Amides of 4-hydroxy-8-methanesulfonylamino-quinoline-2-carboxylic acid as zinc-dependent inhibitors of Lp-PLA2

Article abstract of DOI:10.1016/j.bmcl.2012.11.048

AX10479, the phenyl amide of 4-hydroxy-8-methanesulfonylamino-quinoline-2- carboxylic acid, was identified as a Zn²⁺-dependent, 27 nM inhibitor of human plasma Lp-PLA₂. Structure-activity relationship studies focused on the AX10479 2-phenylamide group identified equipotent cycloaliphatic amides, an enantioselective preference for chiral amides, and phenyl substitution patterns (e.g., 2-methyl-3-fluoro) that increased potency.

Full text of DOI:10.1016/j.bmcl.2012.11.048

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1553–1556
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Amides of 4-hydroxy-8-methanesulfonylamino-quinoline-2-
carboxylic acid as zinc-dependent inhibitors of Lp-PLA2
Yi Hu a, , Emme C.K. Lin a, , Lan M. Pham a, Julia Cajica a, Christopher M. Amantea a, Eric Okerberg a,
Heidi E. Brown a, Allister Fraser a, Lingling Du a, Yasushi Kohno b, Junichi Ishiyama b, John W. Kozarich a,
Kevin R. Shreder a,
⇑
a ActivX Biosciences, Inc., 11025 N. Torrey Pines Road, La Jolla, CA 92037, USA
b Discovery Research Laboratories, Kyorin Pharmaceutical Co., Ltd, 2399-1, Nogi, Nogi-machi, Shimotsuga-gun, Tochigi, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
AX10479, the phenyl amide of 4-hydroxy-8-methanesulfonylamino-quinoline-2-carboxylic acid, was
identified as a Zn2+-dependent, 27 nM inhibitor of human plasma Lp-PLA2. Structure–activity relationship
studies focused on the AX10479 2-phenylamide group identified equipotent cycloaliphatic amides, an
enantioselective preference for chiral amides, and phenyl substitution patterns (e.g., 2-methyl-3-fluoro)
that increased potency.
Received 17 August 2012
Revised 9 November 2012
Accepted 13 November 2012
Available online 8 December 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Heart disease remains the number one cause of mortality in the
world, therefore efforts to pharmacologically reduce atheroscle-
rotic burden continue. Lipoprotein-associated phospholipase A2
(Lp-PLA2) is a calcium-independent phospholipase which produces
proinflammatory and proapoptotic lipid mediators of the athero-
genic process. Lp-PLA2 primarily circulates bound to plasma LDL,
and uptake of these particles into the developing plaque is thought
to contribute to necrotic core progression, smooth muscle cell and
fibrous tissue destruction, and eventually plaque rupture.1 Both
the plasma protein levels and the enzymatic activity of Lp-PLA2
have been established as biomarkers for cardiovascular events,2
and therapeutic inhibition of Lp-PLA2 activity has been shown to
reduce the progression of plaque instability in the clinical setting.3
Pharmacological reduction of Lp-PLA2 activity therefore represents
an exciting new approach to treating this leading unmet need.
Through metal screening experiments and titration studies, we
previously identified AX10185 (Fig. 1) as a Zn2+-dependent, sub-
100 nM inhibitor of Lp-PLA2.4 AX10185 is a member of a class of
compounds that have been identified as being dependent on
exogenous metals for their inhibitory activity.5 Examples of
Zn2+-dependent inhibitors include BABIM (a tryptase inhibitor),6
SB-247464 (a granulocyte colony-stimulating factor agonist),7
and bis-imidazolemethane-based hCMV protease inhibitors.8
Other members with a dependency on multiple metals include
amidine-based inhibitors of thrombin (Cu2+ and Fe3+),9 Schiff base
inhibitors of trypsin (Zn2+, Cu2+, and Fe3+),10 and amidine-benz-
imidazole inhibitors of trypsin (Zn2+, Mn2+, Sc3+, and Hg2+).11
During our optimization of AX10185 as an Lp-PLA2 inhibitor, we
synthesized analogs in which the 8-hydroxy group was replaced by
hydroxy group bioisosteres.12 This effort lead us to the synthesis of
the 8-methanesulfonylamino derivative AX10479 (Fig. 1), using
the general procedure shown in Scheme 1. When examined in an
Lp-PLA2 IC50 assay4 which utilized Lp-PLA2 purified from human
plasma (php-Lp-PLA2), AX10479 was found to be a more potent
Lp-PLA2 inhibitor than AX10185 (php-Lp-PLA2 IC50 values were
determined to be 27 and 74 nM, respectively). As such, we refo-
cused our lead optimization efforts on AX10479, initially through
modification of the 2-phenylamide subunit.
After the realization that AX10185 was a zinc-dependent inhib-
itor, we examined AX10479 using the same battery of tests to
determine whether this compound would demonstrate similar me-
tal-dependent Lp-PLA2 inhibitory activity. Our suspicion that this
might be the case was heightened by the structural similarity of
AX10479 to various 8-para-toluenesulfonamide-quinolines, such
as TSQ and Zinquin-E (Fig. 2), both of which are fluorogenic dyes
capable of detecting free Zn2+ at sub-nM concentrations
under physiological conditions.13 Importantly however, TSQ and
OH
OH
H
H
N
N
N
N
H3C
NH
O
OH
O
S
O O
⇑
Corresponding author.
E-mail address: kevins@activx.com (K.R. Shreder).
AX10185
AX10479
These authors contributed equally to this work.
Figure 1. Structures of AX10185 and AX10479.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.048

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Y. Hu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1553–1556
Table 1
OH
OH
php-Lp-PLA2 IC50 values for cycloaliphatic AX10479 2-amide analogs
a
OH
O
O
N
N
H
N
NO2
O
NH2
O
N
(CH2)
1
2
n-2
NH
O
O O
S
S
O
O
O
5-11
b
c
Compound
n
php-Lp-PLA2IC50 (nM)
O
N
5
6
7
8
9
3
4
5
6
7
8
14,000
230
55
38
32
NH
O
S
O O
3
OH
OH
N
10
11
23
>10,0000
12
H
OH
d
N
N
R
NH
O
NH
O
O
S
S
Table 2
php-Lp-PLA2 IC50 values for chiral AX10479 2-amide analogs
O O
O
4
5 - 47
OH
Scheme 1. Synthesis of 4-hydroxy-8-methanesulfonylamino-quinoline-2-carbox-
ylic acid amides. (a) H2, 10% Pd/C (5 wt %), MeOH, rt, 3 days, quant (b)
methanesulfonyl chloride, pyridine, DCM, rt 16 h, 89%; (c) 2 M NaOH, rt, 2 h then
2 M HCl, 93%; (d) HOBt, EDCI, DMF, rt, 30 min then RNH2, rt, 16 h.
H
N
Ra
H
N
NH
O
Rb
S
O
O
Zinquin-E did not inhibit the activity of Lp-PLA2 (php-Lp-PLA2 IC50
values >100 lM), indicating that the Lp-PLA2 inhibition by
12 20
-
Compound
Ra
Rb
php-Lp-PLA2IC50 (nM)
AX10479 was not due to a general zinc chelation effect. Further-
more, we found that the addition of the well-known metal ion che-
lators EGTA and EDTA to the php-Lp-PLA2 IC50 assay diminished or
extinguished the Lp-PLA2 inhibitory activity of AX10479 (e.g., see
Table 3) without altering the enzymatic activity of Lp-PLA2. In to-
tal, the evidence led us to hypothesize that the inhibition of Lp-
PLA2 by AX10479 was metal dependent, just like AX10185.
To investigate this hypothesis and to address which metal may
be involved, a screening experiment was conducted using both hu-
man plasma-derived php-Lp-PLA2 and an alternate source of en-
zyme, recombinant human Lp-PLA2 (rh-Lp-PLA2).4 IC50 values of
12
13
14
15
16
17
18
19
20
Me
Ph
CH2CN
Et
Me
(CH2)3CH3
Me
Ph
Me
Et
CH2CN
(CH2)3CH3
Me
(CH2)4CH3
(CH2)5CH3
(CH2)6CH3
370
86,000
820
29,000
170
9700
100
150
Me
Me
150
AX10479 were determined in the presence of 50 lM added metal
ence of increasing amounts of added ZnCl2 (Fig. 4). In the absence
of added ZnCl2, the rh-Lp-PLA2 IC50 was determined to be 16 nM.
With added ZnCl2 this value dropped as low as 5 nM, with the
chloride salts (Fig. 3), none of which changed the enzymatic activ-
ity of Lp-PLA2. In the absence of added metal, the php-Lp-PLA2 IC50
value was determined to be 35 nM.4 This value changed depending
on the nature of the metal added. The addition of FeCl3 resulted in
a significant increase in the IC50 value of AX10479 to 46 nM. In
contrast, the addition of other metals resulted in a decrease in
the AX10479 IC50 value. The decrease seen with ZnCl2 was the larg-
est, resulting in a php-Lp-PLA2 IC50 value of 9.9 nM. When these
experiments were repeated with rh-Lp-PLA2, similar trends were
observed (Fig. 3).
maximal inhibitory activity observed at
ꢀ10 M added ZnCl2.
a concentration of
l
To further explore the SAR and improve the potency of this new
8-sulfonamide motif, various 2-amide analogs and derivatives of
AX10479 were evaluated in the php-Lp-PLA2 IC50 assay. All such
compounds were synthesized according to Scheme 1. Conversion
of methyl 4-hydroxy-8-nitro-quinoline-2-carboxylate (1)14 to the
corresponding amine 2 was accomplished using catalytic hydroge-
nation in quantitative yield. Treatment of compound 2 with
methanesulfonyl chloride yielded the methylsulfonamido-meth-
anesulfonate 3. Saponification of 3 with 2 M NaOH resulted in
not only the hydrolysis of the methyl ester but also the deprotec-
tion of the methanesulfonate group to give 4. Amidation of the
resulting carboxylic acid 4 with aromatic or aliphatic amines
yielded the 2-amide derivatives studied herein.
Because Zn2+ resulted in the largest increase of Lp-PLA2 inhibi-
tory activity, this specific metal was examined in greater detail.
The rh-Lp-PLA2 IC50 value of AX10479 was determined in the pres-
O
MeO
O
EtO
When cycloaliphatic 2-amides were examined (Table 1) it was
found that increasing the ring size (n) from cyclopropyl (5: php-
Lp-PLA2 IC50 = 14,000 nM) to cyclooctyl (10: php-Lp-PLA2
IC50 = 23 nM) resulted in a continued increase in potency. How-
ever, jumping to cyclododecyl resulted in a complete loss of Lp-
N
N
NH
NH
S
O
S
O
O O
Zinquin-E
TSQ
PLA2 inhibitory activity (11: php-Lp-PLA2 IC50 >100
lM). In this
Figure 2. Structures of the Zn2+-dependent fluorogenic dyes TSQ and Zinquin-E.
series, no compound was found that was significantly more potent

Y. Hu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1553–1556
1555
Table 3
25
20
15
10
5
php-Lp-PLA2 IC50 values for AX10479 and substituted 2-phenylamide derivatives of
AX10479
OH
R2
H
N
R3
R4
N
NH
O
R6
S
O
R5
O
0
AX10479 and 21 - 47
0.1
1
10
100
[ZnCl2 added] (µM)
Compound
R2
R3
R4
R5
R6
php-Lp-PLA2 IC50 (nM)
Figure 4. Change in rh-Lp-PLA2 IC50 values for AX10479 as a function of added
10479
10479a
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
H
H
Me
H
H
H
H
H
Me
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
Me
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
F
H
H
H
H
H
27
ZnCl2.
>100,000a
16
70
87
44
240
3000
41,000
31
26
13
14
39
49
52
120
250
450
100
39
26
10
55
28
10
9
80
22
Table 4
Et
Comparison of substrate-based Lp-PLA2 and ABP IC50 values for selected Lp-PLA2
inhibitors
nPr
iPr
tBu
H
Compound php-Lp-PLA2IC50
(nM)
rh-Lp-PLA2IC50
(nM)
ABP Lp-PLA2IC50
(nM)
H
F
10479
16
27
170
9700
10
10
9
4
32
980
4
3
3
42
830
11,000
41
CN
NH2
OH
Cl
CF3
CCH
MeO
F
F
F
F
Me
Me
Me
Me
OH
OH
17
41
44
45
59
49
Both rh-Lp-PLA2 and ABP-Lp-PLA2 assays contained 20
lM added ZnCl2.
2-amides derived from (S) and (R)-a-methyl-benzylamine were
H
H
H
F
H
H
H
H
compared, it was found that the (S) isomer (12) was preferred by
a factor of 230-fold. This enantioselective inhibition held for the
two other chiral amide series (14/15 and 16/17) in which one iso-
mer was preferred over the other by a factor of 35- and 57-fold,
respectively. When the butyl chain of 16 was extended (18–20),
there was no significant variation in potency.
H
H
H
H
F
H
H
F
H
H
F
F
H
Finally, derivatization of the phenylamide of AX10479 was
examined (Table 3), and selected ortho-modifications were shown
to improve the Lp-PLA2 inhibitory activity. For example, the ortho-
methyl derivative 21 yielded a twofold increase in potency (php-
Lp-PLA2 IC50 = 16 nM) versus AX10479 whereas the incorporation
of meta-methyl and para-methyl substitutions (22 and 23, respec-
tively) resulted in a relative decrease in potency. Increased homol-
ogation of this ortho-methyl group (24 and 25) or its replacement
with a branched isopropyl (26) or tert-butyl group (27) resulted in
a concomitant decrease in potency with increasing steric bulk.
When fluorine was examined, meta-fluoro (28) and para-fluoro
(29) derivatization had no impact on potency relative to
AX10479 but the ortho-fluoro derivative (30: php-Lp-PLA2
IC50 = 13 nM) was found to be ꢀtwofold more potent than
AX10479. When other ortho-substituted groups were examined,
cyano substitution (31: php-Lp-PLA2 IC50 = 14 nM) improved po-
tency by a factor of 1.9 whereas amino (32), hydroxy (33), chloro
(34), trifluoromethyl (35), alkynyl (36), and methoxy (37) substit-
uents decreased php-Lp-PLA2 inhibitory activity by factors ranging
from 1.3 to 17-fold, all relative to AX10479.
a
Determined in the presence of 10 mM EDTA.
no added metal
Fe(III)Cl3
Ni(II)Cl2
Co(II)Cl2
Cd(II)Cl2
Ca(II)Cl2
Ba(II)Cl2
Mg(II)Cl2
Mn(II)Cl2
Zn(II)Cl2
php-Lp-PLA2
rh-Lp-PLA2
0
20
40
60
Lp-PLA2 IC50 (nM)
AX10479
For selected ortho-substituted derivatives, incorporation of a
fluorine in the 3- or 4-position of the phenyl ring was shown to fur-
ther increase Lp-PLA2 inhibitory activity. Analysis of the four pos-
sible difluoro-phenylamides in which one of the fluorine
substituents was fixed in the ortho-position (38–41) identified
the 2,4-difluoro-phenyl amide 41 as one of the most potent inhib-
itors in this study with a php-Lp-PLA2 IC50 value of 10 nM. A similar
approach was taken in which the methyl group was fixed in the
Figure 3. Influence of 50
values of AX10479.
l
M added metal on the php-Lp-PLA2 and rh-Lp-PLA2 IC50
than AX10479, though SAR was identified indicating that a mini-
mum saturated ring size of 5 was necessary for sub-100 nM
inhibition.
When various chiral 2-amides were examined (Table 2), marked
enantioselective inhibition was seen. When the potency of
ortho-position and
a fluorine was ‘walked’ around the ring

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Y. Hu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1553–1556
2. (a) Koenig, W.; Vossen, C. Y.; Mallat, Z.; Brenner, H.; Benessiano, J.;
Rothenbacher, D. Eur. Heart J. 2009, 30, 2742; (b) Tsimikas, S.; Willeit, J.;
Knoflach, M.; Mayr, M.; Egger, G.; Notdurfter, M.; Witztum, J. L.; Wiedermann,
C. J.; Xu, Q.; Kiechl, S. Eur. Heart J. 2009, 30, 107; (c) Bhatti, S.; Hakeem, A.;
Cilingiroglu, M. Curr. Atheroscler. Rep. 2010, 12, 140.
3. Serruys, P. W.; García-García, H. M.; Buszman, P.; Erne, P.; Verheye, S.;
Aschermann, M.; Duckers, H.; Bleie, O.; Dudek, D.; Bøtker, H. E.; von Birgelen,
C.; D’Amico, D.; Hutchinson, T.; Zambanini, A.; Mastik, F.; van Es, G. A.; van der
Steen, A. F.; Vince, D. G.; Ganz, P.; Hamm, C. W.; Wijns, W.; Zalewski, A.
Circulation 2008, 118, 1172.
(42–45): Versus the singly-substituted ortho-methylated 21, the 4-
fluoro-2-methyl-(44) and 3-fluoro-2-methyl-(45) phenylamides
both showed potency improvement with php-Lp-PLA2 IC50 values
of 10 nM and 9 nM, respectively. Finally, two fluorinated deriva-
tives (46,47) of the ortho-hydroxy 33 were tested, and the 4-flu-
oro-2-hydroxy phenyl amide 47 resulted in a 2.2-fold increase in
potency relative to the parent compound.
To rule out the participation of any thiol-derived small molecule
components used in the substrate-based assay (e.g., 2-thio-PAF or
DTNB) on the observed Zn2+ dependent inhibition, we utilized a
substrate-free and thiol-free assay that relied on the inhibition of
rh-Lp-PLA2 labeling by AX4870, a TAMRA tagged, fluorophospho-
nate activity-based probe (ABP).4 Such probes have been shown
to covalently and irreversibly label the active site of serine hydro-
lases, including lipases,15 and previously have been used to deter-
mine IC50 values for small molecule enzyme inhibitors.16 For
selected Lp-PLA2 inhibitors, ABP IC50 values (Table 4) were calcu-
lated by plotting % inhibition of AX4870 labeling of rh-Lp-PLA2 ver-
sus inhibitor concentration. All inhibitors tested in the ABP assay
exhibited inhibitory activity that paralleled the substrate-based
Lp-PLA2 IC50 assays. For example, the enantiomeric preference for
16 versus 17 was observed in the ABP Lp-PLA2 assay, just as it
was in both substrate-based assays.
4. Lin, E. C. K.; Hu, Y.; Amantea, C. M.; Pham, L. M.; Cajica, J.; Okerberg, E.; Brown,
H. E.; Fraser, A.; Du, L.; Kohno, Y.; Ishiyama, J.; Kozarich, J. W.; Shreder, K. R.
Bioorg. Med. Chem. Lett. 2012, 22, 868.
5. This inhibitor class is different than inhibitors of metalloproteases in which the
metalloprotease inhibitor is designed to chelate active site metals that are
critical for enzymatic activity. For example, see: Kontogiorgis, C. A.;
Papaioannou, P.; Hadjipavlou-Litina, D. J. Curr. Med. Chem. 2005, 12, 339.
6. (a) Katz, B. A.; Luong, C. J. Mol. Biol. 1999, 292, 669; (b) Katz, B. A.; Clark, J. M.;
Finer-Moore, J. S.; Jenkins, T. E.; Johnson, C. R.; Ross, M. J.; Luong, C.; Moore, W.
R.; Stroud, R. M. Nature 1998, 391, 608.
7. Doyle, M. L.; Tian, S. S.; Miller, S. G.; Kessler, L.; Baker, A. E.; Brigham-Burke, M.
R.; Dillon, S. B.; Duffy, K. J.; Keenan, R. M.; Lehr, R.; Rosen, J.; Schneeweis, L. A.;
Trill, J.; Young, P. R.; Luengo, J. I.; Lamb, P. J. Biol. Chem. 2003, 278, 9426.
8. Dhanak, D.; Burton, G.; Christmann, L. T.; Darcy, M. G.; Elrod, K. C.; Kaura, A.;
Keenan, R. M.; Link, J. O.; Peishoff, C. E.; Shah, D. H. Bioorg. Med. Chem. Lett.
2000, 10, 2279.
9. Toyota, E.; Sekizaki, H.; Takahashi, Y. U.; Itoh, K.; Tanizawa, K. Chem. Pharm.
Bull. (Tokyo) 2005, 53, 22.
10. (a) Iyaguchi, D.; Kawano, S.; Takada, K.; Toyota, E. Bioorg. Med. Chem. Lett. 2010,
18, 2076; (b) Toyota, E.; Ng, K. K.; Sekizaki, H.; Itoh, K.; Tanizawa, K.; James, M.
N. J. Mol. Biol. 2001, 305, 471; (c) Toyota, E.; Sekizaki, H.; Itoh, K.; Tanizawa, K.
Chem. Pharm. Bull. (Tokyo) 2003, 51, 625.
11. Paul, J. J.; Kircus, S. R.; Sorrell, T. N.; Ropp, P. A.; Thorp, H. H. Inorg. Chem. 2006,
45, 5126.
12. For a review of bioisosterism which includes examples of phenolic hydroxyl
group replacement with the methylsulfonamide group, see: (a) Lima, L. M.;
Barreiro, E. J. Curr. Med. Chem. 2005, 12, 23; (b) Meanwell, N. A. J. Med. Chem.
2011, 54, 2529.
In conclusion, AX10479 was identified as a Zn2+-dependent,
nanomolar inhibitor of Lp-PLA2. SAR studies focusing on the
AX10479 2-phenylamide subunit identified equipotent cycloali-
phatic amides, an enantiomeric preference for certain chiral
amides, and phenyl substitution patterns that increased potency
(i.e., selected ortho substitutions, and 3- or 4- fluoro substitutions).
Beyond being useful for the development of Lp-PLA2 inhibitors, the
work herein further establishes quinoline metal chelates as poten-
tial enzyme inhibitors. Future work on the continued optimization
of the 8-methanesulfonylamino-quinoline series will be reported
in due course.
13. (a) Nasir, M. S.; Fahrni, C. J.; Suhy, D. A.; Kolodsick, K. J.; Singer, C. P.;
O’Halloran, T. V. J. Biol. Inorg. Chem. 1999, 4, 775; (b) Kimber, M. C.; Mahadevan,
I. B.; Lincoln, S. F.; Ward, A. D.; Tiekink, E. R. J. Org. Chem. 2000, 65, 8204.
14. While a variety of synthetic routes exist in the literature to produce this
compound, in our hands only the following literature procedure gave reliable
purity and yields: Peet, N. P.; Baugh, L. E.; Sunder, S.; Lewis, J. E. J. Med. Chem.
1985, 28, 298.
15. (a) Liu, Y.; Patricelli, M. P.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
14694; (b) Simon, G. M.; Cravatt, B. F. J. Biol. Chem. 2010, 285, 11051.
16. Ahn, K.; Johnson, D. S.; Fitzgerald, L. R.; Liimatta, M.; Arendse, A.; Stevenson, T.;
Lund, E. T.; Nugent, R. A.; Nomanbhoy, T. K.; Alexander, J. P.; Cravatt, B. F.
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References and notes
1. Koenig, W.; Khuseyinova, N. Arterioscler. Thromb. Vasc. Biol. 2007, 27, 15.