The discovery of benzanilides as c-Met receptor tyrosine kinase inhibitors by a directed screening approach

Article abstract of DOI:10.1016/j.bmcl.2011.07.047

A directed screen of a relatively small number of compounds, selected for kinase ATP pocket binding potential, yielded a novel series of hit compounds (1). Hit explosion on two binding residues identified compounds 27 and 43 as the best leads for an optimization program having reduced secondary metabolism, as measured by in vitro rat hepatocytes incubation, leading to oral bio-availability. Structure-activity relationships and molecular modeling have suggested a binding mode for the most potent inhibitor 12.

Full text of DOI:10.1016/j.bmcl.2011.07.047

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5224–5229
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of benzanilides as c-Met receptor tyrosine kinase inhibitors
by a directed screening approach
Joanne V. Allen, Catherine Bardelle, Kevin Blades, Dave Buttar, Louise Chapman, Nicola Colclough,
⇑
Alexander G. Dossetter , Andrew P. Garner, Alan Girdwood, Christine Lambert,
Andrew G. Leach, Brian Law, John Major, Helen Plant, Anthony M. Slater
AstraZeneca R&D, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A directed screen of a relatively small number of compounds, selected for kinase ATP pocket binding
potential, yielded a novel series of hit compounds (1). Hit explosion on two binding residues identified
compounds 27 and 43 as the best leads for an optimization program having reduced secondary metabo-
lism, as measured by in vitro rat hepatocytes incubation, leading to oral bio-availability. Structure–activity
relationships and molecular modeling have suggested a binding mode for the most potent inhibitor 12.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 1 June 2011
Revised 11 July 2011
Accepted 12 July 2011
Available online 23 July 2011
Keywords:
c-Met
Kinase
Directed screening
Ligand efficiency
Secondary metabolism
Bio-isosteres
Decisions
Benzanilides
OH
N
c-Met receptor tyrosine kinase (c-Met) is heavily implicated in
tumour progression and is therefore seen as an important target
for the treatment of cancer.1 Binding of its ligand, Hepatocyte
Growth Factor (HGF, also known as Scatter Factor 1 (SF1)), results
in receptor dimerization, autophosphorylation and recruitment of
Grb2 and Gab1.2,3 Assembly of this signalling complex triggers
downstream signalling through the PI3K/AKT5 and Ras/Raf path-
ways. Significant crosstalk is thought to occur between these two
pathways, and this is strongly speculated to result in cell motility
and proliferation.6 Elevated levels of both HGF/c-Met are observed
in a wide range of tumours types and increased expression is cor-
related with poor clinical outcome.7 In addition, recent research
has indicated that tumour hypoxia can induce over-expression of
c-Met thus triggering cell motility, adding further value to c-Met
as a target.4c-Met inhibitors are now in clinical development for
solid tumours of which several candidates are shown in Figure
1.7 Inhibition of the ATP binding domain with these compounds
has proven an effective method of blocking kinase activity and
down regulation of cell signalling.8 The four candidates all have a
binding group mimicking the purine of ATP; BMS-777607 and
O
N
O
O
N
O
N
N
O
O
O
Cl
O
F
N
O
F
H2N
N
N
BMS-777607
AMG-458
O
S
N
Cl
F
O
N
O
NH
O
N
Cl
O
N
N
H2N
N
N
N
MK-2461
PF-02341066
Figure 1. c-Met tyrosine kinase inhibitors in clinical development.
PF-02341066 have a 2-amino-pyridine.9,10 AMG-458 a quinoline11
and MK-2461 a tricyclic aromatic.12
The use of high throughput screening (HTS) is now widespread in
the pharmaceutical industry as the primary method of obtaining
chemical hits. However, where useful information is available from
x-ray diffraction studies of complexes or past performance of
compounds in previous HTS screens, it should be possible to select
⇑
Corresponding author.
E-mail addresses: al@dossetter.plus.com, al.dossetter@astrazeneca.com (A.G.
Dossetter).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.047

J. V. Allen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5224–5229
5225
a directed compound screening set, reducing time and cost. At the
O
O
O
O
O
b
d
a
OH
OH
Si
O
+
time of screening within the c-Met program both biological and
structural information from other tyrosine kinase inhibitor pro-
grams was available, so we selected a set of eighteen thousand com-
pounds, based on or more of the following criteria: (a) activity in
previous HTS campaigns against other kinase targets (EGFTK, VEG-
FTK for example), (b) ‘privileged’ kinase chemical functionality13 (c)
molecular weight (<400 Da) and c log P (<4.0). Of this set approxi-
mately 10,000 were selected from custom synthesised libraries of
novel templates designed to bind into the kinase ATP pocket.
This directed set of compounds was screened in duplicate at
N
HO
HO
O
O
3
4
5
OH
NO2
O
O
O
O
c
Si
NO2
NH2
e
O
N
O
O
O
K
10 lM concentration using an Alphaquest format employing re-
f,g
N
N
N
O
combinant truncated wild type c-Met enzyme in the presence of
ATP. The activity of the compounds was confirmed in dose response
testing to determine a pIC50 value.14 Within the active compound
set was a small cluster of 27 chemically similar benzanilides, orig-
inally designed to have the carbonyl of the amide providing the
hinge binding group. Hit compound 1 was the most active of this
cluster with a pIC50 value of 6.81 (Table 1). These compounds were
attractive in that they were potent against c-Met enzyme and pos-
sessed a novel kinase template. However, compound 1 had an
undesirable profile of cytochrome-P450 inhibition (Table 1) and
while human microsomal in vitro clearance was low, hepatic cell
clearance was very high, which was attributed to secondary glucu-
ronidation of the 30 -OH group (metabolite found post incubation of
1 with rat hepatocytes). For a discovery program aimed at an oral
administration in the clinic removal of the 30-OH group or replace-
ment was desirable.15 Compound 2 showed that the 2-benzyloxy
group was important as it tested 100 fold lower in activity. Hence
an SAR study program was undertaken to optimise these hits and
determine the developability of the series. Rapid synthesis of
numerous examples involved varying the benzaniline group and
the 2-benzyloxy substituent.
O
+
-
R2
O
HO
SEM
7 - 24
6
Scheme 1. Reagents and conditions: (a) TFAA, TFA, acetone, 0 °C to rt 24 h, 23%; (b)
1-(3-chloropropyl)pyrrolidine, K2CO3, DMF, 80 °C, 4 h, 86%; (c) (2-trimethyl-
silyl)ethoxymethyl chloride (SEM-Cl), DIPEA, CH2Cl2, RT, 2 h, 99%; (d) H2 1 atm,
10% Pd on C, EtOH, RT, 4 h, 47%; (e) NaHMDS, THF, ꢁ78 °C then K2CO3 (aq) workup,
53%; (f) R2-Cl, K2CO3, DMF, 80 °C OR R2-OH, DIAD, PPh3, THF, rt; (g) 1.0 M HCl in
dioxane, 10 min, 39 to 74%.
O
O
O
O
O
O
a
b
OH
O
O
N
N
HO
HO
O
25
26
c OR d
O
O
R1
N
N
O
The compounds with varying 2-alkoxy substitutents were syn-
thesised by the route outlined in Scheme 1. Commercially available
2,4-dihydroxybenzoic acid was protected as the di-isopropylidene
ketal, then the free 4-OH group alkylated to yield intermediate 4.
The hydroxyl bearing benzanilide was introduced using a SEM pro-
tecting group strategy in aniline 5, which was synthesised from
commercially available 3-nitrophenol. Reaction of the aniline 5
with sodium hexamethyldisilylamide produced a reactive nitrogen
nucleophile to attack the carbonyl of 4. Aqueous work up with
potassium carbonate solution yielded the salt 6. Simple alkylation
using alkyl chlorides or via a Mitsonobu protocol with alcohols fol-
lowed by a SEM deprotection yielded the final compounds 7 to 24.
27 - 43
Scheme 2. Reagents and conditions: (a) 1-(3-chloropropyl)pyrrolidine, K2CO3,
DMF, 80 °C, 4 h, 36%; (b) benzyl chloride, K2CO3, DMF, 80 °C OR benzylalcohol,
DIAD, PPh3, THF, RT, 65% (c) (i) 2 M NaOH (aq), MeOH, 50 °C (ii) Ar-NH2, HATU, DMF,
RT, 24 h OR SOCl2, CH2Cl2, RT, 1 hour then R1-NH2, DIPEA, CH2Cl2; 45% (d) Ar-NH2,
AlMe3, toluene, microwave, 150 °C, 15 min, 52%.
Compounds varying the benzanilide group (R1) were synthe-
sized in parallel to R2 variants by the route outlined in Scheme 2.
Commercially available methyl-2,4-dihydroxybenzoate was selec-
tively alkylated on the 4-OH group to yield 25 in good yield.
Table 1
Profiles of benzanilides 1 and 2
O
O
O
O
N
N
N
N
O
H O
HO
HO
1
2
Compound
1
2
c-Met enzyme pIC50
MWt/Da
(lM)
6.81 0.17
446.5
4.86 0.50
356.4
Log D7.4 (c log P)
Efficiency LLEa (LEb)
Aqueous Solubility (
3.0 (5.0)
3.8 (0.29)
6.7
6.00
>300
–(3.75)
1.1 (0.26)
l
M)
HLM clearancec/
l
L/min/kg
Rat Hepatic cell clearance/
P450 inhibitiond/
M (isoform)
—
—
—
l
L/min/kg
l
3.0 (2C19), 1.2 (2C9), 4.2 (3A4)
a
b
c
Ligand Lipophilicity Efficiency LLE = c-Met pIC50–Log D7.4
.
LE (Ligand Efficiency) = (1.4 ꢀ c-Met pIC50)/n(heavy atoms).
HLM–human liver microsomal fraction in vitro incubation.
d
Selected isoform inhibition below 10 lM.

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J. V. Allen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5224–5229
Alkylation under basic conditions with benzylchloride or the
Mitsonobu protocol with benzyl alcohol yielded the intermediate
compound 26. The benzanilide group could either be introduced
via hydrolysis to yield a carboxylic acid then coupling with anilines
using HATU or via the acid chloride. Alternatively the methyl ester
26 could be reacted directly with an aniline using trimethylalu-
minium as an activating agent in toluene to yield final compounds
27–43.16 While several of the reactions were found to proceed at
room temperature or 50 °C, a more rapid and reliable method for
a diverse range of anilines was to heat under microwave conditions
at 150 °C.17
400-F group tolerated. A marked improvement in potency was
observed with small electron withdrawing groups (EWG) at the
200-position (11 and 12) in particular a nitrile group added 10 fold
in activity and a reduced lipophilicity thus is the most efficient
(LLE) compound (12) of the study. A similar trend was observed
in the 300-position with small EWGs (14, 15, 17), although the effect
was not as dramatic. The aromatic phenyl ring can be replaced by a
heterocycle with around 5-fold loss in potency, which can be offset
against the reduction in lipophilicity these groups afford (18, 19,
20). Compounds 21–24 are representative examples of alkyl
groups explored with the aim of reducing molecular size and lipo-
philicity. The results suggested that the R2 group could be simpli-
fied to an n-propyl with an approximate 4 fold drop in potency
(compound 22). However the best saturated alkyl group found
was cyclopentyl (24).
Table 2 outlines the results of SAR exploration around the 2-alk-
oxy substituent R2. A drop in potency was observed with substitu-
tion at the 400-position of the benzyl ring (7, 8 and 9), with only the
Table 2
c-Met inhibition, Log D7.4 and LLE caused by variation to the 2-alkoxy group
O
Table 3
O
c-Met inhibition corresponding to variation of the R1 group
N
N
O
HO
R2
Entry
1
R2
c-Met pIC50 std dev
Log D7.4
LLEc
3.8
6.81 0.16
3.0
7
8
6.96a
3.2
—
3.8
F
6.07 0.04
Cl
9
NAb
—
S
O
O
CH3
10
11
12
6.90 0.42
7.39 0.04
7.76 0.14
—
F
—
CN
F
2.3
5.5
13
7.16 0.57
—
F
F
14
15
16
7.26 0.12
7.01 0.36
7.07a
3.1
—
4.2
4.7
Cl
CN
NO2
2.4
17
18
19
6.90 0.31
6.37 0.08
6.25 0.15
—
N
1.6
1.8
4.8
4.5
N
N
S
20
6.19 0.21
1.6
4.6
21
22
Ethyl
Propyl
5.45 0.01
6.19 0.36
—
2.5
3.7
3.3
23
24
6.04 0.32
6.40 0.24
2.7
—
a
b
c
Compound tested only once.
NA <50% at top dose response concentration of 100
c-Met pIC50–LogD7.4
lM.
.

J. V. Allen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5224–5229
5227
As aforementioned, replacement or removal of the 30-OH was
Table 5
Comparison of Ph-30-OH compared to Ph
considered a key requirement to achieve progress towards orally
bio-available candidates for the clinic. Surprisingly, removal of
the group resulted in only a 2-3 fold drop in potency for compound
27, which suggested it was not a key hydrogen bonding group
(Table 3). Substitution of the R1 aromatic ring with small groups
showed that SAR around the ring was very tight, with only 30 sub-
stituents tolerated with small reductions in inhibition (e.g., 28–
32).18 Compound 31 illustrates that even the addition of a methyl
at the 40 position resulted in a 12 fold drop in potency. Pyridines
(33, 34), nitrile (35) and amides were disappointing as potential
O
N
HO
O
R1
R1 = or
N
O
R2
N
F
R2 =
N
0
acceptor/donor combinations (35, 36 and 37). Traditional0 pheno-
or
N
lic group isosteres were explored by replacement with N–H groups
of varying acidicity without success (39–42).12 So, at first, direct
replacement of the 30-OH to 30-H appeared the best option to move
forward with the group appearing as non-essential.
Compound profiles of 12 and 27 were obtained and in vitro rat
hepatic clearance greatly reduced in the absence of the 30-OH
group yielding a blood level concentration after oral dosing in ro-
dent (Table 4).19 In addition removal of the 30-OH had improved
the cytochrome-P450 profile of the series, with only slight inhibi-
tion of CYP450-2C9 observed. Given the results of our study on
substitution of the benzyl ring (Table 2), we elected to make com-
pound 43, expecting only a small drop in inhibition similar to the
N
F
Compound
R1
R2
c-Met pIC50
Difference
1
Ph-30-OH
Ph
benzyl
6.81
6.47
7.76
6.25
7.07
6.21
7.39
6.30
7.26
6.31
6.37
5.41
6.25
5.02
0.34
1.51
0.85
1.09
0.95
0.96
1.22
27
12
43
16
45
11
44
14
46
18
47
19
48
Ph-30-OH
Ph
Benzyl-200-CN
Benzyl-300-CN
Benzyl-200-F
Benzyl-300-F
3-py-methyl
2-py-methyl
Ph-30-OH
Ph
Ph-30-OH
Ph
Ph-30-OH
Ph
30-OH-Ph
Ph
change from 1 to 27 (D0.34 pIC50). To our surprise the potency
30-OH-Ph
Ph
against the c-Met enzyme was similar to that for 27 (Table 4).
Comparing the profiles of 27 and 43 we see that the 200-CN ap-
peared to have no benefit, except a small reduction in lipophilicity,
which was consistent with analysis of matched pairs for this group
in diverse compounds.20,21
Average difference between matched pairs = 0.99 i.e. 10 fold drop in potency.
Further examples of R2 side chain matched pairs (n = 7) showed
that removal of the 30-OH actually resulted on average in a drop in
potency of an order of magnitude (Table 5). Indeed compound 27
appeared to be unusual in retaining potency after removal of the
30-OH compared to the others and we had been in error to initially
conclude that this group was not vital for binding to c-Met. Re-
examining results from Table 3 we can conclude that the 30-OH
is operating as a donor and acceptor group given that both 30-
OCH3 and 30-NH2 are an order of magnitude less active, and other
acceptors or donor did not function as replacements. The largest
difference in activity of these matched pairs in Table 5 was with
200-CN-benzyl side chain (12 and 43), followed by 2-pyridinemeth-
yl (19 and 48), then 200-F-benzyl (11 and 44) suggesting that the
binding mode of these compounds might involve cooperation be-
tween these apparently distant groups.
We were unable to grow a single crystal suitable for x-ray dif-
fraction of a benzanilide compound and c-Met enzyme to deter-
mine the binding mode of this series. Recently phenolic c-Met
inhibitors have been disclosed by Zang et al.,22 and Albrecht et
al.23 In the case of the latter, a co-crystal complex where a hydrogen
bond with the backbone NH of Tyr 1159 of the hinge region was
made with the O atom of the phenol, and a water molecule formed
a second bond with the carbonyl of Met 1160. If our phenolic ben-
zanilides bind to the hinge in this specific manner this would offer
an explanation for the inactivity of the bio-isosteres tried (33–42)
as they would not present a compact donor/acceptor group.
Table 4
Profiles of benzanilides 27 and 43
O
O
O
O
N
N
N
N
O
O
R3
R3 = OH
R3 = H
12
43
27
N
Compound
12
27
43
c-Met enzyme pIC50
MWt
7.66 0.14
471.6
6.47 0.26
430.5
6.25 0.13
455.6
Log D7.4 (c log P)
2.3 (4.6)
5.5 (0.31)
363
0.1 (2C9), 0.85 (2D6), 1.65 (3A4)
11
3.6 (5.7)
2.9 (0.28)
61.8
8.8 (2C9)
6.0
2.7 (5.2)
3.6 (0.27)
34.4
3.2 (2D6), 4.8 (3A4)
6.5
Efficiency LLEa (LEb)
Aqueous Solubility/
P450 inhibitionc (isoform)/
HLM clearanced/
L/min/kg
Rat Hepatic cell clearance/
lM
l
M
l
l
L/min/kg
89
5.0
8.0
Rat Oral Exposure 2.0 mg/kg dose AUC/
lM.h
< 0.05
0.465
0.201
a
b
c
Ligand Lipophilicity Efficiency LLE = c-Met pIC50–Log D7.4
LE (Ligand Efficiency) = (1.4 ꢀ c-Met pIC50)/n(heavy atoms).
Selected isoform inhibition below 10 M.
HLM–human liver microsomal fraction in vitro incubation.
.
l
d

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J. V. Allen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5224–5229
as a compact donor/acceptor is required in conjunction with an
acceptor on the 20 position of the benzyl.
A directed screen of a relatively small number of compounds,
selected for kinase ATP pocket binding potential, yielded a novel
0
series of hit compounds. These were originally designed as hinge0
binders through the carbonyl group of the benzanilide however
SAR studies demonstrated the importance of the 30-OH-Ph and
200-CN-benzyl working co-operatively to deliver maximum inhibi-
tion. Modeling studies suggest a novel binding mode for compound
12, incorporating a water molecule bound between these two
groups and concomitant binding to the ‘hinge’. The compounds
27 and 43 were identified as optimal for rat bio-availability but
were insufficiently potent and inefficient, in both LLE and LE, to
continue further development.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.07.047.
Figure 2. Compound 12 (green) posed into high resolution structure c-Met
structure 3f66, placing 30-OH as binding to Tyr1159. An internal H-bond between
the amide and ether places the 200-CN-benzyl with the 200-CN pointing toward the
30-OH. A c-Met structure (3ccn) was aligned to evaluate this complex and was found
to have the small molecule phenol bound as shown (yellow).
References and notes
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Starting with the highest resolution co-crystal structure in the
public domain, 3f6624 we sought to model the binding mode of
12 through editing, minimisation and comparison to other c-Met
structures.25 The model has set the 30-OH group as a hinge binder
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NH and benzyl ether oxygen. The central phenyl ring is tight
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Fig. 2). Comparing the modeled complex by alignment with pro-
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sides between carbonyl of met 1160, the 200-CN and the 30-OH sug-
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13. ‘Privileged’ refers to structural motifs that are known to bind into the ATP
binding pocket of kinases, for example H-bond acceptor/donor pairs that
mimic the purine group of ATP. Amides are known kinase inhibitors, for
example: Baldwin, I.; Bamborough, P.; Haslam, C. G.; Hunjan, S. S.; Longstaff, T.;
Mooney, C. J.; Patel, S.; Quinn, J.; Somers, D. O. Bioorg. Med. Chem. Lett. 2008, 18,
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14. Recombinant GST-wild type c-met (kinase domain residues 1056-1371)
enzyme was titrated in the assay by dilution with enzyme diluent (50 mM
MOPS pH6.8, 0.75 mM MnCl2, 0.1 mM sodium orthovanadate, 10 mM DTT &
0.1% BSA). Test compounds (at 10 mM in dimethylsulphoxide (DMSO)) were
diluted in a dose response manner into 384-well low volume assay plates.
‘Total’ control wells contained 6% DMSO instead of compound, ‘blank’ wells
contained 2
(7.2 M ATP, 24 nM biotin-poly EAY (Glu, Ala, Tyr) (CisBio, Product number
6SGATBBO) in 50 mM MOPS pH6.8, 0.75 mM MnCl2, 0.1 mM sodium
orthovanadate, 10 mM DTT & 0.1% BSA) was then added to all test wells. 5
of freshly diluted enzyme was added to all wells to start the reaction and the
plates incubated at room temperature for 60 min. To stop the reaction 5
ll 0.5 M EDTA instead of compound. 5 ll of substrate/ATP mix
l
Figure 3. Alignment of apo-c-Met structure 2g15 with 12/3f66 virtual complex.
Two water molecules present in 2g15, represented by blue spheres, present a
rationale for potency enhancement of 200-CN-benzyl in combination with 30-OH-Ph.
ll
ll

J. V. Allen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5224–5229
5229
‘STOP’ bead solution (50
6760620 M) anti-phosphotyrosine-100 acceptor beads
l
g/ml AlphaScreen (Perkin-Elmer, Product number
analysed using
compound.
a generic HPLC-MS procedure for the presence of parent
&
50 lg/ml
streptavidin-coated AlphaScreen (Perkin-Elmer, Product number 6760620 M)
were added. Plates were then sealed & incubated in the dark for 20 h, before
being read using an AlphaQuest machine (Perkin-Elmer). ‘Blank’ (enzyme
inhibited by EDTA) and ‘total’ (no compound) control values were used to
determine the concentration of test compound which gave 50% inhibition of
enzyme activity.
20. Hajduk, P. J.; Sauer, D. R. J. Med. Chem. 2008, 51, 553.
21. Dossetter, A. G. Bioorg. Med. Chem. 2010, 18, 4405.
22. Jie-F; Vojkovsky, T.; Huang, P.; Liang, C.; Do, S.; Huy, S.; Koenig, M.; Cui, J.
Preparation of triazolotriazines as c-Met modulators for treating cancer. PCT
Int. Appl., WO2005/010005, 2005.
23. Albrecht, B. K.; Harmange, J.-C.; Bauer, D.; Berry, L.; Bode, C.; Boezio, A. A.;
Chen, A.; Choquette, D.; Dussault, I.; Fridrich, C.; Hirai, S.; Hoffman, D.; Larrow,
J. F.; Kaplan-Lefko, P.; Lin, J.; Lohman, J.; Long, A. M.; Moriguchi, J.; O’Connor, A.;
Potashman, M. H.; Reese, M.; Rex, K.; Siegmund, A.; Shah, K.; Shimanovich, R.;
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25. For more detailed description of the molecular modelling see Supplementary
data.
15. For review of bio-isostere replacement of functional groups see: (a) Wermuth,
C. G. The Practice of Medicinal Chemistry; Academic Press, 1996. pp. 204–237;
(b) Chen, X.; Wang, W. Ann. Rep. in Med. Chem. 2003, 38, 333.
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17. Typical condition in Smith Synthesiser microwave was heating to 150 °C for
10 min in toluene solvent. More electron poor amines required prolonged
times, for example 2-aminopyridine required 2 h.
18. In general ortho and para substituents resulted in at least a 20-fold drop in
potency.
19. Five compounds are dosed as a cocktail (2 mg/kg each) with a QC compound
(1 mg/kg) to 2 rats. Plasma samples, obtained at ½, 1, 2, 4 and 6 h post dose are
26. Wang, W.; Marimuthu, A.; Tsai, J.; Kumar, A.; Krupka, H. I.; Zhang, C.; Powell,
B.; Suzuki, Y.; Nguyen, H.; Tabrizizad, M.; Luu, C.; West, B. L. PNAS 2006, 103,
3563.