Highly specific and broadly potent inhibitors of mammalian secreted phospholipases A2

Article abstract of DOI:10.1021/jm800422v

We report a series of inhibitors of secreted phospholipases A₂ (sPLA₂s) based on substituted indoles, 6,7-benzoindoles, and indolizines derived from LY315920, a well-known indole-based sPLA₂ inhibitor. Using the human group X sPLA₂ crystal structure, we prepared a highly potent and selective indole-based inhibitor of this enzyme. Also, we report human and mouse group IIA and IIE specific inhibitors and a substituted 6,7-benzoindole that inhibits nearly all human and mouse sPLA ₂s in the low nanomolar range.

Full text of DOI:10.1021/jm800422v

4708
J. Med. Chem. 2008, 51, 4708–4714
Highly Specific and Broadly Potent Inhibitors of Mammalian Secreted Phospholipases A2
Rob C. Oslund,† Nathan Cermak,† and Michael H. Gelb*,†,‡
Departments of Chemistry and Biochemistry, UniVersity of Washington, Seattle, Washington 98195
ReceiVed April 11, 2008
We report a series of inhibitors of secreted phospholipases A2 (sPLA2s) based on substituted indoles, 6,7-
benzoindoles, and indolizines derived from LY315920, a well-known indole-based sPLA2 inhibitor. Using
the human group X sPLA2 crystal structure, we prepared a highly potent and selective indole-based inhibitor
of this enzyme. Also, we report human and mouse group IIA and IIE specific inhibitors and a substituted
6,7-benzoindole that inhibits nearly all human and mouse sPLA2s in the low nanomolar range.
Introduction
Secreted phospholipases A2 (sPLA2s)a are a family of
disulfide-rich, Ca2+-dependent enzymes that hydrolyze the sn-2
position of glycero-phospholipids to release a fatty acid and a
lysophospholipid.1 The mouse genome encodes 10 sPLA2s
(groups IB, IIA, IIC, IID, IIE, IIF, III, V, X, XIIA), whereas
the human genome encodes all of these except the group IIC
enzyme, which occurs as a pseudogene.2,3 More than a decade
ago there was interest in human group IIA sPLA2 (hGIIA) as
Figure 1. Substituted indole and indolizine sPLA2 inhibitors.
sPLA2s.17 In the current study we have taken a structure-
an anti-inflammatory drug target because it was found at high
concentrations in synovial fluid from arthritis patients,4 although
a clinical trial with an inhibitor against hGIIA failed to show
efficacy in the treatment of rheumatoid arthritis.5 Interest in
inhibitors of sPLA2s has remained because of the possible
involvement of these enzymes in inflammation. For example,
studies with mGX- and mGV-deficient mice show that these
sPLA2s contribute to airway inflammation in a mouse model
of allergic asthma.6,7 Studies with macrophages from mGV-
deficient mice show a partial reduction in eicosanoid production
in response to agonists.8
guided approach using the X-ray structure of hGX16,18 to
obtain inhibitors in the class shown in Figure 1 that are highly
specific for hGX. Along the way we also obtained a highly
specific inhibitor that binds only to hGIIA, mGIIA, hGIIE,
and mGIIE as well as a broadly potent inhibitor that shows
strong inhibition against human and mouse GIB, GIIA, GIID,
GIIE, GIIF, GV and GX sPLA2s. These compounds may be
useful in the study of the role of various mammalian sPLA2s
in cellular and whole animal responses.
Substituted indoles and indolizines first reported by workers
at Lilly and Shionogi are the most potent sPLA2 inhibitors and
the ones with drug potential in terms of pharmacokinetic profiles.
Compounds in this group include the indolizine Indoxam and
the substituted indoles Me-Indoxam and 1 (LY315920; Figure
1).9–12 The development of these compounds is an early example
of structure-guided improvement of binding potency starting
from a lead compound obtained through high-throughput
screening13 and making use of the X-ray structure of hGIIA.14
With the availability of the full set of mouse and human
recombinant sPLA2s, it has been recently possible to explore
the specificity of these compounds against all mammalian
family members.15–17 For example, Me-Indoxam inhibits
hGIIA, mGIIA, mGIIC, hGIIE, mGIIE, hGV, and mGV
sPLA2s with low nanomolar potency, is less potent on hGIB,
mGIB, hGX, and mGX, and inhibits hGIID, mGIID, hGXIIA,
and mGXIIA only at micromolar concentrations.15 Compound
1 potently inhibits hGIIA, mGIIA, hGIIE, mGIIE, hGX, and
mGX enzymes and is less potent on the other mammalian
Chemistry
Reported compounds were prepared using slightly modified
routes.9–12,17,19 The substituted indole and 6,7-benzoindole
inhibitors were prepared using analogous routes starting from
2-carbomethoxy-4-methoxy-indole 4a and 2-carbomethoxy-4-
methoxy-6,7-benzoindole 4b, respectively. However, because
4b could not be purchased commercially, it was prepared from
commercially available 3-methoxy-2-naphthalenemethanol 2a
(Scheme 1). 3-Methoxy-2-naphthalenemethanol (2a) was oxi-
dized with PCC to form the aldehyde 2b. The aldehyde was
treated with methyl azidoacetate and sodium methoxide to form
the azidocinamate 3. Ring closure of 3 was achieved via the
Hemetsberger reaction to give 2-carbomethoxy-4-methoxy-6,7-
benzoindole 4b.
Indole-based inhibitors 11c, 11d, 12a, and 12b were prepared
by N-1 benzylation of commercially available 4a using sodium
hydride as the base to yield 5a (Scheme 2). The methyl ester
was saponified to form the 2-carboxylic acid indole 6a. The
2-acetyl indole 7a was formed by treatment of 6a with
methyllithium. Reduction of the ketone was carried out with
NaBH4 to yield 8a. Deoxygenation of 8a was achieved using a
mixture of NaBH4 and trifluoroacetic acid to give 9a. The
2-isobutyl indole intermediate 9b was prepared in a similar
fashion as 9a except isobutyllithium was used in place of
methyllithium to form 7b with subsequent transformations to
give 9b. Compounds 10a-d were prepared by first deprotecting
* To whom correspondence should be addressed. Tel.: 206-543-7142.
Fax: 206-685-8665. E-mail: gelb@chem.washington.edu.
†
Department of Chemistry.
Department of Biochemistry.
‡
a Abbreviations: hGIIA, human group IIA secreted phospholipase A2
(likewise for other group names); mGIIA, mouse group IIA secreted
phospholipase A2 (likewise for other group names); sPLA2, secreted
phospholipase A2.
10.1021/jm800422v CCC: $40.75
2008 American Chemical Society
Published on Web 07/08/2008

Inhibitors of Phospholipases A2
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4709
Scheme 1a
a Reagents and conditions: (a) PCC, NaAcetate in CH2Cl2; (b) methyl
azidoacetate, NaOMe in THF; (c) xylene or toluene, reflux.
Scheme 2a
Figure 2. An overlay of hGIIA (green) and hGX (magenta) with 12b
docked into the active site. The isoleucine of hGIIA, but not the valine
of hGX, provides extra hydrophobic bulk near the 2-isobutyl group on
the indole ring and presumably excludes the 2-isobutyl indole from
the active site.
hGX.17 We explored this gain in selectivity by docking indole
compounds with larger 2-alkyl groups into the hGIIA and hGX
sPLA2 active sites of existing X-ray crystal structures13,16 using
the FLO/QXP docking program.20 An overlay of the hGIIA and
hGX enzyme structures (rms CR ) 0.98 Å) revealed a region
of extra space in the hGX active site not present in hGIIA. This
difference in hydrophobic space results mostly from a change
in one amino acid residue. hGIIA has an isoleucine whereas
hGX has a valine in the active site region which is contacted
by the 2-position substituent on the indole ring (Figure 2). Larger
2-alkyl substituents would clash with this portion of the hGIIA
active site but not in the case of hGX. Our designs were
supported by data from workers at Shionogi showing that
2-isobutyl indole and indole-like inhibitors selectively inhibited
the hGX enzyme.21 However, this report only included IC50
values for these compounds against hGIB, hGIIA, hGV, and
hGX. As a group X specific inhibitor would be extremely useful,
we wanted to test 2-isobutyl indole derivatives against all human
and mouse sPLA2 enzymes.
Also, in attempts to increase hydrophobicity of these com-
pounds in order to make them more cell permeable, docking
studies revealed that larger substituents such as arylsulfonamides
or alkylsulfonamides could replace the carboxylic acid OH group
on the indole scaffold. In our previous studies, addition of a
methyl group to the 6-position on the indole scaffold did not
affect inhibition potency against the various sPLA2s tested.17
Larger groups including a benzene ring fused to the 6,7-position
of the indole scaffold were also docked into the active site
without affecting key binding interactions.
a Reagents and conditions: (a) NaH, BnBr in DMF; (b) 30% KOH/
MeOH/THF (2:1:1), reflux; (c) MeLi or isopropyllithium in THF; (d) NaBH4
in EtOH/THF; (e) NaBH4, TFA in CH2Cl2; (f) (1) BBr3 in CH2Cl2, (2)
NaH R2COCHR3Br in DMF; (g) (1) oxalyl chloride in CH2Cl2, (2) NH3(g);
(h) 1.5 M NaOH in MeOH/THF or 20% TFA in CH2Cl2.
the 4-methoxy substituent on 9a and 9b using BBr3 followed
by addition of the appropriate alkyl bromoactetate or 2-bromo-
N-(arylsulfonyl)acetamide with sodium hydride as the base.
Addition of the oxalamide group to the indole was carried out
by treating 10a-d with oxalyl chloride followed by addition
of ammonia gas to give compounds 11a-d. Deprotection of
the indole esters 11a and 11b was carried out with NaOH to
give 12a or with trifluoroacetic acid to yield 12b.
Preparation of the 6,7-benzoindole inhibitors 11g, 11h, 12e,
and 12f was done using identical routes described for the
substituted indole inhibitors (Scheme 2). Compounds 14a and
14b, N-methyl amides 15a and 15b, and all 11d derivatives
were prepared using analogous steps to those outlined in Scheme
2. All Indoxam derivatives (15c and 16a-c) were prepared using
similar techniques to those already described.12
In Vitro Inhibiton. Using a fluorometric sPLA2 assay,16 the
substituted indoles, 6,7-benzoindoles, and indolizines were tested
against the full panel of human and mouse sPLA2 enzymes,
with the exception of mGIIC (because humans contain a group
IIC pseudogene) and mGXIIA, which has 94% sequence identity
to hGXIIA.15 All reported compounds in this study except
13a-i, 14b, and 15a-c were tested against hGIII and hGXIIA
sPLA2 enzymes, and gave <50% inhibition for both enzymes
at 1.6 µM concentrations. The active sites of GIII and GXIIA
sPLA2 are predicted to be significantly different than those of
the other mammalian sPLA2s, and this probably explains why
the indole/indolizine set of inhibitors lack potency on GIII and
GXIIA enzymes. IC50 values generated against hGIID were
Results and Discussion
Molecular Modeling. We recently reported that compound
1 was 30 times more potent than the 2-methyl indole against

4710 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Oslund et al.
Table 1. IC50 Values of Substituted Indole Inhibitors against Human and Mouse sPLA2sa
a IC50 values are reported as the mean of duplicate or triplicate analysis with standard deviations. Each compound was screened at 1660 nM and reported
as >1600 nM if the inhibition was <50%. b IC50 values for GIB, GIIA, GIIE, GV, and GX from ref 17. Retest of this compound against hGV and mGV
gave 110 ( 30 and 160 ( 20 nM, respectively. c IC50 values obtained using E. coli membrane assay. Each compound was screened at 1330 nM and reported
as >1300 nM if the inhibition was <50%.
Table 2. IC50 Values of 11d Derivatives against hGX sPLA2a
obtained using the [3H]oleic acid-labeled E. coli membrane
assay, which was preferred for this enzyme because of the higher
sensitivity achieved over the fluorometric assay. Data in Table
1 show that 11d and 12b are highly selective for hGX over all
other human and mouse sPLA2s. Thus, the large isobutyl group
is well tolerated only by hGX, which is consistent with modeling
studies. Interestingly, these compounds lack potency against
mGX despite the fact that hGX and mGX share 72% sequence
identity. Structural alignment reveals that mGX does not contain
a valine in the active site region that contacts the indole
2-position like hGX, but rather a leucine. This extra hydrophobic
bulk sterically excludes the 2-isobutyl indoles from the mGX
active site in similar fashion as with GIIA. Other sPLA2s such
as GIB, GIIE, and GV also have an isoleucine in this region
like the GIIA enzyme. However, GIID and GIIF have a valine
in this region like human GX, which supports the fact that the
2-isobutyl compounds 11h and 12f display somewhat increased
potency against GIID and GIIF enzymes.
A small subset of 11d derivatives were synthesized and tested
against hGX sPLA2 (Table 2). As initial docking studies
predicted that the phenylsulfonamide group would extend out
of the active site, it was surprising to see a 38-fold difference
in inhibition for compounds 13b-d when the phenyl ring was
substituted with a chlorine at the para-, meta-, and ortho-
positions (Table 2). Compounds 13d, and 13f, with substitutions
at the ortho-position with a chloro- or methyl- group, resulted
in higher inhibition potency over 11d (Table 2). It is possible
that the extra methyl or chlorine groups pack into a small pocket
of the active site, which would increase the binding affinity.
However, replacing the phenylsulfonamide on 11d with a
methylsulfonamide (13h) also increases potency against hGX
(Table 2). Without a crystal structure, it is difficult to conclude
how this pheynlsulfonamide is contacting the enzyme active
a IC50 values are reported as the mean of duplicate or triplicate analysis
with standard deviations.
site.
The 6,7-benzoindole inhibitors display general potency
against all tested human and mouse sPLA2 enzymes (Table 3).
Because the extra hydrophobic bulk is predicted not to make
direct contact with the enzyme, the increased potency is likely
due to increased partitioning of the inhibitor into the phospho-

Inhibitors of Phospholipases A2
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4711
Table 3. IC50 Values of Substituted Benzo-Fused Indole Inhibitors against Human and Mouse sPLA2sa
a IC50 values are reported as the mean of duplicate or triplicate analysis with standard deviations. Each compound was screened at 1660 nM and reported
as >1600 nM if the inhibition was <50%. b IC50 values obtained using E. coli membrane assay. Each compound was screened at 1330 nM and reported as
>1300 nM if the inhibition was <50%.
Compound 14a was also potent against hGIIE and mGIIE,
consistent with trends observed for other potent group IIA
indole-based inhibitors. Poor inhibiting control compounds were
successfully designed by introduction of an N-methyl group on
the oxalamide of the indole scaffold to give compounds
15a-15c (Figure 3). Analysis of the co-crystal structure
containing Me-Indoxam in the hGX active site reveals that the
introduced N-methyl group disrupts a key hydrogen bond with
either a histidine or aspartate residue, while also introducing
extra hydrophobic bulk into the active site.16 All N-methyl
oxalamide control compounds had IC50 values that were >30-
fold higher than their parent compound (Figure 3).
The 2-isobutyl Indoxam derivative 16a was synthesized and
found to poorly inhibit sPLA2 enzymatic activity (Table 4).
Since Indoxam does not inhibit hGX in the low nanomolar range
(Table 4), it is not surprising that 16a fails to inhibit hGX. This
result suggests that poor inhibition of hGX activity by Indoxam
Figure 3. Control compounds designed by removing the functionality
or it derivatives has more to do with the indolizine heterocyle
from the 4-postion (14b) or by methylating the oxalamide (15a-c).
and not the substituents present on the ring. Interestingly, the
Control compounds are >30-fold less potent than their parent compound
8-oxopropanone derivative 16b and the 8-methoxy derivative
16c were selectively potent against hGIIA and hGIIE which
was similar to the gain in selectivity displayed by 14a. We also
prepared 15c (Figure 3), which did not significantly inhibit
hGIIA at concentrations below 1600 nM.
(listed below the compound in parenthesis) when tested against hGX
(14b and 15a), human and mouse GIIA, GV, and GX (15b), or human
GIIA (15c).
lipid substrate vesicles, which increases the ratio of XI/KI* (XI
is the mole fraction of inhibitor in the interface and KI* is the
interfacial dissociation constant).22,23 Of particular note is
compound 12e that inhibited human and mouse groups IB, IIA,
IID, IIE, IIF, V, and X sPLA2s with an IC50 of less than 350
nM (Table 3). We also sought structurally similar compounds
that would be devoid of sPLA2 binding activity because such
compounds are useful as controls in cellular studies. The X-ray
structure of an Indoxam analogue bound to hGIIA and Me-
Indoxam bound to hGX show that the carboxyl group of the
substituent at the 4-position of the indole directly coordinates
Conclusion
A series of indole- and indolizine-based compounds were
synthesized and tested against the full set of human and mouse
sPLA2 enzymes. Compound 11d was found to be selectively
potent against hGX over all other human and mouse sPLA2
enzymes. Derivatives of 11d, such as 13h, were also found to
bind with higher affinity to the hGX enzyme active site and
may help in further studies of hGX sPLA2 function. An inhibitor
selective for mouse and human GIIA and GIIE sPLA2 (14a) as
well as selective human GIIA and GIIE inhibitors (16b and 16c)
were also identified from this group of compounds. Compound
12e is potent against human and mouse groups IB, IIA, IID,
IIE, IIF, V, and X and is the most generally potent sPLA2
inhibitor reported to date. It is also the first reported potent
to the active site Ca2+ 16,24
We thus synthesized 14a and 14b
.
with only a methoxy group at the 4-position to remove the
interaction made between the inhibitor and Ca2+. Surprisingly,
while 14b (Figure 3) gave an IC50 of 1000 nM against hGX
(data not included in table), 14a had an IC50 of 14 and 34 nM
against human and mouse GIIA, respectively (Table 3).

4712 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Oslund et al.
Table 4. IC50 Values of Substituted Indolizine Inhibitors against Human and Mouse sPLA2sa
a IC50 values are reported as the mean of duplicate or triplicate analysis with standard deviations. Each compound was screened at 1660 nM and reported
as >1600 nM if the inhibition was <50%. b IC50 values obtained using E. coli membrane assay. Each compound was screened at 1330 nM and reported as
>1300 nM if the inhibition was <50%.
the solvent was removed by rotary evaporation. The crude white
solid was purified by column chromatography on silica gel (20%
EtOAc/80% hexanes) to give a white solid (820 mg, 75% yield).
1H NMR (300 MHz, CDCl3) δ 3.85 (s, 3H), 4.06 (s, 3H), 6.34 (bs,
2H), 6.77 (s, 1H), 7.09 (d, J ) 7.2 Hz, 2H), 7.16-7.31 (m, 4H),
7.37 (t, J ) 7.2 Hz, 1H), 7.68 (s, 1H), 7.78 (d, J ) 8.1 Hz, 1H),
8.06 (d, J ) 8.4 Hz, 1H).
inhibitor of groups IID and IIF sPLA2s. The inhibitors we
describe may be useful in probing the roles of sPLA2s in
inflammatory diseases such as asthma and arthritis.
Experimental Section
Enzyme Inhibition Assays. For compounds with IC50s <1600
nM in the fluorometric assay or <1300 nM in the E. coli membrane
assay, inhibitor concentrations were varied with five different
concentrations used to determine IC50 values. All IC50 values were
obtained by nonlinear regression curve-fitting of percent inhibition
versus log [inhibitor] using the Kaleidagraph software.
Fluorometric Assay. Microtiter plate assay of sPLA2s using
pyrene-labeled phosphatidylglycerol as the substrate was performed
as described previously16 with the exception that seven wells were
used per assay instead of eight.
Preparation of 1-Benzyl-2-carboxylic acid-4-methoxy-6,7-ben-
zoindole (6b). Compound 5b (485 mg, 1.41 mmol) was suspended
in 15 mL of 30% KOH/MeOH/THF (2:1:1) and refluxed for 2.0 h
(all the solid dissolved during reflux). After refluxing, the reaction
was cooled on ice and the pH was made acidic using 2 N HCl,
causing the product to precipitate. The white solid was collected
by vacuum filtration and washed with 1 × 10 mL of cold water
and 2 × 10 mL of cold hexanes to give a white solid (400 mg,
1
86% yield). H NMR (300 MHz, DMSO-d6) δ 4.02 (s, 3H), 6.41
E. coli Membrane Assay. IC50 values calculated for hGIID were
done using a modified procedure from that reported previously.25
See Supporting Information for details.
(bs, 2H), 6.98 (m, 3H), 7.20-7.26 (m, 2H), 7.32 (t, J ) 7.5 Hz,
2H), 7.39 (t, J ) 8.1 Hz, 1H), 7.49 (s, 1H), 7.86 (d, J ) 7.5 Hz,
1H), 8.12 (d, J ) 8.4 Hz, 1H).
Synthesis. All reagents were purchased from Sigma-Aldrich and
used directly unless otherwise stated. Reactions were performed
under an atmosphere of dry nitrogen in oven-dried glassware.
Reactions were monitored for completeness by thin layer chroma-
tography (TLC) using Merck 60F254 silica plates, and column
chromatography was done with 60 Å silica gel purchased from
Silicycle. 1H NMR spectra were recorded on dilute solutions in
CDCl3, CD3OD, or DMSO-d6. NMR spectra were obtained on a
Bruker AC-300 (300 MHz) and electrospray ionization mass spectra
were acquired on a Bruker Esquire LC00066 for all compounds.
Preparative reverse phase HPLC was performed on an automated
Varian Prep Star system using a YMC S5 ODS column (20 × 100
mm, Waters Inc.).
Representative Procedure for Synthesis of Substituted 6,7-
Benzoindole Inhibitors (Compound 12e): Preparation of 1-Benzyl-
2-carbomethoxy-4-methoxy-6,7-benzoindole (5b). Compound 4b
(synthesis described in Supporting Information; 800 mg, 3.14 mmol)
was dissolved in 10 mL dry DMF and stirred at 0 °C and sodium
hydride (140 mg, 5.5 mmol) was added. After stirring for five
minutes at 0 °C, benzylbromide (820 uL, 6.90 mmol) was added
and the reaction was stirred for 30 min at room temperature. The
reaction mixture was poured onto 20 mL of H2O and 20 mL of
EtOAc in a separatory funnel. The layers were separated and the
organic layer was washed with 3 × 10 mL H2O and the combined
aqueous layer was back-extracted with 1 × 20 mL EtOAc. The
combined organic layer was dried over MgSO4 and filtered, and
Preparation of 1-Benzyl-2-acetyl-4-methoxy-6,7-benzoindole (7c).
Compound 6b (920 mg, 1.12 mmol) was dissolved in 40 mL of
dry THF to which 6.6 mL of 1.25 M MeLi in diethyl ether was
added dropwise and stirred at room temperature for 2.5 h. Saturated
NH4Cl (8 mL) was added followed by the addition of 2 N HCl
until the mixture had an acidic pH. The reaction mixture was then
poured onto 30 mL of EtOAc and 30 mL of H2O in a separatory
funnel. The layers were separated and the water phase was
washed with 2 × 20 mL of EtOAc. The organic layers were
combined, dried over MgSO4 and filtered, and the solvent was
removed by rotary evaporation. The crude white solid was
triturated in 15 mL of 1:1 EtOAc/hexanes and separated from
the solvent by vacuum filtration. The white solid collected via
vacuum filtration was washed with 2 × 10 mL of 1:1 EtOAc/
hexanes giving 6b. Additional product could be purified from
the combined filtrate and washings by removing the solvent and
repeating the trituration step described above followed by
chromatography on silica gel (20% EtOAc/80% hexanes) of the
filtrate and washings combined together from the second
trituration step. The purified product was combined to afford a
1
white solid (366 mg, 40% yield). H NMR (300 MHz, CDCl3)
δ 2.63 (s, 3H), 4.08 (s, 3H), 6.35 (bs, 2H), 6.77 (s, 1H), 7.07
(d, J ) 6.9 Hz, 2H), 7.20-7.31 (m, 4H), 7.39 (t, J ) 8.1 Hz,
1H), 7.67 (s, 1H), 7.78 (d, J ) 8.1 Hz, 1H), 8.09 (d, J ) 8.7
Hz, 1H).

Inhibitors of Phospholipases A2
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4713
Preparation of 1-(1-Benzyl-4-methoxy-1H-6,7-benzoindol-2-yl)-
ethanol (8c). Compound 7c (366 mg, 1.11 mmol) was dissolved in
30 mL of 75% EtOH/25% THF, and NaBH4 (100 mg, 3.33 mmol)
was added to the mixture and stirred at room temperature for 16 h.
The reaction mixture was then poured onto 30 mL EtOAc and 30
mL H2O in a separatory funnel. The layers were separated and the
water phase was washed with 2 × 20 mL of EtOAc. The organic
layers were combined and washed with 2 × 20 mL of H2O and 1
× 20 mL of satd NaCl. The organic layer was dried over MgSO4
and filtered, and the solvent was removed by rotary evaporation to
give 8c as a white solid that was used without further purification
(355 mg, 96% yield). 1H NMR (300 MHz, CDCl3) δ 1.72 (d, J )
6.3 Hz 3H), 4.08 (s, 3H), 4.99 (m, 1H), 5.96 (d, J ) 20.7 Hz, 1H),
6.09 (d, J ) 20.7 Hz, 1H), 6.81 (s, 1H), 6.85 (s, 1H), 7.05 (d, J )
6.9 Hz, 2H), 7.15 (t, J ) 6.9 Hz, 1H), 7.20-7.31 (m, 4H), 7.79 (d,
J ) 8.1 Hz, 1H), 7.93 (d, J ) 8.7 Hz, 1H).
Preparation of 1-Benzyl-2-ethyl-4-methoxy-1H-6,7-benzoindole
(9c). Compound 8c (420 mg, 1.26 mmol) was dissolved in 20 mL
of dry CH2Cl2 and added dropwise to a mixture of 14 mL of 99%
trifluoroacetic acid (TFA) and NaBH4 (243 mg, 6.3 mmol) at 20
°C (prepare TFA/NaBH4 mixture in an ice bath by careful dropwise
addition of TFA to NaBH4 and let stir until NaBH4 fully dissolves
before raising temperature). The reaction mixture was stirred at
room temperature for 30 min and then poured onto 30 mL of satd
NaHCO3 and 30 mL of CH2Cl2 in a separatory funnel. Once
bubbling ceased, the layers were separated and the water phase
was washed with 2 × 20 mL of CH2Cl2. The organic layers were
combined, dried over MgSO4 and filtered, and the solvent was
removed by rotary evaporation. The crude material was purified
by column chromatography on silica gel (20% EtOAc/80% hexanes)
to afford a white solid (335 mg, 84% yield). 1H NMR (300 MHz,
CDCl3) δ 1.36 (t, J ) 7.5 Hz 3H), 2.75 (q, J ) 7.5 Hz 2H), 4.07
(s, 3H), 5.77 (s, 2H), 6.56 (s, 1H), 6.80 (s, 1H), 7.05 (d, J ) 6.9
Hz, 2H), 7.13 (t, J ) 6.9 Hz, 1H), 7.20-7.31 (m, 4H), 7.79 (d, J
) 8.1 Hz, 1H), 7.93 (d, J ) 8.7 Hz, 1H).
Preparation of Methyl 2-(1-Benzyl-2-ethyl-1H-6,7-benzoindol-
4-yloxy)acetate (10e). Compound 9c (80 mg, 0.25 mmol) was
dissolved in 8 mL of dry CH2Cl2 and stirred at 0 °C. BBr3 (1.0
M in CH2Cl2; 635 µL, 0.635 mmol) was added in portions over
5 min to the reaction mixture at 0 °C, and the reaction mixture
was stirred for 3 h at room temperature or until product formation
was complete as indicated by TLC. H2O (8 mL) was added to
the reaction mixture to quench excess BBr3, and the reaction
mixture was poured onto 20 mL of CH2Cl2 and 20 mL of H2O
in a separatory funnel. The layers were separated and the water
phase was washed with 2 × 20 mL of CH2Cl2. The organic layers
were combined, dried over MgSO4 and filtered, and the solvent
was removed by rotary evaporation to give the 4-hydroxy-6,7-
benzoindole intermediate, which is unstable and decomposes
giving a purple color upon exposure to air. After drying in vacuo
for 30 min, this compound was then immediately dissolved in 4
mL of DMF and stirred in an ice bath. Sodium hydride (10.3
mg, 0.41 mmol) was added to the reaction mixture and stirred
5 min at 0 °C, with subsequent addition of methyl bromoacetate
(40 µL, 0.456 mmol). The reaction was stirred at room
temperature for 30 min. Additional portions of sodium hydride
were added at 0 °C until the reaction was shown to be complete
by TLC. The reaction mixture was then poured onto 20 mL of
H2O and 20 mL of EtOAc in a separatory funnel. The layers
were separated and the organic layer was washed with 4 × 10
mL of H2O, and the combined aqueous layer was back-extracted
with 1 × 20 mL of EtOAc. The combined organic layers were
dried over MgSO4 and filtered, and the solvent was removed by
rotary evaporation. The crude material was purified by column
chromatography on silica gel (20% EtOAc/80% hexanes) to
afford a white solid (23 mg, 24% yield over two steps). 1H NMR
(300 MHz, CDCl3) δ 1.37 (t, J ) 7.5 Hz 3H), 2.75 (q, J ) 7.5
Hz 2H), 3.85 (s, 3H), 4.90 (s, 2H), 5.76 (s, 2H), 6.69 (s, 1H),
6.76 (s, 1H), 7.05 (d, J ) 6.9 Hz, 2H), 7.15 (t, J ) 6.9 Hz, 1H),
7.20-7.31 (m, 4H), 7.75 (d, J ) 8.1 Hz, 1H), 7.95 (d, J ) 8.4
Hz, 1H).
Preparation of Methyl-2-(3-(2-amino-2-oxoacetyl)-1-benzyl-2-
ethyl-1H-6,7-benzoindol-4-yloxy)acetate (11e). Compound 10e (22.4
mg, 0.06 mmol) was dissolved in 7 mL of dry CH2Cl2 and added
dropwise to 14 mL of dry CH2Cl2 containing oxalyl chloride (26
µL, 0.30 mmol) at room temperature. The reaction mixture was
stirred overnight at room temperature. Ammonia gas was then
bubbled into the reaction mixture for five minutes. The reaction
mixture was then poured into a separatory funnel containing 20
mL of 2 N HCl. The layers were separated and the aqueous layer
was extracted with 2 × 10 mL of CH2Cl2. The organic layers were
combined, dried over MgSO4 and filtered, and the solvent was
removed by rotary evaporation. The crude mixture was purified by
column chromatography over silica gel (70% EtOAc/30% hexanes)
1
to give a white yellow solid (10.9 mg, 41% yield). H NMR (300
MHz, CDCl3) δ 1.23 (t, J ) 7.5 Hz 3H), 2.94 (q, J ) 7.5 Hz 2H),
3.81 (s, 3H), 4.88 (s, 2H), 5.42 (bs, 1H), 5.81 (s, 2H), 6.72 (bs,
1H), 6.81 (s, 1H), 7.10 (d, J ) 6.9 Hz, 2H), 7.17 (t, J ) 6.9 Hz,
1H), 7.20-7.31 (m, 4H), 7.74 (d, J ) 7.8 Hz, 1H), 7.92 (d, J )
8.4 Hz, 1H). MS (ESI, pos. ion) m/z: 467 (M + Na+).
Preparation of 2-(3-(2-Amino-2-oxoacetyl)-1-benzyl-2-ethyl-1H-
6,7-benzoindol-4-yloxy)acetic Acid (12e). Compound 11e (10.9 mg,
0.024 mmol) was dissolved in 5 mL of MeOH/THF (5:1) with 0.5
mL of 1.5 M NaOH added to the reaction mixture and stirred for
2.5 h at room temperature. The reaction mixture was then poured
onto 20 mL of 2 N HCl and 20 mL of CH2Cl2 in a separatory
funnel. The layers were separated and the aqueous layer was
extracted with 2 × 10 mL of CH2Cl2. The combined organic layer
was dried over MgSO4 and filtered, and the solvent was removed
by rotary evaporation to yield 12e quantitatively. A portion of 12e
was purified by HPLC using the following program (eluting solvents
each contained 0.08% TFA): 0-5 min 30% MeOH/70% H2O, 5-30
min 30% MeOH/70% H2O-70% MeOH/30% H2O, 30-32 min
70% MeOH/30% H2O-100% MeOH, 32-35 min 100% MeOH.
The product eluted at 24.5 min and the solvent was removed by
Speed-Vac to afford a white/yellow solid (4.9 mg). 1H NMR (300
MHz, MeOD) δ 1.26 (t, J ) 7.2 Hz 3H), 3.04 (q, J ) 7.5 Hz 2H),
4.93 (s, 2H), 5.99 (s, 2H), 6.96 (s, 1H), 7.15-7.23 (m, 3H),
7.27-7.39 (m, 4H), 7.83 (d, J ) 6.9 Hz, 1H), 8.07 (d, J ) 8.4 Hz,
1H). MS (ESI, pos. ion) m/z: 431 (M+).
Acknowledgment. The authors thank Justin Siegel and Drs.
Brian Smart and Zhanglin Ni for helpful discussions about the
inhibitors used in this work and Dr. Christophe Verlinde for
modeling discussions. This work was supported by an NIH
Molecular Biophysics Training Grant (R.C.O.), the Mary Gates
Foundation (N.C.), and a Merit Award from the National
Institutes of Health (M.H.G.; R37HL036235).
Supporting Information Available: Details of synthetic meth-
ods, including NMR and MS data, for all other described
compounds, HPLC traces showing purity of key target compounds,
molecular modeling details, and E. coli membrane enzyme assay
procedures.This material is available free of charge via the Internet
at http://pubs.acs.org.
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