Conversion of an MMP-potent scaffold to an MMP-selective HER-2 sheddase inhibitor via scaffold hybridization and subtle P1′ permutations

Article abstract of DOI:10.1016/j.bmcl.2007.11.086

A series of β-sulfonamide piperidine hydroxamates were prepared and shown to be potent inhibitors of the human epidermal growth factor receptor-2 (HER-2) sheddase with excellent selectivity against MMP-1, -2, -3, and -9. This was achieved by exploiting su

Full text of DOI:10.1016/j.bmcl.2007.11.086

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 560–564
Conversion of an MMP-potent scaffold to an MMP-selective
HER-2 sheddase inhibitor via scaffold
hybridization and subtle P01 permutations
David M. Burns,a,* Chunhong He,a Yanlong Li,b Peggy Scherle,b Xiangdong Liu,b
Cindy A. Marando,b Mayanne B. Covington,b Gengjie Yang,b Max Pan,b Sharon Turner,b
Jordan S. Fridman,b Gregory Hollis,b Kris Vaddi,b Swamy Yeleswaram,b Robert Newton,b
Steve Friedman,b Brian Metcalf a,b and Wenqing Yaoa
aDepartment of Medicinal Chemistry, Incyte Corporation, Wilmington, DE 19880, USA
bDepartment of Discovery Biology, Incyte Corporation, Wilmington, DE 19880, USA
Received 28 September 2007; revised 19 November 2007; accepted 20 November 2007
Available online 28 November 2007
Abstract—A series of b-sulfonamide piperidine hydroxamates were prepared and shown to be potent inhibitors of the human epi-
dermal growth factor receptor-2 (HER-2) sheddase with excellent selectivity against MMP-1, ꢀ2, ꢀ3, and ꢀ9. This was achieved by
exploiting subtle differences within the otherwise highly conserved S01 binding pocket of the active sites within the metalloprotease
family. In addition, it was discovered that the introduction of polarity to the P1 and P01 groups reduced the projected human
clearance.
Published by Elsevier Ltd.
The human epidermal growth factor receptor-2 (HER-2
or ErbB-2) is a tyrosine kinase receptor that is activated
upon homo- and heterodimerization with another mem-
ber of the HER family or by proteolytic cleavage (shed-
ding) of the extracellular domain (ECD).1 Once
activated, intracellular signal transduction pathways
are initiated that mediate a diverse range of essential cel-
lular activities such as cell proliferation, differentiation,
motility, adhesion, and survival.2 Overexpression of
the oncogene HER-2/neu has been associated with
aggressive pathogenesis, poor prognosis, and decreased
responsiveness to conventional chemotherapeutic and
hormonal treatment regimes in non-small cell lung can-
cer, ovarian cancer, and breast cancer patients.1a In
addition, elevated plasma levels of HER-2 ECD have
been associated with increased metastatic potential and
a decrease in disease-free and overall survival in patients
with breast cancer.1b,3 Therefore, inhibition of the prote-
ase responsible for HER-2 ECD shedding may be ther-
apeutically desirable for treating cancer patients that
overexpress HER-2, particularly breast cancer patients.
Recently we described the design, synthesis, evaluation,
and identification of a novel class of (6S,7S)-N-hydroxy-
6-carboxamide-5-azaspiro[2.5] octane-7-carboxamides
as the first potent and selective inhibitors of HER-2
sheddase.4 From this class of compounds INCB3619
(1) was identified to possess excellent pharmacodynamic
and pharmacokinetic properties and was shown to de-
crease cleaved HER-2 ECD plasma levels, tumor size,
and potentiate the effects of the humanized anti-HER-
2 monoclonal antibody (trastuzumab) in vivo in a
HER-2 overexpressing cancer murine xenograft model.
In an effort to expand upon these results we wanted to
design an alternative novel scaffold that was chemically
more accessible from a synthetic vantage point. Thus,
we desired to eliminate the spiro-cyclopropyl ring as well
as the chirality associated with scaffold 1 (see Fig. 1).
Becker et al. described the facile synthesis of b- and
a-piperidine sulfone hydroxamic acids 2 from readily
available cheap starting materials as inhibitors of
MMP-2, ꢀ9, and ꢀ13.5 Despite our desire to be selective
against these MMPs, we hypothesized that hybridization
of this scaffold 2 with our scaffold 1 to produce
Keywords: HER-2 sheddase; ADAM-10; Beta-sulfonamide piperidine
hydroxamates; Metalloprotease; MMP; P10 substituent; Projected
clearnace; Scaffold hybridization.
*
Corresponding author. Tel.: +1 302 498 6887; fax: +1 302 425
2704; e-mail: dburns@incyte.com
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2007.11.086

D. M. Burns et al. / Bioorg. Med. Chem. Lett. 18 (2008) 560–564
561
Based on our earlier work, we speculated that selectivity
against MMP-2, -3, and -9 may be accomplished by
exploiting the difference at residue 223 within the loop
3 region of the S01 pocket, which is Tyr for MMP-2, -3,
and -9 and Ala for ADAM-10.4
Zn
NH
HO
O
P1
OH
HN
S
N
O
O
N
N
O
O
P1'
O
O
O
We postulated that the addition of a small substituent in
the 2-position of the phenyl ring may be able to fill the
vacancy left by the sterically less demanding Ala residue
of ADAM-10 and have unfavorable steric interactions
with the Tyr residue of MMP-2, ꢀ3, and ꢀ9. Therefore,
a variety of substituents were placed in the 2-position of
the phenyl ring of the 4-phenyl- piperidinyl sulfonamide
analog 8. Introduction of a 2- fluoro- substituent to pro-
duce 9 resulted in a 5-fold increase in selectivity against
MMP-2 and a 2-fold gain in selectivity against MMP-3
and -9 in comparison to the parent phenyl analog 8;
albeit with a concomitant loss of potency. Exchanging
the fluoro substituent of 9 with a cyano group resulted
in a complete loss of potency for both ADAM-10 and
MMP-2, which is in agreement with our hypothesis that
the 2-substituent of the phenyl ring extends into a lipo-
philic region occupied by residue 223, that is, Ala or
Tyr. Replacement of the small fluoro substituent of 9
with a slightly larger methyl group to afford 11 resulted
in a 37-, 13-, and 139-fold increase in selectivity toward
MMP-2, ꢀ3, and ꢀ9. Comparison of compound 11 to
the parent phenyl compound 8 revealed an impressive
166-, 21-, and 278-fold gain in selectivity over MMP-2,
ꢀ3, and ꢀ9, respectively. A minuscule increase in size
of the 2-aryl substituent of 11 from the methyl to the tri-
fluoromethyl group resulted in a significant loss in
potency, indicating that there is limited space available
within this region. Overall, these results insinuate that
the 2-methyl group of compound 11 may be accommo-
dating the space created by the Ala223 of ADAM-10
with optimal lipophilic interactions, while being im-
peded by the Tyr223 of MMP-2, -3, and -9 resulting in
the desired selectivity toward these MMPs, as originally
proposed.
P2'
1
2
Pfizer
INCB3619
MMP-2, 3, & 9 IC50 <1.5 nM
MMP-1 IC50 8660 +/- 1656
HER-2 IC50 68 nM
MMP-1 IC50 >5000 nM
MMP-2 IC50 39 nM
MMP-3 IC50 4000 nM
MMP-9 IC50 428 nM
P1
R
Zn
HO
NH
N
O
N
S
O
O
P1'
3
Figure 1. Hybridization of HER-2 and MMP scaffolds.
compounds of the general structure 3 may produce a
viable novel scaffold that could be attenuated to selec-
tively target the HER-2 sheddase (ADAM-10) by mod-
ifying the P01 substituent. Thus, exploiting potential
differences within the S01 sub-site of the otherwise highly
conserved active sites within the metalloprotease
family.4,6
A
series of tetrahydrofuran-3-yl 4-[(amino-sulfo-
nyl)methyl]-4-[(hydroxyamino)carbonyl] piperidine-1-
carboxylates were prepared and the inhibition of
HER-2 sheddase was evaluated in both a cellular and
an enzymatic assay, denoted as HER-2 and ADAM-10
in Tables 1 and 2, respectively. Selectivity was deter-
mined by comparison of the enzymatic binding to
ADAM-10 to that of MMP-1, ꢀ2, ꢀ3, and ꢀ9.
In an effort to increase the cellular potency of compound
11 a variety of substituents were placed in the 4-posi-
tion.4 Introduction of a 4-fluoro substituent to 11
resulted in a 3-fold loss in HER-2 potency. Replacement
of the 4-fluoro substituent of 13 with the polar N-di-
methyl amide group resulted in complete loss of
potency. Installation of a 4-cyano substituent to com-
pound 11, to afford analog 15, increased the HER-2 cel-
lular potency by 3-fold while maintaining the excellent
selectivity against MMP-1, ꢀ2, ꢀ3, and ꢀ9. Removal
of the 2- methyl substituent of 15 resulted in a com-
pound that had a higher binding affinity for MMP-2
over ADAM-10, which demonstrates the importance
of the 2-methyl substituent in achieving selectivity. The
3,5-dimethyl analogs 17 and 18 exemplify the impor-
tance of both the 2-methyl and the 4-cyano substituents
with regard to both selectivity and potency.
It was found that simple 3-phenyl-2,5-dihydro-1H-pyr-
rol-1-yl 4 and 3-phenyl-pyrrolidin-1-yl 5 sulfonamides
showed promising inhibition of HER-2 sheddase (Table
1). Expanding the heterocycle from a five-membered to
a six-membered ring increased the potency by 5- to 50-
fold. The dramatic increase in potency observed for
compounds 6, 7, and 8 can be explained by examining
the space occupied by the phenyl ring that is attached
to the heterocycle. The six-membered heterocycles orient
the phenyl ring in a linear manner in contrast to the five-
membered heterocycles, which orient the phenyl ring as-
kew approximately 60 °. Since the S01 pocket of the
HER-2 sheddase is believed to be narrow, a linear or
tubular P01 substituent would be preferred.4 The high de-
gree of selectivity of these three compounds against
MMP-1, which has a shallow S01 pocket, and overall lack
of selectivity against MMP-2 and ꢀ9, which has a nar-
row and deep S01 pocket, suggests that the S01 pocket of
the HER-2 sheddase might be similar to that of
MMP-2 and ꢀ9, in agreement with our previous
results.4
The piperazine and tetrahydropiperidine analogs of the
two lead compounds 11 and 15 were prepared and were
shown to have inferior cellular and enzymatic potency in
comparison to the piperidine leads. Comparison of the

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D. M. Burns et al. / Bioorg. Med. Chem. Lett. 18 (2008) 560–564
Table 1. In vitro data for P01 substituents
O
HO
NH
O
N
O
N
O
S
O
O
P1'
b
Enzymatic binding IC50 (nM)
Compound
P10
IC50 (nM)
HER-2a
ADAM-10
MMP-1
MMP-2
MMP-3
MMP-9
4
5
6
7
8
3-Ph-2,5-dihydro-1H-pyrrol-1-yl
3-Ph-pyrrolidin-1-yl
4-Ph-piperazin-1-yl
102
358
13
332
261
15
—
—
52
549
13
4
—
—
—
—
78
25
22
774
259
753
925
237
366
4-Ph-1,2,3,6-tetrahydropyridin-1yl
4-Ph-piperidin-1-yl
7.1
22
10
28
18
9
10
11
12
13
14
15
16
17
18
4-(2-F-Ph)-piperidin-1-yl
4-(2-CN-Ph)-piperidin-1-yl
76
>1000
41
44
>2000
18
>5000
—
135
942
—
100
—
>2000
>2000
>2000
>2000
655
4-(2-Me-Ph)-piperidin-1-yl
4-(2-CF3-Ph)-piperidin-1-yl
>5000
—
—
>5000
—
—
>5000
—
—
>1000
141
>1000
14
1036
92
4-(2-Me-4-F-Ph)-piperidin-1-yl
4-(2-Me-4-C(O)NMe2-Ph)-piperidin-1-yl
4-(2-Me-4-CN-Ph)-piperidin-1-yl
4-(4-CN-Ph)-piperidin-1-yl
>2000
12
39
—
>5000
—
—
>5000
—
—
>5000
—
1999
9
77
96
4-(3,5-di-Me-4-CN-Ph)-piperidin-1-yl
4-(3,5-di-Me-Ph)-piperidin-1-yl
57
286
>5000
>5000
126
315
756
2157
535
955
833
19
20
21
22
4-(2-Me-Ph)-piperazin-1-yl
217
133
25
107
18
—
—
>2000
1999
—
—
—
—
4-(2-Me-Ph)-1,2,3,6-tetrahydropyridin-1yl
4-(2-Me-4-CN-Ph)-piperazin-1-yl
4-(2-Me-4-CN-Ph)-1,2,3,6-tetrahydropyridin-1yl
26
107
>5000
—
>5000
1563
>5000
—
>5000
—
45
a BT-474 cellular proliferation assay, see Ref. 4.
b See Ref. 4.
Table 2. In vitro data for P1 substituents
H
N
O
S
O
X
N
HO
O
bond: 1, 2
X: H, CN
N
O
R
P1
O
Compound
R
Bond
X
IC50 (nM)
HER-2a
Enzymatic binding IC50 (nM)b
Proj. h-Clrc
ADAM-10
MMP-1
MMP-2
L/h/kg (%free)
23
24
25
26
27
28
3-Me-tetrahydrofuran-3-yl
4,4-di-Me-tetrahydrofuran-3-yl
trans-2-OH-cyclopent-1-yl
pyrrolidin-3-yl
1
1
1
1
1
1
H
H
H
H
H
H
36
14
6.2
16
23
30
11
22
16
18
34
19
>5000
>5000
4999
564
574
1.1 (15%)
1.1 (10%)
1.0 (20%)
<0.5 (>60%)
1.1 (10%)
0.7 (40%)
537
546
>5000
>5000
>5000
(2S)-pyrrolidin-2-ylmethyl
(2R)-pyrrolidin-2-ylmethyl
>2000
>2000
29
30
31
(2S)-pyrrolidin-2-ylmethyl
(2R)-pyrrolidin-2-ylmethyl
pyrrolidin-3-yl
1
1
1
CN
CN
CN
6.5
23
13
9.1
11
>5000
>5000
>5000
1272
1380
0.6 (50%)
<0.5 (>60%)
0.6 (50%)
7.8
>2000
32
33
(2S)-pyrrolidin-2-ylmethyl
(2R)-pyrrolidin-2-ylmethyl
2
2
CN
CN
27
19
34
24
>5000
>5000
>2000
>2000
0.7 (40%)
<0.5 (>60%)
a BT-474 cellular proliferation assay, see Ref. 4.
b See Ref. 4.
c See Ref. 7.
2-methyl analogs 11, 19, and 20 to their 2-methyl-4-cya-
no counterparts 15, 21, and 22 reveals the importance of
the 4-cyano group in boosting the cellular inhibition of
HER-2 sheddase. For the analog pairs 17/18 and 19/21
there was a pronounced 9-fold gain in HER-2 inhibi-
tion, which coincided with a 5- and 4-fold gain in
ADAM-10 binding. In contrast, analog pairs 11/15
and 20/22 had a modest 3-fold gain in HER-2 inhibition

D. M. Burns et al. / Bioorg. Med. Chem. Lett. 18 (2008) 560–564
563
that was accompanied by a negligible change and a 6-
fold loss in ADAM-10 binding. These results suggest
that the observed gain in HER-2 cellular potency by
the introduction of a 4-cyano substituent to the P01 phe-
nyl group is a net result of a positive permutation in
physiochemical properties, such as an increase in polar
surface area, taken in combination with either a positive
or negative variance in the enzymatic binding affinity
(ADAM-10).
Schemes 1 and 2.8 The enolate of dimethyl piperidine-
1,4-dicarboxylate 34 was quenched with diiodomethane
followed by SN2 displacement of the primary iodide by
potassium thioacetate. The thioacetate was oxidized to
the corresponding sulfonyl chloride by treatment with
H2O2 and acetic acid followed by reaction with sodium
acetate to form the sodium sulfonate. Reaction with
thionyl chloride afforded 35. Alternatively, the sulfonyl
chloride 35 can be prepared in one step by bubbling
Cl2 (g) into a solution of the thioacetate in methylene
chloride and water. The sulfonamide 37 was easily pre-
pared by treatment of 35 with the appropriate amine,
such as 36, in the presence of a base, such as DIEA. Re-
moval of the methyl carbamate was achieved by reaction
with iodotrimethylsilane in refluxing dichloromethane.
The piperidine N–H was acylated with CDI or p-nitro-
phenyl chloroformate followed by reaction with the
appropriate alcohol, such as 38, in the presence of a
strong base, such as NaH, to afford the desired carba-
mate 39. Basic hydrolysis of the ester 39 followed by
BOP reagent-mediated amide coupling afforded the
hydroxamic acid. Deprotection of the Cbz group with
H2 and 5% Pd/BaSO4 afforded the desired product 30.
Despite the excellent potency and selectivity that com-
pounds 11, 15, and 21 possessed, these compounds suf-
fered from high projected human clearance (proj. h-Clr)
and thus low projected human maximal bioavailability.
Metabolic studies suggested that the carbamate moiety
was being cleaved. It was hypothesized that this cleavage
could be prevented by either sterically blocking the car-
bamate moiety from cleavage or by introducing a polar
group to the P1 substituent, which could reduce the
binding affinity to cytochrome P450 and thus decrease
the clearance. We speculated that modification of the
P1 group could be conducted with minimum disruption
to the binding profile since it was believed that the P1
group was primarily solvent exposed.
3-Methyl-4-piperidine-4-yl-benzonitrile 36 was prepared
from 4-chloro-2-methyl-iodobenzene 40 by mono-lith-
ium halogen exchange and subsequent reaction with
Boc-piperid-4-one 41. The tertiary alcohol is dehydrated
with concomitant loss of the Boc group, which is susbe-
quently reapplied. The tetrahydropiperidine double
bond is reduced in the presence of the aryl chloride using
either a homogeneous catalyst or 5% Pt/C under an
atmosphere of H2 (g). Cyanation of the aryl chloride
was conducted using zinc cyanide and Pd(PBu3)2 and
Zn as catalysts in NMP at 150 °C.9 Removal of the
Boc protecting group with 4 N HCl in 1,4-dioxane affor-
ded the free amine 36.
Attempts to sterically block the tetrahydrofuran carba-
mate of 11 by installation of a 3-methyl group 23 or a
4,4-dimethyl group 24 were both unsuccessful at low-
ering the proj. h-Clr (Table 2). Exchanging the tetra-
hydrofuran carbamate of 11 with the more polar
trans-2-hydroxyl-cyclopent-1-yl carbamate 25 also did
not improve the proj. h-Clr. Replacement of the tetra-
hydrofuran oxygen of 11 with nitrogen to form the
corresponding pyrrolidine 26 resulted in a significant
decrease in proj. h-Clr that may be attributed to a de-
crease in lipophilicity. Despite the encouraging ob-
served decrease in proj. h-Clr the selectivity against
MMP-2, while still good, diminished almost 4-fold in
comparison to 11. Switching the P1 group to the pyrr-
olidin-2-ylmethyl carbamates 27 and 28 resulted in a
minor improvement for only the (2R)-enantiomer 28.
Installation of the 4-cyano group to the P01 phenyl ring
of compounds 27 and 28 to afford analogs 29 and 30
ameliorated the proj. h-Clr while maintaining the
excellent selectivity toward MMP-2. Installation of
the 4-cyano group to the P01 phenyl ring of compound
26 restored the selectivity against MMP-2 while main-
taining the adequate proj. h-Clr. The corresponding 4-
(2-methyl-4-cyano-phenyl)-1,2,3,6-tetrahydropyridin-1yl
analogs 32 and 33 displayed comparable improvements
in the proj. h-Clr in comparison to the (3S)-tetrahy-
drofuran-3-yl parent compound 22. These results
suggest that increasing the polarity at the P1 position
of the molecule by the introduction of a pyrrolidine
N–H may reduce the affinity toward cytochrome
P450. However, it is also reasonable that these ob-
served improvements in Proj. h-Clr may be a manifes-
tation of favorable changes in physiochemical
properties resulting in a reduction in cytochrome
P450 binding.
In conclusion, it has been demonstrated that the b-
piperidine sulfonamide hydroxamic acid core can serve
as a viable scaffold for the selective inhibition of the
HER-2 sheddase, ADAM-10. Selectivity can be
achieved by the installation of a small substituent in
the ortho- position of the P01 phenyl ring, particularly a
methyl group. The cellular inhibition of HER-2 shed-
dase can be enhanced by the installation of a cyano
group in the para- position. Subtle permutations to this
substitution pattern or deviations from these substitu-
ents resulted in a cogent fluctuation in the enzymatic
binding profile. Thus highlighting the high sensitivity
within the S01 pocket of the metalloprotease family. Con-
versely, the P1 substituent can be modified to attenuate
the pharmacokinetic properties of the molecule with
only minor repercussions to the metalloprotease binding
profile. It was found that the introduction of a polar –
NH group to the P1 carbamate substituent decreased
the projected human clearance presumably by diminish-
ing the affinity of the molecule to the active site of the
cytochrome P450. In comparison to the azaspiro-
hydroxamic acid INCB3619, the new lead compounds
29–31 exhibit a 3- to 10-fold gain in cellular inhibition
of HER-2 sheddase with a 125- to 250-fold increase in
selectivity against MMP-2 and a 35- to 125-fold increase
The syntheses of the analogs discussed herein are exem-
plified by the synthesis of compound 30 depicted in

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D. M. Burns et al. / Bioorg. Med. Chem. Lett. 18 (2008) 560–564
O
O
O
O
O
O
O
N
O
O
O
SO2Cl
O
d
S
N
e, f, g
HO
Cbz
a, b, c
O
H
N
N
N
N
O
O
38
34
35
NC
HO
36
CN
37
O
NH
O
S
O
S
O
N
O
N
O
O
h, i, j
N
N
CN
CN
O
O
O
O
N
N
Cbz
H
39
30
Scheme 1. Reagents and conditions: (a) (i) LDA, THF ꢀ78 °C; (ii) CH2I2 (99%); (b) KSAc, DMF (74%); (c) Cl2, H2O, DCM (92%); (d) DIEA, 36
(88%); (e) TMSI, DCM, reflux (100%); (f) CDI or p-NO2-Ph-OC(O)Cl, DIEA, ACN (95%); (g) NaH, 38, THF (90%); (h) LiOH, THF, MeOH, H2O,
rt 2d (99%); (i) BOP, NMM, NH2OH, DMF (84%); (j) H2, 5% Pd/BaSO4 (100%).
H
N
Boc
N
Lluch, A.; Garcia-Conde, J.; Baselga, J.; Clinton, G. M.
Clin. Cancer Res. 2002, 8, 347.
4. Yao, W.; Zhou, J.; Burns, D. M.; Xu, M.; Zhang, C.; Li, Y-
L.; Qian, D-Q.; He, C.; Weng, L.; Shi, E.; Lin, Q.; Agrios,
C.; Burn, T. C.; Caulder, E.; Covington, M. B.; Fridman, J.
S.; Friedman, S.; Katiyar, K.; Hollis, G.; Li, Y.; Liu, C.;
Liu, X.; Marando, C. A.; Newton, R.; Pan, M.; Scherle, P.;
Taylor, N.; Vaddi, K.; Wasserman, Z. R.; Wynn, R.;
Yeleswaram, S.; Jalluri, R.; Bower, M.; Zhou, B-B.;
Metcalf, B. J. Med. Chem. 2007, 50, 603.
5. Becker, D. P.; Villamil, C. I.; Barta, T. E.; Bedell, L. J.;
Boehm, T. L.; DeCrescenzo, G. A.; Freskos, J. N.; Getman,
D. P.; Hockerman, S.; Heintz, R.; Howard, S. C.; Li, M.
H.; McDonald, J. J.; Carron, C. P.; Funckes-Shippy, C. L.;
Mehta, P. P.; Munie, G. E.; Swearingen, C. A. J. Med.
Chem. 2005, 48, 6713.
a, b, c
d, e, f
I
Cl
O
N Boc
40
41
CN
Cl
42
36
Scheme 2. Reagents and conditions: (a) (i) n-BuLi, THF ꢀ78 °C (ii) 41
(68%); (b) TFA; (c) Boc2O, DIEA, DCM (92%); (d) 5% Pt/C, H2,
MeOH (68%); (e) ZnCN2, Zn, Pd(Bu3P)2, NMP 150 °C (76%); (f) 4 N
HCl in 1,4-dioxane, EtOAc (100%).
in selectivity against MMP-9. Further pharmacody-
namic and pharmacokinetic studies are impending to
evaluate the potential leads 29–33.
6. Babine, R. E.; Bender, S. L. Chem. Rev. 1997, 97, 1359.
7. Pooled human liver microsomes were purchased from
XenoTech LLC. (Lenexa, KS, USA) and NADPH was
purchased from Sigma–Aldrich (St. Louis, MO, USA). All
other reagents and solvents used in this study were of the
highest quality commercially available. A 1 lM solution of
compound was preincubated for 3 min at 37 °C with
0.5 mg/mL of human liver microsome protein in 100 mM
potassium phosphate buffer (pH 7.4). Incubations were
initiated with the addition of NADPH (final concentration,
2 mM). Incubations were terminated at 0, 10, 20, and
30 min by the addition of two volumes of acetonitrile.
Following sample centrifugation at 1500g for 10 min
(Allegra 6 Centrifuge, Beckman Coulter, Inc., Fullerton,
CA, USA) the supernatants were analyzed via LC/MS in
positive ion ESI mode on a Thermo Finnigan LCQ Deca
XP Plus.
Acknowledgments
The authors thank Mei Li, Laurine Galya, Karl Blom,
and Jin Liu of the analytical group, Kamna Katiyar,
Jason Boer, and Frank Nedza from the drug discovery
biology group.
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