Discovery of zoniporide: A potent and selective sodium-hydrogen exchanger type 1 (NHE-1) inhibitor with high aqueous solubility

Article abstract of DOI:10.1016/S0960-894X(01)00059-2

Zoniporide (CP-597,396) is a potent and selective inhibitor of NHE-1, which exhibits high aqueous solubility and acceptable pharmacokinetics for intravenous administration. The discovery, synthesis, activities, and rat and dog pharmacokinetics of this compound are presented. The potency and selectivity of zoniporide may be due to the conformation that the molecule adopts due to the presence of a cyclopropyl and a 5-quinolinyl substituent on the central pyrazole ring of the molecule.

Full text of DOI:10.1016/S0960-894X(01)00059-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 803–807
Discovery of Zoniporide: A Potent and Selective
Sodium–Hydrogen Exchanger Type 1 (NHE-1) Inhibitor
with High Aqueous Solubility
Angel Guzman-Perez,* Ronald T. Wester, Mary C. Allen, Janice A. Brown,
Allan R. Buchholz, Ewell R. Cook, Wesley W. Day, Ernest S. Hamanaka,
Scott P. Kennedy, Delvin R. Knight, Paul J. Kowalczyk, Ravi B. Marala,
Christian J. Mularski, William A. Novomisle, Roger B. Ruggeri, W. Ross Tracey
and Roger J. Hill
Pfizer Global Research and Development, Pfizer Inc., Eastern Point Road, Groton CT 06340, USA
Received 20 October 2000; accepted 24 January 2001
Abstract—Zoniporide (CP-597,396) is a potent and selective inhibitor of NHE-1, which exhibits high aqueous solubility and
acceptable pharmacokinetics for intravenous administration. The discovery, synthesis, activities, and rat and dog pharmacokinetics
of this compound are presented. The potency and selectivity of zoniporide may be due to the conformation that the molecule
adopts due to the presence of a cyclopropyl and a 5-quinolinyl substituent on the central pyrazole ring of the molecule. # 2001
Elsevier Science Ltd. All rights reserved.
The sodium–hydrogen exchangers (NHEs) are cell
membrane transport proteins which exchange extra-
The cardioprotective ability of a selective NHE-1 inhi-
bitor could be extremely beneficial in hospital settings;
for example, in patients that are at risk for myocardial
+
+
cellular Na for intracellular H driven by concentra-
tion gradients. NHEs play a key role in intracellular pH
regulation, ion-transport and control of cell volume.
There are at least six known isoforms that are desig-
2
ischemic injury during cardiac surgery. For surgical
settings, an agent with high aqueous solubility that
would allow for intravenous administration would be
preferred.
1
nated NHE-1 through NHE-6. NHE-1 appears to play
a key role in mediating myocardial injury during peri-
ods of ischemia and reperfusion. Although there is some
debate about the degree of NHE-1 activity during
myocardial ischemia, the activation of this exchanger
during reperfusion is clearly detrimental to the heart,
+
due to a cascade of increased intracellular Na , activa-
2+
+
tion of the Na /Ca
exchanger, and intracellular
2
+
Ca overload. Consequently, inhibition of NHE-1, the
sole isoform present in the heart, represents an attrac-
tive approachfor reducing myocardial injury during
ischemia/reperfusion. A selective NHE-1 inhibitor is
desirable due to the important physiological functions
played by the additional NHE isoforms in other tissues.
In particular, NHE-2 and NHE-3 appear to have
Our efforts directed towards the discovery of a selective
NHE-1 inhibitor began with the recognition that car-
4
5
iporide (1a) and eniporide (1b), two NHE inhibitors in
clinical development, bothcontain an acylguanidine moi-
ety. The synthesis and screening of a variety of heteroaryl
acylguanidines led to the identification of pyrazole 3a as a
novel and attractive lead. Herein, we report the modifica-
tions in the structure of 3a that led to the discovery of
zoniporide (CP-597,396) (3d), a potent and selective
NHE-1 inhibitor with the required aqueous solubility
and pharmacokinetics for intravenous administration.
2
,3
1
significant roles in the gastrointestinal and renal systems.
*
0
Corresponding author. Tel.: +1-860-715-0313; fax: +1-860-686-
001; e-mail: angel_guzman-perez@groton.pfizer.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00059-2

804
A. Guzman-Perez et al. / Bioorg. Med. Chem. Lett. 11 (2001) 803–807
The synthesis of 3d is representative of that used to
prepare similar analogues (Scheme 1). Monoethyl
malonic ester (4) was converted to b-ketoester 5 by for-
mation of the corresponding trimethylsilyl ester, fol-
lowed by deprotonation with n-butyllithium and
6
acylation withcyclopropanecarbonyl c hl oride. Treat-
ment of 5 with N,N-dimethylformamide dimethyl acetal
7
provided enamine 6 as a single geometric isomer.
Hydrazine 8 was prepared by diazotization of amine 7
followed by reduction of the diazonium salt with stan-
nous chloride and was isolated as the dihydrochloride
8
salt. This salt was considerably more stable to storage
than the corresponding free base. The condensation of
enamine 6 with hy drazine 8 in the presence of triethyl-
amine afforded pyrazole ester 9 in good yield as the only
7
detectable regioisomer. This ester was converted to the
acylguanidine by two different methods. In one method,
a mixture of the ester and guanidine was heated first in
ethanol, and then neat and in vacuo to provide 3d as the
free base. Alternatively, the ester was hydrolyzed to the
corresponding carboxylic acid 10, which was trans-
formed to the acid chloride using thionyl chloride. The
acid chloride was reacted with guanidine hydrochloride
under Schotten–Baumann conditions to provide 3d as
the free base. In both cases, the free base 3d was con-
verted to the monohydrochloride salt which crystallized
.
.
9
as the monohydrate 3d HCl H O.
2
A fluorescence-imaging plate reader (FLIPR) assay was
used as our primary screen for NHE-1 inhibitory activ-
ity. In this assay, fibroblasts expressing functional
human NHE-1 were exposed to an intracellular acidic
challenge in the presence of varying concentrations or
absence of compound. The rate of NHE-1 mediated
recovery of intracellular pH was determined in a 96-well
format using the FLIPR and a pH sensitive fluorescent
dye. NHE-1 inhibitors reduce the rate of pH recovery.
The IC50 value is the concentration required to inhibit
Scheme 1. Synthesis of zoniporide (3d) (a) (i) TMSCl, pyridine, ether,
ꢀ
ꢀ
2
3 C, 4 h; (ii) n-BuLi (2.5 M in hexanes), ꢁ78 C, 75 min; (iii) cyclo-
ꢀ
propanecarbonyl chloride, DME, ꢁ78–23 C, 18 h, 93%; (b) N,N-
2
dimethylformamide dimethyl acetal, reflux, 1 h, 91%; (c) (i) NaNO ,
ꢀ
2.
O, HCl (aq), 0 C, 1 h; (ii) SnCl 2H
ꢀ
H
2
2
O, HCl (aq), 0–23 C, 3 h; (iii)
HCl (g), EtOAc, 0 C, 15 min, 97%; (d) Et N, EtOH, reflux, 1 h, 88%;
ꢀ
3
ꢀ
(
e) guanidine, EtOH, 80 C, in vacuo, 45 min, 76%; (f) NaOH (2 M
1
0
aq), MeOH, reflux, 1 h, 56%; (g) (i) SOCl
hydrochloride, NaOH (2 M aq), THF, reflux, 1 h, 67%; (h) HCl
conc), THF, 1 h, 82%.
2
, reflux, 1 h; (ii) guanidine
maximal pH recovery by 50%. A similar assay with
fibroblasts expressing human NHE-2 and rat NHE-3
10
was used to determine the inhibition of these isoforms.
(
Table 1. NHE-1 and NHE-2 inhibitory activity and selectivity of zoniporide and analogues in vitro using a FLIPR based assay
Compounda
R
Ar
NHE-1
IC50 (nM)
NHE-2
IC50 (nM)c
NHE-1
versus NHE-2d
b
3
3
3
3
3
a
b
Me
Ph830
Ph350
1-Naphthyl
5-Quinolinyl
5-Quinolinyl
3900
12,800
160
59
860
5
37
Cyclopropyl
Cyclopropyl
Cyclopropyl
Me
c
d (Zoniporide)
e
16,300
12,000
41,600
102
203
48
1
1
a (Cariporide)
b (Eniporide)
—
—
—
—
560
140
28,700
9200
51
66
a
Experiments carried out using the hydrochloride or mesylate salt forms.
Mean of 9–32 experiments.
Mean of 6–13 experiments.
b
c
d
NHE-1 versus NHE-2 selectivity (NHE-2 IC50/NHE-1 IC50).

A. Guzman-Perez et al. / Bioorg. Med. Chem. Lett. 11 (2001) 803–807
805
As indicated in Table 1, the NHE-1 IC50 of lead com-
pound 3a is somewhat higher than that exhibited by
reference compounds cariporide (1a) and eniporide (1b)
in the FLIPR assay, and its NHE-1 versus NHE-2
selectivity is significantly lower. An investigation of the
SAR around the methyl group of 3a led to the identifi-
cation of compound 3b bearing a cyclopropyl group in
its place. Compound 3b has improved potency and
considerably higher selectivity with respect to 3a, and is
comparable to cariporide and eniporide. Replacement
of the phenyl ring with a 1-naphthyl ring provided
compound 3c witheven greater potency and selectivity.
Unfortunately, the moderate aqueous solubility of
compound 3c was not optimal for developing an
intravenous formulation. To overcome this problem,
the 1-naphthyl group in 3c was replaced with he tero-
bicyclic rings. These efforts provided zoniporide (3d),
which contains a 5-quinolinyl ring in place of the 1-
naphthyl group. Zoniporide has excellent NHE-1
potency, which is 2- to 9-fold better than cariporide or
eniporide, and an NHE-1 versus NHE-2 selectivity that
is at least 3-fold higher. Furthermore, it has a rat NHE-
3 IC50 >500,000 nM, which represents a >8000-fold
selectivity against this isoform (Fig. 1). Interestingly,
replacement of the cyclopropyl group of zoniporide
with the methyl group present in the original lead
structure results in a dramatic drop in NHE-1 potency
and selectivity (compound 3e). In fact, the combined
effect of incorporating the cyclopropyl and 5-quinolinyl
groups into this series appears to be synergistic. It is
possible that steric interactions between the 5-quinolinyl
ring and the cyclopropyl ring in zoniporide places both
groups in an optimum conformation for bothpotency
and selectivity.
Computational molecular modeling11 supports this
hypothesis. Modeling of 3a shows that the bond con-
necting the phenyl and pyrazole rings in this compound
is conformationally flexible. As shown in Figure 2, even
the highest energy rotamers around this bond are read-
ily accessible (8–10 kJ/mol) and no particular con-
formation is favored (conformational dilution).
Modeling of zoniporide (3d) provides very different
results for the rotamers about the bond joining the
quinoline and pyrazole rings (Fig. 2). An energy mini-
ꢀ
mum is obtained at about ꢁ120 , and a valley with
relative energy of 4 kJ/mol is observed at approximately
60 . The barriers to rotation of this bond are quite high:
ꢀ
approximately 120 and 60 kJ/mol (at dihedral angles of
Figure 1. Zoniporide inhibition of human NHE-1-, NHE-2-, and rat
NHE-3-mediated pH recovery in vitro using a FLIPR based assay.
Values are meansꢂSE of representative data.
ꢀ
to be considerably more conformationally rigid than 3a.
about 20 and 170 , respectively). Thus, zoniporide appears
The good aqueous solubility of zoniporide makes it well
suited for a clinical target of parenteral administration.
.
Crystalline zoniporide HCl H O displays high solubility
.
2
in several vehicles. For example, it has a solubility at
room temperature of 5.6 and 8.4 mg of active/mL in
sterile water for injection and 5% dextrose for injection,
respectively. This is at least partially due to the presence
of two distinct ionizable sites in the molecule. The pKa
of the acylguanidine and of the quinolinyl moieties are
7.2 and 3.4, respectively.
The pharmacology of zoniporide is very promising. A
2
2
Na uptake assay confirmed the high in vitro potency
IC =14 nM) and selectivity (157-fold and 15,700-fold
vs human NHE-2 and rat NHE-3, respectively) of
zoniporide. Its high selectivity for NHE-1 versus
NHE-2 and NHE-3 diminishes the likelihood of side
(
5
0
Figure 2. Rotational profile of 3a about the phenyl-pyrazole bond,
and of zoniporide (3d) about the quinoline-pyrazole bond. The struc-
ture shown represents the lowest energy conformer obtained for 3d.
1
2
Table 2. Pharmacokinetic parameters of zoniporide after intravenous administration in rat and doga
b
.
Species
C
0
(mg/mL)
AUC0-1 (mg h/mL)
Clp (mL/min/kg)
Vdss (L/kg)
t
1/2 (h)
Plasma protein binding (%)
Rat (n=4)
Dog (n=4)
0.28 (0.08)
0.58 (0.08)
0.07 (0.01)
0.45 (0.15)
237 (28)
40 (13)
7.2 (2.2)
2.3 (0.2)
0.5 (0.1)
0.9 (0.3)
54
55
a
.
Values are expressed as mean (standard deviation). Dose 1 mg/kg. All studies used zoniporide HCl H
.
2
O administered as a solution in normal
saline.
b
Represents the estimated plasma concentration at time zero calculated using log-linear back-extrapolation of the first two measured plasma
concentrations.

806
A. Guzman-Perez et al. / Bioorg. Med. Chem. Lett. 11 (2001) 803–807
effects due to interactions witht eh latter isoforms.
Additionally, zoniporide displays excellent potency in
an ex vivo inhibition of human platelet swelling assay
777. (b) Orlowski, J. Ann. N.Y. Acad. Sci. 1999, 874, 346. (c)
Karmazyn, M. Ann. N.Y. Acad. Sci. 1999, 874, 326. (d)
Gumina, R. J.; Gross, G. J. J. Thromb. Thrombolyis 1999, 8,
3
9. (e) Karmazyn, M. J. Thromb. Thrombolyis 1999, 8, 33. (f)
(
IC =54 nM), which confirms its activity against
50
1
2
Myers, M. L. J. Thromb. Thrombolyis 1999, 8, 53. (g) Erhardt,
L. R. W. Am. J. Cardiol. 1999, 83, 23G. (h) Buerke, M.;
Rupprecht, H.-J.; von Dahl, J.; Terres, W.; Seyfarth, M.;
Schultheiss, H.-P.; Richardt, G.; Sheehan, F. H.; Drexler, H.
Am. J. Cardiol. 1999, 83, 19G. (i) Avkiran, M. Am. J. Cardiol.
endogenous human NHE-1. Furthermore, zoniporide
shows robust cardioprotection in the rabbit heart sub-
jected to 30 min of regional ischemia and 2 h of reper-
fusion, bothin vitro and in vivo (EC 0=0.25 and
5
3
infarct size of 83 and 75%, respectively, in these mod-
49 nM, respectively) witha maximum reduction of
1
999, 83, 10G. (j) Avkiran, M. Eur. Heart J. Suppl. 1999, 1,
K11. (k) Murphy, E.; Cross, H. R.; Steenbergen, C. Eur. Heart
J. Suppl. 1999, 1, K18. (l) Hashimoto, K.; Sugiyama, A.; Xue,
Y. X.; Aye, N. N.; Yamada, C.; Chino, D. Eur. Heart J.
Suppl. 1999, 1, K31. (m) Karmazyn, M. Eur. Heart J. Suppl.
1
3
els.
Zoniporide displays the pharmacokinetics in rat and dog
shown in Table 2, which should allow for intravenous
administration. It is noteworthy that the plasma protein
binding of zoniporide in these species is moderate.
Renal clearance of unchanged drug is low in dogs
1
999, 1, K38. (n) Levitsky, J.; Gurell, D.; Frishman, W. H. J.
Clin. Pharmacol. 1998, 38, 887. (o) Karmazyn, M. Keio J.
Med. 1998, 47, 65. (p) Karmazyn, M. Cur. Res. Ion Channel
Modulat. 1997, 3, 3. (q) Fro
vasc. Res. 1997, 36, 138.
. For a recent review on NHE inhibitors, see: Kleemann,
¨
hlich, O.; Karmazyn, M. Cardio-
(
13%).
3
H. W.; Weichert, A. G. IDrugs 1999, 2, 1009.
4. (a) Weichert, A.; Faber, S.; Jansen, H. W.; Scholz, W.;
Lang, H. J. Arzneim.-Forsch. 1997, 47, 1204. (b) Graul, A.;
In conclusion, zoniporide is a potent and selective inhi-
bitor of NHE-1, which exhibits high aqueous solubility
and acceptable pharmacokinetics for intravenous
administration. The potency and selectivity of zonipor-
ide may be due to the conformation that the molecule
adopts because of the presence of a cyclopropyl and a 5-
quinolinyl substituent on the central pyrazole ring of the
molecule. This compound may be useful in protecting
the myocardium and other tissues from ischemia/reper-
fusion injury, and is currently the subject of further
investigation.
Prous, J.; Castan
A.; Prous, J.; Castan
Dhein, S.; Salameh, A. Cardiovasc. Drug Rev. 1999, 17, 134.
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zyn, M. Expert Opin. Invest. Drugs 2000, 9, 1099.
˜
er, J. Drugs Future 1997, 22, 1197. (c) Graul,
˜
er, J. Drugs Future 1998, 23, 1235. (d)
(
5
4
6
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7. Menozzi, G.; Mosti, L.; Schenone, P. J. Heterocycl. Chem.
1
987, 24, 1669.
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8
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Acknowledgements
1
9
141.
. [1-(Quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]-
The authors are grateful to Leanne M. Buzon, George
C. Chang, Shane A. Eisenbeis, Melissa L. Gillaspy, Lisa
M. Kath, Jaquelyn L. Klug, George Magnus-Aryitey
and Michael R. Makowski for their synthetic contribu-
tions to the NHE-1 program. We thank Mark G. Biron,
Eugene F. Fiese and Joseph E. Mertz for their pharma-
ceutical sciences input. The biology advice of Michael L.
Mangiapane is gratefully acknowledged.
.
.
guanidine hydrochloride monohydrate (3d HCl H O): mp
2
ꢀ
1
1
0
1
54–156 C; H NMR (400 MHz, DMSO-d ) d 0.42 (m, 2H),
6
.59–0.61 (m, 2H), 1.88–1.95 (m, 1H), 7.57 (dd, J=9, 4 Hz,
H), 7.67 (d, J=4 Hz, 1H), 7.82 (d, J=7 Hz, 1H), 7.90 (t,
J=8 Hz, 1H), 8.22 (d, J=8 Hz, 1H), 8.38 (bs, 2H), 8.69 (bs,
1
3
2H), 8.72 (s, 1H), 8.98 (dd, J=4, 1.4 Hz, 1H); C NMR
(100 MHz, DMSO-d ) d 7.8, 8.0, 114.8, 123.7, 125.3, 126.8,
129.6, 131.7, 131.8, 135.7, 141.6, 148.4, 151.2, 152.2, 156.3,
6
+
1
C
63.2; APCIMS 321 [M+H] . Anal. calcd for
.
H N O HCl H O: C, 54.47; H, 5.11; N, 22.42; Cl, 9.46.
.
17 16 6
2
Found: C, 54.36; H, 5.16; N, 22.38; Cl, 9.13.
0. Materials: 2,7-Biscarboxyethyl-5,6-carboxyfluorescein
References and notes
1
acetoxymethyl (BCECF-AM) was purchased from Molecular
Probes (Eugene, OR). All other reagents were of analytical
grade and obtained commercially. Methods: PS120 fibroblast
cells expressing human NHE-1 or rat NHE-3 isoforms were
obtained from Professor J. Pouyssegur (Nice, France). Human
NHE-2 expressing cells were generated at Pfizer Global
Researchand Development (Groton, CT). PS120 fibroblasts
expressing indicated NHE isoform were grown to confluency
in 96-well collagen coated plates. Measurement of intracellular
1
. (a) For recent reviews on the structure, function, regula-
tion, isoforms and distribution of NHE, see: Counillon, L.;
Pouyssegur, J. J. Biol. Chem. 2000, 275, 1. (b) Fliegel, L. J.
Thromb. Thrombolyis 1999, 8, 9. (c) Avkiran, M.; Snabaitis,
A. K. J. Thromb. Thrombolyis 1999, 8, 25. (d) Aharonovitz,
O.; Grinstein, S. Drug News Perspect. 1999, 12, 105. (e) Cou-
´
nillion, L.; Touret, N.; Godart, H.; Pouyssegur, J.Eur. Heart
J. Suppl. 1999, 1, K2. (f) Harris, C.; Fliegel, L. Int. J. Mol.
Med. 1999, 3, 315. (g) Diprov, P.; Fliegel, L. FEBS Lett. 1998,
i
pH (pH ) was based on the methodology of: Russ, U.; Balser,
4
24, 1. (h) Fliegel, L.; Murtazina, R.; Dibrov, P.; Harris, C.;
Moor, A.; Fernandez-Rachubinski, F. A. Biochem. Cell Biol.
998, 76, 735. (i) Kinsella, J. L.; Heller, P.; Froehlich, J. P.
C.; Scholz, W.; Albus, U.; Lang, H. J.; Weichert, A.; Scholk-
ens, B. A.; Gogelein, H. Pflugers Arch. 1996, 433, 26. Cells
were washed three times with assay buffer (10 mM Hepes,
1
Biochem. Cell Biol. 1998, 76, 743. (j) Wakabayashi, S.; Shige-
kawa, M.; Pouyssegur, J. Physiol. Rev. 1997, 77, 51. (k)
Orlowski, J.; Grinstein, S. J. Biol. Chem. 1997, 272, 22373.
2. For recent reviews on the role of NHE-1 in ischemia/
reperfusion injury, see: (a) Karmazyn, M.; Gan, X. T.; Hum-
phreys, R. A.; Yoshida, H.; Kusumoto, K. Circ. Res. 1999, 85,
136 mM NaCl, 5.4 mM KCl, 0.44 mM KH
Na HPO , 1 mM CaCl , 1 mM MgCl and 5.6 mM glucose,
2 4
PO , 0.34 mM
2
4
2
2
pH 7.4) followed by incubation with5 mM BCECF-AM in the
same buffer for 30 min at 37 C. Cells were then washed seven
ꢀ
times withacid loading buffer (30 mM NH
chloride, 5 mM KCl, 1.8 mM CaCl , 1 mM MgCl , 5 mM
Cl, 90 mM choline
4
2
2

A. Guzman-Perez et al. / Bioorg. Med. Chem. Lett. 11 (2001) 803–807
807
glucose, and 15 mM Hepes, pH 7.5). Cells were initially incu-
bated for 1 hwithacid loading buffer followed by an addi-
tional 5 min incubation in the presence or absence of test
compounds. Initial baseline fluorescence was recorded for
eachwell simultaneously using a fluorescence-imaging plate
reader (FLIPR). Intracellular acidification was induced by the
addition of assay buffer withor wit oh ut test compound.
Intracellular acidification was observed as a marked reduction
of BCECF fluorescence. NHE inhibitors prevent recovery
from the acidification (measured as fluorescent units). Data
analysis: The potency of NHE inhibitors was calculated as a
function of its ability to prevent recovery from acidification
11. Conformational analyses were performed using the OPLS/
A force field witht he GBSA solvent approximation as imple-
mented in MacroModel v.7. Low energy conformers were
identified via a Monte Carlo search, followed by energy mini-
mization. Rotations were allowed for all single, acyclic and
non-terminal bonds. All of the low energy conformers place
the cyclopropyl group in the conformation with respect to the
pyrazole ring shown in Figure 2. The rotational profiles in Fig-
ure 2 were generated using the low energy conformers identified
in the Monte Carlo search as the initial structures. The bond of
ꢀ
interest was held fixed at 10 increments and a constrained
energy minimization was performed on eachof these rotamers.
12. Marala, R. B.; Brown, J. A.; Kong, J. X.; Tracey, W. R.;
Knight, D. R.; Wester, R. T.; Sun, D.; Kennedy, S. P.; Hama-
naka, E. S.; Ruggeri R. B.; Hill, R. J., submitted for publication.
13. Knight, D. R.; Smith, A. H.; Flynn, D. M.; MacAndrew,
J. T.; Ellery, S. S.; Kong, J. X.; Marala, R. B.; Wester, R. T.;
Guzman-Perez, A.; Hill, R. J.; Magee, W. P.; Tracey, W. R. J.
Pharmacol. Exp. Ther. 2001, 297, in press.
compared withcontrol. T he IC
(
value for NHE inhibitors
0
i
concentration required to inhibit pH recovery by 50%) was
5
calculated by fitting the data via nonlinear least squares
regression analysis to the equation: % Inhibition=100/
C
X D
[
(
1+(10 /10 ) ], where X=log [drug concentration],
IC50)=log [drug concentration at 50% inhibition], and
D=the Hill coefficient.
C