Synthesis and structure-activity relationship studies of small molecule disruptors of EWS-FLI1 interactions in Ewing's sarcoma

Article abstract of DOI:10.1021/jm501372p

EWS-FLI1 is an oncogenic fusion protein implicated in the development of Ewing's sarcoma family tumors (ESFT). Using our previously reported lead compound 2 (YK-4-279), we designed and synthesized a focused library of analogues. The functional inhibition of the analogues was measured by an EWS-FLI1/NR0B1 reporter luciferase assay and a paired cell screening approach measuring effects on growth inhibition for human cells containing EWS-FLI1 (TC32 and TC71) and control PANC1 cell lines devoid of the oncoprotein. Our data revealed that substitution of electron donating groups at the para-position on the phenyl ring was the most favorable for inhibition of EWS-FLI1 by analogs of 2. Compound 9u (with a dimethylamino substitution) was the most active inhibitor with GI₅₀ = 0.26 ± 0.1 μM. Further, a correlation of growth inhibition (EWS-FLI1 expressing TC32 cells) and the luciferase reporter activity was established (R² = 0.84). Finally, we designed and synthesized a biotinylated analogue and determined the binding affinity for recombinant EWS-FLI1 (K_d = 4.8 ± 2.6 μM).

Full text of DOI:10.1021/jm501372p

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Article
Synthesis and Structure Activity Relationship Studies of Small
Molecule Disruptors of EWS-FLI1 Interactions in Ewing Sarcoma
Perrer N Tosso, Yali Kong, Lauren Scher, Ryan Cummings, Jeffrey Schneider, Said
Rahim, Kevin Travis Holman, Jeffrey Toretsky, Kan Wang, Aykut Uren, and Milton L. Brown
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501372p • Publication Date (Web): 28 Nov 2014
Downloaded from http://pubs.acs.org on November 30, 2014
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Synthesis and Structure Activity Relationship
Studies of Small Molecule Disruptors of EWS-
FLI1 Interactions in Ewing’s Sarcoma
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a,b,c
a,c
c
c
c
Perrer N. Tosso , Yali Kong , Lauren Scher , Ryan Cummins , Jeffrey Schneider , Said
c
d
c
a
c
a,c *
Rahim , K. Travis Holman , Jeffrey Toretsky , Kan Wang , Aykut Üren , Milton L. Brown
a
Georgetown University Medical Center, Center for Drug Discovery, New Research Building
EP07, 3970 Reservoir Road, NW, Washington, D.C. 20057, United States
b
Georgetown University Medical Center, Department of Biochemistry and Molecular Biology,
c
Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, New
Research Building, 3970 Reservoir Road, NW, Washington, D.C. 20057, United States
d
th
Georgetown University, Department of Chemistry, 37 and O Streets NW, Washington D.C .
0057, United States
2
KEYWORDS: Ewing’s Sarcoma, EWSꢀFLI1, YKꢀ4ꢀ279, SAR, Biotinylated Conjugate
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ABSTRACT:
EWSꢀFLI1 is an oncogenic fusion protein implicated in the development of Ewing’s sarcoma
family tumors (ESFT). Using our previously reported lead compound 2 (YKꢀ4ꢀ279), we
designed and synthesized a focused library of analogues. The functional inhibition of the
analogues were measured by an EWSꢀFLI1/ NR0B1 reporter luciferase assay and a paired cell
screening approach measuring effects on growth inhibition for human cells containing EWSꢀ
FLI1 (TC32 and TC71) and control PANC1 cell lines devoid of the oncoprotein. Our data
revealed that substitution of electron donating groups at the paraꢀposition on the phenyl ring was
the most favorable for inhibition of EWSꢀFLI1 by analogs of 2. Compound 9u (with a
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dimethylamino substitution) was the most active inhibitor with a GI = 0.26 ± 0.1 ꢀM. Further, a
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correlation of growth inhibition (EWSꢀFLI1 expressing TC32 cells) and the luciferase reporter
2
activity was established (R = 0.84). Finally, we designed and synthesized a biotinylated
analogue and determined the binding affinity for recombinant EWSꢀFLI1 (K = 4.8 ꢀM ± 2.6
d
ꢀM).
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INTRODUCTION
1
Ewing’s sarcoma (ES) is a malignant tumor in adolescents and young adults and presents with a
high propensity for metastasis, particularly to lung, bone and bone marrow. ES is an orphan
disease with more than 200 new cases diagnosed in the United States each year. ES arises from
the mesenchymal tissues occurring mostly in the bones, however approximately 10% of the
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tumors arise in the soft tissues of the chest wall, pelvis and leg . Currently, adjuvant therapy is
required to treat both the primary tumor and the presumed microscopic metastasis. The intensive
combination of chemotherapy, surgery and radiation has increased the diseaseꢀfree survival rate
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to 70% in patients with localized ES. Patients with metastatic ES have a poor prognosis and thus
the development of specific therapeutic agents targeting ES is urgently needed.
ES are characterized by a nonrandom balanced chromosomal translocation involving the EWS
6
gene and one of the several ETS family of transcription factor genes mainly FLI1 , resulting in
the oncoprotein EWSꢀFLI1. The most common translocation in ES is the t(11;22)(q24;q12)
translocation and occurs in 90ꢀ95% of ES cases. The high frequency of this translocation
7
suggest that the product plays a significant role in the origin of these malignancies .
EWSꢀFLI1 consists of the Nꢀterminal RNAꢀbinding domain of EWS and the Cꢀterminal DNAꢀ
8
,9
binding domain of transcription factors such as ETS family . During this fusion, the
transactivation domain of FLI1 is replaced by the Nꢀterminal region of EWS converting FLI1
into an oncoprotein. Elimination of EWSꢀFLI1 through small interfering and antisense RNA
1
0
results in the prolonged survival of ES xenograftꢀbearing mice . Consequently, this oncoprotein
may provide a unique target in the development of therapeutics against ES.
EWSꢀFLI1 is predicted to be an intrinsically disordered protein (IDP), i.e. lacks secondary and
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tertiary structure under physiological conditions . The disordered nature of EWSꢀFLI1 is
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necessary for optimal transactivation of transcription and the formation of the proteinꢀprotein
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complexes that lead to oncogenesis . Targeting transcription factors requires either the
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disruption of specific DNAꢀprotein or proteinꢀprotein interactions . However, due to their
higher inducedꢀfit sampling properties, IDPs have potential for binding to small molecules and
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may possess potentially multiple binding sites for small molecules .
We previously reported the discovery of NSC635437 (1), identified through screening of small
molecules against recombinant EWSꢀFLI1 using surface plasmon resonance, and we developed
YKꢀ4ꢀ279 (2) with limited SAR (Figure 1A). Both compounds bind EWSꢀFLI1 and disrupt
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EWSꢀFLI1/RHA interaction . We have shown that 2 specifically and stereoselectively inhibits
the function EWSꢀFLI1 and induces apoptosis both in vitro (GI = 0.5ꢀ2.0 ꢀM in ES cells) and
5
0
1
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in vivo .
In this study, we report the full SAR studies of 2 against two Ewing’s Sarcoma cell lines (TC32
and TC71) and the transcriptional inhibition of EWSꢀFLI1. Finally, we designed and synthesized
a novel biotinylated analogue of an active inhibitor to measure binding affinity to EWSꢀFLI1.
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Figure 1: A. Structures of EWSꢀFLI1 inhibitors: 1 = NSC635437, GI = 20 ꢀM in TC32 cells;
5
0
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= YKꢀ4ꢀ279, K = 9.48 ꢀM, GI of 0.5ꢀ2.0 ꢀM in several ES cells. B. Sites for modifications.
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RESULTS
Chemistry
Our previous study led to the discovery of compound 1 (Figure 1), 4,7ꢀdichloroꢀ3ꢀ(2ꢀ(4ꢀ
chlorophenyl)ꢀ2ꢀoxoethyl)ꢀ3ꢀhydroxyindolinꢀ2ꢀone, which displayed antiꢀproliferative activity
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against various ES cell lines with GI of 20 ꢀM in TC32 cells . The aromatic paraꢀmethoxy
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substitution on the phenyl ring afforded compound 2 which exhibited an increased activity with
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GI of 0.9 ꢀM in TC32 cells .
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1
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Since EWSꢀFLI1 is more than 75% disordered , a lack of crystal structure or homology model
2
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of the protein requires a ligand based approach. Thus, we designed a patterned structural study
of 2, to obtain structure activity information and to investigate the electrostatic, steric and
hydrogen bonding requirements for inhibition.
To explore the importance of the 4,7ꢀdichloro substituent group on the indoline ring of 2, we
synthesized indolinꢀ2ꢀone substituted compounds 6a-h (Scheme 1). A corresponding aniline (3a-
e) was reacted with chloral hydrate and hydroxylamine hydrochloride in aqueous sodium sulfate
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to form isonitrosoacetanilide (4a-e) followed by a Sandmeyer reaction with concentrated sulfuric
2
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acid to afford the desired isatin (5a-e) in good yield (55ꢀ90 %)
. We observed that maintaining
°
the temperature at 55 C is necessary for the synthesis of the 4,7ꢀdifluoro and 4,7ꢀdimethoxy
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substituted intermediates. Isatins 5f-h were commercially available and the aldol reaction was
performed in methanol (with Et NH as a catalyst) to give 6a-h in excellent yield (90ꢀ100 %).
2
3
a, R= 3ꢀCl
4a, R= 3ꢀCl
5a, R= 3ꢀCl
6a, R= 4ꢀCl
3b, R= 4ꢀCl
4b, R= 4ꢀCl
5b, R= 4ꢀCl
6b, R= 5ꢀCl
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c, R = 4,5ꢀCl
d, R = 3,6ꢀF
e R = 3,6ꢀOCH
4c, R = 4,5ꢀCl
4d, R = 3,6ꢀF
4e, R = 3,6ꢀOCH
5c, R = 4,5ꢀCl
5d, R = 3,6ꢀF
5e, R = 3,6ꢀOCH
6c, R = 5,6ꢀCl
6d, R = 4,7ꢀF
6e, R = 4,7ꢀOCH
3
3
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f, R = 4,7ꢀCH
6g, R = 7ꢀCl
h, R = H
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°
Scheme 1. Reagents and conditions: (a) Cl CCH(OH) , NH OH.HCl, Na SO , H O, HCl, 55 C,
3
2
2
2
4
2
°
°
overnight, (b) H SO , 55 C to 80 C, (c) 4ꢀmethoxyꢀacetophenone, Et NH, MeOH, rt, 90ꢀ100 %.
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The synthetic strategy for substituting the phenyl ring of 2 is shown in Scheme 2. In general,
commercially available 4,7ꢀdichloroisatin was condensed with a substituted acetophenone (8a-u)
2
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in the presence of a catalytic amount of Et NH to afford 9a-u in excellent yield (90ꢀ100 %) .
2
To understand the importance of the hydroxyl group in the aldol product and to assess the
potential for an in vivo dehydration reaction, we synthesized the dehydrated compound 10. This
was accomplished by treating 2 with concentrated sulfuric acid as shown in Scheme 2 (58 %
23,24
24
yield)
. Of interest was the report that this methodology affords the Z isomer . We confirmed
and assigned the Z configuration of 10 by Xꢀray crystallography (Figure 2).
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H CO
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N
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R
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b
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H
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7a, R= 4ꢀOCH
a, R= 2ꢀOCH
b, R = 3ꢀOCH
c, R = 2,4ꢀOCH
8d, R = 3,5ꢀOCH
8e, R = 2,3ꢀOCH
f, R = 2,3,4ꢀOCH
g, R = 4ꢀH
h, R = 4ꢀF
8i, R = 4ꢀI
j, R = 4ꢀCF
k, R = 4ꢀCN
m, R = 4ꢀCH
n, R = 4ꢀ OC
8p, R = 4ꢀ OiPr
q, R = 4ꢀOPh
r, R = 4ꢀ SCH
s, R = 4ꢀ NH
t, R = 4ꢀ NHCH
8u, R = 4ꢀ N(CH
3
2, R= 4ꢀOCH
9a, R= 2ꢀOCH
9b, R = 3ꢀOCH
9c, R = 2,4ꢀOCH
9d, R = 3,5ꢀOCH
9e, R = 2,3ꢀOCH
9f, R = 2,3,4ꢀOCH
9g, R = 4ꢀH
9h, R = 4ꢀF
9i, R = 4ꢀI
9j, R = 4ꢀCF
3
9k, R = 4ꢀCN
9m, R = 4ꢀCH
9n, R = 4ꢀ OC H
2 5
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10, R= 4ꢀOCH
3
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H
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9p, R = 4ꢀ OiPr
9q, R = 4ꢀOPh
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3
9r, R = 4ꢀ SCH
9s, R = 4ꢀ NH
9t, R = 4ꢀ NHCH
3 2
9u, R = 4ꢀ N(CH )
3
2
2
3
3
3
)
2
Scheme 2. Reagents and conditions: (a) 4,7ꢀdichloroisatin, Et NH, MeOH, rt, 90ꢀ100 %; (b)
2
°
H SO , 0 C, 5 mins, 58 %; (c) Ag O, THF, MeI, rt, 58 %.
2
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Figure 2: Xꢀray single crystal structure of 10, the dehydration product of 2; the structure
determination establishes the Z stereochemistry of 10.
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Hydrogen bonding has an important role in ligand receptor interactions . To check the
involvement of the indoline hydroxyl and amine group in potential hydrogen bonding, we
synthesized the methylated compounds 11, 14a-b and 15. Compound 11 was synthesized
through the Oꢀmethylation of 2 by treating the aldol with methyl iodine in silver oxide to afford
26
1
1 with a yield of 58 % (Scheme 2) . Compound 14a was synthesized by Nꢀmethylating the
isatin (Scheme 3). The double methylated compound 15 was obtained by treating 14a with
methyl iodine in silver oxide.
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13a X= CH3
14a X= CH3
[
14b X= Ph
15
[
1
3b X= Ph
°
Scheme 3. Reagents and conditions: (a) NaH, MeI, DMF, 0 C to rt, 54.5 %; (b) 4ꢀmethoxyꢀ
acetophenone, Et NH, MeOH, rt, 51ꢀ100 %; (c) Ag O, THF, MeI, rt, 56 %.
2
2
We synthesized analog 18 with a pyridine ring instead of the phenyl ring in an attempt to
increase the solubility of the lead compound. The synthesis of 18 began with a bromine lithium
exchange in the reaction of 16 with acetaldehyde in the presence of nꢀBuLi. The resulting
alcohol was oxidized in PDC and 4Å molecular sieves to afford 17. Reaction of 17 with 4,7ꢀ
dichloroisatin afforded the desired compound 18 in 90 % yield (Scheme 4).
o
Scheme 4. Reagents and conditions: (a) 1. Acetaldehyde, nꢀBuLi, THF, ꢀ78 C; 2. PDC, 4Å Mol.
Sieves, CH Cl , 0 °C, (b) 4,7ꢀdichloroisatin, Et NH, MeOH, rt, 90 %.
2
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To further assess binding affinity of these derivatives with EWSꢀFLI1, we synthesized a biotin
conjugate of 9t (Scheme 5). The synthesis started with the Boc (tertꢀbutoxycarbonyl) protection
of the 5ꢀaminopentanoic acid (19) followed by coupling with 4ꢀacetylꢀNꢀmethyl aniline with
DCC to give 21. An aldol addition of 21 with 4,7ꢀdichloroisatin yielded 22 followed by its
deprotection to afford 23. EDCI coupling of biotin to 23 provided the final biotin conjugate
analogue 24 in 49% yield.
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A
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GI = 0.39 ꢀM
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Scheme 5. (A) The design strategy of the biotinylated compound 24. (B) Reagents and
°
conditions (i) (Boc) O, Et N, MeOH, 60 C, overnight, 90 %; (ii) 4ꢀacetylꢀNꢀmethylaniline,
2
3
°
DCC, DMAP, CH Cl , 0 C to rt, 58 %; (iii) 4,7ꢀdichloroisatin, Et NH, MeOH, rt; 95% (iv) 3:1
2
2
2
.
°
CH Cl :TFA, rt, 1.5 h; (v) biotin, EDCI HCl, DMAP, HOBt, CH Cl 0 C to rt, 49%.
2
2
2
2,
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2
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2
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RESULTS AND DISCUSSION
The antiꢀproliferative properties of the derivatives were assayed by monitoring their ability to
inhibit cell growth using a water soluble tetrazolium (WST) assay (see experimental section).
Antiꢀproliferative activities of the small molecules were determined as described in the
experimental section in the two human ES cell lines (TC32 and TC71) and their GI ’s were
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
evaluated. The results are summarized in Tables 1ꢀ5. Compound 2 was used as comparison in the
parallel experiments.
Compounds 6a-h were synthesized to investigate the importance of different substituent groups
in the indoline ring as compared to 2 (Table 1). Of all the compounds in this series, only the
dimethoxy substituted compound (6e) retained the same potency as 2 (GI 1.0 ꢀM) in TC32
50
cells. Since both electron withdrawing (ꢀCl) and donating group substitutions (ꢀOCH ) in the R1
3
and R positions were active, differences in steric contributions were considered. Increased size
4
of the aromatic substitution in the R and R positions (compound 6d versus 2) appears to
1
4
contribute to improved inhibitory activity. Because 6e does not significantly improve the
pharmacological properties of 2, we kept the 4,7ꢀdichloro substitutions on the indoline ring for
the rest of our study.
Compound 2 contains a chiral center. In a previous study, we found that the Sꢀ(ꢀ) was 4ꢀfold
(GI = 0.28 ꢀM) more potent in vitro than the racemic 2. The Rꢀ(+) was almost inactive in
5
0
1
8
vitro . To further understand the influence of the hydroxyl group and the potential dehydration
in vivo, we synthesized the dehydrated analog of 2. The dehydrated compound 10 was as potent
as the parent compound 2 (GI 1.0 ꢀM in TC32), suggesting that the hydroxyl group is not
5
0
required for binding. We prepared derivative 11 in which the hydroxyl group was methylated to
eliminate potential hydrogen bond donation. This chemical transformation resulted in a complete
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loss of inhibitory activity. Further methylations performed are summarized in Table 2. The
methylation of the amide group, represented by compound 14a, resulted in a twoꢀfold reduction
in TC32 cell GI . Furthermore, the replacement of the proton on the amide with a phenyl group
5
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
14b) resulted in the complete loss of activity apparently due to steric effects. The Oꢀmethylated
and Nꢀmethylated compound 15 was inactive.
Table 1. Chemical Structure and Biological Activities of Compounds 6a-6i
Substitutions
GI (ꢀM)
50
PANC 1 TC32
TC71
NI
R1
R2
ꢀH
R3
ꢀH
R4
ꢀH
a
6a
ꢀCl
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
6
b
ꢀCl
ꢀH
ꢀCl
ꢀCl
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
H
ꢀCl
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
NI
6
c
NI
6
d
ꢀF
ꢀF
NI
b
b
6
e
ꢀOCH
3
ꢀOCH
3
1.0
NI
NI
NI
0.94
6f
ꢀCH
ꢀH
3
ꢀCH
ꢀCl
ꢀH
3
NI
NI
NI
6
g
6h
ꢀH
a
b
NI: No significant inhibition (GI > 10 ꢀM). Experimental error is within 20%.
5
0
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Chemical Structure and Biological Activities of OꢀMethylation and NꢀMethylation
Substitutions
GI (ꢀM)
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
b
R1
R2
PANC 1
TC32
TC71
a
2
H
H
NI
0.94
NI
0.92
NI
1
1
ꢀCH
3
H
NI
NI
NI
NI
14a
4b
15
H
ꢀCH
ꢀPh
3
2.2
NI
0.71
NI
1
H
ꢀCH
ꢀCH
NI
NI
3
3
a
b
NI: No significant inhibition (GI > 10 ꢀM). Experimental error is within 20%.
5
0
As previously described, we synthesized 18 by replacing the electron rich phenyl ring by an
electron deficient pyridine ring. This resulted in a complete loss of growth inhibition for the
EWSꢀFLI1 containing cells lines (data not shown).
We further evaluated the positional effect of the methoxy moiety on the phenyl ring, and the
results are summarized in Table 3. The data showed that substitution with 2ꢀmethoxy, 3ꢀmethoxy
and the 2,3ꢀmethoxy (9a, 9b and 9e) reduced inhibition of cell growth. The 3,5ꢀdimethoxy
derivative 9d also resulted in a nearly 10ꢀfold loss of growth activity in TC32 cells. Interestingly,
the 2,4ꢀdimethoxy substitution (9c) resulted in a GI50 of 2.6 ꢀM but the 2,3,4ꢀtrimethoxy
derivative (9f) resulted in a 3ꢀfold loss of the growth activity as compared to 2. Taken together,
these data suggested that the para position contributes more to the potency whereas the meta and
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ortho substitutions decrease the inhibitory activity. We concluded that the mono substitution at
the para position on the aromatic ring plays a significant role in the inhibition of EWSꢀFLI1 in
ES cells.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
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60
Table 3. Chemical Structure and Biological Activities of Compounds 9a-9f
Substitutions
R3
GI (ꢀM)
5
0
R1
R2
R4
b
b
PANC 1 TC32
TC71
2
H
H
H
OCH
3
3
3
H
H
H
H
NI*
NI
NI
NI
NI
NI
NI
0.94
0.92
NI
9a
OCH
H
3
H
NI
NI
9b
OCH
3
H
NI
9c
OCH
H
3
H
OCH
H
2.6
9.0
NI
2.8
NI
9d
OCH
OCH
OCH
3
3
3
OCH
3
9e
OCH
3
3
H
H
NI
9f
OCH
OCH
H
2.70
4.4
a
b
NI: No significant inhibition (GI > 10 ꢀM). Experimental error is within 20%.
5
0
Further SAR studies of the para position on the phenyl ring were performed (9g-u) and the
results are summarized in Table 4. We found that a substitution of 4ꢀmethoxy to an electron
withdrawing groups substituents 9h-k, resulted in the loss of activity. The 4ꢀfluoromethyl
derivative (9j) displayed a 3ꢀfold loss of inhibitory activity in TC32. However, it should be noted
that 9j also inhibited PANC1 cells growth suggesting that its inhibitory action was not selective.
Thus, 9j could have generalized offꢀtarget toxicity.
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2
2
2
2
2
2
2
2
2
3
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4
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5
5
5
6
The 4ꢀisopropyl substitution 9p and the bulky phenyl substitution represented by 9q resulted in
the complete loss of activity. Also, the 4ꢀmethyl derivative (9n) resulted in a complete loss of
activity. The 4ꢀethoxy derivative 9m displayed a 3ꢀfold decrease of potency in the ES cells
suggesting that substitution with the larger group may be unfavorable to the inhibitory activity of
the derivatives. Interestingly, the thiomethyl derivative 9r retained activity comparable to 2 (GI50
of 0.98 ꢀM). The 4ꢀamine substitution 9s on the aromatic ring resulted in the complete loss of
activity. However, the methylamine derivative 9t resulted in more than twoꢀfold increase in
potency (GI 0.34 ꢀM) compared to 2 (GI 0.92 ꢀM). The activity was also improved with the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
dimethylamine derivative 9u with GI of 0.26 ꢀM in TC32 cells. This activity was equal to the
50
1
8
potency of the S-(-) enantiomer of 2 (GI 0.28 ꢀM) in TC32 cells . Antiꢀproliferative results
5
0
in TC32 cells were comparable to those observed in TC71 cells.
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Table 4. Chemical Structure and Biological Activities of Compounds 9g-9u
a,b
Substitution
R1
GI (ꢀM)
5
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
PANC 1
TC32
TC71
2
ꢀOCH
3
NI
0.94 (0.18) 0.92 (0.12)
9
g
ꢀH
NI
NI
NI
NI
NI
9h
ꢀF
NI
9i
ꢀI
NI
NI
NI
9j
ꢀCF
ꢀCN
ꢀCH
ꢀOC
3
11.0 (0.23)
2.9 (0.23)
NI
6.9 (0.19)
NI
9
k
n
NI
NI
NI
NI
NI
NI
NI
NI
9
3
NI
NI
9
m
2
H
5
2.70 (0.21) 3.57 (0.19)
9p
ꢀOiPr
ꢀOPh
NI
NI
NI
NI
9q
9r
ꢀSCH
ꢀNH
ꢀNHCH
ꢀN(CH
3
0.98 (0.17) 0.91 (0.14)
NI NI
0.34 (0.09) 0.39 (0.10)
0.26 (0.1) 0.40 (0.07)
9s
2
9t
3
9u
3
)
2
NI
a
b
NI: No significant inhibition (GI > 10 ꢀM) Experimental error represents S.E.M.
5
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6
Functional Inhibition of EWS-FLI1
Previously, we have demonstrated that compound 2 acted as a functional inhibitor of EWSꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
17
FLI1 . It has been reported that EWSꢀFLI1 binds directly to the microsatellite repeats of the
2
7
promoter NR0B1 to drive oncogenesis in ES . In this study, COS7 cells were transfected with
1
7,28
EWSꢀFLI1 and NR0B1 reporterꢀluciferase plasmids as previously reported (Figure 3A)
.
EWSꢀFLI1ꢀtransfected cells showed a doseꢀdependent decrease in the promoter activity when
treated for 20 h with compound 2 (IC = 0.35 ꢀM) as compared to no transfection (Figure 3BꢀC).
5
0
Control experiments at 48 hours show that the compounds were not cytotoxic to the COS7 cells.
COS7 are fibroblastꢀlike cells derived from monkey kidney that are devoid of the EWSꢀFLI1.
To further assess their inhibitory effects on the transcriptional activities of EWSꢀFLI1, we
selected 6e, 9c, 9m, 9r, 9t, 9u, 10 and 14a which displayed a GI of 2.7 ꢀM or better and 9a, 11
5
0
and 15 which displayed no antiꢀproliferative activity (Figure 4AꢀB). As expected, 9a which
displayed no inhibition on cell growth did not inhibit the transcriptional activities of EWSꢀFLI1.
9r inhibition was comparable to 2 with IC of 0.33 ꢀM whereas 6e has an IC of 0.77 ꢀM.
5
0
50
Compounds 9c and 9m inhibitory effects were lower than 2. Unlike 11 that did not display any
inhibition, 14a and 15 showed modest inhibitory effects on the transcriptional activities of EWSꢀ
FLI1. Consistent with the ES cell growth inhibition data, 9t and 9u exhibited better inhibition of
EWSꢀFLI1 with an IC of 0.18 ꢀM and 0.15 ꢀM respectively. The results strongly support that
5
0
these derivatives inhibit the transcriptional activity of EWSꢀFLI1.
A plot of the IC of the luciferase activity versus the GI (Figure 5) was performed to show that
5
0
50
the cellular growth inhibition effects of the ES cells observed are dependent on the inhibition of
the transcriptional activity of EWSꢀFLI1. A correlation coefficient of 0.84 provides further
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evidence that the effects on the inhibition of the transcriptional activity of EWSꢀFLI1 and the
cell growth inhibition of ES cells are related. This data suggested that the EWSꢀFLI1 mediated
growth of the ES cells can be effectively decreased by inhibiting the transcriptional activity of
EWSꢀFLI1.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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36
37
38
39
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A
EWSꢀFLI1
NROB1 Promoter
Luciferase Gene
COS7
B
No transfection
C
2.0
1
750
1.5
1.0
0.5
1500
1250
1
000
50
500
7
0.0
DMSO
0.1
1
10
30
Concentration (ꢀM)
2
50
0
EWSꢀFLI1
pCiꢀneoꢀEV
NROB1ꢀLuc
DMSO
+
-
-
+
+
-
+
-
+
-
+
-
+
-
+
-
+
-
+
-
-
+
-
+
+
-
+
+
+
+
+
+
1
+
+
3
+
-
+
Compound 2
-
-
-
0.1 0.3
10
Figure 3. (A) Schematic construct of the luciferase reporter assay for the transcription inhibition
of the EWSꢀFLI1 by small molecule. (B) Cell proliferation assay (WST) indicating negligible
effect of compound 2 on COS7 cells. (C) COS7 cells were transfected with NR0B1 luciferase
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2
2
2
2
2
2
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5
5
6
reporter and empty vector (pCiꢀneoꢀEV) or full length EWSꢀFLI1. Diagram shows a mean ± SD
of a single representative experiment performed in quadruplicate
A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
100
2
6
e
75
9a
9c
50
9m
9r
25
9t
9u
0
log(concentration) ꢀM
B
100
2
9a
75
10
5
0
5
0
11
14a
2
15
log(concentration) ꢀM
Figure 4. Luciferase assay showing inhibition of EWSꢀFLI1 transcriptional activity in COS7
cells. (A) Doseꢀresponse profiles of compounds 2, 6e, 9a, 9c, 9m, 9r, 9t, 9u for EWSꢀFLI1
inhibition. (B) Doseꢀresponse profiles of compounds 2, 9a, 10, 11, 14a, 15 for EWSꢀFLI1
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inhibition. Diagrams show a mean ± SD of single representative experiment performed in
quadruplicate, with compound 2 graphed in comparison and 9a as negative control.
10
11
12
13
14
15
16
17
18
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24
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3.5
9c
9
2
R =0.84
3
.0
2.5
.0
1.5
m
2
1
0
0
.0
.5
.0
6e
1
4a
9
u9t
9
r
2
0
.0
0.5
1.0
1.5
2.0
2.5
3.0
GI (ꢀM)
50
Figure 5. Correlation of the analogs on growth inhibition in TC32 cells and Luciferase reporter
activity in COS7 cells.
Design and evaluation of biotinylated probe of compound 24
We envisioned the synthesis of a biotin conjugate that could be used to detect binding of the
derivative and the recombinant EWSꢀFLI1 (Scheme 5A). We developed a synthetic route to
append a five carbon linker terminating with an amine to compound 9t. The amine was exploited
to link biotin to the 9tꢀlinker molecule to afford 24 (Scheme 5A). We evaluated the binding of 24
to recombinant EWSꢀFLI1 as shown in Figure 6A. A 96ꢀwell plate was coated with the
recombinant protein that was allowed to bind with the biotinylated conjugate. The binding of 24
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
to EWSꢀFLI1 was detected with HRP streptavidin conjugate. We detected a dose dependent
binding of 24 to immobilized EWSꢀFLI1 (Figure 6B). No binding was detected when BSA was
immobilized. The saturation was reached at 20ꢀM. Concentrationꢀdependent binding of 24 to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
immobilized EWSꢀFLI1 (Figure 6B) was exhibited by biotinylated 24 over a range of 1ꢀ100 ꢀM
resulting in a K of 4.8 ± 2.6 ꢀM.
d
A
TMB Substrate
HRP
Yellow Color
HRP
Recombinant
Protein
Recombinant
Protein
Recombinant
Protein
Recombinant
EFꢀ24 complex
EFꢀ24-stvnꢀHRP
EWSꢀFLI1 (EF) in well
complex
B
0.3
K = 4.8 ꢀM ± 2.6 ꢀM
d
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0.1
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0
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75
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Concentration (ꢀM)
Figure 6. (A) Schematic overview of ELISAꢀlike assay to probe the binding of the biotin
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conjugate to recombinant EWSꢀFLI1; (B) Saturation curve of biotin conjugate (24) to
recombinant EWSꢀFLI1
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CONCLUSION
This research provides a solid ground for the discovery of new and effective agents to treat
Ewing’s sarcoma. The hit compound 1 was discovered through a surface plasmon resonance
initial screening against recombinant EWSꢀFLI1. Compound 2 with increased activity reduces
1
7
the growth of ES cells and ES orthotopic xenografts . Compound 2 was used as our lead
compound for further detailed SAR studies.
We modified 2 with the aim to clarify the relevant structural determinants for small molecule
inhibition of EWSꢀFLI1. On the indoline of 2, our investigation highlights the importance of the
4,7ꢀposition and the significance of the free amine for optimal inhibition. We found that removal
of the alcohol by dehydration shows that the hydroxyl group is not required for binding. The loss
of activity resulting from the removal of hydrogen bond donation supports the finding that the
alcohol group does not act as a hydrogen bond donor.
Additionally, we have observed that the paraꢀposition on the phenyl ring was the most favorable
for inhibition of EWSꢀFLI1 by 2. Electron donating groups at the paraꢀposition significantly
contribute to a higher inhibitory activity. Compounds 9t and 9u had over 3ꢀfold higher potency
than the lead 2. It appears that the electron density of the substitutions may play a more
significant role in the inhibitory action of the derivatives. The electron density of 4ꢀ
dimethylamine > 4ꢀmethylamine > 4methoxy correlates with increasing inhibitory activity
(compound 9u> compound 9t > compound 2).
We have successfully correlated the cell growth inhibition of the ES cells by the active
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derivatives with the luciferase reporter activity, statistically linking observed activity to the
inhibition of the transcriptional activity of EWSꢀFLI1. Finally, we showed that a derivative binds
to the target using a novel biotin conjugate. The conjugate can be utilized as a screening tool to
better understand the binding of small molecules to the EWSꢀFLI1.
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Experimental Section
Chemistry
General Chemistry Methods
All solvents and reagents were used as obtained from commercial sources unless otherwise
indicated. Commercial reagents and anhydrous solvents were used without further purification.
Analytical thin layer chromatography (TLC) was performed on silica gel 60 F254 aluminum
sheets. Removal of solvents was conducted by using a rotary evaporator, and residual of solvents
was removed from nonvolatile compounds using a vacuum manifold. All reported yields are
1
13
isolated yields. H and C NMR spectra were recorded on a Varian 400MR spectrometer
1
13
operating at 400 MHz for H and 100 MHz for C. Deuterated dimethyl sulfoxide or deuterated
1
methanol or deuterated chloroform was used as the solvent for NMR experiments. H Chemical
shifts values (δ) are referenced to the residual nondeuterated components of the NMR solvents (δ
1
3
=
2.54 ppm for DMSO, δ = 7.26 ppm for CDCl and δ = 3.31 ppm for CD OD). The
C
3
3
chemical shifts (δ) are referenced to CDCl (central peak, δ = 77.16 ppm) or CD OD (δ = 49.00
3
3
ppm) as the internal standard. The HRMS data were obtained on a Waters QꢀTOF Premier mass
spectrometer. Column chromatography was performed using silica gel (35−75 mesh). The purity
of final compounds was evaluated by C, H, N, analysis (Atlantic microlabs). The purity of all the
final compounds was confirmed to be ≥ 95 by combustion.
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General Procedure A. To a solution of choral hydrate in water were added sodium sulfate (6
equiv.), and appropriate aniline (1 equiv.) followed by a solution of hydroxylamine
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°
hydrochloride solution (3 equiv.) in water. The reaction mixture was heated at 55 C with stirring
overnight and then cooled to room temperature. The resulting precipitate was filtered and dried
to obtain the isonitrosoacetanilide intermediates. The isonitrosoacetanilide was added to a
stirring solution of concentrated sulfuric acid slowly to keep the temperature in the range of 60ꢀ
°
°
70 C. Upon completion of the addition, the mixture was heated to 80 C for 10 minutes and then
poured unto crushed ice and stirred for 30 minutes. The precipitated was filtered and washed
three times with water and dried under vacuum to afford the solids 5a-5e.
General Procedure B. To a solution of 4,7ꢀdichloroisatin (1.0 equiv.) was added appropriate
acetophenone (4.0 equiv.) and catalytic amount diethylamine (10 drops) in methanol. The
mixture was stirred at room temperature until the starting material (4,7ꢀdichloroisatin)
disappeared completely. The resulted solution was concentrated and purified in flash
chromatography eluting with Hexane/Ethyl acetate to afford product in quantitative yield. The
product was recrystallized with Hexanes / Ethyl acetate to give a pure product.
4-chloroindoline-2,3-dione (5a). Compound 5a was prepared via general procedure A from 3a
(
500 mg, 3.88 mmoles), chloral hydrate (965 mg, 5.83 mmoles), hydroxylamine hydrochloride
(
808 mg, 11.6 mmoles), sodium sulfate (3.42 g, 23.3 mmoles) and 1N HCl (8mL) following the
1
general procedure A to yield a dark brown solid (580 mg, 82.3 %). H NMR (DMSOꢀd6, 400Hz)
δ 11.17 (s, 1H), 7.53 (t, 1H, J=16.4), 7.06 (d, 1H, J=8), 6.87 (d, 1H, J=8).
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-chloroindoline-2,3-dione (5b), Compound 5b was prepared via general procedure A from 3b
(500 mg, 3.88 mmoles), chloral hydrate (965 mg, 5.83 mmoles), hydroxylamine hydrochloride
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(808 mg, 11.64 mmoles), sodium sulfate (3.42 g, 23.3 mmoles) and 1N HCl (8mL) following the
1
general procedure A to yield a brown solid (600 mg, 85 %). H NMR (DMSOꢀd , 400Hz)
6
δ 11.24 (s, 1H), 7.62ꢀ7.59 (m, 1H), 7.535(s, 1H), 6.92 (d, 1H, J = 8.4).
5
,6-dichloroindoline-2,3-dione (5c); Compound 5c was prepared via general procedure A from
3
c (500 mg, 2.31 mmoles), chloral hydrate (574 mg, 3.47 mmoles), hydroxylamine
hydrochloride (482 mg, 6.94 mmoles), sodium sulfate (2.04 g, 13.9 mmoles) and 1N HCl (4 mL)
1
following the general procedure A to yield a brown solid (370 mg, 74%). H NMR (DMSOꢀd ,
6
4
00Hz) δ 11.24 (s, 1H), 7.7 (d, 1H, J = 9.2), 6.86 (d, 1H, J = 8.4).
4,7-difluoroindoline-2,3-dione (5d); Compound 5d was prepared via general procedure A from
3d (400 ꢀL, 3.96 mmoles), chloral hydrate (722 mg, 4.36 mmoles), hydroxylamine
hydrochloride (827 mg, 11.9 mmoles), sodium sulfate (3.49 g, 23.8 mmoles) and 1N HCl (8 mL)
1
following the general procedure A to yield an orange solid (507 mg, 70 %). H NMR (DMSOꢀd ,
6
19
400Hz) δ 11.704 (s,1H), 7.60ꢀ7.55 (m, 1H), 6.88ꢀ6.23 (m, 1H). F NMR (DMSOꢀd , 367Hz): ꢀ
6
138.21.
4,7-dimethoxyindoline-2,3-dione (5e); Compound 5e was prepared via general procedure A from
3e (400 mg, 2.61 mmoles), chloral hydrate (433.2 mg, 2.61 mmoles), hydroxylamine
hydrochloride (548 mg, 7.83 mmoles), sodium sulfate (2.3 g, 15.7 mmoles) and 1N HCl (5.5
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mL) following the general procedure A to yield a brown solid (184 mg, 34 %). H NMR
(DMSOꢀd , 400Hz) δ 11.70 (s, 1H), 7.32 (d, 1H, J=9.2), 6.62 (d, 1H, J=9.2), 3.80 (s, 3H), 3.78
6
(
s, 3H).
-chloro-3-hydroxy-3-(2-(4-methoxyphenyl)-2-oxoethyl)indolin-2-one (6a) ; Compound 6a was
prepared via general procedure B from 5a (50 mg, 0.27 mmoles) and 4ꢀmethoxyacetophenone
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60
4
°
1
(
165.4 mg, 1.10 mmoles) to yield a white solid (84 mg, 92 %); mp 224ꢀ226 C. H NMR
(DMSOꢀd , 400Hz) δ 10.51 (s, 1H), 7.90 (d, 2H, J=8.8), 7.22 (t, 1H, J=16), 7.04 (d, 2H, J=8.8),
6
1
3
6
.86 (q, 2H), 6.25 (s, 1H), 4.37 (d, 1H, J=18), 3.85 (s, 3H), 3.63 (d, 1H, J=18). C NMR
(
DMSOꢀd , 100Hz): 195.17, 177.98, 163.88, 145.65, 131.06, 130.66 (2C), 130.09, 129.18,
6
128.27, 122.41, 114.47 (2C), 108.97, 74.38, 56.03, 44.35. HRMS (ES) m/z calcd for
+
C H ClNO (M ꢀ H) 330.0533; found, 330.0524. Anal. calcd for C H ClNO C, 61.55; H,
17
14
4
17 14
4
4.25; N, 4.22. Found: C, 61.6; H, 4.34; N, 4.26.
5
-chloro-3-hydroxy-3-(2-(4-methoxyphenyl)-2-oxoethyl)indolin-2-one (6b); Compound 6b was
prepared via general procedure B from 5b (50 mg, 0.27 mmoles) and 4ꢀmethoxyacetophenone
°
1
(
165 mg, 1.10 mmoles) to yield a white solid (85 mg, 93.4 %); mp 191 C. H NMR (DMSOꢀd ,
6
4
00Hz) δ 10.37 (s, 1H), 7.88 (d, 2H, J=8.8), 7.38 (s, 1H), 7.23 (q, 1H), 7.04 (d, 2H, J=8.8), 6.83
1
3
(
d, 1H, J=8.4), 6.16 (s, 1H), 4.11 (d, 1H, J=17.6), 3.84 (s, 3H), 3.59 (d, 1H, J=17.6). C NMR
(
DMSOꢀd , 100Hz): 195.28, 178.57, 163.79, 142.38, 134.57, 130.75 (2C), 129.49, 129.02,
6
1
25.55, 124.35, 114.35 (2C), 111.21, 73.57, 56.04, 45.85. HRMS (ES) m/z calcd for
+
C H ClNO (M ꢀ H) 330.0533; found, 330.0529. Anal. calcd for C H ClNO C, 61.55; H,
17
14
4
17 14
4
4.25; N, 4.22. Found: C, 61.46; H, 4.55; N, 4.09.
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,6-dichloro-3-hydroxy-3-(2-(4-methoxyphenyl)-2-oxoethyl)indolin-2-one (6c); Compound 6c
was prepared via general procedure B from 5c (50 mg, 0.23 mmoles) and 4ꢀ
methoxyacetophenone (139 mg, 0.92 mmoles) to yield a white solid (79 mg, 95 %); mp 214ꢀ215
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C. H NMR (DMSOꢀd , 400Hz) δ 10.64 (s, 1H), 7.91 (d, 2H, J=8.4), 7.47 (d, 1H, J=8.4), 7.04
6
(d, 2H, J=9.2), 6.85 (d, 1H, J=8.4), 6.38 (s, 1H), 4.37 (d, 1H, J=18), 3.85 (s, 3H), 3.68 (d, 1H,
13
J=18). C NMR (DMSOꢀd , 100Hz): 195.18, 177.79, 163.97, 144.21, 131.17, 130.77, 130.73
6
(2C), 130.50, 129.01, 114.50 (2C), 110.15, 75.03, 73.24, 56.05, 44.34. Anal. calcd for
C H Cl NO C, 55.76; H, 3.58; N, 3.82. Found: C, 55.82; H, 3.60; N, 3.88.
17
13
2
4
4,7-difluoro-3-hydroxy-3-(2-(4-methoxyphenyl)-2-oxoethyl)indolin-2-one (6d); Compound 6d
was prepared via general procedure B from 5d (50 mg, 0.27 mmoles) and 4ꢀ
methoxyacetophenone (164 mg, 1.09 mmoles) to yield a light pink solid (83.6 mg, 93 %); mp
°
1
170 C H NMR (DMSOꢀd , 400Hz) δ 10.96 (s,1H), 7.87 (d, 2H, J=8.8), 7.17ꢀ7.11 (m, 1H), 7.01
6
(d, 2H, J=9.2), 6.8ꢀ6,62 (m, 1H), 6.45 (s, 1H), 4.03 (d, 1H, J=17.6), 3.82 (s, 3H), 3.66 (d, 1H,
1
3
J=18). C NMR (DMSOꢀd , 100Hz): 195.18, 177.82, 163.99, 149.24, 130.76, 129.09 (2C),
6
1
9
119.78, 118.11, 114.49, 109.47 (2C), 109.23, 104.99, 73.48, 56.06, 45.17. F NMR (DMSOꢀd ,
6
+
3
67Hz): ꢀ138.62. HRMS (ES) m/z calcd for C H F NO (M ꢀ H) 332.0734; found, 332.0735.
1
7
13
2
4
Anal. calcd for C H F NO C, 61.26; H, 3.93; N, 4.20. Found: 61.45; H, 3.93; N, 4.30
1
7
13
2
4
3
-hydroxy-4,7-dimethoxy-3-(2-(4-methoxyphenyl)-2-oxoethyl)indolin-2-one (6e); Compound 6e
was prepared via general procedure B from 5e (50 mg, 0.24 mmoles) and 4ꢀ
methoxyacetophenone (144 mg, 0.96 mmoles) to yield a brown solid (31.4 mg, 35 %);
1
H NMR (DMSOꢀd , 400Hz) δ 10.16 (s, 1H), 7.82 (d, 2H, J=9.2), 6.99 (d, 2H, J=9.2), 6.81 (d,
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H, J=9.2), 6.39 (d, 2H, J=8.8), 5.85 (s, 1H), 4.17 (d, 1H, J=16.8), 3.80 (s, 3H), 3.73 (s, 3H),
3.59 (s, 3H), 3.41 (d, 1H, J=16.8). Anal. calcd for C H NO : C, 63.86; H, 5.36; N, 3.92. Found:
19 19 6
C, 63.78; H, 5.69; N, 3.90.
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3-hydroxy-3-(2-(4-methoxyphenyl)-2-oxoethyl)-4,7-dimethylindolin-2-one (6f); Compound 6f
was prepared via general procedure B from 5f (50 mg, 0.28 mmoles) and 4ꢀ
methoxyacetophenone (172 mg, 1.14 mmoles) to yield a brown solid (87.4 mg, 96 %); mp 135ꢀ
°
1
136 C. H NMR (DMSOꢀd , 400Hz) δ 10.18 (s, 1H), 7.86 (d, 2H, J=9.2), 6.99 (d, 2H, J=8.8),
6
6.84 (d, 1H, J=7.6), 6.52 (d, 1H, J=7.6), 5.91 (s, 1H), 4.04 (d, 1H, J=17.2), 3.80 (s, 3H), 3.54 (d,
13
1H, J=17.2), 2.20 (s, 3H), 2.13 (s, 3H). C NMR (DMSOꢀd , 100Hz): 195.085, 179.15, 163.72,
6
141.92, 132.01 (2C), 130.68, 130.43, 129.55, 128.73, 123.84, 116.36, 114.37 (2C), 74.70, 56.00,
4
5.03, 17.42, 16.57. Anal. calcd for C H NO .1/4 EtOAc: C, 69.15; H, 6.09; N, 4.03. Found: C,
1
9
19
4
69.10; H, 6.04; N, 4.15.
7-chloro-3-hydroxy-3-(2-(4-methoxyphenyl)-2-oxoethyl)indolin-2-one (6g). Compound 6g was
prepared via procedure B from commercially available 7ꢀchloroisatin (50 mg, 0.27 mmoles) and
1
4
ꢀmethoxyacetophenone (165.4 mg, 1.10 mmoles) to yield a white solid (84 mg, 92 %). H NMR
(DMSOꢀd , 400Hz) δ 10.66 (s,1H), 7.87 (d, 2H, J=8.8), 7.25 (dd, 2H, J=12.4), 7.02 (d, 2H,
6
1
3
J=8.8), 6.90 (t, 1H, J=15.6), 6.18 (s, 1H), 4.06 (d, 1H, J=18), 3.83 (s, 3H), 3.60 (d, 1H, J=18). C
NMR (DMSOꢀd , 100Hz): 195.25, 178.75, 163.82, 141.17, 134.44, 130.85 (2C), 129.43, 129.29,
6
122.94, 122.53, 114.28, 114. 05, 74.03, 56.04, 46.04. HRMS (ES) m/z calcd for C H ClNO (M
1
7
14
4
+
ꢀ
H) 330.0533; found, 330.0330. Anal. calcd for C H ClNO .0.1EtOAc C, 61.37; H, 4.38; N,
17 14 4
4.11. Found: C, 61.27; H, 4.48; N, 4.19.
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