Novel C-4 heteroaryl 13- cis -retinamide Mnk/AR degrading agents inhibit cell proliferation and migration and induce apoptosis in human breast and prostate cancer cells and suppress growth of MDA-MB-231 human breast and CWR22Rv1 human prostate tumor xenografts in mice

Article abstract of DOI:10.1021/jm501792c

The synthesis and in vitro and in vivo antibreast and antiprostate cancers activities of novel C-4 heteroaryl 13-cis-retinamides that modulate Mnk-eIF4E and AR signaling are discussed. Modifications of the C-4 heteroaryl substituents reveal that the 1H-imidazole is essential for high anticancer activity. The most potent compounds against a variety of human breast and prostate cancer (BC/PC) cell lines were compounds 16 (VNHM-1-66), 20 (VNHM-1-81), and 22 (VNHM-1-73). In these cell lines, the compounds induce Mnk1/2 degradation to substantially suppress eIF4E phosphorylation. In PC cells, the compounds induce degradation of both full-length androgen receptor (fAR) and splice variant AR (AR-V7) to inhibit AR transcriptional activity. More importantly, VNHM-1-81 has strong in vivo antibreast and antiprostate cancer activities, while VNHM-1-73 exhibited strong in vivo antibreast cancer activity, with no apparent host toxicity. Clearly, these lead compounds are strong candidates for development for the treatments of human breast and prostate cancers.

Full text of DOI:10.1021/jm501792c

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Article
Novel C-4 Heteroaryl 13-Cis Retinamide Mnk/AR Degrading Agents
Inhibit Cell Proliferation and Migration and Induce Apoptosis in Human
Breast and Prostate Cancer Cells and Suppress Growth of MDA-MB-231
Human Breast and CWR22Rv1 Human Prostate Tumor Xenografts in Mice
Hannah W. Mbatia, Senthilmurugan Ramalingam, Vidya P Ramamurthy,
Marlena S Martin, Andrew K. Kwegyir-Afful, and Vincent C. O. Njar
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501792c • Publication Date (Web): 29 Jan 2015
Downloaded from http://pubs.acs.org on February 6, 2015
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Novel Cꢀ4 Heteroaryl 13ꢀCis Retinamide Mnk/AR Degrading
Agents Inhibit Cell Proliferation and Migration and Induce
Apoptosis in Human Breast and Prostate Cancer Cells and Suppress
Growth of MDAꢀMBꢀ231 Human Breast and CWR22Rv1 Human
Prostate Tumor Xenografts in Mice
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,2,#
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Hannah W. Mbatia , Senthilmurugan Ramalingam , Vidya P. Ramamurthy , Marlena S.
1,2,¥
1,2
1,2,3,*
Martin , Andrew K. KwegyirꢀAfful and Vincent C. O. Njar
1
2
3
Department of Pharmacology, Center for Biomolecular Therapeutics, Maryland, Marlene
Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, 685 West
Baltimore Street, Baltimore, MD 21201ꢀ1559, USA.
¥
Current Address: Bernard J. Dunn School of Pharmacy, Shenandoah University, 45085
University Drive, Suite 202, Ashburn, VA 20147
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ABSTRACT
The synthesis and in vitro and in vivo antiꢀbreast and antiꢀprostate cancers activities of novel Cꢀ4
heteroaryl 13ꢀcis retinamides that modulate MnkꢀeIF4E and AR signaling are discussed.
Modifications of the Cꢀ4 heteroaryl substituents reveal that the 1Hꢀimidazole is essential for high
antiꢀcancer activity. The most potent compounds against a variety of human breast and prostate
cancer (BC/PC) cell lines were compounds 16 (VNHMꢀ1ꢀ66), 20 (VNHMꢀ1ꢀ81) and 22
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(VNHMꢀ1ꢀ73). In these cell lines; the compounds induce Mnk1/2 degradation to substantially
suppress eIF4E phosphorylation. In PC cells, the compounds induce degradation of both fullꢀ
length androgen receptor (fAR) and splice variant AR (ARꢀV7) to inhibit AR transcriptional
activity. More importantly, VNHMꢀ1ꢀ81 has strong in vivo antiꢀbreast and antiꢀprostate cancer
activities, while VNHMꢀ1ꢀ73 exhibited strong in vivo antiꢀbreast cancer activity, with no
apparent host toxicity. Clearly, these lead compounds are strong candidates for development for
the treatments of human breast and prostate cancers.
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INTRODUCTION
Disruption and/or perturbation of capꢀdependent translation is essential for the
1
development of cancers and many fibrotic diseases, the most notable being Alzheimer’s disease.
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Hyperꢀactivation of eukaryotic translation initiation factor 4E (eIF4E), the mRNA 5ꞌ capꢀbinding
protein of capꢀdependent translation promotes exquisite transcriptꢀspecific translation of key
2
mRNAs that are indispensable in cancer initiation, progression and metastases. The oncogenic
potential of eIF4E is dependent on serine 209 phosphorylation by MAPKꢀinteracting kinases 1
and 2 (Mnk1/2). Currently, there are several strategies, directed at disruption of the eukaryotic
initiation factor 4F (eIF4F) complex to inhibit hyperꢀactivity of oncogenic protein translation as
3
a means to effectively treat a variety of cancers.
When targeting a critical process such as translation, a major critical question is
whether the drug will have significant effects on normal cellular processes, leading to limiting
toxicities and too narrow a therapeutic window. This question has been addressed by several
experimental and a few clinical studies. Recently, Graff and colleagues elegantly demonstrated
that downꢀregulation of eIF4E by antisense oligonucleotide therapy caused reduction of in
vivo tumor growth in MDAꢀMBꢀ231 breast and PCꢀ3 prostate cancer models without toxicity
4
despite an ~80% eIF4E knockdown in essential organs. These data provided in vivo evidence
that cancers may be more susceptible to eIF4E inhibition than normal tissue and provided the
basis for advancement of eIF4Eꢀspecific antisense molecule LY2275796 into Phase 1 clinical
5
studies. In other more recent studies, cercosporamide, a potent Mnk1/2 kinase inhibitor was
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reported to exhibit antitumor efficacies against HCT116 colon carcinoma xenograft tumors and
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MV4ꢀ11 acute myeloid leukemia (AML) tumors in animal models, without any toxicity.
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We previously reported that our proprietary novel Cꢀ4 azolyl retinamides (NRs) based on
allꢀtrans retinoic acid (ATRA) (1) (Figure 1) scaffold induced apoptosis, potently inhibited the
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growth, migration and invasion of a variety of human breast and prostate cancer cell lines. With
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respect to the breast cancer cell lines, we demonstrated that the antiꢀbreast cancer activity of our
NRs was due mainly to degradation of Mnk1/2 with subsequent suppression of phosphorylated
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a
eIF4E (peIF4E). However, in the prostate cancer (PC) cell lines, we demonstrated that the antiꢀ
tumor activity of the NRs was due to simultaneous inhibition of the Mnk/eIF4E and androgen
8
b
receptor (AR) signaling pathways. It is important to state here that unlike other reported small
3e, 6, 9
molecules which inhibit Mnk1/2 kinase activities,
our NRs induced Mnk1/2 degradation via
8
the ubiquitinꢀproteasome pathway with resultant depletion of peIF4E. The structures of
1
0
6
promising Mnk 1 and/or 2 kinase inhibitors, including CGP57380 (2), cercosporamide (3),
11
11
MNKIꢀ19 (4) and MNKI85 (5) are presented in Figure 1. In addition, the structures of our
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a
12 12
lead Mnk degrading agent (MNKDA), VNLGꢀ152 (6) and related compounds,
7), VN/66ꢀ1 (8) and Nꢀ(4ꢀhydroxyphenyl) retinamide (4ꢀHPR or fenretinide) (9) are also
depicted in Figure 1.
In the present study, we have synthesized novel compounds based on 13ꢀcis retinoic acid
13ꢀCRA) (10) (Figure 1) scaffold, and their antiꢀcancer activities were evaluated in both in
VN/14ꢀ1
(
(
vitro and in vivo models of human breast and prostate cancers. These novel compounds target
Mnk1/2 degradation in both breast cancer and PC cells and also induce AR degradation in PC
cells, which in turn led to induction of apoptosis, cell cycle arrest, inhibition of cell growth,
colonization, migration, and invasion. Importantly, compounds 20 and 22 significantly inhibited
tumor growth of aggressive MDAꢀMBꢀ213 breast cancer xenografts, while 20 was shown to
substantially suppress castrationꢀresistant prostate cancer CWR2Rv1 xenografts in vivo.
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F
OCHMe2
HO
H
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O
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O
NH2
N
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OH
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2 H
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OCHMe2
N
OH
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H
NHCH CH OMe
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N
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N
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8
6
7
OH
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OH
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N
H
N
H
O
OH
N
9
10
N
Figure 1: Chemical structures of compounds 1 – 10.
RESULTS AND DISCUSSIONS
Design Strategy: Rationale structural modifications of small molecules allow for their
interactions with molecular target(s) in ways that could lead to improved drugꢀlike compounds
13
and possibly enhanced in vivo pharmacokinetic profiles. Because of our desire to discover more
efficacious antiꢀcancer agents, we were eager to exploit compound 10’s scaffold as a strategy to
novel potent/efficacious MNKDAs with improved drugꢀlike properties. The rationale of using
this scaffold is based on experimental and clinical reports that unlike ATRA (1), 13ꢀCRA (10)
1
4
15
has long elimination halfꢀlife in humans and most animal species. In addition, we also
explored rational modification of the terminal amide moiety, Cꢀ4 heterocycles and the
cyclohexene ring (Figure 2). It is relevant to state here that our previous studies with the ATRA
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scaffold MNKDAs identified the Cꢀ4 azolyl retinamides to be superior to the corresponding
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carboxylic acid and esters.
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Cyclohexenyl moiety
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X
Heteroaryl moieties
Terminal Aryl amides
Figure 2: Overall Design Strategy for Novel Cꢀ4 Heteroaryl 13ꢀCis Retinamides
Cꢀ4 Azoyl/terminal Amide Modifications: Based on the continued successes of the Cꢀimidazole
8
retinamides with the ATRA scaffolds as promising antiꢀcancer agents, we designed and
synthesized compounds 16 ꢀ 25 as outlined in Scheme 1
.
Cyclohexene Rigidification/Cꢀ4 Aryl and Heteroaryl Modifications: On the basis of previous
studies which showed that structural rigidification that introduces conformational constrains
around rotatable bonds contributed to higher specificity and potency, greater metabolic stability,
1
6
and improved bioavailability, we designed and synthesized several cyclohexadiene Cꢀ4 aryl
substituted retinamides, compounds 33 ꢀ 40 Scheme 2). A potential advantage of this strategy is
(
that the achiral nature of these compounds would not require the tedious characterization of the
racemates and pure enantiomers as would be required in advanced preclinical development for
1
7
the chiral compounds of Scheme 1
.
Chemistry: Herein, we report, for the first time, the synthesis of 18 novel compounds based on
the structural scaffold of 13ꢀCRA (10), as outlined in Schemes 1 and 2.
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v
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O
OH
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Comp. n X
0 H
Comp. n X
21 1 H
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6
17
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0 p ꢀOH 22
0 o ꢀOH 23
1 p ꢀOH
1 p ꢀF
1 m ꢀF
2 p ꢀOH
(
)
O
N
H
n
X
N
0 p ꢀF
0 m ꢀF
24
25
2
0
N
a
Scheme 1: Synthesis of Cꢀ4 Azoyl/terminal Amide Compounds 17 – 22
a
Reagents and conditions: (i) (CH ) SiCHN , MeOH/benzene; (ii) MnO , CH Cl (iii) NaBH ,
3 3
2
2
2
2;
4
MeOH; (iv) CDI, CH CN; (v) 2M KOH, MeOH, reflux; (vi) EDC, HOBT, DIEA, appropriate
3
anilines, DMF.
The synthesis of (2Z,4E,6E,8E)ꢀ9ꢀ(3ꢀ(1Hꢀimidazolꢀ1ꢀyl)ꢀ2,6,6ꢀtrimethylcyclohexꢀ1ꢀenꢀ1ꢀ
yl)ꢀ3,7ꢀdimethylnonaꢀ2,4,6,8ꢀtetraenoic acid (15) was carried out in five steps from the
commercially available 13ꢀCRA (10) and the procedure was adopted from our reported
12b
procedures of ATRA based retinamides. Protection of the carboxylic acid as the methyl ester
(
11) was done using trimethylsilyldiazomethane in hexanes followed by allylic oxidation using
MnO to give 4ꢀoxo intermediate (12). Reduction of 12 using NaBH gave the (±)ꢀ4ꢀ
2
4
hydroxymethylterionate (13) which was further treated with carbonyldiimidazole (CDI) at
ambient temperature to yield (±)ꢀ(1
H
ꢀimidazolꢀ1ꢀyl)methylterionate (14). Alkaline hydrolysis of
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in refluxing methanol gave the desired free acid 15 in 63% yield. Coupling of the respective
amines with 15 was successfully achieved using ethyl(dimethylaminopropyl) carbodiimide
(
EDC), hydroxybenzotriazole (HOBt), diisopropylethylamine (DIEA) to give compounds 16
Scheme 1), respectively, in average yields of ~50%.
ꢀ
25
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(
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26
1
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1
27, R =
3
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1
2
O
iii
28, R =
1, R =
1
2
O
OH
N
N
N
N
R2
29, R =
2, R =
1
2
iv
N
O
N
(
)
( )
( )
O
N
H
O
N
N
H
n
n
n
X
X
X
H
N
N
N
O
3
3, n = 1, X = p ꢀOH
35, n = 0, X = H
36, n = 1, X = p ꢀOH
7, n = 2, X = p ꢀOH
38, n = 0, X = H
39, n = 1, X = p ꢀOH
40, n = 2, X = p ꢀOH
34, n = 2, X = p ꢀOH
3
a
Scheme 2: Synthesis of Cyclohexen Rigidification/Cꢀ4 Aryl and Heteroaryl Retinamides
a
Reagents and conditions: (i) 5ꢀChloroꢀ2ꢀpyridyl triflimide, NaN(SiMe ) , THF; (ii) 3ꢀ
3
2
methoxybenzylboronic acid, Pd(PPh ) , CeCO , dioxane, reflux; (iii) 2M KOH, MeOH, reflux
3
4
3
(iv) EDC, HOBT, DIEA, appropriate benzylamine, DMF.
The synthesis of achiral Cꢀ4 aryl retinamides 33ꢀ40 (Scheme 2) commenced from the 4ꢀ
oxo intermediate (12). Generation of enolate using sodium bis(trimethylsilyl)amide in THF
solution and trapping of the enolate with Nꢀ(5ꢀchloroꢀ2ꢀpyridyl)bistrifluoromethanesulfonimide
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furnished the vinyl triflate 26 in 75 % yield according to the reported procedure. However, it
should be noted that this reaction was carried out at ꢀ78°C throughout the reaction time, because,
at higher temperatures, decomposition occurred and ≤ 20% of the products were obtained.
10
11
12
13
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19
Regiospecific palladiumꢀcatalyzed crossꢀcoupling reaction with boronic acids provided
compounds 27ꢀ 29 in 88, 69 and 60% yields, respectively. Ester hydrolysis using 2M KOH gave
the corresponding free acids 30 ꢀ 32 in 62, 48 and 70% yields, respectively. Coupling of these
respective acids with appropriate amines was successfully achieved using EDC, HOBt and DIEA
to give compounds 33 – 40, respectively, in average yields of ~50% (Scheme 2).
Biological Studies:
Effects of 13ꢀCis retinamides on the Growth of Breast and Prostate Cancers Cells in Vitro.
We determined the effects of the 13ꢀcis NRs on human cancer cell proliferation using a variety
8
,
of breast and prostate cancer cells lines using our previously described MTT assay procedures.
1
2b, 20
The results (GI values) are summarized in Table 1. Growth inhibitory concentrations
5
0
(
GI values) are the concentrations of compounds that cause 50% growth inhibition obtained
50
from doseꢀresponse curves. The clinically relevant retinoids, ATRA and 4ꢀHPR and the Mnk
inhibitor, cercosporamide were used as comparators in the assays.
Antiꢀproliferative activities in breast cancer cell lines: As stated earlier, the structural
modification focused on modifications of the terminal amide, cyclohexe ring and Cꢀ4
substituents. As presented in Table 1, several important generalizations emerge from the antiꢀ
proliferative data. First, most of our compounds, including compounds 16
–
25, possess higher
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antiꢀproliferative activities towards all four breast cancer cell lines than the positive controls,
ATRA, 4ꢀHPR and the Mnk inhibitors, as evidenced by the lower GI values (Table 1).
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Table 1: Inhibitory concentrations (GI , µM) of NRs and reference compounds on the
5
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0
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2
3
4
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6
7
8
9
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1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Growth of Human Breast Cancer Cells in Vitro
Cpds.
GI s (µM)
GI s (µM)
GI s (µM)
GI s (µM)
5
0
50
50
50
MCFꢀ7
1.90 ± 0.00
1.99 ± 0.26
1.09 ± 0.00
0.40 ± 0.08
5.49 ± 0.11
9.77 ± 0.64
0.16 ± 0.00
1.93 ± 0.16
1.81± 0.15
1.90 ± 0.08
*
MDAꢀMBꢀ231
1.74 ± 0.22
13.05 ± 1.06
4.67 ± 0.14
4.95 ± 0.24
8.51 ± 0.16
5.88 ± 0.27
2.23 ± 0.07
1.41 ± 0.06
1.65 ± 0.04
2.88 ± 0.08
*
MDAꢀMBꢀ468
2.54 ± 0.12
6.83 ± 0.33
3.03 ± 0,39
4.57 ± 0.00
6.76 ± 0.23
7.24 ± 0.14
2.70 ± 0.35
2.45 ± 0.06
3.09 ± 0.18
13.48 ± 0.97
*
SKBRꢀ3
1.73 ± 0.09
3.89 ± 0.12
3.89 ± 0.06
25.11 ± 1.03
16.98 ± 0.79
10.71 ± 0.98
2.45 ± 0.09
5.50 ± 0.00
4.24 ± 0.21
1.41 ± 0.03
*
1
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8
19
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21
2
2
2
2
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2
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3
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*
*
*
*
35
8.32 ±
25.11 ± 0.76
*
39.81 ± 1.33
*
47.40 ±
36
*
*
37
8.75 ±
26.91 ± 0.98
26.91 ± 1.23
50.11 ± 2.40
22.90 ± 1.03
42.24 ±
38
7.01 ±
30.98 ±
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4.96 ±
2.96 ±
11.48 ± 1.07
14.45 ± 0.92
14.12 ± 0.86
11.05 ±
19.05 ± 1.22
11.48 ±
23.99 ±
40
23.44 ± 0.96
14.12 ± 0.55
6.02 ±
b
ATRA
22.90 ± 0.15
3.98 ±
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4ꢀHPR
4.65 ±
b
Cerco.
43.65 ± 0.17
47.86 ± 0.23
26.02 ± 0.87
a
The GI were determined from doseꢀresponse curves (by nonlinear regression analysis using
5
0
GraphPad Prism) compiled from at least three independent experiments using indicated breast
cancer cells, SEM < 10%, and represents the compound concentration required to inhibit cell
growth by 50%.
b
8a
Previously reported in Ramalingam S, et al. * represents
≥ 60% inhibition of breast cancer
cell growth at 10 µM.
Second, for each compound, the antiꢀproliferative activities towards the breast cancer cell lines
varied significantly. In general, the MCFꢀ7 cell line was the most sensitive, while the SKBRꢀ3
were the least sensitive to these compounds. Third, for a given breast cancer cell line, the antiꢀ
proliferative activity depends largely on the structure of the NR, which in turn depends largely
on the nature of the Cꢀ4 heterocycle and cyclohexen/cyclohexadiene ring. [1] Compounds with
the imidazole tethered to Cꢀ4 of the cyclohexene ring (16
proliferative activities than compounds with phenyl (33 and 34), pyridine (35
38 40) tethered to Cꢀ4 of the cyclohexadiene ring. [2] Compared to compound 16 which
–
25) exhibit more potent antiꢀ
–
37) or pyrimidine
(
–
possess an unsubstituted amide phenyl ring, compounds with substituted hydroxyl (17 and 18) or
fluoro (19 and 20) groups displayed mixed antiꢀproliferative activities, but in general were either
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equipotent or less active than compound 16. The only exception was pꢀfluoro compound (19)
with a GI = 0.4 µM against MCFꢀ7 cells was ~4.8 fold more potent than 16 (GI = 1.90 µM).
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[
3] Insertion of a methylene moiety between the amide and phenyl group clearly led to decreased
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antiꢀproliferative activities (compare GI s of 16: 1.74 – 2.54 µM to GI s of 21: 5.88 – 10.71
5
0
50
µM). [4] However, ortho/para hydroxyl or fluoro substitutions to benzyl group of 21 to give
compounds 22 24 led to enhanced antiꢀproliferative activities across the four breast cancer cell
lines. Amongst these three compounds, 22 was the most potent with GI values of 0.16, 2.23,
–
5
0
2.70 and 2.45 µM versus MCFꢀ7, MDAꢀMBꢀ231, MDAꢀMBꢀ468 and SKBRꢀ3 cell lines,
respectively. Related compound 25 with an ethylene moiety between the amide and ꢀhydroxyl
p
group led to varied antiꢀproliferative activities. It is notable that 25 amongst these compounds,
exhibited the best activity (GI = 1.41 µM) against SKBRꢀ3 cell line.
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Antiꢀproliferative activities in prostate cancer cell lines: The GI values of compounds 16
–
50
22
against three human prostate cancer cell lines, including, LNCaP, CWR22Rv1 and PCꢀ3 were
determined and compared to the GI values of clinically relevant drugs, ATRA, 4ꢀHPR, casodex
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and MDV3100 (enzalutamide) (Table 2). In general, the compounds were more potent than
ATRA, but were equipotent to 4ꢀHPR, casodex and MDV3100. The PCꢀ3 cell line was the least
sensitive to all the compounds tested. Compounds 20 and 22 exhibited the most potent antiꢀ
proliferative activities across the three cell lines.
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Table 2:
Inhibitory concentrations (GI , µM) of NRs and reference compounds on the
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Growth of Human Prostate Cancer Cells in Vitro
a
a
a
Cpds.
GI s (µM)
GI s (µM)
GI s (µM)
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50
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16
17
18
19
20
21
22
23
24
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LNCaP
CWR22Rv1
1.28 ± 0.03
3.89 ± 0.08
2.23 ± 0.11
8.12 ± 0.52
2.04 ± 0.01
*
PCꢀ3
16
2.69 ± 0.09
3.54 ± 0.18
3.23 ± 0.19
3.89 ± 0.23
2.69 ± 0.14
*
8.91 ± 0.51
11.74 ± 0.76
15.48 ± 0.87
30.19 ± 0.94
5.62 ± 0.03
*
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2
2
2
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1
2
1.69 ± 0.07
47.86 ± 0.21
2.69 ± 0.14
2.61 ± 0.09
2.88 ± 0.13
1.86 ± 0.06
25.11 ± 0.16
3.23 ± 0.08
3.81 ± 0.18
3.34 ± 0.25
3.54 ± 0.02
36.3 ± 0.27
3.54 ± 0.09
9.15 ± 0.14
9.15 ± 0.26
b
b
ATRA
4
ꢀHPR
b
Casodex
b
MDV3100
a
The GI were determined from doseꢀresponse curves (by nonlinear regression analysis using
5
0
GraphPad Prism) compiled from at least three independent experiments using LNCaP cells, SEM
<
10%, and represents the compound concentration required to inhibit cell growth by 50%.
b
8b
Previously reported in Ramamurthy V et al. * Indicates compound requirement > 100 µM to
induce 50 % inhibition of cell growth in PC cells
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In vitro characterization of compounds 16, 20 and 22 in breast cancer cell lines: We
st
previously reported that the antiꢀproliferative effects of the 1 generation MNKDAs in breast
cancer cells was due to degradation of Mnk1/2 with subsequent depletion of peIF4E and
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induction of apoptosis. To determine if these compounds modulate Mnk1/2 and related
oncogenic proteins, we performed western blot of lysates from MCFꢀ7, MDAꢀMBꢀ231 and
MDAꢀMBꢀ468 human breast cancer cells treated with compounds 16
, 20, and 22 or with vehicle
(
DMSO, negative control) or compound (positive control). Figure 3AꢀC shows that the three
6
compounds significantly and doseꢀdependently, reduced the expressions of Mnk1, Mnk2 and
peIF4E, with no noticeable effects on the expression of total eIF4E. The compounds also caused
doseꢀdependent formation of cꢀPARP which indicates induction of cell death (apoptosis).
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Figure 3: Effect of compounds 16, 20 and 22 on Mnk1 and 2, peIF4E, eIF4E and cPARP
proteins in MCFꢀ7 cells. Equal protein concentrations from MCFꢀ7 (
MDAꢀMBꢀ468 ( ) cells treated for 24 h with 16, 20 and 22 at the various concentrations as
indicated and (20 µmol/L) were separated by SDSꢀPAGE and western blots probed with
A), MDAꢀMBꢀ231 (B) and
C
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antibodies to Mnk1 and 2, peIF4E, eIF4E and cPARP. Vehicle treated cells were included as
control and all blots were reprobed for GAPDH for loading control.
8
Based on previous studies with closely related compound, we evaluated the inhibitory
effects of compounds 16, 20 and 22 on cell migration, using the human metastatic breast cancer
cell line, MDAꢀMBꢀ231 by woundꢀhealing assay. Figure 4 shows that the compounds 20 and 22
significantly inhibited migration of MDAꢀMBꢀ231 cells compared to the control. At 24 h after
cell monolayers were wounded, control cells had completely filled in the scratched area.
Figure 4: Effects of compounds 16, 20 and 22 on MDAꢀMBꢀ231 cell migration: MDAꢀMBꢀ
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31 cells (5 x 10 cells/well) were seeded on Boyden chamber, grown to confluence and
scratches made at experimental time zero. The cells were treated with indicated compounds (5
µM each) for 24h. Representative photomicrographs of initial and final wounds are shown at
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00x magnification.
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In vitro characterization of compounds 16, 20 and 22 in prostate cancer cell lines:
Previously, we demonstrated that the antiꢀtumor activity of the early NRs in prostate cancer cells
was due to simultaneous inhibition of the Mnk/eIF4E and androgen receptor (AR) signaling
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pathways. Similar to studies described above, we performed western blot of lysates from
LNCaP human prostate cancer cells treated with compounds 16
, 20, and 22 or with vehicle
(DMSO, negative control). Figure 5A shows that the three compounds significantly and in a
doseꢀdependent fashion, reduced the expressions of fAR, Mnk1 and Mnk2, with no noticeable
effects on the expression of total eIF4E. In addition, the compounds caused significant depletion
of oncogenic cyclin D1 and they each induced cleaved PARP (cꢀPARP), a reliable marker of
apoptotic cell death (Figure 5B).
Figure 5: Effects of compounds 16, 20 and 22 on the expression of fAR, Mnk, peIF4E,
cyclin D1 and cleaved PARP in LNCaP cells. Equal protein concentrations from LNCaP cells
treated with 16, 20 and 22 at different concentrations (0.6ꢀ20 µM) for 24 h were separated by
SDSꢀPAGE and western blots probed with antibodies to fAR, Mnk1/2, peIF4E, cyclin D1 and
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cleaved PARP. Vehicle treated cells were included as a control and all blots were reprobed for βꢀ
actin for loading control.
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8
Based on previous studies with closely related compound, we evaluated the inhibitory
effects of compounds 16, 20 and 22 on cell migration, using the human metastatic PC cell line,
PCꢀ3 by woundꢀhealing assay. Figure 6 shows that the three compounds significantly inhibited
migration of PCꢀ3 cells compared to the control. At 24 h after cell monolayers were wounded,
control cells had completely filled in the scratched area.
5
Figure 6: Effects of compounds 16, 20 and 22 on PCꢀ3 cell migration: PCꢀ3 cells (5 x 10
cells/well) were seeded on Boyden chamber, grown to confluence and scratches made at
experimental time zero. The cells were treated with indicated compounds (5 µM each) for 24h.
Representative photomicrographs of initial and final wounds are shown at 100x magnification.
21
Since AR is a major driver of proliferation in PCa, we next examined the effect of these
three compounds on AR transcriptional activity in LNCaP cells. As shown in Figure 7, 24 h
exposure of LNCaP cells to lead NRs, 16, 20, and 22 (10 µM) resulted in a 2 – 4ꢀfold dramatic
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inhibition of DHT induced AR transcriptional activity that was however less potent than that
observed upon clinically available MDV3100 treatment. Of these leads, compound 20 was the
most potent.
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Figure 7: Effects of compounds 16, 20, and 22 on DHTꢀinduced AR transactivation in
LNCaP cells. LNCaP cells dual transfected with ARR2ꢀLuc and the Renilla luciferase reporting
vector pRLꢀnull and treated with 10 µM of specified compounds for 18 h in the presence of 10
nmol/L dihydrotestosterone (DHT). Control represents baseline activity without androgen
stimulation. Androgen stimulated luciferase activity (luminescence) was measured in a victor
1420 plate reader. The results are presented as the fold induction (i.e., the relative luciferase
♣
activity of the treated cells divided by that of control) normalized to that of Renilla. *,
<0.01 compared with DHT alone treated cells.
P< 0.05; ,
P
To confirm whether AR and Mnk1 are the major antiꢀcancer targets of these compounds
on LNCaP cells, we analyzed the percentage of cell survival in LNCaP cells by MTT assay
following transient transfection with AR siRNA and/or Mnk1 siRNA for 18h. Here, we present
data for compound 20 which are identical to data for compounds 16 and 22. The efficiency of
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transfection was confirmed by western blot analysis, wherein protein lysates obtained from the
transfected cells after AR siRNA transfection showed a temporal decrease in total AR protein
and cells transfected with Mnk1 siRNA showed a temporal decrease in the expression Mnk1
compared to scrambled (siꢀcontrol) treated controls after 18 h of transfection (Figure 8A). Cells
coꢀtreated with AR and Mnk1 siRNA showed a remarkable decrease in the expression of both
AR and Mnk1 compared to scrambled siRNA treated cells (Figure 8A). MTT assay revealed
that transient transfections with AR and/or Mnk1 siRNA caused a considerable decrease (~ 40%)
in LNCaP cell viability compared to control (Figure 8B, compare lane 1 to lanes 4, 7 and 10).
Treatment of LNCaP cells (unꢀtransfected) with 5 µM concentration of compound 20 for 72 h
showed a robust reduction in cell viability (lane 3). LNCaP cells harboring AR (lane 6) or Mnk1
(lane 9) knockdown also displayed a significant decrease in cell viability upon treatment with 5
µM of compound 20, compared to the Mnk1 and AR siRNA alone treated counterparts (lanes 4
and 7, respectively). However, LNCaP cells with double knockdown of AR and Mnk1 genes
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(
lane 12) did not show any significant decrease in cell viability upon agent treatment compared
to cells coꢀtreated with Mnk1 and AR siRNA (Figure 8B, lane 10). These results strongly
suggest that Mnk1 and AR are prime targets of compounds 16 20 and 22, and that loss of both
,
these targets abolishes the potent growthꢀinhibitory effects mediated by these agents in LNCaP
cells.
We note that although siRNA knockdown and treatment with compound 20 is expected
to cause a similar percent of decrease in cell viability, the effect of siRNA on LNCaP cell
proliferation depends on the treatment period and dose of siRNA used for transfection. In our
study, we have transfected LNCaP with 100 nM of AR/MNK1 siRNA for 18 h. siRNA
22
transfection for 18ꢀ24 h typically results in only 30% of drop in cell viability. Only in cases
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where transfections are carried out for up to 96 h do massive drop in cell proliferation of ~12ꢀ
2
3
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5% (i.e., 85ꢀ88% growth inhibition) occur. Furthermore, the mechanisms by which AR/MNK
siRNA and compound 20 act are different. Whereas siRNA works at the level of transcription,
compound 20 acts at the postꢀtranslational level and hence even though compound 20
recapitulates AR/MNK siRNA affects, the percentage of decrease in cell proliferation caused by
the two agents (i.e., siRNA and NRs) are not alike.
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Figure 8: Effects of NRs on the viability of LNCaP cells transiently knockedꢀdown for AR
and/or Mnk1.
transfected with 100 nM of siAR, siMnk1 and combinations for 18 h. (
in µM concentration) on cell proliferation in LNCaP cells transfected with 100 nM of siAR,
(A) Western blot analysis of the expression of fAR and Mnk1 in LNCaP cells
B
) Effect of compound 20
(
siMNK1 and combinations as determined by MTT assay. The results represent the mean ± SEM
of three independent experiments and are represented as a bar graph after normalizing to control
cells. *,
P< 0.05; ♣, P<0.01 compared with vehicle treated control; aꢀ Significantly different
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from AR siR treated cells,
.05.
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< 0.05; bꢀ Significantly different from MNK1 siR treated cells, P<
0
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Inhibition of breast and prostate cancer tumor growth in vivo: To determine whether the
antiꢀcancer effects exhibited by the lead compounds in cell cultures could be replicated in animal
models, we conducted antiꢀtumor xenograft studies in two wellꢀestablished aggressive models of
human breast (MDAꢀMBꢀ231) and prostate (CWR22Rv1) cancers. As noted above, although
compounds 16, 20 and 22 are equipotent, we selected the latter two compounds for in vivo
studies based on their ease of synthesis, availability and animal cost.
MDAꢀMBꢀ231 antiꢀtumor efficacy study: First, we evaluated the in vivo antitumor efficacy of
compounds 20 and 22 in mice bearing MDAꢀMBꢀ231 triple negative breast cancer (TNBC)
xenografts that were administered intraperitoneal (i.p.) 20 mg/kg compound 20 or 22 5x/week
over 28 days, which resulted in an 85.4% suppression of tumor growth. Indeed, the two
compounds were equipotent (Figure 9A). Thus, the tumor growth inhibition, measured as %T/C
= 14.6%, classified these compounds as highly efficacious according to the National Cancer
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Institutes (NCI’S) criteria. Tumor growth inhibition (%T/C), is defined as the ratio of the
median tumor volume for the treated versus control group. Representative photos of the tumorꢀ
bearing mice in the control and the two treatment groups at termination of experiment (day 28)
are presented in Figure 9B. Importantly, the mice in the treated group did not lose body weight
(Figure 9C) and display any signs of toxicity, suggesting no apparent adverse events of the
compounds.
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For the sake of comparison, with our early lead, compound
6 (based on ATRA scaffold)
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caused 97% suppression of MDAꢀMBꢀ231 tumor growth (data not shown). These data suggest
that compounds 20 and 22 can be classified as strong backꢀup compounds in our novel
retinamides drug discovery and development program. It is important to state here that addition
antiꢀtumor studies are need in several breast cancer models in addition to rigorous
pharmacokinetics/pharmacodynamics and toxicology studies to identify the compound that will
emerge as our Investigation New Drug (IND) candidate. It is also pertinent to note that our
current clinical agent, galeterone that emerged from our prostate cancer drug discovery program
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was a backꢀup compound at the early stage of the development process. Galeterone has
successfully completed phase II clinical trials in castration resistant prostate cancer (CRPC)
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patients and is scheduled to enter pivotal phase III clinical trials in the 1 half of this year.
Molecular analysis of tumors: To assess their in vivo mode of action, we evaluated the
effects of compounds 20 and 22 on Mnkꢀ1, peIF4E and their downstream targets. As expected,
tumors from mice treated with compounds 20 or 22 showed depletion of Mnk1/2, peIF4E,
cyclide D1 and antiꢀapoptotic Breast cancerlꢀ2 with concomitant upꢀregulation of proꢀapoptotic
proteins, Bad and Bax (Figure 9D).
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Figure 9: Compounds 20 and 22 suppress breast xenograft tumor growth in vivo. (A) Nude
female mice bearing MDAꢀMBꢀ231 xenograft tumors (n = 6/group) were treated with 20 or 22
,
administered i.p. 20 mg/kg/day, 5 days per week for 28 days. %T/C values are indicated to the
right of each growth curve and the error bars are the SEM. Compounds 20 and 22 treatments,
each, significantly suppressed tumor growth (for 20, *,
) Representative photos of the tumorꢀbearing mice and excised tumors in the control and the
two treatment groups at termination of experiment (day 28). ( ) Body weight changes of the
mice during the course of treatments. Animals were monitored for changes in body weight as a
p = 0.0001 and for 22, **, p = 0.0001).
(B
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surrogate marker for toxicity in control and treatment groups. (D) The effect of compounds on
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the expression of proteins modulated by Mnk/eIF4E signaling. Total cell lysates in mice treated
with vehicle, compound 20 and 22 were prepared separately using RIPA buffer. Total protein
(50µg/well) from pooled samples (n=6) was run on 10% SDSꢀPAGE and probed with antibodies
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for MNK1, MNK2, pꢀeIF4E, eIF4E, cyclin D1, Breast cancerlꢀ2, Bax, Bad and GAPDH.
CWR22Rv1 antiꢀtumor efficacy study: Similarly, treatment of compound 20 to castrated mice
bearing the aggressive CWR22Rv1 xenografts cause significant inhibition of tumor growth with
% T/C value of 38.8, i.e., 61.2% suppression of tumor growth (Figure 10A). We also found that
the body weights were comparable in mice either from vehicle control or treatment groups
(Figure 10B), suggesting no apparent adverse effects of compound 20.
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Figure 10: Compound 20 suppresses prostate xenograft tumor growth in vivo. (A) Castrated
SCID male mice bearing CWR22Rv1 xenograft tumors (n = 6/group) were treated with 20
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administered i.p. 20 mg/kg/day, 5 days per week for 28 days. %T/C values are indicated to the
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right of each growth curve and the error bars are the SEM. Compounds 20 treatment significantly
suppressed tumor growth (*
of treatments. Animals were monitored for changes in body weight as a surrogate marker for
toxicity in control and treatment groups. ( ) The effect of compounds on the expression of
p = 0.0001). (B) Body weight changes of the mice during the course
C
proteins modulated by AR and Mnk/eIF4E signaling. Total cell lysates in mice treated with
vehicle, compound 20 and 22 were prepared separately using RIPA buffer. Total protein
(50µg/well) from pooled samples (n=6) was run on 10% SDSꢀPAGE and probed with antibodies
for fAR, ARꢀV7, MNK1, MNK2, pꢀeIF4E, eIF4E, cyclin D1, Breast cancerlꢀ2, Bad and
GAPDH.
Molecular analysis of tumors: To assess their in vivo mode of action, we evaluated the
effects of compounds 20 on fAR, ARꢀV7, Mnkꢀ1, peIF4E and their downstream targets. As
expected, tumors from mice treated with compounds 20 showed depletion of fAR, ARꢀV7,
Mnk1/2, peIF4E, cyclin D1 and antiꢀapoptotic Breast cancerlꢀ2 with concomitant upꢀregulation
of proꢀapoptotic proteins, Bad (Figure 10C).
CONCLUSIONS
Our study has shown for the first time that high antiꢀcancer activities can be retained in
Cꢀ4 heteroaryl retinamides based on the rarely utilized 13ꢀcis retinoic acid scaffold. In this study,
we describe a series of novel Cꢀ4 heteroaryl 13ꢀcis retinamides that degrade Mnk 1 and 2 with
concomitant suppression of oncogenic eIF4E phosphorylation in a variety of human breast and
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prostate cancer cell lines. In addition, the compounds also modulate activities of fullꢀlength and
splice variant androgen receptors (fAR and ARꢀV7) via AR antagonism and AR degradation in
prostate cancer cells. Three lead compounds, including 16, 20 and 22 were identified. Of these
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compounds, 20 and 22 exhibited equipotent and strong suppression of the growth of aggressive
human triple negative breast cancer MDAꢀMBꢀ231 tumor xenografts. Compound 20 also proved
to be very effective at inhibiting the growth of castrationꢀresistant CWR22Rv1 human prostate
tumor xenografts. These impressive in vitro and in vivo antiꢀbreast and antiꢀprostate cancers
activities make compounds 20 and 22 strong candidates for further development as potential new
drugs for the treatments of breast and prostate cancers in humans. In addition, given the
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implication of Mnk1/2ꢀeIFE axis in the initiation and progressions of all types of solid tumors
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and hematologic cancers, these and related retinamides warrant evaluation in other types of
cancers.
EXPERIMENTAL SECTION
Chemistry: General: All the materials listed below were of research grade or
spectrophotometric grade in the highest purity commercially available from SigmaꢀAldrich.
Column chromatography was performed on silica (230ꢀ400 mesh, 60 Å) from Silicycle. Silica
gel plates (Merck F254) were used for thin layer chromatography (TLC) and were developed
with mixtures of ethyl acetate (EtOAc)/Petroleum ether or CH Cl /methanol (MeOH) unless
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otherwise specified and were visualized with 254 and 365 nm light and I or Br vapor.
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1
13
Petroleum ether refers to light petroleum, bp 40ꢀ60 °C. H and C NMR spectra were recorded
in CDCl or DMSOꢀd6 using a Brüker 400 MHz NMR instrument and chemical shifts are
3
reported in ppm on the
δ scale relative to tetramethylsilane. High resolution mass spectra were
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obtained on Bruker 12 T APEXꢀQe FTICRꢀMS instrument by positive ion ESI mode by Susan
A. Hatcher, Facility Director, College of Sciences Major Instrumentation Cluster, Old Dominion
University, Norfolk, VA. Melting points (mp) were determined with Fischer Johns melting point
apparatus uncorrected. Purities of the compounds were determined by Waters UPLC BEH C18
1.7µl, 2.1×50mm column using a solvent gradient system of ammonium acetate
buffer/acetonitrile/methanol (100→0, 0→100 and 0→10 respectively) over a period of 10 min.
The purities of all final compounds were determined to be at least 95% pure by a combination of
UPLC, NMR and HRMS.
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(2Z,4E,6E,8E)ꢀmethyl 3,7ꢀdimethylꢀ9ꢀ(2,6,6ꢀtrimethylcyclohexꢀ1ꢀenꢀ1ꢀyl)nonaꢀ2,4,6,8ꢀ
tetraenoate (11)
In a 250 mL round bottomed flask (RBF) equipped with a magnetic stirrer, 13ꢀcisꢀretionoic acid
(10) (5 g, 16.64 mmol) was dissolved in methanol (30 mL) and benzene (100 mL). While
stirring, trimethylsilyldiazomethane solution 2.0M in hexanes (3.730 g, 32.68 mmol) was added
dropꢀwise very slowly and reaction was monitored by the gas evolution on the oil bubbler. The
reaction mixture was concentrated to give an oily yellow crude product. Purification by flash
column chromatography [FCC; petroleum ether/EtOAc (9:1)] afforded compound 11 as a yellow
1
solid. Yield: 4.32 g (83%); mp: 38ꢀ40°C; H NMR (400 MHz, CDCl ) δ 7.77 (d,
J
= 15.3 Hz,
= 16.1 Hz, 1H), 5.64 (s,
= 6.2 Hz, 2H), 1.99 (s, 3H), 1.71 (s, 3H), 1.65 – 1.54
3
1H), 6.99 (dd,
H), 3.70 (s, 3H), 2.07 (s, 3H), 2.03 (t,
m, 2H), 1.47 (dd, = 7.7, 3.9 Hz, 2H), 1.03 (s, 6H); C NMR (100 MHz, CDCl ) δ 166.8,
J = 15.3, 11.5 Hz, 1H), 6.32 – 6.21 (m, 2H), 6.15 (d, J
1
J
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3
(
J
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51.3, 139.8, 137.6, 137.4, 132.2, 130.3, 130.0, 129.2, 128.5, 128.3, 116.0, 77.3, 77.0, 76.7, 50.9,
9.6, 34.2, 33.1, 28.9, 21.7, 20.9, 19.2, 12.8.
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(
2Z,4E,6E,8E)ꢀmethyl 3,7ꢀdimethylꢀ9ꢀ(2,6,6ꢀtrimethylꢀ3ꢀoxocyclohexꢀ1ꢀenꢀ1ꢀyl)nonaꢀ
2
,4,6,8ꢀtetraenoate (12)
In a 500 mL volumetric flask, 11 (4.00 g, 12.7 mmol), MnO (100 g, 1150 mmol) were added
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into 100 mL of dichloromethane. The mixture was allowed to stir on an Innova 2000 platform
shaker at 200 rpm for 12 h. Filtration was done through a pad of celite and the organic reaction
mixture concentrated to give thick yellow oil. This was adsorbed on silica (50 g) and packed on a
column where elution was done gravity with a solvent system of [petroleum ether/EtOAc (9:1)]
1
to yield compound 12 as a yellow solid. Yield: 2.80 g (68%); mp: 83ꢀ85°C; H NMR (400 MHz,
CDCl ) δ 7.85 (d,
J
= 15.4 Hz, 1H), 6.96 (dd,
H), 5.70 (s, 1H), 4.12 (q, = 7.1 Hz, 2H), 3.71 (s, 3H), 2.51 (t,
2.02 (s, 3H), 1.86 (s, 3H), 1.26 (t, 2H), 1.19 (s, 6H). C NMR (100 MHz, CDCl ) δ 199., 166.6,
J
= 15.3, 11.4 Hz, 1H), 6.35 (dd,
J = 12.5, 7.6 Hz,
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J
J
= 6.8 Hz, 2H), 2.08 (s, 3H),
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60.7, 150.7, 140.6, 138.1, 133.6, 131.4, 131.2, 130.1, 125.8, 117.2, 51.0, 37.4, 35.7, 34.2, 27.6,
1.0, 13.7.
(2Z,4E,6E,8E)ꢀmethylꢀ9ꢀ(3ꢀhydroxyꢀ2,6,6ꢀtrimethylcyclohexꢀ1ꢀenꢀ1ꢀyl)ꢀ3,7ꢀdimethylnonaꢀ
2,4,6,8ꢀtetraenoate (13)
In a 25 mL round bottom flask, 12 (2.50 g, 7.61 mmol) was dissolved in methanol (20 mL) and
crystilline sodium borohydride (0.45 g, 11.9 mmol) was added slowly and left to stir at room
temparature for 1 h where TLC analysis shows exhaustion of the starting material. Reaction
mixture was diluted with water (25 mL), extraction done with CH Cl (2 × 20 mL). Organic
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portions were mixed and concentrated to give a thick brownish oil. This was adsorbed on silica
and packed on a column where elution was done via gravity using a solvent system of
1
[
petroleum ether/EtOAc (8:2)] to yield compound 13 as a yellow oil. Yield: 2.01 g (80%); H
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NMR (400 MHz, CDCl ) δ 7.79 (d,
J
= 15.3 Hz, 1H), 7.03 – 6.92 (m, 1H), 6.27 (d,
J = 11.4 Hz,
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1
3
H), 6.19 (d,
.90 (ddd,
J = 6.6 Hz, 2H), 5.66 (s, 1H), 4.01 (s, 1H), 3.71 (s, 3H), 2.07 (s, 3H), 1.99 (s, 3H),
J
= 11.5, 6.9, 3.1 Hz, 1H), 1.84 (s, 3H), 1.76 – 1.57 (m, 2H), 1.43 (ddd, J = 13.2, 7.3,
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.0 Hz, 1H), 1.05 (s, 3H), 1.02 (s, 3H). C NMR (100 MHz, CDCl ) δ 166.7, 151.1, 141.6,
3
139.2, 138.4, 132.0, 131.1, 130.2, 129.8, 127.5, 116.4, 70.2, 50.9, 34.8, 34.5, 29.0, 27.5, 20.9,
8.6, 12.8.
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(2Z,4E,6E,8E)ꢀmethylꢀ9ꢀ(3ꢀ(1Hꢀimidazolꢀ1ꢀyl)ꢀ2,6,6ꢀtrimethylcyclohexꢀ1ꢀenꢀ1ꢀyl)ꢀ3,7ꢀ
dimethylnonaꢀ2,4,6,8ꢀtetraenoate (14)
In a 25 mL round bottom flask 13, (1.90 g, 5.75 mmol) was dissolved in acetonitrile (25 mL )
followed by 1,1'ꢀCarbonyldiimidazole (CDI) (1.21 mg, 7.47 mmol). On adding CDI, reaction
mixture was seen to turn from yellow to dark red with evolution of gas. After 1 hr, TLC analysis
showed exhaustion of starting material and the reaction mixture was therefore concentrated.
Water (20 mL) was added and extraction was effected using with CH Cl (3 × 20 mL). The
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combined organic portion was concentrated, adsorbed on silica and loaded on a column where
elution was done via gravity with a solvent system of [methanol/CH Cl (0.5:9.5)] to yield
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2
1
compound 14 as a red viscous oil. Yield: 1.50 g 69%; H NMR (400 MHz, CDCl ) δ 7.80 (d, J =
3
1
5.4 Hz, 1H), 7.50 (s, 1H), 7.07 (s, 1H), 7.00 – 6.87 (m, 2H), 6.33 – 6.17 (m, 3H), 5.67 (s, 1H),
4.54 (t, = 4.8 Hz, 1H), 3.71 (s, 3H), 2.08 (s, 3H), 2.01 (s, 3H), 1.82 (dt, = 35.3, 18.6 Hz, 2H),
.61 (d, = 10.8 Hz, 3H), 1.57 (d, = 11.2 Hz, 1H), 1.50 (dd, = 6.8, 3.5 Hz, 1H), 1.17 – 1.04
m, 6H). C NMR (100 MHz, CDCl ) δ 166.7, 150.9, 144.8, 139.2, 138.6, 136.8, 131.7, 131.7,
J
J
1
J
J
J
1
3
(
3
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2
30.2, 129.2, 126.5, 125.0, 118.1, 116.7, 77.3, 77.2, 77.0, 76.7, 58.0, 53.4, 51.0, 34.7, 34.6, 29.0,
+
8.1, 27.8, 20.9, 18.8, 12.8; HRMS (ESI) calcd for C H N O Na , 403.2355, Found: 403.2357.
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