The development of 2-benzimidazole substituted pyrimidine based inhibitors of lymphocyte specific kinase (Lck)

Article abstract of DOI:10.1016/j.bmcl.2006.08.132

This communication details the synthesis, biological activity, and binding mode of a novel class of 2-benzimidazole substituted pyrimidines. The most potent analogs disclosed showed low nanomolar activity for the inhibition of Lck kinase and a representat

Full text of DOI:10.1016/j.bmcl.2006.08.132

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5973–5977
The development of 2-benzimidazole substituted pyrimidine
based inhibitors of lymphocyte specific kinase (Lck)
Mark Sabat,a,* John C. VanRens,a Matthew J. Laufersweiler,a Todd A. Brugel,a
Jennifer Maier,a Adam Golebiowski,a Biswanath De,a Vijayasurian Easwaran,a
Lily C. Hsieh,a Richard L. Walter,b Marlene J. Mekel,b Artem Evdokimovb and
Michael J. Janusza
aProcter and Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Rd., Mason, OH 45040, USA
bP&G Analytical Discovery, Miami Valley Innovation Center, PO Box 538707, Cincinnati, OH 45253-8707, USA
Received 7 June 2006; revised 30 August 2006; accepted 31 August 2006
Available online 25 September 2006
Abstract—This communication details the synthesis, biological activity, and binding mode of a novel class of 2-benzimidazole
substituted pyrimidines. The most potent analogs disclosed showed low nanomolar activity for the inhibition of Lck kinase and
a representative analog was co-crystallized with Hck (a structurally related member of the Src family kinases).
ꢀ 2006 Elsevier Ltd. All rights reserved.
Lck is a 56-kD Src family protein tyrosine kinase (PTK)
that plays a critical role in the development and activa-
tion of T cells including T-cell antigen receptor (TCR)
HN
R
2
N
N
phosphorylation (an event necessary for signal transduc-
tion in the T cell signaling cascade of the T-cell recep-
tor).1a,1b Activation of this cascade ultimately results
in the production of cytokines such as interleukin-2
(IL-2) and IFNc.1b,1c,1d The production of these cyto-
kines results in further activation and proliferation of
T lymphocytes to generate an immune response. Unlike
the widespread expression of some other Src family
PTKs, Lck expression is restricted to T-cells and natural
killer (NK) cells.1d As such the inhibition of Lck has
been proposed as a potential treatment for a number
of autoimmune diseases where T-cells are thought to
play an important role such as rheumatoid arthritis
(RA), inflammatory bowel disease (IBD), psoriasis, sys-
temic lupus erythematosus (SLE), and organ graft
rejection.1e
N
4
O
1 (a) R= OH; lck IC50 = 4.2 μM
1 (b) R= NH2; lck IC50 = 6.8 μM
Figure 1. Initial benzo[d]isoxazole containing lead molecule.
the potency of these lead compounds led to the develop-
ment of a facile SAR strategy incorporating benzimid-
azole substituted pyrimidines. This communication
details the synthesis, biological activity, and binding
mode of a novel class of 2,4,6-trisubstituted pyrimidine
derivatives based on the initial lead benzo[d]isoxazole
1. The binding mode of these trisubstituted pyrimidine
inhibitors was also determined from X-ray co-crystal-
lography experiments in the related hematopoietic cell
kinase (Hck), a member of the Src family kinases.1a,b
Screening efforts in our laboratories identified 4-
benzo[d]isoxazole compounds 1a–b as moderate Lck
inhibitors (Fig. 1). Initial work directed at improving
The synthesis of the lead 2,4-disubstituted pyrimidines
(1a–b) is outlined in Scheme 1. 4-Iodo-2-methylthiopyr-
imidine2a (2) was treated with isopropyl magnesium
chloride followed by addition of 2-fluoro-benzaldehyde
to give alcohol 3.2b,2c Oxidation of this material with
MnO2 afforded the corresponding ketone which was
condensed with hydroxyl amine resulting in oxime 4.
Keywords: Kinase; Lck; Hck; Lymphocyte specific kinase; Hemato-
poietic cell kinase; Src family kinase; T cell.
*
Corresponding author. Tel.: +1 858 731 3657; e-mail:
Mark.Sabat@Takedasd.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.132

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M. Sabat et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5973–5977
6e which did not contain the 4-methyl group on the
C2 anilino substituent (compare 6a vs 6e) was devoid
of activity.
a
N
N
N
N
b,c
N
N
S
S
N
2
I
S
S
F
F
OH
N
NOH
3
4
Our attention subsequently focused on understanding
the role of the pyrimidine ring nitrogen atoms (N1 and
N3) in the potency observed. Regioisomeric analogs
7a–c were synthesized using modified literature condi-
tions and the results are presented in Table 1.6
d
e,
f or g
1a-b
N
5
O
Scheme 1. Preparation of compounds 1a–b. Reagents and conditions:
(a) isopropyl magnesium chloride 2 M, THF À40 ꢂC; then 2-fluoro-
benzaldehyde, 34%; (b) MnO2, DCM, 24 h, 97%; (c) hydroxyl-
amineÆHCl, pyridine, 95 ꢂC, 3 h; (d) NaH, DMF 165 ꢂC, 0.5 h, 54%
(2 steps); (e) m-CPBA, DCM, 0 ꢂC, 0.25 h; (f) 3-amino-4-methyl-
phenol, CH3CN, 155 ꢂC, microwave, 15% (2 steps 1a); (g) 4-methyl-
benzene-1,3-diamine, CH3CN, 150 ꢂC, microwave, 0.5 h, HPLC sep-
aration of isomers, 1% (2 steps 1b).
Derivative 7a which transposed the substituents at C2
and C4 relative to original compound 6a displayed
greater potency (Lck IC50 = 24); however, the C4 C6
isomerically substituted compound (8, Fig. 2) was de-
void of any Lck activity.7
With such a potent lead molecule (7a) we again attempt-
ed to introduce alternatively functionalized anilines at
C4. However both the 5-methoxy (7b) and 5-fluoro ani-
line (7c) derivatives showed greatly attenuated activity
(Table 1). The presence of both a 4-methyl and phenolic
hydroxyl on the C2 anilino substituent appeared crucial
for good activity.
Treatment of this crude material with NaH followed by
heat afforded intermediate 5.3 Oxidation of the thio-
group on compound 5 with Oxoneꢁ and displacement
of the resultant sulfone/sulfoxide mixture generated the
final products 1a–b.4
To more quickly expand the SAR of pyrimidines 1 and
to overcome synthetic difficulties with this scaffold, a
benzimidazole group was substituted for the 4-
benzo[d]isoxazole moiety. The resulting phenol 6a
(Table 1) proved to be a significantly more potent
inhibitor (Lck IC50 = 193 nM)5a compared to 1a. Inter-
estingly, the corresponding methyl ether 6b and amides
6c–d displayed greatly attenuated activity. Pyrimidine
Our efforts were next directed at adding functionality to
improve the poor aqueous solubility (4 lg/ml) of scaf-
fold 7a.8 A series of 2,4,6-trisubstituted pyrimidines
were synthesized which maintained both the C2 and
C4 groups found on 7a while introducing various basic
amine substituents to the C6 position of the pyrimidine
core (Tables 2 and 3). We reasoned that basic amine
substituents could be tolerated at position C6 after
examining the active site of Lck in complex with inhib-
itors disclosed in the literature.9 These groups may im-
part greater aqueous solubility and added potency
through interactions with proximal acidic residues.
Table 1. IC50 values for derivatives 6a–m, 7a–c
N
N
H
H
N
N
N
N
N
N
R1
R1
N
N
The synthesis of these 2,4,6-trisubstituted pyrimidines is
outlined in Scheme 2. 4,6-Dichloro-2-methylsulfanyl-py-
rimidine and 5-methoxy-2-methyl-phenylamine were
heated at 140 ꢂC to afford intermediate 10. Oxidation
of the thiol group in 10 with Oxoneꢁ followed by dis-
placement of the resultant sulfone/sulfoxide mixture
with sodium benzimidazolate and the subsequent phenol
deprotection gave 11. The 6-chloro group on this pyrim-
idine was then displaced with various amines and sodi-
um alkoxides to give 12a–p. Analogs 12q–r resulted
from Suzuki–Miyaura coupling of the corresponding vi-
nyl heterocycle and 11.10
7
6
a
Lck IC50 (nM)
Compound
R1
6a
7a
193
24
OH
6b
7b
>10,000
7700
OCH3
6c
5664
NH2
The initially synthesized compounds 12a–e containing
simple substituents at the C6 position were somewhat
O
O
6d
6e
7c
8640
N
H
HN
6
>10,000
>10,000
OH
OH
N
N
N
4
N
F
8
Lck IC50 > 10μM
a IC50’s were determined with a commercial Proflour assay (Promega
corp., Cat. #1271).
Figure 2. Compound 8.

M. Sabat et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5973–5977
5975
Table 2. Lck inhibition data for derivatives 12a–r
Introduction of an ethylmorpholine group at the C6 via
an O (12g) or NH (12i) linker resulted in inhibitors with
better potency in the enzyme assay (Lck IC50 = 3 and
17 nM, respectively). Methylation on the phenolic
hydroxyl (12h) dramatically reduced potency (compare
12g vs 12h).
HN
OR2
N
N
N
N
R1
Substitution of the N-methyl piperizine unit for mor-
pholine (12j vs 12g) gave the first analog with good
aqueous solubility (sol = 70 lg/ml at pH 7.4) with only
a slight decrease in activity (Lck IC50 = 65 nM). Deriva-
tives 12k–n also displayed good kinase inhibition (Lck
IC50 = 3–130 nM). Hydrazino analogs 12o and 12p how-
ever displayed lower potency. Insertion of a methylene
spacer (12q) produced an analog with better enzyme
potency than the corresponding hydrazino analog 12o.
However insertion of an additional ethylene spacer
(12r) in combination with a piperidine substituent (con-
trast 12q, 12m, and 12r) reduced activity.
a
Lck IC50 (nM)
Compound
R1
R2
4
–Cl
–NH2
H
H
H
H
H
1286
294
79
12a
12b
12c
12d
–NHCH3
–N(CH3)2
–OCH3
384
71
O
N
12e
12f
12g
12h
12i
12j
H
137
NCH3
H
3
N
O
Representative compounds that showed promising Lck
inhibition were tested for inhibition of IL-2 production
in a Jurkat cellular assay.11 Analogs displaying cellular
potency were screened for in vitro metabolism12 and
progressed into PK studies (Table 3). Compounds 12g,
12j, and 12q showed moderate to good IL-2 inhibitory
activity (0.054, 0.400, and 0.163 lM, respectively). The
morpholino compound 12g however showed poor aque-
ous solubility. Metabolism studies of the compounds in
Table 3 revealed moderate to high intrinsic clearance
(CLint = 48–124 ml/min kg).13 Inhibitors 12g, 12h, and
12q were subsequently evaluated in a PK study to deter-
mine bioavailability and half-life. Both molecules 12g
and 12q had no oral bioavailability. Only the less potent
methoxy analog 12h was orally bioavailable (% F = 22;
T1/2 = 2.13 h).
H
3
N
O
O
O
O
–CH3
H
867
17
65
N
N
N
H
NCH3
H
N
O
12kb
H
3
N
N
H
N
H
N
12l
H
H
44
N
H
N
12m
130
NH
To delineate the binding mode of these molecules an X-
ray crystallographic structure of 12k with hck (a struc-
turally related member of the Src family kinases) was
obtained (Fig. 3).14 Inhibitor 12k orients itself in the
hck enzyme such that the benzimidazole N hydrogen
bonds to the amide N–H of Met319 and the phenolic
OH hydrogen bonds to carboxyl of Glu 288. An addi-
tional interaction may occur between the O–H of the
Thr316 (gatekeeper residue) and the aniline N–H on
the phenol substituent. The 4-methyl group on the phe-
nol substituent appears to sit in a small hydrophobic
grove formed in part by the CH3 of Thr316. This place-
ment appears to position the phenolic OH for optimal
interaction with Glu288.
NCH3
12n
H
45
N
N
H
NCH3
O
12o
12p
H
H
165
519
N
N
N
H
N
H
NCH3
12q
12r
H
H
43
NH
333
In summary we have reported a class of trisubstituted
pyrimidines that have shown nanomolar activity for
inhibiting Lck kinase activity. The binding mode of this
series of novel Lck inhibitors was determined through
X-ray co-crystallographic studies in the structurally
related Src family kinase Hck (PDB accession code
2HK5). Seven of the most potent analogs were further
tested for inhibition of IL-2 cytokine production, show-
ing a range of potencies (0.054–2.280 lM). The pharma-
cokinetic properties of these pyrimidine analogs were
also evaluated. Compounds 12j–l, 12n, and 12q all had
a IC50’s were determined with a commercial Proflour assay (Promega
corp., Cat. #1271).
b Biological activity of 12k in two related Src family kinases was
determined in
a
similar fashion (Src IC50 = 162 nM; Hck
IC50 = 46 nM).5b,5c
.
less potent inhibitors of Lck compared to the lead 7a
(IC50 = 71–384 nM). The N-methyl piperazine analog
12f, however, displayed better potency but possessed
no aqueous solubility (sol 6 0.1 lg/ml at pH 7.4).

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M. Sabat et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5973–5977
Table 3. IL-2 inhibition and pharmacokinetic data for select compounds
IL-2a IC50 (lM)
Solubility (lg/ml)
Met % lossb
Fd (%)
b
CLint (ml/min kg)
Compound
12g
12hc
12j
0.054
1.270
0.400
0.549
2.195
2.281
0.163
3
27
48
—
35
45
48
39
—
124
48
0
22
—
—
—
—
0
70
83
12k
12l
127
55
107
92
12n
12q
54
40
93
50
a Inhibition of IL-2 synthesis measured from Jurkat cells.
b % loss and intrinsic clearance after 1 h for compounds (1 lM concentration) tested in cryopreserved rat hepatocyte suspensions.
c Compound 12 h had CL_plasma (ml/min kg) = 132, AUC-po (ng-h/ml) = 497, Cmax (ng/ml) = 76.
d Compounds dosed po and iv at 10 mg/kg.
References and notes
Cl
N
HN
O
1. (a) Molina, T. J.; Kishihara, K. I.; Siderovski, D. P.; van
Ewijk, W.; Narendran, A.; Timms, E.; Wakeham, A.;
Paige, C. J.; Hartmann, K. U.; Veillette, A.; Davidson, D.;
Mak, T. W. Nature 1992, 357, 161; (b) Levin, D. S.;
Anderson, S. J.; Forbush, K. A.; Perlmutter, R. M.
EMBO J. 1993, 12, 1671; (c) August, A. J. Biol. Chem.
1996, 271, 10054; (d) Bolen, J. B.; Brugge, J. S. Annu. Rev.
Immunol. 1997, 15, 371; (e) Kamens, J. S.; Ratnofsky, S.
E.; Hirst, G. C. Curr. Opin. Invest. Drugs 2001, 2, 1213.
2. (a) Majeed, A. J.; Antonsen, O.; Benneche, T.; Undheim,
K. Tetrahedron 1989, 45, 993; (b) Tobrman, T.; Dvorak,
D. Org. Lett. 2003, 5, 4289; (c) Boudier, A.; Bromm, L. O.;
Lotz, M.; Knochel, P. Angew. Chem., Int. Ed. 2000, 39,
4414.
HN
OH
12a-r
a
b,c,d
e
N
N
N
Cl
N
S
Cl
S
N
N
Cl
N
10
9
11
Scheme 2. Preparation of compound 12a–r. Reagents and conditions:
(a) 5-methoxy-2-methyl-phenylamine, Hunig’s base, DMF 140 ꢂC,
¨
85%; (b) Oxoneꢁ MeOH, H2O, quantitative; (c) benzimidazole, NaH,
DMF, 0 ꢂC to generate sodium benzimidazolate then MW 60 ꢂC, 67%;
(d) BBr3, DCM, 50%; (e) various amines, anilines, and alkoxides,
NMP, MW 100 ꢂC, 15 min.
3. Shutske, G. M.; Setescak, L. L.; Allen, R. C.; Davis, L.;
Effland, R. C.; Ranbom, K.; Kitzen, J. M.; Wilker, J. C.;
Novick, W. J. J. Med. Chem. 1982, 25, 36.
4. Oxidation with a single equivalent of Oxoneꢁ at 0 ꢂC
favored formation of sulfoxide. However, small amounts
of the sulfone were formed. Both intermediates underwent
nucleophilic displacement.
5. (a) The ability of compounds to inhibit human Lck
enzyme (IC50) was determined using the commercially
available ProFlour Src-family Kinase Assay (Promega
Corporation, Madison, WI; cat. #1271). The assay was
performed according to manufacturer’s instructions, with
2 nM recombinant human active Lck (Upstate Cell
Signaling Solutions, Charlottesville, VA; cat. #14-442) at
an ATP concentration of 10 uM. These IC50 assays were
performed using 10-point dose–response curves, in dupli-
cate (one curve on two separate plates), and on two
separate days (n = 2). Every assay plate included a 10 pt.
dilution curve of 1 benchmark compound. Curve Fitting
was performed using the Sigmoidal Dose Response Model
(4 parameter logistic equation 205 in ExcelFit). 95%
confidence intervals were calculated for each IC50 value.
The assay Quality Control Criteria inlcuded; Signal/
Background: >4; Signal/Noise: >10; % Coefficient of
Variance on No ATP Control (Max. Signal): <10.; (b)
The ability of compounds to inhibit human Hck enzyme
(IC50) was determined using the commercially available
ProFlour Src-family Kinase Assay (Promega Corporation,
Madison, WI; cat. #1271). The assay was performed
according to manufacturer’s instructions, with 2.8 nM
recombinant human active Hck (Invitrogen, Carlsbad, CA
#P2908) at an ATP concentration of 10 mM.; (c) The
ability of compounds to inhibit human Src enzyme (IC50)
was determined using the commercially available Pro-
Flour Src-family Kinase Assay (Promega Corporation,
Madison, WI; cat. #1271). The assay was performed
according to manufacturer’s instructions, with 14.3 nM
Figure 3. Key hydrogen bonds between 12k and Hck.
good aqueous solubility (40–127 lg/ml). Metabolism
studies of the compounds revealed moderate to high
intrinsic clearance (CLint = 48–124 ml/min kg).1 Subse-
quent PK studies revealed the phenol containing phar-
macophore
produced
inhibitors
with
poor
bioavailability. Only the methoxy derivative 12h had
good BA (% F = 22, T1/2 = 2.1 h).
Acknowledgments
We are grateful to A. L. Roe, C. A. Cruze, W. E.
Schwecke, C. R. Dietsch for pharmacokinetic studies,
and M. Buchalova for chemical stability and solubility
studies.

M. Sabat et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5973–5977
5977
recombinant human active Src (Invitrogen, Carlsbad, CA
#P3044) at an ATP concentration of 10 mM.
6. Luo, G.; Chen; Ling, P.; Graham, S. Tetrahedron Lett.
2002, 43, 5739.
7. The authors speculate that repulsive interaction between
pyrimidine C5 proton and the proton on the imidazole
ring forced an unfavorable orientation of this molecule
toward the active site of Lck.
multi-compound assay. Samples from like time-points
containing the different compounds were combined and
an internal standard (1.1 ng/ml Stock) was added. Results
for each compound were expressed as the ratio of the
compound response area over the internal standard
response area. Percent loss was calculated by dividing the
2 and 4 h ratios by the 0 h ratio.
13. (a) In vitro intrinsic clearance (CLint) was calculated from
the first-order depletion rate constant and appropriate
biological scaling factors.13b,13c; (b) Davies, B.; Morris, T.
Pharm. Res. 1993, 10, 1093; (c) Venkatakrishnan, K.; von
Moltke, L. L.; Obach, R. S.; Greenblatt, D. J. Curr. Drug
Metab. 2003, 4, 423.
HN
H
OH
H
5
1
N
N
N
14. (a) Crystallographic methods: The Hck protein herein
described as used for the crystallographic studies is fully
dephosphorylated human Hck kinase domain (HckKD),
residues 225–504. The protein was co-expressed in Esch-
erichia coli with the phosphatase PTPb-90 to maximize
production of soluble HckKD, which was otherwise toxic
to the cells. The HckKD was cloned with a non-cleavable
6-His tag for purification purposes. Briefly, protein for
crystallization studies was prepared by lysing biomass
using a mixture of commercially available surfactant-
based lysis buffers. The resulting lysate was clarified by
centrifugation, loaded onto a Ni-affinity column (Sigma
His-SelectTM), and eluted with a linear imidazole gradient
(0.01–0.250 M). The resulting HckKD containing elution
fractions were pooled and diluted 20· in an appropriate
load buffer (20 mM Tris, pH 8.8, 10 mM DTT) for anion
exchange chromatography (AEC) on MonoQ (GE
Healthcare). A linear NaCl gradient in the AEC resulted
in one large elution peak containing HckKD. Fractions
from this peak were pooled and concentrated to 12–15 mg/
ml and used for crystallization studies. Co-crystals with
inhibitors were grown by incubating the concentrated
HckKD with 0.5–1 mM compound for several hours
before crystallization trials. Crystals suitable for X-ray
diffraction grew in 3–7 days at 15 ꢂC using either 1.9–
2.2 M ammonium sulfate, 0.1 M Tris (pH 8.5) or 3.9–
4.1 M sodium formate. Crystals could be improved by the
addition of several additives to the above conditions, such
as 5–10% glycerol or ethylene glycol. Optimal crystals
were typically grown by streak seeding fresh drops using
previously grown crystals. Crystals grew as hexagonal
rods with average dimension 0.07 · 0.07 · 0.25 mm
(though crystals as small as 0.01 · 0.01 · 0.15 were
successfully used for data collection). X-ray data were
collected at Southeast Regional Collaborative Access
Team (SER-CAT) 22-ID beamline at the Advanced
Photon Source, Argonne National Laboratory. Support-
ing institutions may be found at www.ser-cat.org/mem-
bers.html. The crystals provided typical diffraction of
N
Lck IC50 > 10μM
8. Solubility assay procedure: Solubility of analogs measured
in 50 mM phosphate buffer, pH 7.4, ionic strength 0.15 M.
The solubilities were determined by shake flask method
after 24 h of equilibration. Concentrations in aqueous
solutions were determined for supernatant of centrifuged
and filtered samples by UV–vis spectrophotometry.
Calibration solutions were prepared in acetonitrile/ buffer
in 1:1 volume ratio. The samples were diluted with
acetonitrile to obtain identical media with calibration
standards.
9. Zhu, X.; Kim, J. L.; Newcomb, J. R.; Rose, P. E.; Stover,
D. R.; Toledo, L. M.; Zhao, H.; Morgenstern, K. A.
Structure 1999, 7, 651 (1QPC pdb, Research Collabora-
tory for Structural Bioinformatics).
10. Vice, S.; Bara, T.; Bauer, A.; Evans, C. A.; Ford, J.;
Josien, H.; McCombie, S.; Miller, M.; Nazareno, D.;
Palani, A.; Tagat, J. J. Org. Chem. 2001, 66, 2487.
11. IL-2 release was measured by stimulating the Jurkat E6-1
T cell line (human acute T cell leukemia, ATCC, Manas-
sas, VA) with monoclonal anti-human CD3e antibody and
Phorbol Myristate Acetate (PMA) (Sigma, St. Louis,
MO). Flat bottomed-96-well plates were pre-coated with
400 ng/well of anti-human CD3e mouse antibody UCHT1
(R&D systems, Minneapolis, MN) and incubated for 2 h
at 37 ꢂC. Jurkat cells maintained in RPMI-1640 contain-
ing 10% fetal bovine serum and 1% antibiotics in the log
growth phase (2 · 105 to 1 · 106 cells/ml) were harvested
and incubated in triplicate in 96-well plates for 30 min at
37 ꢂC in the presence or absence of various concentrations
of Lck inhibitors. The cell-inhibitor mixture was then
transferred into the wells of the anti CD3e-coated 96-well
plates, and PMA was added to the wells at a final
concentration of 10 ng/ml (1 ng/well). The plates were
incubated overnight at 37 ꢂC. The amount of IL-2 released
into the culture media was measured by ELISA (R&D
Systems) and the viability of the cells was determined
using the MTS assay (Promega, Madison, WI).
12. (a) Measured as percent loss at 4 hours in rat hepatocytes;
(b) In vitro metabolism assay procedure: In vitro meta-
bolic stability of analogs in plated rat hepatocytes
(Sprague–Dawley) obtained from Cedra Corporation.
Metabolic activity was determined in triplicate using a
total volume of 0.2 ml containing 0.25 lM NCE incubated
in rat hepatocyte and Matrigel blank microtiter plates. The
plates were maintained at 37 ꢂC throughout the study.
Samples were removed from wells at 0, 2, and 4 h, and NCE
samples were analyzed by HPLC/MS/MS with reverse-
phase chromatography. To improve analytical efficiency,
compounds were grouped together (post-incubation) into a
˚
better than 2.0 A resolution. Crystals were indexed and X-
ray data were processed in the P65 space group with
˚
approximate unit cell dimensions a = b = 96.4 A,
˚
c = 70.4 A. Structures were determined by molecular
replacement using a structure previously determined in-
house. Use of the Advanced Photon Source was supported
by the US Department of Energy, Office of Science, Office
of Basic Energy Sciences, under Contract No. W-31-109-
Eng-38; (b) The non-phos Hck construct used in these
studies appears to place the activation loop in a confor-
mation resembling activated Lck (3LCK, 1QPC; phos-
phorylated Y394); the authors speculate inhibitor 12k is
capable of inducing such a conformation through inter-
action with Glu288.