Design, synthesis of novel peptidomimetic derivatives of 4-HPR for rhabdoid tumors

Article abstract of DOI:10.1016/j.bmcl.2008.05.097

Rhabdoid tumors (RTs) are an extremely aggressive pediatric malignancy that results from loss of the INI1/hSNF5 tumor suppressor gene. Loss of INI1 results in aberrant expression of Cyclin D1, which supports rhabdoid tumorigenesis and survival. 4-HPR, a s

Full text of DOI:10.1016/j.bmcl.2008.05.097

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4177–4180
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis of novel peptidomimetic derivatives of 4-HPR
for rhabdoid tumors
c
c,d,e,
Bhaskar C. Das a,b,e, , Melissa E. Smith , Ganjam V. Kalpana
*
*
a Department of Nuclear Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA
b Department of Developmental & Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
c Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA
d Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
e Albert Einstein College Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Rhabdoid tumors (RTs) are an extremely aggressive pediatric malignancy that results from loss of the
INI1/hSNF5 tumor suppressor gene. Loss of INI1 results in aberrant expression of Cyclin D1, which sup-
ports rhabdoid tumorigenesis and survival. 4-HPR, a synthetic retinoid that down-modulates Cyclin
D1, has shown promise in treating various tumors including RTs. In this study, we have generated a
chemical library of peptidomimetic derivatives of 4-HPR in an attempt to create a more biologically
active compound for use as a therapeutic agent against RTs and other tumors. We have synthesized novel
peptidomimetic compound by substituting alkene backbone with a ring structure that retains the biolog-
ical activity in cell culture models of rhabdoid tumors. We further identified derivative of peptidomimetic
Received 2 April 2008
Revised 16 May 2008
Accepted 19 May 2008
Available online 29 May 2008
Keywords:
Rhabdoid tumor
4-HPR
Cyclin D1
Retinoid
compound (11d, IC50 ꢀ 3
l
M) with approximately five times higher potency than 4-HPR (1, IC50 ꢀ 15
lM)
based on a survival assay against rhabdoid tumor cells. These studies indicate that peptidomimetic deriv-
atives that retain the cytotoxic activity are promising novel chemotherapeutic agents against RTs and
other tumors.
Published by Elsevier Ltd.
Rhabdoid tumors of the kidney (Malignant Rhabdoid Tumors,
MRT), central nervous system (Atypical Teratoid and Rhabdoid Tu-
mors, AT/RT), and soft tissues (Extra-renal Rhabdoid Tumors) are
rare pediatric malignancies.1–4 These tumors are aggressive and
mostly incurable as there are no effective or standard treatment
strategies currently available. There is a need to develop novel
therapies preferably based on the understanding of the molecular
basis of genesis of RTs. We previously demonstrated that Cyclin
D1 is essential for the genesis and survival of RTs and that thera-
peutic targeting of Cyclin D1, with agents such as 4-HPR, is effec-
tive in inhibiting RT growth in preclinical models.5,6
potent chemopreventive agent, significantly reducing the risk of
second breast cancer in pre-menopausal women.8
In vitro studies indicate that 4-HPR induces apoptosis in tumor
cell lines by several mechanisms, including, activation of retinoid
receptors RAR b and c, ceremide-dependent cytotoxicity, genera-
tion of free radical oxygen species, increasing NOS expression
resulting in NO-dependent cell cytotoxicity, and increases mito-
chondrial permeability transition resulting in both caspase-depen-
dent and -independent cell death.9–14 4-HPR also induces cell cycle
arrest likely correlated to its ability to down-modulate the expres-
sion or activity of proliferation related targets including c-myc, tel-
omerase, p34/cdc2 and Cyclin D1.15,16 Since RT cells over-express
and are dependent on Cyclin D1, we demonstrated that 4-HPR
exhibits anti-tumor effects on RT cells in vitro and in vivo, with
low toxicity in mouse models.5,6
4-HPR [N-(4-hydroxyphenyl) retinamide, or fenretinide] is a
synthetic retinoid that has a low potential for toxicity and sup-
presses tumor cell growth in vitro at clinically achievable concen-
trations (IC50’s ranging from 1 to 15 l
M).7 It is under phase III
clinical trials for many cancers including neuroblastomas (National
Cancer Institute Ref: 06-C-0227).7 It has also been studied as a che-
mopreventive agent in animal models of carcinogen-induced epi-
thelial tumors and in patients at risk for breast cancer. Compiled
data from a series of phase III trials showed that 4-HPR acts as a
There are several reports indicating that synthetic analogs of
4-HPR are more active as anti-tumor agents or display reduced
toxicity in vitro. For example, a non-hydrolyzable carbon-linked
analog (N-benzyl hydroxyl retinamide, or 4-HBR) no longer sup-
presses plasma vitamin A levels as 4-HPR does, and hence is
safer.17 The sulfur-containing heteroretinoids induce apoptosis
and reactive oxygen species more specifically in malignant cells
versus normal cells therefore reducing unwanted toxicities.18
Conjugations of 4-HPR have also displayed increased anti-tumor
* Corresponding authors. Tel.: +1 7184302422; fax: +1 7184308853.
E-mail address: bdas@aecom.yu.edu (B.C. Das).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.05.097

4178
B. C. Das et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4177–4180
activity. Specifically, glycosyl conjugated mannosyl with 4-HPR
had increased activity in the HL60 promyelocytic leukemia cell
line.19 Notably, 4-oxo-fenretinide has been shown to induce G2-
M cell cycle arrest and apoptosis in both 4-HPR-sensitive and -
resistant cell lines.20
Since substituted or conjugated derivatives of 4-HPR show in-
creased activity or similar activity with more favorable toxicity
profiles, we wanted to further explore modification of this promis-
ing therapeutic agent. We have a long term goal of developing no-
vel therapeutics for RTs within the brain (AT/RTs). We hypothesize
that, structural modification of existing 4-HPR compounds to cre-
ate peptidomimetic derivatives may increase the potency of these
compounds against RTs, may allow for the delivery of these com-
pounds across the blood–brain barrier. We report here design syn-
thesis and biological activity of a novel peptidomimetic compound
of 4-HPR against RT cells in culture.
The generation of peptidomimetics is based on the conforma-
tional, topochemical and electronic properties of the lead peptide
backbone as possible with non-peptide fragments while still main-
taining the pharmacophoric groups of the peptides.21 This makes
the compound more lipophilic, which increases its bioavailability
and possibly facilitates crossing through the blood–brain barrier.22
Replacement of the amide bond with alternative groups also pre-
vents proteolysis and promotes metabolic stability.23 Conforma-
tional flexibility is often required to allow for the pharmacophoric
groups to find their binding sites, but further lead refinement should
favor the formation of moreconformationally restrictedanalogs that
hold appropriate pharmacophoric groups in the bioactive conforma-
tion for binding to the target proteins.24 Based on these principles,
we have attempted to synthesized peptidomimetic derivatives of
4-HPR with substitution of the alkene backbone with a phenyl ring
structure. Duringthesestudies, weobtainedcompounds(11a, Struc-
ture 1), with altered structure compared to 4-HPR, but some of these
compounds remarkably retained biological activity in cell culture
models.
stirred at room temperature under a nitrogen atmosphere for 30 h.
After this analysis, crude ester 7 was purified by flash column
chromatography (hexane/ethyl acetate 98:2) to obtain a brown oil
with 85% yield.27 The ester was saponified to generate a white solid
which was filtered, washed with water, and dried. The product was
recrystallized from hot ethanol and washed with dry hexane to ob-
tain acid 8 as white crystals (87% yield). The structure of he com-
pound was confirmed by 1H, 13C NMR, NOE, HMBC, and by HRMS.
The acid 8 was first converted to acid chloride and coupled with ani-
line derivatives to obtain compounds 11a–11g with 70–90% yield.
The detailedreaction procedureforthe synthesis of lead compound
11d-(E)-4-((3-ethyl-2,4,4-trimethyl-cyclohex-2-enylidene)methyl)-
N-(4-hydroxy-3-iodophenyl)benzamide is provided in Ref. 28.
Cell culture: The MON RT cell line29 was used for in vitro cyto-
toxicity studies. MON cells were maintained in RPMI 1640 supple-
mented with 10% fetal bovine serum, 2mM
penicillin, and 50 g/mL streptomycin at 37 °C with 5% CO2 and
95% humidified air.
L-glutamine, 50 U/mL
l
Compound preparation and storage: Stock solutions of 4-HPR and
its peptidomimetic derivatives were prepared at 10 mM in 100%
ethanol and stored frozen at À80 °C protected from light. Working
solutions (50 lM) and serial dilutions were prepared by diluting
the stock solutions in cell culture medium such that the concentra-
tion of ethanol was <2% in all dilutions.
In vitro cell proliferation assay: Aliquots of 8 Â 103 cells were pla-
ted in 96-well microtiter plates. Twenty-four hours after seeding,
the cells were treated with serial dilutions of each drug and incu-
bated for 3 days. Cell survival was determined using an MTS assay
kit (CellTiter 96Ò Aqueous Non-Radioactive Cell Proliferation Assay
Kit, Promega, Madison, WI). Cell plating, drug treatment and sur-
vival assay were performed using the epMotion 5070 automated li-
quid handling robotic system (Eppenddorff, Westbury, NY).
Statistical analysis and determination of IC50 values: Statistical
analysis of the data was carried out using GraphPad Prism (San
Diego, CA). Relative IC50 values were calculated using the nonlin-
ear regression curve fit with sigmoidal dose–response (variable
slope) function. Relative IC50 is defined as the concentration
giving a response exactly half way between the fitted top and
bottom of the survival curve when graphed as percent inhibition
versus the log of the concentration of compound. The fitting
error, or standard error describes the error involved in fitting
the curve.
OH
H
O
HN
NH
(11a)
O
(1)
HO
)-4-((3-ethyl-2,4,4-trimethylcyclohex-2-enylidene)
methyl)- -(4-hydroxyphenyl)benzamide
Results and discussion: In a parallel study we have reported that
the 4OH group at para-position of the phenyl ring of 4-HPR is nec-
essary for cytotoxic activity against RT cells.30 We wanted to test if
this were to be true if we substitute the para-position in the new
peptidomimetic compound as well. We synthesized several com-
pounds substituting the alkene backbone with a ring structure
and in addition further modifying the 4OH-phenyl moiety. We ob-
tained compound (11a) and its derivatives (11b–11g), as repre-
sented in Scheme 1. These compounds were tested in a biological
survival assay.
4-HPR = 4-hydroxyphenyl retinamide
(E
N
We synthesized further derivatives by additional modifications
in the 4-hydroxy phenylamide groups (compounds 11a–11g in
Scheme 1). These compounds were tested alongside parent com-
pound, 4-HPR, in a biological survival assay to determine their
anti-tumor activity against RT cells.
Previously, we have demonstrated that 4-HPR exhibits cyto-
toxic activity against many RT cells including MON, G401 and
STM.5 The cytotoxicity profiles of 4-HPR against these cells were
Novel peptidomimetic derivatives of 4-HPR were synthesized by
modifying the existing organic reactions. After synthesizing the acid
8 from compound 3 by Witting reaction, we carried out amide cou-
pling reaction with differentaminophenolderivatives. The synthesis
of 8 involves the reaction with methyl magnesium bromide with b-
cyclocitralinTHF thatyields alcohol4 as yellowoil (Scheme1).25 The
alcohol gave satisfactory spectral data and was directly converted to
5 by treatment with triphenylphosphine hydrobromide in metha-
nol. Recrystallization of 5 from methanol/ether (1:6) gave a yellow
crystalline solid.26 Formation of the Witting reagent from 5 in ether
was accomplished with n-butyllithium in hexane at room tempera-
ture (dark-red color), then the Witting reagent was treated with
methyl 4-formybenoate 6 in ether at À78 °C for 10–15 min and then
very similar with IC50s ranging from 5 to 12
l
M.5 MON cells were
least sensitive with an IC50 of ꢀ12
l
M.5 Therefore, we surmised
that screening the activity of novel peptidomimetic compounds
against MON cells will be most effective. If the novel compounds
exhibit improved activity, then we will be encouraged to test these
compounds against other RT cells and other cancer cells in the fu-
ture. Therefore, as a first test of the activity of novel 4-HPR deriv-
ative compounds we chose MON cells to carry out the biological
assay.
Results of the biological survival assay are illustrated in the
Figure 1 and Table 1. The peptidomimetic compound 11a exhibited

B. C. Das et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4177–4180
4179
OH
.
1. n-BuLi, hexane,ether
PPh3 HBr
CH3OH
CHO
CH3MgBr
2. OHC
85%
CO2CH3
THF, 2h, 72%
rt,72h, 78%
+
PPh3
(3)
6
(5)
(4)
NaOH (aq)
(COCl)2
H2N
R3
10 a-e
CH3CH2OH
87%
DMF, CH2Cl2
H
R2
R1
H
H
O
Et3N, DMF, 75-95%
Cl
O
HO
7
( )
O
9
( )
8
( )
O
11a: R1=R2=H. R3= OH
11b: R1=Cl, R2=H, R3= OH
11c: R1=F, R2=H, R3= OH
11d: R1=I, R2=H, R3= OH
11e: R1=H, R2=OH, R3= OH
11f: R1=R2=H, R3=-CH2OH
11g: R1= R2=H, R3= -CH2CH2OH
H
R2
HN
R1
R3
11a-11g
(
)
O
Scheme 1.
pounds 11b and 11c, respectively), exhibited similar level of
activities to that of the parent 4-HPR compound (IC50 values of
120
100
80
60
40
20
0
10–13
stitution of an iodo-moiety at the meta-position of the phenyl ring
demonstrated improved efficacy with IC50’s reduced to 3 M. This
lM, respectively). Interestingly, compound 11d, with sub-
l
compound did not show any precipitation in the culture condi-
tions. These results indicated that compound 11d is a promising
derivative of 4-HPR that shows improved activity in vitro.
4-HPR
11e
11d
8
11c
11b
11f
We have attempted to generate novel derivatives of 4-HPR,
changing the alkene backbone of retinoic acid, substituting it with
a rigid ring structure, and have tested their cytotoxic activity in cell
culture models of RT. We found that substituting the alkene back-
bone of retinoic acid with rigid phenyl group does not change the
activity of the parent compound. Furthermore, substitution of the
para-hydroxy group of the novel compound with ethyl hydroxy
group (11f) resulted in reduced activity, consistent with our paral-
lel findings that 4-OH group at the para-position of the phenyl
group is required for cytotoxic activity. Compounds 11b and 11c
(chloro- and fluoro-substitutions at meta-position of phenyl ring,
respectively) exhibited activity similar to that of 4-HPR. Interest-
ingly, we found that iodo-derivative (compound 11d) was more ac-
0
[compound] μM
Figure 1. Effect of peptidomimetic derivatives of 4-HPR on the survival of rhabdoid
cells. MON (INI1À/À) cells were treated with serial dilutions of the 4-HPR and 4-
HPR derivatives for three days. Survival assay was carried out as described using
MTS assay kit. Percentage of cell survival plotted against concentration of drugs
(mean SEM).
Table 1
tive than the parent compound, with a IC50 of 3 lM.
Relative IC50 values for each drug
The reason for the improved activity of the compound 11d as
compared to 4-HPR and to compounds 11b and 11c is not com-
pletely clear. We propose that perhaps, the bulkiness of the iodo
group (as compared to that of chloro and fluoro groups) may make
it more active. We obtained similar improved results of substitut-
ing the 4-HPR with iodo group reported in a parallel study.30 We
propose that the peptidomimetic derivatives with substitution of
alkene backbone with rigid ring structure is likely to be more sta-
ble in vivo, and hence represents a novel 4-HPR derivative with im-
proved efficacy. Combining with their increased potency, these
compounds are candidates for developing more stable and active
anti-cancer drugs.
Our observation that a derivative of 4-HPR with substitution of
the alkene backbone by a ring structure retains the biological activ-
ity is intriguing and suggests that the functional receptor or target
of 4-HPR could be different from that of RA in rhabdoid cells. We
are currently synthesizing chemical libraries of 4-HPR peptidomi-
metic compounds by changing the amide bonds with different
amide isosteres groups to develop anti-cancer drugs with better
Compound
Relative IC50 in
l
M
Fitting error ( SEM)
4-HPR
8
14.68
>50
.221
NAa
11(b)
11(c)
11(d)
11(e)
11(f)
10.44
12.88
3.149
47.18
29.06
.227
.077
.190
.652
.083
a
IC50 greater than the highest concentration tested.
similar cytotoxicity profile as compared to compound 1 at lower
concentrations of the drugs tested. However, the compounds 11a
and 11g precipitated in cell culture medium and therefore, the
exact IC50 value for these compounds could not be determined.
Compounds 11e, 11f, and 8, exhibited IC50 values of 47.18
lM,
29.06 M, and >50 M, respectively, which are much greater than
l
l
the parent compounds. Two compounds with chloro- and fluoro-
substitutions at the meta-position of the phenyl group (com-

4180
B. C. Das et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4177–4180
14. Pirkmaier, A.; Yuen, K.; Hendley, J.; O’Connell, M. J.; Germain, D. Clin. Cancer
Res. 2003, 9, 1877.
15. DiPietrantonio, A. M.; Hsieh, T. C.; Olson, S. C.; Wu, J. M. Int. J. Cancer 1998, 78,
53.
activity and are using functional genomic approaches to determine
the exact target of active 4-HPR derivatives in rhabdoid cells.
16. Reynolds, C. P.; Wang, Y.; Melton, L. J.; Einhorn, P. A.; Slamon, D. J.;
Maurer, B. J. Med. Pediatr. Oncol. 2000, 35, 597.
Acknowledgments
17. Abou-Issa, H.; Curley, R. W., Jr.; Alshafie, G. A.; Weiss, K. L.; Clagett-Dame, M.;
Chapman, J. S.; Mershon, S. M. Anticancer Res. 2001, 21, 3839.
18. Simoni, D.; Tolomeo, M. Curr. Pharm. Des. 2001, 7, 1823.
19. Winum, J. Y.; Leydet, A.; Seman, M.; Montero, J. L. Farmaco 2001, 56, 319.
20. Villani, M. G.; Appierto, V.; Cavadini, E.; Bettiga, A.; Prinetti, A.; Clagett-Dame,
M.; Curley, R. W.; Formelli, F. Cancer Res. 2006, 66, 3238.
These studies were supported by funding from American Cancer
Society (RSG CCG-10493) and philanthropic funding (Amini foun-
dation) to G.V.K., Institutional training grant (NIGMS T32 GM
007491) to M.E.S., AECOM start up fund to B.D. G.V.K. is a Mark
Trauner faculty scholar and a recipient of the Irma T. Hirschl Career
Scientist Award.
21. Ahn, J. M.; Boyle, N. A.; MacDonald, M. T.; Janda, K. D. Mini Rev. Med. Chem.
2002, 2, 463.
22. Gentilucci, L.; Tolomelli, A.; Squassabia, F. Curr. Med. Chem. 2006, 13, 2449.
23. Perez, J. J.; Corcho, F.; Llorens, O. M. Curr. Med. Chem. 2002, 9, 2209.
24. Reichelt, A.; Martin, S. F. Acc. Chem. Res. 2006, 39, 433.
25. Ferna’ndez-Mateos, A.; Mateos Buro’n, L.; Martı’n de la Nava, E. M.; Rubio
Gonza’lez, R. J. Org. Chem. 2003, 68, 3585.
26. Rosenberger, M.; Neukom, C. J. Org. Chem. 1982, 47, 1782.
27. Waugh, K. M.; Berlin, K. D.; Ford, W. T.; Halt, E. M.; Carrol, J. P.; Schomber,
P. R.; Thompson, M. D.; Schiffl, L. J. J. Med. Chem. 1985, 116.
28. Synthesis of 11d. A mixture of acid 8 (100 mg, 0.352 mmol) in dry DMF (1 mL)
and dry CH2Cl2 (4 mL) was stirred at room temperature under nitrogen
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.05.097.
References and notes
atmosphere for 15 min. Oxalyl chloride (1.31 mmol, 120 lL) was added drop
by drop at 0 °C. The reaction mixture was stirred for another 1.5 h at room
temperature under nitrogen atmosphere. Very carefully removed the solvent,
then added dry DMF (2 mL) at 0 °C under nitrogen atmosphere. Then acid
1. Biegel, J. A. Neurosurg. Focus 2006, 20, E11.
2. Biegel, J. A.; Kalpana, G.; Knudsen, E. S.; Packer, R. J.; Roberts, C. W.; Thiele, C. J.;
Weissman, B.; Smith, M. Cancer Res. 2002, 62, 323.
3. Packer, R. J.; Biegel, J. A.; Blaney, S.; Finlay, J.; Geyer, J. R.; Heideman, R.; Hilden,
J.; Janss, A. J.; Kun, L.; Vezina, G.; Rorke, L. B.; Smith, M. J. Pediatr. Hematol.
Oncol. 2002, 24, 337.
4. Strother, D. Expert Rev. Anticancer Ther. 2005, 5, 907.
5. Alarcon-Vargas, D.; Zhang, Z.; Agarwal, B.; Challagulla, K.; Mani, S.; Kalpana, G.
V. Oncogene 2006, 25, 722.
6. Tsikitis, M.; Zhang, Z.; Edelman, W.; Zagzag, D.; Kalpana, G. V. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 12129.
7. Garaventa, A.; Luksch, R.; Lo Piccolo, M. S.; Cavadini, E.; Montaldo, P. G.;
Pizzitola, M. R.; Boni, L.; Ponzoni, M.; Decensi, A.; DeBernardi, B.; Bellani, F. F.;
Formelli, F. Clin. Cancer Res. 2003, 9, 2032.
8. Bonanni, B.; Lazzeroni, M.; Veronesi, U. Expert Rev. Anticancer Ther. 2007, 7,
423.
9. Fontana, J. A.; Rishi, A. K. Leukemia 2002, 16, 463.
10. Hail, N., Jr.; Kim, H. J.; Lotan, R. Apoptosis 2006, 11, 1677.
11. Lovat, P. E.; Corazzari, M.; Di Sano, F.; Piacentini, M.; Redfern, C. P. Cancer Lett.
2005, 228, 105.
12. Lovat, P. E.; Corazzari, M.; Goranov, B.; Piacentini, M.; Redfern, C. P. Ann. N.Y.
Acad. Sci. 2004, 1028, 81.
13. Reynolds, C. P.; Matthay, K. K.; Villablanca, J. G.; Maurer, B. J. Cancer Lett. 2003,
197, 185.
chloride solution (9) was added dropwise to
iodophenol (0.704 mmol, 165 mg) and triethylamine (2.11 mmol, 300
a
solution of 4-amino-2-
L) in
l
dry DMF (2 mL). The reaction mixture was stirred at room temperature and
progress of the reaction was monitored using TLC. After the reaction
completed 3 h, the reaction was quenched with saturated NH4Cl (8 mL) and
extracted with ethyl acetate (2Â 15 mL). The extracts were washed with (2Â
10 mL) and brine (2Â 10 mL), then dried over Na2SO4, and evaporated. The
residue was purified by flash column chromatography using hexane/ethyl
acetate (4:1) as the eluent to give a pale yellow solid 151.36 mg (86%), mp:
170–171 °C. 1H NMR(300 MHz, DMSO): d10.12 (s, 1H), 10.02 (s, 1H), 8.14 (d,
J = 2.3 Hz, 1H), 7.9 (d, J = 8.3 Hz, 2H), 7.58 (dd, J = 8.7 Hz, 2.3 Hz, 1H), 7.42 (d,
J = 8.3 Hz, 2H), 6.86 (d, J = 8.7 Hz, 1H), 6.47 (s, 1H), 2.60 (m, 2H), 2.20 (q,
J = 7.5 Hz, 2H), 1.88 (s, 3H), 1.44 (m, 2H), and 1.09 (s, 6H) and 1.04 (t,
J = 7.5 Hz, 3H). 13C NMR CDCl3: d 167.4, 149.1, 144.3, 144.2, 142.6, 142.5,
192.6, 129.6, 129.5, 129.5, 127.8, 127.4, 120.9, 120.9, 85.2, 61.1, 52.3, 39.2,
36.2, 28.03, 24.7, 23.3, 15.5, 15.1, and 14.7 ESI MS: Calcd for C25H28INO2
([M+H]+) 502.12. Found: 502.08.
29. Versteege, I.; Sevenet, N.; Lange, J.; Rousseau-Merck, M. F.; Ambros, P.;
Handgretinger, R.; Aurias, A.; Delattre, O. Nature 1998, 394, 203.
30. Das, B. C.; Smith, M. E.; Kalpana, G. V. Bioorg. Med. Chem. Lett. 2008, 18,
3805.