Synthesis of N-alkyl glycine amides as potent inhibitors of leukotriene A4 hydrolase

Article abstract of DOI:10.1016/j.bmcl.2008.06.046

The synthesis and biological evaluation of a series of N-alkyl glycine amide analogs as LTA₄-h inhibitors and the importance of the introduction of a benzoic acid group to the potency and pharmacokinetic parameters of our analogs are described.

Full text of DOI:10.1016/j.bmcl.2008.06.046

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3891–3894
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of N-alkyl glycine amides as potent inhibitors of leukotriene
A4 hydrolase
Bin Ye a, John Bauman a, Ming Chen a, David Davey a, Seock-Kyu Khim a, Beverly King b,c, Thomas Kirkland a,
Monica Kochanny a, Amy Liang a, Dao Lentz b, Karen May a, Lisa Mendoza c, Gary Phillips a, Victor Selchau a,
Sabine Schlyer a, Jih-Lie Tseng b, Robert G. Wei a, Hong Ye a, John Parkinson c, William J. Guilford a,
*
a Department of Medicinal Chemistry, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA
b Department of Molecular Pharmacology, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA
c Department of Immunology, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biological evaluation of a series of N-alkyl glycine amide analogs as LTA4-h inhibitors
and the importance of the introduction of a benzoic acid group to the potency and pharmacokinetic
parameters of our analogs are described. The lead compound in the series, 4q, has excellent potency
and oral bioavailability.
Received 14 March 2008
Revised 11 June 2008
Accepted 12 June 2008
Available online 18 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Leukotriene A4
Leukotriene A4 hydrolase
Anti-inflammatory
Enzyme inhibitor
Leukotriene B4 (LTB4) is a potent pro-inflammatory activator of
inflammatory cells including neutrophils, eosinophils, monocytes,
macrophages, T cells, and B cells, which is a functional link be-
tween early innate and late adaptive immune responses.1 There
is substantial evidence that LTB4 plays a significant role in the
amplification of many inflammatory disease states2 including asth-
ma,3 inflammatory bowel disease (IBD),4 chronic obstructive pul-
LTA4-h is a monomeric, soluble 69 kDa zinc metallohydrolase of
the M1 class which has sequence homology to aminopeptidases M
and B. It can act as a non-specific peptidase but has no known
physiological substrate. The bifunctional nature of LTA4-h allows
for the determination of inhibition constants using either a pepti-
dase or a hydrolase assay.13–15 Since LTA4-h is an intracellular en-
zyme, inhibitors were additionally characterized in a cellular
human whole blood assay (WBA).13 Data from the hydrolase and
WBA are presented in Tables 1–3. In the end the WBA is presumed
to be more relevant, but initially the hydrolase assay was used for
optimization. LTA4-h is considered to be a druggable target with
reports of high-resolution crystal structures with bound inhibi-
tors.16 The search for inhibitors has attracted attention.14,17 In this
letter, we describe our effort to identify a potent, selective, and or-
ally available inhibitor of LTA4-h.
monary
disease
(COPD),5
arthritis,6
psoriasis,7
and
atherosclerosis.8 LTB4 also simulates the production of various
cytokines and may play a role in immunoregulation.9 Therefore,
a therapeutic agent that inhibits the biosynthesis of LTB4 may be
useful for the treatment of these inflammatory conditions.
Three enzymes are involved in the biosynthesis of LTB4 from
arachidonic acid (AA):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 LTB4.10 In addition to being the precursor to LTB4, LTA4
is a substrate of LTC4 synthase to yield the pro-inflammatory cys-
teinyl leukotrienes LTC4, LTD4, and LTE411 and a substrate of lipoox-
ygenases to give the anti-inflammatory mediators, lipoxins A4
(LXA4) and B4 (LXB4).12 Thus, the targeting of LTA4-h would leave
other immune response pathways intact.
Starting from HTS hit 1a with an IC50 of 0.3 lM, a series of ana-
logs were prepared which contained either a benzyloxy or phenoxy
substituent on the aniline and either a cyclic or acyclic diamine.
One of the most potent hits was 2a with IC50 value of 0.16 lM.
Compound 2a was used as the starting point for further
optimization.
O
O
O
O
N
N
NH2
N
N
H
H
* Corresponding author. Fax: +1 650 868 8777.
E-mail address: wguilford@comcast.net (W.J. Guilford).
1a
2a
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.046

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B. Ye et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3891–3894
Table 1
Table 2
Substitution on amine
Variation of the biphenyl ether moiety
H
H
N
N
N R
N
N
N
O
Me
Me
O
Me
Me
O
CO2H
R
a
a
a
a
Compound
R
Hydrolase IC50 (nM)
WBA IC50 (nM)
Compound
R
Hydrolase IC50 (nM)
6
WBA IC50 (nM)
2a
2b
2c
2d
–Me
–CH2Ph
–(CH2)3CO2H
–(CH2)4CO2H
160
90
110
18
135
350
1500
230
2f
120
230
70
O
F
3a
3b
3c
3d
3e
16
3
2e
2f
25
6
430
120
O
CO2H
CO2H
CO2H
F
F
6
120
90
2g
2h
2i
7
15
6
160
290
340
O
Cl
17
19
NO2
O
210
CO2H
F
O
OH
O
CO2H
Br
3f
N
5
60
O
Values are means of at least two experiments.
a
2j
10
28
3
360
440
80
CO2H
CO2H
O
2k
2l
bons (2d and 2e vs 2c) were more potent in the hydrolase assay
than 2a, but not as measured in the WBA. Substitution of a benzene
ring, 2f, for the alkyl chain further increased the potency of the
analog and resulted in a 25-fold increase in the potency in the
hydrolase assay over 2a
CO2H
Although we were unable to obtain crystal structures of bound
inhibitors in this series by either soaking or co-crystallization
experiments, a model of 2f bound to LTA4-h was proposed (Fig.
1). The key interactions are between the diphenyl ether group
and the hydrophobic pocket, the diamine group with Gln136 and
Glu296, and the benzoic acid group and Arg563 and Lys565. The mod-
eled structure rationalizes the 15-fold increase in potency of 2f
over the corresponding phenyl analog, 2b and the importance of
the carboxylate group.
2m
33
4
>1000
250
SO2NH2
N
N
2n
N
N
H
a
Values are means of at least two experiments.
In an attempt to increase the potency further, a variety of sub-
stituents were added to the benzoic acid (Table 1). No significant
improvement in potency in either the hydrolase or WB assays
was observed upon substitution of the benzoic acid ring for 2g,
2h, 2i, and 2j. Replacement of the benzene ring with a heterocycle,
2k or replacement of the carboxylic acid with a sulfonamide, 2m,
showed a slight decrease in potency relative to 2f. The tetrazole
analog, 2n, was equipotent with 2f. The most potent compound
was the extended linker analog, 2l. None of these modifications
had a significant effect on potency in the WBA, which presumably
reflects the difficulty in uptake of charged compounds into cells.
The steric requirement of the hydrophobic pocket was explored
using the linear diamine and modifying the diphenyl ether (Table
2). Neither substitution on the diphenyl ether, 3a, 3c, and 3d, nor
replacement of the ether linkage with a methylene, 3b, resulted
in a significant change in potency compared with 2f. Increasing
the size of the group by the addition of a heterocycle, 3f, or exten-
sion of the linker, 3e, did not significantly affect the activity in
either assay.
The synthesis of analogs is shown in Scheme 1 using 4q as an
example for analog preparation using a chiral reagent. Phenoxyan-
iline is acylated in quantitative yield with bromoacetyl bromide
and one equivalent of diethylaniline in methylene chloride to give
5. Alkylation of 1,1-dimethylethyl (2S)-2-(aminomethyl)-pyrroli-
dinecarboxylate with 5 using Hunig’s base in acetonitrile, followed
by methyl iodide using Hunig’s base in DMF gave the desired ter-
tiary amine 6 in a 25% yield over the two steps. The N-Boc group
was removed with trifluoroacetic acid and the resulting amine
was alkylated with methyl 4-(bromomethyl)benzoate in 40% yield.
Saponification using sodium hydroxide in a mixture of aqueous
methanol and THF then provided 4q in 60% yield. Analog 4p was
prepared similarly to 4q. The enantiomeric pairs 4n and 4o, 4r
and 4s, and 4t and 4u were prepared by separating the racemic
mixtures by chiral chromatography without assignment of abso-
lute configuration.
To further optimize the interactions between 2a and LTA4-h, we
sought to mimic the proposed binding of LTA4 to LTA4-h16 through
the addition of a carbon chain terminating with a carboxylic acid
(Table 1). Alkyl carboxylates having a chain length over three car-
The optimization of the interaction of the diamine group with
the two aspartic acid residues near the zinc was explored next (Ta-

B. Ye et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3891–3894
3893
Table 3 (continued)
Table 3
Variation of the central diamine group
a
a
Compound
X
Hydrolase IC50 (nM)
WBA IC50 (nM)
CO2H
H
Me
Me
N
X
N
4r
26
60
*
*
O
N
N
O
a
a
Compound
X
Hydrolase IC50 (nM)
WBA IC50 (nM)
N
4s
4t
17
60
Me
N
Me
N
2f
6
120
Me
N
H
N
H
N
4a
4b
4c
370
330
19
>1000
>1000
420
120
57
180
220
*
*
N
N
Me
H
N
Me
N
N
4u
Me
N
H
N
a
Values are means of at least two experiments.
Me
Me
N
Me Me
4e
7
70
N
Me
N
O
O
a
O
4f
15
140
N
Br
NH2
N
Me
H
5
4g
1000
>1000
N
N
O
O
Me
N
N
H2N
c
boc
N
N
N
N
4h
4i
70
44
180
240
N
H
b
boc
6
O
O
Me
N
d,e,f
N
N
N
H
N
4q
CO2H
4j
60
270
260
N
Scheme 1. Synthesis of 4q. Reagents and conditions: (a) Bromoacetyl bromide,
diethyl aniline, CH2Cl2; (b) iPrNEt2, CH3CN; (c) MeI, PrNEt2, DMF; (d) TFA, CH2Cl2;
(e) Methyl 4-(bromomethyl)benzoate, K2CO3, DMF; (f) 1 N NaOH, MeOH/THF.
Me
Me
N
4k
490
N
N
N
4l
160
140
380
220
Me
Me
N
4m
N
4n
4o
17
16
100
60
N
*
Figure 1. Model of 2f bound to LTA4-h.
N
Me
Me
ble 3). The relative importance of the N-methyl groups on 2f to
binding was explored with nor-analogs, 4a, 4b, and 4c. Removal
of the N-methyl from the glycine amino group resulted in a 50+-
fold drop in binding as seen in 4a and 4b, but only a 3-fold drop
with removal of the N-methyl from the benzyl amino group, 4c.
The importance of flexibility and the distance between nitrogen
atoms is seen in the 60-fold increase in activity seen by opening
up the piperazine ring, 4g, to the linear ethylene diamine, 4f or
the 10-fold increase in expanding the piperazine ring, 4g, to a
N
4p
4q
100
7
200
30
N
Me
N
N

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B. Ye et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3891–3894
homo-piperazine ring, 4h. The use of cyclic amines introduces
asymmetry into many of the compounds, but potentially allows
for improved binding. For example, the methylaminopiperidine
group can be inserted in two different orientations, 4j and 4k, with
an 8-fold difference in potency in the hydrolase assay. This differ-
ence in potency with orientation was not seen with the 7-mem-
bered ring analogs 4l and 4m. The additional diversity element of
stereochemistry is added with the pyrrolidine or piperidine ana-
logs. Although the importance of the chiral center was minimal
in most cases, as seen in the pairs 4n and 4o, 4r and 4s, and 4t
and 4u, the 10-fold difference in activity between 4p and 4q is
the exception and suggests a tight-binding orientation for the R-
isomer, 4q.
In parallel to the potency assays, the ADME profile of key com-
pounds was evaluated. An added benefit of the benzoic acid group
was the increased stability in rat microsomes from less than 33%
remaining at 1 h for 2a and 2b to >90% remaining at 1 h for 2f.
The clearance value of 38 mL/min/kg and half-life of 0.9 h after iv
administration for 2f was not improved by substitution on the ben-
zoate ring, 2g to 2j, replacement with heterocycles, 2k, or substitu-
tion on the phenoxy group, 3a through 3f (data not shown).
Insertion of a pyrrolidine group in the chain did improve the
half-life of for 4q, 2.6 h after iv administration, but not the clear-
ance value, 28 mL/min/kg. The % F value for both 2f and 4q was
similar at around 45% compared to the negligible oral bioavailabil-
ity of 1a or 2a.
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In this letter, we have described the optimization of a series
of diamines as inhibitors of LTA4-h. Starting with 1a, we have
extended the compound through the addition of a benzoic acid
group to give 4q with increased potency and oral
bioavailability.