Simplified staurosporine analogs as potent JAK3 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.10.062

Simplification of bottom ring and regioselective functionalization of the indolocarbazole unit of staurosporine (2) are described. The modification led to a new series of simplified staurosporine analogs, which exhibited significant inhibitory activity ag

Full text of DOI:10.1016/j.bmcl.2006.10.062

Bioorganic & Medicinal Chemistry Letters 17 (2007) 326–331
Simplified staurosporine analogs as potent JAK3 inhibitors
Shyh-Ming Yang,* Ravi Malaviya, Lawrence J. Wilson, Rochelle Argentieri, Xin Chen,
Cangming Yang, Bingbing Wang, Druie Cavender and William V. Murray
Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 8 Clarke Drive, Cranbury, NJ 08512, USA
Received 10 August 2006; revised 13 October 2006; accepted 23 October 2006
Available online 26 October 2006
Abstract—Simplification of bottom ring and regioselective functionalization of the indolocarbazole unit of staurosporine (2) are
described. The modification led to a new series of simplified staurosporine analogs, which exhibited significant inhibitory activity
against Janus kinase 3 (JAK3). The structure–activity relationships (SAR) are discussed and a proposed binding model is also
highlighted.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Janus kinases (JAK), including JAK1, JAK2, Tyk2, and
JAK3, are cytoplasmic protein tyrosine kinases that play
pivotal roles in cytokine mediated biological responses
by activating the cytoplasmic latent forms of STAT pro-
teins.1 Among the four members of the JAK family,
JAK3 is abundantly expressed in lymphoid cells and
plays a crucial role in normal lymphocyte development
and function, as evidenced by qualitative and quantita-
tive deficiencies in the B-cell as well as T-cell compart-
ments of the immune system of JAK3-deficient mice2
and development of severe combined immunodeficiency
in JAK3-deficient patients.3 Furthermore, it has been
demonstrated that JAK3 plays a key role in the regula-
tion of mast cell mediated allergic and asthmatic
responses.4 Therefore, a potent and specific chemical
inhibitor of JAK3 may be useful for the treatment of im-
mune-mediated diseases including rejection of organ
transplant, atopic dermatitis, allergy, and asthma.4,5
H
N
O
Me
CN
N
Me
N
N
N
O
N
O
H
Me
N
H
N
MeO
NHMe
2, staurosporine
1, CP-690550
H
N
H
N
O
O
iPrO
EtS
SEt
9
3
N
N
N
O
O
H
Me
MeO2C
N
O
OH
4, CEP-7055
3, CEP-1347
Several potent JAK3 inhibitors have been recently
identified,6 including naphthyl ketones,6d oxindole
derivatives,6e pyridine-containing tetracycles,6f and
CP-690550 (1,6g Fig. 1). In particular, 1 has shown selec-
tivity for JAK3 over JAK1 and JAK2 (100- and 20-fold,
respectively) and has demonstrated efficacy in animal or-
gan transplant models.
Figure 1. Chemical structures of CP-690550 (1), staurosporine (2),
CEP-1347 (3), and CEP-7055 (4).
Staurosporine (2) is a potent inhibitor of several ki-
nases.7 Despite the nonselective kinase activities, stauro-
sporine’s ability to inhibit phosphorylation and to
modulate expression of JAK proteins has been demon-
strated.8 In addition, introduction of a suitable substitu-
ent at the C-3 and/or C-9 positions of the
indolocarbazole unit of staurosporine provided potent
kinase inhibitors CEP-1347 (3, for ChAT, JNK, and
MLKs)9 and CEP-7055 (4, for VEGF-R)10 that entered
Keywords: Staurosporine; JAK3; Janus kinases 3.
*
Corresponding author. Tel.: +1 609 409 3491; fax: +1 609 655
6930; e-mail: syang9@prdus.jnj.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.062

S.-M. Yang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 326–331
327
O
H
N
H
N
H
N
O
O
O
O
O
NH2
O
N
H
6
d
b,c
OMe
a
N
H
N
N
N
H
N
H
N
H
N
H
7
5
8
OH
9
g,h
i
e,f
H
N
H
N
H
N
H
N
O
O
O
O
j or k
N
N
N
N
N
N
N
N
X
OH
HO
13, X= OH
14, X= H
HO
OH
OH
12
10
11
Scheme 1. Reagents and conditions: (a) t-BuOK, THF, 0 ꢁC to rt, 4 h, then HCl, 89%; (b) PdCl2, DMF, 90 ꢁC, 3 h, 80%; (c) Sn, HCl, HOAc,
110 ꢁC, 5 h, 96%; (d) epibromohydrin, Cs2CO3, DMF, rt, 24 h, 74%; (e) 5,5-dibromomethyl-2,2-dimethyl-1,3-dioxane, Cs2CO3, MeCN, microwave,
160 ꢁC, 90 min, 35%; (f) THF, MeOH, cat. p-TSA, 45 ꢁC, 3 h, 55%; (g) 1,1-bis(tert-butyldiphenylsiloxy)-2,2-bis(chloromethyl)ethane, Cs2CO3, DMF,
50 ꢁC, 24 h, 50%; (h) TBAF, THF, rt, 15 min, 78%; (i) 3-chloro-2-chloromethyl-1-propene, Cs2CO3, DMF, rt, 24 h, 80%; (j) OsCl3, Me3NO, THF,
CHCl3, rt, 48 h, 13 (83%); (k) 9-BBN, THF, 65 ꢁC, 3 h, then H2O2, 10% NaOH(aq), 65 ꢁC, 14 (81%).
Table 1. IC50 of 9–14 against JAK3
clinical trials. Herein we describe the discovery of novel
JAK3 inhibitors related to staurosporine. Our synthetic
a
IC50 (nM)
Compound
efforts focused on the simplifying the bottom ring and
introducing regioselective functionality on the indole
rings. This led to a new series of potent JAK3 inhibitors.
9
10
11
12
13
14
2
142b
13.5 ( 1.5)
36 ( 1)
72 ( 10)
27 ( 2)
17.5 ( 4.5)
6 ( 3)
The initial focus was to reduce the synthetic complexity
of the bottom piece of 2, by replacing the amino-sugar
ring with a medium size alkyl ring. The hydroxyl group
was appended to further mimic the H-bonding interac-
tion of the methylamino group of AFN941 with
Arg953 of the JAK3 backbone.11 The synthesis of the
key intermediate 8 started with the condensation of 5
and 6 under t-BuOK conditions to form the bis-indole
7 (Scheme 1).12 Subsequent oxidative cyclization medi-
ated by PdCl2 followed by monoreduction of the biscar-
bonyl using tin metal afforded 8 in excellent yield. With
the intermediate 8 in hand, subsequent alkylation with
suitable electrophiles rapidly provided diverse hydroxyl
compounds with a simplified bottom ring. For example,
double alkylation of 8 with dibromide or dichloride fol-
lowed by a suitable deprotective process gave diols 10
and 11. Furthermore, alkylation with epibromohydrin
accompanied by a ring opening of the epoxide afforded
9. Dialkylation with 3-chloro-2-chloromethyl-1-propene
gave alkene 12 that could be further converted to 14 or
diol 13 by hydroboration or dihydroxylation,
respectively.
a Values are means of 3–5 experiments, standard deviation is given in
parentheses.
b Single experiment.
the hydroxymethyl group. This modification enhanced
activity, presumably by providing a crucial H-bonding
interaction with the Arg953 residue. Furthermore, the
introduction of a second hydroxymethyl group (10) gave
a slight improvement in potency. Compound 10 was
only 2-fold less potent than staurosporine (2). The slight
decrease in potency of the diol 11 (IC50 of 36 nM) could
be due to an unfavorable positioning of the hydroxyl
group by the planar alkene configuration. Compound
12, which was devoid of the hydroxyl group, still gave
good activity at 72 nM.
After completing the investigation of the simplified
bottom pieces, the next effort was focused on regiose-
lective functionalization at the C-3 and/or C-9 position
of the indolecarbazole unit to explore the SAR of the
new lead structure 14. Synthesis was initiated with
the protection of 14 to afford acetyl-protected 15 or
16 depending on reaction temperature (Scheme 2). Sub-
sequent Lewis acid promoted bisformylation9c with
dichloromethyl methyl ether gave 17, which could be
further manipulated to provide bis-substituted analogs
22j and 22h. Further conversion of 22h in isopropanol
The JAK3 inhibitory activities (IC50) for the hydroxyl
compounds 9–14 compared to
2 are shown in
Table 1.13 These results, in general, revealed that these
simplified staurosporine analogs with hydroxyl groups
in the bottom ring exhibited good activity with IC50’s
below 40 nM with the exception of 9 (142 nM). Exten-
sion of the OH group by one methylene (9 vs 14) result-
ed in an 8-fold increase in potency. Moreover,
comparing 9 and 13 clearly shows the importance of

328
S.-M. Yang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 326–331
H
N
X
Ac
H
N
O
N
N
O
O
O
R1
R2
3
9
Br
a or b
g
N
N
N
N
N
N
N
N
22
OH
i
h
OAc
OH
OAc
14
15, X= Ac
16, X=H
18
22d
22k–q
c (X= H)
c
22a
22r
m
i
Ac
N
H
N
Ac
N
H
N
O
O
O
O
OH
R1
Br OHC
j
Br
OHC
CHO
Br
l
N
N
N
N
N
N
N
N
f
h
OAc
OH
OAc
OAc
17
20, R1= CH2NMe2
21
19
22j
22e
o
22g, R1= CH2-(4-morpholine)
d
n
k (from 20)
H
N
H
N
H
N
O
H
N
O
OiPr
O
OiPr
O
R1
R2
N
Br
N
N
N
N
N
N
N
N
OH
OH
OH
22b
22h, R1, R2= CH2OH
22i, R1, R2= CH2OiPr
22f
OH
e
22c
Scheme 2. Reagents and conditions: (a) Ac2O, DMAP, THF, 50 ꢁC, 15 (82%); (b) Ac2O, DMAP, THF, rt, 16 (47%); (c) Cl2CHOMe, TiCl4, CH2Cl2,
rt, 24 h, 17 (83%), 19 (83%); (d) NaBH4, THF, rt, then MeOH, 82%; (e) PrOH, camphorsulfonic acid (CSA), rt, 3 days, 17%; (f) i—morpholine,
i
NaBH(OAc)3, AcOH, THF, CH2Cl2; ii—NaOMe, MeOH, 19%; (g) NBS, CHCl3, MeOH, rt, 1 h, 81%; (h) NaOMe, MeOH, rt, 1 h, 22d (80%), 22e
(75%); (i) alkene, Pd(OAc)2 (15 mol %), (o-tolyl)3P (30 mol %), H2O/DMF (1:19), microwave, 150 ꢁC, 30 min, then NaOMe, MeOH, ca. 20–40% (two
steps); (j) morpholine or dimethyl amine, NaBH(OAc)3, THF, rt, 4 h, then NaOMe, MeOH, 22g (80%), 20 (72%); (k) N,N,N0,N0-
tetramethylethylenediamine, NaBH4, PdCl2(dppf) (10mol%), DMF, rt to 45 ꢁC, 6 h, 35%; (l) NaBH4, THF, rt, 1 h, 72%; (m) PdCl2(dppf)
(10 mol %), HCO2Na, DMF, 100 ꢁC, 1.5 h, then NaOMe, MeOH, 58%; (n) i—(CF3CO)2O, Et3N, CH2Cl2, 0 ꢁC to rt, 1 h, 81%; ii—iPrOH, CSA,
microwave, 150 ꢁC, 1 h, then NaOMe, MeOH, 75%; (o) i—PdCl2(dppf) (10 mol %), HCO2Na, DMF, 100 ꢁC, 1.5 h, 75%; ii—iPrOH, CSA, 70 ꢁC,
36 h, then NaOMe, MeOH, 29%.
in the presence of camphorsulfonic acid9c gave 22i.
Regioselective bromination at the C-3 position of the
more electron rich indole ring was achieved to afford
intermediate 18 and 22d after deprotection. The bro-
mine substituent, in fact, offered more options as a
protecting group or for further installation of a C-3
substituent. For example, the Heck coupling of 18 with
different substituted alkenes afforded 22k–q.14 Formy-
lation of 18 gave 19 and 22e after typical deprotection.
Further elaboration of 19 by reductive amination gave
22g and 20, which could undergo debromination to af-
ford 22c with a dimethylaminomethyl group at the C-9
position.15a Moreover, the hydroxyl compound 21,
formed by reduction of 19, was converted to 22a by
debromination with PdCl2(dppf) and sodium for-
mate,15b and 22r by a Heck coupling reaction. Finally,
compounds 22b and 22f were obtained in a similar
manner from intermediate 21.
The effect of these compounds was tested in vitro against
JAK3 enzyme and compared to that of the parent
hydroxyl compound 14 (Table 2). The IC50 fluctuated
considerably depending on the position and pattern of
the C-3 and C-9 substituents. For instance, introduction
of a hydroxymethyl at C-9 position (R1, 22a) resulted in
a marked improvement in potency (ca. 6-fold) compared
to the parent 14. However, a bulkier substituent such as
an isopropoxymethyl (22b) or dimethylaminomethyl
(22c) caused a 27- and 83-fold loss of potency, respec-
tively. This observation differed slightly from that
reported for compound 4 (Fig. 1) for which the isoprop-
oxide group was well tolerated.10 Moreover, the activity
dropped dramatically to the micromolar range when the
morpholinylmethyl group was attached (22g). The steric
influence of R1 was consistent and could also be
observed in compounds 22d–g, suggesting the exis-
tence of a small pocket around the C-9 position.

S.-M. Yang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 326–331
329
Table 2. Substituent effect and SAR of 22
R1 (C-9)
R2 (C-3)
a
IC50 (nM)
Compound
14
–(H)
–(H)
H
17.5 ( 4.5)
3 ( 0.5)
22a
22b
22c
22d
22e
22f
CH2OH
CH2OiPr
CH2NMe2
H
H
80 ( 27)
250 ( 10)
23.5 ( 1.5)
15 ( 3)
H
Br
Br
Br
CHO
CH2OiPr
70 ( 9)
O
22g
Br
3.13b lM
N
22h
22i
CH2OH
CH2OiPr
CH2OH
CH2OiPr
14 ( 6)
1.05b lM
O
O
N
22j
>5b lM
354 ( 40)
592b
N
N
22k
22l
H
Me
N
H
S
N
22m
22n
22o
22p
22q
22r
H
109 ( 19)
11 ( 3)
31 ( 1)
686b
N
N
H
N
N
H
N
H
N
H
CO2H
H
O
H
237b
N
H
N
N
CH2OH
3 ( 0.5)
N
a Values are means of 3–5 experiments, standard deviation is given in parentheses.
b Single experiment.
A comparison between 14 and 22d as well as 22a and
22h shows how well a small substituent (R2), such as
bromine or hydroxylmethyl, could be tolerated at the
C-3 position without significantly altering potency.
Increasing the size of the hydrophobic substituent at
C-3, for example isopropoxymethyl (22i), was unfavor-
able, causing a 13-fold decrease in potency. The loss of
potency of compound 22j (IC50 > 5 lM) might be attrib-
uted to the morpholinomethyl substitution at C-9. Heck
coupling of the intermediate bromide, as described
above, provided several C-3 substituted compounds that
gave moderate to good potency depending on the nature
of the aromatic ring and the length of the linker. For
example, the activity of the pyridine or thiazole substi-
tuted alkenes (22k–l) was greater than 300 nM. Howev-
er, the imidazole substituted alkene (22m) provided a
compound with an IC50 of 109 nM. Notably, an elon-
gated linker, such as allyl (22n), increased the activity
to 11 nM, which was a slight improvement over the par-
ent compound 14 and ca. 10- and 54-fold better than
22m and 22l, respectively. Moreover, the allyl guanidine
substituted compound 22o also provided an IC50 value
in the double-digit range (31 nM). These results suggest
that the imidazole ring or guanidine unit can be tethered
with a suitable linker to provide a crucial interaction
with the JAK3 enzyme. Further introduction of a car-
boxylic acid (22p) or amide with a longer solubilizing
group (22q) gave no improvement. Finally, the combi-
nation of both optimized R1 and R2 substituents, name-
ly the hydroxymethyl at C-9 and the allyl imidazole at
C-3, led to compound 22r having an IC50 value of
3 nM, which is more potent than staurosporine.
The possible binding mode of 22r in the ATP-binding
site of JAK3 was investigated by molecular docking,16
based on the newly published JAK3 crystal structure11

330
S.-M. Yang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 326–331
Acknowledgment
S.-M. Yang thanks Dr. Maud Urbanski for helpful dis-
cussion during the preparation of the manuscript.
References and notes
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Glu871
H
Glu903
H
Tyr904
O
9
N
O
Leu905
O
Phe968
H
N
N
3
N
N
O
H
Arg953
22r
Figure 2. Docking study of 22r into ATP-binding site of JAK3.
(Fig. 2). The docking result indicated that 22r may
adopt a typical bidentate hydrogen-bonding mode with
the JAK3 backbone at the hinge region the lactam
nitrogen with the carbonyl oxygen of Glu903 and the
carbonyl oxygen with the amide nitrogen of Leu905.
The hydroxymethyl on the bottom ring may form a
hydrogen bond with the side chain of Arg953. The
hydroxymethyl side chain at the C-9 position may point
to the inside of a small binding pocket and form
hydrogen bonds with Glu871 and Phe968 explaining
the large contribution of the hydroxyl group to the
potency. The imidazole side chain at the C-3 position
extends into the solvent. Interestingly, the OH of the
phenol side chain of the Tyr904 residue is perpendicular
to the imidazole ring, which could potentially act as a
hydrogen bond acceptor.17 This may explain the
observed activities.
6. For recent reviews, see: (a) Thompson, J. E. Drug News
Perspect. 2005, 18, 305; (b) Sudbeck, E. A.; Uckun, F. M.
IDrugs 1999, 2, 1026; (c) Papageorgiou, A. C.; Wikman, L.
E. K. Trends Pharmacol. Sci. 2004, 25, 558; For some
recent JAK3 inhibitors, see: (d) Brown, G. R.; Bamford,
A. M.; Bowyer, J.; James, D. S.; Rankine, N.; Tang, E.;
Torr, V.; Culbert, E. J. Bioorg. Med. Chem. Lett. 2000, 10,
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Bamborough, P.; Bright, C.; Crowe, S.; Eastwood, P.;
Fenton, G.; Foster, M.; Harrison, T. K. P.; King, S.; Lai,
J.; Lawrence, C.; Letallec, J.-P.; McCarthy, C.; Moorcroft,
N.; Page, K.; Rao, S.; Redford, J.; Sadiq, S.; Smith, K.;
Souness, J. E.; Thurairatnam, S.; Vine, M.; Wyman, B.
Bioorg. Med. Chem. Lett. 2003, 13, 3105; For selective
JAKs inhibitor, see: (f) Thompson, J. E.; Cubbon, R. M.;
Cummings, R. T.; Wicker, L. S.; Frankshun, R.; Cunn-
ingham, B. R.; Cameron, P. M.; Meinke, P. T.; Liverton,
N.; Weng, Y.; DeMartino, J. A. Bioorg. Med. Chem. Lett.
2002, 12, 1219; For selective JAK3 inhibitor, see: (g)
Changelian, P. S.; Flanagan, M. E.; Ball, D. J.; Kent, C.
R.; Magnuson, K. S.; Martin, W. H.; Rizzuti, B. J.;
Sawyer, P. S.; Perry, B. D.; Brissette, W. H.; McCurdy, S.
P.; Kudlacz, E. M.; Conklyn, M. J.; Elliott, E. A.; Koslov,
E. R.; Fisher, M. B.; Strelevitz, T. J.; Yoon, K.; Whipple,
D. A.; Sun, J.; Munchhof, M. J.; Doty, J. L.; Casavant, J.
M.; Blumenkopf, T. A.; Hines, M.; Brown, M. F.; Lillie,
B. M.; Subramanyam, C.; Chang, S. P.; Milici, A. J.;
Beckius, G. E.; Moyer, J. D.; Su, C.; Woodworth, T. G.;
Gaweco, A. S.; Beals, C. R.; Littman, B. H.; Fisher, D. A.;
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S.; Kawabata, T. T.; Finco-Kent, D.; Baker, D. L.;
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In summary, a series of simplified staurosporine analogs
with different substituted groups attached at C-3 and/or
C-9 position have been synthesized. These compounds
exhibited excellent inhibitory activity against JAK3.
The SAR indicated that a small group with H-bonding
capability, such as a hydroxymethyl, at the C-9 position
is preferred to lower the IC50 to a single-digit nanomolar
range. Moreover, an allyl linker with an imidazole unit
was well tolerated at the C-3 position. Unfortunately,
22n and 22r exhibited poor solubility precluding them
from further development. However 22o had moderate
solubility at pH 2.0 (0.03 mg/mL). Introduction of a
suitable group at the C-3 position may provide a com-
pound with improved solubility.

S.-M. Yang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 326–331
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plate. Next, 5 lL of diluted compounds in DMSO and
48 lL of biotinylated peptide enzyme substrate (5 lg
diluted in TK buffer containing 10 lM ATP) were added
to each well. Control wells received equal volume of
vehicle. The contents of the wells were mixed and the
reaction mixture was incubated for 1 h at room temper-
ature. Post incubation, 90 lL of reaction mixture was
transferred into a washed Neutravidin-coated plate (Pierce
Neutravidin Biotin-binding Protein 31000; 1 mg/mL
1:100). The plate was then incubated for 15 min at room
temperature and washed three times with PBS-T. PY99
anti-phosphotyrosine antibody (Santa Cruz #sc-
7020HRP) was added into each well and the plate was
incubated for 40 min at room temperature. After incuba-
tions, the plate was washed three times and 100 lL TMB
(Sigma, St. Louis, MO) was added to each well. The plate
was incubated for another 40 min at room temperature in
the dark. The reaction was stopped by the addition of 1 M
H2SO4 (50 lL/well) and the optical density was read at
450/650 nm.
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Hudkins, R. L. Bioorg. Med. Chem. Lett. 2002, 12, 147; (c)
Kaneko, M.; Saito, Y.; Saito, H.; Matsumoto, T.; Mat-
suda, Y.; Vaught, J. L.; Dionne, C. A.; Angeles, T. S.;
Glicksman, M. A.; Neff, N. T.; Rotella, D. P.; Kauer, J.
C.; Mallamo, J. P.; Hudkins, R. L.; Murakata, C. J. Med.
Chem. 1997, 40, 1863.
10. Gingrich, D. E.; Reddy, D. R.; Iqbal, M. A.; Singh, J.;
Aimone, L. D.; Angeles, T. S.; Albom, M.; Yang, S.; Ator,
M. A.; Meyer, S. L.; Robinson, C.; Ruggeri, B. A.;
Dionne, C. A.; Vaught, J. L.; Mallamo, J. P.; Hudkins, R.
L. J. Med. Chem. 2003, 46, 5375.
11. Boggon, T. J.; Li, Y.; Manley, P. W.; Eck, M. J. Blood
2005, 106, 996, The structure of AFN941 is presented
below
14. (a) Alterman, M.; Andersson, H. O.; Garg, N.; Ahlse˘n, G.;
Lo¨vgren, S.; Classon, B.; Danielson, U. H.; Kvarnstro¨m,
I.; Vrang, L.; Unge, T.; Samuelsson, B.; Hallberg, A. J.
Med. Chem. 1999, 42, 3835; (b) Lawson, E. C.; Kinney, W.
A.; Luci, D. K.; Yabut, S. C.; Wisnoski, D.; Maryanoff, B.
E. Tetrahedron Lett. 2002, 43, 1951, For compound 22o,
the acetic acid salt of 2-(3-propenyl)amino-1-ethoxycar-
bonyl-4,5-dihydroimidazole was used as the cross-cou-
pling partner.
H
N
O
N
N
O
H
Me
15. (a) Wei, B.; Hor, T. S. A. J. Mol. Cat. A: Chem. 1998,
132, 223; (b) Helquist, P. Tetrahedron Lett. 1978, 19,
1913.
MeO
NHMe
AFN941
16. The compound 22r was docked, using the docking
program Glide (Glide, Schrodinger, 1500 S. W. First
Avenue, Suite 1180, Portland, OR 97201). The molecular
mechanism calculations were done with MacroModel
(MacroModel, Schrodinger, 1500 S. W. First Avenue,
Suite 1180, Portland, OR 97201), using OPLS2001 force
field. The effect of aqueous solution was treated by GB/SA
model. Polak–Ribiere conjugate gradient method was
used for energy minimization, and the derivative conver-
.
12. Faul, M. M.; Winneroski, L. L.; Krumrich, C. A. J. Org.
Chem. 1998, 63, 6053.
13. JAK3 enzyme was purified from Sf21 cells (San Diego,
CA) infected with a baculovirus expression vector for
JAK3 (JH1 and JH2 domain). Enzyme activity of JAK3
was determined in terms of enzyme phosphorylation by
the following method. JAK3 enzyme solution (48 lL) in
1.25· TK buffer (62.5 mM HEPES, pH 7.5, 12.5 mM
MgCl2) containing 42 mM DTT (Sigma, St. Louis, MO)
was added into the each well of a polypropylene 96-well
´
˚
gence criterion was set at 0.05 KJ/A-Mol.
17. Levitt, M.; Perutz, M. F. J. Mol. Biol. 1988, 201, 751.