Discovery of novel and potent aryl diamines as leukotriene A4 hydrolase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.06.041

The synthesis and biological evaluation of a series of aryl diamines as inhibitors of LTA₄-h inhibitors are described. The optimization which led to the identification of the optimal para-substitution on the diphenyl ether moiety and diamine spacer is discussed. The resulting compounds such as 3l have excellent enzyme and cellular potency as well as desirable pharmacokinetic properties.

Full text of DOI:10.1016/j.bmcl.2008.06.041

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3895–3898
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel and potent aryl diamines as leukotriene
A4 hydrolase inhibitors
Seock-Kyu Khim, John Bauman, Jarred Evans, Beverly Freeman, Beverly King, Thomas Kirkland,
Monica Kochanny, Dao Lentz, Amy Liang, Lisa Mendoza, Gary Phillips, Jih-Lie Tseng,
*
Robert G. Wei, Hong Ye, Limei Yu, John Parkinson, William J. Guilford
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 aryl diamines as inhibitors of LTA4-h inhibitors are
described. The optimization which led to the identification of the optimal para-substitution on the diphe-
nyl ether moiety and diamine spacer is discussed. The resulting compounds such as 3l have excellent
enzyme and cellular potency as well as desirable pharmacokinetic properties.
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
LTA4
Leukotriene A4 hydrolase
Enzyme inhibitor
Antiinflammatory
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
asthma,3 inflammatory bowel disease (IBD),4 chronic obstructive
pulmonary disease (COPD),5 arthritis,6 psoriasis,7 and atherosclero-
sis.8 LTB4 also stimulates 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.
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 and 2. In the end, the WBA is pre-
sumed to be more relevant, but initially the hydrolase assay was
used for optimization. LTA4-h is considered to be a druggable tar-
get with reports of high-resolution crystal structures with bound
inhibitors.16 The search for inhibitors has attracted attention,14,17
including our report on N-alkyl glycine amides.18 In this letter,
we describe the transition of N-alkyl glycine amide 1 to the aryl
diamine template of potent, selective, and orally available inhibi-
tors of LTA4-h.
As part of a general strategy to decrease the molecular weight,
basicity, and overall length of the compound, we reduced the cen-
tral diamine group in our N-alkyl glycine analog series to yield our
aryl diamine template described here. The transformation is out-
lined in Figure 1 with truncation of the glycine group in 1 by re-
moval of the aminomethyl group to give 2. The 6-fold loss in
activity could be reversed by further truncation of 2 to give an aryl
diamine analog represented by 3. Unfortunately, we were unable
to obtain X-ray crystal structures of our inhibitors in LTA4-h and
relied on SAR to direct our optimization effort.
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-inflam-
11
matory cysteinyl leukotrienes LTC4, LTD4 and LTE4 and a sub-
strate of lipoxygenases 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.
LTA4-h is a monomeric, soluble 69-kDa zinc metallohydrolase of
the M1 class which has sequence homology to aminopeptidases M
The preparation of analogs in the aryl diamine series is shown
with the synthesis of 3l (Scheme 1). Conversion of 4-carboxyani-
* Corresponding author. Tel.: +1 650 868 8777.
E-mail address: wguilford@comcast.net (W.J. Guilford).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.041

3896
S.-K. Khim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3895–3898
Table 1
Table 2
Optimization of spacer of 4-(2-oxazolyl)phenyl phenyl ether analogs
Optimization of phenoxy moiety of 3l
O
O
H
H
N
N
N
X
OH
O
N
R
O
OH
a
a
Compound
R
LTA4-h IC50 (nM)
WBA IC50 (nM)
a
a
Compound
X
LTA4-h IC50 (nM)
WBA IC50 (nM)
O
3l
5
40
20
3a
30
80
18
N
N
O
Me
N
O
3b
3c
3d
3e
3f
3
130
3
4a
4b
4c
4d
4e
4f
14
6
Me
N
O
560
50
310
870
30
Cl
F
O
320
40
6
N
Cl
N
O
5
1500
220
100
200
70
O
F
11
9
N
H
40
O
3g
3h
3i
N
Me
23
13
4
170
230
30
MeO
F3C
O
60
2
4g
4h
4i
N
N
O
O
N
3j
7
60
N
8
19
N
S
O
O
3k
3l
15
5
30
N
4j
5
25
Ph
N
40
N
N
4k
4l
9
100
220
40
S
3m
1
40
O
47
3
a
Values are means of at least two experiments.
4n
sole, 6, to the corresponding acid chloride with POCl3 followed by
treatment with aminoacetaldehyde dimethyl acetal gave the
amide intermediate 7. Cyclization using Eaton’s reagent to the de-
sired oxazole followed by treatment with an aqueous methanolic
sodium hydroxide solution gave the desired 4-(2-oxazolyl)phenol,
8. Nucleophilic displacement of the fluorine on nitrofluorobenzene
by 8 gave aminodiphenyl ether 9 after hydrogenolysis. Reductive
alkylation of 9 with N-Boc-nortropinone followed by removal of
the Boc protecting group with 4.0 M HCl solution in THF gave a sin-
gle endo diastereomer, 10. Alkylation of 10 with methyl bro-
momethylbenzoate followed by saponification gave 3l.
Since the N-methyl tertiary aniline analog of 2 was inactive in
the hydrolase assay (IC50 >1000 nM), the piperidine group on 2
was systematically modified to optimize the inhibitor to LTA4-h
interactions (Table 1). Several cyclic, bicyclic, and acyclic amines
were prepared and assayed to rank the importance of the distance
between the aromatic groups and the placement of the amine in
4o
75
440
N
a
Values are means of at least two experiments.
the chain. When a flexible chain is used as in the acyclic series,
the optimal distance between the aromatic groups is set at 6-
atoms, 3b, with a 40-fold decrease in activity seen with the corre-
sponding 5-atom chain 3c. In the piperidine series, the addition of
a carbon to either end of the piperidine group increased the po-
tency at least 6-fold, 3d and 3e, compared to 3a. Shifting of the
nitrogen out of the ring, as seen in the aminocyclohexane analogs
3f and 3g, resulted in a loss of activity compared to 3d. Shifting of
the anilino group from C-4 to C-3 of the piperidine, 3h, resulted in
a 2-fold drop in activity compared to 3a, but a similar shift in the

S.-K. Khim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3895–3898
3897
Table 3
N
H
N
DMPK profile of selected inhibitors
O
IC50 = 60 nM (LTA4-h)
IC50 = 270 nM (hWBA)
N
Me
Compound Cl
(mL/min/kg)
Cmax
t1/2
%F
Metabolic stabilitya
OH
O
O
(
lg/mL) (h)
O
O
Rat
Dog
Human
1
3a
3b
3l
4a
4b
4d
1
23
6
36
4
37
4
7
1
2
1.5 99
1.9 67
2.9 99
0.9 62
12
4.8 99
91
81
96
90
81
75
91
91
100
94
92
81
82
94
99
97
Removal of Aminomethyl group
N
96 100
99
H
N
O
IC50 = 360 nM (LTA4-h)
IC50 = 230 nM (hWBA)
9
2
OH
a
metabolic stability;
% remaining after 1.0 h in the corresponding liver
2
microsomes.
Removal of C=O Group
O
similar activity to 3l. Introduction of chlorine at the para position,
4b, did not improve potency, whereas at the ortho position, 4c, was
50-fold less potent. The potency of the corresponding fluorine ana-
logs, 4d and 4e, reversed the trend and suggests steric bulk is
favorable at the para position but not at the ortho position. The ste-
ric argument is supported by the lack of a significant impact of
shifting from electron-donating substituent as methoxy (4f) to an
electron-withdrawing substituent, as trifluoromethyl (4g) or by
substitution of a pyrrole (4h), thiazole (4i), or phenyl (4j) group.
Although the benzothiazole (4k) group maintained potency, the
benzyl analog (4l) was about 10-fold less potent and may indicate
the importance of the shape of the diphenyl ether group to binding
in the hydrophobic pocket. The increase in potency upon substitu-
tion of carbon for the ether oxygen, 4n versus 4a, suggests that the
oxygen does not interact with the protein. The loss of activity upon
introduction of a pyridine group, 4o, suggests that polar groups are
not tolerated in the hydrophobic pocket.
H
N
IC50 = 46 nM (LTA4-h)
IC50 = 350 nM (hWBA)
OH
N
O
3
Figure 1. Optimization strategy.
O
O
c,d
MeO
a,b
HO
N
H
OMe
OMe
OMe
6
7
O
O
N
The WBA does not follow the activity in the hydrolase assay due
to the need for analogs to enter the cell to inhibit the target. Cellu-
lar uptake favors the less polar analogs as 4h, 4i, and 4j over polar
substituents as methoxy (4f) or trifluoromethyl (4g). A similar ef-
fect was seen with the pharmacokinetic profile (PK) of the analogs
after additional factors such as molecular rigidity and accessibility
are considered. Flexible analogs such as 3b had higher clearance
and shorter half-lives (data not shown) than the endo-nortropane
analogs 3l, 4b, and 4d (Table 3) which had low clearance, signifi-
cant Cmax, long half-lives, metabolic stability, and good oral
bioavailability.
In summary, a series of aryl diamines were designed and pre-
pared which showed significant activity in both the enzyme and
cell-based assays. The optimum amine, nortropane group, and
the optimum phenoxy substituent, 2-oxazole, were combined in
analog 3l. Further studies are planned and will be reported on in
due course.
e,f
NH2
N
OH
N
O
8
9
O
H
g,h
N
i,,j
NH
O
O
10
O
H
N
OH
N
N
O
3l
Scheme 1. Reagents and conditions: (a) POCl3, DMF; (b) protected aminal, CH2Cl2;
(c) Eaton’s reagent, 180 °C; (d) NaOH, MeOH–H2O; (e) 4-nitrofluorobenzene, DIEA,
DMF; (f) H2, Pd/C, MeOH; (g) N-Boc-nortropinone, NaBH(OAc)3, AcOH, CH2Cl2; (h)
4.0 M HCl, THF; (i) methyl 4-bromomethylbenzoate, K2CO3, MeCN; (j) LiOHÁH2O,
H2O–THF, reflux.
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