Structural optimization of azadipeptide nitriles strongly increases association rates and allows the development of selective cathepsin inhibitors

Article abstract of DOI:10.1021/jm101272p

Using the example of cathepsin K, we demonstrate the design of highly potent and selective azadipeptide nitrile inhibitors. A systematic scan with respect to P2 and P3 substituents was carried out. Structural modifications strongly affected the enzyme-inh

Full text of DOI:10.1021/jm101272p

396 J. Med. Chem. 2011, 54, 396–400
DOI: 10.1021/jm101272p
Structural Optimization of Azadipeptide Nitriles Strongly Increases Association Rates and Allows the
Development of Selective Cathepsin Inhibitors
€
Maxim Frizler, Friederike Lohr, Norbert Furtmann, Julia Klas, and Michael Gutschow*
€
Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
Received October 6, 2010
Using the example of cathepsin K, we demonstrate the design of highly potent and selective azadipeptide
nitrile inhibitors. A systematic scan with respect to P2 and P3 substituents was carried out. Structural
modifications strongly affected the enzyme-inhibitor association (but not dissociation) rate. A combina-
tion of optimized P2 and P3 substituents with a methylation of the P3-P2 amide linker resulted in the
picomolar cathepsin K inhibitor 19 with remarkable selectivity over cathepsins L, B, and S.
Introduction
Results and Discussion
Cysteine cathepsins show a high degree of similarity and
play a significant role in many (patho)physiological processes
such as bone remodeling, antigen presentation, osteoporosis,
autoimmune disorders, and cancer.1 Therefore, cathepsins
represent important targets for new therapeutic strategies.2
Inhibitors of cysteine cathepsins are mainly peptidic or pepti-
domimetic structures containing electrophilic groups prone
to covalent interactions with the active-site cysteine. Among
them, nitrile-based inhibitors receive the most attention in
current drug discovery. Nitriles inhibit cysteine proteases by
the reversible formation of a thioimidate adduct resulting
from the nucleophilic attack of the active-site cysteine at the
nitrile carbon.2 The design of peptidomimetic cathepsin inhib-
itors has usually been based on the structure of their natural
substrates. In substrates of proteases, amino acid residues to
the N-terminus of the scissile bond are designated P1, P2, etc.
The corresponding complementary regions of the enzyme’s
active site are numbered S1, S2, etc. The S2 pocket represents
the best defined subsite of cysteine cathepsins with endopepti-
dase activity and determines their primary substrate specificity.
Azapeptides have attracted much interest due to their
unique properties and applications as peptidomimetics in a
variety of biological systems.3 Recently, we have reported on
proteolytically stable azadipeptide nitriles as a novel class of
cysteine protease inhibitors with picomolar Ki values toward
therapeutically relevant cathepsins K, S, and L that form
reversible isothiosemicarbazide adducts with the target en-
zymes. These azadipeptide nitriles were composed of a Cbz-
protected P2 amino acid and a P1 aza-amino nitrile, whose
nitrogens are essentially alkylated for reasons of the synthetic
access.4 Despitetheirexcellent inhibitoryactivity, thereported
azadipeptide nitriles were nonselective and it remained un-
clear whether selectivity for a single target cathepsin can be
achieved at all with this class of inhibitors. Using the example
of cathepsin K, an approach for the development of selective
azadipeptide nitrile inhibitors is reported herein.
We initiated our study with a systematic scan for P2 sub-
stituents of the azadipeptide nitrile scaffold. For this purpose,
the target compounds 1-3 and 6-8 were prepared as depicted
in Table 1 and, together with the known inhibitors 4 and 5,4
tested on human cathepsins L, S, K, and B. Regardless of
the substitution pattern, compounds 1-8 displayed a time-
depended slow-binding behavior. Analysis of the progress
curves then allows for the determination of the second-order
association rate constants (kon) and the first-order dissociation
rate constants (koff).5 The L-leucine derivative 4 showed strong
inhibitory activity and a weak preference for cathepsin K
(Table 1). This inhibitor was selected for further optimization of
the P3 substituent, while its P2 residue, interacting with the S2
subsite of the proteases, was maintained in all further com-
pounds of this study. The kon values of compounds 1-8 varied
considerably depending on the substitution at the P2 position,
while the corresponding koff values were in the same range (see
Supporting Information, Tables S4, S7). For example, the
17000-fold higher kon value of 4 for cathepsin K, compared to
that of the glycine derivative 1 (Table 4), is reflected by the
improved potency of 4 (64 pM versus 260 nM, Table 1).
Next, the carbamate group of 4 was replaced by a urea
moiety to permit additional hydrogen bond formation. The
corresponding azadipeptide nitriles9 and 10 (Table 2) differ in
the length of the P3-P2 linker. The route to the urea-based
inhibitors 9 and 10 rests upon the finding that the carbamoyl-
protected L-leucine can be converted via mixed anhydride
method without racemization.6 L-Leucine tert-butyl ester 22
(Table 2) was reacted with 1,10-carbonyldiimidazole (CDIa)
and benzylamine or aniline, respectively. The resulting deriv-
atives 23 were treated with TFA to cleave the tert-butyl ester,
transformed into the corresponding 1,2-dimethylhydrazides
and reacted with cyanogen bromide to obtain 9 and 10
(Table 2). With Ki values of 11 and 17 pM, respectively, com-
pounds 9 and 10 were even more potent for cathepsin K than
a Abbreviations: CDI, 1,10-carbonyldiimidazole; cath, cathepsin;
DIPEA, N-ethyldiisopropylamine; DMAP, 4-dimethylaminopyridine;
EDC, 1-ethyl-3-(dimethylaminopropyl)carbodiimide; NMM, N-methyl-
morpholine; rt, room temperature.
*To whom correspondence should be addressed. Phone: þ49 228
732317. Fax: þ49 228 732567. E-mail: guetschow@uni-bonn.de.
r
pubs.acs.org/jmc
Published on Web 12/03/2010
2010 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 397
Table 2. Synthesis of Urea-Based Compounds 9-15 and Ki Values
Table 1. Synthesis of Carbamate-Based Compounds 1-8 and Ki Values
Ki (nM)a
compd
cath L
cath S
cath K
cath B
1
860
480
1.5
800
9.1
260
33
840
55
2
3
1.5
0.87
0.064
0.14
0.46
0.071
0.16
4.3
4b
5b
6
0.90
0.16
2.6
0.33
0.51
0.83
0.20
0.86
0.43
0.68
0.88
0.48
0.38
7
0.40
0.36
8
a The corresponding standard errors are noted in the Supporting
Information(Table S1). b Literature Ki values4 for cathepsins L, S, and K
have been obtained by a different method.
the corresponding carbamate-based derivative 4 (Tables 1
and 2).
On the basis of recent results on cathepsin K-selective di-
peptide nitriles with large biaryl P3 substituents,7 we extended
the P3 moiety of compounds 9 and 10 to improve the selec-
tivity for cathepsin K over cathepsins L, S, and B. A 1,2,4-
oxadiazole heterocycle as a common bioisoster of amide and
ester groups8 was chosen for the extension. Two benzylurea
derivatives bearing a methyl group (11) and a 2-thienyl
substituent (12) in position 5 of the 1,2,4-oxadiazole ring, as
well as their phenylurea counterparts 13 and 14, were synthe-
sized (Table 2). The synthetic route includes the conversion of
aromatic nitriles 24 to acyloxyamidines 25, and the oxadiazole
ring closure carried out in acetic acid at 80 °C followed by
deprotection with TFA. The final transformations to 11-15
were performed asin the case of9 and10. The exemplary route
to 13 is outlined in the Experimental Section.
Inhibitors 11-14 exhibited similar binding affinities with
picomolar Ki values on cathepsin K. Triaryl derivatives 12
and 14 were slightly more potent than the methyl substituted
biaryl compounds 11 and 13. Among the six urea-based azadi-
peptide nitriles, 14 displayed the most promising selectivity
profile for cathepsin K over cathepsins L, S, and B (Table 2).
The corresponding carba-analogue 15, prepared from 26
(n = 0, R = 2-thienyl) and aminoacetonitrile showed the same
trend to selectively inhibit cathepsin K, but with 3 orders of
magnitude higher Ki values. A fast-binding inhibition behavior
was observed for the carba-analogue 15, in contrast to the
azadipeptide nitriles 9-14. The kon values of the latter com-
pounds for the four cathepsins reflect the different Ki values
(for kon and koff values of 9-14 see Supporting Information,
Tables S5, S8).
Ki (nM)a
compd
cath L
cath S
cath K
cath B
9
0.39
0.045
1.4
0.20
0.16
0.17
0.16
0.19
0.32
180
0.011
0.017
0.11
0.65
1.3
10
11
12
13
14
15
2.8
1.3
1.1
2.0
0.072
0.045
0.022
34
2.4
2.4
4.4
4700
>22000
a The corresponding standard errors are noted in the Supporting
Information (Table S2).
As it became obvious with inhibitor 14, the combination of
the extended P3 triaryl motif with a short P3-P2 linker was
advantageous, and we decided to further reduce the linker,
leading to the design of amide-based azadipeptide nitriles 18
and 19 (Table 3). Because of the racemization of N-acylamino
acids known to occur during the activation of their carboxylic
groups, we developed a convergent synthesis for the amide-based
compounds. It was first employed for the carba-analogues 16

398 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Frizler et al.
Table 3. Synthesis of Amide-Based Compounds 16-19 and Ki Values
Table 4. kon Values of Selected Azadipeptide Nitriles
kon (103 M-1 s-1 a
)
compd
cath L
cath S
cath K
cath B
1
0.51
620
1.7
2000
800
ndb,d
0.10
1700
3300
63
0.11
130
4
18
19
800
ndb,c
1600
0.62
a The corresponding standard errors are noted in the Supporting
Information (Tables S4-S6). b Not determined. c For [19] = 20 μM,
a kobs value could not be obtained by nonlinear regression of the progress
curves. A limit kobs(1 þ [S]/Km)/[I]<1.0 ꢀ 103 M-1 s-1 was therefore
estimated. d For [19] = 350 nM, a kobs value could not be obtained by
nonlinear regression of the progress curves. A limit kobs(1þ [S]/Km)/[I]<
20 ꢀ 103 M-1 s-1 was therefore estimated.
with a fast-binding behavior exhibited a 300-7600-fold selec-
tivity for cathepsin K over cathepsins L and B and a moderate
selectivity over cathepsin S, the corresponding azadipeptide
nitrile 18, a slow-binding inhibitor, was more potent but much
less selective (Table 3). We expected the hydrogen-bond donat-
ing CONH linker of 18 to contribute to binding to the four
cathepsins studied, whereas the P2 and P3 moieties are already
optimized for cathepsin K. Therefore, a methylation to obtain a
CONMe linker might be better tolerated by cathepsin K than
by the other cathepsins. This was indeed the case. The methy-
lated azadipeptide nitrile 19 still exhibited a picomolar inhibi-
tion constant for cathepsin K and a remarkable selectivity over
cathepsins L, B, and S (4300-fold, 800-fold, and 200-fold,
respectively).
As expected, P3-P2 linker methylation affected the enzyme-
inhibitor association rate (19 versus 18, Table 4). The N-methyl
compound 19 showed clearly decreased kon values, reflecting a
delayed approach to steady-state. This effect was less pro-
nounced at cathepsin K, for which the optimized substructures,
common in 18 and 19, attenuate the lack of a hydrogen bond
formation to the backbone amide of Gly 667 in the case of 19.
Conclusions
In this study, using the example of cathepsin K, we have
demonstrated an approach to design highly potent and selective
azadipeptide nitrile inhibitors. Whereas the carba-analogous
dipeptide nitriles typically show fast-binding kinetics, a different,
slow-binding behavior was observed for azadipeptide nitriles.
Thus, it was possible to determine the influence of structural
features on association and dissociation rate constants for our
series of peptidic nitrile inhibitors. A strong impact of structural
variations in azadipeptide nitriles on the enzyme-inhibitor asso-
ciation rate was demonstrated. Although reversibly (see Sup-
porting Information, Figure S3) forming isothiosemicarbazide
adducts, these compounds gain their inhibitory activity not only
from the covalent attraction but also from specific noncovalent
interactions to the active site, as demonstrated herein for azadi-
peptide nitrile inhibitors for the first time. The dissociation rate,
however, was not affected by the compounds’ structure. This
finding reflects the difficulty in delivering significant binding
energy from noncovalent interactions, as discussed for cathep-
sins and concluded from the large and relatively shallow active
site of these target enzymes.7,9
Ki (nM)a
compd
cath L
cath S
cath K
cath B
16
17
18
19
940
>22000
0.22
140
>22000
0.15
2.9
270
>22000
>22000
0.36
0.032
0.63
2700
140
510
a The corresponding standard errors are noted in the Supporting
Information (Table S3).
and 17 (Table 3). The P3 building block 30 was prepared in five
steps with an overall yield of 49% (see Supporting Information).
Boc-protected L-leucine (31, R = H) and N-methyl-L-leucine
(31, R = Me) were reacted with aminoacetonitrile, followed by
removal of the protecting group using methanesulfonic acid
and basic extraction to produce 32 (R = H, R = Me). In the
final step, building block 30 was activated and coupled with 32
to obtain dipeptide nitriles 16 and 17.
Similarly, Cbz-protected L-leucine derivatives 33 (R=H,
R = Me) were converted into the corresponding 1,2-dimethyl-
hydrazides, whereupon the deprotection was carried out hy-
drogenolytically. The coupling products 36 were purified on
silica gel and subjected to the final reaction step with cyanogen
bromide leading to 18 and 19. Compounds 16 and 18, both
possessing a CONH linker, showed clear differences in potency,
selectivity, and kinetic properties. While the carba-analogue 16
Experimental Section
€
General. Melting points were determined on a Buchi 510 oil
bath apparatus and are uncorrected. Thin layer chromatogra-
phy was performed on Merck aluminum sheets. Preparative
column chromatography was performed on silica gel 60, 0.060-
0.200 mm. 13C NMR (125 MHz) and 1H NMR (500 MHz)

Brief Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 399
spectra were recorded on a Bruker Avance DRX 500 spectrometer.
Elemental analyses were performed with a Vario EL apparatus.
LC-DAD chromatograms and ESI-MS spectra were recorded on
an Agilent 1100 HPLC system with Applied Biosystems API-2000
mass spectrometer. Optical rotation was determined on a Perkin-
Elmer 241 polarimeter. IR spectra were recorded on a Bruker
Tensor 27 FT-IR spectrometer. Enzymatic assays were performed
on a Varian Cary 50 Bio spectrophotometer and a Monaco Safas
flx spectrofluorometer. Amino acid derivatives were from Bachem
(Bubendorf, Switzerland), Acros (Geel, Belgium), and Aldrich
(Steinheim, Germany), and chromogenic and fluorogenic sub-
strates as well as enzymes were obtained from Bachem
(Bubendorf, Switzerland), Calbiochem (Darmstadt, Germany),
solvent was dried (Na2SO4) and removed under reduced pres-
sure. The solid product was recrystallized from ethyl acetate/
n-hexane to obtain 26 (n = 0, R = Me) as a white solid (2.80 g,
66% from 25); mp 156-158 °C. 1H NMR (500 MHz, DMSO-d6)
δ 0.89-0.92 (m, 6H), 1.48-1.58 (m, 2H), 1.65-1.73 (m, 1H),
2.61 (s, 3H), 4.17-4.23 (m, 1H), 6.52 (d, 3J = 8.2 Hz, 1H), 7.54
(d, 3J = 8.8 Hz, 2H), 7.85 (d, 3J = 8.8 Hz, 2H), 8.89 (s, 1H), 12.62
(bs, 1H). 13C NMR (125 MHz, DMSO-d6) δ 12.09, 21.76, 22.94,
24.55, 41.05, 50.90, 117.70, 119.00, 127.92, 143.23, 154.70,
167.53, 174.80, 177.05.
N-[4-(5-Methyl-1,2,4-oxadiazol-3-yl)phenylcarbamoyl]-leucyl-
methylazaalanine-nitrile (13). Compound 26 (n = 0, R = Me;
1.50 g, 4.51 mmol) was dissolved in dry THF (40 mL) and cooled
to -25 °C. To the stirred solution, N-methylmorpholine (NMM)
(0.54 mL, 4.91 mmol) and isobutyl chloroformate (0.65 mL, 4.99
mmol) were added consecutively. 1,2-Dimethylhydrazine dihy-
drochloride (3.00 g, 22.6 mmol) was suspended in H2O (3 mL),
and 10 N NaOH (5.00 mL) was added under ice-cooling. This
solution was given to the reaction mixture when the precipitation
of N-methylmorpholine hydrochloride occurred. It was allowed
to warm to room temperature within 30 min and stirred for
additional 90 min. After evaporation of the solvent, the resulting
aqueous residue was extracted with ethyl acetate (3 ꢀ 30 mL).
The combined organic layers were washed with H2O (30 mL),
satd NaHCO3 (30 mL), H2O (30 mL), and brine (30 mL). The
solvent was dried (Na2SO4) and evaporated. The crude product
was purified by column chromatography on silica gel using ethyl
acetate to obtain N-[4-(5-methyl-1,2,4-oxadiazol-3-yl)phenylcar-
bamoyl]-leucine 1,2-dimethylhydrazide as a colorless oil. Sodium
acetate (0.48 g, 5.85 mmol) and cyanogen bromide (0.47 g, 4.44
mmol) were added to a solution of N-[4-(5-methyl-1,2,4-oxadiazol-
3-yl)phenylcarbamoyl]-leucine 1,2-dimethylhydrazide (1.10 g, 2.94
mmol) in MeOH (30 mL). The mixture was stirred at room
temperature for 48 h, and the solvent was removed under reduced
pressure. The residue was suspended in H2O (10 mL), a pH of ∼2
was adjusted (10% KHSO4), and it was extracted with ethyl acetate
(3 ꢀ 30 mL). The combined organic layers were washed with H2O
(20 mL), satd NaHCO3 (2 ꢀ 30 mL), H2O (30 mL), and brine
(30 mL). The solvent was dried (Na2SO4) and removed in vacuo.
The oily residue was purified by column chromatography on silica
gel using MeOH/CH2Cl2 (40:1) as eluent. Additionally, the product
was recrystallized from ethyl acetate/petroleum ether to obtain 13
as a white solid (0.66 g, 37% from 26); mp 185 °C; [R]20D = þ65.6
€
and Enzo Life Sciences (Lorrach, Germany). All tested com-
pounds possessed a purity of not less than 95%.
N-(4-Cyanophenylcarbamoyl)-leucine tert-Butyl Ester (24,
n = 0). The hydrochloride salt of leucine tert-butyl ester 22
(5.00 g, 22.3 mmol) was dissolved in dry THF (50 mL) and
treated with N-ethyldiisopropylamine (DIPEA) (5.70 mL, 33.3
mmol). The resulting reaction mixture was treated with 4-cya-
nophenyl isocyanate (3.55 g, 24.6 mmol) and stirred for 4 h at
room temperature (rt). After evaporation of the solvent, the
residue was suspended in H2O. The aqueous suspension was
adjusted with 10% KHSO4 to pH ∼2 and extracted with ethyl
acetate (3 ꢀ 30 mL). The combined organic layers were washed
with H2O (20 mL), 10% KHSO4 (20 mL), H2O (20 mL), and
brine (20 mL). The solvent was dried (Na2SO4) and evaporated
under reduced pressure. The crude product was recrystallized
from ethyl acetate/petroleum ether to obtain 24 (n = 0) as a
1
white solid (6.70 g, 91%); mp 113 °C. H NMR (500 MHz,
3
3
DMSO-d6) δ 0.88 (d, J = 6.6 Hz, 3H), 0.91 (d, J = 6.7 Hz,
3H), 1.40 (s, 9H), 1.46-1.54 (m, 2H), 1.62-1.70 (m, 1H),
3
3
4.10-4.14 (m, 1H), 6.61 (d, J = 8.2 Hz, 1H), 7.54 (d, J =
8.9 Hz, 2H), 7.65 (d, 3J = 8.8 Hz, 2H), 9.07 (s, 1H). 13C NMR
(125 MHz, DMSO-d6) δ 21.82, 22.77, 24.56, 27.78, 40.91, 51.64,
80.85, 102.88, 117.68, 119.48, 133.36, 144.69, 154.40, 172.36.
N-[(4-Acetoxyamidino)phenylcarbamoyl]-leucine tert-Butyl
Ester (25, n = 0, R = Me). Compound 24 (n = 0; 6.30 g, 19.0
mmol) was dissolved in EtOH (50 mL) and treated with DIPEA
(6.46 mL, 37.7 mmol) and hydroxylamine hydrochloride (2.64 g,
38.0 mmol). The resulting solution was heated to reflux over-
night. After evaporation of the solvent, the oily residue was
dissolved in dry MeCN (50 mL) and treated with acetic anhy-
dride (5.39 mL, 57.4 mmol). The solution was stirred at room
temperature for 5 h. The solvent was removed in vacuo, and the
oily residue was suspended in H2O. The aqueous suspension was
adjusted with 10% KHSO4 to pH ∼2 and extracted with ethyl
acetate (3 ꢀ 30 mL). The combined organic layers were washed
with 5% KHSO4 (2 ꢀ 30 mL), H2O (30 mL), satd NaHCO3
(3 ꢀ 30 mL), H2O (30 mL), and brine (30 mL). The solvent was
dried (Na2SO4) and removed under reduced pressure. The crude
product was recrystallized from ethyl acetate to obtain 25 (n=0,
R = Me) as a white solid (5.40 g, 70% from 24); mp 95 °C. 1H
NMR (500 MHz, DMSO-d6) δ 0.89 (d, 3J = 6.6 Hz, 3H), 0.92
(d, 3J = 6.6 Hz, 3H), 1.41 (s, 9H), 1.44-1.54 (m, 2H), 1.63-1.71
(m, 1H), 2.11 (s, 3H), 4.11-4.15 (m, 1H), 6.45 (d, 3J = 8.2 Hz,
1H), 6.62 (s, 2H), 7.42 (d, 3J = 8.9 Hz, 2H) 7.59 (d, 3J = 8.9 Hz,
2H), 8.74 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ 20.00,
21.85, 22.77, 24.55, 27.78, 41.07, 51.56, 80.73, 116.98, 124.26,
127.42, 142.28, 154.66, 156.24, 168.65, 172.56.
1
(c = 0.32, CHCl3). H NMR (500 MHz, DMSO-d6) mixture of
rotamers (only the data of the major rotational isomer are noted) δ
0.93-0.97 (m, 6H), 1.36-1.53 (m, 2H), 1.73 (bs, 1H), 2.62 (s, 3H),
3.11 (s, 3H), 3.27 (s, 3H), 4.80-4.83 (m, 1H), 6.64 (d, 3J = 8.2 Hz,
1H), 7.53 (d, 3J = 8.5 Hz, 2H), 7.85 (d, 3J = 8.8 Hz, 2H), 8.89 (s,
1H). 13C NMR (125 MHz, DMSO-d6) δ 12.09, 21.40, 23.29, 24.57,
30.56, 40.46, 40.94, 47.91, 114.22, 117.79, 119.15, 127.92, 142.98,
154.93, 167.50, 174.67, 177.07. FTIR (KBr, cm-1) 2219 (CꢁN).
LC-ESI/MS (90% H2O to 100% MeOH in 20 min, then 100%
MeOH to 30 min, DAD 239.8-340.8 nm): tr = 21.83, 98% purity,
m/z = 400.3 ([M þ H]þ).
€
Acknowledgment. We thank Michael Lulsdorff and
Patrick Jim Kuppers for assistance. This work was supported
€
by the NRW International Research School Biotech-Pharma,
Germany.
N-[4-(5-Methyl-1,2,4-oxadiazol-3-yl)phenylcarbamoyl]-leucine
(26, n = 0, R = Me). Compound 25 (n = 0, R = Me; 5.20 g, 12.8
mmol) was dissolved in concentrated acetic acid (40 mL) and
stirred at 80 °C for 5 h. After evaporation of the acetic acid, the
oily product was dissolved in CH2Cl2 (40 mL) and treated with
TFA (20 mL). The resulted solution was stirred for 5 h at room
temperature. The solvent was evaporated, and the oily residue
was suspended in H2O. The resulting aqueous suspension was
extracted with ethyl acetate (3 ꢀ 20 mL). The combined organic
layers were washed with H2O (2 ꢀ 30 mL) and brine (30 mL). The
Supporting Information Available: Inhibition assays and
equations, representative plots and detailed kinetic parameters,
preparation of compounds 1-36, as well as 1H and 13C NMR
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
Note Added after ASAP Publication. This paper was pub-
lished ASAP on December 3, 2010 with errors in the footnotes
of Table 4, and the Supporting Information file. The correct
version was reposted on December 8, 2010.

400 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Frizler et al.
€
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