1-(5-Carboxyindol-1-yl)propan-2-ones as inhibitors of human cytosolic phospholipase A2α: Synthesis and properties of bioisosteric benzimidazole, benzotriazole and indazole analogues

Article abstract of DOI:10.1016/j.bmcl.2009.03.019

The indole ring systems of the cytosolic phospholipase A₂α (cPLA₂α) inhibitor 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid (2) and the isomeric 6-carboxylic acid (3) were replaced by benzimidazole, benzotriazole and ind

Full text of DOI:10.1016/j.bmcl.2009.03.019

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2107–2111
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1-(5-Carboxyindol-1-yl)propan-2-ones as inhibitors of human cytosolic
phospholipase A2a: Synthesis and properties of bioisosteric benzimidazole,
benzotriazole and indazole analogues
*
Stefanie Bovens, Martina Kaptur, Alwine Schulze Elfringhoff, Matthias Lehr
Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Hittorfstrasse 58-62, D-48149 Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The indole ring systems of the cytosolic phospholipase A2
oxopropyl]indole-5-carboxylic acid (2) and the isomeric 6-carboxylic acid (3) were replaced by benz-
imidazole, benzotriazole and indazole scaffolds, respectively. The effect of the structural variations on
a
(cPLA2a) inhibitor 1-[3-(4-octylphenoxy)-2-
Received 12 February 2009
Revised 4 March 2009
Accepted 5 March 2009
Available online 10 March 2009
cPLA2a inhibitory potency, metabolic stability and solubility was studied. The lead 2 and the indazole-
5-carboxylic acid 28 were the metabolically most stable compounds in an assay with rat liver micro-
somes, while the benzimidazole-5-carboxylic acid derivative 13 possessed the best water solubility
(22 lg/mL at pH 7.4). The indazole-5-carboxylic acid 28 revealed the highest cPLA2a inhibitory potency
of the compounds in this series. With an IC50-value of 0.005
the lead 2.
Keywords:
Indole
Bioisosters
Cytosolic phospholipase A2
Metabolic stability
Solubility
l
M it was about sevenfold more active than
a inhibitors
Ó 2009 Elsevier Ltd. All rights reserved.
Cytosolic phospholipase A2
a
(cPLA2
a
) is an esterase that selec-
3, and also at position 2 of the indole scaffold by nitrogen. There-
fore, the heteroanalogous benzimidazole, benzotriazole and inda-
tively cleaves the sn-2 position of arachidonoyl glycerophospholip-
ids of biomembranes to produce free arachidonic acid and
lysophospholipids.1 Arachidonic acid is subsequently metabolized
to a variety of inflammatory mediators including prostaglandins
zole derivatives of 2 were synthesized and evaluated for cPLA2
a
inhibitory potency. Furthermore, the stability of the electrophilic
ketone groups of these compounds against metabolic reduction
was investigated in a test system applying rat liver microsomes,
since conversion of the ketone moiety of 2 to an alcohol function-
and leukotrienes. When the phospholipid substrate of cPLA2a is a
phosphatidylcholine with an ether function at the sn-1 position,
the lysophospholipid generated is the direct precursor of platelet
activating factor (PAF), another mediator of inflammation.2 Thus,
ality causes loss of cPLA2a
inhibition.15,16 Finally, also the aqueous
solubility of the target compounds was measured, since a sufficient
dissolution is one important constraint for oral bioavailability.
The synthesis of the benzimidazole analogue of 2 started from
methyl 1H-benzimidazole-5-carboxylate (4) (Scheme 1). N-Alkyl-
inhibition of cPLA2a would lead to the blockade of the cellular pro-
duction of all these proinflammatory lipid mediators. Therefore,
this enzyme is considered to be an attractive target for the design
of new anti-inflammatory drugs.3–5
Despite several potent inhibitors of cPLA2a having been devel-
oped, such as thiazolidinediones of Shionogi,6,7 benzhydrylindoles
of Wyeth8,9 and 1,3-diaryloxypropan-2-ones of AstraZeneca (1,
AR-C70484XX),10 only the compounds of Wyeth have undergone
clinical development.11 Recently, we have published a series of
cPLA2a inhibitors with indole scaffold like 2 and 3, structurally re-
lated to 1 (Fig. 1).12–14
Structure–activity relationship studies on indole-5-carboxylic
acid 2 have revealed that substitution of the indole 3-position
strongly influences the activity of the compound.12 We wanted
to investigate the effect of a replacement of the carbon at position
* Corresponding author. Tel.: +49 251 83 33331; fax: +49 251 83 32144.
E-mail address: lehrm@uni-muenster.de (M. Lehr).
Figure 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.019

2108
S. Bovens et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2107–2111
Scheme 1. Reagents and conditions: (a) Epichlorohydrin, KOH, Bu4 N+BrÀ, rt, 2 h; (b) 4-octylphenol, 4-dimethylaminopyridine, 120 °C, 30 min; (c) Dess–Martin periodinane
reagent, CH2Cl2, rt, 3 h; (d) triethyl orthoformate, ethanol, H2SO4, reflux, 12 h; (e) (1) aq NaOH, methanol, reflux, 1,5 h, (2) aq HCl, THF, reflux, 3 h.
ation with epichlorohydrin yielded two isomeric products, which
were shown to be the benzimidazole-5-carboxylic and -6-carbox-
ylic ester derivatives 5 and 6 (ratio about 1:1 evaluated by 1H
NMR spectroscopy). Since 5 and 6 as well as the following methyl
ester derivatives 7, 8–11, 12 could not be separated successfully
by chromatography on silica gel, mixtures of the isomers were ap-
plied as reactants in the subsequent reaction steps. Regioselective
opening of the oxirane ring of 5, 6 was achieved without solvent
in the presence of catalytic amounts of 4-dimethylaminopyridine.
Oxidation of the resulting alcohol intermediates 7, 8 to the desired
ketones 9, 10 was carried out with Dess–Martin periodinane re-
agent. Next the ketone groups of 9, 10 were acetalized with tri-
ethyl orthoformate in the presence of catalytic amounts of
H2SO4. The methyl ester groups of obtained compounds 11, 12
were saponified with aqueous NaOH. Finally, the acetal protecting
group was removed by aqueous HCl in THF to yield a mixture of
benzimidazole-5- and -6-carboxylic acid derivatives 13 and 14.
These two acids could be separated by reversed phase HPLC.
The structures of 13 and 14 were assigned by NOE experiments.
Irradiation of the N–CH2–CO protons of 13 gave NOE-enhance-
ments of the doublet signal of the proton at C-7 of the benzimid-
azole at 7.17–7.19 ppm, confirming that 13 is the benzimidazole-
5-carboxylic acid derivative. The structure of the other isomer
conclusively was established as being benzimidazole-6-carboxylic
acid 14.
Reaction of methyl 1H-benzotriazole-5-carboxylate (15) with
2-(4-octylphenoxymethyl)oxirane12 also led to a mixture of two
isomers (16, 17) (Scheme 2). From these, the target benzotria-
zole-5- and -6-carboxylic acids 22 and 2317 were afforded, using
the reaction sequence described above for the synthesis of 13
and 14 from 7 and 8. Separation of the target compounds 22 and
23 was performed by normal phase chromatography on silica gel.
Scheme 2. Reagents and conditions: (a) 2-(4-Octylphenoxymethyl)oxirane, NaH, DMF, 90 °C, 2 h; (b) Dess–Martin periodinane reagent, CH2Cl2, rt, 3 h; (c) triethyl
orthoformate, ethanol, H2SO4, reflux, 6.5 h; (d) (1) aq NaOH, methanol, reflux, 1.5 h, (2) aq HCl, THF, reflux, 8 h.

S. Bovens et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2107–2111
2109
Scheme 3. Reagents and conditions: (a) Epichlorohydrin, KOH, Bu4 N+BrÀ, rt, 2 h; (b) 4-octylphenol, 4-dimethylaminopyridine, 120 °C, 45 min; (c) Dess–Martin periodinane
reagent, CH2Cl2, rt, 3 h; (d) aq KOH, ethanol, rt, 18 h.
Scheme 3 outlines the synthesis of indazole-5-carboxylic acid
28. The 2-oxopropyl substituent was introduced in position 1 of
methyl indazole-5-carboxylate (24) applying the reaction se-
quence described for the synthesis of 9 and 10 (Scheme 1). The
methyl ester group of obtained compound 27 was hydrolyzed with
aqueous KOH in ethanol to give the target compound 28.18 Inda-
zole-6-carboxylic acid derivative 29 was synthesized in the same
way starting from methyl indazole-6-carboxylate.
Evaluation of the inhibitory activity against cPLA2a19 showed
that introduction of a nitrogen atom at position 3 of indole-5-car-
boxylic acid 2 led to decrease of activity. With an IC50 of 0.15 lM
the benzimidazole-5-carboxylic acid 13 is about fourfold less po-
tent than indole-5-carboxylic acid 2 (Table 1). In contrast, shifting
the nitrogen from position 3 to position 2 significantly increased
enzyme inhibitory potency, as seen by the inhibition data of the
indazole-5-carboxylic acid 28 (IC50: 0.005
lM). The benzotria-
Indole-5-carboxylic acid 2 has been found to be a potent inhib-
zole-5-carboxylic acid 22 (IC50: 0.026 M) again has about the
l
itor of cPLA2
a
with an IC50-value of 0.035
l
M against the enzyme
same activity as the indole lead 2. Here the negative effect of nitro-
gen in position 3 seems to be compensated by the positive effect of
nitrogen in position 2 of the heterocycle.
The structure–activity relationships of the 6-carboxylic acid ser-
ies synthesized differ remarkably from that of the corresponding 5-
carboxlic acids. Here, as well nitrogen in position 3 as nitrogen in
position 2 led to a significant increase of enzyme inhibition. While
isolated from human platelets.12 In the course of further struc-
ture–activity relationship studies, one aim was to replace the in-
dole scaffold of 2 by benzimidazole, benzotriazole, and indazole,
respectively. During the synthesis of the benzimidazole and the
benzotriazole bioisosters of 2, not only the desired 5-carboxylic
acids 13 and 22, but also the isomeric 6-carboxylic acids 14 and
23 were obtained. Therefore, we included carboxylic acid deriva-
tives with carboxylic acid group in position 6 of the heterocycles
in our investigations too.
the indole-6-carboxylic acid 3 possesses an IC50 of 1.1
lM, the IC50-
values of the benzimidazole- and indazole-6-carboxylic acids 14
and 29 are 0.085
lM and 0.026 lM, respectively. Concomitant
Table 1
cPLA2
a
-inhibitory potency, metabolic stability and thermodynamic solubility
N
N
O
O
O
O
N
COOH
C8H17
COOH
C8H17
13,14
2,3
N
O
O
N
N
O
N
O
N
COOH
COOH
C8H17
C8H17
28,29
22,23
Metabolic stabilityb (%)
Thermodynamic solubilityc
(lg/mL)
a
Compd
Position of the COOH-group
Inhibition of cPLA2
a
IC50
(l
M)
2
3
5
6
5
6
5
6
5
6
0.035
1.1
0.15
0.085
0.026
0.016
0.005
0.026
65
39
49
40
43
44
64
36
5
<1
<1
22
7
<1
2
<1
5
13
14
22
23
28
29
8
0.8
0.2
0.8
a
Values are the means of at least two independent determinations, errors are within 20%; IC50-value of reference inhibitor 1 (AR-C70484XX): 0.011
l
M.10,12
Percentage of parent compound remaining after metabolism by rat liver microsomes; values are the means of at least two independent determinations; in case of 2:
b
mean standard deviation, n = 5.
c
Mean standard deviation, n = 3, in case of values <1 lg/mL: n = 2.

2110
S. Bovens et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2107–2111
introduction of two nitrogen atoms in position 3 and 2 had an addi-
tive effect. Thus, the activity of the benzotriazole-6-carboxylic acid
comitantly possessing high cPLA2a-inhibitory potency, adequate
water solubility and good stability against metabolic reduction of
23 (IC50: 0.016 lM) is higher than that of 14 and 29, respectively.
the pharmacophoric ketone group.
Structure–activity relationship studies have revealed that an
important pharmacophoric element of 2 is the activated ketone
References and notes
moiety.12,15 Its reduction to an alcohol results in a loss of cPLA2
a
inhibitory potency. Like other compounds with electrophilic ke-
tone groups,20,21 2 is metabolically susceptible to keto reduc-
tion.14,16 To investigate metabolic stability of the ketone group of
the new compounds, their metabolisation by rat liver microsomes
was also studied.19 After incubation of the microsomes in presence
of NADPH still about 65% of the indole-5-carboxylic acid 2 could be
measured by HPLC and UV-detection in comparison with reference
incubations in absence of NADPH. The main metabolite formed
was the corresponding inactive alcohol as shown by LC–MS exper-
iments performed as described recently.14,16 Indazole-5-carboxylic
acid 28 showed about the same metabolic stability (64%) as the
corresponding indole derivative 2, while all other compounds eval-
uated were less stable (Table 1). At the end of the metabolic reac-
tions only 30–50% of these substances were detectable yet.
Comparing the metabolism of the 5-carboxylic acids with that of
the 6-carboxylic acids, one striking difference could be observed.
In case of the 5-carboxylic acid derivatives 2, 13, 22 and 28, LC–
MS experiments showed that the corresponding alcohols of the
compounds were the main metabolites. Contrary, in case of the
6-carboxylic acid derivatives 3, 14, 23 and 29 only small amounts
of their alcohol forms could be detected. Instead several more polar
metabolites occurred.
1. Kudo, I.; Murakami, M. Prostaglandins Other Lipid Mediators 2002, 68-69, 3.
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3. Connolly, S.; Robinson, D. H. Expert Opin. Ther. Pat. 1995, 5, 673.
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M.; Fujii, Y.; Yamada, M.; Ogawa, T.; Okada, T.; Hashizume, H.; Kii, M.; Hara, S.;
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McKew, J. C. J. Med. Chem. 2007, 50, 1380.
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B.; Sum, F. W.; Tam, S.; Hu, Y.; Chen, L.; Kirincich, S. J.; Michalak, R.; Thomason,
J.; Ipek, M.; Wu, K.; Wooder, L.; Ramarao, M. K.; Murphy, E. A.; Goodwin, D. G.;
Albert, L.; Xu, X.; Donahue, F.; Ku, M. S.; Keith, J.; Nickerson-Nutter, C. L.;
Abraham, W. M.; Williams, C.; Hegen, M.; Clark, J. D. J. Med. Chem. 2008, 51,
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10. Connolly, S.; Bennion, C.; Botterell, S.; Croshaw, P. J.; Hallam, C.; Hardy, K.;
Hartopp, P.; Jackson, C. G.; King, S. J.; Lawrence, L.; Mete, A.; Murray, D.;
Robinson, D. H.; Smith, G. M.; Stein, L.; Walters, I.; Wells, E.; Withnall, W. J. J.
Med. Chem. 2002, 45, 1348.
11. Khurdayan, V.; Cullell-Young, M. Drug News Perspect. 2005, 18, 277.
12. Ludwig, J.; Bovens, S.; Brauch, C.; Schulze Elfringhoff, A.; Lehr, M. J. Med. Chem.
2006, 49, 2611.
13. Hess, M.; Schulze Elfringhoff, A.; Lehr, M. Bioorg. Med. Chem. 2007, 15, 2883.
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16, 3489.
Due to the two long acyl chains, the phospholipid substrates of
cPLA2a possess a substantial lipophilicity. Therefore it can be ex-
15. Ludwig, J. PhD Thesis, University of Münster, Germany, 2004.
16. Fabian, J.; Lehr, M. J. Pharm. Biomed. Anal. 2007, 43, 601.
pected that inhibitors, which shall bind competitively to the active
site of the enzyme, must possess similar properties. This assump-
17. Final step of the synthesis of 1-[3-(4-octylphenoxy)-2-oxopropyl]-1H-
benzotriazole-6-carboxylic acid 23: To solution of a mixture of 20 and 21
(170 mg, 0.33 mmol) in methanol (10 mL) was added a solution of NaOH
(1.0 g) in water (120 mL). The mixture was heated under reflux for 1.5 h. After
acidifying with 6 M HCl, the mixture was concentrated to about 20 mL, treated
with THF (30 mL) and heated under reflux for additional 8 h. After
concentrating until some precipitates appeared, the reaction mixture was
extracted exhaustively with ethyl acetate. The organic layers were dried
(Na2SO4) and the solvent was evaporated. The two isomers 22 and 23 were
separated and purified by chromatography on silica gel (0.015–0.040 mm)
eluting with hexane/ethyl acetate/formic acid (1. 8:2:0.5; 2. 7:3:0.5) to yield 22
(67 mg) and 23 (12 mg) as solids; mp 211–212 °C and 174–176 °C,
respectively. Compound 23: 1H NMR (400 MHz, DMSO-d6): d (ppm) 0.82 (t,
J = 6.8 Hz, 3H), 1.21–1.24 (m, 10H), 1.50–1.52 (m, 2H), 2.48–2.49 (m, 2H), 5.12
(s, 2H), 6.11 (s, 2H), 6.90 (d, J = 8.6 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H), 7.94 (d,
J = 8.6 Hz, 1H), 8.13 (d, J = 8.6 Hz, 1H), 8.52 (s, 1H), 13.26 (s, broad, 1H). 1D NOE
(600 MHz, DMSO-d6): d (ppm) 1H(irr)/1H(res) 6.11 (COCH2 N)/8.52 (benzotriazole
H-7) and 5.12 (OCH2CO); 8.52 (benzotriazole H-7)/6.11 (COCH2 N). MS (ESI+):
m/z 424 [M+H]+.
tion is confirmed by the fact that all known cPLA2a inhibitors with
pronounced potency bear larger lipophilic residues,6–10,12 which
lead to a high total lipophilicity of the compounds. Such a high
lipophilicity can cause a low water solubility, which may result
in poor drug absorption, because the drug does not dissolve suffi-
ciently in the aqueous content of the gastrointestinal tract. Thus,
we also measured the aqueous solubility of all new compounds un-
der thermodynamic conditions19,22 by equilibrating the solids in
phosphate buffer (pH 7.4) for 20 h at room temperature, separating
non-soluble material by centrifugation and measuring the soluble
aqueous concentration by HPLC. Both indolecarboxylic acid deriv-
atives (2,3) possess a water solubility of less than 1
1). Replacement of the indole scaffold by a benzimidazole resulted
in more soluble compounds (13: 22 g/mL; 14: 7 g/mL). With sol-
ubility values of less than 1 g/mL, benzotriazole- and indazole-5-
lg/mL (Table
l
l
18. Final step of the synthesis of 1-[3-(4-octylphenoxy)-2-oxopropyl]indazole-5-
carboxylic acid 28: A solution of 27 (68 mg, 0.16 mmol) in ethanol (6 mL) was
treated with a solution of 10% aqueous KOH (2 mL). The mixture was stirred at
room temperature for 18 h. After acidification with 1 M HCl, the reaction
mixture was extracted exhaustively with ethyl acetate. The combined organic
layers were dried (Na2SO4) and the solvent was distilled off. Chromatography
on silica gel (hexane/ethyl acetate/formic acid, 8:2:0.1) yielded 28 (28 mg,
43%). For further purification, an aliquot of the product (11 mg) was subjected
to semi-preparative RP-HPLC applying acetonitrile/H2O/formic acid
(800:200:0.4) as mobile phase. The eluates were concentrated under reduced
pressure until most of the acetonitrile was removed. Freeze drying of the
l
carboxylic acids 22 and 28 again have only poor water solubility.
The dissolution values of the benzotriazole- and indazole-6-car-
boxylic 23 (2
of 1
lg/mL) and 29 (5 lg/mL) slightly exceed the amount
l
g/mL. Solubility guidelines for drugs under development are
given by Lipinski and co-workers.22 According to those, com-
pounds with mid-range permeability and average potency should
possess a minimum thermodynamic solubility of 50
lg/mL. With
a
solid; mp 160–162 °C. 1H NMR
a solubility of 22 g/mL, 13 comes closest to this limit.
l
remaining solution gave 28 (8 mg) as
(400 MHz, DMSO-d6): d 0.83 (t, J = 6.8 Hz, 3H), 1.16–1.28 (m, 10H), 1.45–1.55
(m, 2H), 2.45–2.50 (m, 2H), 5.01 (s, 2H), 5.68 (s, 2H), 6.85 (d, J = 8.6 Hz, 2H),
7.09 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 8.9 Hz, 1H), 7.93 (dd, J = 8.8 Hz and J = 1.5 Hz,
1H), 8.27–8.28 (m, 1H), 8.44–8.45 (m, 1H). MS (ESI+): m/z 423 [M+H]+.
In conclusion, we have studied the effect of the replacement of
the indole scaffold of 2 and 3 by benzimidazole, benzotriazole and
indazole on cPLA2a inhibitory potency, metabolic stability and sol-
ubility. The compounds with the highest metabolic stability were
indole-5- and indazole-5-carboxylic acids 2 and 28. Substitution
of indole by benzimidazole increased water solubility significantly.
The indazole-5-carboxylic acid 28 showed the highest activity
19. Inhibition of cPLA2
a: The target compounds were evaluated in an assay applying
cPLA2a
isolated from human platelets.23 Enzyme activity was measured in a
solution containing covesicles of 1-stearoyl-2-arachidonoyl-sn-glycero-3-
phosphocholine (SAPC) and 1,2-dioleoyl-sn-glycerol (DOG). Inhibitory
potency of the test compounds was assessed by comparing the amount of
arachidonic acid released from SAPC in their absence and presence after an
incubation time of 60 min with reversed phase HPLC and UV-detection at
200 nm. Metabolic stability: Test compounds were incubated with rat liver
microsomes under aerobic conditions in absence and presence of the cofactor
NADPH as described previously.14 The metabolic reactions were terminated
against cPLA2
fold more active than the lead 2 and represents one of the most po-
tent inhibitors of cPLA2
known today.3–5 The aim of further
studies will be to develop derivatives of these compounds, con-
a. With an IC50-value of 0.005 lM, 28 is about seven-
a

S. Bovens et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2107–2111
2111
after 30 min. The extent of metabolism was evaluated with reversed phase
HPLC and UV-detection. The structures of the main metabolites were
confirmed by LC/MS analysis.14,16 Water Solubility: Water solubility was
determined experimentally according to published procedures.14,24 Briefly,
sodium phosphate buffer (pH 7.4) was added to a test compound, and the
suspension obtained was equilibrated by sonication (10 min) and shaking at
room temperature (20 h), followed by centrifugation. An aliquot of the
supernatant was diluted with acetonitrile, and the concentration of the test
compound was determined by reversed phase HPLC and UV-detection using a
regression curve.
20. Riendeau, D.; Guay, J.; Weech, P. K.; Laliberté, F.; Yergey, L.; Li, C.;
Desmarais, S.; Perrier, H.; Liu, S.; Nicoll-Griffith, D.; Street, I. P. J. Biol. Chem.
1994, 269, 15619.
21. Connolly, S. Abstract book; 10th Mainzer Forum Medicinal Chemistry; Mainz,
Germany, 2002.
22. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
1997, 23, 3.
23. Schmitt, M.; Lehr, M. J. Pharm. Biomed. Anal. 2004, 35, 135.
24. Kim, I. H.; Heirtzler, F. R.; Morisseau, C.; Nishi, K.; Tsai, H. J.; Hammock, B. D. J.
Med. Chem. 2005, 48, 3621.