Potent, exceptionally selective, orally bioavailable inhibitors of TNF-α Converting Enzyme (TACE): Novel 2-substituted-1H-benzo[d]imidazol-1-yl)methyl)benzamide P1′ substituents

Article abstract of DOI:10.1016/j.bmcl.2008.01.075

Novel ((2-substituted-1H-benzo[d]imidazol-1-yl)methyl)benzamides were found to be excellent P1′ substituents in conjunction with unique constrained β-amino hydroxamic acid scaffolds for the discovery of potent selective inhibitors of TNF-α Converting Enzyme (TACE). Optimized examples proved potent for TACE, exceptionally selective over a wide panel of MMP and ADAM proteases, potent in the suppression of LPS-induced TNF-α in human whole blood and orally bioavailable.

Full text of DOI:10.1016/j.bmcl.2008.01.075

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1577–1582
Potent, exceptionally selective, orally bioavailable inhibitors
of TNF-a Converting Enzyme (TACE): Novel 2-substituted-
1H-benzo[d]imidazol-1-yl)methyl)benzamide P10 substituents
Gregory R. Ott,* Naoyuki Asakawa, Zhonghui Lu, Rajan Anand, Rui-Qin Liu,
Maryanne B. Covington, Krishna Vaddi, Mingxin Qian, Robert C. Newton,
David D. Christ, James M. Trzaskos and James J.-W. Duan
Departments of Discovery Chemistry and Discovery Biology, Bristol-Myers Squibb Research and Development,
Rte 206 and Province Line Road, Princeton, NJ 08543, USA
Received 17 December 2007; revised 17 January 2008; accepted 18 January 2008
Available online 26 January 2008
Abstract—Novel ((2-substituted-1H-benzo[d]imidazol-1-yl)methyl)benzamides were found to be excellent P10 substituents in con-
junction with unique constrained b-amino hydroxamic acid scaffolds for the discovery of potent selective inhibitors of TNF-a Con-
verting Enzyme (TACE). Optimized examples proved potent for TACE, exceptionally selective over a wide panel of MMP and
ADAM proteases, potent in the suppression of LPS-induced TNF-a in human whole blood and orally bioavailable.
ꢀ 2008 Elsevier Ltd. All rights reserved.
The overexpression of the pro-inflammatory cytokine
Tumor Necrosis Factor-a (TNF-a) has been implicated
in numerous pathological conditions.1 The anti-TNF-a
biological therapeutics entaneracept, infliximab, and
adalimumab have demonstrated clinical success in
inflammatory and autoimmune diseases and as such
have validated the modulation of TNF-a as a drug dis-
covery paradigm.2 TNF-a Converting Enzyme (TACE
or ADAM-17) is the principle sheddase that governs
the cleavage of membrane bound pro-TNF-a to the sol-
uble form.3 TACE is a member of the ADAM (A Disin-
tegrin And Metalloprotease) family in the metzincin
superfamily of metalloproteases. We and others have
targeted TACE for the development of orally adminis-
tered, small molecule modulators of TNF-a.4 In partic-
ular, structural motifs including c-lactam5 (1, Fig. 1),
cyclic succinate6 (2), b,b-disubstituted-b-amino7 (3),
a,b-cyclized-b-amino8 (4, 5), and b-sulfone9 (6) hydroxa-
mic acids have been shown to inhibit TACE as well as
modulate TNF-a production in both cell-based assays
and animal models. Common binding elements among
these inhibitors include the classical bidentate coordina-
tion to the active site zinc by the hydroxamic acid,
hydrogen bonds from the hydroxamic acid NH and
OH to the protein backbone as well as a key hydrogen
bond acceptor from the amide carbonyl or sulfone.
Common among this group is the 4-(2-quinolinylmeth-
oxy)phenyl P10 substituent.
This unique 4-(2-quinolinylmethoxy)phenyl P10 element
provided selectivity against the structurally related ma-
trix metalloproteases (MMPs); selectivity against the
MMPs was desired since broad based MMP inhibitors
were shown to have musculoskeletal side effects in clin-
ical trials.10 The P10 component was integral to achiev-
ing potency in a cellular assay which measures the
suppression of LPS-induced TNF-a in human whole
blood (WBA).11 Acceptable oral pharmacokinetic pro-
files were also achieved on examples containing this moi-
ety. Importantly, this group translated across scaffolds
with a high degree of success imparting similar proper-
ties to each. Though important for achieving selectivity
against related MMP and ADAM proteases, there was
still room for improvement in this area especially against
MMPs-3,-7,-8, and -12. Thus, the goals for the next gen-
eration of TACE inhibitors required increased selectiv-
ity against MMPs and further structural diversification
Keywords: TACE; TNF-a Converting Enzyme; MMP; Matrix metal-
loprotease; TNF modulator.
*
Corresponding author at present address: Cephalon, Inc. 145
Brandywine Parkway, West Chester, PA 19380, USA. Tel.: +1 610
738 6861; fax: +1 610 738 6558; e-mail: gott@cephalon.com
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.075

1578
G. R. Ott et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1577–1582
Figure 1. Scaffold translation of 4-(2-methyl-quinolinylmethoxy)phenyl P10 group.
that included replacement of the 4-(2-methyl-quinoli-
nyl)methoxy substituent while retaining cell potency
and acceptable oral pharmacokinetics.
TACE potency/selectivity profiles and optimized exam-
ples proved to have excellent oral pharmacokinetic
properties.8 We envisioned the 2-substituted-1H-
benzo[d]imidazol-1-yl)methyl)benzamide P10 substitu-
ents would provide a promising commencement to fur-
ther optimize the factors listed above.
Toward this end, reexamination of the P10 moieties from
the c-lactam scaffold studies revealed examples that
demonstrated suitable attributes such as cell permeabil-
ity, selectivity, and potential for increased structural
diversity. Of note, c-lactam 7 (Fig. 2), containing the
2-(methylthio)benzimidazolemethyl phenyl moiety,
stood out as exhibiting cell potency (WBA
IC50 = 316 nM) and modest selectivity against MMPs-
1,-2, and -9. The benzimidazole group was especially
attractive for drug-like properties, ease of incorporation,
and numerous positions to integrate structural changes.
Thus, alkylation of 2-methylthio[1H]benzimidazole with
methyl 4-(bromomethyl)benzoate provided ester 10
(Scheme 1). Saponification and coupling to the BOC-
protected pyrrolidine 128 provided the amide 13. Depro-
tection and derivatization at this stage were followed by
conversion to the hydroxamic acid. This chemistry was
applicable to all analogues synthesized throughout this
study.12
The recently discovered cis-a,b-substituted-b-benzamido
hydroxamic acid scaffolds were characterized by good
The initial examples were tested in vitro using semi-puri-
fied porcine TACE (pTACE) to evaluate enzyme
Figure 2. ((2-Substituted-1H-benzo[d]imidazol-1-yl)methyl)phenyl P10 moieties.

G. R. Ott et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1577–1582
1579
Scheme 1. Reagents: (a) Cs2CO3, DMSO (99%); (b) LiOH, MeOH, H2O (72%); (c) BOP Reagent, DIPEA, DMF (99%); (d) TFA, CH2Cl2 (99%); (e)
NH2OH, NaOMe, MeOH.
potency.11 The primary selectivity profile was evaluated
against MMPs-1,-2, and -9. Cellular suppression of
LPS-induced TNF-a was measured using human whole
blood (WBA). Membrane permeability [Papp (A ! B)],
was measured using Caco-2 cells.13 Pharmacokinetic
studies were done in a discrete or cassette-dose fashion
(n-in-1) administered intravenously and orally to Spra-
gue–Dawley rats and beagle dogs; the plasma samples
were analyzed by LC/MS/MS.14
<1 and 2.6 nM. Interestingly, pTACE potency was
unaffected by increasing steric bulk at the 2-position of
the benzimidazole. Improved selectivity was observed
against MMP-2 and -9, as compared to the c-lactam
7. Importantly, potent activity in the cellular assay was
realized (WBA IC50s <100 nM). Caco-2 permeability
values for these inhibitors suggested low potential for
oral absorption with Papp 6 0.2 · 10ꢀ6 cm/s. Indeed,
pharmacokinetic studies in rats revealed less than 1%
oral bioavailability for 16 and 20.
We were gratified to find that the 2-substituted benzim-
idazolyl P10 substituents translated well to the b-amino
acid scaffolds (Table 1); analogues 15–20 displayed po-
tent activity against pTACE with IC50s ranging between
With the realization of excellent enzyme and cell
potency for these analogues, further profiling against
a wider panel of MMPs was undertaken (Table 2).
Table 1. In vitro properties of inhibitors 15–20
Compound
X
R
pTACEa (IC50 nM)
WBAb (IC50 nM)
MMP-1,-2,-9 Ki (nM)
Caco-2c
15
16
17
18
19
20
NBOC
NH
SCH3
SCH3
CH3
CH3
i-Pr
<1.0
1.6
1.0
2.6
1.0
1.6
62
34
92
56
70
64
>2128
>2128
>2128
>2128
>2128
>2128
0.2
0.2
0.1
0.1
0.2
0.1
NBOC
NH
NBOC
NH
i-Pr
a pTACE IC50 and MMP Ki values are from single determination.
b Inhibition of TNF-a release in WBA was determined with three donors.
c Papp (A ! B) · 10ꢀ6 cm/s.

1580
G. R. Ott et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1577–1582
drofuran8b (A, X@O), oxaspiro[4.4]nonane15 (B), and
tetrahydopyran8a (C).
Table 2. MMP profile of inhibitors 15, 17, and 19
Enzymea
15
17
19
TACEb
<1
1.0
1.0
In conjunction with the cyclopentane-b-benzamido
hydroxamic acid, we were pleased to find that both the
isopropyl and cyclopropyl derivatives 21 and 22 retained
potency in both the enzymatic and cellular assays. Con-
sistent with the improvement in permeability in the
Caco-2 assay as well as the more modest clearance, the
cyclopropyl derivative 22 displayed better oral bioavail-
ability (22%) as compared to isopropyl derivative 21
(8%). The N-(2-propynyl)-pyrrolidine analogues 23
and 24 gave favorable potency in the enzyme assay,
though we observed a drop in cell potency that appeared
to coincide with the bulkier tert-butylbenzimidazole
derivative 24. Minimal oral bioavailability was observed
for 23 and 24 (3% and 8%, respectively) with higher
intrinsic clearance and low permeability contributing
to these results.
MMP-1
MMP-2
MMP-3
MMP-7
MMP-8
MMP-9
MMP-10b
MMP-12b
MMP-13
MMP-14
MMP-15
MMP-16
>4949
>3333
>4501
4075
>4946
>3333
2609
>4946
>3333
3915
5858
1865
3502
1957
>3058
>2128
4600
>2128
>10,000
540
>2128
1600
250
390
>5025
>5290
>7088
>5454
>5025
>5290
>7088
>5554
>5025
>5290
>7088
>5554
a Ki (nM).
b IC50 (nM).
Representative of this class, inhibitors 15, 17, and 19 dis-
played exceptional selectivity against the panel of
MMPs. Notably, superb selectivity over MMP-3,-7,-8,
and -12 was achieved; selectivity over this subset was,
in general, modest with the 4-(2-methylquinoli-
nyl)methoxyphenyl P10.
Turning to the oxaspiro[4.4]nonane scaffold, the isopro-
pyl derivative 25 maintained cell potency (WBA
IC50 = 69 nM) and provided acceptable oral bioavail-
ability (F = 22%), though this was in contrast to both
the Caco-2 value and the higher clearance. Unlike the
corresponding analogue on the cyclopentyl scaffold,
the cyclopropylbenzimidazole analogue 26 did not offer
any advantage over the isopropyl derivative. Turning to-
ward larger and more lipophilic benzimidazole substitu-
ents, the tert-butyl derivative 27 gave better oral
bioavailability (F = 29%), which, in this instance, was
consistent with the enhanced Caco-2 value; again, a
slight erosion in cell potency was observed (WBA
To further evaluate in vitro and in vivo properties with
the new benzimidazole P10 motif, a more thorough
exploration of b-benzamido hydroxamic acid scaffolds
was initiated. These scaffolds were effective for con-
structing cell potent, orally bioavailable TACE inhibi-
tors. The b-benzamido hydroxamic acid scaffolds of
particular interest included the cyclopentane8a (Table
3, A, X@CH2), pyrollidine8b (A, X@N-alkyl), tetrahy-
Table 3. In vitro and in vivo results for 21–33
Compound Scaffold
A,B,C
X
R
pTACEa
(IC50 nM) (IC50 nM) Ki (nM)
WBAb
MMP-1,-2,-9
Caco-2c Cld(L/h/kg) Fd (%) rat
21
22
23
24
25
26
27
28
29
30
31
32
33
A
A
A
A
B
B
B
B
C
C
C
C
A
CH2
CH2
i-Pr
c-Pr
1.0
2.7
1.8
2.3
1.1
1.3
2.0
65
54
>4948, >3333, >2128 0.1
>4948, >3333, >2128 2.0
>4948, >3333, >2128 0.1
>4948, >3333, >2128 0.4
>4948, >3333, >2128 0.1
>4948, >3333, >2128 0.1
>4948, >3333, >2128 1.4
>4948, >3333, >2128 2.4
>4948, >3333, >2128 0.5
>4948, >3333, >2128 0.3
>4948, >3333, >2128 0.7
>4948, >3333, >2128 0.7
>4948, >3333, >2128 0.1
3.0
1.5
9.3
6.8
6.9
1.4
2.9
2.4
3.8
1.9
1.2
1.6
3.2
8
22
3
N(2-propynyl) i-Pr
N(2-propynyl) t-Bu
137
317
69
8
—
—
—
—
—
—
—
—
O
i-Pr
c-Pr
t-Bu
22
14
29
33
8
42
252
140
105
153
188
232
102
CF(Me)2 <1.0
i-Pr 1.6
CF(Me)2 2.2
36
55
53
20
CF2CH3
CF3
CF3
2.6
1.4
1.0
a pTACE IC50 and MMP Ki values are from single determination.
b Inhibition of TNF-a release in WBA was determined with three donors.
c Papp (A ! B) · 10ꢀ6 cm/s.
d Determination of 3 for each dosing group, avg. value.

G. R. Ott et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1577–1582
1581
Table 5. Pharmacokinetic parameters of 32a
IC50 = 252 nM). In an effort to maintain cell potency
and provide enough lipophilicity to boost oral absorp-
tion, addition of a fluorine atom in place of the methine
hydrogen of the isopropyl provided 28. Importantly, a
boost to the Caco-2 permeability was observed and
indicative of that oral bioavailability improved to 33%.
PK parameters
Rat
Dog
IV
Dose (mg/kg)
t1/2 (h)
5.0
3.5
2.0
4.2
0.6
1.0
Cl (L/h/kg)
Vss (L/kg)
AUC (nM h)
1.6
1.5
6849
9967
The previously described tetrahydropyranyl and tetrahy-
drofuranyl scaffolds were shown to possess favorable po-
tency/pharmacokinetic properties;8 this trend continued
with the benzimidazole P10 substituents. Relative to the
prior analogues, improved clearance values, though still
in the moderate range, were observed for 30–32 (1.2–
1.9 L/h/kg). The fluoroisopropyl analogue 30 displayed
favorable cell potency (WBA IC50 = 153 nM) with good
oral bioavailability in rat (F = 36%). It appeared that
substituting fluorine for hydrogen in the 2-alkyl position
of the benzimidazole led to favorable pharmacokinetics
without sacrificing potency or selectivity. Indeed, reten-
tion of cell potency and a boost in oral bioavailability
were observed for both the difluoroethylbenzimidazole
derivative 31 (WBA IC50 = 188 nM, F = 55%) and the tri-
fluoromethyl derivative 32 (WBA IC50 = 232 nM,
F = 53%). The tetrahydrofuranyl derivative 33 proved
to be more potent in the WBA (IC50 = 102 nM) as com-
pared to 32 but this resulted in a less favorable pharmaco-
kinetic profile (F = 20%). Interestingly, for the
tetrahydropyranyl analogues, increasing oral bioavail-
ability paralleled increasing Caco-2 values, though the
Papp values were modest in magnitude.
PO
Dose (mg/kg)
tmax (h)
t1/2 (h)
8.0
0.3
2.4
5840
53
8.0
0.5
4.8
39,466
99
AUC (nM h)
F (%)
a Determination of 3 for each dosing group, avg. value.
Further in vivo profiling for 32 in a second species (dog)
was performed and the full pharmacokinetic profile in
both rat and dog is shown in Table 5. Following iv
and oral dosing in rats, 32 is exemplified by low to
moderate clearance (1.6 L/h/kg), rapid absorption
(tmax = 0.3 h), and high oral exposure (AUC =
5840 nMh). The oral bioavailability is 53%. Pharmaco-
kinetic analysis in beagle dogs showed lower clearance
(0.6 L/h/kg) as compared to rats and longer oral half-life
(t1/2 = 4.8 h). Near complete oral bioavailability
(F = 99%) was observed in dogs.
Compound 32, when analyzed for serum protein bind-
ing, displayed acceptable free fraction in rodents (rat,
20% unbound) as well as higher order species, (dog,
20% unbound, human, 21% unbound).16
Oral efficacy of 32 was determined using the LPS-TNF
model of endotoxemia in mouse.11 Lipopolysaccharride
was administered along with different doses of 32 in
vehicle, by oral gavage administration at t = 0 h. One
hour post dose, the mice were euthanized, whole blood
collected by cardiac puncture, and the plasma used for
the measurement of TNF-a concentration. 32 displayed
a dose dependent suppression of TNF-a with a calcu-
lated ED50 1.9 mg/kg.
Inhibitor 32 was profiled against a wider panel of MMP
and ADAM proteases (Table 4). Importantly, 32 dis-
played outstanding selectivity, especially against
MMP-3,-7,-12, and ADAMTS-4 and -5.
Table 4. MMP and ADAM profile of inhibitor 32
In summary, novel ((2-substituted-1H-benzo[d]imidazol-
1-yl)methyl)benzamides were found to be excellent P10
substituents in conjunction with unique b-amino
hydroxamic scaffolds for the discovery of potent, selec-
tive inhibitors of TACE. Optimized examples demon-
strated oral bioavailability. In particular, 32 proved
potent for pTACE, selective over a wide panel of MMPs
and ADAM proteases as well as potent in the suppres-
sion of LPS-induced TNF-a in human whole blood.
Importantly, following oral dosing, 32 displayed an
ED50 of 1.9 mg/kg in an acute model of inflammation.
Enzymea
Ki (nM)
pTACE (IC50
MMP-1
MMP-2
MMP-3
MMP-7
MMP-8
MMP-9
)
1.4
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
8000
MMP-10 (IC50
MMP-12 (IC50
MMP-13
)
)
Acknowledgments
>10,000
>10,000
>10,000
>10,000
>1000
MMP-14
MMP-15
MMP-16
We thank John Giannaras, Sherrill Nurnberg and Paul
Strzemienski for running in vitro assays, Robert J. Col-
lins for LPS study in animals, Bernice Brogdon, Bing
Lu, Hang Zheng and Jingtao Wu for assistance in phar-
macokinetic studies and Maria Ribadeneira for Caco-2
assay. We thank George Emmett and Joerg Deerberg
for providing synthetic intermediates.
ADAMTS-1
ADAMTS-4
ADAMTS-5
ADAM-10
6700
1000
>10,000
a pTACE IC50 and MMP Ki values are from single determination.

1582
G. R. Ott et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1577–1582
C.; Duan, J. J.-W.; Decicco, C. P. Bioorg. Med. Chem.
Lett. 2004, 14, 4453.
References and notes
10. Levitt, N. C.; Eskens, F. A. L. M.; O’Byrne, K. J.;
Propper, D. J.; Denis, L. J.; Owen, S. J.; Choi, L.;
Foekens, J. A.; Wilner, S.; Wood, J. M.; Nakajima, M.;
Talbot, D. C.; Steward, W. P.; Harris, A. L.; Verweij, J.
Clin. Cancer Res. 2001, 7, 1912.
11. Details of the pTACE (porcine TACE), MMPs, human
whole blood (WBA) assays as well as LPS-mouse
efficacy model are reported in previous publication, see
Xue, C.-B.; Voss, M. E.; Nelson, D. J.; Duan, J. J.-W.;
Cherney, R. J.; Jacobson, I. C.; He, X.; Roderick, J.;
Chen, L.; Corbett, R. L.; Wang, L.; Meyer, D. T.;
Kennedy, K.; DeGrado, W. F.; Hardman, K. D.;
Teleha, C. A.; Jaffee, B. D.; Liu, R.-Q.; Copeland, R.
A.; Covington, M. B.; Christ, D. D.; Trzaskos, J. M.;
Newton, R. C.; Magolda, R. L.; Wexler, R. R.;
Decicco, C. P. J. Med. Chem. 2001, 44, 2636.
12. Unless otherwise noted, all starting materials are com-
mercially available and all synthesized compounds are in
accordance with their 1H NMR and mass spectral data.
2-(1,1-difluoro-ethyl)-1H-benzo[d]imidazole and 2-(1-flu-
oro-1-methyl-ethyl)-1H-benzo[d]imidazole used to prepare
compounds 28, 30, and 31 were synthesized from o-phen-
ylenediamine and the corresponding carboxylic acids, 2,2-
difluoropropanoic acid, and 2-fluoroisobutyric acid, via
two-step procedure involving BOP reagent coupling to
form the mono-amide and cyclization with AcOH. For a
similar benzimidazole cyclization procedure see Fonseca,
T.; Gigante, B.; Gilchrist, T. L. Tetrahedron 2001, 57,
1793.
1. Newton, R. C.; Decicco, C. P. J. Med. Chem. 1999, 42,
2295.
2. (a) Moreland, L. W.; Baumgartner, S. W.; Schiff, M. H.;
Tindall, E. A.; Fleischmann, R. M.; Weaver, A. L.;
Ettlinger, R. E.; Cohen, S.; Koopman, W. J.; Mohler, K.;
Widmer, M. B.; Blosch, C. M. N. Engl. J. Med. 1997, 337,
141; (b) Lipsky, P. E.; van der Heijde, D. M. F. M. E.; St.
Clair, W.; Furst, D. E.; Breedveld, F. C.; Kalden, J. R.;
Smolen, J. S.; Weisman, M.; Emery, P.; Feldmann, M.;
Harriman, G. R.; Maini, R. N. N. Engl. J. Med. 2000, 343,
1594; (c) Machold, K. P.; Smolen, J. S. Exp. Opin. Bio.
Ther. 2003, 3, 351.
3. Black, R. A. Int. J. Biochem. Cell Biol. 2002, 34, 1.
4. For a recent review see Skotnicki, J. S.; Levin, J. I. Ann.
Rep. Med. Chem. 2003, 38, 153.
5. Duan, J. J.-W.; Chen, L.; Wasserman, Z. R.; Lu, Z.; Liu,
R.-Q.; Covington, M. B.; Qian, M.; Hardman, K. D.;
Magolda, R. L.; Newton, R. C.; Christ, D. D.; Wexlre, R.
R.; Decicco, C. P. J. Med. Chem. 2002, 45, 4954.
6. (a) Xue, C.-B.; He, X.; Roderick, J.; Corbett, R. L.; Duan,
J. J.-W.; Liu, R.-Q.; Covington, M. B.; Newton, R. C.;
Trzaskos, J. M.; Magolda, R. L.; Wexler, R. R.; Decicco,
C. P. Bioorg. Med. Chem. Lett. 2003, 13, 4293; (b) Xue,
C.-B.; He, X.; Roderick, J.; Corbett, R. L.; Duan, J. J.-W.;
Liu, R.-Q.; Covington, M. B.; Qian, M.; Ribadeneira, M.
D.; Vaddi, K.; Christ, D. D.; Newton, R. C.; Trzaskos, J.
M.; Magolda, R. L.; Wexler, R. R.; Decicco, C. P. Bioorg.
Med. Chem. Lett. 2003, 13, 4299.
7. Gilmore, J. L.; King, B. W.; Harris, C.; Maduskuie, T.;
Mercer, S. E.; Liu, R.; Covington, M. B.; Qian, M.;
Ribadeneria, M. D.; Vaddi, K.; Trzaskos, J. M.; Newton,
R. C.; Duan, J. J.-W.; Decicco, C. P. Bioorg. Med. Chem.
Lett. 2006, 16, 2699.
13. Artursson, P.; Palm, K.; Luthman, K. Adv. Drug Delivery
Rev. 1996, 22, 67.
14. Wu, J.-T.; Zeng, H.; Qian, M.; Brogdon, B. L.; Unger, S.
E. Anal. Chem. 2000, 72, 61.
15. Ott, G. R.; Asakawa, N.; Liu, R.; Covington, M. B.; Qian,
M.; Vaddi, K.; Newton, R. C.; Christ, D. D.; Trzaskos, J.
M.; Gayla, L.; Scholz, T.; Marshall, M.; Duan, J. J.-W.
Bioorg. Med. Chem. Lett. 2008, 18, 1288.
16. For details of serum protein binding measurement, see
Cherney, R. J.; Duan, J. J.-W.; Voss, M. E.; Chen, L.;
Wang, L.; Meyer, D. T.; Wasserman, Z. R.; Hardman, K.
D.; Liu, R.-Q.; Covington, M. B.; Qian, M.; Mandlekar,
S.; Christ, D. D.; Trzaskos, J. M.; Newton, R. C.;
Magolda, R. L.; Wexler, R. R.; Decicco, C. P. J. Med.
Chem. 2003, 46, 1811.
8. (a) Duan, J. J.-W.; Chen, L.; Lu, Z.; Xue, C.-B.; Liu, R.-
Q.; Covington, M. B.; Qian, M.; Wasserman, Z. R.;
Vaddi, K.; Christ, D. D.; Trzaskos, J. M.; Newton, R. C.;
Decicco, C. P. Bioorg. Med. Chem. Lett. 2008, 18, 241; (b)
Ott, G. R.; Asakawa, N.; Lu, Z.; Liu, R.-Q.; Covington,
M. B.; Vaddi, K.; Qian, M.; Newton, R. C.; Christ, D. D.;
Trzaskos, J. M.; Decicco, C. P. Bioorg. Med. Chem. Lett.
2008, 18, 694.
9. Xue, C.-B.; Chen, X. T.; He, X.; Roderick, J.; Corbett, R.
L.; Ghavimi, B.; Liu, R.-Q.; Covington, M. B.; Qian, M.;
Ribadeneira, M. D.; Vaddi, K.; Trzaskos, J.; Newton, R.