Synthesis and SAR of novel benzoxaboroles as a new class of β-lactamase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.02.024

A new class of benzoxaborole β-lactamase inhibitors were designed and synthesized. 6-Aryloxy benzoxaborole 22 inhibited AmpC P99 and CMY-2 with K _i values in the low nanomolar range. Compound 22 restored antibacterial activity of ceftazidime against Enterobacter cloacae P99 expressing AmpC, a class C β-lactamase enzyme. The SAR around the arylbenzoxaboroles, which included the influence of linker and substitutions was also established.

Full text of DOI:10.1016/j.bmcl.2011.02.024

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2533–2536
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of novel benzoxaboroles as a new class
of b-lactamase inhibitors
Yi Xia a, , Kathy Cao a, Yasheen Zhou a, M. R. K. Alley a, Fernando Rock a, Manisha Mohan a,
⇑
Maliwan Meewan a, Stephen J. Baker a, Sarah Lux a, Charles Z. Ding a, Guofeng Jia b,
Maureen Kully b, Jacob J. Plattner a
a Anacor Pharmaceuticals, Inc., 1020 E. Meadow Circle, Palo Alto, CA 94303, USA
b Naeja Pharmaceuticals, Inc., 4290-91A Street, Edmonton, Alberta, Canada T6E5V2
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of benzoxaborole b-lactamase inhibitors were designed and synthesized. 6-Aryloxy benzox-
aborole 22 inhibited AmpC P99 and CMY-2 with Ki values in the low nanomolar range. Compound 22
restored antibacterial activity of ceftazidime against Enterobacter cloacae P99 expressing AmpC, a class
C b-lactamase enzyme. The SAR around the arylbenzoxaboroles, which included the influence of linker
and substitutions was also established.
Received 11 January 2011
Revised 4 February 2011
Accepted 8 February 2011
Available online 12 February 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
b-lactamase inhibitors
Benzoxaborole
Antibacterial
The b-lactam antibiotics represent the drug of choice to treat
Gram-negative bacterial infections. Resistance to the b-lactams in
Gram-negative bacteria is predominately through the acquisition
of b-lactamases,1 which act by catalyzing the hydrolysis of the
amide bond of the b-lactam ring. The combination of the prolifer-
ation of b-lactamases and a poor drug pipeline pose a serious
threat to the future treatment of Gram-negative bacterial infec-
tions.2 The b-lactamases can be classified by their structure into
four classes (A, B, C and D).3 Class A, C and D are serine proteases,
while class B are metallo-hydrolases. Inhibition of b-lactamases is a
common method of treatment in the clinic with the currently mar-
keted inhibitors clavulanic acid, tazobactam, and sulbactam pre-
dominately inhibiting class A enzymes. The class C enzymes,
AmpC and CMY, which are resistant to these inhibitors, are a signif-
icant problem in the clinic. Furthermore, b-lactam-based inhibitors
can induce the expression of the chromosomal encoded class C
b-lactamase, AmpC, especially in Enterobacteria spp., which can
lead to development of resistance during therapy.4 The use of a
non-b-lactam based b-lactamase inhibitor would avoid this signifi-
cant handicap.
therefore locking the enzyme in this inactive state. Since boronic
acids are often associated with poor drug-like properties, we have
evaluated other boronic acid derivatives as enzyme inhibitors. We
have previously reported that benzoxaboroles, a 5-membered bor-
on containing heterocycle fused to an aromatic ring, showed selec-
tive inhibition of leucyl-tRNA synthetase by coordinating to cis-
diols of substrate tRNA in the editing active site.7 These benzoxab-
oroles posses significantly better pharmacokinetic properties than
boronic acids. Therefore, we set about to use the oxaborole chem-
ical scaffold to design potent and specific b-lactamase inhibitors.
Screening our chemical library of boron compounds against
the class A enzyme TEM-1 and the class C enzyme AmpC from
Enterobacter cloacae P99 identified benzoxaborole, compound 1,
with promising broad-spectrum inhibitory activity (Table 1).
Compound 1 is the benzoxaborole with an ether linker at C6 posi-
tion. Subsequently, we explored the effect of a variety of linkage
groups and different substitution pattern at C6 of benzoxaboroles
on b-lactamase inhibition. We also synthesized the heteroaryl ana-
logs to investigate the effect of lipophilicity on anti-bacterial
activity.
Boronic acids inhibitors of b-lactamases have been known since
the late 1970s5 where its empty p orbital forms a covalent bond
with the catalytic serine residue of a b-lactamase, thus acting
as a serine trap.6 The boronic acid-serine adduct mimics the
transition state and is stabilized by the enzyme active site,
The synthesis of benzoxaboroles with thioether, sulfoxide, and
sulfone, amino, amide and carbamate linkage groups (1–10) at
C6 position has been reported previously.8 The synthesis of substi-
tuted aryloxy benzoxaboroles is outlined in Scheme 1. Nucleophilic
substitution of 2-bromo-4-fluorobenzaldehyde (12) by phenol
gave ether (13). Palladium-mediated boronylation provided
aldehyde (14), followed by reduction with NaBH4 and acid-
catalyzed cyclization to the phenoxybenzoxaboroles (15a–m).
⇑
Corresponding author.
E-mail address: axia@anacor.com (Y. Xia).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.024

2534
Y. Xia et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2533–2536
Table 1
b-lactamase inhibition of benzoxaboroles with different linkers
O
B
O
OTf
O
OH
OH
O
a
b
O
X
B
O
O
O
O
16
17
18
Compd
X
Kia
(l
M)
OH
B
OH
c,d
CTX-M-9a
TEM-1
AmpC P99
CMY-2
e
O
HO
B
O
O
1
2
3
4
5
6
7
8
9
O
S
1.89
>32
>32
>32
>32
>32
>32
>32
>32
>32
1.02
1.76
>37
10.6
>37
4.87
11.23
>37
0.71
9.09
1.51
12.65
25.63
46.94
8.42
59.29
108
>138
13.8
13.8
19
CH2
CO
CH(OH)
SO
19.79
28.47
7.28
57.98
63.22
46.56
16
20
f
f
SO2
OH
B
O
OH
B
O
CONH
NHOCO
NH
N
O
N
O
>37
>37
O
O
N
10
4.73
N
a
21
23
O
All b-lactamases were tested as described in Ref. 11.
O
g
g
OH
OH
As shown in Scheme 2, benzoxaboroles with heteroaryl at C6
N
N
O
N
O
B
B
were also synthesized. 2-Hydroxy-4-methoxybenzaldehyde was
converted to triflate (17) followed by catalytic boronylation to pro-
vide compound 18. Reduction with NaBH4 and acid-catalyzed
cyclization to the benzoxaboroles (19). Treatment of BBr3 at À10
to 0 °C for demethylation afforded 20. Compound 20 was reacted
with a series of heteroaryl chloride by nucleophilic substitution.
For example, treatment of 20 with methyl 5-chloropyrazine-
2-carboxylate in basic condition provided 21. Subsequently, hydro-
lysis of the ester with LiOH in methanol and water yielded the
corresponding carboxylic acid (22).9 Compounds 23 and 2410 were
synthesized in similar method as shown in Scheme 2.
To probe the effect of different linker groups on anti-b-lacta-
mase inhibitory effects, those benzoxaboroles were tested for their
ability to inhibit both class A (CTX-M-9a and TEM-1) and class C
(AmpC P99, CMY-2) b-lactamases. The data shown in Table 1 sum-
marizes the Ki values for those compounds on b-lactamase
inhibition.
O
O
HO
HO
N
O
22
O
24
Scheme 2. Synthesis of C6-heteroaryl benzoxaboroles. Reagents and conditions: (a)
(CF3SO2)2O, pyridine, À10 to 0 °C; (b) bis(pinacolato-diboron), PdCl2(dppf), KOAc,
dioxane, 80 °C; (c) NaBH4, MeOH-THF, 0 °C; (d) HCl; (e) BBr3, DCM, À10 to 0 °C; (f)
methyl-5-chloropyrazine-2-carboxylate or methyl 2-chloropyrimidine-5-carboxyl-
ate, K2CO3, DMF, 80 °C; (g) LiOH, MeOH-H2O, 0 °C to rt.
Based on these results, C6 phenoxy-benzoxaboroles (1) was se-
lected as a template for further SAR exploration. The effect on affin-
ity of different substitution group of 1 on b-lactamase inhibition is
summarized in Table 2.
In general, compounds with small substitution at meta-position
exhibited better inhibitory activity against CTX-M-9a, while main-
taining class C activities. Compounds 15a–c had Ki values about
2–3-fold more potent than 1 in CTX-M-9a, suggesting that small
polar group contributed on inhibitory binding. Adding a methylene
to amino or hydroxyl group to hydroxymethyl (15d) and amino-
methyl (15e) reduced affinity suggesting the binding geometry
was not optimum by increasing the distance from the polar OH
or NH2 group to the enzyme residues. Bulky groups (15f, 15g)
decreased activity further which is consistent with the above
findings.
On the contrary, substitution on para-position reduced affinity
in CTX-M-9a as compared to meta-position. Compounds 15i and
15j were about 7-fold less potent than 1, while corresponding
meta-substituted analogs (15a, 15c) improved affinity by 2–3-fold,
respectively. Interestingly, anionic charged moiety, such as
carboxylate on either meta- (15h) or para-position (15n) was not
As shown in Table 1, compound 1 with ether linker showed
moderate broad-spectrum inhibition of class
A and class C
b-lactamases. Converting the hydrogen bond acceptor oxygen to
methylene linker (3), thioether (2) or hydrogen bond donor amino
linker (10) diminished potency in CTX-M-9a and TEM-1. Carbonyl
(4), carbinol (5), sulfoxide (6), and sulfone (7) represent the
category of linkage groups with a hydrogen bond acceptor at an
increased distance from the boron of the benzoxaborole and
showed decreased potency. The linker SAR indicated that oxygen
is essential for good potency and it may contribute to the potency
through a hydrogen bonding interaction with the enzyme. Also the
SAR suggests that the length and hydrogen-bonding properties of
the linkage group had a significant effect on b-lactamase inhibitory
activity.
O
B
R1
R2
OH
F
Br
R1
R2
R1
O
O
Br
a
O
b
+
CHO
R2
CHO
CHO
11
12
13
14
OH
R1
R2
O
c, d
B
O
15
Scheme 1. Synthesis of phenoxy benzoxaboroles. Reagents and conditions: (a) Cs2CO3, DMF, 80 °C; (b) bis(pinacolato-diboron), PdCl2(dppf), KOAc, dioxane, 80 °C; (c) NaBH4,
MeOH-THF, 0 °C; (d) HCl.

Y. Xia et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2533–2536
2535
tolerated in CTX-M-9a and TEM-1. However, they demonstrated
improved affinity in class C b-lactamase. Compound 15n was ex-
tremely potent against AmpC P99 and CMY-2 with a Ki value in
carboxylic acid at p-position, however, resulted in diminished class
A b-lactamase activity. The SAR described here would be very
useful for further design and development of broad-spectrum
b-lactamase inhibitors. Further studies to improve spectrum of
b-lactamase inhibition are underway and will be reported in due
course.
0.02 lM, suggesting a canonical anion recognition residue around
the carboxylate in AmpC P99 and CMY-2, which revealed the
importance of this group for recognition by AmpC P99, CMY-2
b-lactamases.
With promising inhibitory activity of 15n in AmpC P99 and
CMY-2, we further replaced the phenyl ring with heterocyclic rings
to investigate the effects of lipophilicity as lipophilic compounds
tend to be good substrates for efflux pumps. As expected, pyrazine
(22) and pyrimidine (24) carboxylic acids showed very potent inhi-
bition on AmpC P99 and CMY-2 with Ki values in the low nanomo-
lar range. These potent class C b-lactamase inhibitors were then
tested for their anti-bacterial inhibition in E. cloacae and Escherichia
coli with a plasmid bearing the CMY-2 class C b-lactamase. As
shown in Table 3, compound 22 restored antibacterial activity of
References and notes
1. Livermore, D. M. J. Antimicrob. Chemother. 1998, 41, 25–41.
2. Ho, J.; Tambyah, P. A.; Paterson, D. L. Curr. Opin. Infect. Dis. 2010, 23,
546.
3. Bush, K.; Jacoby, G. A.; Medeiros, A. A. Antimicrob. Agents Chemother. 1995, 39,
1211–1233.
4. Tondi, D.; Calo, S.; Shoichet, B.; Costi, M. P. Bioorg. Med. Chem. 2010, 3416–
3419.
5. (a) Ambler, R. P. Philos. Trans. R. Soc. Lond. (Biol.) 1980, 289, 321–331; (b) Kiener,
P. A.; Waley, S. G. Biochem. J. 1978, 169, 197–204.
6. Morandi, S.; Morandi, F.; Casell, E.; Schoichet, B.; Prati, F. Bioorg. Med. Chem.
Lett. 2008, 1195–1205.
7. Rock, F. L.; Mao, W.; Yaremchuk, A.; Tukalo, M.; Crepin, T.; Zhou, H.; Zhang, Y.-
K.; Hernandez, V.; Akama, T.; Baker, S. J.; Plattner, J. J.; Shapiro, L.; Martinis, S.
A.; Benkovic, S. J.; Cusack, S.; Alley, M. R. K. Science 2007, 316, 1759.
8. Ding, D.; Zhao, Y.; Meng, Q.; Xie, D.; Nare, B.; Chen, D.; Bacchi, C. J.; Yarlett,
N.; Zhang, Y. K.; Hernandez, V.; Xia, Y.; Freund, Y.; Abdulla, M.; Ang, K.;
Ratnam, J.; McKerrow, J.; Jacobs, R. T.; Zhou, H.; Plattner, J. Med. Chem Lett.
2010, 1, 165.
9. Experimental procedure for the preparation of compound 22: To a solution of 2-
hydroxy-4-methoxy-benzaldehyde 16 (30.0 g, 0.197 mol) and pyridine
(77.98 g, 0.986 mol) in dichloromethane (120 mL) was slowly added Tf2O
(83.44 g, 0.296 mol) at À10 to 0 °C over a 2.5-h period. The mixture was stirred
at 0 °C for 30 min. Ice-water (150 mL) was added, and the mixture was
acidified with diluted hydrochloric acid to pH 2. The resulting mixture was
extract with 50% EtOAc/hexanes (2 Â 400 mL). The extract was washed with
brine, dried and concentrated to dryness to give 51.01 g (91.1%) of compound
17 as pale-yellow oil. 1H NMR (400 MHz, CDCl3) d 10.13 (s, 1 H), 7.95 (d,
J = 8.79 Hz, 1 H), 7.03 (dd, J = 8.79, 2.34 Hz, 1 H), 6.88 (d, J = 2.34 Hz, 1 H), 3.93
(s, 3 H). MS (ESI) m/z = 285 [M+H]+. To a solution of bis(pinacolato)diborane
(58.66 g, 0.231 mol) in dioxane (600 mL) was added KOAc (52.33 g, 0.533 mol).
After degassed for 15 min, PdCl2(dppf) (13.0 g, 0.0178 mol) and 17 (50.51 g,
0.178 mol) were added to the reaction mixture. The mixture was stirred at
80 °C for 45 min. The reaction was quenched by adding ice-water (400 mL). The
resulting mixture was extract with 50% EtOAc/hexanes (2 Â 600 mL). The
extract was washed with brine, dried and concentrated to dryness. The residue
was purified by chromatography on silica gel (EtOAc/hexanes = 1:3) to give
43.48 g (93.2%) of 18 as pale-yellow waxy solid.1H NMR (400 MHz, CDCl3) d
10.88 (s, 1 H), 8.44 (d, J = 8.50 Hz, 1 H), 7.80 (d, J = 2.64 Hz, 1 H), 7.54 (dd,
J = 8.50, 2.64 Hz, 1 H), 4.41 (s, 3 H), 1.91 (s, 12 H). MS (ESI) m/z = 263 [M+H]+. To
a solution of 18 (25.0 g, 95.4 mmol) in methanol (160 mL) was slowly added
NaBH4 powder (10.82 g, 0.286 mol) at 0–10 °C. After stirred for 1 h at room
temperature, the mixture was concentrated to remove one-third of methanol.
The resulting mixture was cooled to 0 °C, acidified to pH 3 using diluted
hydrochloric acid and diluted to 2-fold with cold water. The white precipitate
was collected, washed with 30% MeOH/H2O, water, and dried to give 11.5 g
(73.5%) of 19 as white solid. 1H NMR (400 MHz, DMSO-d6) d 9.11 (s, 1 H), 7.29
(d, J = 8.21 Hz, 1 H), 7.23 (d, J = 2.34 Hz, 1 H), 7.03 (dd, J = 8.21, 2.34 Hz, 1 H),
ceftazidime from >128
expressing AmpC P99 at the concentration of 8
In addition, 22 restored antibacterial activity of ceftazidime to
0.5 g/mL against E. coli expressing CMY-2.
In conclusion, we have synthesized and discovered a series of
lg/mL to 1
l
g/mL against E. cloacae P99
lg/mL of inhibitor.
l
novel benzoxaborole b-lactamase inhibitors. The SAR around C6
benzoxaboroles, which included the influence of linkage, ring and
substitution patterns, was established. The most potent class C
b-lactamase inhibitor 22 may form productive hydrogen bonding
interactions with AmpC though its p-carboxylic acid. The same
Table 2
b-lactamase inhibition of substituted C6 phenoxybenzoxaboroles
OH
R1
R2
O
B
O
Compound R1
R2
Ki (lM)
CTX-
M-9a
TEM- AmpC
CMY-
2
1
P99
1
H
OCH3
OH
NH2
CH2OH
CH2NH2
OCH2Ph
CH2N(CH3)2
H
H
H
H
H
H
H
H
1.89
1.1
1.02
1.4
0.71
1.35
nta
2.75
8.4
3.33
2.27
7.72
1.44
6
1.51
2.65
3.12
6.09
8.43
nta
74.5
15.9
2.53
>46
14.7
12.3
46.8
nta
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
15n
0.816
0.69
1.92
4.18
8.75
6.02
22.6
14.1
14.8
15.1
nta
1.01
1.36
0.56
1.08
0.51
>37
4.34
nta
COOH
H
4.90 (s,
2 H), 3.75 (s, 3 H). To a solution of 19 (10.0 g, 61.0 mmol) in
H
H
H
H
H
H
OCH3
NH2
CH2NH2
CH2N(CH3)2 21.1
COOEt
COOH
dichloromethane (400 mL) was slowly added BBr3 (134 mL, 1 M in DCM,
0.134 mol) at À10 to À5 °C. The mixture was stirred at 0 °C to room
temperature for 3 h. The reaction mixture was poured into ice-water
(300 mL). The resulting mixture was extract with EtOAc (600 mL). The
extract was washed with brine, dried and concentrated to dryness to give
9.11 g (99.6%) of 20 as off-white foam. 1H NMR (400 MHz, DMSO-d6) d 9.27 (br
s, 1 H), 9.03 (br s, 1 H), 7.16 (d, J = 8.20 Hz, 1 H), 7.08 (d, J = 2.34 Hz, 1 H), 6.86
(dd, J = 8.20, 2.34 Hz, 1 H), 4.90 (s, 2H). MS (ESI) m/z = 151 [M+H]+. To a solution
of 20 (0.37 g, 2.47 mmol) in anhydrous DMF (8 mL) were added Cs2CO3 (2.01 g,
2.71 mmol) and 5-chloro-pyrazine-2-carboxylic acid methyl ester (0.468 g,
2.71 mmol) at room temperature. After stirring at 90 °C for 1.5 h, the reaction
mixture was cooled to 0 °C, diluted with water (10 mL) and acidified to pH 3
using diluted hydrochloric acid. The off-white precipitate was collected,
washed with water and dried to give the crude product which was purified
by chromatography on silica gel (DCM/MeOH = 40:3) to give 0.47 g (66.5%) of
21. MS (ESI) m/z = 287 [M+H]+. To a solution of 21 (0.47 g, 1.64 mmol) in
methanol (16 mL) was added aqueous LiOH-H2O (0.345 g in 12 mL of water,
8.21 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 1 h. The
reaction mixture was acidified to pH 2 using diluted hydrochloric acid. The
white precipitate was collected, washed with water and 30% of EtOAc/hexanes
and dried to give 0.392 g (87.9%) of 22; mp 202–204 °C. 1H NMR (400 MHz,
DMSO-d6) d 9.28 (s, 1 H), 8.74 (d, J = 1.2 Hz, 1 H), 8.66 (d, J = 1.2 Hz, 1 H),
7.53–7.50 (m, 2 H), 7.37 (dd, J = 8.4 Hz, 2.0 Hz, 1 H), 5.03 (s, 2 H). MS (ESI) m/
z = 271 [MÀH]À.
8.73
0.812 13.3
3.14
3.11
18.9
40.63
5.55
0.02
>32
>32
0.02
a
Not tested.
Table 3
Inhibition of b-lactamases by aryl- and heteroarylbenzoxaboroles and Minimum
Inhibitory Concentration (MIC; lg/mL) of ceftazidime in presence of 8 lg/mL of these
b-lactamase inhibitors
Ki (l
M)
MIC (lg/mL)
CTX-M-9a TEM-1 AmpC CMY-2 E. cloacae
E. coli SYN2549
P99
P99AmpC CMY-2
1
1.89
1.02
18.9
20.4
14.7
0.71
0.02
0.02
0.08
1.51
0.02
0.02
0.07
32
8
1–-2
4–16
8
1
<0.5
<0.5
15n >32
22
24
>32
>32
10. Experimental procedure for the preparation of compound 24: To a solution of 20
(0.5 g, 3.33 mmol) in anhydrous DMF (15 mL) were added K2CO3 (1.382 g,

2536
Y. Xia et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2533–2536
10.0 mmol) and 2-chloro-pyrimidine-5-carboxylic acid methyl ester (0.575 g,
3.33 mmol) at room temperature. After stirring at room temperature for 25 h,
(m, 2H), 7.35 (dd, J = 8.4 Hz, 2.0 Hz, 1 H), 5.03 (s, 2 H). MS (ESI) m/z = 273
[M+H]+.
the reaction mixture was cooled to 0 °C diluted with water (20 mL) and
acidified to pH 2 using diluted hydrochloric acid. The white precipitate was
collected, washed with water and dried to give 0.678 g of 23; mp 117–118 °C.
1H NMR (400 MHz, DMSO-d6) d 9.26 (s, 1 H), 9.08 (s, 2 H), 7.44–7.57 (m, 2 H),
7.31–7.40 (m, 1 H), 5.03 (s, 2 H), 3.88 (s, 3 H). MS (ESI) m/z = 287 [M+H]+. To a
solution of 23 (0.5 g, 1.75 mmol) in methanol (20 mL) was added aqueous LiOH
(0.419 g in 15 mL of water, 17.5 mmol) at 0 °C. The resulting mixture was
stirred at room temperature for 1.5 h. After removed most of the methanol, the
reaction mixture was cooled to 0 °C and acidified to pH 2 using diluted
hydrochloric acid. The white precipitate was collected, washed with water and
dried to give the crude product which was purified by chromatography on
silica gel (hexane/THF/AcOH = 2:1:trace) to give 0.102 g (21%) of 24; mp 195–
196 °C. 1H NMR (400 MHz, DMSO-d6) d 9.27 (br s, 1H), 9.04 (s, 2H), 7.51–7.49
11. All b-lactamases were tested as essentially described by Payne et al
J. Antimicrob. Chemother. 1991, 28, 775–776. with a few modifications. The
buffer was 50 mM potassium phosphate pH 7 with 0.2% Triton x-100, and the
concentration of nitrocefin was 500 lM for class A b-lactamases and 200 lM
for class C b-lactamases. Kinetic data is collected by measuring the rate of
change in A486 over 30 min. The fraction of enzyme inhibited is determined by
dividing the reaction rates in the presence of inhibitor by the reaction rate
determined in the absence of inhibitor. Dose–response curves are then
generated by plotting log [inhibitor] versus fraction inhibited. IC50 values
were determined from the dose-response curves by determining the inhibitor
concentration required to reduce the maximum inhibitory activity of the
compound by 50%. The Ki values were calculated from the IC50 using the Km for
nitrocefin for each enzyme and the following equation. Ki = IC50/(1 + [S]/Km).