the amino group might not be critical for binding
of the inhibitor. However, the simple carboxylic acid, 5,
was completely inactive in the peptidase assay (Table 3).
Analogs of 4f, both ester 6a or 6b and amide 6c have
similar activity to 4f in the peptidase assay but are signif-
icantly more potent than 4f in the whole blood activity.
The hydroxamic acid 6d has a similar profile. The po-
tency difference in the whole blood assay can be attrib-
uted to an improvement in cell permeability of the
ester and amide analogs over the zwitterion 4f, but
the similar potency in the peptidase assay suggests that
the amides and esters are cleaved by LTA4-h during
binding. Unfortunately due to technical difficulties, we
were unable to confirm that analogs 6a–d are acting as
pro-drugs and more importantly if binding of the acid
to the zinc is critical for potency.
ND
NH2
NH2
ND
O
O
N
NH2
NH2
NH2
NH2
ND
N
H
O
O
3g
4a
4b
ND
NH2
680
>10,000
ND
CO2H
O
O
CO2H
5400
IC50 values (nM) are means of at least two independent determina-
tions. ND = Not determined.
Removal of the carboxylic acid from 4f yielded 7a which
is threefold less potent in the peptidase assay, but 20-
fold more potent in the whole blood assay than 4f. As
can be seen in Figure 5, the crystal structure 7a with
LTA4-h can be superimposed on the corresponding
structure with 4f with the exception for chelation to
the zinc. In retrospect, the minimal increase in binding
affinity associated with the carboxylic acid to zinc inter-
action is consistent with the fact that a free carboxylic
acid is the product of LTA4-h acting as a peptidase.
According to the Circe effect,24 the enzyme is designed
to have reduced affinity for the products versus the reac-
tants. The relative unimportance of the carboxylic acid
to binding affinity has been observed earlier.25 For our
˚
sphere (<2.4 A). The loss of potency may be explained
by observation that the amine does not form any H-
bonds with the protein. This implies that there is an
energetic penalty for desolvating the basic amine when
it binds to the protein. It was anticipated that simple
modifications of these Glu and Asp derivatives would
improve interactions with the protein and increase the
binding affinity.
Following this logic, we explored the orientation of the
amine group using D-Glu and D-Asp (4c–d) without

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T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
Figure 3. Compound 4b bound to rhLTA4-h (PDB entry 3CHP).
Table 2. Glu/Asp analogs
studies based on the LTA4-h/4f complex indicate that
steric interactions are also a factor.
H
N
R
With the glutamic acid on the right side of the molecule,
we explored modification to the aryl region (Table 4).
For this set of analogs, no significant difference in po-
tency in the peptidase assay was observed between the
corresponding primary amide, the ester or the carbox-
ylic acid. Removal of the benzyl group, 8a, or removal
of a flexible tether, 8b, gave inactive analogs. Substitu-
tion of pyridine for benzene, 8d, or changing the substi-
tution pattern on the central ring to meta, 8c, resulted in
a significant loss in potency. The linker between the two
aryl groups could be shortened to an oxygen, 8e, or car-
bon, 8f, without loss in potency. A secondary amine, 8g,
was tolerated as a linker but not a tertiary amine, 8h.
Lengthening the tether to a three or four atom chain
(8i–j) or placing substituents on the tether (8k–l) was
tolerated.
O
Number
R
Peptidase IC50
680
WBA IC50
>10,000
NH2
NH2
4a
CO2H
O
O
CO2H
4b
4c
5400
7200
ND
ND
NH2
NH2
CO2H
O
The deep, relatively narrow pocket accessed by these
flexible, hydrophobic moieties was predicted from the
crystal structure. As shown in the structure of 8l
(Fig. 6), the second aromatic unit simply extends down
into the pocket of the enzyme. This pocket is believed
to bind the long hydrophobic tail of the natural sub-
strate LTA4,17 and the best binding results were always
achieved with hydrophobic residues in this region that
possessed some flexibility.
CO2H
4d
4e
4f
9300
1100
20
ND
O
O
CO2H
NH2
>10,000
8000
ND
NH2
CO2H
For analogs of the terminal aryl group, the simple ether
linkage was preferred for its potency and ease of synthe-
sis (Table 5). Changing the terminal aryl group to an al-
kyl group such as methyl or cyclohexyl (9a–b) removed
potency altogether. A methyl scan on the terminal phe-
nyl ring (9c–e) showed that ortho and meta substituents
reduced activity, while the para substituent was well tol-
erated. Introduction of heterocyclic substituents in the
para position such as pyrrole or furan (9f–g) is tolerated,
but does not improve the potency relative to the simple
phenoxyphenyl substitution (8e). There was a potency
difference in the WBA.
O
O
NH2
CO2H
4g
1600
IC50 values (nM) are means of at least two independent determina-
tions. ND = Not determined.
inhibitors, the carboxylic acid of 4f appears to be toler-
ated, but it does not noticeably improve binding. The
significant decrease in affinity upon the addition of one
or two methyl groups to the amine (7b–c) can be ex-
plained by the loss of a hydrogen bond, but modeling
With the selection of the phenoxyphenyl group on the
left-hand side, modification of the glutamic acid portion

T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
4969
Figure 4. The rhLTA4/4f crystal structure (PDB entry 3CHQ).
Table 3. Initial acid modification
of 8e was investigated. The initial goal was to extend the
inhibitor in order to make a strong interaction with
Arg563. This interaction was intended to mimic the nat-
ural interaction of LTA4 with this amino acid, and the
interaction is shown clearly with the structure of
hydroxamic acid 1 with LTA4-h in Figure 1. Modifying
compound 8e by extending the acid one carbon gave 10,
the most potent compound in the peptidase assay (Table
6). However, the carboxylic acid is still interacting with
the zinc rather than the arginine according to the crystal
structure (not shown PDB entry XXX7).
O
O
R1
N
H
R2
Number R1
R2
Peptidase IC50 WBA IC50
4f
5
CO2H
CO2H
CO2Me
NH2
H
20
>18,000
39
8000
ND
48
6a
NH2
O
Seeking a simple way to further separate the carboxylic
acid from the hydrophobic tail, a series of amido acids
was synthesized (11a–c). Although these compounds
were all potent in the primary assay, they were uni-
formly weak in the whole blood assay. In addition, no
change in potency was observed on variation of the
chain length, a clear sign that no specific interaction
was being made with the terminal acid. Furthermore,
none of these compounds were more potent than our
benchmark compound, 8e, in either assay. Even more
compelling, replacing the carboxylic acid with a hydro-
xyl group (11d) or an amine (11e) provided compounds
that were equipotent. Once again, these compounds
could all be pro-drugs, in which the amide bond is
6b
6c
6d
NH2
NH2
NH2
NH2
60
23
25
61
27
O
O
85
NH2
O
130
OH
N
H
7a
7b
7c
H
H
H
380
>10,000
ND
NHMe 11,000
NMe2 9400
IC50 values (nM) are means of at least two independent determina-
tions. ND = Not determined.
Figure 5. 7a (cyan atoms) superimposed on the crystal structure of 4f (green atoms) bound to rhLTA4-h (PDB entries 3CHR and 3CHQ,
respectively).

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T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
Table 4. Central ring and aryl tether modifications
O
R2
NH2
R1
N
H
Number
R1
R2
Peptidase IC50
WBA IC50
8000
4f
CO2H
20
O
O
8a
8b
>18,000
9700
ND
ND
NH2
NH2
O
N
O
8c
8d
8e
8f
8g
8h
8i
CO2Me
730
2600
19
1300
O
O
O
N
ND
NH2
CO2H
3300
O
21
110
NH2
H
N
CO2H
CO2Me
CO2H
CO2H
CO2H
CO2H
210
8000
46
180
Me
N
ND
O
>10,000
>10,000
>10,000
>10,000
O
8j
31
O
O
O
8k
8l
18
OH
17
IC50 values (nM) are means of at least two independent determinations. ND = Not determined.

T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
4971
Figure 6. The rhLTA4-h/8l complex (PDB entry 3CHS). The view emphasizes the hydrophobic pocket that binds the aromatic rings of the inhibitors.
The figure includes several residues that were omitted from previous figures in order to simplify the view. These include Ala137, Trp311, and Tyr378.
Table 5. Terminal aromatic ring modifications
Table 6. Attempts to extend the acid
O
NH2
R
R1
O
H
N
R2
NH2
Peptidase WBA
N
O
H
O
Number R1
R2
ZK Number
R
Peptidase
IC50
WBA
IC50
IC50
IC50
8e
10
CO2H
CO2H
19
6
3300
7000
8e
CO2H
19
3300
ND
ND
9a
9b
Me
CO2Me
>18,000
>18,000
H
N
O
CO2H
11a
11b
11c
11d
11e
22
17
19
20
22
>1000
3400
>1000
72
NH2
O
O
H
N
O
CO2H
9c
2800
ND
NH2
Me
H
N
CO2H
O
O
9d
9e
9f
150
13
570
110
4700
92
O
O
O
NH2
NH2
H
Me
N
OH
Me
H
N
NH2
3100
CO2H
22
N
IC50 values (nM) are means of at least two independent determina-
tions. ND = Not determined.
O
9g
29
NH2
O
felt that removing the zwitterionic character of these
compounds would improve their cell permeability, and
thus bring the whole blood potency closer to the pepti-
dase potency. This theory was supported by the high
whole blood activity of esters and amides such as com-
pound 6b. Converting the carboxylic acid to a primary
alcohol (12a) or an amine (12b) provided some of the
most potent compounds in the whole blood assay (Table
7). However, a compound with a methoxy group in this
position (12c) had low activity in the peptidase assay,
perhaps due to the lack of a hydrogen donating group.
The sulfonic acid analog of compound 10 (12d) was a
IC50 values (nM) are means of at least two independent determina-
tions. ND = Not determined.
cleaved by LTA4-h resulting in the formation of 8e.
Compound 11d even had excellent whole blood potency,
a property not shared by any of the extended acids.
Therefore, attempts to make a positive interaction with
Arg563 with a terminal carboxylic acid were abandoned.
Instead, attention was turned to replacing the carboxylic
acid in order to improve the whole blood potency. It was

4972
T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
Table 7. Attempts to replace the acid
hydrolase was measured by inhibition of the hydrolysis
of L-alanine-p-nitroanilide to L-alanine and highly col-
ored nitroaniline. In brief, LTA4-h (29 nM) was incu-
bated with L-alanine-p-nitroanilide (1 mM = Km) in
50 mM HEPES (pH 7.5), 100 mM KCl, 1% DMSO in
the absence or presence of test compound for 1 h at
ambient temperature. Eight concentrations (at 1:3.16
dilution) of test compound were run for IC50 determina-
tion. Reaction was terminated by addition of acetic acid
(final concentration 1%). Formation of colored nitroan-
iline was measured by the increase in absorbance at
405 nm in a Victor 2 plate reader (Wallac). Spontaneous
hydrolysis of the substrate was corrected for by sub-
tracting the absorbance of control incubations without
enzyme. A standard compound was included in each
assay plate. The IC50 values were determined from
dose–response curves by non-linear fit of data to the
4-parameter fit equation. At least two independent
determinations were made for each IC50 value.
NH2
R
H
N
O
O
Number
12a
R
Peptidase IC50 WBA IC50
14
110
OH
12b
21
440
NH2
12c
5600
130
ND
OMe
12d
>10,000
SO3H
H
N
12e
12f
12g
740
350
390
2000
ND
ND
N
N
S
S
NH2
5.1.2. Whole blood assay. The whole blood assay was
performed as described previously.21
N
N
N
5.1.3. Crystallography. The protein was purified and
crystallized according to the previously established pro-
tocols.16 Protein crystallization was performed in melt-
ing point capillaries using liquid–liquid diffusion.
Shortly for this 5 lL protein 5 mg/mL in 10 mm Tris
buffer, pH 7.5 was layered on top of 5 lL precipitate
solution containing 28% (w/v) polyethylene glycol
(MW 8000), 100 mM imidazole pH 7.2 and 5 mM
YbCl3. Capillaries were closed and equilibrated at room
temperature. Plate-like crystals appear within one to
three weeks. The space group of these crystals is
O
N
12h
85
7000
O
IC50 values (nM) are means of at least two independent determina-
tions. ND = Not determined.
˚
˚
P21212 with cell dimensions a = 67 A, b = 133 A, and
˚
c = 84 A. The inhibitors were introduced to the crystals
relatively potent compound, but had no activity in
whole blood. A number of heterocyclic replacements
for the acid were also synthesized (12e–h). However,
all of these compounds had lower activity than the sim-
ple hydroxyl or amine containing compounds.
by using soaking experiments. The synthesized com-
pounds were dissolved in 100% DMSO to make stock
solutions with a concentration of 10 mM or 20 mM.
The crystals were soaked overnight in 1 mM inhibitor,
10 or 5% DMSO, 50 mM imidazole, pH 7.2 and
2.5 mM YbCl3. The soaking solution was refreshed
three times.
4. Conclusion
A series of glutamic acid based compounds that are po-
tent inhibitors of LTA4-h has been identified. Starting
from the weakly active HTS hit 2; compound 4f was
identified with excellent activity in the primary peptidase
assay. The poor activity of this compound in the second-
ary whole blood assay was believed to be caused by poor
cell permeability. Several derivatives of this compound
were quite active in suppressing LTB4 production in
whole blood, particularly the amino alcohol 12a and
the tert-butyl ester 6b.
Data for the several inhibitor-LTA4h complexes were
collected at stations I711 or I911-2 at the MaxLab syn-
chrotron, Lund Sweden. Prior to data collection crystals
were briefly soaked in 1 mM inhibitor, 10 or 5% DMSO,
50 mM imidazole pH 7.2, 2.5 mM YbCl3 and 25% glyc-
erol and flash cooled in a cooled nitrogen gas-stream at
100 K. Data were collected on a 165 mm Mar-research
CCD detector at 100 K. Data processing was done by
using Mosflm26 and subsequent scaling and merging of
the data were performed by using programs from the
CCP4 software package.27 Coordinates were refined
using Refmac28 using the LTA4-h bestatin complex
(PDB entry 1HS6) as a starting model with water mole-
cules and inhibitor removed but not the coordinates for
the co-crystallized Yb ions or a bound imidazole mole-
cule. In each case about 2000 reflections were used for
monitoring (Rfree) during the refinement cycles. Dictio-
naries for the different inhibitors were created using
the ‘monomer library sketcher’ in the CCP4 suite. Water
5. Experimental
5.1. Biological methods
5.1.1. Peptidase assay. This assay was modified from the
previously described protocol20 as described: Com-
pound potency against the peptidase activity of LTA4

T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
Table 8. Crystallographic data and refinement statistics
4973
Compound
2
4b
4f
7a
8i
˚
A (A)
67.37
67.77
67.23
67.35
67.62
133.27
83.64
2.55
˚
B (A)
133.18
84.18
1.8
133.57
83.63
2.1
133.26
84.59
2.09
133.21
83.10
2.2
˚
C (A)
˚
Resolution (A)
Completeness %
Rmerge (%)
I/rI
Multiplicity
Rfactor (%)
Rfree (%)
99.1 (99.6)
5.8 (25.2)
10.4 (2.9)
3.9 (3.7)
18.81
99.8 (99.8)
5.1 (27.5)
11.0 (2.4)
4 (3.8)
23.2
99.7 (99.8)
8.2 (30.6)
13.1 (4.4)
3.6 (3.7)
20.6
99.6 (99.6)
7.1 (33.2)
6.9 (2.0)
4.1 (3.9)
20.68
99.9 (100)
6.9 (30.9)
21.12
24.75
0.008
1.05
68
22.32
0.014
27.8
0.007
24.0
0.005
26.69
0.015
˚
Rsmd bonds (A)
Rsmd angles (ꢁ)
No. of water molecules
1.68
541
0.80
430
0.80
344
1.41
360
molecules were added by using Arp/wArp.29 Manual
model building as well as interpretation of electron den-
sity maps was performed using the program XtalView
(McRee, 1999).30 Details on the processing and refine-
ment of the data can be found in Table 8. The occupan-
cies of the various inhibitors were judged by a
comparison between the B-factors of the inhibitors
and those of the surrounding protein atoms.
J2 = 3.7 Hz, 2 H), 7.40 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz,
2H), 7.35 (t, J = 6.9 Hz, 2H), 7.29 (tt, J1 = 6.9 Hz,
J2 = 2.9 Hz, 1 H), 6.92 (dt, J1 = 9.2 Hz, J2 = 3.3 Hz,
2 H), 5.03 (s, 2H), 3.06 (d, J = 5.8 Hz, 1H), 1.75 (br s,
1H), 1.63–1.58 (m, 1H), 1.49–1.41 (m, 1H), 1.12–1.06
(m, 1H), 0.84 (d, J = 6.9 Hz, 3H), 0.80 (t, J = 7.3 Hz,
3H); Anal. (C19H24N2O2) C, H, N.
5.2.4. 2S-2-Amino-N-[4-(phenylmethoxy)phenyl]benzene-
propanamide hydrochloride (3c). The synthesis of 3c was
carried out in an analogous manner to 2 using Boc-Phe
and deprotection with 4 M hydrogen chloride in
dioxane: 1H NMR (400 MHz, DMSO-d6) d 10.41 (s,
1H), 8.01 (br s, 2 H), 7.41 (dt, J1 = 8.8 Hz, J2 = 3.7 Hz,
4H), 7.36 (tt, J1 = 7.0 Hz, J2 = 1.8 Hz, 2H), 7.32–7.19
(m, 6H), 6.95 (dt, J1 = 9.1 Hz, J2 = 2.2 Hz, 2H), 5.03 (s,
2H), 4.09 (t, J = 7.3 Hz, 1H), 3.12 (dd, J1 = 13.6 Hz,
J2 = 6.2 Hz, 1H), 3.04 (dd, J1 = 13.5 Hz, J2 = 7.3 Hz,
1H); Anal. (C22H22N2O2Æ0.4 H2OÆ1HCl) C, H, N, Cl.
5.2. Chemistry
5.2.1.2-Amino-N-[4-(phenylmethoxy)phenyl]-acetamide(2).
To a solution of Boc-Gly (6.7 g, 29 mmol) in CH2Cl2 was
added carbonyl diimidazole (4.6 g, 1 equiv). After stirring
for 1 h, 4-benzyloxyaniline hydrochloride (5 g, 1 equiv)
was added and the reaction was heated to reflux. After
16 h, the reaction was brought to room temperature,
washed with K2CO3 (satd aq), dried and concentrated to
give a brown solid. This crude product was dissolved in
CH2Cl2 and TFA (10 equiv) was added. After stirring for
16 h, solvent was removed and the residue was partitioned
between NaOH (1 M aq) and CH2Cl2. The layers were sep-
arated, the aqueous layers were extracted with 2· CH2Cl2,
and the combined organic layers were dried and concen-
trated. The resulting brown solid was recrystallized from
EtOAc/hexanes to provide 2 (2.1 g, 29%) as a white solid:
1H NMR (400 MHz, CDCl3) d 9.23 (s, 1 H), 7.49 (dt,
J1 = 9.2 Hz, J2 = 2.4 Hz, 2H), 7.42–7.30 (m, 5H), 6.93 (dt,
J1 = 8.8 Hz, J2 = 2.4 Hz, 2H), 5.03 (s, 2H), 3.44 (s, 2H);
Anal. (C15H16N2O2) C, H, N.
5.2.5. 2S-N-[4-(Phenylmethoxy)phenyl]-2-pyrrolidinecarb-
oxamide hydrochloride (3d). The synthesis of 3d was
carried out in an analogous manner to 2 using Boc-Pro
and deprotection with 4 M hydrogen chloride in
dioxane: 1H NMR (400 MHz, DMSO-d6) d 10.61 (s,
1H), 7.50 (dt, J1 = 9.1 Hz, J2 = 3.3 Hz, 2H), 7.41 (dd,
J1 = 8.4 Hz, J2 = 1.8 Hz, 2H), 7.36 (t, J = 7.0 Hz, 2H),
7.29 (tt, J1 = 7.0 Hz, J2 = 2.9 Hz, 1H), 6.98 (dt,
J1 = 9.1 Hz, J2 = 3.3 Hz, 2H), 5.05 (s, 2H), 4.29 (t,
J = 7.0 Hz, 1H), 3.26–3.17 (m, 1H), 2.39–2.32 (m, 1H),
1.94–1.86 (m, 4H); Anal. (C18H20N2O2Æ1HCl) C, H, N, Cl.
5.2.2. 2S-2-Amino-N-[4-(phenylmethoxy)phenyl]-propan-
amide hydrochloride (3a). The synthesis of 3a was carried
out in an analogous manner to 2 using Boc-Ala and
deprotection with 4 M hydrogen chloride in dioxane: 1H
NMR (400 MHz, DMSO-d6) d 10.51 (s, 1H), 8.25 (br s,
2H), 7.51 (dt, J1 = 9.1 Hz, J2 = 3.3 Hz, 2H), 7.41 (dd,
J1 = 8.5 Hz, J2 = 1.9 Hz, 2H), 7.36 (t, J = 7.0 Hz, 2H),
7.29 (tt, J1 = 7.0 Hz, J2 = 1.4 Hz, 1H), 6.97 (dt,
J1 = 8.8 Hz, J2 = 3.7 Hz, 2H), 5.04 (s, 2H), 3.98 (q,
J = 7.3 Hz, 1H), 1.42 (d, J = 6.9 Hz, 3H); Anal.
(C16H18N2O2Æ1HCl) C, H, N, Cl.
5.2.6.N1-[4-(Phenylmethoxy)phenyl]-L-glutamamidehydro-
chloride (3e). The synthesis of 3e was carried out in an anal-
ogous manner to 2 using Boc-Gln and deprotection with
1
4 M hydrogen chloride in dioxane: H NMR (400 MHz,
DMSO-d6) d 10.63 (s, 1H), 8.36 (br s, 2H), 7.52 (dt,
J1 = 9.2 Hz, J2 = 3.3 Hz, 2 H), 7.46 (br s, 1H), 7.42 (d,
J = 6.6 Hz, 2H), 7.36 (t, J = 7.3 Hz, 2H), 7.30 (tt,
J1 = 7.4 Hz, J2 = 2.6 Hz, 1H), 6.98 (dt, J1 = 9.1 Hz,
J2 = 3.3 Hz, 2H), 6.91, (br s, 1H), 5.06 (s, 2H), 3.96 (br s,
1H), 2.21 (t, J = 7.7 Hz, 2H), 2.06–1.96 (m, 2H); Anal.
(C18H21N3O3Æ0.9H2OÆ1.5HCl) C, H, N, Cl.
5.2.3. 2S,3S-2-Amino-3-methyl-N-[4-(phenylmethoxy)-
phenyl]pentanamide (3b). The synthesis of 3b was carried
out in an analogous manner to 2 using Boc-Ile: 1H
NMR (400 MHz, DMSO-d6) d 7.50 (dt, J1 = 9.1 Hz,
5.2.7. 2S-2-Amino-N-[4-(phenylmethoxy)phenyl]-3-[4-(1H-
imidazole)]propanamide dihydrochloride (3f). The synthe-
sis of 3f was carried out in an analogous manner to 2

4974
T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
using Boc-His and deprotection with 4 M hydrogen
chloride in dioxane: H NMR (400 MHz, DMSO-d6) d
5.2.10. N1-[4-(Phenylmethoxy)phenyl]-L-aspartamine tri-
fluoroacetate (4b). Synthesis of 4b was carried out in an
analogous manner to 4a using Boc-Asp(OMe)-OH: H
NMR (400 MHz, DMSO-d6) d 10.09 (s, 1H), 8.21 (br
s, 2H), 7.45 (d, J = 9.2 Hz, 2H), 7.42–7.34 (m, 4H),
7.30 (t, J = 6.8 Hz, 1H), 6.95 (d, J = 9.2 Hz, 2H), 5.04
(s, 2H), 4.24 (br s, 1H), 2.96–2.82 (m, 2H); Anal.
(C17H18N2O4Æ1.05CF3CO2H) C, H, N, F.
1
1
10.81 (s, 1H), 8.99 (s, 1H), 8.55 (br s, 2H), 7.49 (d,
J = 8.8 Hz, 2H), 7.41 (dt, J1 = 6.8 Hz, J2 = 1.6 Hz,
2H), 7.37 (tt, J1 = 7.2 Hz, J2 = 1.6 Hz, 2H), 7.30 (tt,
J1 = 7.2 Hz, J2 = 2.8 Hz, 1H), 6.98 (dt, J1 = 9.2 Hz,
J2 = 2.4 Hz, 2H), 5.05 (s, 2H), 4.37 (br s, 1H), 3.32–
3.20 (m, 2H); Anal. (C19H20N4O2Æ0.4H2OÆ2HCl) C, H,
N, Cl.
5.2.11. N1-[4-(Phenylmethoxy)phenyl]-D-glutamine tri-
fluoroacetate (4c). Synthesis of 4c was carried out in an
analogous manner to 4a using D-Boc-Glu(OBn)-OH: 1H
NMR (400 MHz, DMSO-d6) d 10.31 (s, 1H), 8.20 (br s,
2H), 7.47 (d, J = 8.8 Hz, 2H), 7.43 (t, J = 7.2 Hz, 2H),
7.37 (t, J = 8.0 Hz, 2H), 7.31 (t, J = 6.8 Hz, 1H), 6.99 (d,
J = 8.8 Hz, 2H), 5.06 (s, 2H), 3.89 (br s, 1H), 2.34 (t,
5.2.8. 2S-2,6-Diamino-N-[4-(phenylmethoxy)phenyl]hex-
anamide dihydrochloride (3g). To a solution of Diboc-
Lys (1.7 g, 5 mmol) and N-methylmorpholine (1.1 mL,
2 equiv) in THF (10 mL) at À20 ꢁC was added isobutyl
chloroformate (0.65 mL, 1 equiv). After stirring at this
temperature for 30 min, 4-benzyloxyaniline (1 g, 1
equiv) was added and the turbid reaction was allowed
to warm to room temperature over 16 h. The reaction
was filtered and concentrated. The residue was dissolved
in EtOAc, washed with NaHSO4 (satd aq), NaHCO3
(satd aq) and brine, dried and concentrated. The crude
coupled product was dissolved in CH2Cl2 and HCl
(4 M in dioxane, 10 equiv) was added. After stirring
for 24 h, solvent was removed. The resulting pink solid
was washed with EtOAc/MeOH (4:1) to provide pure
3g (10%) as a white solid: 1H NMR (400 MHz,
DMSO-d6) d 10.76 (s, 1H), 8.36 (br s, 2H), 7.92 (br s,
2H), 7.55 (d, d, J = 8.8 Hz, 2H), 7.41 (d, J = 6.8 Hz,
2H), 7.37 (tt, J1 = 6.8 Hz, J2 = 1.2 Hz, 2H), 7.30 (tt,
J1 = 7.2 Hz, J2 = 1.6 Hz, 1H), 6.98 (d, J = 9.2 Hz, 2H),
5.05 (s, 2H), 3.97 (br s, 1H), 2.74 (br s, 2H), 1.87–1.75
(m, 2H), 1.60–1.53 (m, 2H), 1.43–1.35 (m, 2H); Anal.
(C19H25N3O2Æ0.6 EtOAcÆ2HCl) C, H, N, Cl.
J = 6.4 Hz,
2H),
2.05–2.00
(m,
2H);
Anal.
(C18H20N2O4Æ0.5H2OÆ1.2CF3CO2H) C, H, N, F.
5.2.12. N1-[4-(Phenylmethoxy)phenyl]-D-aspartamine (4d).
Synthesis of 4d was carried out in an analogous manner to
4a using Boc-Asp(OBn)-OH: 1H NMR (400 MHz,
DMSO-d6) d 10.04 (s, 1H), 8.19 (br s, 2H), 7.48–7.36 (m,
4H), 7.28 (t, J = 7.6 Hz, 2H), 7.23 (t, J = 8.4 Hz, 1H),
6.93 (d, J = 7.6 Hz, 2H), 5.02 (s, 2H), 4.24–4.20 (m, 1H),
2.93–2.88 (m, 2H); Anal. (C17H18N2O4Æ0.2CF3CO2H) C,
H, N, F.
5.2.13. N4-[4-(Phenylmethoxy)phenyl]-L-aspartamine tri-
fluoroacetate (4e). Synthesis of 4e was carried out in an
analogous manner to 4a using Boc-Asp(OH)-OBn: H
1
NMR (400 MHz, DMSO-d6) d 10.09 (s, 1H), 8.22 (br
s, 2H), 7.45 (d, J = 8.8 Hz, 2H), 7.40 (t, J = 6.8 Hz,
2H), 7.36 (t, J = 7.2 Hz, 2H), 7.30 (t, J = 6.8 Hz, 1H),
6.95 (d, J = 8.8 Hz, 2H), 5.04 (s, 2H), 4.24 (br s, 1H),
2.96–2.85 (m, 2H); Anal. (C17H18N2O4Æ1.15CF3CO2H)
C, H, N, F.
5.2.9. N1-[4-(Phenylmethoxy)phenyl]-L-glutamine trifluo-
roacetate (4a). To a solution of Boc-Glu(OBn)-OH
(1.7 g, 5 mmol) and N-methylmorpholine (1.1 mL, 2
equiv) in THF (10 mL) at À20 ꢁC was added isobutyl
chloroformate (0.65 mL, 1 equiv). After stirring at this
temperature for 30 min, 4-benzyloxyaniline (1 g, 1
equiv) was added and the turbid reaction was allowed
to warm to room temperature over 16 h. The reaction
was filtered and concentrated. The residue was dissolved
in EtOAc, washed with NaHSO4 (satd aq), NaHCO3
(satd aq) and brine, dried and concentrated. The cou-
pled product was dissolved in MeOH and NaOH
(1 M, aq) was added. After stirring for 24 h, the solvent
was removed and the residue was partitioned between
CH2Cl2 and NaHSO4 (satd aq) and the layers were sep-
arated. The aqueous layers were extracted with 2·
CH2Cl2 and the combined organic layers were dried
(MgSO4) and concentrated to give the free carboxylic
acid. This acid was dissolved in CH2Cl2 and TFA (10
equiv) was added. After stirring for 4 days, solvent
was removed. The resulting residue was purified by re-
verse phase HPLC using a gradient of MeCN in water
5.2.14. N5-[4-(Phenylmethoxy)phenyl]-L-glutamine hemi-
trifluoroacetate (4f)
5.2.14.1. Step 1: Preparation of N2-[(1,1-dimethyleth-
oxy)carbonyl]-N5-[4-(phenylmethoxy)phenyl]-L-glutamine
(14). To a solution of Boc-Glu(OH)-OBn (3.5 g,
10 mmol) and N-methylmorpholine (2.2 mL, 2 equiv)
in THF (20 mL) at À20 ꢁC was added isobutyl chloro-
formate (1.2 mL, 1 equiv). After stirring at this temper-
ature for 30 min, 4-benzyloxyaniline (2 g, 1 equiv) was
added and the turbid reaction was allowed to warm to
room temperature for over 16 h. The reaction was fil-
tered and concentrated. The residue was dissolved in
EtOAc, washed with NaHSO4 (satd aq), NaHCO3 (satd
aq) and brine, dried, and concentrated. The coupled
product was dissolved in MeOH and NaOH (1 M, aq)
was added. After stirring for 24 h, the solvent was re-
moved and the residue was partitioned between CH2Cl2
and NaHSO4 (satd aq) and the layers were separated.
The aqueous layers were extracted with 2· CH2Cl2
and the combined organic layers were dried (MgSO4)
and concentrated. The residue was recrystallized from
EtOAc/hexanes to provide compound 14 (56%) as a pale
1
to provide pure 4a (13%) as a white solid: H NMR
(400 MHz, DMSO-d6) d 6.66 (d, J = 7,6 Hz, 2H), 6.62–
6.58 (m, 2H), 6.54 (t, J = 7.6 Hz, 2H), 6.50–6.46 (m,
1H), 6.16 (d, J = 8.0 Hz, 2H), 4.26 (s, 2H), 3.19 (t,
J = 6.4 Hz, 1H), 1.71 (t, J = 7.2 Hz, 2H), 1.40(hp, J =
7.2 Hz, 2H); Anal. (C18H20N2O4Æ0.22H2OÆ1.1CF3
CO2H) C, H, N, F.
1
pink solid: H NMR (400 MHz, DMSO-d6) d 9.73 (s,
1H), 7.45 (d, J = 9.1 Hz, 2H), 7.41 (d, J = 6.6 Hz, 2H),
7.36 (t, J = 7.0 Hz, 2H), 7.29 (tt, J1 = 6.9 Hz,

T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
4975
J2 = 2.5 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.91 (dt,
J1 = 9.1 Hz, J2 = 2.2 Hz, 2H), 5.03 (s, 2H), 3.92–3.86
(m, 1H), 2.33 (t, J = 7.6 Hz, 2H), 2.03–1.95 (m, 1H),
1.82–1.74 (m, 1H), 1.35 (s, 9H).
J = 7.3 Hz, 2H), 2.03–1.96 (m, 1H), 1.83–1.75 (m, 1H),
1.35 (s, 9H).
5.2.17.2. Step 2: Synthesis of N5-[4-(phenylmetho-
xy)phenyl]-L-glutamine methyl ester hydrochloride (6a).
Compound 17 was slurried in CH2Cl2 and HCl (4 M in
dioxane, 10 equiv) was added to give a clear solution.
After stirring for 16 h, solvent was removed. The resulting
solid was washed with EtOAc/MeOH (4:1) to provide
5.2.14.2. Step 2: Preparation of N5-[4-(phenylmetho-
xy)phenyl]-L-glutamine hemitrifluoroacetate (4f). Com-
pound 14 was slurried in CH2Cl2 and TFA (10 equiv)
was added to give a clear solution. After stirring for
16 h, solvent was removed. The resulting oil was tritu-
rated with EtOAc/hexanes and washed with MeOH to
provide pure 4f (69%) as a white solid: 1H NMR
1
pure 6a (59%) as a white solid: H NMR (400 MHz,
DMSO-d6) d 9.95 (s, 1H), 8.51 (s, 2H), 7.47 (dt,
J1 = 9.2 Hz, J2 = 2 Hz, 2H), 7.42–7.34 (m, 4H), 7.29 (tt,
J1 = 7.6 Hz, J2 = 1.2 Hz, 1H), 6.92 (dt, J1 = 6.8 Hz,
J2 = 2 Hz, 2H), 5.03 (s, 2H), 4.05 (t, J = 6.4 Hz, 1H),
3.71 (s, 3H), 2.55–2.39 (m, 2H), 2.12–2.01 (m, 2H); Anal.
(C19H22N2O4Æ1HCl) C, H, N, Cl.
(400 MHz, DMSO-d6)
d 9.82 (s, 1H), 8.22 (d,
J = 4.4 Hz, 2H), 7.44 (dt, J1 = 9.2 Hz, J2 = 2.0 Hz, 2H),
7.37 (d, J = 6.8 Hz, 2H), 7.31 (t, J = 6.8 Hz, 2H), 7.24
(t, J = 7.2 Hz, 1H), 6.88 (d, J = 8.8 Hz, 2H), 5.00 (s,
2H), 3.96–3.90 (m, 1H), 2.54–2.39 (m, 2H), 2.06 (hp,
J = 7.6 Hz, 2H); Anal. (C18H20N2O4Æ1H2OÆ0.5CF3CO2H)
C, H, N, F.
5.2.18. N5-[4-(Phenylmethoxy)phenyl]-L-glutamine 1,1-
dimethylethyl ester (6b). Synthesis of 6b was carried
out in an analogous manner to 3g using Boc-
Glu(OH)-OtBu: 1H NMR (400 MHz, CDCl3) d 8.49
(s, 1H), 7.44–7.30 (m, 6H), 7.25 (d, J = 4.4 Hz, 1H),
6.91–6.87 (m, 2H), 5.01 (d, J = 3.2 Hz, 2H), 3.52–3.49
(m, 1H), 2.58- 2.48 (m, 2H), 1.93–1.89 (m, 2H), 1.44
(d, J = 3.6 Hz, 9H); Anal. (C22H28N2O4) C, H, N.
5.2.15. N5-[4-(Phenylmethoxy)phenyl]-D-glutamine hydro-
chloride (4g). Synthesis of 4g was carried out in an analo-
gous manner to 4a using D-Boc-Glu(OH)-OBn and
deprotection with 4 M hydrogen chloride in dioxane: 1H
NMR (400 MHz, DMSO-d6) d 9.95 (s, 1H), 8.33 (s,
2H), 7.46 (dt, J1 = 9.2 Hz, J2 = 2.0 Hz, 2H), 7.42–7.4
(m, 2H), 7.38–7.34 (m, 2H), 7.29 (tt, J1 = 7.6 Hz,
J2 = 1.2 Hz, 1H), 6.92 (dt, J1 = 8.8 Hz, J2 = 3.6 Hz, 2H),
5.03 (s, 2H), 3.91 (t, J = 6.4 Hz, 1H ), 2.56–2.39 (m, 2H),
2.11–1.96 (m, 2H); Anal. (C18H20N2O4Æ0.5H2OÆ0.9HCl)
C, H, N, Cl.
5.2.19. N5-[4-(Phenylmethoxy)phenyl]-L-glutamamide tri-
fluoroacetate (6c). To a solution of 14 (0.3 g) in acetoni-
trile (10 mL) were added Boc anhydride (0.18 g, 1.2
equiv), pyridine (0.05 mL, 1 equiv), and (NH4)2CO3
(0.2 g, 3 equiv). After stirring for 3 h, the reaction was
partitioned between EtOAc and water and the layers
were separated. The aqueous layer was extracted with
2· EtOAc and the combined organic layers were washed
with HCl (1N, aq), water, and brine, then dried
(MgSO4), and concentrated. This product was dissolved
in CH2Cl2 and TFA (10 equiv) was added. After stirring
for 2 h, solvent was removed. The resulting white solid
was recrystallized from MeOH/Et2O to provide 6c
(0.2g, 62%) as a white solid: 1H NMR (400 MHz,
DMSO-d6) d 9.88 (s, 1H), 8.06 (s, 2H), 7.85 (s, 1H),
7.62 (s, 1H), 7.45 (dt, J1 = 8.8 Hz, J2 = 2.0 Hz, 2H),
7.40 (dt, J1= 6.4 Hz, J2 = 2.0 Hz, 2H), 7.38–7.34
(m, 2H), 7.29 (tt, J1 = 7.6 Hz, J2 = 1.2 Hz, 1H), 6.92
(dt, J1 = 8.8 Hz, J2 = 2.0 Hz, 2H), 5.03 (s, 2H), 3.75
(t, J = 6.4 Hz, 1H), 2.24–2.30 (m, 2H), 2.03–1.97
(m, 2H); Anal. (C18H21N3O3Æ0.5H2OÆ1.1CF3CO2H) C,
H, N, F.
5.2.16. 5-Oxo-5-[[4-(phenylmethoxy)phenyl]amino]penta-
noic acid (5). Synthesis of 5 was carried out in an analo-
1
gous manner to 14 using monomethyl glutaric acid: H
NMR (400 MHz, DMSO-d6) d 9.72 (s, 1H), 7.45 (d,
J = 9.2 Hz, 2H), 7.41 (d, J = 6.8 Hz, 2H), 7.36 (tt,
J1 = 6.8 Hz, J2 = 1.2 Hz, 2H), 7.30 (tt, J1 = 7.2 Hz,
J2 = 1.6 Hz, 1H), 6.91 (d, J = 9.2 Hz, 2H), 2.28 (t,
J = 7.2 Hz, 2H), 2.24 (t, J = 7.6 Hz, 2H), 1.76 (tt, J1 =
8.0 Hz, J2 = 7.2 Hz, 2H); Anal. (C18H19NO4Æ0.1H2O)
C, H, N.
5.2.17. N5-[4-(Phenylmethoxy)phenyl]-L-glutamine methyl
ester hydrochloride (6a)
5.2.17.1. Step 1: Synthesis of N2-[(1,1-dimethyleth-
oxy)carbonyl]-N5-[4-(phenylmethoxy)phenyl]-L-gluta-
mine methyl ester (17). To a solution of Boc-Glu(OH)-
OMe (1.7 g, 5 mmol) and N-methylmorpholine (1.1 mL,
2 equiv) in THF (20 mL) at À20 ꢁC was added isobutyl
chloroformate (0.65 mL, 1 equiv). After stirring at this
temperature for 30 min, 4-benzyloxyaniline (1 g, 1 equiv)
was added and the turbid reaction was allowed to warm
to room temperature for over 16 h. The reaction was fil-
tered and concentrated. The residue was dissolved in
EtOAc, washed with NaHSO4 (satd aq), NaHCO3 (satd
aq) and brine, dried and concentrated. The resulting solid
was recrystallized from EtOAc/hexanes to provide com-
5.2.20. N1-Hydroxy-N5-[4-(phenylmethoxy)phenyl]-L-glu-
tamamide trifluoroacetate (6d). To a solution of 17 (1 g)
in MeOH (25 mL) was added hydroxylamine (50% in
water, 4 mL, 20 equiv) over four days of stirring. Sol-
vent was removed, and the resulting white solid was
washed with EtOAc, water, and NaHSO4 (satd aq)
and dried to provide the desired product as a mixture
with 4-benzyloxyaniline sulfate. This mixture was slur-
ried in CH2Cl2 and TFA (10 equiv) was added. After
stirring for 4 h, the solid was filtered off and the mother
liquor was concentrated. The resulting white solid was
purified by reverse phase HPLC using a gradient of
MeCN in water to provide 6d (1%) as a white solid:
1H NMR (400 MHz, CD3OD) d 7.44–7.40 (m, 4H),
1
pound 17 (54%) as a white solid: H NMR (400 MHz,
DMSO-d6) 9.73 (s, 1H), 7.45 (d, J = 9.2 Hz, 2H), 7.41
(d, J = 7.0 Hz, 2H), 7.36 (t, J = 7.3 Hz, 2H), 7.31–7.26
(m, 1H), 6.91 (dt, J1 = 9.2 Hz, J2 = 3.3 Hz, 2H), 5.03 (s,
2H), 4.00–3.95 (m, 1H), 3.60 (s, 3H), 2.33 (t,

4976
T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
7.34 (t, J = 7.2 Hz, 2H), 7.30–7.27 (m, 1H), 6.93 (d,
J = 7.6 Hz, 2H), 5.04 (s, 2H), 3.84 (t, J = 5.2 Hz, 1H),
2.63–2.49 (m, 2H), 2.25–2.13 (m, 2H); Anal.
(C18H21N3O4Æ1.1H2OÆ1CF3CO2H) C, H, N, F.
(br s, 1H), 2.46–2.38 (m, 2H), 2.07–2.01 (m, 2H); Anal.
(C15H18N4O2Æ1.45HCl) C, N, H.
5.2.26. N5-[3-(phenylmethoxy)phenyl]-L-glutamine, methyl
ester (8c). Synthesis of 8c was carried out in an analogous
manner to 3g using Boc-Glu(OH)-OMe and 3-benzyloxy-
aniline: 1H NMR (400 MHz, CDCl3) d 8.37 (s, 1H), 7.43–
7.39 (m, 4H), 7.37 (t, J = 8.0 Hz, 2H), 7.31 (tt, J1 = 7.2 Hz,
J2 = 2.8 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 6.99 (d,
J = 8.0 Hz, 1H), 6.70 (dd, J1 = 8.0 Hz, J2 = 2.0 Hz, 1H),
5.29 (s, 2H), 3.71 (s, 3H), 3.57 (dd, J1 = 8.8 Hz,
J2 = 4.8 Hz, 1H), 2.61–2.45 (m, 2H), 2.27–2.19 (m, 1H),
1.96–1.87 (m, 1H); Anal. (C19H22N2O4Æ0.8H2O) C, H, N.
5.2.21. 4-Amino-N-[4-(phenylmethoxy)phenyl]butanamide
hydrochloride (7a). Synthesis of 7a was carried out in an
analogous manner to 3g using N-Boc-4-aminobutanoic
1
acid: H NMR (400 MHz, DMSO-d6) d 10.00 (s, 1H),
7.98 (s, 2H), 7.49 (dt, J1 = 9.2 Hz, J2 = 2 Hz, 2H), 7.42–
7.33 (m, 4H), 7.29 (tt, J1 = 7.2 Hz, J2 = 1.6 Hz, 1H),
6.91 (dt, J1 = 9.2 Hz, J2 = 2.4 Hz, 2H), 5.02 (s, 2H), 2.79
(t, J = 7.6 Hz, 2H), 2.38 (t, J = 7.6 Hz, 2H), 1.87–1.79
(m, 2H); Anal. (C17H20N2O2Æ1HCl) C, H, N, Cl.
5.2.27. N5-[(4-Phenoxy)-3-pyridyl]-L-glutamamide tristri-
fluoroacetate (8d)
5.2.22. 4-Methylamino-N-[4-(phenylmethoxy)phenyl]butana-
mide trifluoroacetate (7b). Synthesis of 7b was carried out in
an analogous manner to 3g using N-Boc-N-methyl-4-amin-
obutanoic acid with purification by preparative HPLC: 1H
NMR (400 MHz, DMSO-d6) d 9.84 (s, 1H), 8.38 (s, 2H),
7.46 (d, J = 7.6 Hz, 2H), 7.40 (d, J = 7.2 Hz, 2H), 7.36 (tt,
J1 = 6.0 Hz, J2 = 1.6 Hz, 2H), 7.30 (tt, J1 = 5.6 Hz,
J2 = 2.4 Hz, 1H), 6.92 (dt, J1 = 6.4 Hz, J2 = 2.8 Hz, 2H),
5.03 (s, 2H), 2.90 (t, J = 6.4 Hz, 2H), 2.54 (d, J = 3.2 Hz,
3H), 2.36 (t, J = 5.6 Hz, 2H), 1.87–1.79 (m, 2H); Anal.
(C18H22N2O2Æ1CF3CO2H) C, H, N, F.
5.2.27.1. Step 1: Synthesis of 2-Phenyloxy-5-nitropyr-
idine (18). To a solution of phenol (2 g, 21.2 mmol) in
DMSO (20 mL) was added KOtBu (2.4 g, 21.2 mmol) at
rt. After 30 min, 2-chloro-5-nitropyridine (2.1 g,
19.3 mmol) was added. The reaction was heated to
120 ꢁC, then after 2 h of stirring the heat was removed.
The reaction mixture was poured into ice water and the
resulting solid was collected by filtration. This solid was
purified by column chromatography using a gradient of
MeOH in CH2Cl2 to afford 18 (96%) as a yellow solid:
1H NMR (400 MHz, DMSO-d6) d 9.03 (1H, s), 8.45 (d,
J = 3.2 Hz, 1H), 7.54 (m, 2H), 7.36 (m, 1H), 7.18 (m,
2H), 7.06 (d, J = 8.8 Hz, 1H).
5.2.23. 4-Dimethylamino-N-[4-(phenylmethoxy)phenyl]-
butanamide trifluoroacetate (7c). Synthesis of 7c was
carried out in an analogous manner to 17 using N,N-di-
methyl-4-aminobutanoic acid hydrochloride and purifi-
cation by preparative HPLC: 1H NMR (400 MHz,
DMSO-d6) d 9.87 (s, 1H), 9.50 (br s, 1H), 7.46 (d,
J = 5.2 Hz, 2H), 7.40 (d, J = 6.4 Hz, 2H), 7.36 (tt,
J1 = 6.0 Hz, J2 = 1.6 Hz, 2H), 7.30 (t, 5.6 Hz, 1H), 6.93
(d, J = 5.6 Hz, 2H), 5.03 (s, 2H), 3.08–3.02 (m, 2H), 2.76
(s, 6H), 2.38–2.32 (m, 2H), 1.93–1.85 (m, 2H); Anal.
(C19H24N2O2Æ0.2H2OÆ0.1CH2Cl2Æ1.4CF3CO2H) C, H,
N, F.
5.2.27.2. Step 2: Synthesis of N5-[(4-phenoxy)-3-pyri-
dyl]-L-glutamamide (8d). To a solution of SnCl2 H2O
(16.7 g, 74 mmol) in EtOAc (250 mL) was added 19 (4 g,
8.1 mmol) in EtOAc (10 mL). The reaction mixture was
kept at reflux for 3 h, and cooled. The reaction mixture
was quenched with 50% NaOH to pH 12, and resulting so-
lid was filtered off. The solution phase was extracted with
ether, dried, and concentrated to provide the aminopyri-
dine. To a solution of Boc-Gln (505 mg, 3 mmol) and
HOBt (460 mg, 3 mmol) in DMF (10 mL) was added
EDC (593 mg, 3 mmol) at rt. After 30 min, the aminopyr-
idine (465.2 mg, 2.5 mmol) was added. The reaction mix-
ture was stirred at rt overnight, and quenched with water.
The reaction mixture was extracted with EtOAc, washed
with brine, dried, and concentrated. The residue was slur-
ried in CH2Cl2 and TFA (10 equiv) was added to give a
clear solution. After stirring for 16 h, solvent was re-
moved. The resulting solid was purified by reverse phase
HPLC using a gradient of MeCN in water to provide pure
8d (33%) as a white solid: 1H NMR (400 MHz, DMSO-d6)
d 10.23 (s, 1H), 8.32 (d, J = 2.7 Hz, 1H), 8.17 (br s, 3H),
8.03 (dd, J1 = 9.2 Hz, J2 = 2.9 Hz, 1H), 7.91 (s, 1H),
7.62 (s, 1H), 7.37 (t, J = 7.4 Hz, 2H), 7.15 (t, J = 7.4 Hz,
1H), 7.05 (d, J = 7.7 Hz, 2H), 6.98 (d, J = 9.1 Hz, 1H),
3.79 (q, J = 5.5 Hz, 1H), 2.50–2.36 (m, 2H), 2.07–1.98
(m, 2H); Anal. (C16H18N4O3Æ2.65CF3CO2H) C, H, N.
5.2.24. N5-phenyl-L-glutamamide dihydrochloride (8a).
To a solution of Boc-Gln-OH (246 mg, 1 mmol) and
HATU (456 mg, 1.2 mmol) in DMF (10 mL) was added
Et3N (152 mg, 1.5 mmol) at rt. After 30 min, aniline
(93 mg, 1 equiv) was added. The reaction was stirred
at rt overnight, and quenched with water. The reaction
mixture was extracted with EtOAc, washed with brine,
dried and concentrated. To a solution of this residue
in CH2Cl2 was added HCl (4 M in dioxane, 10 equiv).
After stirring for 16 h, the resulting white solid was fil-
tered off, washing with water, to provide 8a (87%) as a
white solid: 1H NMR (400 MHz, DMSO-d6) d 10.18
(s, 1H), 8.29 (br s, 3H), 7.99 (s, 1H), 7.60–7.56 (m,
3H), 7.26 (t, J = 7.3 Hz, 2H), 7.00 (t, J = 7.7 Hz, 1H),
3.77 (br s, 1H), 2.51–2.40 (m, 2H), 2.10–1.99 (m, 2H);
Anal. (C11H15N3O2Æ0.07 H2OÆ2.1HCl) C, H, N.
5.2.25. N5-[4-(1H-pyrrol-1-yl)phenyl]-L-glutamamide hydro-
chloride (8b). Synthesis of 8b was carried out in an analogous
manner to 8a using 4-(1H-pyrrol-1-yl)-aniline: H NMR
(400 MHz, DMSO-d6) d 10.17 (s, 1H), 8.17 (br s, 3H),
7.92 (br s, 1H), 7.67–7.63 (m, 3H), 7.48 (d, J = 8.8 Hz,
2H), 7.27 (t, J = 2.1 Hz, 2H), 6.21 (t, J = 2.2 Hz, 1H), 3.77
5.2.28. N5-(4-Phenoxyphenyl)-L-glutamine (8e)
5.2.28.1. Step 1: N2-[(1,1-dimethylethoxy)carbonyl]-
N5-(4-phenoxyphenyl)-L-glutamine (19). Synthesis of 19
was carried out in an analogous manner to 14 using
Boc-Glu(OH)-OMe and 4-phenoxyaniline: 1H NMR
1
(400 MHz, DMSO-d6)
d 9.90 (s, 1H), 7.56 (d,

T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
4977
J = 8.8 Hz, 2H), 7.32 (tt, J1 = 7.3 Hz, J2 = 2.0 Hz, 2H),
7.05 (tt, J1 = 7.3 Hz, J2 = 1.1 Hz, 1H), 6.92 (t,
J = 8.9 Hz, 4H), 3.91–3.86 (m, 1H), 2.35 (t, 7.5 Hz,
2H), 2.06–1.96 (m, 1H), 1.83–1.72 (m, 1H), 1.34 (s, 9H).
resulting solid was purified by reverse phase HPLC
using a gradient of MeCN in water to provide 8h
1
(40%) as a white solid: H NMR (400 MHz, DMSO-
d6) d 9.94 (s, 1H), 8.38 (br s, 3H), 7.50 (d, J = 8.8 Hz,
2H), 7.19 (t, J = 8.8 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H),
6.84 (d, J = 7.7 Hz, 2H), 6.81 (t, J = 7.3 Hz, 1H), 4.10
(br s, 1H), 3.74 (s, 3H), 3.19 (s, 3H), 2.53–2.40 (m, 2H),
2.10–2.03 (m, 2H); Anal. (C19H23 N3O3Æ1.35CF3CO2H)
C, N, H.
5.2.28.2. Step 2: N5-(4-Phenoxyphenyl)-L-glutamine
trifluoroacetate (8e). Compound 19 was dissolved in
CH2Cl2 and TFA (10 equiv) was added. After stirring
for 16 h the reaction was concentrated. The residue was
purified by reverse phase HPLC using a gradient of
MeCN in water to provide 8e (29%) as a white solid: 1H
NMR (400 MHz, DMSO) d 10.04 (s, 1H), 8.26 (s, 2H),
7.57 (dt, J1 = 8.8 Hz, J2 = 2.4 Hz, 2H), 7.36–7.31 (m,
2H), 7.09–7.05 (m, 1H), 6.98–6.90 (m, 2H), 3.95 (t,
J = 6 Hz, 1H), 2.57–2.42 (m, 2H), 2.13–1.99 (m, 2H);
Anal. (C17H18N2O4Æ1.05CF3CO2H) C, H, N, F.
5.2.32. N5-[4-(2-Phenylethoxy)phenyl]-L-glutamine (8i)
5.2.32.1. Step 1: Synthesis of N2-[(1,1-dimethylethoxy)
carbonyl]-N5-(4-hydroxyphenyl)-L-glutamine, 1,1-dimeth-
ylethyl ester (20). To a solution of Boc-Glu(OH)-OtBu
(1.7 g, 5 mmol) and N-methylmorpholine (1.1 mL, 2
equiv) in THF (20 mL) at À20 ꢁC was added isobutyl
chloroformate (0.65 mL, 1 equiv). After stirring at this
temperature for 30 min, 4-benzyloxyaniline (1 g, 1
equiv) was added and the turbid reaction was allowed
to warm to room temperature over 16 h. The reaction
was filtered and concentrated. The residue was dissolved
in EtOAc, washed with NaHSO4 (satd aq), NaHCO3
(satd aq), and brine, dried and concentrated. The cou-
pled product was dissolved in THF, 10% Pd/C
(5 mol%) was added, and the reaction mixture stirred
under 40 psi H2 overnight. The reaction mixture was
then filtered over Celite and concentrated. The resulting
white solid was purified by silica gel chromatography
using a gradient of EtOAc in hexanes to provide 20
5.2.29. N5-[4-Benzylphenyl]-L-glutamamide trifluoroace-
tate (8f). To a solution of Boc-Gln-OH (784 mg, 3 mmol)
and HOBt (552 mg, 1.2 equiv) in DMF (10 mL) was
added EDC (712 mg, 1.2 equiv) at rt. After 30 min, 4-ben-
zyl aniline (550 mg, 1 equiv) was added. The reaction was
stirred at rt overnight, and quenched with water. The reac-
tion mixture was extracted with EtOAc, washed with
0.1N HCl, NaHCO3 (satd aq), and brine. The organic
layer was dried and concentrated. To a solution of this res-
idue in CH2Cl2 was added TFA (10 equiv). After stirring
for 16 h, reaction was concentrated. The residue was sus-
pended in cold ether and filtered to provide 8f (76%) as a
1
1
white solid: H NMR (400 MHz, DMSO-d6) d 9.97 (s,
(27%) as a white solid: H NMR (400 MHz, DMSO-
1H), 8.13 (br s, 3H), 7.87 (s, 1H), 7.61 (s, 1H), 7.47 (d,
J = 8.4 Hz, 2H), 7.25 (t, J = 7.3 Hz, 2H), 7.18–7.11 (m,
5H), 3.85 (s, 2H), 3.76 (t, J = 6.2 Hz, 1H), 2.44–2.34 (m,
2H), 2.01 (q, J = 7.0 Hz, 2H); Anal. (C18H21N3O2Æ
0.5H2OÆ1.23CF3CO2H) C, H, N.
d6) d 9.58 (s, 1 H), 9.10 (s, 1H), 7.34 (d, J = 9.0 Hz,
2H), 7.15 (d, J = 6.4 Hz, 1H), 6.65 (d, J = 7.0 Hz, 2H),
3.85–3.76 (m, 1H), 2.34 (t, J = 8.1 Hz, 2H), 1.98–1.90
(m, 1H), 1.81–1.74 (m, 1H), 1.41 (s, 9H), 1.39
(s, 9H).
5.2.30. N5-[4-(N-Phenylamino)phenyl]-L-glutamine bistri-
fluoroacetate (8g). Synthesis of 8g was carried out in an
analogous manner to 8f using N-phenyl-p-phenylenedi-
5.2.32.2. Step 2: Synthesis of N5-[4-(2-phenyleth-
oxy)phenyl]-L-glutamine (8i). To a solution of 20
(600 mg, 1.52 mmol) in DMF (10 mL) was added
K2CO3 (525 mg, 4 equiv). After stirring for 30 min phen-
ethyl bromide (310 mg, 1.67 mmol) was added. The reac-
tion mixture was kept at rt overnight, and quenched with
water. The reaction mixture was extracted with EtOAc,
washed with brine, and dried and concentrated. The resi-
due was dissolved in CH2Cl2 and HCl (4 M in dioxane, 10
equiv) was added. After stirring for 16 h, solvent was
removed. The resulting solid was purified by reverse phase
HPLC using a gradient of MeCN in water to provide pure
8i (21%) as a white solid: 1H NMR (400 MHz, DMSO-d6)
d 9.89 (s, 1H), 7.44 (d, J = 8.8 Hz, 2H), 7.38–7.30 (m,
5H), 6.85 (d, J = 8.5 Hz, 2H), 4.12 (t, J = 6.3 Hz, 2H),
3.79 (br s, 1H), 2.97 (t, J = 6.5 Hz, 2H), 2.38–2.33 (m,
2H),2.06–1.95(m, 2H);Anal. (C19H22N2O4Æ0.4CF3CO2H)
C, H, N.
1
amine: H NMR (400 MHz, DMSO-d6) d 9.84 (s, 1H),
8.27 (br s, 3H), 7.47–7.39 (m, 2H), 7.18–7.12 (m, 2H),
7.01–6.93 (m, 5H), 6.76–6.69 (m, 1H), 3.95 (br s, 1H),
2.49–2.36 (m, 2H), 2.06–2.02 (m, 2H); Anal.
(C17H19N3O3Æ1.9CF3CO2H) C, H, N.
5.2.31. N5-[4-(N-Methyl-N-phenylamino)phenyl]-L-gluta-
mine methyl ester trifluoroacetate (8h). To a solution of
Boc-Glu(OH)-OMe (260 mg, 1 mmol) and HOBt
(200 mg, 1 equiv) in DMF (10 mL) was added EDC
(210 mg, 1.1 equiv). After stirring for 30 min, N-phenyl-
p-phenylenediamine (184 mg, 1 equiv) was added. The
reaction was stirred for 16 h and then quenched with
water. The reaction mixture was extracted with EtOAc,
washed with 0.1 N HCl, NaHCO3 (satd aq), and brine.
The organic layer was dried and concentrated. To a
solution of the coupled product in CH2Cl2/MeOH/
AcOH (10 mL, 5:1:0.2) were added formaldehyde (40%
in water, 10 equiv) and NaBH3CN (4 equiv). The cloudy
reaction was stirred for 1 h, then diluted with EtOAc,
and washed with brine. The organic layer was dried
and concentrated. To a solution of this residue in
CH2Cl2 was added HCl (4 M in dioxane, 10 equiv).
After stirring for 16 h, reaction was concentrated. The
5.2.33. N5-[4-(3-Phenylpropoxy)phenyl]-L-glutamine (8j).
Synthesis of 8j was carried out in an analogous manner to
8i using 1-phenyl-3-bromopropane: 1H NMR (400 MHz,
DMSO-d6) d 9.91 (s, 1H), 7.46 (d, J = 8.8 Hz, 2H), 7.28–
7.13 (m, 5H), 6.82 (d, J = 8.7 Hz, 2H), 3.86 (t,
J = 7.0 Hz, 2H), 3.76 (br s, 1H), 2.71 (t, J = 6.2 Hz, 2H),
2.30–2.21 (m, 2H), 2.04–1.95 (m, 4H); Anal.
(C20H24N2O4Æ0.18CF3CO2H) C, H, N.

4978
T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
5.2.34. N5-[4-(2-Oxo-3-phenylpropoxy)phenyl]-L-gluta-
mine trifluoroacetate (8k). Synthesis of 8k was carried
out in an analogous manner to 8i using phenacetyl bro-
mixture was then filtered over Celite and concentrated to
provide the desired 4-cyclohexyloxyaniline. To a solution
of Cbz-Gln (505 mg, 1.2 equiv) and HOBt (280 mg, 1.2
equiv) in DMF (10 mL) was added EDC (360 mg, 1.2
equiv) at rt. After 30 min, the cyclohexyloxyaniline
(287 mg, 1.5 mmol) was added. The reaction mixture was
stirred at rt for 16 h, and quenched with water. The reac-
tion mixture was extracted with EtOAc, washed with
0.1 N HCl, NaHCO3 (satd aq) and brine. The organiclayer
was dried and concentrated. This product was dissolved in
MeOH (10 mL), 10% Pd/C (5 mol%) was added and the
reaction stirred under 40 psi H2 for 2 h. The reaction mix-
ture was then filtered over Celite and concentrated to pro-
vide pure 9b (65%) as a white solid: 1H NMR (400 MHz,
DMSO-d6) d 9.72 (s, 1H), 7.43 (d, J = 9.2 Hz, 2H), 7.38
(s, 1H), 7.00 (s, 1H), 6.82 (d, J = 9.2 Hz, 2H), 4.23–4.17
(m, 1H), 3.01 (t, J = 7.7 Hz, 1H), 2.65–2.56 (m, 1H),
2.39–2.23 (m, 2H), 1.88–1.61 (m, 3H), 1.50–1.30 (m, 3H),
0.94–0.88 (m, 5H); Anal. (C17H25N3O3Æ0.8H2O) C, H, N.
1
mide: H NMR (400 MHz, DMSO-d6) d 9.89 (s, 1H),
8.27 (br s, 3H), 7.93 (d, J = 7.2 Hz, 2H), 7.67 (t,
J = 7.0 Hz, 1H), 7.55 (t, J = 7.2 Hz, 2H), 7.46 (d,
J = 8.8 Hz, 2H), 6.88 (d, J = 9.1 Hz, 2H), 5.51 (s, 2H),
3.94 (br s, 1H), 2.54–2.41 (m, 2H), 2.09–2.00 (m, 2H);
Anal. (C19H20N2O5Æ1.4CF3CO2H) C, H, N.
5.2.35. N5-[4-(2-Hydroxy-3-phenylpropoxy)phenyl]-L-glu-
tamine trifluoroacetate (8l). To a solution of 20 (3 g,
7.6 mmol) in DMF (40 mL) was added K2CO3 (2.6 g,
2.5 equiv). After stirring for 30 min, phenacetyl bromide
(1.7 g, 1.1 equiv) was added. The reaction mixture was
kept at rt overnight, and quenched with water. The reac-
tion mixture was extracted with EtOAc, washed with
brine, and dried and concentrated. The residue was dis-
solved in MeOH, cooled to 0 ꢁC, and NaBH4 was added.
After stirring for 30 min the reaction was quenched with
brine, extracted with EtOAc, and the organic layer was
dried and concentrated. The residue was dissolved in
CH2Cl2 and HCl (4 M in dioxane, 10 equiv) was added.
After stirring for 16 h, solvent was removed. The result-
ing solid was purified by reverse phase HPLC using a gra-
dient of MeCN in water to provide pure 8l (30%) as a
5.2.38. N5-[4-(2-methylphenoxy)phenyl]-L-glutamamide
hydrochloride (9c). Synthesis of 9c was carried out in an
analogous manner to 9b using o-cresol: 1H NMR
(400 MHz, DMSO-d6) d 10.12 (s, 1H), 8.20 (br s, 3H),
7.94 (s, 1H), 7.60 (s, 1H), 7.55 (dt, J1 = 9.2 Hz,
J2 = 2.8 Hz, 2H), 7.27 (d, J = 7.6 Hz, 1H), 7.16 (td,
J1 = 7.6 Hz, J2 = 1.6 Hz, 1H), 7.04 (td, J1 = 7.2 Hz,
J2 = 1.2 Hz, 1H), 6.84 (dt, J1 = 9.2 Hz, J2 = 2.4 Hz, 2H),
6.78 (d, J = 7.2 Hz, 1H), 3.79–3.74 (m, 1H), 2.45–2.34
(m, 2H), 2.16 (s, 3H), 2.06–2.00 (m, 2H); Anal.
(C18H21N3O3Æ0.6H2OÆ1HCl) C, H, N.
1
white solid: H NMR (400 MHz, DMSO-d6) d 9.88 (s,
1H), 8.33 (br s, 3H), 7.48–7.40 (m, 4H), 7.36 (t,
J = 7.4 Hz, 2H), 7.26 (t, J = 7.1 Hz, 1H), 6.85 (d,
J = 8.2 Hz, 2H), 4.86 (t, J = 2.6 Hz, 1H), 3.99–3.92 (m,
3H), 2.55–2.42 (m, 2H), 2.12–2.03 (m, 2H); Anal.
(C19H22N2O5Æ0.6H2OÆ1.3CF3CO2H) C, H, N.
5.2.39. N5-[4-(3-methylphenoxy)phenyl]-L-glutamamide
hydrochloride (9d). Synthesis of 9d was carried out in
an analogous manner to 9b using m-cresol: H NMR
5.2.36. N5-[4-Methoxyphenyl]-L-glutamine methyl ester
trifluoroacetate (9a). To a solution of Boc-Glu(OH)-OMe
(940 mg, 1.2 equiv) and HOBt (552 mg, 1.2 equiv) in
DMF (10 mL) was added EDC (710 mg, 1.2 equiv) at rt.
After 30 min, p-methoxyaniline (370 mg, 3 mmol) was
added. The reaction was stirred at rt for 16 h, and then
quenched with water. The reaction mixture was extracted
with EtOAc, washed with 0.1 N HCl, NaHCO3 (satd aq),
and brine. The organic layer was dried and concentrated.
To a solution of this residue in CH2Cl2 was added TFA
(10 equiv). After stirring for 1 h, the reaction mixture was
concentrated. The residue was purified by reverse phase
HPLC using a gradient of MeCN in water to provide 9a
(92%) as a white solid: 1H NMR (400 MHz, DMSO-d6) d
9.85 (s, 1 H), 8.42 (br s, 3H), 7.46 (d, J = 9.2 Hz, 2H),
6.84 (d, J = 9.1 Hz, 2H), 4.08 (br s, 1H), 3.72 (s, 3H), 3.68
(s, 3H), 2.52–2.40 (m, 2H), 2.10–2.03 (m, 2H); Anal.
(C13H18N2O4Æ1.32CF3CO2H) C, H, N.
1
(400 MHz, DMSO-d6) d 10.31 (s, 1H), 8.37 (br s, 3H),
8.06 (s, 1H), 7.66–7.54 (m, 3H), 7.19 (s, 1H), 6.94–6.72
(m, 5H), 3.79 (br s, 1H), 2.54–2.40 (m, 2H), 2.23 (s,
3H), 2.10–2.00 (m, 2H); Anal. (C18H21N3O3Æ0.6H2OÆ
1HClÆ0.25Et2O) C, H, N.
5.2.40. N5-[4-(4-methylphenoxy)phenyl]-L-glutamamide
hydrochloride (9e). Synthesis of 9e was carried out in
an analogous manner to 9b using p-cresol: H NMR
1
(400 MHz, DMSO-d6) d 10.12 (s, 1H), 8.20 (br s, 3H),
7.93 (s, 1H), 7.61 (s, 1H), 7.57 (d, J = 9.2 Hz, 2H),
7.14 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 9.2 Hz, 2H), 6.83
(d, J = 8.4 Hz, 2H), 3.76 (br s, 1H), 2.46–2.39 (m, 2H),
2.25 (s, 3H), 2.06–2.00 (m, 2H); Anal. (C18H21N3O3Æ0.7-
H2OÆ1HClÆ0.25Et2O) C, H, N.
5.2.41. N5-[4-[4-(1H-pyrrol-1-yl)phenoxy]phenyl]-)-L-glu-
tamine hemitrifluoroacetate (9f). Synthesis of 9f was
carried out in an analogous manner to 9b using 4-(1H-
pyrrol-1-yl)-phenol: 1H NMR (400 MHz, DMSO) d
10.10 (s, 1H), 8.11 (s, 2H), 7.59–7.51 (m, 4H), 7.26 (t,
J = 2 Hz, 2H), 7.00 (t, J = 9.2 Hz, 4H), 6.22 (t, J = 2 Hz,
2H), 3.78 (s, 1H), 2.48–2.46 (m, 2H), 2.04–2.02 (m, 2H);
Anal. (C21H21N3O4Æ0.5H2OÆ0.5CF3CO2H) C, H, N, F.
5.2.37. N5-[4-(Cyclohexyloxy)phenyl]-L-glutamamide (9b).
To a solution of cyclohexylamine (2.1 g, 21.2 mmol) and 4-
fluoronitrobenzene (3 g, 1 equiv) in DMSO (30 mL) was
added K2CO3 (5.8 g, 2 equiv). The reaction mixture was
heated to 110 ꢁC, then after 3 h of stirring the heat was re-
moved. Thereactionmixturewaspouredintoicewaterand
the water was extracted with EtOAc. The organic layer was
dried and concentrated to provide the crude nitro com-
pound. This product was dissolved in MeOH/EtOAc
(60 mL, 2/1), 10% Pd/C (5 mol%) was added, and the reac-
tion mixture stirred under 40 psi H2 for 3 h. The reaction
5.2.42. N5-[4-(4-(3-Furyl)phenoxy)phenyl]-L-glutamamide
trifluoroacetate (9g). A mixture of 4-(4-bromophenoxy)-
aniline (2.2 g, 8.2 mmol), 3-furylboronic acid (915 mg, 1

T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
4979
equiv), Pd(PPh3)2Cl2 (576 mg, 10 mol%), and Na2CO3
(2N aq, 9 mL) in i-PrOH (30 mL) was kept at 80 ꢁC
overnight. The reaction mixture was cooled to rt, diluted
with water, and extracted with EtOAc. The organic
phase was washed with brine, dried, and concentrated
to provide crude aniline which was carried on to the next
step. To a solution of Boc-Gln-OH (296 mg, 1.2 equiv)
and HOBt (184 mg, 1.2 equiv) in DMF (5 mL) was
added EDC (238 mg, 1.2 equiv) at rt. After 30 min,
the aniline (250 mg, 1 mmol) was added. The reaction
mixture was stirred at rt overnight, and quenched with
water. The reaction mixture was extracted with EtOAc,
and the organic layer was washed with brine. The organ-
ic layer was dried and concentrated. To a solution of this
residue in CH2Cl2 was added HCl (4 M in dioxane, 10
equiv). After stirring for 16 h, reaction mixture was con-
centrated. The resulting solid was purified by reverse
phase HPLC using a gradient of MeCN in water to pro-
5.2.44. N1-(2-Carboxyethyl)-N5-(4-Phenoxyphenyl)-L-glu-
tamamide trifluoroacetate (11a). To a solution of 18
(0.83 g, 2.0 mmol) and HOBt hydrate (0.4 g, 1.5 equiv)
in DMF (10 mL) were added EDC (0.4 g, 1.1 equiv)
and triethylamine (1 mL, 1.5 equiv). After stirring for
30 min, H-Gly-OMe (0.18 g, 1 equiv) was added and
the reaction mixture was stirred overnight. Partitioned
the reaction between EtOAc and water, separated the lay-
ers, washed the organic layer with NaHSO4 (satd aq),
NaHCO3 (satd aq) and brine, dried (MgSO4) and concen-
trated. The residue was dissolved in MeOH and NaOH
(1 M, aq) was added. After stirring for 24 h, the solvent
was removed, the residue was partitioned between
CH2Cl2 and NaHSO4 (satd aq), and the layers were sep-
arated. The aqueous layers were extracted with 2·
CH2Cl2 and the combined organic layers were dried
(MgSO4) and concentrated to give the free carboxylic
acid This acid was slurried in CH2Cl2 and HCl (4 M in
dioxane, 10 equiv) was added to give a clear solution.
After stirring for 16 h, solvent was removed. The result-
ing solid was purified by reverse phase HPLC using a gra-
dient of MeCN in water to provide pure 11a (36%) as a
1
vide 9g (36%) as a white solid: H NMR (400 MHz,
DMSO-d6) d 10.07 (s, 1H), 8.14 (br s, 3H), 7.89 (s,
1H), 7.70–7.57 (m, 5H, 6.99–6.77 (m, 6H), 3.77 (br s,
1H), 2.45–2.33 (m, 2H), 2.05–1.98 (m, 2H); Anal.
(C21H21N3O4Æ1.48CF3CO2H) C, H, N.
1
white solid: H NMR (400 MHz, DMSO-d6) d 10.02 (s,
1H), 8.80 (t, J = 5.9 Hz, 1H), 8.18 (br s, 3H), 7.58 (d,
J = 9.1 Hz, 2H), 7.34 (t, J = 8.8 Hz, 2H), 7.08 (t,
J = 7.3 Hz, 1H), 6.96 (d, J = 8.8 Hz, 2H), 6.93 (d,
J = 7.7 Hz, 2H), 3.93–3.86 (m, 3H), 2.50–2.43 (m, 2H),
2.04 (q, J = 6.6 Hz, 2H); Anal. (C19H21N3O5Æ0.5-
H2OÆ1.4CF3CO2H) C, H, N, F.
5.2.43. N6-[4-(4-methylphenoxy)phenyl]-L-homoglutamine
trifluoroacetate (10). To a solution of Boc-homo-
glu(OBz)-OH (1 g, 2.8 mmol) in MeOH/PhCH3 (1/1,
30 mL) was added TMSCHN2 (2 M in hexanes, 10 mL
total) dropwise until bubbling was not observed and
the yellow color persisted for more than 1 minute. The
solution was then concentrated. A slurry of this product
and Pd/C (0.2 g, 20 w/w%) in MeOH (100 mL) was stir-
red under 40 psi H2 for 24 h. The reaction mixture was
filtered over Celite and the mother liquor was concen-
trated to give the desired acid as a yellow oil. To a
solution of this acid (0.8 g, 2.5 mmol) and N-methylmor-
pholine (0.7 mL, 2 equiv) in THF (10 mL) at À20 ꢁC
was added isobutyl chloroformate (0.33 mL, 1 equiv).
After stirring at this temperature for 30 min, 4-phenox-
yaniline (0.5 g, 1 equiv) was added and the turbid reac-
tion mixture was allowed to warm to room temperature
for over 16 h. The reaction mixture was filtered and con-
centrated. The residue was dissolved in EtOAc, washed
with NaHSO4 (satd aq), NaHCO3 (satd aq) and brine,
dried and concentrated. The coupled product was dis-
solved in MeOH and NaOH (1 M, aq) was added. After
stirring for 24 h, the solvent was removed and the resi-
due was partitioned between CH2Cl2 and NaHSO4 (satd
aq), and the layers were separated. The aqueous layers
were extracted with 2· CH2Cl2 and the combined organ-
ic layers were dried (MgSO4) and concentrated to give
the free carboxylic acid. This acid was slurried in
CH2Cl2 and HCl (4 M in dioxane, 10 equiv) was added
to give a clear solution. After stirring for 16 h, solvent
was removed. The resulting solid was purified by reverse
phase HPLC using a gradient of MeCN in water to pro-
vide pure 10 (22%) as a white solid: 1H NMR (400 MHz,
DMSO-d6) d 10.03 (s, 1H), 7.91 (br s, 3H), 7.58 (d,
J = 8.8 Hz, 2H), 7.34 (t, J = 7.3 Hz, 2H), 7.08 (t,
J = 7.3 Hz, 1H), 6.97 (d, J = 8.8 Hz, 2H), 6.93 (d,
J = 8.4 Hz, 2H), 3.64–3.58 (m, 1H), 2.68–2.41 (m, 4H),
1.92–1.83 (m, 2H); Anal. (C18H20N2O4Æ1H2OÆ1.1CF3
CO2H) C, H, N, F.
5.2.45. N1-(3-Carboxypropyl)-N5-(4-phenoxyphenyl)-L-glu-
tamamide trifluoroacetate (11b). Synthesis of 11b was car-
ried out in an analogous manner to 11a using b-alanine
1
ethyl ester: H NMR (400 MHz, DMSO-d6) d 9.96 (s,
1H), 8.54 (s, 1H), 8.20–8.08 (m, 2H), 7.56 (d, J =
7.2 Hz, 2H), 7.34 (t, J = 8.4 Hz, 2H), 7.08 (t, J = 6.8 Hz,
1H), 6.97 (d, J = 8.0 Hz, 2H), 6.92 (d, J = 7.2 Hz, 2H),
3.77 (br s, 1H), 3.55–3.45 (m, 4H), 2.38–2.33 (m, 2H),
2.06–1.95 (m, 2H); Anal. (C20H23N3O5Æ1H2OÆ1.25CF3-
CO2H) C, H, N, F.
5.2.46. N1-(4-Carboxybutyl)-N5-(4-phenoxyphenyl)-L-glu-
tamamide bistrifluoroacetate (11c). Synthesis of 11c was
carried out in an analogous manner to 11a using
ethyl 3-aminopropionate: 1H NMR (400 MHz,
DMSO-d6) d 10.04 (s, 1H), 8.48 (t, J = 5.5 Hz, 1H),
8.17 (br s, 3H), 7.58 (d, J = 9.2 Hz, 2H), 7.34 (t,
J = 7.3 Hz, 2H), 7.07 (t, J = 7.3 Hz, 1H), 6.96 (d,
J = 9.1 Hz, 2H), 6.92 (d, J = 7.7 Hz, 2H), 3.76 (br s,
1H), 3.17–3.11 (m, 2H), 2.37 (q, J = 7.7 Hz, 2H),
2.25 (t, J = 7.4 Hz, 2H), 2.03–1.97 (m, 2H), 1.66 (t,
J = 7.3 Hz, 2H); Anal. (C21H25N3O5Æ0.3H2OÆ1.6CF3CO2H)
C, H, N, F.
5.2.47. N1-(2-Hydroxyethyl)-N5-(4-phenoxyphenyl)-L-glu-
tamamide trifluoroacetate (11d). A solution of 18 (2.7 g,
6.5 mmol) and HOBt hydrate (1.3 g, 1.5 equiv) in DMF
(30 mL) was cooled to 0 ꢁC and EDC (1.4 g, 1.1 equiv)
and triethylamine (1 mL, 1.1 equiv) were added. After
stirring for 30 min, 2-aminoethanol (0.4 g, 1 equiv) was
added and the reaction mixture was allowed to warm
to rt overnight. Partitioned the reaction mixture between
EtOAc and water, separated the layers, washed the

4980
T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
organic layer with NaHSO4 (satd aq), NaHCO3 (satd aq)
and brine, dried (MgSO4) and concentrated. The result-
ing solid was purified by reverse phase HPLC using a gra-
dient of MeCN in water to provide pure 11d (16%) as a
5.2.49.2. Step 2: Synthesis of 4S-4-amino-5-hydroxy-
N-(4-phenoxyphenyl)pentanamide (12a). Compound 21
was slurried in CH2Cl2 and HCl (4 M in dioxane, 10
equiv) was added to give a clear solution. After stirring
for 16 h, solvent was removed. The resulting solid was
partitioned between EtOAc and NaOH (1M aq), the
layers were separated, the aqueous layer was extracted
with EtOAc, and the combined organic layers were dried
(MgSO4) and concentrated. The resulting solid was puri-
fied by silica gel chromatography using a gradient of
MeOH in CH2Cl2 to provide pure 12a (30%) as a white
1
white solid: H NMR (400 MHz, DMSO-d6) d 10.00 (s,
1H), 8.47 (t, J = 5.6 Hz, 1H), 8.18 (d, J = 10.8 Hz, 2H),
7.58–7.56 (m, 2H), 7.36–7.32 (m, 2H), 7.07 (t,
J = 7.6 Hz, 1H), 6.97–6.91 (m, 4H), 3.79 (s, 1H), 3.45–
3.42 (m, 2H), 3.24–3.17 (m, 2H), 2.40–2.33 (m, 2H),
2.00–1.98 (m, 2H); Anal. (C19H23N3O4Æ1.5CF3CO2H) C,
H, N, F.
1
solid: H NMR (400 MHz, DMSO-d6) d 10.02 (s, 1H),
5.2.48. N1-(2-Aminoethyl)-N5-(4-phenoxyphenyl)-L-glu-
tamamide hydrochloride (11e). A solution of 18 (0.52 g,
1.2 mmol) and HOBt hydrate (0.25 g, 1.5 equiv) in
DMF (10 mL) was cooled to 0 ꢁC and EDC (0.26 g,
1.1 equiv) and triethylamine (0.19 mL, 1.1 equiv) were
added. After stirring for 30 min, N-Boc ethylenediamine
(0.2 g, 1 equiv) was added and the reaction was allowed
to warm to rt overnight. Partitioned the reaction mix-
ture between EtOAc and water, separated the layers,
washed the organic layer with NaHSO4 (satd aq), NaH-
CO3 (satd aq) and brine, dried (MgSO4) and concen-
trated. The residue was dissolved in CH2Cl2 and HCl
(4 M in dioxane, 10 equiv) was added to give a clear
solution. After stirring for 48 h, the precipitated solid
was filtered to provide pure 11 e (2%) as a white solid:
1H NMR (400 MHz, DMSO-d6) d 10.01 (s, 1H),
8.60 (t, J = 3.2 Hz, 1H), 8.17 (br s, 3H), 7.75 (br s,
2H), 7.58 (d, J = 7.2 Hz, 2H), 7.28 (t, J = 8.0 Hz, 2H),
7.03 (t, J = 7.2 Hz, 1H), 6.95 (d, J = 8.4 Hz, 2H), 6.89
(d, J = 7.6 Hz, 2H), 3.82–3.77 (m, 1H), 3.41–3.34
(m, 2H), 2.94–2.85 (m, 2H), 2.42–2.39 (m, 2H), 2.08–
2.01 (m, 2H); Anal. (C19H24N4O3Æ0.2EtOAcÆ0.8HCl)
C, H, N.
7.82 (br s, 2H), 7.58 (d, J = 9.2 Hz, 2H), 7.33 (tt,
J1 = 8.7 Hz, J2 = 2.2 Hz, 2H), 7.07 (t, J = 7.4 Hz, 1H),
6.96 (d, J = 9.2 Hz, 2H), 6.92 (d, J = 7.7 Hz, 2H), 3.60
(dd, J1 = 11.3 Hz, J2 = 3.6 Hz, 1H), 3.44 (dd, J1 =
11.4 Hz, J2 = 6.2 Hz, 1H), 3.17–3.11 (m, 1H), 2.42 (q,
J = 7.7 Hz, 2H), 1.84–1.78 (m, 2H); Anal. (C17H20N2O3Æ
0.6CH2Cl2) C, H, N.
5.2.50.
mide (12b)
4S-4,5-Diamino-N-(4-phenoxyphenyl)pentana-
5.2.50.1. Step 1: Synthesis of 4S-4-(1,1-dimethyl-
ethylcarbamoylamino)-5-methansulfonyloxy-N-(4-phenoxy-
phenyl)pentanamide (16). To a solution of 21 (3.6 g,
9 mmol) in pyridine (60 mL) was added MsCl (3.5 mL,
5 equiv) which caused a substantial exotherm and a pre-
cipitate formed. The reaction mixture was stirred for
1 h, then MeOH (20 mL) was added until all of the solid
dissolved. The reaction mixture was poured into iced
HCl (0.5 M aq) and the resulting solid was filtered to pro-
vide pure 16 (99%) as a white solid: 1H NMR (400 MHz,
CDCl3) d 8.42 (br s, 1H), 7.54 (d, J = 8.9 Hz, 2H), 7.33 (tt,
J1 = 8.8 Hz, J2 = 3.1 Hz, 2H), 7.09 (t, J = 7.8 Hz, 1H),
6.99–6.94 (m, 4H), 5.01 (d, J = 8.1 Hz, 1H), 4.27 (dd,
J1 = 11.0 Hz, J2 = 3.2 Hz, 1H), 4.22 (dd, J1 = 11.5 Hz,
J2 = 4.9 Hz, 1H), 4.01–3.96 (m, 1H), 3.04 (s, 3H), 2.45
(t, J = 8.1 Hz, 2H), 1.97–1.91 (m, 2H), 1.43 (s, 9H).
5.2.49. 4S-4-Amino-5-hydroxy-N-(4-phenoxyphenyl)pen-
tanamide (12a)
5.2.49.1. Step 1: Synthesis of S-4-(1,1-dimethylethyl-
carbamoylamino)-5-hydroxy-N-(4-phenoxyphenyl)pentana-
mide (21). To a solution of Boc-Glu(OH)-OBn (8 g,
25 mmol) and N-methylmorpholine (7 mL, 2 equiv) in
THF (100 mL) at À20 ꢁC was added isobutyl chlorofor-
mate (3.3 mL, 1 equiv). After stirring at this temperature
for 30 min, 4-phenoxyaniline (5 g, 1 equiv) was added
and the turbid reaction mixture was allowed to warm
to room temperature for over 16 h. The reaction mixture
was filtered and concentrated. The residue was dissolved
in EtOAc, washed with NaHSO4 (satd aq), NaHCO3
(satd aq) and brine, dried and concentrated. This product
was dissolved in MeOH/THF (3/1) and heated to 50 ꢁC.
NaBH4 (4 equiv) was added and the reaction was al-
lowed to self-reflux for 20 min. The reaction was stirred
for another 4 h, then it was poured into iced HCl
(0.5 M, aq). The resulting solid was filtered, then recrys-
tallized from MeOH/water to provide compound 21
5.2.50.2. Step 2: Synthesis of 4S-4-(1,1-dimethyl-
ethylcarbamoylamino)-5-amino-N-(4-phenoxyphenyl)pen-
tanamide trifluoroacetate (22). To a solution of 16 (0.48 g,
1.0 mmol) in DMF (5 mL) was added NaN3 (0.2 g, 3
equiv). This reaction mixture was stirred at 60 ꢁC for
6 h, and then poured into ice water. The water was ex-
tracted with 2· EtOAc and the combined organic layers
were dried (MgSO4) and concentrated to give the azide
as a white solid. This azide (0.1 g) was dissolved in
MeOH/EtOAc (1/1, 10 mL) and Pd/C was added. The
black suspension was agitated under 50 psi H2 pressure
for 2.5 h. The reaction mixture was filtered over Celite
and concentrated. This residue was purified by reverse
phase HPLC using a gradient of MeCN in H2O to provide
22 (60%) as a white solid: 1H NMR (400 MHz, CDCl3) d
8.61 (br s, 1 H), 7.47 (d, J = 9.1 Hz, 2H), 7.29 (tt,
J1 = 8.7 Hz, J2 = 2.3 Hz, 2H), 7.06 (t, J = 7.6 Hz, 1H),
6.96–6.91 (m, 4H), 5.86 (d, J = 8.1 Hz, 1H), 3.88–3.81
(m, 1H), 3.18–3.02 (m, 2H), 2.44 (t, J = 9.1 Hz, 2H),
2.01–1.94 (m, 2H), 1.37 (s, 9H).
1
(80%) as a white solid: H NMR (400 MHz, DMSO-
d6) d 9.88 (s, 1 H), 7.58 (d, J = 8.8 Hz, 2H), 7.33 (tt,
J1 = 8.8 Hz, J2 = 2.2 Hz, 2H), 7.08 (t, J = 7.3 Hz, 1H),
6.96–6.90 (m, 4H), 6.48 (d, J = 8.5 Hz, 1H), 4.61 (t,
J = 5.9 Hz, 1H), 3.38–3.28 (m, 1H), 3.25–3.19 (m, 1H),
2.31–2.25 (m, 2H), 1.86–1.80 (m, 1H), 1.55–1.50 (m,
1H), 1.35 (s, 9H).
5.2.50.3. Step 3: Synthesis of 4S-4,5-diamino-N-(4-
phenoxyphenyl)pentanamide bistrifluoroacetate (12b).
Compound 22 was dissolved in CH2Cl2 and TFA (10

T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
4981
equiv) was added. After stirring for 4 h the reaction mix-
ture was concentrated. The resulting yellow oil was puri-
fied by reverse phase HPLC using a gradient of MeCN in
concentrated. The residue was dissolved in EtOAc,
washed with NaHSO4 (satd aq), NaHCO3 (satd aq) and
brine, dried and concentrated. A solution of the resulting
amide (6 g, 13 mmol) in DMF (250 mL) was cooled to
0 ꢁC and cyanuric chloride (2.5 g, 1 equiv) was added.
The reaction mixture was allowed to warm to room tem-
perature and stirred for 16 h, and then the reaction was
poured into water. The solid was filtered off to provide
1
water to provide 12b (16%) as a white solid: H NMR
(400 MHz, CD3OD) d 7.55–7.53 (m, 2H), 7.32 (t,
J = 7.6 Hz, 2H), 7.07 (t, J = 7.6 Hz, 1H), 6.94 (d,
J = 8.8 Hz, 4H), 3.687–3.624 (m, 1H), 3.30–3.26 (m,
2H), 2.70–2.66 (m, 2H), 2.18–1.98 (m, 2H); Anal.
(C17H21N3O2Æ2.3CF3CO2H) C, N, F. H: Calcd, 4.18,
found, 3.50.
1
15 (66%) as a pale yellow solid: H NMR (400 MHz,
CDCl3) d 7.86 (br s, 1H), 7.45 (d, J = 9.0 Hz, 2H), 7.26
(tt, J1 = 8.4 Hz, J2 = 2.1 Hz, 2H), 7.05 (t, J = 7.8 Hz,
1H), 6.96–6.91 (m, 4H), 5.6 (br s, 1H), 4.64 (br s, 1H),
2.65–2.53 (m, 2H), 2.30–2.23 (m, 2H), 1.43 (s, 9H).
5.2.51. 4S-4-Amino-5-methoxy-N-(4-phenoxyphenyl)-pen-
tanamide (12c). Compound 21 (0.5 g, 1.3 mmol) was dis-
solved in MeCN/THF (3/1, 20 mL) and Ag2O (1.5 g, 5
equiv) and MeI (1.2 mL, 10 equiv) were added to give a
black suspension. After stirring for 7 days, the reaction
was filtered over Celite and the mother liquor was con-
centrated. This product was dissolved in CH2Cl2 and
TFA (10 equiv) was added. After stirring for 2 h, solvent
was removed. The residue was partitioned between
NaOH (1 M aq) and CH2Cl2. The layers were separated,
the aqueous layers were extracted with 2· CH2Cl2, and
the combined organic layers were dried and concentrated.
The resulting solid was purified by silica gel chromatogra-
phy using a gradient of MeOH in CH2Cl2 to provide pure
12c (16%) as a white solid: 1H NMR (400 MHz, DMSO-
d6) d 9.93 (s, 1H), 7.58 (d, J = 9.1 Hz, 2H), 7.33 (tt,
J1 = 7.3 Hz, J2 = 2.2 Hz, 2H), 7.06 (t, J = 7.3 Hz, 1H),
6.97–6.91 (m, 4H), 3.23 (s, 3H), 3.02 (dd, J1 = 9.2 Hz,
J2 = 5.2 Hz, 1H), 3,13 (dd, J1 = 9.5 Hz, J2 = 6.6 Hz,
1H), 2.81–2.75 (m, 1H), 2.44–2.28 (m, 2H), 1.74–1.65
(m, 1H), 1.47–1.38 (m, 1H); Anal. (C18H22N2O3Æ
0.1CH2Cl2) C, H, N.
5.2.53.2. Step 2: Synthesis of 4S-4-amino-4-(2-(1,3,4-
triazolyl))-N-(4-phenoxyphenyl)butanamide trifluoroace-
tate (12e). A slurry of cyanide (2 g, 4.7 mmol), formic
hydrazide (0.56 g, 2 equiv) and K2CO3 (0.14 g, 0.2
equiv) in EtOH was stirred at reflux for 24 h. The reac-
tion mixture was then poured into water and the result-
ing solid was isolated by filtration. This product was
slurried in CH2Cl2 and TFA (10 equiv) was added to
give a solution. After stirring for 2 h, solvent was re-
moved. The residue was purified by reverse phase HPLC
using a gradient of MeCN in H2O to provide 12e (12%)
as a white solid: 1H NMR (400 MHz, DMSO-d6) d 9.93
(s, 1H), 8.70 (d, J = 2.5 Hz, 1H), 8.50 (br s, 2H), 7.55 (d,
J = 9.1 Hz, 2H), 7.34 (t, J = 7.3 Hz, 2H), 7.07 (t, J =
7.3 Hz, 1H), 6.95 (d, J = 9.2 Hz, 2H), 6.92 (d, J =
7.7 Hz, 2H), 4.47 (br s, 1H), 2.35 (t, J = 6.9 Hz, 2H),
2.27–2.11 (m, 2H); Anal. (C18H19N5O2Æ0.2H2OÆ1CF3CO2H)
C, H, N, F.
5.2.54. 4S-4-Amino-5-(2-thio-5-amino-1,3,4-thiadiazol-2-
yl)-N-(4-phenoxyphenyl)pentanamide bistrifluoroacetate
(12f). To a solution of 5-amino-1,3,4-thiadiazole-2-thiol
(0.06 g, 1.1 equiv) in DMF (5 mL) was added NaH
(0.02 g, 1.1 equiv) giving a strong exotherm. After stirring
for 3 h, compound 16 (0.2 g, 0.4 mmol) was added. This
reaction mixture was stirred for 16 h, and then poured
into ice water. The solid was filtered off and the mother li-
quor was concentrated to provide crude product. This
product was dissolved in CH2Cl2 and HCl (4 M in diox-
ane, 10 equiv) was added. After stirring for 24 h the reac-
tion mixture was concentrated. The resulting yellow oil
was purified by reverse phase HPLC using a gradient of
MeCN in water to provide 12f (11%) as a yellow oil: 1H
NMR (400 MHz, CD3OD) d 7.52 (dd, J1 = 8.8 Hz,
J2 = 2.2 Hz, 2H), 7.32 (tt, J1 = 7.3 Hz, J2 = 2.2 Hz, 2H),
7.08 (t, J = 7.4 Hz, 1H), 6.96–6.92 (m, 4H), 3.68–3.64
(m, 3H), 3.56 (dd, J1 = 14.6 Hz, J2 = 4.8 Hz, 1H), 3.34
(J1 = 15.0 Hz, J2 = 9.8 Hz, 1H), 2.68–2.57 (m, 2H),
2.13–2.08 (m, 2H); Anal. (C19H21N5O2S2Æ2.5CF3CO2H)
C, H, N, S.
5.2.52. 2S-2-Amino-5-oxo-5-[(4-phenoxyphenyl)amino]-1-
pentanesulfonic acid (12d). A solution of 16 (0.4 g,
0.8 mmol) and potassium thioacetate (0.2 g, 2 equiv) in
acetone (10 mL) was stirred for 48 h. The suspension
was filtered over Celite and concentrated to provide the
thioacetate. To a solution of this product in AcOH
(1 mL) was added H2O2 (30% in water, 0.5 mL) in AcOH
(1 mL). After stirring for 24 h, the reaction mixture was
diluted with Et2O and the resulting precipitate was filtered
and washed with MeOH and water to provide 12d (28%)
as a white solid: 1H NMR (400 MHz, DMSO-d6) d 9.97 (s,
1H), 7.84 (s, 2H), 7.56 (dt, J1 = 8.8 Hz, J2 = 2 Hz, 2H),
7.36–7.31 (m, 2H), 7.07 (tt, J1 = 7.2 Hz, J2 = 1.2 Hz,
1H), 6.97–6.91 (m, 4 H), 3.38 (s, 1H), 2.84 (dd,
J1 = 12 Hz, J2 = 2.4 Hz, 1H), 2.65–2.59 (m, 1H), 2.48–
2.29 (m, 2H), 1.99–1.79 (m, 2H); Anal. (C17H20N2O5
SÆ0. 15H2OÆ0.1 CH2Cl2) C, H, N, S.
5.2.53. 4S-4-Amino-4-(2-(1,3,4-triazolyl))-N-(4-phenoxy-
phenyl)butanamide (12e)
5.2.53.1. Step 1: Synthesis of 4S-4-(1,1-dimethyl-
ethylcarbamoylamino)-4-cyano-N-(4-phenoxyphenyl)butana-
mide (15). To a solution of Boc-Gln (8 g, 25 mmol) and N-
methylmorpholine (7 mL, 2 equiv) in THF (100 mL) at
À20 ꢁC was added isobutyl chloroformate (3.3 mL, 1
equiv). After stirring at this temperature for 30 min, 4-
phenoxyaniline (5 g, 1 equiv) was added and the turbid
reaction mixture was allowed to warm to room tempera-
ture for over 16 h. The reaction mixture was filtered and
5.2.55. 4S-4-Amino-5-(1H-pyrrol-1-yl)-N-(4-phenoxyphe-
nyl)pentanamide bistrifluoroacetate (12g). A solution of
compound 22 (0.3 g, 0.75 mmol) and NaOAc (0.06 g, 1
equiv) in AcOH (250 mL) was heated to 80 ꢁC and
2,5-dimethoxytetrahydrofuran (0.13 mL, 1.3 equiv) was
added dropwise to the solution. After stirring for 2 h,
the reaction was diluted with water and washed with
2· CH2Cl2. The water layer was then brought to pH

4982
T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
8. (a) Friedrich, E. B.; Tager, A. M.; Liu, E.; Pettersson, A.;
Owman, C.; Munn, L.; Luster, A. D.; Gerstzen, R. E.
Arterioscler. Thromb. Vasc. Biol. 2003, 23, 1761; (b)
Subbarao, K.; Jala, V. R.; Mathis, S.; Suttles, J.; Zach-
arias, W.; Ahamed, J.; Ali, H.; Tseng, M. T.; Haribabu, B.
Arterioscler. Thromb. Vasc. Biol. 2004, 24, 369; (c)
13 with K2CO3 (satd aq) and extracted with 2· CH2Cl2.
The organic fractions were dried and concentrated to
provide crude pyrrole. This pyrrole was dissolved in
CH2Cl2 and HCl (4 M in dioxane, 10 equiv) was added.
After stirring for 6 h the reaction mixture was concen-
trated. The residue was purified by reverse phase HPLC
using a gradient of MeCN in water to provide 12g (1%)
as a yellow oil: 1H NMR (400 MHz, CD3OD) d 7.53 (d,
J = 8.8 Hz, 2 H), 7.32 (t, J = 7.3 Hz, 2H), 7.08 (t,
J = 7.4 Hz, 1H), 6.94 (d, J = 8.8 Hz, 4H), 6.76 (t,
J = 1.8 Hz, 2H), 6.16 (t, J = 1.8 Hz, 2H), 4.22 (dd,
J1 = 14.6 Hz, J2 = 4.8 Hz, 1H), 4.12 (J1 = 14.7 Hz,
J2 = 7.3 Hz, 1H), 2.66–2.51 (m, 2H), 1.96 (q,
J = 6.6 Hz, 2H); Anal. (C21H23N3O2Æ2.1CF3CO2H) C,
N. H: Calcd, 4.30, found, 3.83.
´
Helgadottir, A.; Manolescu, A.; Thorleifsson, G.; Gret-
arsdottir, S.; Jonsdottir, H.; Thorsteinsdottir, U.; Samani,
´
´
´
N. J.; Gudmundsson, G.; Grant, S. F. A.; Thorgeirsson,
´
G.; Sveinbjornsdottir, S.; Valdimarsson, E. M.; Matthias-
son, S. E.; Johannsson, H.; Gudmundsdottir, O.; Gurney,
´
´
´
M. E.; Sainz, J.; Thorhallsdottir, M.; Andresdottir, M.;
Frigge, M. L.; Topol, E. J.; Kong, A.; Gudnason, V.;
Hakonarson, H.; Gulcher, J. R.; Stefansson, K. Nat.
Genet. 2004, 36, 233; (d) Jala, V. R.; Haribabu, B. Trends
Immun. 2004, 25, 315.
9. Ford-Hutchinson, A. W. Crit. Rev. Immunol. 1990, 10, 1.
10. Yokomizo, T.; Izumi, T.; Chang, K.; Takuwa, Y.;
Shimizu, T. Nature 1997, 387, 620.
11. Yokomizo, T.; Kato, K.; Terawaki, K.; Izumi, T.;
Shimizu, T. J. Exp. Med. 2000, 192, 421.
12. Ford-Hutchinson, A. W.; Gresser, M.; Young, R. N.
Annu. Rev. Biochem. 1994, 63, 383.
13. Aharony, D. Am. J. Respir. Crit. Care Med. 1998, 157,
S214.
14. Serhan, C. N. Prostaglandins 1997, 53, 107.
15. Haeggstro¨m, J. Z. Am. J. Respir. Crit. Care Med. 2000,
161, S25.
16. Thunnissen, M. M.; Nordlund, P.; Haeggstro¨m, J. Z. Nat.
Struct. Biol. 2001, 8, 131.
17. Haeggstroem, J. Z. J. Biol. Chem. 2004, 279, 50639.
18. Orning, L.; Gierse, J. K.; Fitzpatrick, F. A. J. Biol. Chem.
1994, 269, 11269.
19. Thunnissen, M. M.; Andersson, B.; Samuelsson, B.;
Wong, C.-H.; Haeggstro¨m, J. Z. FASEB J. 2002, 16,
1648.
20. (a) Penning, T. D. Curr. Pharm. Des. 2001, 7, 163; (b)
Penning, T. D.; Russell, M. A.; Chen, B. B.; Chen, H. Y.;
Liang, C.-D.; Mahoney, M. W.; Malecha, J. W.; Miyas-
hiro, J. M.; Yu, S. S.; Askonas, L. J.; Gierse, J. K.;
Harding, E. I.; Highkin, M. K.; Kachur, J. F.; Kim, S. H.;
Villani-Price, D.; Pyla, E. Y.; Ghoreishi-Haack, N. S.;
Smith, W. G. J. Med. Chem. 2002, 45, 3482.
21. Sandanayaka, V.; Mamat, B.; Yu, P.; Zhao, L.; Bedell, L.;
Bhagat, N.; Winger, J.; Keyvan, M.; Bock, B.; Krohn, M.;
Chandrasekar, P.; Mo, X.; Zhou, L.; Mishra, R.; Onua,
5.2.56. 4S-4-Amino-5-(1-phthalimido)-N-(4-phenoxyphe-
nyl)pentanamide trifluoroacetate (12h). To a solution of
16 (1.2 g, 2.5 mmol) in DMF (25 mL) was added potas-
sium phthalimidate (0.5 g, 1.1 equiv). This slurry was
stirred at 100 ꢁC for 4 h, and then poured into NaHCO3
(satd aq). The water layer was extracted with 2· EtOAc
and the combined organic layers were dried (MgSO4)
and concentrated to give the phthalimide as a white so-
lid. This product was dissolved in CH2Cl2 and TFA (10
equiv) was added. After stirring for 16 h the reaction
was concentrated. The residue was purified by reverse
phase HPLC using a gradient of MeCN in water to pro-
1
vide 12h (14%) as a white solid: H NMR (400 MHz,
DMSO-d6) d 9.93 (s, 1H), 7.86–7.80 (m, 4H), 7.56 (d,
J = 9.1 Hz, 2H), 7.33 (t, J = 8.8 Hz, 2H), 7.06 (t,
J = 7.3 Hz, 1H), 6.96–6.91 (m, 4H), 3.50 (dd,
J1 = 7.4 Hz, J2 = 2.2 Hz, 2H), 2.98–2.91 (m, 1H), 2.45–
2.33 (m, 2H), 1.77–1.68 (m, 1H), 1.51–1.44 (m, 1H);
Anal. (C25H23N3O4Æ0.51CF3CO2H) C, H, N, F.
References and notes
1. (a) Goodarzi, K.; Goodarzi, M.; Tager, A. M.; Luster, A.
D.; von Andrian, U. H. Nat. Immunol. 2003, 4, 965; (b)
Ott, V. L.; Cambier, J. C.; Kappler, J.; Marrack, P.;
Swanson, B. J. Nat. Immunol. 2003, 4, 974; (c) Tager, A.
M.; Bromley, S. K.; Medoff, B. D.; Islam, S. A.; Bercury,
S. D.; Friedrich, E. B.; Carafone, A. D.; Gerszten, R. E.;
Luster, A. D. Nat. Immunol. 2003, 4, 982.
2. (a) Lewis, R. A.; Austen, K. F.; Soberman, R. J. N. Engl.
J. Med. 1990, 323, 645; (b) Henderson, W. R. Ann. Intern.
Med. 1994, 121, 684.
´
E.; Zhang, J.; Porsteinsdottir, M.; Halldorsdottir, G.;
Sigporsdottir, H.; Friedman, M.; Zembower, D.; Andres-
´
´
son, P.; Singh, J.; Gurney, M. Abstracts of Papers, 233rd
National Meeting of the American Chemical Society,
Chicago, IL, March 25–29, 2007; American Chemical
Society: Washington, DC, 2007; MEDI 236.
22. Ollmann, I. R.; Hogg, J. H.; Mun˜oz, B.; Haeggstro¨m, J.
Z.; Samuelsson, B.; Wong, C.-H. Bioorg. Med. Chem.
1995, 3, 969.
3. Munafo, D. A.; Shindo, K.; Baker, J. R.; Bigby, T. D.
J. Clin. Invest. 1994, 93, 1042.
4. Sharon, P.; Stenson, W. F. Gastroenterology 1984, 86, 453.
5. Barnes, P. J. Respiration 2001, 68, 441.
23. Askonas, L. J.; Kachur, J. F.; Villani-Price, D.; Liang, C.-D.
D.; Russell, M.; Smith, W. G. J. Pharmacol. Exp. Ther.
2002, 300, 577.
6. (a) Griffiths, R. J.; Pettipher, E. R.; Koch, K.; Farrell, C.
A.; Breslow, R.; Conklyn, M. J.; Smith, M. A.; Hackman,
B. C.; Wimberly, D. J.; Milici, A. J.; Scampoli, D. N.;
Cheng, J. B.; Pillar, J. S.; Pazoles, C. J.; Doherty, N. S.;
Melvin, L. S.; Reiter, L. A.; Biggars, M. S.; Falkner, F. C.;
Mitchell, D. Y.; Liston, T. E.; Showell, H. J. Proc. Natl.
Acad. Sci. U.S.A. 1995, 92, 517; (b) Tsuji, F.; Oki, K.;
Fujisawa, K.; Okahara, A.; Horiuchi, M.; Mita, S. Life
Sci. 1998, 64, PL51.
24. Jenks, W. P. Adv. Enzymol. Areas Mol. Biol. 1975, 43, 219.
25. Penning, T. D.; Chandrakumar, N. S.; Chen, B. B.; Chen,
H. Y.; Desai, B. N.; Djuric, S. W.; Docter, S. H.; Gasiecki,
A. F.; Haack, R. A.; Miyashiro, J. M.; Russell, M. A.; Yu,
S. S.; Corley, D. G.; Durley, R. C.; Kilpatrick, B. F.;
Parnas, B. L.; Askonas, L. J.; Gierse, J. K.; Harding, E. I.;
Highkin, M. K.; Kachur, J. F.; Kim, S. H.; Krivi, G. G.;
Villani-Price, D.; Pyla, E. Y.; Smith, W. G.; Ghoreishi-
Haack, N. S. J. Med. Chem. 2000, 43, 721.
7. (a) Ikai, K. J. Dermatol. Sci. 1999, 21, 135; (b) Zhu, Y. I.;
Stiller, M. J. Skin Pharmacol. Appl. Skin Physiol. 2000, 13,
235.
26. Leslie, A. G. W. Joint CCP4 + ESF-EAMCB Newsletter
on Protein Crystallography, No. 26, 1992.

T. A. Kirkland et al. / Bioorg. Med. Chem. 16 (2008) 4963–4983
4983
27. Collaborative Computational Project, Number 4 Acta
Crystallogr., Sect. D 1994, 50, 760–763.
29. Perrakis, A.; Sixma, T. K.; Wilson, K. S.; Lamzin, V. S.
Acta Crystallogr., Sect. D 1997, 53, 448.
28. Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta
Crystallogr., Sect. D 1997, 53, 240.
30. McRee, D. E. Practical Protein Crystallography, 2nd ed.;
Academic Press: New York, 1999.