Oxindole-based inhibitors of cyclin-dependent kinase 2 (CDK2): Design, synthesis, enzymatic activities, and X-ray crystallographic analysis

Article abstract of DOI:10.1021/jm010117d

Two closely related classes of oxindole-based compounds, 1H-indole-2,3-dione 3-phenylhydrazones and 3-(anilinomethylene)-1,3-dihydro-2H-indol-2-ones, were shown to potently inhibit cyclin-dependent kinase 2 (CDK2). The initial lead compound was prepared as a homologue of the 3-benzylidene-1,3-dihydro-2H-indol-2-one class of kinase inhibitor. Crystallographic analysis of the lead compound bound to CDK2 provided the basis for analogue design. A semiautomated method of ligand docking was used to select compounds for synthesis, and a number of compounds with low nanomolar inhibitory activity versus CDK2 were identified. Enzyme binding determinants for several analogues were evaluated by X-ray crystallography. Compounds in this series inhibited CDK2 with a potency ～10-fold greater than that for CDK1. Members of this class of inhibitor cause an arrest of the cell cycle and have shown potential utility in the prevention of chemotherapy-induced alopecia.

Full text of DOI:10.1021/jm010117d

J . Med. Chem. 2001, 44, 4339-4358
4339
Oxin d ole-Ba sed In h ibitor s of Cyclin -Dep en d en t Kin a se 2 (CDK2): Design ,
Syn th esis, En zym a tic Activities, a n d X-r a y Cr ysta llogr a p h ic An a lysis
H. Neal Bramson, J ohn Corona, Stephen T. Davis, Scott H. Dickerson, Mark Edelstein, Stephen V. Frye,
Robert T. Gampe, J r., Phil A. Harris, Anne Hassell, William D. Holmes, Robert N. Hunter, Karen E. Lackey,
Brett Lovejoy,§ Michael J . Luzzio,# Val Montana, Warren J . Rocque, David Rusnak, Lisa Shewchuk,
J ames M. Veal, Duncan H. Walker,† and Lee F. Kuyper*
GlaxoSmithKline Inc., Five Moore Drive, Research Triangle Park, North Carolina 27709
Received March 15, 2001
Two closely related classes of oxindole-based compounds, 1H-indole-2,3-dione 3-phenylhydra-
zones and 3-(anilinomethylene)-1,3-dihydro-2H-indol-2-ones, were shown to potently inhibit
cyclin-dependent kinase 2 (CDK2). The initial lead compound was prepared as a homologue of
the 3-benzylidene-1,3-dihydro-2H-indol-2-one class of kinase inhibitor. Crystallographic analysis
of the lead compound bound to CDK2 provided the basis for analogue design. A semiautomated
method of ligand docking was used to select compounds for synthesis, and a number of
compounds with low nanomolar inhibitory activity versus CDK2 were identified. Enzyme
binding determinants for several analogues were evaluated by X-ray crystallography. Com-
pounds in this series inhibited CDK2 with a potency ∼10-fold greater than that for CDK1.
Members of this class of inhibitor cause an arrest of the cell cycle and have shown potential
utility in the prevention of chemotherapy-induced alopecia.
In tr od u ction
synthesis; and cyclin B controls the activity of CDK1
during mitosis. Other mechanisms for CDK regulation,
such as phosphorylation-dephosphorylation and endog-
enous protein inhibitors, also play important roles in
cell cycle control.5,6
The cyclin-dependent kinases (CDKs) are key regula-
tors of the cell cycle,1 the complex process by which cells
divide.2 The cell division cycle is commonly viewed as
an orderly progression through four distinct phases: (1)
G1 (gap 1), the phase in which the cell prepares for DNA
synthesis, (2) S (synthesis), the stage in which DNA is
replicated, (3) G2 (gap 2), the phase in which the cell
prepares for mitosis, and (4) M (mitosis), the stage that
leads to chromosome segregation and daughter cell
formation. CDKs provide much of the control that is
required for the cell to move through these phases in a
coordinated manner.
As their name implies, the activity of CDKs is
dependent upon a regulatory subunit called a cyclin.
Members of the cyclin family bind to and activate their
CDK partners.3 For example, cyclins A and B activate
CDK1, cyclins A and E regulate the activity of CDK2,
and the D-type cyclins are associated with CDK4. While
the concentration of the CDKs remains relatively con-
stant throughout the cell cycle, cyclin expression and
degradation occur in a periodic fashion.4 The rise and
fall of cyclin concentrations are timed to provide specific
CDK activities as they are needed for progression
through the various stages of the cell cycle. Thus, cyclin
D is expressed in G1, leading to the phosphorylation of
the retinoblastoma protein by CDK4; CDK2 is activated
by cyclin E in late G1 and by cyclin A during DNA
The recognition of the importance of CDKs to the
process of cell division has stimulated an interest in
them as potential targets for proliferative diseases such
as cancer, psoriasis, and restenosis,7-9 and for the
prevention of chemotherapy-associated side effects such
as alopecia.10,11 A number of small-molecule inhibitors
of CDKs have been identified12-17 and are described in
recent reviews.18-21 Flavopiridol is the first CDK inhibi-
tor to progress into clinical trials and is being evaluated
as an anticancer agent.22,23 In addition, peptide inhibi-
tors of CDK2 were recently shown to preferentially
induce transformed cells to undergo programmed cell
death (apoptosis) relative to untransformed cells.24
Here we describe the discovery of a new class of CDK
inhibitor, represented by structure 1, that stops cell
* To whom correspondence should be addressed. Phone: (919) 483-
2114; Fax: (919) 483-3411; e-mail: lfk30687@gsk.com.
§ Present address: Brett Lovejoy, Pennie & Edmonds LLP, 3300
Hillview Avenue, Palo Alto, CA 94304.
cycle progression by inhibiting CDK2 activity in cells
and has shown potential utility in the prevention of
chemotherapy-induced alopecia.11 The initial lead iden-
tification, structure-based analogue design, compound
synthesis, kinase inhibition data, and X-ray crystal-
#
Present address: M. J . Luzzio, Pfizer Inc., Pfizer Global Research
and Development, Groton, CT 06340.
† Present address: Duncan H. Walker, Hoffman-La Roche Inc., 340
Kingsland Street, Nutley, NJ 07110-1199.
10.1021/jm010117d CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/02/2001

4340 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Bramson et al.
Sch em e 1a
Sch em e 2a
a
Conditions: (i) Pd(OAc)2, Et3N, P(o-Tol)3, CH3CN; (ii) EtOH,
cat HCl(aq); (iii) H2 Pd/C, MeOH/THF.
sis.28 This latter procedure involved sequential treat-
ment of the aniline with tert-butyl hypochlorite, ethyl
(methylthio)acetate, triethylamine, and hydrochloric
acid to give the 3-methylthiooxindole 11, which was
desulfurized with zinc or Raney nickel to give the
corresponding oxindole 12. A modification of the Gas-
sman procedure29 involving initial mixing of ethyl
(methylthio)acetate and sulfuryl chloride, followed by
addition of the aniline with one equivalent of Proton
Sponge and finally triethylamine treatment, was em-
ployed to prepare compounds that were not accessible
via the original Gassman procedure, e.g., the oxindole
ester (R ) 5-CO2Me). Conversion of the oxindole to the
dimethylaminomethinyloxindole or ethoxymethinylox-
indole 13 (X ) H) was achieved with dimethylforma-
mide-N,N-di-tert-butyl acetal or diethoxymethyl acetate,
respectively, and subsequent condensation with an
aniline yielded the enamine 14 (X ) H). The corre-
sponding methyl-substituted enamines 14 (X ) Me)
were obtained using dimethylacetamide-N,N-dimethyl
acetal following the same procedure.
a
Conditions: (i) chloral hydrate, NH2OH HCl, Na2SO4, EtOH/
HCl(aq); (ii) H2SO4(conc) or BF3OEt2; (iii) (tBuOCO)2O, THF; (iv) 2.5
equiv t-BuLi, THF; (v) EtO2CCO2Et, THF; (vi) HCl(aq); (vii) 1 equiv
HCl, EtOH.
lographic structures of CDK2/inhibitor complexes are
reported. Compounds with low nanomolar IC50 values
for CDK2 were identified, and CDK2 binding determi-
nants were evaluated through crystallographic analyses.
Ch em istr y
Isa tin s a n d Isa tin Hyd r a zon es. Substituted isatins
4 typically were prepared via the classical Sandmeyer
reaction25 involving conversion of the substituted aniline
with hydroxylamine and chloral hydrate to the inter-
mediate isonitrosoacetanilide 2, followed by cyclization
in either concentrated sulfuric acid or boron trifluoride
etherate as shown in Scheme 1. Alternatively, for some
meta-substituted anilines (R ) iPr, Ph, OEt) where this
methodolgy failed, the isatin synthesis of Hewawasam
and Meanwell26 was followed. In that procedure, the
aniline was protected as the tert-butylcarbonate (3).
Ortho-lithiation of intermediate 3 and treatment with
diethyl oxalate gave the R-ketoester, which upon expo-
sure to aqueous acid underwent cyclization to the isatin.
Subsequent condensation of the substituted isatin 4
with an arylhydrazine in ethanol, either using the
hydrazine salt or one equivalence of hydrochloric acid,
yielded the isatin hydrazone 1.
A number of 4-alkylisatin hydrazones 7 were prepared
as shown in Scheme 2. Heck coupling of 4-iodoisatin
with alkenes produced 4-alkenylisatins 5, which were
converted to the corresponding hydrazones 6. Subse-
quent reduction furnished the 4-alkylisatin hydrazones
7.
A series of 5-carboxamides 10 were prepared from the
pentafluorophenyl ester 9, which was derived from the
corresponding carboxylic acid 8 by treatment with
pentafluorophenyl trifuoroacteamide (Scheme 3).
Oxin d oles. Oxindoles 12 were obtained, as delin-
eated in Scheme 4, either directly from the correspond-
ing isatin 4 by Wolf Kishner reduction27 or alternatively
from the substituted aniline via the Gassman synthe-
Ster eoch em istr y. As viewed by their 1H NMR
spectra, the isatin hydrazones and corresponding enam-
ines exist predominately as the Z (cis) conformation in
solution, presumably due to the intramolecular hydro-
gen bonding between the NH of the hydrazone/enamine
linkage and the carbonyl group of the indolinone.
Confirmation of this stereochemistry was achieved for
the enamine by observation of a nuclear Overhauser
effect between the 4-position proton of the oxindole ring
system and the vinyl hydrogen of the enamine linkage
i
for the 5-carboxylic ester (R ) 5-CO2 Bu, compound 55,
Table 1). In some analogues where a heteroatom (O or
N) containing functionality was substituted at the
4-position of the oxindole, allowing formation of an
alternative hydrogen bond with the linker NH in the E
1
(trans) conformation, H NMR spectra of equilibrated
samples corresponded to mixtures of Z and E isomers.
For example, the 4-phenoxy derivative 32 was observed
as a 5:2 Z:E mixture after 10 days of equilibration, and
the 4-carboxamido analogue 40 was found as a 1:7 Z:E
mixture. Occasionally, compounds in the series precipi-
tated from the reaction solution as a mixture of Z and

Oxindole-Based Inhibitors of CDK2
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4341
Sch em e 3a
a
Conditions: (i) CF3CO2C6F5; (ii) RR′NH, pyridine, CH3CN.
Sch em e 4a
E isomers, as indicated by NMR. However, in the
absence of a hydrogen bond acceptor at position-4,
equilibration led to disappearance of signals for the E
isomer.
Resu lts a n d Discu ssion
Lea d Discover y. As part of an ongoing effort to
identify selective inhibitors of protein kinases, ana-
logues of the 3-(benzylidene)indolin-2-ones 15 were
examined. The indolin-2-ones 15 are known inhibitors
of receptor tyrosine kinases, such as epidermal growth
factor receptor and Her-2,30 and a closely related
compound shows selective inhibition of vascular endot-
helial growth factor receptor 2 kinase.31 Compounds
16-19, available in one synthetic step from commercial
materials, were prepared as homologues of compounds
15 and assayed for inhibition of various kinases. Com-
pounds 17-19 were inactive (IC50 >10 µM) against all
kinases tested, but compound 16 selectively inhibited
CDK2 (IC50 ) 60 nM) and served as the lead compound
for the work described here.
a
Conditions: (i) t-BuOCl, THF; (ii) MeSCO2Et; (iii) Et3N; (iv)
HCl(aq); (v) MeSCO2Et; (vi) SO2Cl2; (vii) Proton Sponge; (viii) Et3N;
(ix) Zn, HOAc or RaNi; (x) NH2NH2, EtOH; (xi) NaOEt, EtOH;
(xii) Me2NCH(Ot-Bu)2, DMF or MeCO2CH(OEt)2, AcOH; (xiii)
Me2NC(Me)(Ot-Bu)2; (xiv) 1 equiv HCl, EtOH.
discussed below, the amino group of Lys-33 was con-
sidered to be a potential interaction site for inhibitors.
Thus, structures of inhibitor complexes with inactive
CDK2 should be viewed with some caution, especially
regarding inhibitor-protein interactions involving the
Lys-33 region of the enzyme.
Inhibitor 16 was found to occupy the ATP binding site
of CDK2 as shown in Figure 1. The oxindole ring system
of compound 16 interacted with CDK2 in a manner
analogous to that observed for compounds in inhibitor
class 15 bound to fibroblast growth factor receptor
kinase.39 Two hydrogen bonds were formed between the
lactam moiety of compound 16 and CDK2. Specifically,
the amide NH was H-bonded to the backbone carbonyl
of Glu-81 and the amide carbonyl oxygen was H-bonded
with the backbone NH of Leu-83. Those interactions
were analogous to hydrogen bonds between ATP and
CDK2.
Inspection of the CDK2/compound 16 structure fur-
nished a number of guidelines for analogue design. The
7-position of the oxindole ring system was positioned
close to the side chain of Phe-80 and appeared to be too
sterically crowded to permit substitution. The 6-position
projected toward a small cavity at the back of the
binding cleft and into the region affected by cyclin A
association. This cavity is smaller in size, relative to
An a logu e Design . A structure-based selection pro-
cess was used to explore analogues in this series of
compounds. CDK232 and its complexes with ATP,32-34
cyclin A,35,36 the protein inhibitor p27,37 and small
molecule inhibitors12,20,38 have been studied by X-ray
crystallography.5 To evaluate and select potential ana-
logues for synthesis, we utilized the crystal structure
of compound 16 bound to the inactive form of CDK2
(Figure 1). This monomeric form of the kinase offers
crystallographic advantages over the activated cyclin A
complex, including higher resolution, a smaller struc-
ture, and one molecule in the asymmetric unit versus
two for the cyclin A complex. However, the activation
of CDK2 by complexing with cyclin A induces confor-
mational changes in the protein that affect the ATP
binding site to some degree. The most significant effect
involves a rotation of the C-helix, which alters the
active-site geometry in the region of the triad of catalytic
active-site residues Lys-33, Glu-51, and Asp-145. As

4342 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Ta ble 1. CDK1 and CDK2 Inhibitory Activities of Compounds 16-81
Bramson et al.
kinase IC50 (nM)
cmpd no.
R4
R5
R6
R7
H
X
CDK1
CDK2
Lead
16
H
Br
H
N
N
780
60
Linker
17
18
19
20
21
22
23
24
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1300
3000
2300
300
220
10
120
690
360
43
22
2.3
CH
CCH3
N
CCH3
N
CH
Cl
5-oxazolyl
5-oxazolyl
5-oxazolyl
17.
7.1
2.5
2.0
CCH3
4-Substituents
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
I
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
N
N
N
N
N
N
N
N
N
N
N
N
CH
CH
N
110
46
37
19
15
4.6
7.9
2.5
1.2
1.5
93
3.4
13
21
9.3
-CH2CH3
-CH(CH3)2
-CH2CH(CH3)2
-CHdC(CH3)2
-OCH2CH3
-OCH(CH3)2
-OPh
-(CH2)2-(4-pyridyl)
-CHdCH-(4-phenol)
-(CH2)2-(4-phenol)
3-pyrazolyl
-CO2CH2CH3
-CH2OH
550
41
290
290
170
150
250
130
1700
>1000
>1000
12
19
8.9
54
2400
>1000
-NO2
-CONH2
N
5-Substituents
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
I
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
N
N
N
N
N
N
N
CH
N
290
95
330
77
210
710
1400
2800
350
170
130
150
12
19
16
2.8
130
69
230
74
51
60
9.3
34
11
46
10
12
15
74
310
16
43
15
28
-CH3
-OH
-OCH3
-NO2
-NH2
-N(CH3)2
-SO2CH3
-SO2NH2
-SO3H
-CO2H
-CO2CH3
-CO2CH2CH(CH3)2
-COCH2CH(CH3)2
-CONH2
N
N
CH
CH
CH
CH
N
N
N
N
N
2.1
3.0
1.9
4.5
17
12
25
-CON(CH3)2
-CONH(CH2)2-(1H-imidazol-4-yl)
-CONH(CH2)3-(1H-imidazol-1-yl)
-CONHCH2-(4-pyridyl)
-CONHCH2-(3-pyridyl)
-CONHCH2C(CH3)2CH2OH
-CONHCH2-(2,6-dimethoxyphenyl)
8.9
N
N
N
2.1
6.8
1.7
6-Substituents
64
65
66
67
68
69
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
H
H
H
H
H
N
N
N
N
CH
N
520
660
790
43
21
75
-CH2CH3
-CH(CH3)2
-C(CH3)3
-CH2OH
-OPh
>10 000
>10 000
740
61
>10 000
>10 000

Oxindole-Based Inhibitors of CDK2
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4343
Ta ble 1 (Continued)
kinase IC50 (nM)
cmpd no.
R4
R5
R6
R7
X
CDK1
CDK2
7-Substituents
70
71
72
H
H
H
H
-CH3
Cl
H
H
H
-CH3
-CH3
-CH3
CH
CH
CH
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
4,5-Substituents
73
74
75
76
77
78
79
80
81
82
Cl
Cl
-CH3
-CH3
-OCH3
-NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
N
N
N
N
N
N
N
CH
N
250
1700
87
120
83
150
43
29
13
54
4.6
13
2.2
9.5
7.1
2.8
1.6
1.5
-CHdN-NH-
-C(Cl)dN-NH-
-NdN-NH-
-S-CHdN-
-S-CHdN-
-CHdCH-CHdN-
-CHdCH-CHdN-
12
15
CH
F igu r e 1. Crystal structure of compound 16 bound to CDK2. (A) Surface representation of CDK2 illustrating the binding site
cavity. The oxindole portion of compound 16 was bound deep inside the pocket, and the sulfonamide functionality was positioned
at the opening to the cleft. Inhibitor atoms are colored as follows: C, white; N, blue; O, red; S, yellow; Br, purple. (B) The binding
cavity is illustrated with a dot surface. Hydrogen bonds between compound 16 and protein are shown as yellow lines. Atom
coloring is as in A except the inhibitor carbon atoms are green and hydrogen atoms are light blue.
many other protein kinases, due to the bulkiness of the
Phe-80 side chain. That part of the cleft appeared to be
compatible with small substituents. The juxtaposition
of the 5-position and Lys-33 suggested hydrogen-bond
acceptors at that location on the oxindole might enhance
inhibitor affinity. Lipophilic substituents at position-4
appeared to be appropriate for the relatively hydropho-
bic environment of that region of the protein (Val-18
and Leu-134). In addition to these observations regard-
ing potential oxindole substitution, the structure also
suggested that the sulfonamide group positioned at the
cleft opening might provide a site for substitution that
could be used to alter compound properties such as
solubility and pharmacokinetics, without negatively
affecting enzyme affinity and possibly increasing affinity
and selectivity.
employed generally utilized anilines as starting materi-
als (see Chemistry section). A set of 410 anilines,
considered compatible with synthesis conditions, was
selected from commercially available anilines using a
molecular weight cutoff of 250. Each of the selected
aniline structures was then electronically converted to
the corresponding analogue of compound 16, and the
hypothetical analogue was docked into the ATP binding
site of the CDK2/cyclin A structure. The details of this
docking procedure are provided in the experimental
section. Synthetic targets were selected using a combi-
nation of molecular mechanics energy scores and careful
visual inspection of each docked structure. The initial
set of prepared compounds resulting from this protocol,
e.g., compounds 22, 27, 31, 49, 50, 56, 79, and 81 (Table
1), were potent inhibitors of CDK2 and provided the
basis for subsequent synthetic efforts. The use of model-
ing for designing substituents on the pendant hydrazone
or enamine portion of the inhibitors was not pursued
extensively because of the complexities associated with
evaluating the effects of solvent at the protein-solvent
interface.
Molecular modeling was used to evaluate and select
potential substituents at the 4-7 positions of the
oxindole template. A semiautomated computational
procedure was developed to generate and dock virtual
analogues in this class of inhibitor to CDK2. The
synthetic routes to the oxindole ring system that we

4344 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Bramson et al.
were potent inhibitors of CDK2 with IC50 values in the
range of 1-8 nM.
The 4-position projected toward the opening of the
binding site, and large hydrophobic substituents were
accommodated, as illustrated by compounds 33-35. In
contrast, hydrophilic groups at the 4-position generally
were detrimental to binding. For example, the 4-nitro
and 4-amido derivatives 39 and 40 showed relatively
weak inhibition of CDK2.
5-Su bstitu en ts. Many of the compounds containing
substituents at the 5-position were designed to interact
with CDK2 via hydrogen bonding to the side chain of
Lys-33 and/or the backbone NH of Asp-145. Carboxylic
esters were particularly active inhibitors (Table 1), an
observation consistent with those substituents being
capable of forming a hydrogen bond with Lys-33 and
positioning a hydrophobic group in the lipophilic region
near position 4. The 5-ozazolyl-substituted derivatives
22-24 also fit that paradigm and were potent inhibitors
of the enzyme. In addition, the amide group was found
to be a favorable 5-substituent and provided a conve-
nient handle for introducing water-solubilizing substit-
uents (compounds 56-62). Modeling suggested a par-
ticularly favorable steric fit for the dimethoxyphenyl
group of amide 63, consistent with its effective inhibition
of CDK2. Charged substituents such as sulfonic and
carboxylic acids provided no advantage over similar
neutral substituents. For example, the carboxylic acid
52 was 10-fold weaker than the corresponding methyl
ester 53 as an inhibitor of CDK2. Fluorine, iodine, and
methyl substituents at the 5-position were also compat-
ible with kinase binding. However, the dimethylamino-
containing compound 48 was a relatively poor inhibitor.
6-Su bstitu en ts. The inhibition activities of 6-substi-
tuted analogues (Table 1) were in accord with the
relatively small CDK2 pocket adjacent to that position
in the enzyme structure. Smaller substituents such as
ethyl (65), isopropyl (66), and hydroxymethyl (68)
moderately contribute to affinity as compared to the
corresponding 6-hydrogen derivatives 17 and 18. In
contrast, no measurable inhibition was observed for
compounds containing larger substituents such as tert-
butyl (67) and phenoxy (69).
7-Su bstitu en ts. In the crystal structure of compound
16 in complex with CDK2 (Figure 1), the 7-position of
the inhibitor was oriented toward the side chain meth-
ylene group of Phe-80 with a C-7 to Câ distance of 3.3
Å. Therefore, substituents at position-7 would be ex-
pected to encounter unfavorable steric interactions with
the enzyme. The lack of activity found for 7-methyl
derivatives (see Table 1) was consistent with that
expectation. For example, comparison of the CDK2 IC50
values of the 5,7-dimethyl derivative 71 and the 5-me-
thyl analogue 43 (see Table 1) indicated that 7-methyl
substitution produced a >200-fold decrease in affinity.
4,5-Disu bstitu en ts. Disubstitution at the 4- and
5-positions can provide highly potent inhibitors of
CDK2. As shown in Table 1, compounds with 4,5-fused
heterocycles that contain a hydrogen bond acceptor at
the 5-position and hydrophobic character at the 4-posi-
tion, such as compounds 80 and 81, display high affinity
for CDK2. The structure of compound 91 bound to the
CDK2/cyclin A complex illustrates the potential for
hydrogen bonds to the side chain of Lys-33 and the
F igu r e 2. A comparison of the two complexes in the asym-
metric unit of the crystal structure of compound 91 bound to
CDK2/cyclin (A) Hydrogen bonds between inhibitor and pro-
tein are shown as purple lines. Atom coloring: H, white;
protein C, green; inhibitor (complex 1) C, orange; inhibitor
(complex 2) C, green; N, blue; O, red; S, yellow.
Results from compounds prepared in this study are
discussed below and organized by regions of the core
inhibitor template.
Hyd r a zon e/En a m in e Lin k er . The hydrazone link-
age between the inhibitor’s indolinone ring and phenyl
group imposed a number of synthetic restrictions on the
accessibility of potential inhibitors, limiting the diversity
of phenyl substituents that could be explored. Replace-
ment of the hydrazone connection with an enamine
would provide significantly expanded access to substit-
uents on both the indolinone and phenyl rings through
well-established synthetic routes. This synthetic flex-
ibility permitted the preparation of a diverse set of
inhibitors that were used to develop an understanding
of CDK2 inhibition and selectivity. Preparation and
evaluation of hydrazone-enamine pairs of compounds
demonstrated that the enamine substitution for hydra-
zone was essentially inconsequential to enzyme binding
(compare compounds 17 and 18, 22 and 23, 79 and 80,
and 81 and 82, Table 1). Molecular modeling evaluation
was concordant with that observation.
Also consistent with structural and modeling consid-
erations was the observation that the carbon atom of
the enamine linkage could be substituted with groups
larger than hydrogen, such as methyl, without nega-
tively affecting inhibition of CDK2 (compare CDK2 IC50
values for compounds 18 versus 19, 20 versus 21, and
23 versus 24). In recent patent literature, a group from
Boerhringer Ingelheim describe related CDK inhibitors
containing an aryl-substituted enamine linkage.40
4-Su bstitu en ts. As pointed out above, binding of
inhibitor 16 to CDK2 placed the 4-position in a hydro-
phobic region of the enzyme active site. Thus, hydro-
phobic substituents were expected to enhance affinity
for CDK2, while hydrophilic groups would be detrimen-
tal. These expectations were realized as evidenced by
the inhibitory activities of compounds 25-40 (Table 1).
Compounds with lipophilic substituents such as ethyl,
isopropyl, isobutyl, and isobutenyl (compounds 26-29)

Oxindole-Based Inhibitors of CDK2
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4345
Ta ble 2. CDK1 and CDK2 Inhibitory Activities for Compounds 83-109
kinase IC50 (nM)
cmpd no.
R4
R5
-S-CHdN-
Y
Z
X
CDK1
CDK2
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
-SO2NHCH3
-SO2N(CH3)2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
64
50
41
38
12
72
45
41
100
64
5.6
4.6
4.7
3.6
0.54
4.5
1.0
4.3
9.7
5.6
281
5.6
9.5
3.3
75
560
1000
1000
660
5.7
7.5
26
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-SO2NH(CH2)2OH
-SO2NH(CH2)2-imidazol-5-yl
-SO2NH(CH2)2O(CH2)2OH
-SO2NH[(CH2)2O]4CH3
-SO2N(CH3)[(CH2)2O]4CH3
-SO2NH-phenyl
-SO2NH-pyrid-2-yl
-SO2N(CH3)-pyrid-2-yl
-SO2NH-2,6-dimethylphenyl
-SO2NHCH2-phenyl
-SO2NH-isoquinol-2-yl
-SO2NHC(NH2)dNH
-SO2NHCOCH3
14 000
52
42
45
910
2900
5500
3800
2500
39
100
170
56
H
H
H
H
H
H
H
H
-SO2NHCH3
-SO2NH(CH2)2N(CH3)2
-SO2NH-thiazol-2-yl
-SO2NHC(NH2)dNH
-SO2CH3
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-S-CHdN-
-CH2SO2NH2
-CH2SO2NHCH3
-CH2SO2NHCH3
-CH2SO2NH(CH2)2OH
-CH2SO2CH3
5.7
13
50
14
8.9
CH
CH
CH
CH
200
1200
31
-CH2SO2CH2-
-CH2SO2CH2-
H
oxazol-5-yl
19
backbone NH of Asp-145, although the inhibitor was
found to bind somewhat differently in the two complexes
within the asymmetric unit. The two complexes are
compared in Figure 2. In complex 1, the thiazole
nitrogen atom of compound 91 was 3.1 Å from the amino
nitrogen of Lys-33 and 3.5 Å from the backbone nitrogen
of Asp-145. The corresponding distances in complex 2
were 3.3 and 3.8 Å, respectively. Complex 1 appeared
to be the more relevant of the two complexes on the
basis of other CDK2/cyclin A/inhibitor crystal structures
that often showed no electron density for the inhibitor
in complex 2.
N-Su bstitu ted Su lfon a m id es. As shown in Figure
1, the sulfonamide moiety of compound 16 interacts with
Asp-86 at the opening to the binding cleft of CDK2. The
sulfonamide moiety forms hydrogen bonds with the
backbone NH and the side chain carboxylate functional-
ity of Asp-86. The position of the sulfonamide at the
solvent interface suggested that sulfonamide substitu-
ents might have little interaction with the enzyme and
would project into solution. This orientation at the
mouth of the binding cleft suggested that sulfonamide
substituents might be used to modify properties such
as solubility and pharmacokinetic parameters without
detrimentally affecting enzyme affinity.
sulfonamides. The poor activity of this compound pre-
sumably was related to the expected steric constraints
imposed by the ortho-methyl groups of the phenyl
moiety.
Our initial assumption was that both hydrogen bonds
between the sulfonamide and protein were important
to affinity. However, the inhibitory activities of disub-
stituted sulfonamide derivatives and crystal structures
of several monosubstituted sulfonamide analogues bound
to CDK2 indicated that the hydrogen bond between
sulfonamide and backbone NH played the more impor-
tant role in binding. As shown in Table 2, similar
inhibition activities were observed for -SO2N(CH3)R
and -SO2N(H)R pairs of compounds: 83 versus 84, 88
versus 89, and 91 versus 92. These data suggest that
the sulfonamide NH-carboxylate (Asp-86) hydrogen
bond observed in the crystal structure of the CDK2
complex with compound 16 was not significantly con-
tributing to the protein/ligand affinity. This conclusion
was supported by additional crystallographic data. For
example, the N-methylsulfonamide derivative 83, which
is equipotent to the corresponding unsubstituted sul-
fonamide 80, was found to bind to CDK2 with a
sulfonamide oxygen H-bonded to the backbone NH of
Asp-86 but with its N-methyl group, not its NH, oriented
toward the carboxylate of Asp-86 (see Figure 3). The
amino(imino)methyl substituted sulfonamide analogue
101 was found to bind to CDK2 with its sulfonamide
NH positioned away from Asp-86 (see Figure 3). The
heterocyclic-substituted sulfonamide derivatives 92 and
The inhibition data shown in Table 2 illustrates the
relative insensitivity of CDK2 to substituents on the
sulfonamide group. One exception was the 2,6-dimeth-
ylphenyl analogue 93, which was significantly weaker
as an inhibitor of CDK2 than the other substituted

4346 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Bramson et al.
F igu r e 3. Crystal structures of CDK2 bound to compounds (A) 98, (B) 101, (C), 91, and (D) 100, illustrating the interaction
between inhibitor and Asp-86. Hydrogen bonds are indicated as purple lines. Atom coloring: H, white; C, green; N, blue; O, red;
S, yellow.
F igu r e 4. Crystal structure comparison of compounds 105
F igu r e 5. Crystal structure of compound 109 bound to CDK2.
and 16 bound to CDK2. Coloring as in Figure 3, except carbon
atoms of compound 16 are orange.
Coloring as in Figure 3.
100 both bound to the kinase with the sulfonamide NH
oriented toward the carboxylate of Asp-86 but at an
N-to-O distance of 5 and 4 Å, respectively. In each of
the above structures, the sulfonamide oxygen was
H-bonded to the backbone NH of Asp-86, suggesting an
importance to that interaction. As will be discussed
below, the latter H-bond was a consistent feature in two
other related sulfone-containing compounds.
Su lfon a m id e R ep la cem en t s. In concert with the
conclusion that the sulfonamide NH was not important
to binding, the methyl sulfone analogue 102 was found
to strongly inhibit CDK2, as shown in Table 2. Interest-
ingly, compounds in which the sulfonamide was linked
to the phenyl ring by a methylene group were also
significantly active, as exemplified by compounds 103
and 105. An X-ray crystal structure of compound 105
bound to CDK2 showed that the inhibitor was able to
adopt a conformation that provided interactions between
the sulfonamide and Asp-86 similar to those observed
for compound 16, as illustrated in Figure 4.
coefficient relating pIC50 values for CDK2 versus CDK1
was 0.90. This observation is in accord with the rela-
tively high overall sequence similarity (69% identity)
between the two kinases and the essentially identical
composition of the ATP binding sites (residues 79, 84,
and 85 differ but their side chains project away from
the ATP site). The compounds displayed a range of
activity against a panel of other protein kinases but
were generally selective for CDK2. A typical example
is compound 91, which showed the following inhibition
activities [kinase, IC50 (nM)]: CDK4, 130; c-fms, 950;
erbB-2, >10000; erk-2, 21000; gsk-3, 56; lck, 980; mek,
25000; p38, 32000; src, 440; raf, 7600; tie-2, 3500;
VEGFR-2, 22.
Cellu la r Activities. The most potent and CDK2-
selective compounds were evaluated in two cellular
assays: an anti-proliferation assay and a mechanistic
assay that measures progression from G1- to S-phase
(example data shown in Table 3). The compounds
inhibited proliferation of tumor cells, with IC50 values
consistent with CDK2 inhibitors that are competitive
with respect to ATP. The anti-proliferative activity of
the compounds was up to 8-fold selective for the tumor
cells relative to the HFF normal cell line. The com-
pounds were also active in blocking progression of cells
into S-phase, consistent with the role of CDK2 activity
in the regulation of G1/S phase progression. The anti-
proliferative activity of compound 91 has been shown
A crystal structure of the fused cyclic sulfone 109
bound to CDK2 showed a hydrogen bond from the
sulfone to the backbone NH of Asp-86 (see Figure 5),
similar to that observed in the other CDK2 complexes
described above.
Kin a se Selectivity. The compounds in the series
described here were generally about 10-fold more potent
as inhibitors of CDK2 than of CDK1. The correlation

Oxindole-Based Inhibitors of CDK2
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4347
Ta ble 3. Cell-Based Data for a Selected Set of Compounds
(IC50, µM)
was stirred as the product precipitated from solution. The
precipitate was collected by suction filtration, washed thor-
oughly with water, filtered, and air-dried to provide 6.9 g (94%)
of N-benzothiazol-6-yl-2-hydroxyimino-acetamide, 1H NMR
(DMSO-d6): δ 12.2 (s, 1H), 10.4 (s, 1H), 9.2 (s, 1H), 8.5 (s, 1H),
7.9 (d, 1H), 7.7 (m, 1H), 7.7 (s, 1H); APCI m/z 220 (M-H)-. To
a 1-L three-neck round-bottom flask was placed a magnetic
stir bar and 100 mL of concentrated sulfuric acid. The flask
was fitted with a thermometer to monitor the temperature of
the reaction. The sulfuric acid was heated to 100 °C, and 10.0
g (45.2 mmol) of N-benzothiazol-6-yl-2-hydroxyimino-aceta-
mide was added slowly. The solution was heated for ∼1 h, and
the reaction mixture was poured into 750 g of ice and water.
The residual reaction mixture in the reaction vessel was
washed out with an additional 20 mL of cold water. The
aqueous slurry was stirred for about 1 h and filtered. The solid
was washed thoroughly with water, filtered, and air-dried to
yield 4.3 g (46%) of 6H-[1,3]thiazolo[5,4-e]indole-7,8-dione: 1H
NMR (DMSO-d6): δ 11.1 (s, 1H), 9.2 (s, 1H), 8.2 (d, 1H), 7.0
(d, 1H); APCI m/z 203 (M-H)-.
cmpd no. G1/S HFF HT29 MDAMB468 RKO SW620
27
54
63
81
83
88
90
91
94
1.8
2.3
1.1
0.29
0.65
5.4
3.3
2.6
7.8
8.3
1.5
5.9 14.
4.4
2.0
0.17
0.81
1.8
2.4
1.4
6.8
2.2
4.5
6.0
1.4
0.89
1.3
0.39
0.59
1.5
0.91
1.7
4.8
1.4
0.79
0.87
2.1
2.9
1.1
10.
3.6
1.0
1.9
3.2
5.0
4.3
2.0
0.66
3.1
3.0
4.4
2.2
5.1
1.8
2.7
6.2
13.
2.5
5.9
7.9
0.65
0.95
1.1
2.3
2.8
4.7
102
108
to arrest cells and protect normal cells from chemo-
therapy-induced toxicity.11 These data were consistent
with an anti-proliferative mechanism expected for in-
hibition of CDK2 and suggested that this series of
inhibitors has therapeutic potential for treating prolif-
erative disorders, including chemotherapy-induced alope-
cia.
P r oced u r e B. A second method for 1H-indol-2,3-dione
(isatin) synthesis42 is exemplified by the preparation of 6-phe-
noxy-1H-indole-2,3-dione. To a stirred solution of 1.0 g (6.0
mmol) of chloral hydrate in 25 mL of water was added 7.0 g
(22 mmol) of sodium sulfate decahydrate, followed by a solution
of 1.18 g (17.0 mmol) of hydroxylamine hydrochloride in 10
mL of water. A solution of 1.0 g (5.4 mmol) of 3-phenoxyaniline
in 10 mL of 1.0 N HCl was then added with stirring. The
resulting suspension was warmed, and 40 mL of 95% EtOH
was added to dissolve the suspension. The solution was
refluxed for 0.75 h and then cooled to ambient temperature.
The resulting solid was collected by vacuum filtration and air-
dried to afford 0.95 g (67%) of 2-hydroxyimino-N-(3-phen-
oxyphenyl)acetamide as a solid: 1H NMR (DMSO-d6): δ 6.42
(d, J ) 8.4 Hz, 1H), 7.06 (d, J ) 7.9 Hz, 2H), 7.18 (t, J ) 7.3
Hz, 1H), 7.25-7.50 (m, 5H), 7.64 (s, 1H), 10.29 (s, 1H), 12.21
(s, 1H); APCI: m/z 255 (M-H)-. A suspension of 0.15 g (0.58
mmol) of 2-hydroxyimino-N-(3-phenoxyphenyl)acetamide in 0.4
mL of BF3 etherate was heated to 85 °C for 0.75 h. The mixture
was cooled to room temperature, and 10 g of crushed ice was
added. The resulting solid was collected by vacuum filtration
and subjected to flash chromatography on silica gel (hexane/
EtOAc 1.5:1) to afford 6-phenoxy-1H-indole-2,3-dione as a solid
(0.018 g, 13%): 1H NMR (DMSO-d6): δ 6.44 (d, J ) 2.0 Hz,
1H), 6.56 (dd, J ) 2.0, 8.4 Hz, 1H), 7.08 (d, J ) 8.2 Hz, 1H),
7.22-7.29 (m, 1H), 7.38-7.46 (m, 2H), 7.52 (d, J ) 8.4 Hz,
1H), 9.05 (s, 1H); APCI: m/z 255 (M+Na)+.
Con clu sion s
The compounds described here represent a novel class
of CDK2 inhibitor. Crystallographic analysis showed
that the initial lead compound was bound in the ATP
site of the kinase, and structure-assisted methods
provided an effective means of developing a large set of
compounds with low nanomolar CDK2 IC50 values.
Binding interactions were studied through analogue
synthesis and X-ray crystallographic analysis of several
CDK2/inhibitor complexes. A hydrogen bond between
inhibitors and the backbone NH of Asp-86 near the
opening of the ATP binding cleft of CDK2 was of
particular interest and appeared to play a significant
role in affinity. Members of this series of inhibitors
cause an arrest of the cell cycle and exhibit a selective
killing effect on several tumor cell lines.41 In addition,
these compounds may have utility in the prevention of
chemotherapy-induced hair loss.11
Exp er im en ta l Section
P r oced u r e C. A third method for 1H-indol-2,3-dione (isatin)
synthesis:26 preparation of 4-isopropoxy-1H-indol-2,3-dione and
conversion to 4-[N′-(4-isopropoxy-2-oxo-1,2-dihydro-indol-3-
ylidene)-hydrazino]-benzenesulfonamide (31). A solution of
3.78 g (25.0 mmol) of 3-isopropoxy aniline and 5.46 g (25.0
mmol) of di-tert-butyl dicarbonate in 25 mL of THF was heated
to reflux for 2 h. The solution was cooled to ambient temper-
ature, and solvent was removed in vacuo. The residue was
dissolved in 100 mL of EtOAc, and the solution was washed
with three 50-mL portions of 0.5 M citric acid and 50 mL of
brine. The solution was dried over MgSO4, and removal of
solvent in vacuo afforded N-(tert-butyloxy-carbonyl)-3-iso-
propoxyaniline as a white solid (5.75 g, 92%): mp 79-81 °C;
1H NMR (DMSO-d6): δ 1.21 (d, J ) 6.0 Hz, 6H), 1.43 (s, 9H),
4.46 (septet, J ) 6 Hz, 1H), 6.47 (dd, J ) 2.1, 8.1 Hz, 1H),
6.94 (d, J ) 8.1 Hz, 1H), 7.0-7.1 (m, 2H), 9.23 (s, 1H); APCI:
m/z 274 (M+Na)+. To a solution of 2.5 g (10 mmol) of N-(tert-
butyloxycarbonyl)-3-isopropoxyaniline in 15 mL of dry THF
at -78 °C was added 15 mL (25 mmol) of 1.7 M tert-
butyllithium in hexanes. The mixture was stirred at -20 °C
for 2 h. A solution of 1.84 g (12.5 mmol) of diethyl oxalate in
10 mL of dry THF was added slowly over 5 min, and the
mixture was stirred at -20 °C for 2 h. The reaction mixture
was then poured into 100 mL of 1.0 N HCl and extracted with
two 100-mL portions of EtOAc. Solvent was removed in vacuo,
and the residue was dissolved in 100 mL of a 1:1 mixture of
EtOH and 6 N HCl and heated to reflux for 1 h. The mixture
Gen er a l Meth od s. 1H NMR spectra were obtained on
VARIAN Unity Plus NMR spectrophotometers at 300 or 400
MHz. Mass spectra were obtained on Micromass Platform II
mass spectrometers from Micromass Ltd., Altrincham, UK,
using either atmospheric chemical ionization (APCI) or elec-
trospray ionization (ESI). Analytical thin-layer chromatogra-
phy (TLC) was used to verify the purity of some intermediates
that could not be isolated or that were too unstable for full
characterization and to follow the progress of reactions. Merck
Silica gel 60 (230-400 mesh) was employed for the flash
chromatographic purification of some compounds.
P r oced u r e A. A typical procedure for the synthesis of
isatin25 is exemplified by the preparation of 6H-[1,3]thiazolo-
[5,4-e]indole-7,8-dione. To a 1-L flask was added a magnetic
stir bar, 85 g of sodium sulfate, and 100 mL of water. The
mixture was magnetically stirred until all the solids were
dissolved. To the resultant aqueous solution was added a
solution of 6-aminobenzothiazole (4.96 g, 33.0 mmol) in 50 mL
of 1 N aqueous hydrochloric acid and 10 mL of ethanol. The
mixture was stirred, and chloral hydrate (6.0 g, 36 mmol) was
added. To the resultant solution was added a solution of
hydroxylamine hydrochloride (7.50 g, 108 mmol) in 30 mL of
water. The final mixture was heated with stirring to a gentle
boil until all solids dissappeared, and heating was continued
for an additional 15 min. The flask was removed from the heat,
and the solution was poured onto 500 g of ice. The mixture

4348 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Bramson et al.
was cooled to ambient temperature and was extracted with
four 100-mL portions of EtOAc. The combined extracts were
evaporated to dryness to provide crude 4-isopropoxy-1H-indol-
2,3-dione, which was dissolved in 10 mL of EtOH containing
0.50 g (2.2 mmol) of 4-hydrazinobenzenesulfonamide hydro-
chloride. The solution was heated to 80 °C for 1 h and cooled
to ambient temperature. The resulting solid was collected by
vacuum filtration and purified by flash chromatography on
silica gel (EtOAc/hexane 3:2) to afford the title compound as
a yellow solid (0.052 g, 1.4%): mp >250 °C; 1H NMR (DMSO-
d6): δ 3.35 (d, J ) 6 Hz, 6H), 4.74 (septet, J ) 6 Hz, 1H), 6.48
(d, J ) 7.7 Hz, 1H), 6.69 (d, J ) 8 Hz, 1H), 7.14-7.2 (m, 3H),
7.47 (d, J ) 8.7 Hz, 2H), 7.75 (d, J ) 8.7 Hz, 2H), 11.01 (s,
1H), 12.79 (s, 1H); APCI: m/z 373 (M-H)-. Anal. (C17H18N4O4S)
C, H, N, S.
P r oced u r e F . A method for 3-[(dimethylamino)methylene]-
1,3-dihydro-2H-indol-2-one formation: preparation of 8-[(di-
methylamino)methylene]-6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-
7-one. To a suspension of 1.0 g (5.3 mmol) of 6,8-dihydro-7H-
[1,3]thiazolo[5,4-e]indol-7-one in 7.5 mL of DMF was added
1.38 g (6.80 mmol) of N,N-dimethylformamide-di-tert-butyl
acetal. The mixture was stirred at ambient temperature for 1
h and diluted with 7.5 mL of Et2O. The resulting precipitate
was isolated by filtration to afford the title compound as a tan
solid (1.0 g, 77%): 1H NMR (DMSO-d6): δ 3.33 (bs, 3H),
3.59 (bs, 3H), 6.97 (d, J ) 8.4, 1H), 7.33 (s, 1H), 7.62 (d, J )
8.4, 1H), 9.13 (s,1H), 10.29 (s, 1H); APCI- MS: m/z 246
(M+H)+.
P r oced u r e G. A general method for enamine formation:
preparation of 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]-
indol-8-ylidene)methyl]amino}-N-(2-pyridinyl)benzenesulfona-
mide (91). To a 25-mL round-bottom flask was added a stir
bar, 246 mg (1.00 mmol) of 8-(ethoxymethylene)-6,8-dihydro-
7H-[1,3]thiazolo[5,4-e]indol-7-one, 249 mg (1.00 mmol) of
sulfapyridine, and 10 mL of ethanol. The flask was fitted with
a water-cooled reflux condenser, and the mixture was heated
to reflux using an oil bath with stirring for 18 h. The reaction
was allowed to cool and was filtered. The precipitate was
washed with excess ethanol and dried under vacuum to yield
321 mg (71%) of the title compound: 1H NMR (DMSO-d6): δ
6.87 (br t, 1H), 7.12 (d, J ) 8.4 Hz, 1H), 7.16 (br d, J ) 8.2 Hz,
1H), 7.54 (d, J ) 8.7 Hz, 2H), 7.72 (dt, J ) 1, 7.3 Hz, 1H), 7.82
(d, J ) 8.4 Hz, 1H), 7.87 (d, J ) 8.7 Hz, 2H), 8.04 (br d, 1H),
8.06 (d, J ) 12.0 Hz, 1H), 9.27 (s, 1H), 10.91 (s, 1H), 11.14 (d,
P r oced u r e D. A general method for 1,3-dihydro-indol-2-
one (oxindole) synthesis:29 preparation of 2-oxo-2,3-dihydro-
1H-indole-5-carboxylic acid methyl ester and conversion to
2-oxo-3-(4-sulfamoyl-phenylamino-methylene)-2,3-dihydro-1H-
indole-5-carboxylic acid methyl ester (53). A solution of 2.66 g
(20.0 mmol) of ethyl (methylthio)acetate dissolved in 200 mL
of dichloromethane was cooled with stirring to -70 °C and 2.7
g (20 mmol) of sulfuryl chloride was added. The reaction was
stirred for 30 min at -70 °C, and a solution of 3.0 g (20 mmol)
of methyl 4-aminobenzoate and 4.3 g (20 mmol) of Proton
Sponge in 250 mL of dichloromethane was added dropwise over
1 h. The resulting pink slurry was treated with 2.3 g (23 mmol)
of TEA in one portion, and the solution was allowed to warm
to room temperature. The solution was washed with three 250-
mL portions of water, dried over MgSO4, and concentrated to
give an oil, which was subjected to chromatography on silica
gel eluting with hexane:EtOAc (1:1) to yield 2.0 g (42% yield)
of 3-methylthio-2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid
methyl ester: 1H NMR (DMSO-d6): δ 1.97 (s, 3H), 3.35 (s, 3H),
4.67 (s, 1H), 6.97 (d, J ) 8.2 Hz, 1H), 7.84 (s, 1H), 7.91 (d, J
) 8.2 Hz, 1H), 10.97 (s, 1H). A solution of 2.0 g (8.4 mmol) of
3-methylthio-2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid meth-
yl ester in 20 mL of acetic acid was treated with 10 g of zinc
powder. The reaction mixture was stirred for 2 h at room
temperature, filtered through Celite, and concentrated to
dryness. The residue was chromatographed on silica gel
eluting with hexane:EtOAc (1:1) to yield 1.6 g (99% yield) of
2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid methyl ester as
a pink solid: 1H NMR (DMSO-d6): δ 3.52 (s, 2H), 3.77 (s, 3H),
6.87 (d, J ) 8.2 Hz, 1H), 7.74 (s, J ) 1H), 7.80 (d, J ) 8.2 Hz,
1H), 10.72 (br s, 1H). Conversion to the 3-dimethylamino-
methylene-2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid meth-
yl ester (mixture of E and Z isomers) was carried out via
procedure F in 49% yield: 1H NMR (DMSO-d6): δ 3.29 Z (s,
6H), 3.31 E (s, 6H), 3.76 Z (s, 3H), 3.76 E (s, 3H), 6.74 Z (d, J
) 8.1 Hz, 1H), 6.81 E (d, J ) 8.2 Hz, 1H), 7.47-7.50 Z (m,
1H), 7.50-7.52 E (m, 1H), 7.57 E (dd, J ) 1.3, 8.2 Hz, 1H),
7.74 Z (s, 1H), 7.89 Z (s, 1H), 7.94 E (s, 1H), 10.33 Z (bs, 1H),
10.43 E (bs, 1H). The title compound was prepared in 41% yield
from 3-[(dimethylamino)methylene]oxindole-5-carboxylic acid
methyl ester and 4-aminobenzenesulfonamide according to
procedure G: 1H NMR (DMSO-d6): δ 3.81 (s, 3H), 6.92 (d, J
) 8.2 Hz, 1H), 7.26 (s, 2H), 7.60 (d, J ) 8.4 Hz, 2H), 7.69 (d,
J ) 8.2 Hz, 1H), 7.75 (d, J ) 8.4 Hz, 2H), 8.29 (s, 1H), 8.86 (d,
J ) 12.4 Hz, 1H), 10.80 (d, J ) 12.4 Hz, 1H), 10.94 (s, 1H);
APCI m/z 372 (M-1)-. Anal. (C17H15N3O5S) C, H, N, S.
J
) 12.0 Hz, 1H); APCI+ MS m/z 450 (M+H)+. Anal.
(C21H15N5O3S2) C, H, N, S. Note: One equivalent of strong acid,
e.g., HCl or methanesulfonic acid, is generally required in this
reaction. The acid can be supplied as the aniline salt or as a
separate component. Similar conditions can be used for
condensing anilines with 3-dimethylaminomethylene-, 3-tert-
butoxymethylene-, and 3-hydroxymethylene-substituted 2,3-
dihydro-1H-indol-2-ones.
P r oced u r e H. A method for 5-N-substituted amide forma-
tion: preparation of 2-oxo-3[(4-sulfamoyl-phenyl)-hydrazono]-
2,3-dihydro-1H-indole-5-carboxylic acid dimethylamide (57).
2-Oxo-3-[(4-sulfamoyl-phenyl)-hydrazono]-2,3-dihydro-1H-in-
dole-5-carboxylic acid was prepared from 1H-indole-2,3-dione-
5-carboxylic acid and 4-sulfonamidophenyl-hydrazine hydro-
chloride according to procedure E. To a suspension of 2.75 g
(7.63 mmol) of the 2-oxo-3[(4-sulfamoyl-phenyl)-hydrazono]-
2,3-dihydro-1H-indole-5-carboxylic acid in 20 mL of DMF was
added 1.38 mL (8.03 mmol) pentafluorophenyltrifluoroacetate
(PFPTFA), 0.69 mL (8.53 mmol) pyridine, and the suspension
was stirred under N2 for 20 min. TLC (silica gel, 20% MeOH/
CH2Cl2) indicated residual starting material remained, and
the reaction was treated with 10 mL DMF and additional
PFPTFA and pyridine (equal portions to above). The reaction
was stirred overnight and then poured into 400 mL of ether.
The solution was washed with two 500-mL portions of water,
and 300 mL of EtOAc was added to dissolve precipitate. The
solution was washed with 500 mL of water, dried over Na2-
SO4, filtered through silica gel and concentrated to remove
ether. The resulting solid was collected by filtration, washed
with 50 mL of 1:1 ethyl acetate:hexanes, and dried overnight
in a vacuum oven at 70 °C to give the title compound as a
bright yellow solid (2.30 g, 57%): mp >230 °C; 1H NMR
(DMSO-d6): δ 12.77 (s, 1H), 11.68 (s, 1H), 8.32 (d, J ) 1.9 Hz,
1H), 8.11 (dd, J ) 1.9 Hz, J ) 8.2 Hz, 1H), 7.79 (d, J ) 8.9 Hz,
2H), 7.67 (d, J ) 8.9 Hz, 2H), 7.28 (s, 2H), 7.16 (d, J ) 8.4 Hz,
1H); APCI: m/z 525 (M-H)-. Anal. (C21H11N4O5SF5) C, H, N.
To 100 mg (0.190 mmol) 2-oxo-3[(4-sulfamoyl-phenyl)-hydra-
zono]-2,3-dihydro-1H-indole-5-carboxylic acid pentafluorophe-
nyl ester in 5 mL of acetonitrile was added 50 µL (5.6 M in
ethanol, 0.28 mmol) of a solution of dimethylamine and 20 µL
(0.25 mmol) of pyridine, and the reaction was stirred overnight.
The solution was concentrated, and the resulting solid was
triturated under EtOAc to give the title compound as a yellow
solid (39 mg, 53%): mp >230 °C; 1H NMR (DMSO-d6): δ 12.71
(s, 1H), 11.22 (s, 1H), 7.75 (d, J ) 8.8 Hz, 2H), 7.60 (s, 1H),
P r oced u r e E. A general method for condensing isatins and
phenylhydrazines: preparation of 3-{[4-(aminosulfonyl)phenyl]-
hydrazono}-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide (50). A
solution of 2,3-dioxo-5-indolinesulfonamide (0.29 g, 1.3 mmol)
and 4-hydrazinobenzenesulfonamide hydrochloride (0.32 g, 1.4
mmol) in 26 mL of ethanol was stirred at reflux for 2 h. Solid
was collected from the hot suspension by filtration and was
air-dried to give 0.43 g (83%) of the title compound, mp >250
1
°C; H NMR (DMSO-d6): δ 7.04 (d, J ) 8.4 Hz, 1H), 7.25 (s,
2H), 7.26 (s, 2H), 7.60 (d, J ) 8.9 Hz, 2H), 7.70 (dd, J ) 8.2,
1.9 Hz, 1H), 7.78 (d, J ) 8.7 Hz, 2H), 7.98 (d, J ) 1.6 Hz, 1H),
11.43 (s, 1H), 12.75 (s, 1H); APCI m/z 395 (M)-. Anal.
(C14H13N5O5S2‚1/2H2O) C, H, N, S.

Oxindole-Based Inhibitors of CDK2
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4349
7.58 (d, J ) 8.8 Hz, 2H), 7.31 (dd, J ) 1.7, 8.1 Hz, 1H), 7.23
(s, 2H), 6.93 (d, J ) 8.0 Hz, 1H), 2.95 (s, 6H); APCI: m/z 386
(M-H). Anal. (C17H17N5O5S‚1/2H2O) C, H, N.
787 mL (701 g, 4.73 mol) of triethylorthoformate, and 2.4 L of
acetic acid was heated at reflux for 1.5 h. The dark brown
solution was cooled to 50 °C over a period of 1 h, and the
solution was concentrated to an oil under vacuum at 45 °C.
The oil was taken up in 3.9 L of ethanol and concentrated to
half volume. The resulting solid was collected by filtration,
washed with two 600-mL portions of ethanol, and dried under
vacuum at 60 °C to furnish 97 g (64%) of the title compound
as an orange solid, 1H NMR (DMSO-d6): δ 1.42 (t, J ) 7.1 Hz,
3H), 4.42 (q, J ) 7.1 Hz, 2H), 7.02 (d, J ) 8.6 Hz, 1H), 7.68 (s,
1H), 7.82 (d, J ) 8.6 Hz, 1H), 9.07 (s, 1H), 10.50 (s, 1H). Anal.
(C12H10N2O2S‚0.4 C2H4O2) C, H, N, S. Concentration of filtrate
provided an additional 27 g of desired product for an overall
yield of 82%.
4-[2-(5-Br om o-2-oxo-1,2-d ih yd r o-3H -in d ol-3-ylid en e)-
h yd r a zin o]ben zen esu lfon a m id e (16). 4-Hydrazinobenzene-
sulfonamide hydrochloride (0.60 g, 2.6 mmol) was dissolved
in EtOH (80 mL). 5-Bromoisatin (0.50 g, 2.2 mmol) was added,
and the reaction temperature was increased to reflux for 2 h.
The reaction mixture was cooled to room temperature, and
solids were collected by filtration. The solids were washed twice
with EtOH (15 mL each) and once with diethyl ether (20 mL)
and were dried under vacuum for 16 h to afford an orange
solid (0.87 g 100%): 1H NMR (DMSO-d6): δ 12.74 (s, 1H); 11.20
(s, 1H); 7.78 (d, J ) 8.7 Hz, 2H); 7.74 (d, J ) 1.8 Hz, 1H); 7.63
(d, J ) 8.7 Hz, 2H); 7.42 (dd, J ) 8.4, 1.8 Hz, 1H); 7.27 (bs,
2H); 6.87 (d, J ) 8.4 Hz, 1H); ES-MS: 394 (M-H)-. Anal.
(C14H11N4O3SBr) C, H, N, S.
4-[2-(2-Oxo-1,2-d ih yd r o-3H-in d ol-3-ylid en e)h yd r a zin o]-
ben zen esu lfon a m id e (17). The title compound was prepared
from isatin and 4-hydrazinobenzenesulfonamide hydrochloride
according to procedure E: 1H NMR (DMSO-d6): δ 6.88 (d, J
) 7.8 Hz, 1H), 7.02 (t, J ) 7.5 Hz, 1H), 7.20 (s, 2H), 7.23 (t, J
) 7.8 Hz, 1H), 7.51 (d, J ) 8.7 Hz, 2H), 7.53 (d, J ) 7.5 Hz,
1H), 7.74 (d, 8.7 Hz, 2H), 11.04 (s, 1H), 12.74 (s, 1H); APCI
m/z 315 (M-H)-.
4-{[(2-Oxo-1,2-d ih yd r o-3H -in d ol-3-ylid e n e )m e t h yl]-
a m in o}ben zen esu lfon a m id e (18). The title compound was
prepared from 3-(ethoxymethylene)-1,3-dihydro-2H-indol-2-one
and sulfanilamide according to procedure G: 1H NMR (DMSO-
d6): δ 6.80 (m, 1H), 6.90 (m, 1H), 6.99 (m, 1H), 7.21 (s, 2H),
7.49 (d, J ) 8.7 Hz, 2H), 7.55 (d, J ) 7.5 Hz, 1H), 7.73 (d, J )
8.7 Hz, 2H), 8.58 (d, J ) 12.3 Hz, 1H), 10.52 (s, 1H), 10.78 (d,
J ) 12.3 Hz, 1H); APCI m/z 314 (M-H)-. Anal. (C15H13N3O3S)
C, H, N, S.
4-{[1-(2-Oxo-1,2-d ih yd r o-3H -in d ol-3-ylid e n e )e t h yl]-
a m in o}ben zen esu lfon a m id e (19). 3-(1-Dimethylaminoeth-
ylidene)-1,3-dihydroindol-2-one was prepared from 1,3-dihy-
droindol-2-one and N,N-dimethylacetamide dimethyl acetal
according to procedure F. Condensation of 3-(1-dimethylami-
noethylidene)-1,3-dihydroindol-2-one and sulfanilamide ac-
cording to procedure G provided the title compound: 1H NMR
(DMSO-d6): δ 2.61 (s, 3H), 6.94 (d, J ) 7.5 Hz, 1H), 6.99 (t, J
) 7.5 Hz, 1H), 7.07 (t, J ) 7.5 Hz, 1H), 7.41 (s, 2H), 7.42 (d, J
) 7.5 Hz, 1H), 7.48 (d, J ) 8.6 Hz, 2H), 7.87 (d, J ) 8.6 Hz,
2H), 10.70 (s, 1H), 12.34 (s, 1H); ES-MS m/z 328 (M-H)-. Anal.
(C16H14N3O3S‚1/2H2O) C, H, N, S.
4-[2-(5-Ch lor o-2-oxo-1,2-d ih yd r o-3H -in d ol-3-ylid en e)-
h yd r a zin o]b en zen esu lfon a m id e (20). Condensation of
5-chloroisatin and 4-hydrazinobenzenesulfonamide hydrochlo-
ride according to procedure E afforded the title compound: 1H
NMR (DMSO-d6): δ 6.90 (d, J ) 8.2 Hz, 1H), 7.24 (m, 3H),
7.60 (m, 3H), 7.76 (d, J ) 8.7 Hz, 2H), 11.17 (s, 1H), 12.73 (s,
1H); APCI m/z 350 (M)-. Anal. (C14H11N4O3SCl) C, H, N, S,
Cl.
4-{[1-(5-Ch lor o-2-oxo-1,2-d ih yd r o-3H-in d ol-3-ylid en e)-
eth yl]a m in o}ben zen esu lfon a m id e (21). 3-(1-Dimethylami-
noethylidene)-5-chloro-1,3-dihydroindol-2-one was prepared
from 5-chloro-1,3-dihydroindol-2-one and N,N-dimethylaceta-
mide dimethyl acetal according to procedure F. Condensation
of 3-(1-dimethylaminoethylidene)-5-chloro-1,3-dihydroindol-2-
one and sulfanilamide according to procedure G provided the
title compound: 1H NMR (DMSO-d6): δ 2.53 (s, 3H), 6.87 (d,
J ) 8.2 Hz, 1H), 7.03 (dd, J ) 1.5, 8.2 Hz, 1H), 7.35 (d, J )
P r oced u r e I. A general method for introducing 4-substit-
uents via palladium-catalyzed coupling: preparation of 4-(N′-
{4-[2-(4-hydroxyphenyl)-vinyl]-2-oxo-1,2-dihydro-indol-3-ylidene}-
hydrazino)benzenesulfonamide (34). A mixture of 1.0 g (3.6
mmol) of 4-iodo-1H-indole-2,3-dione,43 0.42 g (4.2 mmol) of
TEA, 0.06 g (0.27 mmol) of palladium(II) acetate, 0.16 g (0.54
mmol) of tri-o-tolylphosphine, and 5.0 g (4.2 mmol) of a 10%
solution of 4-vinylphenol in propylene glycol was suspended
in 15 mL of dry acetonitrile in a Pyrex sealed tube and heated
to 100 °C for 4 h. The mixture was cooled to room temperature,
quenched with 50 mL of 10% hydrochloric acid, and extracted
with two 100-mL portions of EtOAc. The combined extracts
were dried over MgSO4 and concentrated to give a brown solid,
which was subjected to chromatography on silica gel, eluting
with hexane:EtOAc (3:1), to yield 0.125 g (13%) of trans-4-[2-
(4-hydroxyphenyl)-vinyl]-1H-indole-2,3-dione as a red solid: 1H
NMR (DMSO-d6): δ 6.6-7.6 (m, 8H), 7.77 (d, J ) 16.4 Hz,
1H), 9.85 (bs, 1H), 11.00 (bs, 1H); APCI m/z 264 (M-1)-.
Condensation of trans-4-[2-(4-hydroxyphenyl)-vinyl]-1H-indole-
2,3-dione and 4-hydrazinobenzenesulfonamide hydrochloride
according to procedure E gave the title compound in 27% yield
as an orange solid: 1H NMR (DMSO-d6): δ 6.78 (d, J ) 7.8
Hz, 1H), 6.88 (d, J ) 8.7 Hz, 2H), 7.26 (t, J ) 7.8 Hz, 1H),),
7.29 (s, 2H), 7.36 (d, J ) 16.5 Hz, 1H), 7.47 (d, J ) 7.8 Hz,
1H), 7.53 (d, J ) 8.7 Hz, 2H), 7.57 (d, J ) 8.7 Hz, 2H),), 7.81
(d, J ) 8.7 Hz, 2H), 8.03 (d, J ) 16.5 Hz, 1H), 9.78 (s, 1H),
11.17 (s, 1H), 13.02 (s, 1H); APCI m/z 433 (M-1)-. Anal.
(C22H18N4O4S‚1/2H2O) C, H, N, S.
8-(Meth ylsu lfa n yl)-6,8-d ih yd r o-7H-[1,3]th ia zolo[5,4-e]-
in d ol-7-on e (11). Under a nitrogen atmosphere, a solution of
116 mL (121 g, 0.900 mol) of ethyl methylthioacetate and 1.33
L of dichloromethane was cooled to -74 °C with a dry ice/
acetone bath. Sulfuryl chloride (70.0 mL, 118 g, 0.870 mol)
was added over a 5 min period with temperature maintained
between -70 and -74 °C, and the clear, colorless solution was
stirred at -70 to -74 °C for 25 min. A solution of 157 mL
(117 g, 0.900 mol) of N-ethyl-diisopropylamine, 131 g (0.870
mol) of 6-aminobenzothiazole, and 1 L of dichloromethane was
added over a period of 100 min with temperature maintained
between -69 and -74 °C. An additional 0.9 L of dichlo-
romethane was added to dissolve a cream-colored precipitate,
and the solution was stirred for 30 min. Triethylamine was
added over a 2-min period, causing a temperature rise to -48
°C. After 15 min the cooling bath was removed, and the brown-
orange solution was warmed to -30 °C over a 45-min period
with a heat gun. The solution was concentrated under vacuum
at 20 °C, and the resulting dark orange oil was treated with
1.4 L of 0.5 N HCl and 1.3 L of tert-butylmethyl ether. The
mixture was stirred for 2.5 h at room temperature. The golden
precipitate was isolated by filtration and dried in a vacuum
at room temperature to furnish 92 g (44%) of the title
1
compound, H NMR (DMSO-d6): δ 1.78 (s, 3H), 4.87 (s, 1H),
7.07 (d, J ) 8.5 Hz, 1H), 7.97 (d, J ) 8.5 Hz, 1H), 9.19 (s, 1H),
10.79 (br s, 1H). Anal. (C10H8N2OS2‚0.1 H2O‚C5H12O) C, H, N,
S.
6,8-Dih yd r o-7H-[1,3]th ia zolo[5,4-e]in d ol-7-on e (12). A
mixture of 2.7 g (11 mmol) of 8-(methylsulfanyl)-6,8-dihydro-
7H-[1,3]thiazolo[5,4-e]indol-7-one, 3.0 g of activated Zn, 41 mL
of acetic acid, and 41 mL of THF was heated at 60-65 °C for
3.5 h. The gray-green mixture was filtered through a sintered
glass funnel containing a 0.5 in. pad of Celite, and the pad
was washed with 40 mL of hot 1:1 THF:acetic acid. Water (200
mL) was added to the filtrate, and the pale yellow mixture
was stirred for 10 min. The resulting solid was isolated by
filtration, washed with 25 mL of water, and dried under
vacuum at 70 °C to furnish 1.05 g (49%) of the title compound
as a pale yellow solid, 1H NMR (DMSO-d6): δ 3.75 (s, 2H),
7.04 (d, 8.5 Hz, 1H), 7.90 (d, J ) 8.5 Hz, 1H), 9.17 (s, 1H),
10.59 (s, 1H). Anal. (C9H6N2OS) C, H, N, S.
8-(Eth oxym eth ylen e)-6,8-d ih yd r o-7H-[1,3]th ia zolo[5,4-
e]in d ol-7-on e (13). A mixture of 116.7 g (0.61 mol) of oxindole,

4350 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Bramson et al.
1.5 Hz, 1H), 7.37 (s, 2H), 7.44 (d, J ) 8.6 Hz, 2H), 7.83 (d, J
) 8.6 Hz, 2H), 10.78 (s, 1H), 12.35 (s, 1H); ES-MS m/z 362
(M-H)-. Anal. (C16H14N3O3ClS‚1/2H2O) C, H, N, S.
chromatography on silica gel, eluting with hexane:EtOAc (4:
1), to furnish 0.027 g (13%) of 4-isobutyl-1H-indole-2,3-dione:
1H NMR (DMSO-d6): δ 0.89 (d, J ) 6.7 Hz, 6H), 1.86 (nonet,
J ) 6.7 Hz, 1H), 2.72 (d, J ) 6.7 Hz, 1H), 6.74 (d, J ) 7.8 Hz,
1H), 6.86 (d, J ) 7.8 Hz, 1H), 7.48 (t, J ) 7.8 Hz, 1H), 11.03
(s, 1H). Condensation of 4-isobutyl-1H-indole-2,3-dione and
4-sulfonamido- phenylhydrazine hydrochloride according to
procedure E gave the title compound in 65% yield: 1H NMR
(DMSO-d6): δ 0.96 (d, J ) 6.4 Hz, 6H), 2.05 (m, 1H), 2.87 (d,
J ) 7.0 Hz, 2H), 6.79 (d, J ) 7.6 Hz, 1H), 6.85 (d, J ) 7.6 Hz,
1H), 7.20 (t, J ) 7.6 Hz, 1H), 7.26 (s, 2H), 7.51 (d, J ) 8.5 Hz,
2H), 7.81 (d, J ) 8.5 Hz, 2H), 11.13 (s, 1H), 13.03 (s, 1H); APCI
m/z 371 (M-H)-. Anal. (C18H20N4O3S‚1/2H2O) C, H, N, S.
4-[1-(5-Oxa zol-5-yl-2-oxo-1,2-d ih yd r o-in d ol-3-ylid en e)-
h yd r a zin o]ben zen esu lfon a m id e (5:1 E:Z isom er m ixtu r e)
(22). The title mixture of isomers was prepared from 5-(oxazol-
5-yl)-1H-indole-2,3-dione and 4-hydrazinobenzenesulfonamide
hydrochloride according to procedure E: mp >250 °C; 1H NMR
(DMSO-d6): δ (5:1 ratio of Z:E isomers), E 6.97 (d, J ) 8.2
Hz, 1H), Z 7.00 (d, J ) 8.2 Hz, 1H), E 7.23 (s, 2H), Z 7.25 (s,
2H), Z 7.61 (d, J ) 9.1 Hz, 2H), E 7.61 (d, J ) 9.1 Hz, 2H), Z
7.62 (dd, J ) 8.2, 1.7 Hz, 1H), Z 7.65 (s, 1H), E 7.65 (s, 1H), E
7.65 (dd, J ) 8.2, 1.5 Hz, 1H), Z 7.78 (d, J ) 8.9 Hz, 2H), E
7.81 (d, J ) 8.9 Hz, 2H), Z 7.90 (d, J ) 1.7 Hz, 1H), Z 8.40 (s,
1H), E 8.43 (s, 1H), E 8.47 (d, J ) 1.3 Hz, 1H), E 10.83 (s,
1H), E 10.98 (s, 1H), Z 11.25 (s, 1H), Z 12.78 (s, 1H); ESI m/z
382 (M-H)-. Anal. (C17H13N5O4S‚1.2 H2O‚0.4 C2H6O) C, H, N.
4-({[5-(1,3-Oxa zol-5-yl)-2-oxo-1,2-d ih yd r o-3H -in d ol-3-
yliden e]m eth yl}am in o)ben zen esu lfon am ide (23). The title
compound was prepared in 68% yield from ethoxymethylene-
5-oxazol-5-yl-1,3-dihydro-indol-2-one and 4-aminobenzene-
sulfonamide hydrochloride according to procedure G: 1H NMR
(DMSO-d6): δ 10.79 (d,1H), 10.73 (s, 1H), 8.76 (d, 1H), 8.38
(s, 1H), 8.0 (s, 1H), 7.77 (d, 2H), 7.56 (d, 2H), 7.43 (s, 1H), 7.40
(d, 1H), 7.26 (s, 2H), 6.91 (d, 1H); APCI: m/z 381 (M-H)-.
4-[1-(5-Oxa zol-5-yl-2-oxo-1,2-d ih yd r o-in d ol-3-ylid en e)-
eth yla m in o]ben zen esu lfon a m id e (24). 3-(1-Dimethylami-
noethylidene)-5-(oxazol-5-yl)-1,3-dihydroindol-2-one was pre-
pared from 5-(oxazol-5-yl)-1,3-dihydroindol-2-one and N,N-
dimethylacetamide dimethyl acetal according to procedure F.
Condensation of 3-(1-dimethylaminoethylidene)-5-(oxazol-5-yl)-
1,3-dihydroindol-2-one and sulfanilamide according to proce-
4-{N′-[4-(2-Meth yl-p r op en yl)-2-oxo-1,2-d ih yd r o-in d ol-
3-ylid en e]h yd r a zin o}ben zen esu lfon a m id e (29). By meth-
ods described in procedure I, 4-(2-methyl-propenyl)-1H-indole-
2,3-dione was prepared from 4-iodo-1H-indole-2,3-dione and
isobutylene in 34% yield: 1H NMR (DMSO-d6): δ 1.82 (s, 3H),
1.90 (s, 3H), 6.79 (d, J ) 7.9 Hz, 1H), 6.94 (d, J ) 7.9 Hz, 1H),
7.47 (t, J ) 7.9 Hz, 1H), 10.97 (s, 1H); APCI m/z 200 (M-1)-.
Condensation of 4-(2-methyl-propenyl)-1H-indole-2,3-dione and
4-hydrazinobenzenesulfonamide hydrochloride according to
procedure E gave the title compound as a yellow solid (51%
1
yield): H NMR (DMSO-d6): δ 1.84 (s, 3H), 2.04 (s, 3H), 6.78
(s, 1H), 6.79 (d, J ) 7.8 Hz, 1H), 6.96 (d, J ) 7.8 Hz, 1H), 7.24
(t, J ) 7.8 Hz, 1H), 7.24 (s, 2H), 7.48 (d, J ) 8.8 Hz, 2H), 7.80
(d, J ) 8.8 Hz, 2H), 11.11 (s, 1H), 12.91 (s, 1H); APCI m/z 369
(M-1)-. Anal. (C18H18N4O3S) C, H, N, S.
4-[2-(4-Eth oxy-2-oxo-1,2-d ih yd r o-3H-in d ol-3-ylid en e)-
h yd r a zin o]b en zen esu lfon a m id e (30). Condensation of
4-ethoxy-1H-indole-2,3-dione and 4-hydrazinobenzenesulfona-
mide hydrochloride using procedure E provided the title
compound: 1H NMR (DMSO-d6): δ 1.44 (t, J ) 7.0, 3H), 4.13
(q, J ) 7.0 Hz, 2H), 6.50 (d, J ) 7.5 Hz, 1H), 6.68 (d, J ) 8.4
Hz, 1H), 7.15-7.21 (m, 3H), 7.46 (d, J ) 8.8 Hz, 2H), 7.74 (d,
J ) 8.8, 2H), 11.03 (s, 1H), 12.78 (s, 1H); APCI: m/z 359 (M-
H)-. Anal. (C16H16N4O4S) C, H, N, S.
4-[N′-(2-Oxo-4-p h en oxy-1,2-d ih yd r o-in d ol-3-ylid en e)-
h yd r a zin o]ben zen esu lfon a m id e (4:10 m ixtu r e of E a n d
Z isom er s) (32). The title compound was prepared from
3-phenoxyaniline and 4-sulfonamidophenyl-hydrazine hydro-
chloride according to procedure C: mp >250 °C;1H NMR
(DMSO-d6, equilibrated for 10 days at room temperature): δ
6.42 E (d, J ) 8.4 Hz, 1H), 6.70 E (d, J ) 7.7 Hz, 1H), 6.76 Z
(d, J ) 8.2 Hz, 1H), 6.82 Z (d, J ) 7.8 Hz, 1H), 6.99 Z (d, J )
8.1 Hz, 2H), 7.06 Z (d, J ) 8.8 Hz, 2H), 7.1-7.6 E (m, 10H),
7.1-7.6 Z (m, 6H), 7.62 Z (d, J ) 8.8 Hz, 2H), 7.74 E (d, J )
8.7 Hz, 2H), 10.88 E (s, 1H), 11.18 E (s, 1H), 11.27 Z (s, 1H),
12.77 Z (s, 1H); APCI: m/z 407 (M-H)-. Anal. (C20H16N4O4S)
C, H, N, S.
4-{N′-[2-Oxo-4-(2-p yr id in -4-yl-eth yl)-1,2-d ih yd r o-in d ol-
3-ylid en e]h yd r a zin o}ben zen esu lfon a m id e (33). A mixture
of 3.0 g (20 mmol) of 3-nitroiodobenzene, 3.5 mL (25 mmol) of
TEA, 0.045 g (0.20 mmol) of palladium(II) acetate, and 2.77 g
(25.0 mmol) of 4-vinylpyridine was suspended in 4 mL of dry
acetonitrile in a Pyrex sealed tube and heated to 100 °C for
48 h. The mixture was cooled to room temperature and was
quenched with 200 mL of 10% hydrochloric acid. The resulting
yellow solid was isolated by filtration and partitioned between
250 mL of EtOAc and 250 mL of 1 N aqueous sodium
hydroxide. The organic phase was dried over MgSO4 and
concentrated to give 3.0 g (66%) of 4-[2-(3-nitrophenyl)ethenyl]-
pyridine as a yellow solid: 1H NMR (DMSO-d6): δ 3.0-4.6
(br s, 1H), 7.71-7.78 (m, 2H), 8.07 (d, J ) 15.8 Hz, 1H), 8.13-
8.16 (m, 3H), 8.24 (d, J ) 8.0 Hz, 1H), 8.56 (s, 1H), 8.84 (d, J
) 5.7 Hz, 2H); ESI m/z 227 (M+1)+. A portion (1.3 g, 7.1 mmol)
of this solid was dissolved in 100 mL of EtOAc, and 0.5 g of
10% palladium on charcoal was added. The mixture was
hydrogenated on a Parr apparatus at 40 psi for 1.5 h. Another
0.5 g batch of 10% palladium on charcoal was added, and the
mixture was subjected to further hydrogenation for 1 h. The
palladium catalyst was removed by filtration through a pad
of Celite, and the filtrate was concentrated to give 1.13 g
(100%) of 3-(4-pyridinyl)ethylaniline: 1H NMR (DMSO-d6): δ
1
dure G provided the title compound: mp >250 °C; H NMR
(DMSO-d6): δ 2.51 (s, 0.8H, DMSO), 2.61 (s, 3H), 6.97 (d, J )
8.2 Hz, 1H), 7.37 (s, 2H), 7.40 (dd, J ) 8.0, 1.5 Hz, 1H), 7.45
(d, J ) 8.8 Hz, 2H), 7.56 (s, 1H), 7.66 (d, J ) 1.2 Hz, 1H), 7.83
(d, J ) 8.5 Hz, 2H), 8.34 (s, 1H), 10.85 (s, 1H), 12.33 (s, 1H);
APCI m/z 395 (M-H)-. Anal. (C19H16N4O4S‚0.1 C2H6OS‚0.6
H2O) C, H, N, S.
4-[N′-(4-Iodo-2-oxo-1,2-dih ydr o-in dol-3-yliden e)-h ydr azi-
n o]ben zen esu lfon a m id e (25). The title compound was pre-
pared from 4-iodo-1H-indole-2,3-dione43 and 4-sulfonamidophe-
nyl-hydrazine hydrochloride according to procedure E in 87%
overall yield: 1H NMR (DMSO-d6): δ 6.93 (d, J ) 7.6 Hz, 1H),
6.99 (t, J ) 7.6 Hz, 1H), 7.25 (s, 2H), 7.50 (d, J ) 7.6 Hz, 1H),
7.66 (d, J ) 8.7 Hz, 2H), 7.77 (d, J ) 8.7 Hz, 2H), 11.17 (s,
1H), 12.94 (s, 1H); APCI m/z 441 (M-H)-. Anal. (C14H11IN4O3S)
C, H, N, S.
4-[2-(4-Eth yl-2-oxo-1,2-d ih yd r o-3H-in d ol-3-ylid en e)h y-
d r a zin o]ben zen esu lfon a m id e (26). The title compound was
prepared from 4-ethyl-1H-indole-2,3-dione and 4-hydrazi-
nobenzenesulfonamide hydrochloride according to procedure
E: 1H NMR (DMSO-d6): δ 1.24 (t, J ) 7.4 Hz, 3H), 2.94 (q, J
) 7.4 Hz, 2H), 6.73 (d, J ) 7.7 Hz, 1H), 6.88 (d, J ) 7.7 Hz,
1H), 7.17 (t, J ) 7.7 Hz, 1H), 7.22 (s, 2H), 7.46 (d, J ) 8.7 Hz,
2H), 7.76 (d, J ) 8.7 Hz, 2H), 11.07 (s, 1H), 12.94 (s, 1H); APCI
m/z 343 (M-H)-. Anal. (C16H16N4O3S‚2/3H2O) C, H, N, S.
4-[N′-(4-Isop r op yl-2-oxo-1,2-d ih yd r o-in d ol-3-ylid en e)-
h yd r a zin o]ben zen esu lfon a m id e (27). The title compound
was prepared from 4-isopropyl-1H-indole-2,3-dione44 and 4-hy-
drazinobenzenesulfonamide hydrochloride according to pro-
cedure E in 73% yield: 1H NMR (DMSO-d6): δ 1.30 (d, J )
6.7 Hz, 6H), 3.82 (septet, J ) 6.7 Hz, 1H), 6.76 (d, J ) 7.8 Hz,
1H), 7.01 (d, J ) 7.8 Hz, 1H), 7.23 (t, J ) 7.8 Hz, 1H), 7.24 (s,
2H), 7.48 (d, J ) 8.7 Hz, 2H), 7.79 (d, J ) 8.7 Hz, 2H), 11.10
(s, 1H), 13.05 (s, 1H); APCI m/z 357 (M-1)-. Anal. (C17H18N4O3S)
C, H, N, S.
4-[N′-(4-Isobu tyl-2-oxo-1,2-d ih yd r o-in d ol-3-ylid en e)h y-
d r a zin o]ben zen esu lfon a m id e (28). A mixture of 0.20 g (1.0
mmol) of 4-(2-methyl-propenyl)-1H-indole-2,3-dione and 0.05
g of 10% palladium on charcoal in 25 mL of EtOAc was
subjected to hydrogenation on a Parr apparatus at 46 psi for
1 h. The mixture was filtered through Celite, and the filtrate
was concentrated to dryness. The solid was purified by

Oxindole-Based Inhibitors of CDK2
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4351
2.69 (m, 2H), 2.80 (m, 2H), 4.9 (bs, 2H), 6.33 (d, J ) 7.7 Hz,
2H), 6.38 (s, 1H), 6.86 (t, J ) 7.7 Hz, 1H), 7.20 (d, J ) 5.8 Hz,
2H), 8.41 (d, J ) 5.8 Hz, 2H). Conversion of 3-[2-(4-pyridinyl)-
ethyl]-aniline to 4-(2-pyridin-4-yl-ethyl)-1H-indole-2,3-dione
was accomplished according to procedure A in 24% overall
yield: 1H NMR (DMSO-d6): δ 2.80 (m, 2H), 3.10 (m, 2H), 6.70
(d, J ) 8.0 Hz, 1H), 6.81 (d, J ) 8.0 Hz, 1H), 7.24 (m, 2H),
7.40 (t, J ) 8.0 Hz, 1H), 8.42 (bs, 2H), 11.00 (s, 1H). Conversion
of 4-(2-pyridin-4-yl-ethyl)-1H-indole-2,3-dione to the title com-
pound was accomplished according to procedure E in 40%
overall yield: 1H NMR (DMSO-d6): δ 2.98 (t, J ) 7.9 Hz, 2H),
3.30 (m, 2H, underneath water peak), 6.78 (d, J ) 7.7 Hz, 1H),
6.88 (d, J ) 7.6 Hz, 1H), 7.17 (t, J ) 7.6 Hz, 1H), 7.25 (s, 2H),
7.29 (d, J ) 6.0 Hz, 2H), 7.37 (d, J ) 8.8 Hz, 2H), 7.66 (d, J )
8.8 Hz, 2H), 8.47 (d, J ) 6.0 Hz, 2H), 11.13 (s, 1H), 12.98 (s,
1H); APCI m/z 420 (M-1)-. Anal. (C21H19N5O3S‚0.15 HCl) C,
H, N, S.
4-(N′-{4-[2-(4-Hydr oxyph en yl)-eth yl]-2-oxo-1,2-dih ydr o-
in d ol-3-ylid en e}h yd r a zin o)ben zen esu lfon a m id e (35). A
mixture of 0.028 g (0.64 mmol) of 4-(N′-{4-[2-(4-hydroxyphe-
nyl)-vinyl]-2-oxo-1,2-dihydro-indol-3-ylidene}-hydrazino)-ben-
zenesulfonamide and 0.015 g of 10% palladium on charcoal in
60 mL of MeOH:THF (4:1) was subjected to hydrogenation on
a Parr apparatus at 50 psi for 1 h. The mixture was filtered
through Celite, and the filtrate was concentrated to give 0.026
g (93%) of the title compound as a yellow solid: 1H NMR
(DMSO-d6): δ 2.82 (t, J ) 8.0 Hz, 2H), 3.23 (t, J ) 8.0 Hz,
2H), 6.69 (d, J ) 8.4 Hz, 2H), 6.78 (d, J ) 7.7 Hz, 1H), 6.89 (d,
J ) 7.7 Hz, 1H), 7.07 (d, J ) 8.4 Hz, 2H), 7.18 (t, J ) 7.7 Hz,
1H), 7.26 (s, 2H), 7.45 (d, J ) 8.8 Hz, 2H), 7.71 (d, J ) 8.8 Hz,
2H), 9.20 (bs, 1H), 11.12 (s, 1H), 13.02 (s, 1H); APCI m/z 435
(M-1)-. Anal. (C22H20N4O4S‚H2O) C, H, N, S.
4-{N′-[2-Oxo-4-(1H -p yr a zol-3-yl)-1,2-d ih yd r o-in d ol-3-
ylid en e]h yd r a zin o}ben zen esu lfon a m id e (36). 4-(1H-Pyra-
zol-3-yl)-1H-indole-2,3-dione was prepared from 3-(1H-pyrazol-
3-yl)aniline according to procedure A. The title compound was
prepared from 4-(1H-pyrazol-3-yl)-1H-indole-2,3-dione and
4-hydrazinobenzenesulfonamide hydrochloride according to
procedure E: 1H NMR (DMSO-d6): δ 6.72 (s, 1H), 7.22 (s, 2H),
7.39 (s, 1H), 7.48-7.60 (m, 4H), 7.76 (d, J ) 8.7 Hz, 2H), 7.77
(s, 1H), 11.11 (s, 1H), 12.93 (s, 1H); ESI: m/z 381 (M-H)-.
2-Oxo-3-(4-su lfa m oyl-p h en yla m in o)-m eth ylen e]-2,3-d i-
h yd r o-1H-in d ole-4-ca r boxylic a cid eth yl ester (37). The
title compound was prepared from 2-oxo-2,3-dihydro-1H-
indole-4-carboxylic acid ethyl ester45,46 and sulfanilamide
according to procedure G in 14% overall yield: 1H NMR
(DMSO-d6): δ 1.33 (t, J ) 7.1 Hz, 3H), 4.37 (q, J ) 7.1 Hz,
2H), 7.10 (d, J ) 7.6 Hz, 1H), 7.15 (t, J ) 7.6 Hz, 1H), 7.30 (s,
2H), 7.41 (d, J ) 8.6 Hz, 2H), 7.57 (d, J ) 7.6 Hz, 1H), 7.82 (d,
J ) 8.6 Hz, 2H), 9.50 (d, J ) 12.6 Hz, 1H), 10.96 (s, 1H), 11.75
(d, J ) 12.6 Hz, 1H); APCI m/z 386 (M-1)-.
(DMSO-d6): δ 7.17 (d, J ) 8.1 Hz, 1H), 7.32 (d, J ) 8.1 Hz,
1H), 7.56 (t, J ) 8.1 Hz, 1H), 8.02 (bs, 2H), 11.86 (bs, 1H);
APCI+MS m/z 191 (M+1)+. Condensation of 1H-indole-2,3-
dione-4-carboxamide with 4-hydrazinobenzenesulfonamide hy-
drochloride according to procedure E gave the title compound
in 31% yield: 1H NMR (DMSO-d6, equilibrated for 10 days at
room temperature): E isomer, δ 7.11 (d, J ) 8.3 Hz, 1H), 7.18
(s, 2H), 7.27 (d, J ) 8.8 Hz, 2H), 7.32 (d, J ) 7.0 Hz, 1H), 7.51
(d, J ) 7.4 Hz, 1H), 7.75 (d, J ) 8.8 Hz, 2H), 8.0 (bs, 2H),
10.40 (s, 1H), 10.80 (s, 1H); Z isomer, δ 11.10 (s, 1H), 13.11 (s,
1H); APCI m/z 359 (M)-. Anal. (C15H13N5O4S‚0.12 H2O) C, H,
N, S.
4-[2-(5-F lu or o-2-oxo-1,2-d ih yd r o-3H -in d ol-3-ylid en e)-
h yd r a zin o]ben zen esu lfon a m id e (41). The title compound
was prepared from 5-fluoro-1H-indole-2,3-dione and 4-hydrazi-
nobenzenesulfonamide hydrochloride using procedure E: 1H
NMR (DMSO-d6): δ 6.87 (dd, J ) 4.3, 8.4 Hz, 1H), 7.05 (dt, J
) 2.6, 9.1 Hz, 1H), 7.21 (s, 2H), 7.38 (dd, J ) 2.6, 8.4 Hz, 1H),
7.56 (d, J ) 8.8 Hz, 2H), 7.74 (d, J ) 8.8 Hz, 2H), 11.05 (s,
1H), 12.75 (s, 1H); APCI m/z 333 (M-H)-. Anal. (C14H11N4O3-
FS) C, H, N, S.
4-[2-(5-Iod o-2-oxo-1,2-d ih yd r o-3H-in d ol-3-ylid en e)h y-
d r a zin o]ben zen esu lfon a m id e (42). Condensation of 5-iodo-
1H-indole-2,3-dione and 4-hydrazino-benzenesulfonamide hy-
drochloride according to procedure E provided the title
compound: 1H NMR (DMSO-d6): δ 6.74 (d, J ) 8.2 Hz, 1H),
7.24 (s, 2H), 7.56 (dd, J 1 ) 1.7, J 2 ) 8.2 Hz, 1H), 7.60 (d, J )
8.8 Hz, 2H), 7.75 (d, J ) 8.8 Hz, 2H), 7.86 (d, J ) 1.7, 1H),
11.15 (s, 1H), 12.70 (s, 1H); APCI: m/z 441 (M-H)-. Anal.
(C14H11N4O3SI) C, H, N, S, I.
4-[N′-(5-Meth yl-2-oxo-1,2-d ih yd r o-in d ol-3-ylid en e)h y-
d r a zin o]ben zen esu lfon a m id e (43). The title compound was
prepared from 5-methyl-1H-indole-2,3-dione47 and 4-hydrazi-
nobenzenesulfonamide hydrochloride according to procedure
E in 86% yield: 1H NMR (DMSO-d6): δ 2.3 (s, 3H), 6.76 (d, J
) 7.9 Hz, 1H), 7.11 (d, J ) 7.9 Hz, 1H), 7.20 (s, 2H), 7.57 (d,
J ) 8.8 Hz, 2H), 7.77 (d, J ) 8.8 Hz, 2H), 8.02 (s, 1H), 10.51
(s, 1H), 10.62 (s, 1H); APCI m/z 329 (M-1)-. Anal. (C15H14N4O3S)
C, H, N, S.
4-[N′-(5-Hyd r oxy-2-oxo-1,2-d ih yd r o-in d ol-3-ylid en e)h y-
d r a zin o]ben zen esu lfon a m id e (44). The title compound was
prepared from 5-hydroxy-1H-indole-2,3-dione48 and 4-hydrazi-
nobenzenesulfonamide hydrochloride according to procedure
E in 30% yield: 1H NMR (DMSO-d6): δ 6.79 (dd, J ) 2.2, 8.3
Hz, 1H), 6.72 (d, J ) 8.3 Hz, 1H), 6.98 (d, J ) 2.2 Hz, 1H),
7.25 (s, 2H), 7.53 (d, J ) 8.7 Hz, 2H), 7.78 (d, J ) 8.7 Hz, 2H),
9.20 (s, 1H), 10.80 (s, 1H), 12.82 (s, 1H); APCI m/z 331 (M-
H)-.
4-[N′-(5-Meth oxy-2-oxo-1,2-d ih yd r o-in d ol-3-ylid en e)h y-
d r a zin o]ben zen esu lfon a m id e (45). The title compound was
prepared from 5-methoxy-1H-indole-2,3-dione47 and 4-hydrazi-
nobenzenesulfonamide hydrochloride according to procedure
4-({[4-(Hyd r oxym eth yl)-2-oxo-1,2-d ih yd r o-3H-in d ol-3-
ylid en e]m eth yl}a m in o)ben zen esu lfon a m id e (38). Proce-
dures analogous to those used in the preparation of the
corresponding 6-hydroxymethyl isomer provided the title
compound: 1H NMR (DMSO-d6): δ 2.37 (d, J ) 5.0 Hz, 3H),
4.68 (s, 2H), 5.31 (br s, 1H), 6.78 (d, J ) 7.5 Hz, 1H), 6.92 (d,
J ) 7.5 Hz, 1H), 7.00 (t, J ) 7.5 Hz, 1H), 7.34 (q, J ) 5.0 Hz,
1H), 7.44 (d, J ) 8.6 Hz, 2H), 7.71 (d, J ) 8.6 Hz, 2H), 8.32 (d,
J ) 12.2 Hz, 1H), 10.65 (s, 1H), 11.26 (d, J ) 12.2 Hz, 1H); ES
m/z 358 (M-1)-. Anal. (C17H17N3O4S) C, H, N, S.
4-[N′-(4-Nit r o-2-oxo-1,2-d ih yd r o-in d ol-3-ylid en e)-h y-
d r a zin o]ben zen esu lfon a m id e (39). The title compound was
prepared from 4-nitro-1H-indole-2,3-dione47 and 4-hydrazi-
nobenzenesulfonamide hydrochloride according to procedure
E in 33% yield: 1H NMR (DMSO-d6): δ 7.23 (d, J ) 7.7 Hz,
1H), 7.31 (s, 2H), 7.47 (t, J ) 7.9 Hz, 1H), 7.56 (d, J ) 7.9 Hz,
2H), 7.59 (d, J ) 7.2 Hz, 1H), 7.83 (d, J ) 7.7 Hz, 2H), 11.59
(s, 1H), 13.20 (s, 1H); APCI m/z 361 (M)-. Anal. (C14H11N5O5S)
C, H, N, S.
1
E: mp >250 °C; H NMR (DMSO-d6): δ 3.80 (s, 3H), 6.87 (s,
2H), 7.20 (s, 1H), 7.28 (s, 2H), 7.60 (d, J ) 8.8 Hz, 2H), 7.81
(d, J ) 8.8 Hz, 2H), 10.93 (s, 1H), 12.85 (s, 1H); APCI m/z
344.9 (M-H)-.
4-[N′-(5-Nit r o-2-oxo-1,2-d ih yd r o-in d ol-3-ylid en e)-h y-
d r a zin o]ben zen esu lfon a m id e (46). The title compound was
prepared from 5-nitro-1H-indole-2,3-dione47 and 4-hydrazi-
nobenzenesulfonamide hydrochloride according to procedure
E in 94% yield: 1H NMR (DMSO-d6): δ 7.14 (d, J ) 8.6 Hz,
1H), 7.33 (s, 2H), 7.75 (d, J ) 8.8 Hz, 2H), 7.84 (d, J ) 8.8 Hz,
2H), 8.23 (dd, J ) 2.2, 8.6 Hz, 1H), 8.42 (d, J ) 2.2 Hz, 1H),
11.76 (s, 1H), 12.78 (s, 1H). Anal. (C14H11N5O5S) C, H, N.
4-[N′-(5-Am in o-2-oxo-1,2-d ih yd r o-in d ol-3-ylid en e)-h y-
d r a zin o]ben zen esu lfon a m id e h yd r och lor id e (47). The
title compound was prepared from 5-amino-1H-indole-2,3-
dione and 4-hydrazinobenzenesulfonamide hydrochloride ac-
cording to procedure E: 1H NMR (DMSO-d6): δ 6.95 (d, J )
8 Hz, 1H), 7.2 (d, J ) 8 Hz, 1H), 7.26 (s, 2H), 7.46 (s, 1H), 7.5
(d, J ) 8 Hz, 2H), 7.8 (d, J ) 8 Hz, 2H), 9.7 (br s, 3H), 11.2 (s,
1H), 12.8 (s, 1H); APCI m/z 330.2 (M-H)-.
3-{[4-(Am in osu lfon yl)p h en yl]h yd r a zon o}-2-oxo-4-in -
d olin eca r boxa m id e (20:3 E:Z isom er r a tio) (40). 1H-
Indole-2,3-dione-4-carboxamide was prepared from aniline-3-
carboxamide according to procedure A in 3% yield: 1H NMR
4-({[5-(Dim eth yla m in o)-2-oxo-1,2-d ih yd r o-3H-in d ol-3-
yliden e]m eth yl}am in o)ben zen esu lfon am ide (48). The title

4352 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Bramson et al.
compound was prepared from 5-(dimethylamino)-3-(ethoxy-
methylene)-1,3-dihydro-2H-indol-2-one and sulfanilimide using
procedure G: 1H NMR (DMSO-d6): δ 10.78 (d, J ) 12.2 Hz,-
1H), 10.20 (s, 1H), 8.57 (d, J ) 12.1 Hz, 1H), 7.75 (d, J ) 8.6
Hz, 2H), 7.48 (d, J ) 8.8 Hz, 2H), 7.23 (s, 2H), 7.18 (s, 1H),
6.66 (d, J ) 8.4 Hz, 1H), 6.48 (d, J ) 8.5 Hz, 1H), 2.82 (s, 6H).
APCI m/z 357 (M-1). Anal. (C17H18N4O3S‚1.4H2O) C, H, N.
4-[N ′-(5-Me t h ylsu lfon yl-2-oxo-1,2-d ih yd r o-in d ol-3-
ylid en e)-h yd r a zin o]ben zen esu lfon a m id e (49). 5-Methyl-
sulfonyl-1H-indole-2,3-dione was prepared from 4-methylsul-
fonylaniline according to procedure A: 1H NMR (DMSO-d6):
δ 3.21 (s, 3H), 7.07 (d, J ) 8.3 Hz, 1H), 7.92 (d, J ) 1.7 Hz,
1H), 8.05 (dd, J ) 8.2, 2.0 Hz, 1H), 11.46 (s,1H); APCI m/z
225 (M)-. The title compound was prepared from 5-methyl-
sulfonyl-1H-indole-2,3-dione and 4-hydrazinobenzenesulfona-
mide hydrochloride according to procedure E: mp >250 °C;
1H NMR (DMSO-d6): δ 3.20 (s, 3H), 7.11 (d, J ) 8.3 Hz, 1H),
7.26 (s, 2H), 7.65 (d, J ) 8.9 Hz, 2H), 7.78 (d, J ) 8.7 Hz, 2H),
7.79 (dd, J ) 8.2, 1.9 Hz, 1H), 8.06 (d, J ) 1.6 Hz, 1H), 11.54
(s, 1H), 12.75 (s, 1H); APCI m/z 394 (M)-. Anal. (C15H14N4O5S2‚
0.9 H2O) C, H, N, S.
2-Oxo-3-[(4-su lfam oyl-ph en yl)-h ydr azon o]-2,3-dih ydr o-
1H-in d ole-5-su lfon ic a cid sod iu m sa lt (51). The title
compound was prepared from 1H-indole-2,3-dione-5-sulfonic
acid and 4-hydrazinobenzenesulfonamide according to proce-
dure E: 1H NMR (DMSO-d6): δ 6.83 (d, J ) 8.0 Hz, 1H), 7.22
(s, 2H), 7.50 (dd, J ) 1.7, 8.0 Hz, 1H), 7.56 (d, J ) 8.7 Hz,
2H), 7.76 (d, J ) 8.7 Hz, 2H), 7.77 (d, J ) 1.7 Hz, 1H), 11.12
(s, 1H), 12.70 (s, 1H); APCI: m/z 395 (M-H)-. Anal. (C14H11N4-
O6S2Na‚0.9 H2O‚0.2 C2H6O) C, H, N S.
3-{[4-(Am in osu lfon yl)a n ilin o]m eth ylen e}-2-oxo-2,3-d i-
h yd r o-1H-in d ole-5-ca r boxylic a cid (52). The title com-
pound was prepared from 3-[(dimethylamino)methylene]-2-
oxo-2,3-dihydro-1H-indole-5-carboxylic acid and sulfanilamide
according to procedure G: 1H NMR (DMSO-d6): δ 6.91 (d, J
) 8.1 Hz, 1H), 7.26 (s, 2H), 7.60 (d, J ) 8.8 Hz, 2H), 7.68 (d,
J ) 8.1 Hz, 1H), 7.77 (d, J ) 8.8 Hz, 2H), 8.29 (s, 1H), 8.85 (d,
J ) 12.3 Hz, 1H), 10.79 (d, J ) 12.3 Hz, 1H), 10.90 (s, 1H),
12.4 (br s, 1H).
correlation was observed between the C-4 proton at 8.27 ppm
and the enamino vinylic proton at 8.86 ppm, consistent with
the Z configuration of the enamino double bond; APCI m/z 414.
4-({[5-(3-Meth ylbu ta n oyl)-2-oxo-1,2-d ih yd r o-3H-in d ol-
3-ylid en e]m et h yl}a m in o)b en zen esu lfon a m id e (55). 3-
[(Dimethylamino)methylene]-5-(3-methylbutanoyl)-1,3-dihydro-
2H-indol-2-one was prepared according to procedure F and
condensed with sulfanilamide as in procedure G to provide the
title compound: 1H NMR (DMSO-d6): 10.93 (s, 1H), 10.79 (d,
J ) 12.5 Hz, 1H), 8.83 (d, J ) 12.5 Hz, 1H), 8.28 (s, 1H), 7.77
(d, J ) 8.8 Hz, 2H), 7.71 (dd, J ) 8.1, 1.3 Hz, 1H), 7.59 (d, J
) 8.8 Hz, 2H), 7.26 (s, 2H), 6.91 (d, J ) 8.1 Hz, 1H), 2.84 (d,
J ) 6.8 Hz, 2H), 2.16 (m, 1H), 0.92 (d, J ) 6.8 Hz, 6H). APCI
m/z 398 (M-H)-. Anal. (C20H21N3O4S) C, H, N.
3-{[4-(Am in osu lfon yl)p h en yl]h yd r a zon o}-2-oxo-2,3-d i-
h yd r o-1H-in d ole-5-ca r boxa m id e (56). The title compound
was prepared from 2,3-dioxo-5-indolinecarboxamide and 4-hy-
drazinobenzenesulfonamide according to procedure E: 1H
NMR (DMSO-d6): δ 6.90 (d, J ) 8.2 Hz, 1H), 7.22 (s, 2H), 7.36
(s, 1H), 7.57 (d, J ) 8.7 Hz, 2H), 7.79 (d, J ) 8.7, 2H), 7.84 (d,
J ) 7.4 Hz, 2H), 8.57 (s, 1H), 10.88 (s, 2H); APCI: m/z 358
(M-H)-. Anal. (C15H13N5O4S) C, H, N, S.
3-{[4-(Am in osu lfon yl)p h en yl]h yd r a zon o}-N-[2-(1H-im -
id a zol-4-yl)eth yl]-2-oxo-2,3-d ih yd r o-1H-in d ole-5-ca r box-
a m id e (58). The title compound was prepared from 2-oxo-3[(4-
su lfa m oyl-ph en yl)-h ydr a zon o]-2,3-dih ydr o-1H -in dole-5-
carboxylic acid pentafluorophenyl ester and 2-(1H-imidazol-
4-yl)ethylamine according to procedure H: mp >230 °C; 1H
NMR (DMSO-d6): δ 2.75 (t, J ) 7.4 Hz, 2H), 3.48 (m, 2H),
6.81 (s, 1H), 6.97 (d, J ) 7.2 Hz, 1H), 7.25 (s, 2H), 7.54 (s,
1H), 7.60 (d, J ) 8.6 Hz, 2H), 7.79 (m, 3H), 8.08 (s, 1H), 8.57
(br t, J ) 6 Hz, 1H), 11.31 (s, 1H), 11.88 (br s, 1H), 12.76 (s,
1H). Anal. (C20H18N7O4S) C, H, N.
3-{[4-(Am in osu lfon yl)p h en yl]h yd r a zon o}-N-[3-(1H-im -
id a zol-1-yl)p r op yl]-2-oxo-2,3-d ih yd r o-1H -in d ole-5-ca r -
boxa m id e (59). The title compound was prepared from 2-oxo-
3[(4-sulfamoyl-phenyl)-hydrazono]-2,3-dihydro-1H-indole-5-
carboxylic acid pentafluorophenyl ester and 3-(1H-imidazol-
1
1-yl)propylamine according to procedure H: mp >230 °C; H
NMR (DMSO-d6): δ 1.96 (p, J ) 6.7 Hz, 2H), 3.24 (m, 2H),
4.01 (t, J ) 6.8 Hz, 2H), 6.88 (s, 1H), 6.97 (d, J ) 8.1 Hz, 1H),
7.21 (s, 1H), 7.25 (s, 2H), 7.60 (d, J ) 8.7 Hz, 2H), 7.66 (s,
1H), 7.79 (d, J ) 8.7 Hz, 2H), 7.81 (d, J ) 8.1 Hz, 1H), 8.08 (s,
1H), 8.51 (t, J ) 5.5 Hz, 1H), 11.32 (s, 1H), 12.77 (s, 1H). Anal.
(C21H21N7O4S‚1/2H2O) C, H, N.
Isobu tyl 3-{[4-(a m in osu lfon yl)a n ilin o]m eth ylen e}-2-
oxo-2,3-dih ydr o-1H-in dole-5-car boxylate (54). 3-Methylthio-
2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid isobutyl ester
was prepared in 59% yield from isobutyl 4-aminobenzoate
using the procedures of Gassman and van Bergen:28 1H NMR
(DMSO-d6): δ 0.93 (d, J ) 6.6 Hz, 6H), 1.93 (s, 3H), 1.98
(septet, J ) 6.6 Hz, 1H), 4.02 (m, 2H), 4.62 (s, 1H), 6.92 (d, J
) 8.2 Hz, 1H), 7.79 (s, 1H), 7.86 (d, J ) 8.2 Hz, 1H), 10.91 (s,
1H); ESI m/z 302 (M+Na)+. Zinc reduction of 3-methylthio-2-
oxo-2,3-dihydro-1H-indole-5-carboxylic acid isobutyl ester ac-
cording to the method of Gassman and van Bergen28 provided
2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid isobutyl ester in
99% yield: 1H NMR (DMSO-d6): δ 0.93 (d, J ) 6.6 Hz, 6H),
1.97 (septet, J ) 6.6 Hz, 1H), 3.53 (s, 2H), 3.99 (d, J ) 6.6 Hz,
2H), 6.88 (d, J ) 8.2 Hz, 1H), 7.75 (s, 1H), 7.82 (d, J ) 8.2 Hz,
1H), 10.72 (s, 1H); ESI m/z 256 (M+Na)+. Conversion of 2-oxo-
2,3-dihydro-1H-indole-5-carboxylic acid isobutyl ester to 3-[(dim-
ethylamino)methylene]-2-oxo-2,3-dihydro-1H-indole-5-carbox-
ylic acid isobutyl ester (mixture of E and Z isomers) was
accomplished in 75% yield according to procedure E: 1H NMR
(DMSO-d6): δ 0.94 Z (d, J ) 8.8 Hz, 6H), 0.94 E (d, J ) 8.8
Hz, 6H), 1.94-2.01 Z and E (m, 2H), 3.30 Z (s, 6H), 3.32 E (s,
6H), 3.97-3.99 Z and E (m, 4H), 6.75 Z (d, J ) 8.2 Hz, 1H),
6.83 E (d, J ) 8.2 Hz, 1H), 7.47 E (s, 1H), 7.53 Z (d, J ) 0.8.2
Hz, 1H), 7.59 E (d, J ) 8.2 Hz, 1H), 7.73 Z (s, 1H), 7.88 Z (s,
1H), 7.98 E (s, 1H), 10.34 Z (bs, 1H), 10.44 E (bs, 1H); ESI
m/z 289 (M+H)+. The title compound was prepared in 66%
yield from 3-[(dimethylamino)methylene]-2-oxo-2,3-dihydro-
1H-indole-5-carboxylic acid isobutyl ester and 4-aminobenzene-
sulfonamide hydrochloride according to procedure G: 1H NMR
(DMSO-d6): δ 0.96 (d, J ) 6.6 Hz, 6H), 2.01 (septet, J ) 6.6
Hz, 1H), 4.04 (d, J ) 6.6 Hz, 2H), 6.93 (d, J ) 8.2 Hz, 1H),
7.26 (s, 2H), 7.60 (d, J ) 8.7 Hz, 2H), 7.71 (dd, J ) 1.6, 8.2
Hz, 1H), 7.76 (d, J ) 8.7 Hz, 2H), 8.27 (s, 1H), 8.86 (d, J )
12.5 Hz, 1H), 10.83 (d, J ) 12.5 Hz, 1H), 10.95 (s, 1H). NOESY
3-{[4-(Am in osu lfon yl)p h en yl]h yd r a zon o}-2-oxo-N-(4-
p y r id in y lm e t h y l)-2,3-d ih y d r o -1H -in d o le -5-c a r b o x -
a m id e) (60). The title compound was prepared from 2-oxo-
3[(4-sulfamoyl-phenyl)-hydrazono]-2,3-dihydro-1H-indole-5-
carboxylic acid pentafluorophenyl ester and (4-pyridyl)-
1
methylamine according to procedure H: mp 211-215 °C; H
NMR (DMSO-d6): δ 4.46 (d, J ) 5.9 Hz, 2H), 6.97 (d, J ) 8.2
Hz, 1H), 7.21 (s, 2H), 7.27 (d, J ) 5.6 Hz, 2H), 7.56 (d, J ) 8.7
Hz, 2H), 7.75 (d, J ) 8.7 Hz, 2H), 7.83 (dd, J ) 1.3, 8.2 Hz,
1H), 8.13 (s, 1H), 8.46 (d, J ) 5.6 Hz, 2H), 9.09 (t, J ) 5.9 Hz,
1H), 11.31 (s, 1H), 12.73 (s, 1H). Anal. (C21H18N6O4S‚H2O) C,
H, N.
3-{[4-(Am in osu lfon yl)p h en yl]h yd r a zon o}-2-oxo-N-(3-
p y r id in y lm e t h y l)-2,3-d ih y d r o -1H -in d o le -5-c a r b o x -
a m id e (61). The title compound was prepared from 2-oxo-3[(4-
sulfamoyl-phenyl)-hydrazono]-2,3-dihydro-1H-indole-5-carbox-
ylic acid pentafluorophenyl ester and (3-pyridyl)methylamine
according to procedure H: mp 211-215 °C; 1H NMR (DMSO-
d6): δ 4.46 (d, J ) 5.7 Hz, 2H), 6.95 (d, J ) 8.2 Hz, 1H), 7.21
(s, 2H), 7.31 (dd, J ) 4.5, 7.8 Hz, 1H), 7.56 (d, J ) 8.7 Hz,
2H), 7.68 (d, J ) 7.8 Hz, 1H), 7.75 (d, J ) 8.7 Hz, 2H), 7.81
(dd, J ) 1.5, 8.2 Hz, 1H), 8.10 (d, J ) 1.5 Hz, 1H), 8.41 (d, J
) 4.5 Hz, 1H), 8.51 (s, 1H), 9.06 (t, J ) 5.7 Hz, 1H), 11.30 (s,
1H), 12.73 (s, 1H). Anal. (C21H18N6O4S‚H2O) C, H, N.
3-{[4-(Aa m i n o s u lfo n y l)p h e n y l]h y d r a z o n o }-N -(3-
h yd r oxy-2,2-d im et h ylp r op yl)-2-oxo-5-in d olin eca r b ox-
a m id e (62). The title compound was prepared from 2-oxo-3[(4-
sulfamoyl-phenyl)-hydrazono]-2,3-dihydro-1H-indole-5-carbox-
ylic acid pentafluorophenyl ester and 3-hydroxy-2,2-dimeth-

Oxindole-Based Inhibitors of CDK2
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4353
ylpropylamine according to procedure H: mp >230 °C; 1H
NMR (DMSO-d6): δ 0.79 (s, 6H), 3.07 (d, J ) 6.2 Hz, 2H), 3.10
(d, J ) 6.2 Hz, 2H), 4.59 (t, J ) 6.2, 1H), 6.94 (d, J ) 8.2 Hz,
1H), 7.21 (s, 2H), 7.56 (d, J ) 8.8 Hz, 2H), 7.76 (d, J ) 8.8 Hz,
2H), 7.79 (d, J ) 1.3 Hz, 1H), 8.05 (d, J ) 1.3 Hz, 1H), 8.39 (t,
J ) 6.2 Hz, 1H), 11.29 (s, 1H), 12.75 (s, 1H). Anal. (C20H23N5O5S)
C, H, N.
silyloxy)methyl-1,3-dihydro-indol-2-one as a gummy white
solid: 1H NMR (DMSO-d6): δ 0.04 (s, 6H), 0.87 (s, 9H), 3.39
(s, 2H), 4.62 (s, 2H), 6.75 (s, 1H), 6.81 (d, J ) 7.5 Hz, 1H),
7.10 (d, J ) 7.5 Hz, 1H), 10.30 (bs, 1H). A solution of 0.32 g
(1.2 mmol) of 4-(tert-butyldimethylsilyloxy)methyl-1,3-dihydro-
indol-2-one in DMF dimethylacetal (3 mL) was heated to 100
°C for 0.75 h. The excess DMF dimethylacetal was removed
under high vacuum, and the resulting dark oil was chromato-
graphed on silica gel, eluting with EtOAc/MeOH (98:2), to give
0.16 g (41%) of 3-dimethylaminomethylene-6-(tert-butyldim-
ethylsilyloxy)methyl-1,3-dihydro-indol-2-one (11:9 mixture of
E and Z isomers) as a yellow solid: 1H NMR (DMSO-d6, peak
areas normalized using the combined peak areas for δ 9.88
and 9.66 as 1H): δ 0.21 (s, 2.70H), 0.34 (s, 3.3H), 0.85 (s,
4.05H), 0.86 (s, 4.95H), 3.25 (s, 2.70H), 3.30 (s, 3.30H), 4.58
(s, 0.9H), 4.59 (s, 1.1H), 6.64-6.71 (m, 2H), 7.16 (d, J ) 7.7
Hz, 0.45H), 7.29 (d, J ) 8.3 Hz, 0.55H), 7.33 (s, 0.55H), 7.47
(s, 0.45H), 9.88 (s,0.55H) 9.96 (s, 0.45H); APCI m/z 331 (M+1)+.
A solution of 0.334 g (1.00 mmol) of 3-(dimethylamino)-
methylene-6-(tert-butyldimethylsilyloxy)methyl-1,3-dihydro-in-
dol-2-one in 2-methylpropanol (3 mL) was treated with 0.174
g (1.00 mmol) of sulfanilamide and 0.25 g (4.0 mmol) of acetic
acid. The solution was refluxed for 3 h and cooled to room
temperature. The resulting yellow precipitate was isolated by
filtration, washed with ethanol, and dried to yield 0.134 g
(29%) of 6-([tert-butyldimethyl-silyloxy]methyl-2-oxo-1,2-dihy-
dro-indol-3-ylidenemethyl)-amino]-benzenesulfonamide (Z iso-
mer): 1H NMR (DMSO-d6): δ 0.05 (s, 6H), 0.87 (s, 9H), 4.65
(s, 2H), 6.81 (s, 1H), 6.85 (d, J ) 8.0 Hz, 1H), 7.23 (s, 2H),
7.49-7.51 (m, 3H), 7.75 (d, J ) 8.4 Hz, 2H), 8.56 (d, J ) 12.3
Hz, 1H), 10.52 (s, 1H), 10.76 (d, J ) 12.3 Hz, 1H); APCI m/z
458 (M-H)-. To a solution of 0.125 g (2.80 mmol) of 6-([tert-
butyldimethylsilyloxy]methyl-2-oxo-1,2-dihydro-indol-3-yli-
denemethyl)-amino]-benzenesulfonamide in THF (5 mL) was
added 0.27 mL of a 1 M solution of tert-butylammonium
fluoride in THF, and the mixture was stirred at room tem-
perature for 1 h. The resulting yellow precipitate was isolated
by filtration, washed with THF, and dried. Chromatographic
purification of the solid on silica gel, eluting with a hexane to
EtOAc gradient, gave 0.053 g (55%) of the title compound: 1H
NMR (DMSO-d6): δ 4.43 (d, J ) 5.8 Hz, 2H), 5.08 (t, J ) 5.8
Hz, 1H), 6.82 (s, 1H), 6.85 (d, J ) 8.2 Hz, 1H), 7.23 (s, 2H),
7.50 (d, J ) 7.5 Hz, 2H), 7.74 (d, J ) 8.7 Hz, 3H), 8.56 (d, J )
12.2 Hz, 1H), 10.54 (s, 1H), 10.75 (d, J ) 12.1 Hz, 1H); APCI
m/z 345 (M-H)-. Anal. (C16H15N3O4S‚1/2H2O) C, H, N, S.
3-{[4-(Am in o s u lfo n y l)p h e n y l]h y d r a zo n o }-N -(2,6-
dim eth oxyben zyl)-2-oxo-5-in dolin ecar boxam ide (63). The
title compound was prepared from 2-oxo-3[(4-sulfamoyl-phen-
yl)-hydrazono]-2,3-dihydro-1H-indole-5-carboxylic acid pen-
tafluorophenyl ester and 2,6-dimethoxybenzylamine according
to procedure H: mp > 250 °C; 1H NMR (DMSO-d6): δ 3.76 (s,
6H), 4.43 (d, J ) 4.2 Hz, 2H), 6.65 (d, J ) 8.4 Hz, 2H), 6.91 (d,
J ) 8.2 Hz, 1H), 7.23 (s, 2H), 7.25 (d, J ) 8.2 Hz, 1H), 7.56 (d,
J ) 8.6 Hz, 2H), 7.79 (m, 3H), 8.07 (s, 1H), 8.13 (br s, 1H),
11.27 (s, 1H), 12.76 (s, 1H); APCI m/z 532 (M+Na)+; Anal.
(C24H23N5O6S‚1/2H2O)
C, H, N, S.
4-[N′-(6-Br om o-2-oxo-1,2-d ih yd r o-in d ol-3-ylid en e)-h y-
d r a zin o]ben zen esu lfon a m id e (64). The title compound was
prepared from 6-bromo-1H-indole-2,3-dione49 and 4-hydrazi-
nobenzenesulfonamide hydrochloride according to procedure
1
E: mp >250 °C; H NMR (DMSO-d6): δ 7.05 (s, 1H), 7.23 (d,
J ) 8.1 Hz, 1H), 7.50 (d, J ) 8.1 Hz, 1H), 7.56 (d, J ) 8.7 Hz,
2H), 7.75 (d, J ) 8.7 Hz, 2H), 11.2 (s, 1H), 12.7 (s, 1H); APCI
m/z 395 (M-H)-.
4-[N′-(6-E t h yl-2-oxo-1,2-d ih yd r o-in d ol-3-ylid en e)-h y-
d r a zin o]ben zen esu lfon a m id e (65). The title compound was
prepared from 6-ethyl-1H-indole-2,3-dione44 and 4-hydrazino-
benzenesulfonamide hydrochloride according to procedure E
in 79% yield: 1H NMR (DMSO-d6): δ 1.16 (t, J ) 7.5 Hz, 3H),
2.60 (q, J ) 7.5 Hz, 2H), 6.74 (s, 1H), 6.89 (d, J ) 7.5 Hz, 1H),
7.22 (s, 2H), 7.46 (d, J ) 7.5 Hz, 1H), 7.50 (d, J ) 8.7 Hz, 1H),
7.75 (d, J ) 8.7 Hz, 2H), 11.02 (s, 1H), 12.70 (s, 1H); APCI
m/z 343 (M-H)-. Anal. (C16H16N4O3S‚0.32 H2O) C, H, N, S.
4-[2-(6-Isopr opyl-2-oxo-1,2-dih ydr o-3H-in dol-3-yliden e)-
h yd r a zin o]ben zen esu lfon a m id e (66). The title compound
was prepared from 6-isopropyl-1H-indole-2,3-dione and 4-hy-
drazinobenzenesulfonamide hydrochloride according to pro-
cedure E: 1H NMR (DMSO-d6): δ 1.17 (d, J ) 6.9 Hz, 6H),
2.88 (septet, J ) 6.9 Hz, 1H), 6.75 (s, 1H), 6.92 (d, J ) 7.8 Hz,
1H), 7.21 (s, 2H), 7.46 (d, J ) 7.8 Hz, 1H), 7.50 (d, J ) 8.7 Hz,
2H), 7.74 (d, J ) 8.7 Hz, 2H), 11.01 (s, 1H), 12.71 (s, 1H); APCI
m/z 357 (M-H)-.
4-[2-(6-ter t-Bu tyl-2-oxo-1,2-dih ydr o-3H-in dol-3-yliden e)-
h yd r a zin o]ben zen esu lfon a m id e (67). The title compound
was prepared from 6-tert-butyl-1H-indole-2,3-dione and 4-hy-
drazinobenzenesulfonamide hydrochloride according to pro-
cedure F: 1H NMR (DMSO-d6): δ 1.26 (s, 9H), 6.88 (d, J )
1.4 Hz, 1H), 7.09 (dd, J ) 1.4, 8.0 Hz, 1H), 7.21 (s, 2H), 7.47
(d, J ) 8.0 Hz, 1H), 7.50 (d, J ) 8.7 Hz, 2H), 7.75 (d, J ) 8.7
Hz, 2H), 10.97 (s, 1H), 12.72 (s, 1H); AP-MS m/z 371 (M-H)-.
Anal.(C18H20N4O3S) C, H, N, S.
4-[N′-(2-Oxo-6-p h en oxy-1,2-d ih yd r o-in d ol-3-ylid en e)-
h yd r a zin o]ben zen esu lfon a m id e (69). The title compound
was prepared from 6-phenoxy- and 4-hydrazinobenzenesulfona-
mide according to procedure E in 87% yield: mp >250 °C; 1H
NMR (DMSO-d6): δ 6.42 (d, J ) 2.2 Hz, 1H), 6.73 (dd, J 1
)
2.2 Hz, J 2 ) 8.5 Hz, 1H), 7.17 (d, J ) 8 Hz, 2H), 7.25 (s, 1H),
7.28 (d, J ) 7.4 Hz, 2H), 7.49 (t, J ) 7.9 Hz, 2H), 7.73 (d, J )
8.8 Hz, 2H), 7.82 (d, J ) 8.8 Hz, 2H), 8.25 (d, J ) 8.5 Hz, 2H),
10.61 (s, 1H), 10.65 (s, 1H); APCI: m/z 431 (M+Na)+. Anal.
(C20H16N4O4S‚1/4H2O) C, H, N, S.
4-[(6-H yd r oxym e t h yl-2-oxo-1,2-d ih yd r o-in d ol-3-yli-
d en em eth yl)-a m in o]ben zen esu lfon a m id e (68). A solution
of 0.42 g (2.0 mmol) of 6-hydroxymethyl-3-methysulfanyl-1,3-
dihydro-indol-2-one in DMF (10 mL) was treated with 0.32 g
(2.1 mmol) of tert-butyldimethylsilyl chloride and 0.15 g (2.2
mmol) of imidazole and stirred for 16 h. The solution was
diluted with 50 mL of hexane and 50 mL of EtOAc, washed
with brine, dried over MgSO4 and concentrated to give 0.28 g
(43%) of 3-methylsulfanyl-6-(tert-butyldimethylsilyloxy)methyl-
1,3-dihydro-indol-2-one as a clear oil which crystallized upon
storage at room temperature: 1H NMR (DMSO-d6): δ 0.01 (s,
6H), 0.97 (s, 9H), 2.00 (s, 3H), 4.52 (s, 1H), 4.72 (s, 2H), 6.85
(s, 1H), 6.96 (d, J ) 7.7 Hz, 1H), 7.25 (d, J ) 7.7 Hz, 1H),
10.54 (s, 1H). A solution of 0.28 g (0.86 mmol) of 3-methylsul-
fanyl-4-(tert-butyldimethylsilyloxy)methyl-1,3-dihydro-indol-2-
one in THF (10 mL) was stirred with saturated ammonium
chloride solution (10 mL), and activated zinc dust (2 g) was
added. The mixture was stirred 16 h at room temperature.
The organic phase was separated, dried over MgSO4, and
concentrated to give 0.32 g of impure 4-(tert-butyldimethyl-
4-[2-(7-Met h yl-2-oxo-1,2-d ih yd r o-3H-in d ol-3-ylid en e)-
h yd r a zin o]ben zen esu lfon a m id e (70). The title compound
was prepared from 7-methyl-1H-indole-2,3-dione and 4-hy-
drazinobenzenesulfonamide hydrochloride according to pro-
cedure E: 1H NMR (DMSO-d6): δ 2.21 (s, 3H), 6.98 (t, J ) 7.5
Hz, 1H), 7.10 (d, J ) 7.5 Hz, 1H), 7.24 (s, 2H), 7.41 (d, J ) 7.5
Hz, 1H), 7.56 (d, J ) 8.8 Hz, 2H), 7.78 (d, J ) 8.8 Hz, 2H),
11.14 (s, 1H), 12.81 (s, 1H); APCI: m/z 329 (M-H)-. Anal.
(C15H14N4O3S) C, H, N, S.
4-[2-(5,7-Dim eth yl-2-oxo-1,2-dih ydr o-3H-in dol-3-yliden e)-
h yd r a zin o]ben zen esu lfon a m id e (71). The title compound
was prepared from 5,7-dimethyl-1H-indole-2,3-dione and 4-hy-
drazinobenzenesulfonamide hydrochloride according to pro-
cedure E: 1H NMR (DMSO-d6): δ 2.19 (s, 3H), 2.27 (s, 3H),
6.92 (s, 1H), 7.24 (s, 3H), 7.55 (d, J ) 8.8 Hz, 2H), 7.77 (d, J
) 8.8 Hz, 2H), 11.03 (s, 1H), 12.79 (s, 1H); APCI: m/z 343
(M-H)-. Anal. (C16H16N4O3S) C, H, N, S.
4-[2-(5-Ch lor o-7-m eth yl-2-oxo-1,2-d ih yd r o-3H-in d ol-3-
ylid en e)h yd r a zin o]ben zen esu lfon a m id e (72). The title

4354 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Bramson et al.
compound was prepared from 5-chloro-7-methyl-1H-indole-2,3-
dione and 4-hydrazinobenzenesulfonamide hydrochloride ac-
cording to procedure E: 1H NMR (DMSO-d6): δ 2.23 (s, 3H),
7.17 (d, J ) 1.8 Hz, 1H), 7.26 (s, 2H), 7.45 (d, J ) 1.8 Hz, 1H),
7.62 (d, J ) 8.7 Hz, 2H), 7.78 (d, J ) 8.7 Hz, 2H), 11.26 (s,
1H), 12.78 (s, 1H); APCI: m/z 364 M-. Anal. (C15H13N4O3SCl)
C, H, N, Cl, S.
4-[2-(4-Ch lor o-5-m eth yl-2-oxo-1,2-d ih yd r o-3H-in d ol-3-
ylid en e)h yd r a zin o]ben zen esu lfon a m id e (73). The title
compound was prepared from 4-chloro-5-methyl-1H-indole-2,3-
dione and 4-hydrazinobenzenesulfonamide hydrochloride ac-
cording to procedure E: 1H NMR (DMSO-d6): δ 2.32 (s, 3H),
6.80 (d, J ) 8 Hz, 1H), 7.22 (d, J ) 8 Hz, 1H), 7.25 (s, 2H),
7.56 (d, J ) 8.6 Hz, 2H), 7.79 (d, J ) 8.6 Hz, 2H), 11.20 (s,
1H), 13.00 (s, 1H); APCI: m/z 363 (m-H)-. Anal. (C15H13N4O3-
SCl) C, H, N, S, Cl.
4-[2-(4-Ch lor o-5-m eth oxy-2-oxo-1,2-d ih yd r o-3H-in d ol-
3-ylid en e)h yd r a zin o]ben zen esu lfon a m id e (74). The title
compound was prepared from 4-chloro-5-methoxy-1H-indole-
2,3-dione and 4-hydrazinobenzenesulfonamide hydrochloride
according to procedure E: 1H NMR (DMSO-d6): δ 3.86 (s, 3H),
6.87 (d, J ) 8.5 Hz, 1H), 7.07 (d, J ) 8.5 Hz, 1H), 7.30 (s, 2H),
7.60 (d, J ) 8.6 Hz, 2H), 7.83 (d, J ) 8.6 Hz, 2H), 11.15 (s,
1H), 13.09 (s, 1H); APCI: m/z 379 (M-H)-. Anal. (C15H13N4O4-
SCl) C, H, N, S, Cl.
4-[2-(4-Met h yl-5-n it r o-2-oxo-1,2-d ih yd r o-3H -in d ol-3-
ylid en e)h yd r a zin o]ben zen esu lfon a m id e (75). The title
compound was prepared from 4-methyl-5-nitro-1H-indole-2,3-
dione and 4-hydrazinobenzenesulfonamide hydrochloride ac-
cording to procedure E: 1H NMR (DMSO-d6): δ 2.79 (s, 3H),
6.90 (d, J ) 8.6 Hz, 1H), 7.24 (s, 2H), 7.52 (d, J ) 8.7 Hz, 2H),
7.62 (d, J ) 8.7 Hz, 2H), 7.77 (d, J ) 8.6 Hz, 1H), 7.90 (d, J )
8.6 Hz, 1H), 11.60 (s, 1H), 13.03 (s, 1H); ES-MS m/z 374 (M-
H)-. Anal. (C15H13N5O5S‚1/2H2O) C, H, N, S.
4-[N′-(7-Oxo-6,7-d ih yd r o-3H -p yr r olo[3,2-e]in d a zol-8-
ylid en e)-h yd r a zin o]ben zen esu lfon a m id e (76). The title
compound was prepared from 3,6-dihydro-pyrrolo[3,2-e]inda-
zole-7,8-dione50 and 4-hydrazinobenzenesulfonamide hydro-
chloride according to procedure E in 8% yield: 1H NMR
(DMSO-d6): δ 7.02 (d, J ) 8.7 Hz, 1H), 7.28 Z (s, 2H), 7.51 (d,
J ) 8.6 Hz, 2H), 7.68 (d, J ) 8.8 Hz, 1H), 7.82 (d, J ) 8.7 Hz,
2H), 8.34 (s, 1H), 10.98 (s, 1H), 12.90 (s, 1H), 13.20 (s, 1H);
APCI m/z 356 (M)-. Anal. (C15H12N6O3S‚1.46 H2O‚0.2 C4H8O2)
C, H, N, S.
4-[N′-(1-Ch lor o-7-oxo-6,7-dih ydr o-3H-pyr r olo[3,2-e]in da-
zol-8-yliden e)-h ydr azin o]ben zen esu lfon am ide (77). 1-Chlo-
ro-3,6-dihydro-pyrrolo[3,2-e]indazole-7,8-dione was prepared
from 5-amino-3-chloroindazole according to procedure A in 38%
yield: 1H NMR (DMSO-d6): δ 7.08 (d, J ) 7.9 Hz, 1H), 7.92
(d, J ) 7.9 Hz, 1H), 10.95 (s, 1H), 13.70 (s, 1H). Condensation
of 1-chloro-3,6-dihydro-pyrrolo[3,2-e]indazole-7,8-dione and
4-hydrazinobenzenesulfonamide hydrochloride according to
procedure E gave the title compound in 45% yield: 1H NMR
(DMSO-d6): δ 7.11 (d, J ) 8.8 Hz, 1H), 7.26 (s, 2H), 7.51 (d, J
) 8.8 Hz, 1H), 7.64 (d, J ) 8.8 Hz, 2H), 7.82 (d, J ) 8.8 Hz,
2H), 11.17 (s, 1H), 13.25 (s, 1H), 13.41 (s, 1H): APCI m/z 389/
391 (M-H)-. Anal. (C15H11ClN6O3S) C, H, N, S.
4-[2-(7-Oxo-6,7-dih ydr o[1,2,3]tr iazolo[4,5-e]in dol-8(3H)-
ylid en e)h yd r a zin o]b en zen esu lfon a m id e (m ixt u r e of E
a n d Z isom er s) (78). 1,6-Dihydro[1,2,3]triazolo[4,5-e]indole-
7,8-dione was prepared according to procedure A in 56%
yield: 1H NMR (DMSO-d6): δ 6.93 (d, J ) 8.6 Hz, 1H), 8.32
(d, J ) 8.6 Hz, 1H), 11.14 (s, 1H); APCI m/z 189 (M+1)+.
Condensation of 1,6-dihydro[1,2,3]triazolo[4,5-e]indole-7,8-di-
one with 4-hydrazinobenzenesulfonamide hydrochloride ac-
cording to procedure E gave the title compound in 15% yield:
1H NMR (DMSO-d6): δ 7.06 Z (d, J ) 8.4 Hz, 1H), 7.24 E (d,
J ) 8.4 Hz, 1H), 7.30 Z (s, 2H), 7.30 E (s, 2H), 7.55 E (d, J )
8.5 Hz, 2H), 7.82 Z (d, J ) 8.5 Hz, 2H), 7.82 E (d, J ) 8.5 Hz,
1H), 7.90 E (d, J ) 8.7 Hz, 2H), 7.90 Z (d, J ) 8.8 Hz, 2H),
7.98 Z (d, J ) 8.4 Hz, 1H), 10.86 E (s, 1H), 11.35 Z (s, 1H),
12.87 Z (s, 1H), 12.95 E (s, 1H), 16.00 Z (s, 1H), 16.25 E (s,
1H); APCI m/z 356 (M-H)-. Anal. (C14H11N7O3S‚H2O) C, H, N,
S.
4-[2-(7-Oxo-6,7-d ih yd r o-8H-[1,3]th ia zolo[5,4-e]in d ol-8-
ylid en e)h yd r a zin o]ben zen esu lfon a m id e (79). The title
compound was prepared from 6H-[1,3]thiazolo[5,4-e]indole-7,8-
dione and 4-hydrazinobenzene sulfonamide hydrochloride ac-
cording to procedure E: 1H NMR (DMSO-d6): δ 7.11 (d, J )
8.6 Hz,1H), 7.26 (s, 2H), 7.59 (d, J ) 8.7 Hz, 2H), 7.81 (d, J )
8.7 Hz, 2H), 7.97 (d, J ) 8.4 Hz, 1H), 9.29 (s, 1H), 11.24 (s,
1H), 12.67 (s, 1H); APCI m/z 372 (M-H)-. Anal. (C15H11N5O3S2)
C, H, N, S.
4-{[(7-oxo-6,7-d ih yd r o-8H -[1,3]t h ia zolo[5,4-e]in d ol-8-
yliden e)m eth yl]am in o}ben zen esu lfon am ide (80). The title
compound was prepared from 8-(ethoxymethylene)-6,8-dihy-
dro-7H-[1,3]thiazolo[5,4-e]indol-7-one and sulfanilimide in 94%
yield (contaminated with 8% 8-(ethoxymethylene)-6,8-dihydro-
7H-[1,3]thiazolo[5,4-e]indol-7-one) according to procedure G:
1H NMR (DMSO-d6): δ 7.12 (d, J ) 8.3 Hz, 1H), 7.31 (s, 2H),
7.58 (d, J ) 9.0 Hz, 2H), 7.82 (m, 3H), 8.07 (d, J ) 8.3 Hz,
1H), 9.26 (s, 1H), 10.92 (s, 1H), 11.16 (d, J ) 12.1 Hz, 1H).
Anal. (C16H12N4O3S‚0.08 C12H10N2O2S‚1/3C2H6O) C, H, N, S.
4-[N′-2-Oxo-2,3-dih ydr opyr r olo[3,2-f]qu in olin -1-yliden e)-
h yd r a zin o]ben zen esu lfon a m id e (81). The title compound
was prepared in 24% yield from 3-H-pyrrolo[3,2-f]quinoline-
1,2-dione and 4-hydrazinobenzene sulfonamide hydrochloride
according to procedure E: 1H NMR (DMSO-d6): δ 7.27 (s, 2H),
7.53 (d, J ) 8.8 Hz, 1H), 7.67 (d, J ) 8.8 Hz, 2H), 7.76 (dd, J
) 4.7, 8.4 Hz, 1H), 7.80 (d, J ) 8.8 Hz, 2H), 8.00 (d, J ) 8.9
Hz, 1H), 8.89 (d, J ) 3.7, 1H), 9.16 (d, J ) 8.2 Hz, 1H), 11.46
(s, 1H), 13.10 (s, 1H). APCI m/z 368 (M+H)+. Anal. (C17H13N5-
O3S‚3/4 HCl‚2/3H2O) C, H, N, S.
4-{[(2-Oxo-2,3-d ih yd r o-1H -p yr r olo[3,2-f]q u in olin -1-
yliden e)m eth yl]am in o}ben zen esu lfon am ide (82). The title
compound was prepared from 1-[(dimethylamino)methylene]-
1,3-dihydro-2H-pyrrolo[3,2-f]quinolin-2-one and sulfanilamide
according to procedure G: 1H NMR (DMSO-d6): δ 7.32 (s, 2H),
7.47 (d, J ) 8.8 Hz, 1H), 7.55 (dd, J ) 8.8, 4.2 Hz, 1H), 7.70
(d, J ) 8.7 Hz, 2H), 7.79 (d, J ) 8.8 Hz, 1H), 7.82 (d, J ) 8.7
Hz, 2H), 8.78 (d, J ) 3.4 Hz, 1H), 8.90 (m, 2H), 11.09 (s, 1H),
11.91 (d, J ) 11.8 Hz, 1H); APCI m/z 365 (M-H)-. Anal.
(C18H14N4O3S‚0.4 H2O) C, H, N.
N-Meth yl-4-{[(7-oxo-6,7-d ih yd r o-8H-[1,3]th ia zolo[5,4-e]-
in d ol-8-ylid en e)m eth yl]a m in o}ben zen esu lfon a m id e (83).
The title compound was prepared from 4-amino-N-methylben-
zenesulfonamide and 8-(ethoxymethylene)-6,8-dihydro-7H-
[1,3]thiazolo[5,4-e]indol-7-one according to procedure G: 1H
NMR (DMSO-d6): δ 2.36 (d, J ) 5.1 Hz, 3H), 7.08 (d, J ) 8.4
Hz, 1H), 7.34 (q, J ) 5.1 Hz, 1H), 7.58 (d, J ) 8.7 Hz, 2H),
7.72 (d, J ) 8.7 Hz, 2H), 7.79 (d, J ) 8.4 Hz, 1H), 8.05 (d, J )
12.1 Hz, 1H), 9.23 (s, 1H), 10.89 (s, 1H), 11.14 (d, J ) 12.1
Hz, 1H); APCI m/z 385 (M-H)-. Anal. (C17H14N4O3S2‚1/2H2O)
C, H, N, S.
N,N-Dim eth yl-4-{[(7-oxo-6,7-d ih yd r o-8H-[1,3]th ia zolo-
[5,4-e]in dol-8-yliden e)m eth yl]am in o}ben zen esu lfon am ide
(84). The title compound was prepared from 4-amino-N,N-
dimethylbenzenesulfonamide and 8-(ethoxymethylene)-6,8-
dihydro-7H-[1,3]thiazolo[5,4-e]indol-7-one according to proce-
dure G: 1H NMR (DMSO-d6): δ 2.56 (s, 6H), 7.08 (d, J ) 8.4
Hz, 1H), 7.61 (d, J ) 8.7 Hz, 2H), 7.70 (d, J ) 8.7 Hz, 2H),
7.79 (d, J ) 8.4 Hz, 1H), 8.05 (d, J ) 12.1 Hz, 1H), 9.23 (s,
1H), 10.89 (s, 1H), 11.16 (d, J ) 12.1 Hz, 1H); APCI m/z 399
(M-H)-. Anal. (C18H16N4O3S2‚1/2H2O) C, H, N, S.
N-(2-Hydr oxyeth yl)-4-{[(7-oxo-6,7-dih ydr o-8H-[1,3]th ia-
zolo[5,4-e]in d ol-8-ylid en e)m eth yl]a m in o}ben zen esu lfon -
a m id e (85). The title compound was prepared in 51% yield
from N-(2-hydroxyethyl)-4-aminobenzenesulfonamide and
8-(ethoxymethylene)-6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-
1
7-one according to procedure G: H NMR (DMSO-d6): δ 2.74
(q, J ) 6.0 Hz, 2H), 3.32 (q, J ) 6.0 Hz, 2H), 4.64 (t, J ) 6.0
Hz, 1H), 7.08 (d, J ) 8.5 Hz, 1H), 7.50 (t, J ) 6.0 Hz, 1H),
7.56 (d, J ) 8.7, 2H), 7.74 (d, J ) 8.7 Hz, 2H), 7.79 (d, J ) 8.5
Hz, 1H), 8.05 (d, J ) 12.0 Hz, 1H), 9.23 (s, 1H), 10.88 (s, 1H),
11.14 (d, J ) 12.0 Hz, 1H); APCI m/z 415 (M-H)-. Anal.
(C18H16N4O4S2‚2 H2O) C, H, N, S.
N-[2-(1H-Im id a zol-5-yl)eth yl]-4-{[(7-oxo-6,7-d ih yd r o-
8H -[1,3]t h ia zolo[5,4-e]in d ol-8-ylid en e)m et h yl]a m in o}-

Oxindole-Based Inhibitors of CDK2
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4355
ben zen esu lfon a m id e h yd r och lor id e (86). The title com-
pound was prepared from 4-amino-N-[2-(1H-imidazol-5-
yl)ethyl]benzene sulfonamide and 8-(ethoxymethylene)-6,8-
dihydro-7H-[1,3]thiazolo[5,4-e]indol-7-one according to procedure
G: 1H NMR (DMSO-d6): δ 2.77 (t, J ) 6.6 Hz, 2H), 3.05 (q, J
) 6.6 Hz, 2H), 7.13 (d, J ) 8.4 Hz, 1H), 7.40 (s, 1H), 7.59 (d,
J ) 8.8 Hz, 2H), 7.73-7.85 (m, 3H), 8.07 (d, J ) 12.0 Hz, 1H),
8.96 (s, 1H), 9.27 (s, 1H), 10.95 (s, 1H), 11.18 (d, J ) 12.0 Hz,
1H), 14.20 (bs, 2H); ESI: m/z 467 (M+H)+. Anal. (C21H18N6O3S2‚
HCl‚2 H2O) C, H, N, S, Cl.
N-[2-(2-Hyd r oxyet h oxy)et h yl]-4-{[(7-oxo-6,7-d ih yd r o-
8H -[1,3]t h ia zolo[5,4-e]in d ol-8-ylid en e)m et h yl]a m in o}-
ben zen esu lfon a m id e (87). The title compound was prepared
from 4-amino-N-(2-(2-hydroxyethoxy)ethyl)-benzenesulfona-
mide and 8-(ethoxymethylene)-6,8-dihydro-7H-[1,3]thiazolo-
[5,4-e]indol-7-one according to procedure G: 1H NMR (DMSO-
d6): (2.88 (q, J ) 6.0 Hz, 2H), 3.31 (t, J ) 5.0 Hz, 2H), 3.36 (t,
J ) 5.8 Hz, 2H), 3.42 (t, J ) 5.1 Hz, 2 Hz), 4.5 (br s, 1H), 7.10
(d, J ) 8.4 Hz, 1H), 7.59 (d, J ) 8.8 Hz, 2H), 7.60 (t, J ) 6.0
Hz, 1H), 7.77 (d, J ) 8.7 Hz, 2H), 7.81 (d, J ) 8.6 Hz, 1H),
8.07 (d, J ) 12.2 Hz, 1H), 9.25 (s, 1H), 10.91 (s, 1H), 11.16 (d,
J ) 12.2 Hz, 1H); APCI m/z 459 (M-H)-. Anal. (C20H20N4O5S2‚
H2O) C, H, N.
1H), 9.22 (s, 1H), 10.18 (s, 1H), 10.87 (s, 1H), 11.09 (d, J )
12.0 Hz, 1H); ES-MS m/z 447 (M-H)-. Anal. (C22H16N4O3S2)
C, H, N, S.
N-Meth yl-4-{[(7-oxo-6,7-d ih yd r o-8H-[1,3]th ia zolo[5,4-e]-
in d ol-8-ylid en e)m eth yl]a m in o}-N-(2-p yr id in yl)ben zen e-
su lfon a m id e (92). The title compound was prepared from
4-amino-N-(2-pyridinyl)benzenesulfonamide and 8-(ethoxy-
methylene)-6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-7-one ac-
cording to procedure G: 1H NMR (DMSO-d6): δ 3.18 (s, 3H),
7.08 (d, J ) 8.5 Hz, 1H), 7.21 (dd, J ) 5.0, 7.3 Hz, 1 H), 7.52
(m, 4H), 7.79 (d, J ) 8.5 Hz, 1H), 7.82 (m, 1H), 8.02 (d, J )
12.0 Hz, 1H), 8.28 (d, J ) 3.2 Hz, 1H), 9.23 (s, 1H), 10.89 (s,
1H), 11.13 (d, J ) 12.0 Hz, 1H); APCI m/z 462 (M-H)-. Anal.
(C22H17N5O3S2‚0.7 HCl) C, H, N, S.
N-(2,6-Dim eth ylph en yl)-4-{[(7-oxo-6,7-dih ydr o-8H-[1,3]-
th iazolo[5,4-e]in dol-8-yliden e)m eth yl]am in o}ben zen esu l-
fon a m id e (93). The title compound was prepared from
4-amino-N-(2,6-dimethylphenyl)benzenesulfonamide and 8-(eth-
oxymethylene)-6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-7-one
according to procedure G: 1H NMR (DMSO-d6): δ 1.94 (s, 6H),
6.97 (m, 3H), 7.08 (d, J ) 8.4 Hz, 1H), 7.54 (d, J ) 8.9 Hz,
2H), 7.79 (d, J ) 8.9 Hz, 2H), 8.06 (d, J ) 12.1 Hz, 1H), 9.23
(s, 1H), 10.90 (s, 1H), 11.18 (d, J ) 12.1 Hz, 1H). Anal.
(C24H20N4O3S2) C, H, N, S.
4-{[(7-Oxo-6,7-d ih yd r o-8H -[1,3]t h ia zolo[5,4-e]in d ol-8-
ylid en e)m eth yl]a m in o}-N-(3,6,9,12-tetr a oxa tr id ec-1-yl)-
ben zen esu lfon a m id e (Z-isom er ) (88). A solution of 2.3 g
(6.3 mmol) of toluene-4-sulfonic acid 2-{2-[2-(2-methoxy-ethoxy)-
ethoxy]-ethoxy}-ethyl ester and ∼4 mL (∼60 mmol) of am-
monium hydroxide in 10 mL of ethanol was stirred overnight
at ∼60 °C. The solvent was removed on a rotary evaporator,
and the residue was sequentially redissolved in ethanol and
concentrated several times. The residue was then dissolved
in ethanol, treated with ∼1.5 mL of TEA, and concentrated
on a rotary evaporator. This residue was dissolved in 10 mL
of THF, and 1.4 g (6.0 mmol) of 4-N-acetylsulfanilyl chloride
and 1 mL (7 mmol) of TEA were added. The reaction mixture
was stirred 1.5 h at room temperature and then 30 min at
reflux. The solution was concentrated onto silica gel and
chromatographed with an EtOAc to 5% MeOH/EtOAc gradient
to give 4-N-(2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethyl)-
sulfonamidophenyl]acetamide as an oil (1.92 g, 79%): 1H NMR
(DMSO-d6): δ 2.05 (s, 3H), 2.83 (q, J ) 5.9 Hz, 2H), 3.19 (s,
3H), 3.30-3.48 (m, 14H), 7.52 (t, J ) 5.8 Hz, 1H), 7.68 (d, J )
8.8 Hz, 2H), 7.72 (d, J ) 8.8 Hz, 2H), 10.27 (s, 1H); APCI m/z
403 (M-H)-. A solution of 1.9 g (4.7 mmol) of N-[4-(2-{2-[2-(2-
methoxy-ethoxy)-ethoxy]-ethoxy}-ethylsulfamoyl)-phenyl]-ac-
etamide and 0.45 g (4.7 mmol) of methanesulfonic acid in 15
mL of ethanol was stirred at ∼70 °C for 24 h. Excess TEA
was added, and the solvent was removed on a rotary evapora-
tor. The residue was applied to a short column of silica gel
and eluted with EtOAc to give 4-(N-(2-{2-[2-(2-methoxy-
ethoxy)-ethoxy]ethoxy}ethyl)-sulfonamidoaniline as an oil (1.2
g, 70%): 1H NMR (DMSO-d6): δ 2.76 (q, J ) 6.0 Hz, 2H), 3.20
(s, 3H), 3.32 (t, J ) 6.2 Hz, 2H), 3.37-3.48 (m, 12H), 5.88 (s,
2H), 6.56 (d, J ) 8.7 Hz, 2H), 7.11 (t, J ) 6.0 Hz, 1H), 7.37 (d,
J ) 8.7 Hz, 2H); APCI m/z 361 (M-H)-. The title compound
was prepared from 4-(N-(2-{2-[2-(2-methoxyethoxy)-ethoxy]-
ethoxy}ethyl)sulfonamidoaniline and (8Z)-8-(ethoxymethyl-
ene)-6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-7-one according to
procedure G: mp 158-159 °C; 1H NMR (DMSO-d6): δ 2.87
(dt, J ) 5.6, 5.6 Hz, 2H), 3.17 (s, 3H), 3.33-3.38 (m, 4H), 3.38-
3.47 (m, 10H), 7.10 (d, J ) 8.3 Hz, 1H), 7.58 (d, J ) 8.7 Hz,
2H), 7.63 (t, J ) 5.7 Hz, 1H), 7.77 (d, J ) 8.7 Hz, 2H), 7.81 (d,
J ) 8.5 Hz, 1H), 8.06 (br d, J ) 8.9 Hz, 1H), 9.25 (s, 1H), 10.91
(s, 1H), 11.16 (br d, J ) 10.8 Hz, 1H); APCI m/z 561 (M-H)-.
Anal. (C25H30N4O7S2‚1/3H2O) C, H, N.
N-Ben zyl-4-{[(7-oxo-6,7-d ih yd r o-8H-[1,3]th ia zolo[5,4-e]-
in d ol-8-ylid en e)m eth yl]a m in o}ben zen esu lfon a m id e (94).
The title compound was prepared from 4-amino-N-benzylben-
zenesulfonamide and 8-(ethoxymethylene)-6,8-dihydro-7H-
[1,3]thiazolo[5,4-e]indol-7-one according to procedure G: 1H
NMR (DMSO-d6): δ 3.98 (d, J ) 6.2 Hz, 2H), 7.13 (d, J ) 8.4
Hz, 1H), 7.25 (m, 5H), 7.59 (d, J ) 8.7 Hz, 2H), 7.80 (d, J )
8.7 Hz, 2H), 7.84 (d, J ) 8.4 Hz, 1H), 8.09 (m, 2H), 9.28 (s,
1H), 10.94 (s, 1H), 11.18 (d, J ) 12.1 Hz, 1H); APCI m/z 461
(M-H)-. Anal. (C23H18N4O3S2‚3/2H2O) C, H, N, S.
8-{[4-({[Am in o(im in o)m eth yl]a m in o}su lfon yl)a n ilin o]-
m eth ylen e}-7-oxo-7,8-d ih yd r o-6H-[1,3]th ia zolo[5,4-e]in -
d ole (96). The title compound was prepared in 26% yield from
8-(ethoxymethylene)-6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-
7-one and 4-amino-N-(amino-imino-methyl)-benzenesulfona-
mide according to procedure G: 1H NMR (DMSO-d6): δ 6.64
(br s, 4H), 7.08 (d, J ) 8.4 Hz, 1H), 7.47 (d, J ) 8.6 Hz, 2H),
7.71 (d, J ) 8.6 Hz, 2H), 7.78 (d, J ) 8.4 Hz, 1H), 8.03 (d, J )
12.2, 1H), 9.22 (s, 1H), 10.86 (s, 1H), 11.10 (d, J ) 12.2 Hz,
1H); APCI+MS m/z 415 (M+H)+. Anal. (C17H14N6O3S2‚2H2O)
C, H, N, S.
N-Acetyl-4-{[(7-oxo-6,7-d ih yd r o-8H-[1,3]th ia zolo[5,4-e]-
in d ol-8-ylid en e)m eth yl]a m in o}ben zen esu lfon a m id e (97).
The title compound was prepared in 26% yield from 8-(ethoxy-
methylene)-6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-7-one and
N-acetyl-4-aminobenzenesulfonamide according to procedure
G: 1H NMR (DMSO-d6): δ 1.87 (s, 3H), 7.08 (d, J ) 8.4 Hz,
1H), 7.57 (d, J ) 8.8 Hz, 2H), 7.80 (d, J ) 8.4 Hz, 1H), 7.85 (d,
J ) 8.8 Hz, 2H), 8.04 (d, J ) 12.0, 1H), 9.24 (s, 1H), 10.90 (s,
1H), 11.16 (d, J ) 12.0, 1H), 11.98 (s, 1H); ESI m/z 413 (M-
H)-. Anal. (C18H14N4O4S2) C, H, N, S.
N-Met h yl-4-{[(2-oxo-1,2-d ih yd r o-3H -in d ol-3-ylid en e)-
m eth yl]a m in o}ben zen esu lfon a m id e (98). The title com-
pound was prepared from 3-(ethoxymethylene)-1,3-dihydro-
2H-indol-2-one and 4-amino-N-methylbenzenesulfonamide
according to procedure G: 1H NMR (DMSO-d6): δ 2.40 (d, J
) 5.2 Hz, 3H), 6.85 (d, J ) 7.6 Hz, 1H), 6.94 (t, J ) 7.6 Hz,
1H), 7.04 (t, J ) 7.6 Hz, 1H), 7.33 (q, J ) 5.2 Hz, 1H), 7.57 (d,
J ) 8.7 Hz, 2H), 7.60 (d, J ) 7.6 Hz, 1H), 7.72 (d, J ) 8.7 Hz,
2H), 8.63 (d, J ) 12.4 Hz, 1H), 10.57 (s, 1H), 10.84 (d, J )
12.4 Hz, 1H); APCI m/z 328 (M-H)-. Anal. (C16H15N3O3S) C,
H, N, S.
4-{[(7-Oxo-6,7-d ih yd r o-8H -[1,3]t h ia zolo[5,4-e]in d ol-8-
ylid en e)m et h yl]a m in o}-N-p h en ylb en zen esu lfon a m id e
(90). The title compound was prepared from 4-amino-N-
phenylbenzenesulfonamide and 8-(ethoxymethylene)-6,8-di-
hydro-7H-[1,3]thiazolo[5,4-e]indol-7-one according to procedure
G: 1H NMR (DMSO-d6): δ 6.97 (t, J ) 7.5 Hz, 1H), 7.07 (m,
3H), 7.18 (t, J ) 7.5 Hz, 2H), 7.50 (d, J ) 8.7 Hz, 2H), 7.69 (d,
J ) 8.7 Hz, 2H), 7.78 (d, J ) 8.6 Hz, 1H), 8.00 (d, J ) 12.0 Hz,
4-{[(2-Oxo-1,2-d ih yd r o-3H-in d ol-3-ylid en e)m eth yl]a m i-
n o}-N-(1,3-th ia zol-2-yl)ben zen esu lfon a m id e (100). The
title compound was prepared from 4-amino-N-(1,3-thiazol-2-
yl)benzenesulfonamide and 3-(ethoxymethylene)-1,3-dihydro-
2H-indol-2-one according to procedure G: 1H NMR (DMSO-
d6): δ 6.82 (d, J ) 4.6 Hz, 1H), 6.84 (d, J ) 7.8 Hz, 1H), 6.93
(d, J ) 7.8 Hz, 1H), 7.03 (t, J ) 7.4 Hz, 1H), 7.25 (d, J ) 4.6
Hz, 1H), 7.49 (d, J ) 8.8 Hz, 2H), 7.59 (d, J ) 7.4 Hz, 1H),

4356 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Bramson et al.
7.75 (d, J ) 8.8 Hz, 2H), 8.60 (d, J ) 12.3 Hz, 1H), 10.56 (s,
1H), 10.81 (d, J ) 12.3 Hz, 1H), 12.68 (s, 1H); ES-MS m/z 397
(M-H)-. Anal. (C18H14N4O3S2) C, H, N, S.
8-{[(2,2-Dioxid o-1,3-d ih yd r o-2-ben zoth ien -5-yl)a m in o]-
m et h ylen e}-6,8-d ih yd r o-7H -[1,3]t h ia zolo[5,4-e]in d ol-7-
on e (108). The title compound was prepared in 37% yield from
8-(ethoxymethylene)-6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-
7-one and 2,2-dioxo-1,3-dihydrobenzo[c]thiophene-5-ylamine
according to procedure G: 1H NMR (DMSO-d6): δ 11.11 (d,-
1H), 10.89 (s, 1H), 9.27 (s, 1H), 8.06 (d, 1H), 7.82 (d, 1H), 7.47
(m, 2H), 7.13 (d, 1H), 6.98 (d, 1H), 6.5 (m, 2H); APCI m/z 384
(M+H)+. Anal. (C18H13N3O3S2‚H2O) C, H, N, S: C, 53.85; H,
3.77; N, 10.47; S, 15.97. Found: C, 53.84; H, 3.84; N, 10.36; S,
15.95.
3-{[(2,2-Dioxid o-1,3-d ih yd r o-2-ben zoth ien -5-yl)a m in o]-
m et h ylen e}-5-(1,3-oxa zol-5-yl)-1,3-d ih yd r o-2H -in d ol-2-
on e (109). The title compound was prepared from 2,2-dioxido-
1,3-dihydro-2-benzothien-5-ylamine and 3-(ethoxymethylene)-
5-(1,3-oxazol-5-yl)-1,3-dihydro-2H-indol-2-one according to
procedure G: 1H NMR (DMSO-d6): δ 4.45 (s, 2H), 4.50 (s, 2H),
6.93 (d, J ) 8.4 Hz, 1H), 7.37-7.51 (m, 5H), 7.99 (s, 1H), 8.39
(s, 1H), 8.73 (d, J ) 12.3 Hz, 1H), 10.70 (s, 1H), 10.75 (d, J )
12.3 Hz, 1H); APCI m/z 392 (M-H)-. Anal. (C20H15N3O4S‚H2O)
C, H, N, S.
Molecu la r Mod elin g. Favorable substitutions on the core
oxindole template (positions 4-7) were identified via a ligand-
protein docking protocol. Initially, commercially available
anilines were identified and converted to their corresponding
SMILES51 strings. These anilines were prefiltered for chemi-
cally reactive groups and molecular weight criteria to yield
an initial starting set of 410 anilines. In-house virtual chem-
istry algorithms were then used to convert the anilines to the
corresponding 4-[2-(2-oxo-1,2-dihydro-3H-indol-3-ylidene)hy-
drazino]benzene-sulfonamides. In the case of asymmetric
anilines containing a meta-substituent, two possible oxindole
products could result from the chemistry (yielding 4- or
6-substituted oxindoles), and both products were investigated
via the docking algorithms.
The SMILES strings were then converted to the X-PLOR52
psf/pdb molecule format via in-house algorithms. Initially, the
atomic coordinates for each molecule were assigned dummy
values. The molecule psf/pdb’s served as input for an X-PLOR
high-temperature dynamics script for conformational search-
ing. Each molecule was heated at 2000 °C for 0.5 ps, followed
by a 0.2 ps simulation at 100 °C, followed by 200 steps of
minimization. This process was repeated 100 times for each
molecule to yield 100 starting conformations. Conformations
with energy greater than 7.5 kcal/mol above the lowest energy
structure identified were rejected. Remaining conformations
were rms superimposed, and, in cases where two structures
had a heavy atom rms deviation of less than 0.25 Å, the higher
energy structure was rejected. The lowest energy conformation
for each molecule was used as input for a Gaussian53 quantum
mechanics calculation to obtain partial atomic charges. Mol-
ecules were optimized using the semiempirical PM3 basis set
followed by a Hartree-Fock 3-21G* single point calcula-
tion.54,55 In the latter step, charges were obtained using the
CHELPG methodology.56
3-{[4-({[Am in o(im in o)m eth yl]a m in o}su lfon yl)a n ilin o]-
m eth ylen e}-2-oxo-2,3-d ih yd r o-1H-in d ole (101). The title
compound was prepared from 1-amino-4-({[amino(imino)-
methyl]amino}sulfonyl)benzene and 3-(ethoxymethylene)-1,3-
dihydro-2H-indol-2-one according to procedure G: 1H NMR
(DMSO-d6): δ 6.7 (br s, 4H), 6.84 (d, J ) 7.6 Hz, 1H), 6.93 (t,
J ) 7.6 Hz, 1H), 7.03 (t, J ) 7.6 Hz, 1H), 7.47 (d, J ) 8.7 Hz,
2H), 7.59 (d, J ) 7.6 Hz, 1H), 7.71 (d, J ) 8.7 Hz, 2H), 8.61 (d,
J ) 12.2 Hz, 1H), 10.55 (s, 1H), 10.80 (d, J ) 12.2 Hz, 1H);
APCI m/z 356 (M-H)-. Anal. (C16H15N5O3S‚H2O) C, H, N, S.
8-{[4-(Meth ylsu lfon yl)a n ilin o]m eth ylen e}-6,8-d ih yd r o-
7H-[1,3]th ia zolo[5,4-e]in d ol-7-on e (102). The title com-
pound was prepared from 4-(methylsulfonyl)aniline and
8-(ethoxymethylene)-6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-
7-one according to procedure G: 1H NMR (DMSO-d6): δ 3.21
(s, 3H), 7.13 (d, J ) 8.4 Hz, 1H), 7.66 (d, J ) 8.7 Hz, 2H), 7.84
(d, J ) 8.4 Hz, 1H), 7.91 (d, J ) 8.7 Hz, 2H), 8.10 (d, J ) 12.0
Hz, 1H), 9.28 (s, 1H), 10.95 (s, 1H), 11.21 (d, J ) 12.0 Hz, 1H);
APCI m/z 370 (M-H)-. Anal. (C17H13N3O3S2) C, H, N, S.
(4-{[(7-Oxo-6,7-d ih yd r o-8H-[1,3]th ia zolo[5,4-e]in d ol-8-
yliden e)m eth yl]am in o}ph en yl)m eth an esu lfon am ide (103).
The title compound was prepared in 25% yield from 8-(ethoxy-
methylene)-6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-7-one and
4-aminophenylmethane sulfonamide according to procedure
G: 1H NMR (DMSO-d6): δ 11.1 (d,1H), 10.9 (s, 1H), 9.3 (s,
1H), 8.1 (d, 1H), 7.8 (d, 1H), 7.5 (q, 4H), 7.2 (d, 1H), 6.9 (s,
2H), 4.2 (s, 2H); APCI m/z 387 (M+H)+.
N-Meth yl-(4-{[(7-oxo-6,7-d ih yd r o-8H-[1,3]th ia zolo[5,4-
e]i n d o l-8-y li d e n e )m e t h y l]a m i n o }p h e n y l)m e t h a n e -
su lfon a m id e (104). The title compound was prepared from
4-[(methylsulfonyl)methyl]aniline and 8-(ethoxymethylene)-
6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-7-one according to pro-
cedure G: 1H NMR (DMSO-d6): δ 2.58 (d, J ) 4.8 Hz, 3H),
4.32 (s, 2H), 6.90 (q, J ) 4.8 Hz, 1H), 7.12 (d, J ) 8.4 Hz, 1H),
7.40 (d, J ) 8.4 Hz, 2H), 7.45 (d, J ) 8.4 Hz, 2H), 7.80 (d, J )
8.4 Hz, 1H), 8.05 (d, J ) 12.3 Hz, 1H), 9.26 (s, 1H), 10.86 (s,
1H), 11.05 (d, J ) 12.3 Hz, 1H); APCI m/z 399 (M-H)-. Anal.
(C18H16N4O3S2‚H2O) C, H, N, S.
N-Meth yl-{4-[2-(7-oxo-6,7-d ih yd r o-8H-[1,3]th ia zolo[5,4-
e ]in d ol-8-ylid e n e )h yd r a zin o]p h e n yl}m e t h a n e su lfon -
a m id e (105). The title compound was prepared from 4-hy-
drazinobenzyl methyl sulfone and 6H-[1,3]thiazolo[5,4-e]in-
dole-7,8-dione according to procedure E: 1H NMR (DMSO-d6):
δ 2.54 (d, J ) 4.8 Hz, 3H), 4.29 (s, 2H), 6.87 (q, J ) 4.8 Hz,
1H), 7.12 (d, J ) 8.5 Hz, 1H), 7.38 (d, J ) 8.5 Hz, 2H), 7.49 (d,
J ) 8.5 Hz, 2H), 7.95 (d, J ) 8.5 Hz, 1H), 9.27 (s, 1H), 11.20
(s,1H),12.63 (s,1H);APCI: m/z400 (M-H)-.Anal.(C17H15N5O3S2‚
1/2H2O) C, H, N, S.
N-(2-H yd r oxyet h yl)(4-{[(7-oxo-6,7-d ih yd r o-8H -[1,3]-
t h ia zolo[5,4-e]in d ol-8-ylid en e)m et h yl]a m in o}p h en yl)-
m eth a n esu lfon a m id e (106). The title compound was pre-
pared from (4-aminophenyl)-N-(2-hydroxyethyl)methanesulfona-
mide and 8-(hydroxymethylene)-6,8-dihydro-7H-[1,3]thiazolo-
[5,4-e]indol-7-one according to procedure G: 1H NMR (DMSO-
d6): δ 2.95 (q, J ) 6.0 Hz, 2H), 3.40 (q, J ) 6.0 Hz, 2H), 4.32
(s, 2H), 4.72 (t, J ) 6.0 Hz, 1H), 7.02 (t, J ) 6.0 Hz, 1H), 7.11
(d, J ) 8.4 Hz, 1H), 7.40 (d, J ) 8.8 Hz, 2H), 7.44 (d, J ) 8.8
Hz, 2H), 7.46 (d, J ) 8.8 Hz, 2H), 7.79 (d, J ) 8.5, 1H), 8.05
(d, J ) 12.3, 1H), 9.25 (s, 1H), 10.86 (s, 1H), 11.05 (d, J ) 12.3
Hz, 1H), 12.78 (s, 1H); APCI: m/z 431 (M+H)+. Anal.
(C19H18N4O4S2‚H2O) C, H, N S.
Molecule topologies and conformations were then converted
into a format suitable for use in docking calculations employing
the Macromodel software package.57 The initial protein con-
formation was taken from the CDK2-cyclin A-flavopiridol
complex solved in-house. Additionally, the X-ray structure of
CDK2 in complex with compound 16 was used to generate an
initial positioning for the oxindole template. Initial docking
was accomplished by rms fitting to the coordinates for the
oxindole moiety of compound 16. These coordinates were also
used as positional restraints for the conformations of the
derivative molecules. Energy minimization calculations were
then performed using the GB/SA solvation model58 with 12 Å
cutoffs for nonbonded interactions. Residues within an 8 Å
sphere of the ATP binding site were allowed to adjust to the
presence of the ligand while other atoms within the protein
were restrained.
8-({4-[(Meth ylsu lfon yl)m eth yl]a n ilin o}m eth ylen e)-6,8-
d ih yd r o-7H-[1,3]th ia zolo[5,4-e]in d ol-7-on e (107). The title
compound was prepared in 66% yield from 8-(ethoxymethyl-
ene)-6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-7-one and 4-me-
thylsulfonylmethylaniline according to procedure G: 1H NMR
(DMSO-d6): δ 2.90 (s, 3H), 4.47 (s, 2H), 7.12 (d, J ) 8.4 Hz,
1H), 7.44 (d, J ) 8.8 Hz, 2H), 7.47 (d, J ) 8.8 Hz, 2H), 7.80 (d,
J ) 8.4 Hz, 1H), 8.07 (d, J ) 12.3 Hz, 1H), 9.26 (s, 1H), 10.87
(s, 1H), 11.07 (d, J ) 12.3 Hz, 1H); APCI m/z 384 (M-H)-. Anal.
(C18H15N3O3S2‚1/3 H2O) C, H, N, S.
Following minimization, the molecules were scored and
sorted according to “binding” energy. This energy included
ligand internal energy differences (relative to the lowest energy

Oxindole-Based Inhibitors of CDK2
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4357
Ta ble 4. Crystallographic Data
compd no.
space group
cell: a
16
98
105
100
101
109
91
P212121
54.14
72.71
72.93
500-2.2
157 652
13 325
87.9
P212121
72.39
73.96
54.06
500-2.2
51 192
13 376
87.6
P212121
72.76
73.78
54.00
500-2.0
104 211
19 360
95.6
P212121
72.53
74.04
54.08
500-2.0
44 454
17 232
85.0
P212121
72.45
73.90
54.10
500-2.0
60 211
19 110
93.8
P212121
72.68
73.72
54.17
500-2.0
98 375
19 650
96.9
P6222
184.95
184.95
212.83
500-2.8
50 936
541 713
95.5
b
c
resolution (Å)
no. of obs
no. of unique reflns
completeness (%)
Rmerge (%)
Rfactor (%)
Rfree (%)
7.1
20.0
23.9
4.3
18.4
22.4
4.2
19.1
23.5
6.0
18.7
22.8
5.2
20.3
23.8
5.2
19.1
22.2
7.0
21.8
26.3
ave B
37.1
32.7
31.5
36.0
32.5
36.7
55.3
Rms bonds (Å)
Rms angles (°)
0.009
1.4
0.009
1.4
0.009
1.3
0.009
1.3
0.011
2.0
0.009
1.4
0.010
1.4
conformation), protein-ligand interaction energies, and sol-
vation energy differences. Internal energy differences within
the protein were excluded. The coordinates for the lowest
energy bound conformation of each molecule were then saved
to file. Each final docked molecule structure was visually
inspected. This inspection, in conjunction with a consideration
of the binding energy scores, was used to select molecules for
synthesis.
Cr ysta llogr a p h y. The procedures for protein expression,
purification, crystallization, and crystal structure determina-
tion of the CDK2 complexes reported here have been described
previously.11,38,59 All structures were derived from CDK2 or
CDK2/cyclin A crystals that were soaked in solutions contain-
ing inhibitor. Crystallographic data is shown in Table 4.
En zym ology. Human CDK2/cyclin A was expressed in
insect cells and purified as previously described for the
production of CDK2,59 except for the following changes. Human
CDK2 and human cyclin A were coexpressed in Baculovirus-
infected T.ni cells. An HS (Poros) column was equilibrated in
25 mM HEPES (pH 7.5), 25 mM NaCl, 2 mM EDTA, and
human CDK2/cyclin A was eluted from this column with 0.3
M NaCl. Final purification was accomplished using a Phar-
macia S-200 Superdex column in place of the S-75 column used
for the CDK2 preparation. CDK2/cyclin A was stored at -80
°C prior to use.
tumor cell lines (RKO, HT29, SW620, MDAMB468) and cells
isolated from human foreskin fibroblasts (HFF). Briefly, cells
were seeded (2500 to 5000 cells/well) in 96-well plates and
allowed to attach for ∼24 h. Cells were incubated with
compound for 72 h in DMEM media containing 10% fetal
bovine serum. The cellular metabolic activity, an indicator of
cell viability, was assessed using MTT.60 Cells were treated
with a range of compound concentrations in duplicate to
generate dose-response curves. The compound concentration
required to inhibit 50% of cell growth (IC50) was determined
using a nonlinear regression analysis.
G1/S P h a se P r ogr ession Assa y. Inhibition of progression
of cells from G1 into S-phase was measured using a BrdU
incorporation assay. HFF cells were grown to confluency for
synchronization. Cells were then plated in 96-well plates for
∼18 h, compound was added for 2 h, then cells were co-treated
with BrdU for 4 h. BrdU incorporated into cellular DNA was
detected and quantitated using an antibody sandwich BrdU
ELISA. Dose response curves were generated by varying the
compound concentration (in triplicate). The compound con-
centration required to inhibit 50% of BrdU incorporation (IC50
was determined using a nonlinear regression analysis.
)
Ack n ow led gm en t. We thank R. Rutowski for as-
sistance with NMR analyses.
Human CDK1 was expressed and purified using the proce-
dures noted above for the production of CDK2 except for the
following changes. Human CDK1/cyclin A was expressed in
baculovirus-infected Sf9 cells. The complex was step-eluted
from the HS column (Poros) with 0.4 M NaCl. Enzyme-
containing fractions were aliquoted and stored at -80 °C.
Enzyme assays were performed in 96-well plates. Briefly,
compounds were diluted in 100% DMSO and transferred in
25-100% DMSO. Serial 2- or 3-fold dilutions were made.
Assays contained 100 mM HEPES (pH 7.5), 10 mM MgCl2,
0.1% BSA, 5% DMSO (with and without inhibitor), 1.4 µM
[γ-32P]ATP (0.07 µCi/well), 1.5 µM of the peptide Biotin-
aminohexyl-Ala-Arg-Arg-Pro-Met-Ser-Pro-Lys-Lys-
Lys-Ala-CONH2 and enzyme. Reactions were allowed to
proceed for 20-30 min (CDK2) or 30-60 min (CDK1). The
DMSO solutions were added to the assay plate first, and the
reactions were initiated by the addition of enzyme. Enzyme
concentrations in the assays were 5-10 nM for both CDK1/
cyclin A and CDK2/cyclin A as determined by protein titration.
The enzyme catalyzed reactions were terminated by the
addition of four assay volumes of 100 mM EDTA in PBS (pH
7.0) containing 0.5 mg of Neutravidin-coated SPA beads
(Amersham Pharmacia). The plates were allowed to sit
undisturbed for 8-24 h prior to quantitation using a scintil-
lation counter.
Refer en ces
(1) Morgan, D. O. Cyclin-dependent kinases: Engines, clocks, and
microprocessors. Annu. Rev. Cell Dev. Biol. 1997, 13, 261-291.
(2) Stein, G.; Baserga, R.; Giordano, A.; Denhardt, D. T. The
Molecular Basis of Cell Cycle and Growth Control; J ohn Wiley
& Sons: New York, 1998; p 389.
(3) Pines, J . Cyclins and cyclin-dependent kinases: take your
partners. Trends Biochem. Sci. 1993, 18, 195-197.
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