Syntheses and antiproliferative evaluation of oxyphenisatin derivatives

Article abstract of DOI:10.1016/j.bmcl.2007.02.060

Syntheses and structure-antiproliferative relationship for oxyphenisatin analogues are described. The cell proliferation data showed that the presence of substituents (especially F, Cl, Me, CF₃, and OMe) in the 6- and 7-position of oxyphenisati

Full text of DOI:10.1016/j.bmcl.2007.02.060

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2854–2857
Syntheses and antiproliferative evaluation
of oxyphenisatin derivatives
Muhammed K. Uddin,a Serge G. Reignier,a Tom Coulter,a Christian Montalbetti,a,*
b
b
b
b,
*
˚
Charlotta Grana¨s, Steven Butcher, Christian Krog-Jensen and Jakob Felding
aEvotec(UK) Ltd, 111 Milton Park, Abingdon, Oxon OX14 4RX, UK
bBioImage A/S Mørkhøj Bygade 28, DK-2860 Søborg, Denmark
Received 15 January 2007; revised 16 February 2007; accepted 21 February 2007
Available online 25 February 2007
Abstract—Syntheses and structure–antiproliferative relationship for oxyphenisatin analogues are described. The cell proliferation
data showed that the presence of substituents (especially F, Cl, Me, CF3, and OMe) in the 6- and 7-position of oxyphenisatin
markedly enhanced the potency in the MDA-468 cell line without affecting the MDA-231 cell line. The best compounds from this
series showed low nanomolar antiproliferative activity towards the MDA-468 cell line and a 1000-fold selectivity over the MDA-231
cell line.
Ó 2007 Elsevier Ltd. All rights reserved.
Cancer is the second leading cause of death in developed
countries, accounting for nearly one in five deaths. One
characteristic of cancer cells is their highly proliferative
nature. Consequently, inhibition of proliferative path-
ways is considered an effective strategy to fight cancer.
One way to identify compounds with antiproliferative
effects is to screen for compounds which reduce prolifer-
ation in one cancer cell line and counter screen against
another cancer cell line. Important advantages of this
approach are that the active compounds are reasonably
cell permeable and water soluble. Furthermore, com-
pounds with nonspecific toxicity are eliminated and, as
the functional end-effect is measured in whole cells, the
risk of observing compensatory mechanisms at a later
stage is significantly reduced. However, in contrast to
screening against an isolated molecular cancer target
this approach requires further studies to elucidate the
exact mode of action.
ing approach was designed to identify compounds with
a cell selectivity profile similar to CCI-779, a novel
mTOR inhibitor.1 Natarajan et al. recently published
cell growth inhibition data on a similar series of which
the lead compound is the unresolved mixture of chiral
compound 2.2 They compared the activity of these com-
pounds to that of clotrimazole 3 and suggested the
HO
OH
*
OH
OH
O
O
N
H
N
H
Cl
Me
2 (+/-)
1
HO
In an anticancer project we identified 6-chloro-3,3-bis-
(40-hydroxyphenyl)-7-methyl-1,3-dihydroindole-2-one 1
(see Fig. 1) as a potent inhibitor of the cell proliferation
in the MDA-468 cell line (IC50 = 20 nM). Furthermore,
the compound was more than 100-fold less active to-
wards the MDA-231 cell line (IC50 > 3 lM). This screen-
Cl
N
N
O
N
H
3
4
Keywords: Antiproliferative; Anticancer; MDA-468 cell line; Pheni-
satin; Whole cell assay.
Figure 1. Structure of compound 1, Lead compound of reference 1b 2,
Clotrimazole 3, and Oxyphenisatin 4.
*
Corresponding authors. E-mail: christian.montalbetti@evotec.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.060

M. K. Uddin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2854–2857
Table 1. Role of the hydrogen donor groups of compound 1
2855
O
a
b
X
O
3'
4'
1.
2.
3.
NH2
N
2'
H
R
X
R
O
O
N
R
Cl
O
NH2
N
Me
H
Compound
MDA-468 MDA-231
a
IC50 (lM) IC50 (lM)
X
R
a
O
c
4
(oxyphenisatin)
0.112
na
—
—
O
NHBoc
N
H
1
0.020
na
na
na
na
na
na
na
na
na
na
na
40-OH
40-OH
40-H
40-F
40-OMe
40-NH2
40-NMe2
40-NHAc
40-NHSO2Me
20-OH
H
Me
H
H
H
H
H
H
H
H
R
R
1a
1b
1c
1d
1e
1f
1g
1h
1i
0.238
0.291
1.8
Scheme 1. Reagents and conditions: (a) CCl3CHO, H2NOH, 60 °C
then H2SO4, 90 °C; (b) AcOH, diethyl ketomalonate, then KOH(aq);
(c) tBuLi, diethyl oxalate, then 180 °C, <5 mmHg.
2.6
na
na
na
a
OH
O
R
O
N
na
a)
b)
O
O
a Values are means of two experiments (na, not active (>3 lM)).
N
O
R
N
H
R
N
H
H
b
Figure 2.
antiproliferative effect to be mediated by translation ini-
tiation inhibition.
a mixture of two isomers (see pathway a or pathway b
in Fig. 2).7
However, in our two cell lines, clotrimazole 3 showed
no selectivity and an IC50 of more than 10 lM in both
assays. Since compound 1 is a substituted oxyphenisa-
tin (4), the latter was included in the screen (see
Fig. 1). The cell proliferation data showed oxyphenisa-
tin 4 to be equally selective, but ꢀ5-fold less potent
(see Table 1). Oxyphenisatin acetate is a laxative
which was used for more than 40 years as a non-
prescription drug before it was taken off the market
in 1972 due to hepatotoxicity.3 The hepatic reaction
to oxyphenisatin is, however, presumably due to a
hypersensitivity response rather than a nonspecific
toxic effect.4
We controlled the regiochemistry of the product forma-
tion by use of a chlorine atom to block one of two
potential cyclisation positions and then subsequently
removed the halogen by a catalytic dehalogenation with
a Pd/C/H2 system affording either the 4 or the 6-substi-
tuted oxyphenisatin (several examples are shown in
Scheme 2).8
HO
HO
HO
In the present work, we describe the syntheses and struc-
ture–activity relationship of these symmetrical oxyphe-
nisatin derivatives using antiproliferative data from the
MDA-468 cell line. We specifically excluded chiral mol-
ecules to avoid potential resolution problems during fur-
ther development phases.
OH
OH
OH
OH
a
O
O
NH2
N
H
N
H
80%
2g
Cl
Cl
4i
Cl
HO
Isatins are commonly used intermediates for accessing
oxyphenisatins and several procedures exist in the lit-
erature to form this particular ring system.5 In this
study, we have primarily used the synthetic methods
shown in Scheme 1. The Sandmeyer methodology6
(see example 1 in Scheme 1) is the oldest and most
frequently used synthetic approach towards isatins.
The key step involves the cyclisation of the intermedi-
ate obtained by reacting anilines, chloral hydrate and
hydroxylamine.
a
O
O
70%
N
NH2
N
H
H
Cl
HO
HO
OH
OH
a
O
O
50%
N
F
NH2
N
F
F
H
H
Cl
Cl
2b
In the case of meta-substituted anilines, the cyclisation
step in the Sandmeyer procedure can potentially afford
Scheme 2. Reagents and condition: (a) Pd/C, H2, AcOK, rt, MeOH.

2856
M. K. Uddin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2854–2857
The Martinet isatin procedure9 (see example 2 in
Scheme 1) involves the reaction of diethyl keto-malonate
hydrate with anilines in the presence of acid, followed
by the oxidative decarboxylation of the resulting
intermediate.
Table 2. Substituents in the 5- and 6-position of oxyphenisatin
HO
OH
5
R
O
The ortho-lithiation procedure10 (see example 3 in
Scheme 1) proceeds by an a-directed deprotonation of
N-Boc protected aniline, followed by trapping of the
aryllithium intermediate with diethyl oxalate, and cycli-
sation under electrophilic conditions results in the isatin
formation.
6
N
H
Compound
MDA-468
a
IC50 (lM)
MDA-231
a
IC50 (lM)
R
2a
2b
2c
2d
2e
2f
0.170
0.028
0.152
0.190
0.208
0.183
0.077
0.173
0.191
na
na
na
na
na
na
na
na
na
na
na
na
na
na
5-F
6-F
5-Cl
5-Br
We have obtained the desired oxyphenisatin derivatives,
quickly and in large numbers via a double Friedel–
Crafts reaction11,12 on the keto function of the isatins
5-I
5-Me
6-Me
2g
2h
2i
using substituted phenyl derivatives containing
a
5-OMe
6-OMe
5-Ph
combination of ortho- or para-orientating groups (see
Scheme 3).
2j
2k
2l
na
na
5-(2-Thienyl)
5-(4-Pyridyl)
COOH
Further functionalisations were also performed on
the prepared oxyphenisatins. For example Suzuki
reactions13 were carried out on 5/7-bromo and
5/7-iodo-3,3-bis-(4-hydroxy-phenyl)-1,3-dihydro-indol-
2-one with boronic acid and palladium tetrakis tri-
phenyl phosphine, better results were obtained
from the bromo precursors (see Scheme 4).
2m
na
a Values are means of two experiments (na, not active (>3lM)).
From Table 1 it is evident that the OH-functions
attached to the 4-position of the phenyl ring systems
are crucial as removing or methylating the OH function
(1b and 1d), moving the OH-function to the 2-position
(1i) or replacing the 4-OH-function with phenol bioiso-
sters in the same position (1e, 1g–h) all resulted
in compounds with a significantly reduced potency.
14
Finally, a Sonogashira coupling of derivative 3d with
TMS acetylene, followed by TBAF treatment, afforded
compound 3h in a low yield of 5% (see Table 3).
To explore the structural–antiproliferative activity rela-
tionship of compound 1, we performed a systematic
investigation of the functionalities present. Initially we
evaluated the importance of the hydrogen bond donor
moieties.
Table 3. Substituents in the 7-position of oxyphenisatin
HO
OH
O
R'
N
H
R
O
R'
O
Compound
MDA-468
a
MDA-231
a
R
N
9-38%
IC50 (lM)
IC50 (lM)
R
H
O
N
H
3a
3b
3c
3d
3e
3f
0.034
0.006
0.006
0.062
0.027
0.004
0.057
0.090
0.262
0.330
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
F
Cl
R
Scheme 3. Reagents and condition: Ph-R0, AcOH, H2SO4 or TfOH,
90 °C.
Br
I
Me
CF3
CN
3g
3h
3i
HO
HO
C „ CH
OMe
Et
OH
OH
3j
3k
3l
i-Pr
Ar
O
I/Br
O
5-40%
N
H
N
H
na
na
t-Bu
Ph
3m
3n
3o
3p
3q
Ar = 5-phenyl
Ar = 5-(2-thienyl)
Ar = 5-(4-pyridyl)
3m
Ar = 7-(2-thienyl) 3n
Ar = 7-(4-pyridyl)
Ar = 7-phenyl
2j
na
na
2-Thienyl
4-Pyridyl
COOH
CONMe2
2k
2l
3o
na
na
Scheme 4. Reagents and condition: Pd(PPh3)4, Ar–B(OH)2, K2CO3,
DME, 80 °C.
a Values are means of two experiments (na, not active (>3 lM)).

M. K. Uddin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2854–2857
2857
Table 4. Disubstituted oxyphenisatin derivatives
moderate inhibition (1–2 lM) of CYP2C9 and 2C19
probably due to the presence of the phenol moieties.15
HO
OH
In conclusion, we have used a cell-based screening ap-
proach to assess structure–activity relationships and
identified two low nanomolar compounds with reason-
able physicochemical and in vitro ADME profiles. The
in vivo activity of this compound series has also been
confirmed in appropriate xenograft models.12
R2
5
O
6
N
7
H
R1
Compound
MDA-468
a
IC50 (lM)
MDA-231
a
IC50 (lM)
R1
R2
References and notes
4a
4b
4c
4d
4e
4f
0.037
0.100
0.162
0.160
0.003
0.006
0.089
0.007
0.006
0.009
0.021
0.055
na
na
na
na
na
na
na
na
na
na
na
na
na
na
5-F
5-F
7-F
7-Me
7-Me
7-Me
7-F
1. Yu, K.; Toral-Barza, L.; Discafani, C.; Zhang, W-G.;
Skotnicki, J.; Frost, P.; Gibbons, J. J. Endo-Related
Cancer 2001, 8, 249.
2. (a) Natarajan, A.; Fan, Y.-H.; Chen, H.; Guo, Y.; Iyasere,
J.; Harbinski, F.; Christ, W. J.; Aktas, H.; Halperin, J. A.
J. Med. Chem. 2004, 47, 1882; (b) Natarajan, A.; Guo, Y.;
Harbinski, F.; Fan, Y.-H.; Chen, H.; Luus, L.; Diercks, J.;
Aktas, H.; Halperin, J. A. J. Med. Chem. 2004, 47, 4979;
(c) Halperin, J. A.; Natarajan, A.; Aktas, H.; Fan, Y.-H.;
Chen, H. WO2005/080335A1.
5-Me
5-OMe
6-F
6-F
6-Br
7-Me
7-Me
7-Me
7-Cl
4g
4h
4i
6-Me
6-Me
6-OMe
6-Cl
4j
7-Me
7-F
4k
4l
Cyclo-
Cyclo-
Pentyl
Hexyl
3. (a) Pearson, A. J.; Grainger, J. M.; Scheuer, P. J.; McIntyre,
N. Lancet 1971, 1, 994; (b) Mallory, A.; Frank, B. W.; Kern,
F., Jr. N. Engl. J. Med. 1971, 285, 1266; Kotha, P.; Rake, M.
O.; Williat, D. Br. J. Med. 1980, 281, 1530.
4m
a Values are means of two experiments (na, not active (>3lM)).
4. Reynolds, T. B.; Peters, R. L.; Yamada, S. N. Engl.
J. Med. 1971, 285, 813.
5. da Silva, J. F. M.; Garden, S. J.; Pinto, A. C. J. Braz.
Chem. Soc. 2001, 12, 273.
Interestingly, methylating the amide function (1a) also
resulted in complete loss of antiproliferative activity.
6. (a) Alam, M.; Younas, M.; Zafar, M. A.; Naeem Pak.
J. Sci. Ind. Res. 1989, 32, 246 (CA 112:7313u); (b)
Smolders, R. R.; Waefelaer, A.; Francart, D. Ing. Chim.
(Brussels) 1982, 64, 5 (CA 97:182148n); (c) Loloiu, G.;
Loloiu, T.; Maior, O. Khim. Geterosilk. Soedin. 1998, 396
(Web of Science); (d) Garden, S. J.; Torres, J. C.; Ferriera,
A. A.; Silva, R. B.; Pinto, A. C. Tetrahedron Lett. 1997,
38, 1501.
We then explored the structure antiproliferative rela-
tionship of monosubstituted oxyphenisatin derivatives
carrying different substituents in either the 5-, 6- or 7-po-
sition (see Tables 2 and 3). The oxyphenisatins with a
substituent at the 4-position did not show any relevant
activity.
7. Varma, R. S.; Singh, A. P. Indian J. Chem., Sect. B 1990,
B, 578.
8. Hauser, C. R.; Manyik, R. M. J. Org. Chem. 1953, 18,
588.
9. (a) Taylor, A. J. Chem. Res., Synop. 1980, 347; (b) Rice,
K. C.; Boone, B. J.; Rubin, A. B.; Rauls, T. J. J. Med.
Chem. 1976, 19, 887.
The data indicate that halogens and small lipophilic sub-
stituents in the 6- and 7-position (2b, 2g, 3a, 3b, and 3f)
are important for the antiproliferative activity. Whereas,
larger substituents (3k, 3l, and 3m) or polar substituents
(2m, 3i, 3p and 3q) reduce the potency.
10. (a) Hewawasam, P.; Meanwell, N. Tetrahedron Lett. 1994,
35, 7303; (b) Rivalle, C.; Bisogni, E. J. Heterocycl. Chem.
1997, 34, 441.
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M. P.; Osso, F. J. Ann. Quim. 1976, 72, 571; (c) Song, H.
N.; Lee, H. J.; Kim, H. R.; Ryu, E. K.; Kim, J. N. Synth.
Commun. 1999, 29, 3303; (d) Wexler, H.; Barboiu, V. Rev.
Roum. Chim. 1976, 21, 127 (CA 85 :5447r).
Finally, we evaluated disubstituted oxyphenisatin
derivatives carrying two small and lipophilic substitu-
ents. Table 4 summarizes the results and confirms the
importance of having relatively small and lipophilic sub-
stituents in the 6- and/or 7-position.
The two lead compounds (3f and 4e) were further pro-
filed and both showed acceptable Caco-2 permeability
(19.6 and 43.1 nm/s, respectively) and aqueous solubility
(0.64 and 0.23 mg/mL, respectively, at pH 7.0). They are
both highly bound to serum protein (99% and 98%,
respectively), but show no evidence of P-gp mediated
efflux using Vinblastine as internal control and Verapa-
mil as P-gp inhibitor. Both compounds showed poor
inhibition (>10 lM) of CYP1A2, 2D6 and 3A4, but
´
12. Felding, J.; Pedersen, H. C.; Krog-Jensen, C.; Prætegaard,
M.; Butcher, S. P.; Linde, V.; Coulter, T. S.; Montalbetti,
C.; Uddin, M.; Reignier, S. WO2005/097107A2.
13. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
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Lett. 1975, 50, 4467.
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