Simplified YM-26734 inhibitors of secreted phospholipase A2 group IIA

Article abstract of DOI:10.1016/j.bmcl.2008.09.041

Simplified analogs of YM-26734, a known inhibitor of secreted phospholipase A₂ (sPLA₂) group IIA, were synthesized and found to also display potent inhibition at low nanomolar concentrations. Analogs were based on the didodecanoylphl

Full text of DOI:10.1016/j.bmcl.2008.09.041

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5415–5419
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Simplified YM-26734 inhibitors of secreted phospholipase A2 group IIA
Rob C. Oslund a, Nathan Cermak a, Christophe L. M. J. Verlinde b, Michael H. Gelb a,b,
*
a Department of Chemistry, Campus Box 351700, University of Washington, Seattle, WA 98195, USA
b Department of Biochemistry, Campus Box 357350, University of Washington, Seattle, WA 98195, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Simplified analogs of YM-26734, a known inhibitor of secreted phospholipase A2 (sPLA2) group IIA, were
synthesized and found to also display potent inhibition at low nanomolar concentrations. Analogs were
based on the didodecanoylphloroglucinol portion of YM-26734 which contains the predicted active site
calcium binding group.
Received 15 August 2008
Revised 5 September 2008
Accepted 9 September 2008
Available online 12 September 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Eicosanoids
Lipid mediators
Inflammation
Structure-based drug design
Phospholipases
OH
Secreted phospholipases A2 (sPLA2s) are a family of interfacial
enzymes that catalyze the sn-2 esterolysis of glycerophospholip-
ids.1 Products of sPLA2 enzymatic activity include arachidonic acid,
a well-known precursor of pro-inflammatory eicosanoids.1 Cur-
rently, there are ten known mammalian sPLA2s, (groups IB, IIA,
IIC (pseudo gene in humans), IID, IIE, IIF, III, V, X, XIIA) all of which
contain a conserved active site that consists of a Ca2+ binding loop
and an Asp-His catalytic dyad.2,3 Over the past two decades there
has been an increasing body of research implicating the involve-
ment of sPLA2s, especially groups IIA (GIIA), V, and X, in different
inflammatory processes including arthritis,4 asthma,5 atheroscle-
rosis,6 and sepsis.7 The involvement of one or more sPLA2s in these
diseases and other processes highlights a critical need to under-
stand their biological functions.
OH
HO
O
HO
O
HO
OH
C11H23
HO
C11H23
OH
C11H23
OH O
O
OH O
2
1
Figure 1. Potent GIIA sPLA2 inhibitor YM-26734 (compound 1) derived from YM-
26567-1 (compound 2). Data to assign chirality have not been published.
One powerful approach towards elucidating sPLA2 function is
through the use of small molecule inhibitors. A number of different
academic and industrial laboratories have explored small molecule
active site inhibitors of sPLA2.8 Some of the most potent inhibitors,
displaying low nanomolar potency, include the functionalized in-
The natural product, YM-26567-1 (compound 2, Fig. 1) iso-
lated by Yamanouchi Pharmaceuticals from the fruit of Horsfiel-
dia amygdaline was identified as
a micromolar inhibitor of
rabbit platelet sPLA2.13 Further development of this compound
led to 1, which had increased potency against rabbit platelet
sPLA2 with an IC50 of 85 nM.12 Here, we further explore 1 as
an inhibitor against mammalian sPLA2 enzymes and elucidate
its active site binding mode. This process led us to simplified
analogs of 1 that displayed nearly identical inhibition potency
against GIIA sPLA2.
dole9 and pyrazole10 scaffolds from Eli Lilly,
D-tyrosine analogs re-
ported by Hansford and co-workers,11 and the natural product
analog YM-26734 (compound 1, Fig. 1) from Yamanouchi Pharma-
ceuticals12 (all reviewed in Ref. 1). With a number of potent inhib-
itor scaffolds available, our focus has turned towards the
optimization of these inhibitors to increase cell permeability and/
or selectivity among the sPLA2 groups.
Compound 1 was prepared using a modified version of a previ-
ously reported procedure (Scheme 1).14 Commercially available 3
was benzyl protected under basic conditions to yield 4 and subse-
quently refluxed with commercially available 5 in KOH to form the
chalcone 6. Compound 6 was refluxed in H2SO4/MeOH to form the
* Corresponding author. Tel.: +1 206 543 7142; fax: +1 206 685 8665.
E-mail address: gelb@chem.washington.edu (M.H. Gelb).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.041

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R. C. Oslund et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5415–5419
OBn
O
O
O
OH
b
OH
c
H
OBn
OR
OBn
5
6
3: R = H
4: R = Bn
a
OBn
OBn
HO
OH
C11H23
BnO
O
BnO
O
C11H23
d
O
OH O
O
OH
7
8
9
OR
RO
O
HO
C11H23
OH
C11H23
e
O
OH O
10: R =Bn
1: R =H
f
Scheme 1. Reagents: (a) BnBr, K2CO3; (b) 40% KOH; (c) 10% H2SO4/MeOH; (d) NaBH4, MeOH; (e) 4 N HCl/dioxane; (f) 1 atm H2, Pd(OH)2/C.
flavanone 7 which was reduced with NaBH4 to yield 8. Compound
8 was condensed with 9 in HCl/dioxane to give 10. Deprotection of
10 using Pd(OH)2/C under H2 afforded 1 as a mixture of four ster-
eoisomers. Diastereomers were separated by HPLC using a reverse
phase C18 column, and the enantiomers were isolated using a Dai-
cel Chirex column (see Supplementary data). In addition, we pre-
pared 7,40-dihydroxyflavan from 7 under reducing conditions in
H2 and Pd(OH)2/C (Scheme 2).
Di-acylation of phloroglucinol 11a and resorcinol 11b was done
in either dodecanoic anhydride and BF3ꢀOEt2 or dodecanoic acid
and ZnCl2 to give 9 and 12b, respectively (Scheme 3). Formation
of 15 was done by monoacylating 11a in dodecanoic acid chloride
and AlCl3 followed by complete reduction of the acyl group under
Wolf Kishner conditions to give 14. Monoacylation and di-acetyla-
tion of 14 were performed using dodecanoic acid chloride and
AlCl3 and acetic anhydride and BF3ꢀOEt2 to yield 15 and 16, respec-
tively. Compounds 17b–d, f, and 18b (Scheme 4) were prepared
using similar chemistry as shown in Scheme 3.
We found that 1 inhibited all GIIA enzymes and human GV at
low nanomolar concentrations, displayed moderately potent inhi-
bition against mouse GV and showed no inhibition of human and
mouse GX at low micromolar concentrations. These results are
consistent with the 85 nM IC50 value previously reported for rabbit
GIIA sPLA2.12 However, Hamaguchi and co-workers recently re-
ported IC50 values of 1 lM and 0.2 lM for 1 against GIIA and GX,
respectively (the authors did not disclose whether this was human
or mouse sPLA2).15 These discrepancies in potency are probably
due to the differences in substrate and assay conditions used to ob-
tain IC50 values.
In order to assess if one stereoisomer is more potent over the
others, we isolated all four stereoisomers of 1 and tested them
individually against rat GIIA sPLA2 (Table 2). Interestingly, all four
isomers of 1 had IC50s between 60 and 120 nM. We found this sur-
prising because one would expect the dramatic structural diversity
between the four isomers to result in different binding affinities.
Intrigued by this result, we decided to model the binding of 1 in
the active site of human GIIA sPLA2. We manually positioned 1 into
the GIIA active site by overlaying it onto Indole 8 from the co-crys-
tal structure reported by Schevitz and co-workers (PDB code:
1DB4).16 Compound 1 was oriented into the active site by creating
a binding interaction between the oxygen from one of the dodec-
anoyl chains and the para-hydroxy group with the active site cal-
cium ion involved in stabilizing the transition state (Fig. 2). We
chose this binding pose to mimic the acetylacetonate bidentate li-
gands that coordinate to metals including calcium.17 This posi-
tioned the other dodecanoyl oxygen and its vicinal hydroxyl
group to contact a neighboring lysine residue (Fig. 2). In this orien-
tation, the dodecanoyl alkyl chains are able to fill the active site as
they extend out into the solvent.
Initially we tested 1 as a four-isomer mixture against human,
mouse and rat GIIA, and human and mouse GV and GX sPLA2 en-
zymes (Table 1) (see Supplementary data for all assay details).
OBn
OH
BnO
O
HO
O
1atm H2
Pd(OH)2/C
O
7
7,4'-dihydroxyflavan
Scheme 2. Preparation of 7,40-dihydroxyflavan.

R. C. Oslund et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5415–5419
5417
R2
O
R2
O
C11H23
C11H23
a
R1
R3
R1
R3
9 R1 = R2 = R3 = OH
11a R1 = R2 = R3 = OH
12b R1 = R3 = OH, R2 = H
11b R1 = R3 = OH, R2 = H
11a
b
OH
O
OH
OH O
C11H23
HO
C11H23
HO
C11H23
C11H23
OH
c
d
OH
OH
HO
13
14
15
e
OH O
C11H23
HO
Me
Me
OH
O
16
Scheme 3. Reagents: (a) dodecanoic anhydride, BF3ꢀEt2O or dodecanoic acid, ZnCl2; (b) C11H23COCl, AlCl3; (c) ZnHg, HCl; (d) C11H23COCl, AlCl3; (e) acetic anhydride, BF3ꢀEt2O.
R2
O
R2
O
R4
R5
a
R1
R3
R1
R3
Me
Me
17b R1 = R2 = R3 = OH, R4 = R5 = C11H23
17c R1 = R2 = R3 = OH, R4 = R5 = Me
17a R1 = R2 = R3 = OH
18a R1 = R3 = H, R2 = OH
17d R1 = R2 = R3 = OH, R4 = R5 = Benzyl
18b R1 = R3 = H, R2 = OH, R4 = R5 = C11H23
17a
b
O
OH
O
OH O
c
Me
HO
Me
HO
C11H23
OH
OH
Me
Me
17f
17e
Scheme 4. Reagents: (a) alkyl or benzyl anhydride, BF3ꢀEt2O or C11H23COCl, AlCl3; (b) acetic anhydride, BF3ꢀEt2O; (c) C11H23COCl, AlCl3.
Since the large structural variations of the four isomers of 1 are
all located on the flavanoid moiety which is predicted to extend
out of the active site, the binding affinity would not change among
the isomers of 1. The IC50 values of the four stereoisomers in Table
2 support this binding orientation.
Didodecanoylphloroglucinol (9) and 7,40-dihydroxyflavan were
prepared as two separate moieties and tested against human,
mouse and rat GIIA sPLA2s. 7,40-Dihydroxyflavan displayed IC50
s
of >3300 nM against human, mouse and rat GIIA sPLA2s (data not
shown), whereas 9 inhibited mouse and rat GIIA with low nanomo-

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R. C. Oslund et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5415–5419
Table 1
Inhibition of compound 1 against sPLA2s
sPLA2
IC50 (nM)
80 20
hGIIA
mGIIA
rGIIA
hGV
mGV
hGX
30
5
120
5
110 20
520 140
>1600
mGX
>1600
IC50 values are reported as the mean of triplicate analysis with standard deviations.
Table 2
Inhibition of compound 1 stereoisomersa against rat GIIA sPLA2s
OH
OH
HO
O
HO
O
HO
C11H23
OH
C11H23
HO
C11H23
OH
C11H23
O
OH
O
O
OH O
(- -)
(+ +)
OH
OH
HO
O
HO
O
Figure 2. (a) Compound 1 (green) overlaid onto Indole 8 (light blue) in the human
GIIA sPLA2 active site (PDB code: 1DB4). (b) The predicted interactions of compound
1 made with the active site Ca2+ and lys 62 models the acetylacetonate bidentate
ligand (the dodecanoyl chains were shortened to propanoyl chains to simplify
docking).
HO
C11H23
OH
HO
OH
C11H23 C11H23
C11H23
O
OH
O
O
OH O
Table 3
Inhibition of compounds 9 and 17b against mammalian sPLA2s
(+ -)
(- +)
IC50 (nM)
Inh. hGIIA
mGIIA
110 40 90 30 ꢂ1600
60 10 80 20
530 100 ꢂ1600 >1600 >1600
rGIIA
hGV
mGV
hGX
mGX
9
ꢂ1600
690 130
ꢂ1600 >1600 >1600
17b
Compound 1 stereoisomer
IC50 (nM)
Compounds were screened in duplicate at 1660 nM and reported as either
ꢂ1600 nM or >1600 nM if the inhibition was 650%. IC50 values below 1600 nM are
reported as the mean of triplicate analysis using four to five inhibitor concentra-
tions per analysis with standard deviations.
+ +
60 10
70 10
110 10
120 30
ꢁ ꢁ
+ ꢁ
ꢁ +
IC50 values are reported as the mean of duplicate analysis with standard deviations.
a
hydroxyl and the carbonyl groups are most likely involved in coor-
dinating the calcium ion and neighboring lysine residue.
Absolute stereochemistry was not determined.
We also prepared 16, 17c, 17d, and 17f. All four of these com-
pounds exhibited IC50 values of >1600 nM against human and
mouse GIIA, GV, and GX sPLA2s. The loss of extra hydrophobicity
most likely lowers the amount of inhibitor partitioned onto the
vesicle surface which lowers the amount of inhibitor that the en-
zyme ‘sees’ for inhibition. The fact that 16 does not give potent
inhibition suggests that the dodecanoyl groups also play an impor-
tant role in binding the active site of the enzyme.
lar potency. Interestingly, we found that 9 does not inhibit human
GIIA and GV sPLA2s with the same level of potency as 1 for reasons
we do not fully understand. Notwithstanding the lack of potency of
9 on human GIIA and GV sPLA2s, this result is consistent with pat-
ent work previously reported by Yamanouchi Pharmaceuticals.18
They reported that 9 and other diacylphloroglucinol derivatives
inhibit rabbit platelet sPLA2 with IC50’s somewhere in the range
of 0.010–30
l
M. Our work shows that 9 and 17b, a methyl deriva-
In summary, we have tested a class of inhibitors derived from
YM-26734 (compound 1) against GIIA, GV, and GX sPLA2s. The pre-
dicted binding pose of compound 1 in the active site of human GIIA
sPLA2 led to compounds 9 and 17b. These two compounds inhib-
ited mouse and rat GIIA sPLA2 with similar potency observed for
compound 1. This class of hydrophobic inhibitors may be useful
for mouse and rat cellular studies of sPLA2 function where cell-
permeable inhibitors with low nanomolar inhibition are needed.
tive of 9, inhibit mouse and rat GIIA with low nanomolar potency
(Table 3).
To investigate whether or not the carbonyl oxygens or hydroxyl
groups are important for sPLA2 inhibition, we synthesized 12b, 15,
and 18b. The inhibition potency for these three compounds was
>1600 nM against human and mouse GIIA, GV, and GX sPLA2s (data
not shown). This supports our predicted binding mode where the

R. C. Oslund et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5415–5419
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Acknowledgments
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This work was supported by an NIH Molecular Biophysics Train-
ing Grant (R.C.O.), the Mary Gates Foundation (N.C.) and a Merit
Award from the National Institutes of Health (M.H.G.)
(R37HL036235).
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Supplementary data
Synthesis of all compounds, enantiomeric separation of com-
pound 1, and enzyme assay details are available as Supplementary
data. Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2008.09.041.
14. Hamaguchi, K.; Koda, A.; Yamamoto, H.; Miyake, A.; Isogai, A.; Suzuki, A.; Pei,
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