Potent and selective phosphopeptide mimetic prodrugs targeted to the Src homology 2 (SH2) domain of signal transducer and activator of transcription 3

Article abstract of DOI:10.1021/jm2000882

Signal transducer and activator of transcription 3 (Stat3), a target for anticancer drug design, is activated by recruitment to phosphotyrosine residues on growth factor and cytokine receptors via its SH2 domain. We report here structure-activity relation

Full text of DOI:10.1021/jm2000882

ARTICLE
pubs.acs.org/jmc
Potent and Selective Phosphopeptide Mimetic Prodrugs Targeted to
the Src Homology 2 (SH2) Domain of Signal Transducer and Activator
of Transcription 3
||
Pijus K. Mandal,† Fengqin Gao,† Zhen Lu,† Zhiyong Ren,§, Rajagopal Ramesh,‡,^ J. Sanderson Birtwistle,†
Kumaralal K. Kaluarachchi,† Xiaomin Chen,§,# Robert C. Bast, Jr.,† Warren S. Liao,*,† and
John S. McMurray*,†
†Department of Experimental Therapeutics, ‡Department of Thoracic and Cardiovascular Surgery, and §Department of Biochemistry
and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030,
United States
S Supporting Information
b
ABSTRACT: Signal transducer and activator of transcription 3
(Stat3), a target for anticancer drug design, is activated by
recruitment to phosphotyrosine residues on growth factor and
cytokine receptors via its SH2 domain. We report here structureꢀ
activity relationship studies on phosphopeptide mimics targeted
to the SH2 domain of Stat3. Inclusion of a methyl group on the
β-position of the pTyr mimic 4-phosphocinnamide enhanced
affinity 2- to 3-fold. Bis-pivaloyloxymethyl prodrugs containing
β-methylcinnamide, dipeptide scaffolds Haic and Nle-cis-3,4-methanoproline, and glutamine surrogates were highly potent,
completely inhibiting phosphorylation of Stat3 Tyr705 at 0.5ꢀ1 μM in a variety of cancer cell lines. The inhibitors were selective for
Stat3 over Stat1, Stat5, Src, and p85 of PI3K, indicating ability to discriminate individual SH2 domains in intact cells. At
concentrations that completely inhibited Stat3 phosphorylation, the prodrugs were not cytotoxic to a panel of tumor cells, thereby
showing clear distinction between cytotoxicity and effects downstream of activated Stat3.
’ INTRODUCTION
of Stat3 is a key event that contributes to increased survival and
proliferation of cancer cells. Small molecule inhibitors targeted to
the SH2 domain of Stat3 would be potential chemotherapeutic
agents for the treatment of cancer by inhibiting receptor binding,
Tyr705 phosphorylation, nuclear translocation, and transcrip-
tional activity, resulting in decreased cell cycling and survival and
increased tumor cell death by apoptosis.5
Contrary to this hypothesis, two recent reports showed that
JAK kinase inhibitors P6 (1)6 and AZD1480 (2), at concentra-
tions that completely eliminated Tyr705 phosphorylation, were
not cytotoxic to a variety of cultured melanoma,7 breast, prostate,
and pancreatic tumor cell lines.8 These results suggest that tumor
cells grown in culture do not require pStat3 for survival and call
into question the above hypotheses. Morevover, these studies
suggest that if a compound were cytotoxic to cells grown in 2D
cultures, it likely has off-target activities with respect to Stat3.8
Caveats must also be acknowledged concerning the biological
activities of Stat3. Unphosphorylated Stat3 (U-Stat3) complexes
with unphosphorylated NF-κB, resulting in the transcription of
κB-dependent genes.9 In nontranscriptional roles, Ser727-phos-
phorylated Stat3 has been found in electron transport complexes
in mitochondria10 and in this capacity supports the growth of Ras
Signal transducer and activator of transcription 3 (Stat3) is a
member of the STAT family of transcription factors that transmits
extracellular signals from receptors on the plasma membrane
directly to the nucleus where it binds to various promoters and
initiates gene transcription.1 In the canonical mechanism, when
cytokines such as interleukin-6 (IL-6) or growth factors such as
vascular endothelial growth factor (VEGF), epidermal growth
factor (EGF), or platelet-derived growth factor (PDGF) bind to
their receptors, Stat3, via its Src homology 2 (SH2) domain, is
recruited to phosphotyrosine residues on the receptor and
becomes phosphorylated on Tyr705 by JAK kinases, Src kinase,
or the kinase activity of the receptor. Phosphorylated Stat3 (pStat3)
dimerizes via reciprocal pTyr705-SH2 domain interactions and is
then translocated to the nucleus, where it initiates transcription
of downstream genes. Introduction of antisense, dominant negative,
and decoy oligonucleotides against Stat3 into tumor cells lines
has been shown to reduce transcription of antiapoptotic genes
such as Bcl-2, Bcl-xL, Mcl-1, and survivin, cell cycle progression
genes such as cyclin D1 and c-Myc, metastasis supporting genes
including MMP-2,2,3 and VEGF3,4 and to result in apoptosis.
Stat3 is constitutively activated (i.e., phosphorylated on Tyr705)
in several cancer types, such as breast, lung, prostate, ovarian,
leukemia, multiple myeloma, and others.5 Taken together, these
findings support the hypothesis that phosphorylation of Tyr705
Received: January 26, 2011
Published: April 12, 2011
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2011 American Chemical Society
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Chart 1a
aNames for some of the prodrugs are given after their compound number.
transformed cells by sustaining glycolytic and oxidative phos-
phorylation.11 Thus, the reported cytotoxicity and alterations in
gene transcription ensuing from Stat3 knockdown and domi-
nant-negative overexpression may, in part, be due to mechanisms
not related to pTyr705-driven transcription. Therefore, highly
potent and selective inhibitors of Stat3 phosphorylation are needed
to understand the requirements of Tyr705 phosphorylation in
cancer cell growth.
manual solid phase synthesis on Rink amide resin by first coupling
Fmoc-Glu-NHBn27 or modified Fmoc glutamic acids30 via the
side chain. After addition of the remaining amino acids, peptidomi-
metics 4a, 5a, 6a, and 7a (Figure 1) were prepared by capping
with 4-(di-tert-butoxyphosphoryloxy)cinnamic acid.29 Inhibitors
4b, 5b, 6b, 7b, and 8ꢀ19 (Table 1) were capped with penta-
chlorophenyl (2E)-4-phosphoryloxyphenylbutenoic acid (25,
Scheme 1). Peptides and mimetics were cleaved and purified
by reverse phase HPLC.
The SH2 domain of Stat3 has been targeted in several labora-
tories by a variety of phosphopeptides,12ꢀ16 peptidomimetics,17ꢀ22
and small molecules.23ꢀ25 We are targeting the SH2 domain of
Stat3 with inhibitors based on our lead peptide Ac-pTyr-Leu-
Pro-Gln-Thr-Val-NH2.26ꢀ31 We recently reported the conver-
sion of a conformationally constrained version of the lead peptide29
to a cell-permeable, phosphatase-stable peptidomimetic, BP-PM6
(3, Chart 1), that completely inhibited constitutive phosphoryla-
tion of Stat3 Tyr705 (pStat3) in MDA-MB-468 breast cancer
cells at 10 μM.32 The X-ray structure33 and molecular models of
peptides bound to the SH2 domain29,34 suggest that a methyl
group on the β-carbon of phosphotyrosine or a suitable mimic
might increase affinity because of increased hydrophobic inter-
action. In this communication we demonstrate that a β-methyl
group on the phosphocinnamate pTyr mimic enhances affinity
for Stat3. This modification and recently described glutamine
analogues30 were incorporated into a series of peptidomimetic
prodrugs that displayed >10-fold enhanced potency over 3,
inhibiting pStat3 at 0.1ꢀ0.5 μM. We show that these prodrugs
are selective for the SH2 domain of Stat3 over those of Stat1,
Stat5, Src, and the p85 regulatory domain of the phosphatidyli-
nositol 3-kinase in intact cells. There was no effect on p38MAPK
or S473Akt phosphorylation. However, as reported for the JAK
inhibitors,7,8 they are not cytotoxic to a panel of tumor cells in 2D
culture on plastic plates at concentrations that inhibit Stat3 phos-
phorylation.
Synthesis of the Phosphotyrosine Mimic (2E)-4-Phos-
phoryloxyphenylbutenoic Acid. The phenolic hydroxyl group
of 4-hydroxyacetophenone (20) was phosphorylated with diethyl-
chlorophosphate at the beginning of the synthesis to install the
phosphate. The modified acetophenone (21) was elaborated
by HornerꢀEmmons vinylogation with tert-butyl (diethylphos-
phono)acetate. The use of EtOH as a solvent resulted in 100%
stereoselectivity for the trans isomer. Unfortunately, transester-
ification of the carboxyl group to an ethyl ester occurred and
selective cleavage of the carboxy ester could not be achieved, as
cleavage of one or more ethyl groups on the phosphate was ob-
served. However, the use of tert-butanol as the solvent avoided
the side reaction. The stereoselectivity was not as high as with
ethanol and resulted in approximately 25% of the cis isomer,
which could readily be separated using silica gel chromatography.
The resulting tert-butyl ester (22) was cleaved with TFA to give
23, which was esterified with pentachlorophenol (24). Removal
of the ethyl groups with trimethylsilyl iodide (TMSI) gave the
phosphate 25 ready for coupling to amino acid sequences.
Synthesis of Prodrugs. To inhibit Stat3 in intact cells, we
employed the same prodrug strategy as with 3 (Chart 1).32 The
phosphate group of β-methyl cinnamate was substituted with the
isosteric difluoromethylphosphonate (F2Pm) group to render
inhibitors stable to phosphatases.32,35 The negatively charged
oxygen atoms on the F2Pm group were capped with carboxyes-
terase-labile pivaloyloxymethyl (POM)36 groups to facilitate cell
penetration. The active ester bis-POM building block approach32
was used to assemble the prodrugs. Starting from iodoacetophe-
none (26), HornerꢀEmmons coupling with tert-butyl (diethyl-
phosphono)acetate gave the iodocinnamate, 27 (Scheme 2).
’ RESULTS
Chemistry. Phosphopeptide inhibitors were synthesized using
a convergent strategy. Amino acid sequences were assembled by
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Figure 1. Left: Model of pYLPQ-NHBn34 docked to the SH2 domain of Stat3 showing space between inhibitor and Glu638. Surface electrostatic
potential was calculated with APBS tools in PyMol65. Right: Incorporation of methyl group on the β-position of 4-phosphoryloxycinnamide enhances
affinity for Stat3, as judged by a fluorescence polarization assay. 4a and 7a were from ref 29. 5a was from ref 30.
Table 1. Effect of Substitution of Gln-NHBn with Glutamine
Scheme 1a
Mimics on Peptidomimetics 3b, 4b, 5b, and 6ba
a KI values were determined by fluorescence polarization.27
a Reagents and condition: (i) diethyl chlorophosphate, TEA, CH2Cl2,
t
0 ꢀC to room temp overnight; (ii) LitOBu,
tBuOH, (EtO)2POCH2CO2 Bu,
As in the case of 22, t-BuOH was used as the solvent and the cis
and trans isomers were separated by silica gel chromatography.
Copperꢀcadmium cross-coupling with diethyl bromodifluoro-
methylphosphonate37 provided phosphonate 28. Acidolytic re-
moval of the tert-butyl ester followed by esterification with penta-
chlorophenol gave intermediate 29a. Trimethylsilyl iodide treatment
removed the phosphonate ethyl groups, resulting in phosphonic
acid 30a. The phosphonate was neutralized with 2 equiv of
NaOH, and the sodium counterions were exchanged with silver.
The silver salt was alkylated with 2 equiv of pivaloyloxymethyl
iodide in toluene to give prodrug building block 31a. 4-Nitrophenyl
room temp overnight; (iii) TFA/CH Cl2 (95:5) 1 h; (iv) C6Cl5OH,
DCC, DMAP (cat.) EtOAc, room tem2p 24 h; (v) TMSI, BSTFA, CH2
Cl2, 0 ꢀC 1 h, then room temp 1 h.
esters were also synthesized using identical reaction schemes (29b,
30b, 31b).
Prodrugs were formed by solution phase coupling of 31a or
31b to Haic-XXX or Nle-mPro-XXX intermediates, which were
synthesized on Rink resin and were purified by reverse phase
HPLC before use. Acylation of dipeptides was accomplished with
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Scheme 2. Synthesis of Bis-POM-Protected
Difluoromethylphosphonocinnamates for
Incorporation into Prodrugsa
mixture of enantiomers, and peptides incorporating them can be
separated into the individual diastereomers, one of which exhibits
higher affinity than the other.27 The results presented for 5a, 5b,
6a, and 6b in Figure 1 are from the more active stereoisomers.
Unfortunately, we have not been able to obtain a crystal structure
of Stat3 complexed with any of the β-methylcinnamide-contain-
ing inhibitors to determine the nature of the increase in affinity.
To gain an understanding of the effect of β-methyl substitution
on the conformation of the cinnamate, we determined the crystal
structure of a model compound, 4-iodo-β-methylcinnamoylleu-
cine tert-butyl ester (manuscript in preparation). In this structure
the aromatic ring deviates 27ꢀ30ꢀ from the plane of the Rꢀβ
double bond to avoid steric clash with the β-methyl group
(Figure S1). The cinnamide carbonyl oxygen is on the same side
of the CꢀCR bond as the double bond, which was observed in
the crystal structure of several cinnamides.38,39 The double bond
is slightly distorted because of collision between the carbonyl
oxygen and the β-methyl group. This conformation was sup-
ported by 1H ROESY NMR spectroscopy of the dipeptide mimic
in DMSO-d6 in which there was a strong cross-peak between the
Leu NH and the R-proton of the cinnamate. The corresponding
cross-peak was observed in ROESY spectra of the non-POM
version of prodrug 33 (Chart 1), βMF2PmCinn-Haic-Gln-NHBn,
as well as 3, F2PmCinn-Haic-Gln-NHBn,32 possessing no methyl
group on the β-position of the cinnamate. It is uncertain if the
increases in affinity of the β-methylcinnamide-possessing inhibi-
tors are the result of the extra hydrophobic interaction between
the β-methyl group and Glu638, a more favorable conformation
of the aromatic ring, or both.
Modifications of Glutamine. Glutamine at pY þ 3 is an
essential part of the recognition determinant for Stat3.31,40,41 To
further reduce the peptide nature of our inhibitors, we replaced
the C-terminal Gln-NHBn groups of compounds 4b, 5b, 6b, and
7b with glutamine mimics Apa, 2-aminoethyl carbamate (Aec),
and 2-aminoethylurea (Aeu), recently reported by our laboratory
(Table 1).30 Overall, the Gln mimics were 1.2- to 4-fold less avid
than the Gln-NHBn leads. In cases of proline or methanoproline,
the urea analogues (10, 13, and 16) were the highest affinity
inhibitors, displaying KI of 39ꢀ94 nM. The (R)-4-aminopenta-
mides (8, 11, and 13) were approximately 2-fold less potent than
the corresponding Gln-NHBn-containing inhibitors, and the carba-
mates (9, 11, and 15) were the least tolerated. The pattern with
the Haic-containing compounds was slightly different. The urea,
19, showed the least affinity and the amino pentanamide, 17, the
greatest.
Effect of the β-Methyl Group and C-Terminal Substitution
on the Inhibition of Constitutive Stat3 Phosphorylation in
Intact Breast Tumor Cells. To determine the effect of the
β-methyl group on the inhibition of Stat3 phosphorylation in intact
cells, prodrug 3 was compared with 32, possessing a methyl group
on the β-position of the cinnamate (Chart 1 and Figure 2). The
MDA-MB-468 breast cancer cell line was utilized, as these cells
possess constitutively active Stat3.42 Cells were treated with com-
pounds for 2 h, and total and Tyr705-phosphorylated Stat3 levels
were estimated by Western blotting of cell lysates (Figure 2,
column A). Both compounds reduced the level of pStat3 in a
dose-dependent manner, suggesting that the prodrugs enter cells
and are stripped of the POM groups and the resulting phospho-
nates bind to the SH2 domain of Stat3, resulting in (1) breakup
of preformed dimers followed by dephosphorylation and/or
(2) prevention of binding to growth factor or cytokine receptors
and the subsequent phosphorylation of Tyr705. Addition of the
a Reagents and conditions (i) (EtO)2POCH2CO2tBu; (ii) BrCdCF2PO3Et2,
CuCl; (iii) (a) TFA, (b) C6Cl5OH, DCC; (iv) TMS-I; (v) (a) NaOH,
(b) AgNO3, (c) POM-I.
catalytic amounts of dimethylaminopyridine under anhydrous
conditions. Prodrugs were purified by RP-HPLC using gradients
of MeCN in water with no TFA or other additives. All prodrugs
were >98% pure by reverse phase HPLC and gave the correct
mass by high resolution mass spectrometry.
Unprotected difluoromethylphosphonates were prepared by
acylation of amino acid sequences by intermediate 30a on solid
supports, followed by cleavage with TFA and HPLC purification.
To prepare mono-POM-protected prodrugs, 31a was coupled to
presynthesized peptides in solution using DMF and HOBt
hydrate, conditions that result in premature hydrolysis of one
of the POM groups.32 HPLC purification yielded both mono-
and bis-POM prodrugs. To prepare 40 (Chart 1), the diethyl
ester analogue of 34, 29a was coupled to resin-bound Haic-Apa
(Apa = (R)-4-aminopentamide) followed by TFA cleavage and
HPLC purification.
Inclusion of a Methyl Group on the β-Position of a pTyr
Mimic Increases Affinity. Examination of the original crystal
structure of Stat333 and molecular models developed by us29,34
showed that there was space between the β-carbon of phospho-
tyrosine or pCinn and the side chain methylene groups of Glu638
that could be filled to increase hydrophobic interaction between
the inhibitor and protein (Figure 1). Addition of a methyl group
to the β-position of phosphocinnamate resulted in 1.5- to 3-fold
increases in affinity in a series of phosphopeptide mimetics, as
judged by a fluorescence polarization assay (Figure 1).27 Note
that commercially available 3,4-cis-methanoproline is sold as a
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Figure 2. Inhibition of the constitutive phosphorylation of Tyr705 of Stat3 in MDA-MB-468 breast tumor cells. (A) Effect of substitution on the
cinnamide β-position and the C-terminus. (B) Inhibition of Nle-mPro-based prodrugs. (C) Time course of inhibition. Prodrug concentrations were
5 μM. (D) Effect of zero, one, or two POM groups on inhibition. Prodrug concentrations were 5 μM. Cells were treated with the indicated
concentrations for 2 h (parts A, B, and D) or for the indicated time intervals (part C). After cell lysis pStat3 and total Stat3 were determined by Western
blots. For each pair of gels the top is pStat3 and the lower is total Stat3.
β-methyl group provided a slight but detectable enhancement in
the inhibition of the phosphorylation of Stat3.
respectively, were isolated during the HPLC runs and were tested
for their abilities to inhibit Stat3 phosphorylation in MDA-MB-
468 breast cancer cells (Figure 2, column B). In each case, the
first eluting isomers from HPLC purification runs were very
potent inhibitors. Inhibition of pStat3 was evident at 10 nM and
was nearly complete at 100 nM. As expected, the second stereo-
isomers were very poor inhibitors: the intensity of the band at
25 μM was only partially reduced. This corresponds to the
reduced affinity for isolated Stat3 measured for the diastereo-
meric mPro containing peptidomimetics.27 Compound 35 has a
benzyloxyethyl group at the R-position of the glutamine surrogate,
whereas 37 possesses a methyl group. The high potency of the
latter as well as 33 and 34 suggests that in intact cells, C-terminal
benzyl appendages are not necessary for efficient inhibition of
Stat3 phosphorylation.
Compound 16, possessing 2-aminomethylurea in place of
glutamine, was a very high affinity inhibitor in the fluorescence
polarization assay (KI = 49 nM). In the case of the corresponding
prodrug, 39, we were unable to separate the diastereomers, so the
compound was tested as a mixture of stereoisomers. In spite of
the high affinity of the parent compound, there was little
inhibition of the phosphorylation of Stat3 in the breast tumor
cells up to 1 μM (Figure 2, column B). At 5 μM complete in-
hibition was observed. The alkylcarboxamides impart greater
cellular potency than the ethylurea.
Even though the C-terminal methyl group resulted in 2-fold
lower affinity than the benzylamide in the phosphate series (17
and 7b, respectively, Table 1 and Figure 1), the potencies of
prodrugs incorporating the simpler structure were substantially
enhanced (34 and 33, Figure 2A). There was noticeable inhibi-
tion of pStat3 at 0.1 μM, and the signal was completely gone at
0.5 μM. It is unclear whether this is due to reduced cell
penetration, increased clearance, or degradation of the benzylamide-
containing compounds.
Additional prodrugs incorporating the Nle-mPro and Haic
scaffolds and glutamine mimics in Table 1 were synthesized. For
the first group, compounds 6b, 14, 15, and 16 were converted to
their corresponding prodrugs. In a previous study from our lab-
oratory30 it was discovered that the glutamine surrogate (4R,5S)-
4-amino-5-benzyloxyhexanamide, in addition to being isosteric
to Gln-NHBn, was equipotent in the context of the pCinn-Leu-
mPro (but not pCinn-Haic). Therefore, this mimic was included
in the Nle-mPro prodrug series. In addition to the Haic contain-
ing prodrugs 32ꢀ34, compounds incorporating the urea and
carbamate groups of 18 and 19 in the Haic series were converted
to their corresponding prodrugs. This series was screened for the
ability to inhibit constitutive phosphorylation of Stat3 in BT20
breast tumor cells. From this series, 35 and 37 stood out as having
considerable potency (Chart 1). This pair of prodrugs, posses-
sing the Nle-cis-3,4-methanoproline dipeptide scaffold, was also
highly potent and completely inhibited pStat3 at 0.5 μM (Figure 2,
column B).
The time course of inhibition of constitutive phosphorylation
of Stat3 in MDA-MB-468 cells is shown in Figure 2, column C. A
single dose of 5 μM of the high affinity prodrugs 34, 35, and 37
completely inhibited pStat3 formation at 30 min, and the effect
was sustained for 4 h. Partial recovery was evident at 8 h, and
recovery was complete at 16 h.
During the synthesis of 35 and 37, diastereomers possessing
the opposite enantiomers of cis-3,4-methanoproline, 36 and 38,
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Inhibition of pStat3 Nuclear Translocation. MDA-MB-468
cells were treated with 34 for 2 h and were then stained with
fluorescent antibodies for pTyr705 Stat3 (Figure 3). In vehicle-
treated controls, pStat3 had a strong presence in the nucleus.
Treatment with the prodrug not only greatly reduced the level of
pStat3 but also abrogated nuclear localization.
Two POM Groups Are Required for Inhibition of Stat3.
Analogues of 34, 35, and 37 possessing zero and one POM group
were assayed for their ability to inhibit Stat3 phosphorylation in
MDA-MB-468 cells. At 5 μM and 2 h treatment the unprotected
(NP) and mono-POM (MP) esters did not inhibit pStat3 for-
mation, whereas the bis-POM (BP) prodrugs did (Figure 2,
column D). Thus, consistent with the first generation prodrug,
3,32 two POM groups are required for efficient cell penetration
and inhibition of Stat3 phosphorylation.
Selectivity for Stat3. Ladbury et al. argued that selective dis-
ruption of individual signaling pathways with phosphopeptide
mimics in intact cells may be difficult to achieve because the
differences in measured affinities of phosphopeptides for the SH2
domains of different proteins are relatively small.43,44 To study
the selectivity of our prodrugs, we assayed for the inhibition of
the phosphorylation of Tyr701 of Stat1, Tyr694 of Stat5, Ser473
of Akt, and Tyr861 of the focal adhesion kinase (FAK), which are
all mediated directly or indirectly by SH2 domains binding to
pTyr residues on proteins. MDA-MB-468 cells were treated with
5 μM 34, 35, and 37 for 1.5 h and were then stimulated with EGF.
After 30 min the cells were lysed and pStat3, phosphoTyr694
Stat5, and phosphoSer473 Akt levels were analyzed by Western
blots (Figure 4A). The prodrugs completely inhibited the increase
in Stat3 phosphorylation induced by EGF. Like Stat3, Stat5 binds
to pTyr residues on receptors via its SH2 domains and becomes
phosphorylated on Tyr694. Stat5 phosphorylation was not inhibited
by our prodrugs. Phosphatidylinositol 3-kinase (PI3K) is recruited
to EGFR via the SH2 domains of p85, the regulatory subunit,
which activates the kinase domain, resulting in the phosphoryla-
tion of phosphatidylinositol 2,4-diphosphate on the 3-position.
Phosphatidylinositol 2,3,4-triphosphate recruits both phosphati-
dylinositol-dependent kinase (PDK) and Akt via their plekstrin
homology domains. Akt is then phosphorylated on Ser473 by
PDK. PI3K is constitutively activated in MDA-MB-468 cells
Figure 3. Fluorescence immunohistochemical monitoring of the un-
hibition of Stat3 phosphorylation by compound 34 in MDA-MB-468
breast tumor cells. Cells were treated with 5 μM 34 for 2 h, fixed,
permeabilized, stained with anti pTyr705 Stat3 antibodies, and exam-
ined with confocal microscopy. All photos were taken with the same
microscope settings.
Figure 4. Selectivity of Stat3 inhibitors. (A) MDA-MB-468 cells were treated with prodrugs for 1.5 h and were stimulated with EGF. After 30 min cells
were lysed and levels of pStat3, pSer473 Akt, and pTyr699 Stat5 were estimated by Western blots. (B) Inhibiton of constitutive phosphorylation of FAK
Y861. (C) Inhibition of IFN-γ stimulated phsphorylation of Stat1. (D, E) Effect of 34 (5 μM) on downstream genes in HCC-827 NSCLC cells.
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Figure 5. Inhibition of Stat3 phosphorylation by prodrug 34 in ovarian cancer (SKOV3-ip, Hey) and melanoma (MeWo and A375) cell lines. For each
pair of blots, the upper is pStat3 and the lower is Stat3.
Figure 6. Effect of prodrugs on the survival of cultured cancer cell lines. (A) MDA-MB-468 cells were treated with a single dose of the indicated
compounds for 72 h. (B) MDA-MB-468 cells were treated daily with 34 and 35 for 3 days. Cell viability in parts A and B was determined by MTT assays.
(C) Cancer cell lines were treated daily with 34 for 3 days. (D) Cancer cell lines were treated daily with 40 for 3 days. Cell viability in parts C and D was
determined by SRB assays.
because of loss of PTEN, and ATP competitive inhibitors of this
enzyme have been shown to reduce phosphorylation of Akt.45,46
The fact that our prodrugs do not inhibit Akt phosphorylation
suggests that they do not bind to the SH2 domains of p85 and
prevent downstream signaling of PI3K. Via its SH2 domain, Src
kinase binds to FAK and phosphorylates the unique substrate
Tyr861.47 MDA-MB-468 cells express constitutive phosphoryla-
tion of Tyr861 of FAK,48 and levels of Tyr861 phosphorylation
have been shown to decrease on treatment of tumor cells with the
Src inhibitor dasatinib.49,50 After 2 h of treatment with the prodrugs
no reduction of Tyr861 phosphorylation was observed (Figure 4B).
Therefore, we conclude that our prodrugs do not bind to the SH2
domain of Src. To test for effects on Stat1, cells were treated with
increasing concentrations of the prodrugs for 1.5 h followed by
30 min of stimulation with interferon γ (IFNγ). Tyr701 phos-
phorylation of Stat1 was determined by Western blotting. There
was a dose dependent inhibition of Stat1 phosphorylation with
complete inhibition at 5 μM, ∼10-fold higher than that required
for Stat3 (Figure 4C). In HCC-827 NSCLC cells, 34 had no
effect on the phosphorylation of p38 MAPK and Ser473 of Akt.
No inhibition of the expression of the canonical downstream
genes, cyclin D1 and Bcl-xL, was observed in MDA-MB-468 cells
on treatment with 5 μM 34 (data not shown). Cyclin D1 was not
inhibited in HCC-827 cells. However, survivin was reduced in
the lung line (Figure 4E) and the breast line (data not shown).
Stat3 Phosphorylation Is Inhibited in Other Tumor Cell
Lines. A panel of cell lines was tested for the inhibition of Stat3
phosphorylation by 34 (Figure 5). Melanoma lines MeWo and
A375 and the ovarian cancer line HEY have no or very little basal
pStat3 levels. However, these cell lines are very responsive to IL-6,
which induces high levels of Stat3 phosphorylation on Tyr705.
After 1.5 h of exposure to prodrugs, cells were stimulated with IL-6.
As shown in Figure 5, 34 inhibited pStat3 formation, but slightly
higher concentrations were required to completely abrogate
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phosphorylation. The constitutively activated Stat3 in SKOV3ip
ovarian cancer cells was completely inhibited at 1 μM. At 5 μM,
34 inhibited pStat3 in 10 min in HCC-827 non-small-cell lung
cancer cells (Figure 4D). The effect lasted for at least 4 h, and at
24 h pStat3 had not returned to pretreatment levels (Figure 4E).
Prodrugs Are Weakly Cytotoxic to Cultured Cell Lines.
Compound 34 and the diastereomeric pairs 35, 36 and 37, 38
were assayed for cytotoxicity to MDA-MB-468 cells using the
MTT assay at 72 h. As shown in Figure 6A, 34 and diastereois-
meric pair 37 and 38 exhibited IC50 of ∼30 μM. Prodrug 35 was
more potent, with an IC50 of 10ꢀ15 μM. Interestingly, 36, con-
taining the opposite stereoisomer of mPro that does not inhibit
pStat3 formation until 25 μM, also inhibited growth at 10ꢀ15 μM.
Because both diastereoisomers inhibited growth at equal con-
centrations and 34, 37, and 38 were not inhibitory until 30 μM,
we cannot conclude that the observed cytotoxicity of 35 was
mediated through its effects on Stat3 inhibition.
Knowing that pStat3 levels recover after 8 h, the experiment
was repeated with daily dosing of 34 and 35 (Figure 6B). There
was little change in the survival curves. Similar studies were con-
ducted with daily treatment of MCF-7 breast cancer cells, which
do not harbor constitutively phosphorylated Stat3, and on SKOV3-
ip ovarian cancer cells and HCC-827 lung cancer cells, both of
which have constitutively phosphorylated Stat3 (Figure 6C).
In all of these lines, 34 elicited very weak cytotoxicities, with
IC50 > 30 μM. The most sensitive cell line was MDA-MB-468,
and intermediate sensitivity was observed in HCC-827 cells.
Both MCF7 and SKVO3-ip cells were equally insensitive. Thus,
no strong correlation between cytotoxicity and constitutive Stat3
phosphorylation was observed. Note that the concentrations of
prodrugs in these experiments are much higher than those required
to completely inhibit the phosphorylation of Tyr705 of Stat3.
Additional cancer cell lines harboring constitutive Stat3 phos-
phorylation, melanoma cells MeWO and A375, and NSCLC cells
H1299, H1819, H520 H528, and A549, all showed <20%
inhibition at 5ꢀ10 μM 34 and 35 (data not shown), concentra-
tions that completely abrogate pStat3 levels.
affinity than the benzylcarbamoyl group for the isolated protein
(Table 1), the former resulted in much greater potency in intact
cells. The C-terminal ethyl benzyl ether of 35 likely produces off-
target cytotoxicity, since 36 exhibited the same degree of growth
inhibition but it was 20- to 25-fold less potent at inhibiting Stat3
phosphorylation. In addition, in intact cells, incorporation of the
glutamine mimic 4-aminopentamide into either of the Haic or
Nle-mPro scaffolds resulted in higher potency inhibition of Stat3
phosphorylation than 2-aminoethylurea and 2-aminoethylcarba-
mate, two surrogates that increased affinity for Stat3 protein.
Two POM esters are required for efficient inhibition of Stat3
phosphorylation. This is consistent with observations that nega-
tively charged compounds are not cell permeable.
Selectivity of inhibitors for SH2 domains in intact cells has not
received much attention presumably because there have not been
many reported cell-permeable antagonists of these domains. Our
prodrugs were selective for the SH2 domain of Stat3 in breast
tumor cells at 10 times the concentration that completely inhibited
Stat3 phosphorylation. The fact that the prodrugs do not inhibit
PI3K and Src function is not surprising, since the SH2 domains of
these proteins accommodate the hydrophobic amino acids Met
and Ile and their analogues at position pY þ 3, respectively.52,53
At this position, our inhibitors have hydrophilic glutamine mimics
that would not bind in the hydrophobic pockets of p85 and Src.
The 3ꢀ structures of the SH2 domains of Stat333 and Stat554 are
remarkably similar.34 However, their amino acid sequences are
dissimilar in the peptide binding regions which would account for
the difference in binding. It has been observed that the IL-6 response
includes weak and transient activation of Stat1 (reviewed by
Regis et al.64). Reciprocally, IFNγ promotes weak stimulation of
Stat3. Indeed Gerhartz et al. showed that Stat1 could be recruited
to pTyr-Xxx-Pro-Gln sequences on the IL-6 coreceptor, gp130,
centered on Tyr905 and Tyr915.55 Our peptidomimetics are
derived from the former binding site. The SH2 domains of Stat1
and Stat3 are highly similar both in sequence and in 3ꢀ structure.34
Therefore, cross-reactivity for these two proteins both by biolo-
gical stimulation and by our peptidomimetics is not surprising.
However, since these Stats are activated by different cytokines
and growth factors, it remains to be seen if the reduced inhibition
of Stat1 is significant. Although this is not an exhaustive survey of
SH2 domains, the results are very encouraging. Selectivity for
Stat3 over Stat1 and Stat5 cannot be achieved by inhibitors of the
JAK kinases. Thus, our compounds are the most selective inhibitors
of Stat3 phosphorylation reported to date.
To assess the effect of the phosphonate group on cytotoxicity,
compound 40, which retained diethyl protection on the phos-
phonate oxygens, was examined (Figure 6D). Trialkylphosphates
and dialkylphosphonates are known to be biologically stable,51
and indeed at 25 μM, the highest concentration examined, this
compound had no effect on Stat3 phosphorylation in MDA-MB-
468 cells (data not shown). Growth inhibition was not apparent
until well above 50 μM. These results suggest that the observed
cytotoxicities of 34 were not due to the β-methylcinnamate, Haic, or
4-aminopentamide moieties but rather to the phosphonate group.
The lack of cytotoxicity of our prodrugs and small molecule,
ATP-competitive JAK2 inhibitors,7,8 at concentrations that com-
pletely inhibit Tyr705 phosphorylation, runs counter to the hy-
pothesis that pStat3 is essential for tumor cell growth and that
inhibition of Stat3 results in apoptosis in cultured cells.5,56ꢀ58 It
is possible that knockdown of total Stat3 with antisense or siRNA
abrogates the recently reported mitochondrial function of this
protein10,11 and cotranscriptional activity of U-Stat3 which may
contribute to the apoptosis reported by these treatments. Our
results and those of Kreiss et al.7 and Hedvat et al.8 suggest that
survival assays of cancer cell lines grown in two-dimensional cell
cultures on plastic dishes are not effective models of the efficacy
of Stat3 inhibitors.
’ DISCUSSION
In this report we show that peptidomimetic phosphopeptide
prodrugs targeting the SH2 domain of Stat3 can potently inhibit
the phosphorylation of Stat3 in intact tumor cells. Compounds
34, 35, and 37 are some of the highest potency SH2 domain-
targeted compounds reported to date, as regards to inhibiting
their target. The β-methyl group on the cinnamide-based pTyr
mimic resulted in 2- to 3-fold increases in affinity and slight en-
hancement for inhibition of Stat3 phosphorylation in intact cells.
Two central dipeptide scaffolds, Haic and Nle-mPro, were evaluated
and found to behave identically in potency for Stat3 inhibition in
intact breast tumor cells. The C-terminus of the peptide was
quite important. Even though the methyl group resulted in lower
At concentrations greater than 25 μM, ∼50-fold that which
inhibited Stat3 phosphorylation, the prodrugs did indeed exhibit
cytoxicity. The nonhydrolyzable bis ethyl ester indicated that
growth inhibition was due to the phosphonate group. At 25 μM,
2 h treatment with 34 inhibited phosphorylation of FAK, Akt,
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and Stat5 in MDA-MB-468 cells, indicating off-target effects
(Figure S2). It is unknown whether other pathways are perturbed
by high concentrations of our phosphopeptide mimetics. It appears
that at the high concentrations of the bis-POM prodrugs that
result in cytotoxicity, the phosphonate group is reacting non-
selectively with other SH2 domains and possibly phosphotyr-
osine binding domains or the active sites of protein tyrosine
phosphatases which might impact survival, even of cells such as
MCF-7 which do not harbor constitutively phosphorylated Stat3.
At 5 μM, ∼10-fold the concentration at which pStat3 is in-
hibited, the prodrugs reported here did not reduce the expression
of the canonical downstream genes Bcl-xL in the MDA-MB-468
breast cancer line or cyclin D1 in this or the NSCLC line HCC-
827. Other transcription factors and pathways, for example, NF-
κB59 and PI3K/Akt,60,61 are involved in the expression of these
genes. We conclude that inhibition of Stat3 phosphorylation
alone is not sufficient to inhibit downstream gene expression and
that disruption of more than one transcripition factor may be re-
quired. At high concentration of prodrug, the observed off-target
inhibition of other pathways would make attributing reduction in
the expression of canonical downstream genes solely to inhibi-
tion of Stat3 Tyr705 phosphorylation tenuous.
coupled to Rink resin by addition of 3 equiv plus 3 equiv of DIEA in
8ꢀ10 mL of DMF/CH2Cl2 until ninhydrin tests were negative.28 For
Fmoc-Haic, Fmoc-cis-3,4-methanoproline, and phosphorylated cin-
namic acid derivatives, couplings were performed with 1.5ꢀ2 equiv of
each of acid, DIC, and HOBt in DMF/CH2Cl2 overnight or until
ninhydrin tests were negative. After coupling and deprotection steps,
resins were washed with 5 ꢁ 10 mL of DMF/CH2Cl2. On completion of
the peptide chain, resins were washed with CH2Cl2 (3 ꢁ 10 mL) and
were treated with TFA/TIS/H2O (95:2.5:2.5).63 (3 ꢁ 5 mL) for 15 min
each. The combined filtrates sat at room temperature for 1ꢀ2 h, and the
volumes were reduced in vacuo. Peptides were precipitated in ice cold
Et2O, collected by centrifugation, and washed 2ꢁ more with the same
solvent and centifiged. After drying, peptides were purified by reverse
phase HPLC on a Rainin Rabbit HPLC or a Varian Dynamax HPLC
instrument using a Phenomenex Luna C18(2) 10 μm, 2.1 cm ꢁ 25 cm
column. Gradients of MeCN in H2O or MeCN in 0.01 M NH4OAc
(pH 6.5) at 10ꢀ20 mL/min were employed. For phosphopeptides,
solvents contained 0.1% TFA. For prodrugs, no TFA was used in the
mobile phase. Peptides were tested for purity by reverse phase HPLC on
a Hewlett-Packard 1090 HPLC or an Agilent 1100 HPLC using a
Phenomenex Luna C18(2) 5 μm, 4.6 ꢁ 250 mm column. A gradient of
0ꢀ40% MeCN/30 min was used for posphopeptides and peptide
intermediates. For prodrugs the gradient was 10ꢀ80% MeCN/
30 min. Phosphopeptides and prodrug intermediates were dried in
vacuo over P2O5 at 37ꢀ for 24 h prior to use.27 All compounds were
>95% pure (HPLC) before evaluation. Purities, yields, and mass spectral
characteristics of phosphopeptides and prodrugs are provided in the
Supporting Information.
In summary, the availability of these highly potent and selective
inhibitors of Stat3 phosphorylation has allowed dissection of
pathways downstream of this key effector molecule from off-
target, cytotoxic responses. Evaluation and development of 34
and analogues as potential antitumor agents in tumor xenograft
and tumor microenvironment models are in progress and will be
reported under separate cover.
Synthesis of 4-Diethylphosphoryloxyacetophenone, 21.
To an ice cold stirred solution of 4-hydroxyacetophenone (2.0 g,
14.7 mmol) and TEA (4.1 mL, 29.4 mmol) in 30 mL of CH2Cl2 under
argon, diethyl chlorophosphate (2.5 mL, 17.6 mmol) was added dropwise.
The mixture was stirred overnight and was quenched by the addition of
30 mL of 5% aqueous HCl. The layers were separated, and the aqueous
phase was extracted with CH2Cl2 (2 ꢁ 30 mL). The combined organic
layers were washed with brine and dried (MgSO4). The solvent was
removed under vacuum, and the crude product was purified by silica gel
column chromatography, eluting with EtOAcꢀhexanes. Yield 3.6 g
(90%). 1H NMR (CDCl3, 300 MHz) δ1.31ꢀ1.38 (m, 6H), 2.58 (s, 3H),
4.17ꢀ4.28 (m, 4H), 7.31 (d, J = 8.7 Hz, 2H), 7.97 (d, J = 8.7 Hz, 2H).
13C NMR (CDCl3, 75.0 MHz) δ 16.0, 26.5, 64.8, 82.5, 119.9, 130.3,
133.9, 154.4, 196.6. HRMS (M þ H) calcd, 273.0892; found, 273.0965.
Synthesis of tert-Butyl 3-(4-Diethylphosphoryloxyphe-
nyl)butenoate, 22. n-BuLi in hexane (4.0 mL of 2.5 M, 9.5 mmol)
’ METHODS
NR-Protected amino acids were purchased from NovaBiochem, Che-
mImpex, or Anaspec. HOBt was from ChemImpex. Anhydrous DMF for
amino acid solutions was from Aldrich. Other solvents were reagent
grade and were used without further purification. NMR spectra were
obtained on either a Bruker DPX 300 MHz spectrometer or a Bruker
DRX 500 MHz spectrometer. Fmoc-Glu-NHBn was prepared as described
by Coleman et al.27 4-(Di-tert-butoxyphosphoryloxy)cinnamic acid was
synthesized as described in Mandal et al.29 (R)-4-(9-fluorenylmethoxy-
carbonlyamino)pentanoate, 4-nitrophenyl 2-(9-fluorenylmethoxycarbonly-
amino)ethyl carbamate, 4-nitrophenyl 2-(9-fluorenyloxycarbonlyamino)
ethylcarbonate, and (4R,5S)-4-(9-fluorenyloxycarbonlyamino)-5-benzyloxy-
hexanoate were prepared as described by Mandal et al.30 Racemic Fmoc-cis-
3,4-methanoproline was purchased from EMD Biosciences (Novabiochem).
Haic was synthesized as described in Mandal et al.29 Peptides were assayed for
affinity to Stat3 using fluorescence polarization as described by Coleman
et al.27 Stat3 was expressed and purified as described.62 For the synthesis of
phosphopeptides, Rink resin with a loading of 0.6 mmol/g was employed.
For thesynthesisofprodrugs, Rink resin with a loading of 1.2 mmol/g was
used. Resins were obtained from Advanced Chemtech, Inc. Antibodies
used in the Western blots are described in a table in the Supporting
Information.
General Procedure for the Synthesis of Phosphopeptides
and Peptidomimetics, 4ꢀ19. Solid phase syntheses were carried
out manually using commercially available Rink resin. Resin, 0.2 g, was
placed in a manual reactor and swollen and washed with 5 ꢁ 10 mL of
DMF/CH2Cl2. Fmoc groups were removed with 3 ꢁ 6 mL of 20%
piperidine/DMF for 5 min each. For coupling, 3-fold excesses of Fmoc-
amino acids, DIC, and HOBt were used in 8ꢀ10 mL of DMF/CH2Cl2 and
were allowed to proceed until resin samples tested negative with ninhydrin
tests. 4-Nitrophenyl 2-(9-fluorenylmethoxycarbonlyamino)ethylcarbamate
and 4-nitrophenyl 2-(9-fluorenyloxycarbonlyamino)ethylcarbonate were
t
was added carefully to dry BuOH (10 mL) via a syringe under argon
atmosphere. After 30 min, a solution of tert-butyl diethylphosphonoa-
t
cetate (2.00 g, 8 mmol) in 10 mL of dry BuOH was added at room
temperature and the solution was stirred for 1.0 h. A solution of 21 (2 g,
7.4 mmol) in 5 mL of tBuOH was added and the mixture stirred overnight
at room temperature. The reaction was quenched with 30 mL of saturated
NH4Cl (aq) and extracted with ether (4 ꢁ 40 mL). The combined
organic extracts were washed with water (2 ꢁ 20 mL), brine (1 ꢁ 30 mL)
and dried (MgSO4). The solvent was removed and the crude product
was purified by silica gel column chromatography, eluting with 20%
1
EtOAcꢀhexane (v/v), yielding 2.0 g (74%) of 22. H NMR (CDCl3,
300 MHz) δ1.32ꢀ1.37 (m, 6H), 1.51 (s, 9H), 2.50 (s, 3H), 4.16ꢀ4.27
(m, 4H), 6.0 (s, 1H), 7.2 (d, J = 8.7 Hz, 2H), 7.43 (d, J = 8.7 Hz, 2H). 13C
NMR (CDCl3, 75.0 MHz) δ 16.0,16.1, 17.6, 26.2, 28.3, 64.7, 64.83, 80.1,
115.3, 119.1, 119.8, 127.7, 130.7, 139.3, 151.0, 152.7, 161.8, 166.3, 196.9.
Anal. Calcd for C18H27O6P: C, 58.37; H, 7.35; O, 25.92; P, 8.36. Found:
C, 58.62; H, 7.33. HRMS (M þ H) calcd, 371.1624; found, 371.1558.
Synthesis of (2E)-3-(4-Diethylphosphoryloxyphenyl)but-
enoic Acid, 23. Compound 22 (1.0 g) was treated with 10 mL of TFA/
CH2Cl2 (95:5) for 1.0 h. The solvents were removed in vacuo, and
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residual TFA was removed by the addition and evaporation of toluene
(3 ꢁ 5 mL). Compound 23 was used without further purification. 1H
NMR (CDCl3, 300 MHz) δ 1.34ꢀ1.39 (m, 6H), 2.51 (s, 3H), 6.13
(s, 1H), 7.22 (d, J = 8.7 Hz, 2H), 7.47 (d, J = 8.7 Hz, 2H).
were removed in vacuo and stripped of residual TFA by addition and
evaporation of toluene (2 ꢁ 5 mL), and addition of ether resulted in an
off-white precipitate. The solid was collected by centrifugation, dried,
and purified by reverse phase HPLC to give 22 mg of 9. HRMS (M þ H)
calcd, 555.2220; found, 555.2186.
Synthesis of Pentachlorophenyl (E)-3-(4-Diethoxypho-
sphorylphenyl)but-2-enoate, 24. A solution of 23 (2.00 g, 6.4
mmol), pentachlorophenol (1.9 g, 7.0 mmol), DCC (1.6 g, 7.7 mmol),
and DMAP (0.08 g, 0.64 mmol) in 100 mL of ethyl acetate was stirred at
room temperature for 24 h. The mixture was filtered through Celite and
the solvent removed in vacuuo. The crude product was purified by silica
gel chromatography, eluting with 25% ethyl acetateꢀhexanes to give 2.6 g
(72%) of 24 as white solid. 1H NMR (CDCl3, 300 MHz) δ 1.26ꢀ1.32
(m, 6H), 2.54 (d, J = 1.2 Hz, 3H), 4.12ꢀ4.21 (m, 4H), 6.34 (d, J = 1.2 Hz,
1H), 7.2 (d, J = 8.7 Hz, 2H), 7.49 (d, J = 8.7 Hz, 2H). 13C NMR (CDCl3,
75.0 MHz) δ 16.0, 16.1, 18.4, 64.8, 64.8, 68.9, 113.4, 120.2, 120.3, 128.0,
128.1, 131.2, 131.9, 137.8, 141.9, 144.3, 152.1, 160.6, 161.9. HRMS (M þ
H) calcd, 560.9362; found, 560.7184.
Synthesis of Pentachlorophenyl (E)-3-(4-Phosphorylphe-
nyl)but-2-enoate, 25. Iodotrimethylsilane (2.0 mL, 14.2 mmol) in
5 mL of dry CH2Cl2 was added dropwise to a solution of 24 (2.0 g,
3.55 mmol) and bis(trimethylsilyl)trifluoroacetamide (1.8 mL, 7.1 mmol)
in 20 mL of dry CH2Cl2 at 0 ꢀC under argon. Stirring was continued for
1 h at 0 ꢀC and 1 h at room temperature. The solution was concentrated
in vacuo. The residue was treated with 20 mL of MeCN/H2O (9:1) and
5 drops of concentrated HCl for 30 min, and the solvents were removed
in vacuo. Toluene (5 mL) was added and evaporated twice. On addition
of Et2O solids separated, which were collected by filtration and washed
with the same solvent to give 1.6 g of 25 as a white powder (88%) which
was used without further purification. 1H NMR (DMSO-d6, 300 MHz)
δ 2.6 (s, 3H), 6.6 (s, 1H), 7.24 (d, J = 8.7 Hz, 2H), 7.75 (d, J = 8.7 Hz,
2H). HRMS (M þ H) calcd, 503.8658; found, 503.5797.
Synthesis of βMpCinn-Leu-Pro-AEU, 10. Rink resin (0.2 g,
0.15 mmol) was swollen in DMF/CH2Cl2 (1:1) and was washed with
2 ꢁ 10 mL of the same solvent. The Fmoc group was removed by
treatment with 20% piperidine in DMF for 5 min (repeated 3 times).
Coupling of p-nitrophenyl 2-(9-fluorenylmethoxycarbonyl)aminoethyl
urea30 was accomplished with 3-fold excesses of urea, HOBt, and DIPEA
in 10 mL of DMF/CH2Cl2 (1:1). For coupling of Fmoc-proline and
Fmoc-leucine, 3-fold excesses of Fmoc-amino acids, DIC, and HOBt
were used in 10 mL of DMF/CH2Cl2 (1:1). The final coupling was
carried out with 2-fold excess of pentachlorophenyl-3-methyl-40-phos-
phoryloxy cinnamate (23), triethylamine, and HOBt in 10 mL of DMF/
CH2Cl2. After all couplings, resins were washed with 3 ꢁ 10 mL of
DMF/CH2Cl2 followed by only CH2Cl2 (3 ꢁ 10 mL). Resins were
cleaved with three treatments of 10 mL of TFA/TIS/H2O (95:2.5:2.5)
for 10 min each, and combined TFA solutions were removed in vacuum.
The remainder TFA was stripped off by addition of toluene (2 ꢁ 5 mL),
and addition of ether resulted in an off-white precipitate. The solid was
collected by centrifugation, dried, and purified by reverse phase HPLC
to give 26 mg of 10. HRMS (M þ H) calcd, 555.2186; found, 554.2402.
Synthesis of tert-Butyl (E)-3-(4-Iodophenyl)but-2-enoate,
27. Asolutionoftert-butyl diethylphosphonoacetate (10.0 g, 39.6 mmol) in
30 mL of dry THF was added slowly to a freshly prepared solution of
19 mL of 2.5 M (hexanes) lithium tert-butoxide and tBuOH (30 mL) and
stirred for 1 h. A solution of 4-iodoacetophenone (26) (9.0g, 36.6mmol) in
20 mL of dry THF was added to the reaction mixture, and stirring was
continued overnight. The solvents were removed under vacuum. The
residue was dissolved in 400 mL of ether and was washed with water
(2 ꢁ 30 mL) followed by brine (30 mL) and dried over MgSO4. After
filtration and evaporation of the solvent, the crude product was then
purified by silica gel column chromatography, eluting with 1% EtOAc in
hexane. The product 27 was obtained as an oil (10.0 g, 79% yield). 1H
NMR (CDCl3, 300 MHz) δ 1.515 (s, 9H), 2.48 (s, 3H), 6.03 (s, 1H), 7.18
(d, 2H, J = 8.4 Hz), 7.68 (d, 2H, J =8.4 Hz). 13CNMR(CDCl3, 75.0 MHz)
δ 17.5, 28.3, 80.2, 94.6, 119.5, 128.0, 137.6, 141.9, 152.6, 166.1. Anal. Calcd
for C14H17IO2: C, 48.85; H, 4.98; I, 36.87; O, 9.3. Found: C, 49.40; H, 5.00,
I, 35.97. HRMS (M þ H) calcd, 345.0351; found, 345.0319.
Synthesis of βMpCinn-Leu-Pro-Apa, 8. Rink resin (0.2 g,
0.15 mmol) was swollen in DMF/CH2Cl2 (1:1) and was washed with
2 ꢁ 10 mL of the same solvent. The Fmoc group was removed by
treatment with 20% piperidine in DMF (3 ꢁ 5 min). Coupling of 4-(9-
fluorenylmethyloxycarbonylamino)pentanoic acid30 was accomplished
with 3-fold excesses of amino acid, PyBop, HOBt, and DIPEA in 10 mL
of DMF/CH2Cl2 (1:1). For proline and leucine, 3-fold excesses of
Fmoc-amino acids, DIC, and HOBt were used. The final coupling was
carried out with a 2-fold excess of pentachlorophenyl-3-methyl-4-phos-
phoryloxycinnamate (25), triethylamine, and HOBt in 10 mL of DMF/
CH2Cl2. After completion of the synthesis, the resin was washed with
3 ꢁ 10 mL of DMF/CH2Cl2 followed by CH2Cl2 (3 ꢁ 10 mL). The
resins was cleaved with three treatments of 10 mL of TFA/TIS/H2O
(95:2.5:2.5) for 10 min each, and the combined TFA solutions were
removed in vacuum. The remainder TFA was stripped off by addition of
toluene (2 ꢁ 5 mL), and addition of ether resulted in an off-white
precipitate. The solid was collected by centrifugation, dried, and purified
by reverse phase HPLC to give 34 mg of 8. ES-MS (M þ H) calcd,
567.26; found, 567.25.
Synthesis of βMpCinn-Leu-Pro-AEC, 9. Rink resin (0.2 g,
0.15 mmol) was swollen in DMF/CH2Cl2 (1:1) and was washed with
2 ꢁ 10 mL of the same solvent. The Fmoc group was removed by
treatment with 20% piperidine in DMF (3 ꢁ 5 min). Coupling of
p-nitrophenyl 2-(9-fluorenylmethoxycarbonylamino)ethylcarbonate30
was accomplished with 3-fold excesses of carbonate, HOBt, and DIPEA
in 10 mL of DMF/CH2Cl2 (1:1). For coupling of Fmoc-proline and
Fmoc-leucine, 3-fold excesses of Fmoc-amino acids, DIC, and HOBt
were used. The final coupling was carried out with 2-fold excess of
pentachlorophenyl-3-methyl-4-phosphoryloxycinnamate (25), triethy-
lamine, and HOBt in 10 mL of DMF/CH2Cl2. After all coupling, resins
were washed with 3 ꢁ 10 mL of DMF/CH2Cl2 followed by CH2Cl2
(3 ꢁ 10 mL). Resins were cleaved with three treatments of 10 mL of
TFA/TIS/H2O (95:2.5:2.5) for 10 min each. The combined filtrates
Synthesis of tert-Butyl (2E)-3-[4-[(Diethoxyphosphinyl)di-
fluoromethyl]phenyl]but-2-enoate, 28. To a solution of diethyl
bromodifluoromethylphosphonate (6.45 g, 24.1 mmol) in dry DMF
(100 mL), cadmium powder (5.41 g, 48.2 mmol) was added. The sus-
pension was stirred for 8 h under argon. Unreacted cadmium was re-
moved by filtration under argon, and the filtrate was treated with CuCl
(2.86 g, 28.9 mmol) and 27 (5.00 g, 15.1 mmol) at room temperature for
24 h. Et2O, 400 mL, was added, and the mixture was stirred for 5 min and
filtered. The organic layer was washed with saturated NH4Cl (2 ꢁ 40 mL)
and water (4 ꢁ 40 mL), dried (MgSO4), and evaporated to give an oily
residue. The crude product was purified by silica gel column chroma-
tography with 40% EtOAcꢀhexane (v/v) as the eluent to give 4.0 g
(68%) of 28 as a colorless oil. 1H NMR (CDCl3, 300 MHz) δ 1.3ꢀ1.35
(m, 6H), 1.52 (s, 9H), 2.54 (s, 3H), 4.13ꢀ4.28 (m, 4H), 6.07 (s, 1H)
7.53 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.4 Hz, 2H). 13C NMR (CDCl3, 75.0
MHz) δ 16.3, 16.4, 17.7, 28.3, 64.8, 64.9, 80.3, 120.4, 126.4, 145.0, 152.7,
166.0. 19F NMR (CDCl3, 282.0 MHz) δ ꢀ108.57 (d, J = 115.6 Hz, 2F). 31
P
NMR (CDCl3, 202.0 MHz) δ 6.15 (t, J = 115.1 Hz, 1P). Anal. Calcd for
C19H27F2O5P: C, 56.43; H, 6.73; F, 9.4; O, 19.78; P, 7.66. Found: C, 56.13;
H, 6.73; F, 9.15. HRMS (M þ H) calcd, 405.1642; found, 405.1698.
Synthesis of Pentachlorophenyl (2E)-3-[4-[(Diethoxy-
phosphinyl)difluoromethyl]phenyl]but-2-enoate, 29a.
A solution of 28 (4.00 g, 9.9 mmol) in 5 mL of dry CH2Cl2 was treated
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with 20 mL of TFA for 1 h at room temperature. The TFA was removed
in vacuo, and residual acid was removed by addition and evaporation of
toluene (2 ꢁ 10 mL). The crude cinnamic acid derivative (3.5 g, 9.8 mmol),
pentachlorophenol (2.8 g, 10.7 mmol), DCC (3.0 g, 14.7 mmol), and
DMAP (0.120 g, 0.98 mmol) in 100 mL of EtOAc was stirred at room
temperature for 24 h. The mixture was filtered through Celite and the
solvent removed in vacuuo. The crude product was purified by silica gel
chromatography, eluting with 25% EtOAcꢀhexanes to give 5.1 g (84%)
of 29a as a white solid. 1H NMR (CDCl3, 300 MHz) δ 1.34ꢀ1.4 (m,
6H), 2.67 (s, 3H), 4.18ꢀ4.32 (m, 4H), 6.5 (s, 1H), 7.65ꢀ7.72 (m, 4H).
13C NMR (CDCl3, 75.0 MHz) δ 16.3, 16.4, 18.5, 64.8, 64.9, 114.9,
126.56, 126.58, 126.65, 126.71, 126.74, 126.8, 127.93, 131.4, 132.0,
143.63, 144.24, 160.5, 161.73. 19F NMR (CDCl3, 282.0 MHz) δ ꢀ108.8
(d, J = 112.8 Hz, 2F). Anal. Calcd for C21H18Cl5F2O5P: C, 42.28; H,
3.04; Cl, 29.71; F, 6.37. Found: C, 42.48; H, 3.15; Cl, 29.45; F, 6.25.
HRMS (M þ H) calcd, 594.9381; found, 594.9357.
18.4, 64.8, 64.9, 116.3, 122.5, 125.2, 126.5, 126.6, 126.7, 126.8, 143.9,
145.2, 155.5, 159.2, 163.5. 19F NMR (CDCl3, 282.0 MHz) δ ꢀ108.8 (d,
J = 112.8 Hz, 2F). 31P NMR (CDCl3, 202.0 MHz) δ 5.94 (t, J = 113.1
Hz, 1P). Anal. Calcd for C21H22F2NO7P: C, 53.74; H, 4.72; F, 8.1; N,
2.98; O, 23.86; P, 6.6. Found: C, 53.84; H, 4.72; F, 8.12; N, 3.12.
Synthesis of 4-Nitrophenyl (2E)-3-[4-(Phosphoryldifluo-
romethyl)phenyl]but-2-enoate, 30b. Iodotrimethylsilane (2.5 mL,
17.0 mmol) in 10 mL of dry CH2Cl2 was added dropwise to a solution of
27b (2.0 g, 4.26 mmol) in 20 mL of dry CH2Cl2 at 0 ꢀC under argon.
Stirring was continued for 1 h at 0 ꢀC and 1 h at room temperature. The
solution was concentrated in vacuo. The residue was taken up in 20 mL
of MeCN/H2O/AcOH (8:1:1), stirred for 45 min, and concentrated in
vacuo. Toluene (5 mL) was added and evaporated twice. On addition of
Et2O solids separated, which were collected by filtration and washed
with the same solvent to give 1.5 g of 28b as a white powder (85%),
which was used without purification. 1H NMR (DMSO-d6, 300 MHz)
δ 2.6 (s, 3H), 6.53 (s, 3H), 7.53 (d, J = 10.2 Hz, 2H), 7.61 (d, J = 8.1 Hz,
2H), 7.80 (d, J = 8.1 Hz, 2H), 8.32 (d, J = 10.2 Hz, 2H). 19F NMR
(DMSO-d6, 282.0 MHz) δ ꢀ1108.4 (d, J = 104.3 Hz, 2F).
Synthesis of Pentachlorophenyl (2E)-3-[4-(Phosphoryldi-
fluoromethyl)phenyl]but-2-enoate, 30a. Iodotrimethylsilane
(2.0 mL, 13.4 mmol) in 5 mL of dry CH2Cl2 was added dropwise to a
solution of 29a (2.0 g, 3.35 mmol) and bis(trimethylsilyl)trifluoro-
acetamide (1.8 mL, 6.8 mmol) in 20 mL of dry CH2Cl2 at 0 ꢀC under
argon. Stirring was continued for 1 h at 0 ꢀC and 1 h at room tem-
perature. The solution was concentrated in vacuo. The residue was
taken up in 20 mL of MeCN/H2O/AcOH (8:1:1), stirred for 45 min,
and concentrated in vacuo. Toluene (5 mL) was added and evapo-
rated twice. On addition of ether solids separated, which were col-
lected by filtration and washed with the same solvent to give 1.6 g
of 30a as a white powder (89%). It was used directly in the next step
with no purification. HRMS (M þ H) calcd, 538.8755; found,
538.8773.
Synthesis of p-Nitrophenyl (2E)-3-[4-[[Bis[(2,2-dimethyl-
1-oxopropoxy)methoxy]phosphinyl]difluoromethyl]phen-
yl]but-2-enoate (31b). NaOH (174 mg, 4.3 mmol) in 2 mL of water
was added dropwise to a stirred suspension of 30b (1.0 g, 2.4 mmol) in
5 mL of water. When the mixture became clear (pH ∼9), AgNO3 (910 mg,
5.32 mmol) was added. After 2 h at 4 ꢀC the gray precipitate was
collected by filtration, dried, and pulverized in a mortar and pestle. The
powder was suspended in dry toluene (10 mL), and pivaloyloxymethyl
iodide (1.8 g, 7.2 mmol) was added. The mixture was stirred for 48 h at
room temperature. After filtration the solvent was removed in vacuo and
the crude product was purified by silica gel column chromatography,
eluting with 30% EtOAcꢀhexanes to give 31b (0.9 g, 58%) as an oil. 1H
NMR (CDCl3, 300 MHz) δ 1.24 (s, 18H), 2.66 (s, 3H), 5.66ꢀ5.8 (m,
4H), 6.38 (s, 1H), 7.36 (d, J = ꢀ7.72 (m, 4H). 19F NMR (CDCl3, 282.0
MHz) δ ꢀ108.8 (d, J = 112.8 Hz, 2F). Anal. Calcd for C29H34F2NO11P:
C, 54.29; H, 5.34; F, 5.92; N, 2.18; O, 27.43; P, 4.83. Found: C, 54.00; H,
5.47; F, 6.10; N, 2.20.
Synthesis of βMF2Pm(POM2)Cinn-Haic-Apa, 34. Method A.
Rink resin (0.3 g, 0.225 mmol) was swollen in DMF/CH2Cl2 (1:1) and
was washed with 2 ꢁ 5 mL of the same solvent. The Fmoc group was
removed by treatment with 20% piperidine in DMF for 3 min (repeated
3 times). For coupling of the next two amino acids, Fmoc-(R)-4-amino-
pentanoic acid and Fmoc-Haic-OH, 3-fold excesses of the Fmoc-amino
acids, PyBOP, and HOBt were used along with 6-fold excesses of DIPEA
in 4 mL of DMF/CH2Cl2. After assembly of the amino acid chain, the
Fmoc group was removed by treatment with 20% piperidine in DMF
and the resins were washed with 3 ꢁ 10 mL of DMF/CH2Cl2 (1:1).
Cleavage was accomplished with three treatments of the resins with
5 mL of TFA/TIS/H2O (95:2.5:2.5) for 10 min each. The solvents were
removed in vacuo, and residual acid was removed by addition and
evaporation of toluene (3 ꢁ 5 mL). Et2O was added, and the precipitate
was collected by centrifugation. The crude product was purified by reverse
phase HPLC using a gradient of MeCN in H2O. HRMS (M þ H) calcd,
345.1927; found, 345.1101. Pure H-Haic-NHCH(CH3)CH2CH2CONH2
(100 mg, 0.29 mmol), 31b (0.223 g, 0.29 mmol), dry and distilled
DIPEA (0.1 mL, 0.58 mmol), and HOBt (0.045 g, 0.29 mmol) in 4 mL
of dry N-methylpyrrolidone and CH2Cl2 (1:1) were mixed together and
stirred for 2 h. The reaction was monitored by HPLC. After completion,
the solvent was removed and the crude product was purified by reverse
phase HPLC using a gradient of MeCN in H2O to yield 27 mg of 34. 1H
NMR (acetonitrile-d3 500 MHz) δ 1.1 (d, J = 6.5 Hz, 3H), 1.2 (s, 18H),
1.6ꢀ1.73 (m, 2H), 2.03ꢀ2.26 (m, 8H), 2.52 (s, 3H), 3.0ꢀ3.17 (m,
2H), 3.2ꢀ3.26 (m, 1H), 3.38 (m, 1H), 3.8 (m, 1H), 4.5 (m, 1H), 5.03 (m,
1H), 5.46 (s, 1H), 5.62ꢀ5.7 (m, 4H), 6.2 (s, 1H), 6.36 (s, 1H), 6.74 (d,
J = 8.0 Hz, 1H), 7.00 (m, 1H), 7.09ꢀ7.11 (m, 2H), 7.32 (d, J = 6.5 Hz,
Synthesis of Pentachlorophenyl (2E)-3-[4-[[Bis[(2,2-dime-
thyl-1-oxopropoxy)methoxy]phosphinyl]difluoromethyl]-
phenyl]but-2-enoate, 31a. NaOH (144 mg, 3.6 mmol) in 2 mL of
H2O was added dropwise to a stirred suspension of 30a (1 g, 1.9 mmol)
in 5 mL of H2O. When the mixture became clear (pH ∼9), AgNO3 (807mg,
4.75 mmol) was added. After 2 h at 4 ꢀC the gray precipitate was col-
lected by filtration, dried, and pulverized in a mortar and pestle. The
powder was suspended in dry toluene (10 mL), and pivyloxymethyl iodide
(1.4 g, 5.7 mmol) was added. The mixture was stirred for 48 h at room
temperature. After filtration the solvent was removed in vacuo and the
crude product was purified by silica gel column chromatography, eluting
with 30% EtOAcꢀhexanes to give a colorless sticky liquid of 31a (0.9 g,
1
64%). H NMR (CDCl3, 500 MHz) δ 1.23 (s, 18H), 2.65 (s, 3H),
5.66ꢀ5.76 (m, 4H), 6.47 (s, IH), 7.66 (m, 4H). 13C NMR (CDCl3, 125
MHz) δ 18.5, 26.8, 38.8, 82.4, 82.5, 115.1, 126.6, 126.7, 126.8, 127.9,
131.4, 132.0, 144.0, 144.2, 160.3, 161.7, 176.5. 19F NMR (CDCl3, 282.0
MHz) δ ꢀ109.22 (d, J = 124.0 Hz, 2F). 31P NMR (CDCl3, 202.0 MHz)
δ 4.81 (t, J = 123.2 Hz, 1P). Anal. Calcd for C29H30Cl5F2O9P: C, 45.31;
H, 3.93; Cl, 23.06; F, 4.94. Found: C, 45.12; H, 3.93; Cl, 22.90; F, 5.08.
HRMS (M þ H) calcd, 767.0116; found, 767.0124.
Synthesis of 4-Nitrophenyl (2E)-3-[4-[(Diethoxyphosphin-
yl)difluoromethyl]phenyl]but-2-enoate, 29b. A solution of 28
(4.00 g, 9.9 mmol) in 5 mL of dry CH2Cl2 was treated with 20 mL of
trifluoroacetic acid for 1 h at room temperature. The TFA was removed
in vacuo, and residual acid was removed by addition and evaporation of
toluene (2 ꢁ 10 mL). The crude cinnamic acid derivative (3.5 g, 10.0 mmol),
p-nitrophenol (1.7 g, 12.0 mmol), and DCC (3.0 g, 14.7 mmol) in
100 mL of EtOAc were stirred at room temperature for 24 h. The
mixture was filtered through Celite and the solvent removed in vacuuo.
The crude product was purified by silica gel chromatography, eluting
with 25% EtOAc in hexanes to give 3.8 g (80%) of 29b as a white solid.
1H NMR (CDCl3, 300 MHz) δ 1.32ꢀ1.4 (m, 6H), 2.66 (s, 3H),
4.16ꢀ4.3 (m, 4H), 6.37 (s, 1H), 7.35 (d, J = 9.0 Hz, 4H), 7.6ꢀ7.7 (m,
4H), 8.3 (d, J = 9.0 Hz, 2H). 13C NMR (CDCl3, 75.0 MHz) δ16.3, 16.4,
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Journal of Medicinal Chemistry
ARTICLE
1H), 7.58 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H). 13C NMR
(acetonitrile-d3 125 MHz) δ 0.2, 0.3, 0.5, 0.6, 0.8, 16.4, 20.1, 26.0, 29.6,
31.1, 31.7, 31.9, 38.4, 44.9, 53.3, 61.45, 82.8, 121.5, 123.0, 124.2, 126.4,
126.6, 129.5, 133.2, 138.7, 145.6, 148.7, 165.5, 170.3, 170.4, 174.8, 176.4.
HRMS (M þ H) calcd 847.3495 found 847.3489.
SKOV3-ip cells were cultured in McCoy’s 5A medium at 3 ꢁ 105 cells/
well. Inhibition of pStat3 was assayed identically as in the case of MDA-
MB-468 cells. Hey ovarian tumor cells and MeWo and A375 melanoma
cells were cultured at a density of 3 ꢁ 105 cells/well in RPMI1640 media.
After overnight culture and media change, cells were treated with
increasing concentrations of 32 for 2 h. IL-6 (10 ng/mL) was added
at the last 30 min of incubations. Cells were lysed, and pStat3 and total
Stat3 were determined as above.
Effect of Prodrug on the Phosphorylation of Stat3, Stat5,
and Akt in Response to Epidermal Growth Factor Stimula-
tion. MDA-MB-468 cells were prepared, and prodrugs were added to
the culture media to give the correct final concentrations as above. After
1.5 h epidermal growth factor (EGF) was added at 100 ng/mL. After 30
min cells were collected and lysed and proteins were separated by PAGE
and transferred to three PVDF filters as above. The first filter was blocked
with 5% bovine serum albumin and probed for total and phosphoStat3 as
above. The second filter was probed for phosphoSer473Akt and total
Akt using appropriate antibodies and similar detection procedures to those
used for Stat3. The third filter was probed for phosphoTyr699 Stat5 and
total Stat5 using appropriate antibodies and similar detection procedures
to those used for Stat3.
Effect of Prodrugs on the Phosphorylation of Stat1. MDA-
MB-468 cells were prepared, and prodrugs were added to the culture
media to give the correct final concentrations as above. After 1.5 h
interferon γ was added at 25 ng/mL. After 30 min cells were collected
and lysed and proteins were separated by PAGE and transferred to
PVDF filters as above. Filters were probed for phosphoTyr701 Stat1 and
total Stat1 using appropriate antibodies and similar detection procedures
to those used for Stat3.
Effect of Prodrugs on the Phosphorylation of Focal Adhe-
sion Kinase. MDA-MB-468 cells were prepared, and prodrugs (10 mM/
DMSO) were added to the culture media to give the correct final
concentrations as above. After 2 h cells were lysed and total FAK and
pTyr861FAK was assayed by Western blots as described above.
Inhibition of Growth of MDA-MB-468 Breast Cancer Cells.
MDA-MB-468 cells were cultured in DMEM with 10% FBS. Cells were
plated into 96-well plates (1500 cells per well) in triplicate. The next day
the medium was changed. Immediately before use, prodrugs were dissolved
in ethanol to 10 mM. This stock solution was then diluted to the ap-
propriate concentrations for addition to the wells containing the MDA-
MB-468 cells. MTT assays were performed at 72 h. These assays were
run three times.
Method B. To a stirred solution of TFA H-Haic-NHCH(CH3)-
3
CH2CH2CONH2 (0.050 g, 0.11 mmol), N-methylmorpholine (0.036 mL,
0.33 mmol), and DMAP (0.005 g, 0.033 mmol) in 3 mL of dry NMP was
added a solution of 31a (0.085 g, 0.11 mmol) in 2 mL of dry MeCN
under inert atmosphere. The reaction was monitored by HPLC. After
completion, about 1 h, the reaction mixture was concentrated under
vacuum and then purified by reverse phase HPLC using MeCNꢀwater
system. Yield: 0.070 g (76%) of 34. HRMS (M þ H) calcd 847.3495,
found 847.3489.
Synthesis of Synthesis of βMF2PmCinn-Haic-Apa, 34-NP.
Rink resin (0.15 g, 0.18 mmol) was swollen in DMF/CH2Cl2 (1:1) and
was washed with 2 ꢁ 10 mL of the same solvent. The Fmoc group was
removed by treatment with 20% piperidine in DMF (3 ꢁ 5 min). For
coupling of 4-(9-fluorenylmethyloxycarbonylamino)pentanoic acid,30
3-fold excesses of amino acid, PyBop, HOBt, and DIPEA in 10 mL of
DMF/CH2Cl2 (1:1) were used. Coupling of Fmoc-Haic-OH was done
with 2-fold excesses of Fmoc-amino acid, DIC, and HOBt. The final
coupling was carried out with 2-fold excess of 30a, Et3N, and HOBt in
10 mL of DMF/CH2Cl2. After all couplings, resins were washed with
3 ꢁ 10 mL of DMF/CH2Cl2 followed by only CH2Cl2 (3 ꢁ 10 mL).
Resins were cleaved with TFA/TIS/H2O (95:2.5:2.5) (3 ꢁ 10 mL) for
10 min each, and combined TFA solutions were removed in vacuum.
The remainder TFA was stripped off by addition of toluene (2 ꢁ 5 mL),
and addition of ether resulted in an off-white precipitate. The solid was
collected by centrifugation, dried, and purified by reverse phase HPLC
to give 34 mg of the desired material. HRMS (M þ H) calcd, 619.2133;
found, 619.2139.
Synthesis of βMF2Pm(POM)Cinn-Haic-Apa, 34-MP. To a
stirred solution of TFA H-Haic-NHCH(CH3)CH2CH2CONH2 (0.050 g,
3
0.11 mmol), HOBt H2O (0.019 g, 0.12 mmol), and DIPEA (0.04 mL,
3
0.22 mmol) in 2 mL of DMF was added a solution of 31a (0.085 g,
0.11 mmol) in 2 mL of CH2Cl2 under inert atmosphere. The reaction
was monitored by HPLC. After completion, about 1 h, the reaction
mixture was concentrated under vacuum and triturated with hexaneꢀ
ether. The solid residue (0.089 g) was purified by reverse phase HPLC
using MeCNꢀwater system. Yield: 0.027 g (33%) of 34-MP. HRMS
(M þ H) calcd 733.2814, found 733.2814 and 0.016 g (22%) of 34.
HRMS (M þ H) calcd 847.3495, found 847.3489.
For daily treatment, cells were plated and treated with prodrugs as
above. At 24 and 48 h, prodrug was added to the same media. Total
EtOH concentration was less than 1% in all wells. Cell viability was
determined with the MTT assay.
Inhibition of Stat3 Tyrosine 705 Phosphorylation in
Tumor Cells. MDA-MB-468 breast tumor cells (4 ꢁ 105) were plated
in six-well culture dishes in DMEM media containing 10% FCS and were
allowed to grow overnight. Prodrugs were prepared as 10 mM stock
solutions in DMSO immediately before use, and aliquots were added to
the culture media to give the correct final concentrations. After 2 h the
cells were washed with ice cold phosphate buffered saline. Washed cells
were treated with lysis buffer (50 mM Hepes, pH 7.4, 150 mM NaCl,
1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyropho-
sphate, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM Na3VO4,
10 μg/mL leupeptin, and 10 μg/mL aprotinin). Cell-free detergent
extracts were centrifuged at 15 000 rpm in a microcentrifuge for 30 min
at 4 ꢀC and the protein concentrations of the supernatants determined.
Aliquots containing 12 μg of protein were separated on 8% SDSꢀPAGE
and were transferred to PVDF filters. The filters were blocked with 5%
bovine serum albumin and were probed with pStat3Y705 antibody followed
by secondary antibody, whose signal was detected with an enhanced
chemiluminescence kit (ECL, Amersham, Chicago, IL). Filters were
stripped with stripping buffer (62.5 mM Tris, pH 6.8, 2% SDS, and 0.1 M
2-mercaptoethanol) at 50 ꢀC for 30 min. Filters were then probed with
total Stat3 antibody and visualized with chemiluminescence as above.
Immunofluorescence Microcroscopy. MDA-MB-468 cells
were plated onto slides and treated with DMSO or 34 (5 μM). After
2 h, cells were fixed in 4% paraformaldehyde and permeabilized using
0.5% Triton X-100. Washed cells were blocked with 3% BSA and in-
cubated with antibodies against pTyr705Stat3. After the cells were
washed, they were incubated with secondary antibodies conjugated with
Alexa Fluor 594 (Molecular Probe, Invitrogen). Finally, slides were
mounted and examined using confocal microscopy (Olympus FluoView
500 or 1000, Olympus, Inc., Melville, NY). All images were obtained
with the same microscope settings.
’ ASSOCIATED CONTENT
S
Supporting Information. Tables of peptide characteriza-
b
tion, prodrug characterization, and antibodies used in Western
blots; figure of the crystal structure of iodophenylbut-2-enoyl-
leucine tert-butyl ester and NOE pattern of peptidomimetics 3-
NP and 34-NP; NME spectra of 3-NP and 34-NP; and figure of
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Journal of Medicinal Chemistry
ARTICLE
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’ AUTHOR INFORMATION
Corresponding Author
*For W.S.L.: phone, 713-834-6278; fax, 713-745-2107; e-mail,
wsliao@mdanderson.org. For J.S.M. phone, 713-745-3763; fax,
713-792-1204; e-mail, jmcmurra@mdanderson.org.
Present Addresses
Department of Pathology, University of Alabama at Birmingham,
West Pavilion P220, 619 South 19th Street, Birmingham, AL 35233.
^Department of Pathology, University of Oklahoma Health
Sciences Center, 975 N.E., 10th Street, Oklahoma City, OK 73104.
#Pfizer Global Research and Development, Eastern Point Road,
Groton, CT 06340.
’ ACKNOWLEDGMENT
We express our gratitude to Manish Shanker and Ailing
W. Scott for technical assistance. We acknowledge the National
Cancer Institute (Grant CA096652), the MDACC SPORE in
Ovarian Cancer (Grant P50 CA083639), and the CTT/TI-3D
Chemistry and Molecularly-Targeted Therapeutic Development
Grant Program for support of this work. We also acknowledge
the NCI Cancer Center Support Grant CA016672 for the
support of our NMR facility and the Translational Chemistry
Core Facility for mass spectrometry and X-ray crystallography.
Lung tumor and melanoma analyses were supported by the UT
SPORE in Lung Cancer (Grant P50 CA070907) and the
MDACC SPORE in Melanoma (Grant P50 CA093459).
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’ ABBREVIATIONS USED
Aec, 2-aminoethylcarbamate; Aeu, 2-aminoethylurea; Apa, (R)-
4-aminopentamide; DIEA, diisopropylethylamine; DIPCDI, dii-
sopropylcarbodiimide; EGF, epidermal growth factor; Fmoc,
9-fluorenylmethoxycarbonyl;Haic, 5-(amino)-1,2,4,5,6,7-hexahy-
dro-4-oxo-(2S,5S)-azepino[3,2,1-hi]indole-2-carboxylic acid; HOBt,
1-hydroxybenzotriazole; IL-6, interleukin 6; JAK, Janus kinase; mPro,
cis-3,4-methanoproline; MMP-2, matrix metalloproteinase-2;
pCinn, 4-phosphoryloxycinnamide;βMpCinn, β-methyl-4-phos-
phoryloxycinnamide or (2E)-3-(4-phosphoryloxyphenyl)-2-bu-
tenamide; βMF2PmCinn, (2E)-3-[4-[(phosphinyl)difluorome-
thyl]phenyl]-2-butenamide; PDGF, platelet derived growth factor;
POM, pivaloyloxymethyl; PyBOP, 1H-benzotriazol-1-yloxytri-
pyrrolidinophosphonium hexafluorophosphate; SAR, structureꢀ
activity relationship; SH2, Src homology 2; Stat3, signal trans-
ducer and activator of transcription 3; TES, triethylsilane; TIS,
triisopropylsilane; U-Stat3, unphosphorylated signal transducer
and activator of transcription 3; VEGF, vascular endothelial growth
factor
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