Synthesis of phosphatase-stable, cell-permeable peptidomimetic prodrugs that target the SH2 domain of Stat3

Article abstract of DOI:10.1021/ol9012662

The synthesis of prodrugs targeted to the SH2 domain of Stat3 is reported. Using a convergent strategy, the pivaloyloxymethyl phosphonodiester of pentachlorophenyl 4-phosphonodifluoromethylcinnamate, a phosphotyrosine surrogate, was synthesized and used t

Full text of DOI:10.1021/ol9012662

ORGANIC
LETTERS
2009
Vol. 11, No. 15
3394-3397
Synthesis of Phosphatase-Stable,
Cell-Permeable Peptidomimetic Prodrugs
That Target the SH2 Domain of Stat3
Pijus K. Mandal, Warren S.-L. Liao, and John S. McMurray*
Department of Experimental Therapeutics, The UniVersity of Texas M. D. Anderson
Cancer Center, 1515 Holcombe BouleVard, Houston, Texas 77030
jmcmurra@mdanderson.org
Received June 5, 2009
ABSTRACT
The synthesis of prodrugs targeted to the SH2 domain of Stat3 is reported. Using a convergent strategy, the pivaloyloxymethyl phosphonodiester
of pentachlorophenyl 4-phosphonodifluoromethylcinnamate, a phosphotyrosine surrogate, was synthesized and used to acylate peptidomimetic
fragments that were prepared on solid supports. Two prodrugs described here inhibited the phosphorylation of Stat3 in breast tumor cells.
Signal transducer and activator of transcription 3 (Stat3) is
a cytosolic transcription factor that has elevated, constitutive
activity in several cancer types.1,2 Via its SH2 domain, Stat3
is recruited to phosphotyrosine residues on activated growth
factor or cytokine receptors and is then phosphorylated on
tyrosine 705. Phosphorylated Stat3 (pStat3) dimerizes by
reciprocal interactions between phosphotyrosine 705 and SH2
domains, and the dimer is translocated to the nucleus where
it binds to promoters and initiates transcription of genes
involved in unchecked cell division, invasion, and angio-
genesis, characteristic of cancer.1,2 Inhibitors targeting the
SH2 domain would split dimers and would prevent receptor
docking, thereby preventing phosphorylation on Tyr705 and
subsequent gene expression.
SH2 domains recognize phosphotyrosine residues and are
integral components of intracellular signaling complexes. The
development of inhibitors targeting these domains is com-
plicated because the negatively charged phosphate impedes
crossing of cell membranes. In addition, phosphate esters of
tyrosine are susceptible to cleavage by phosphatases, thereby
(3) Garcia-Echeverria, C. Protein Tyrosine Kinases 2006, 31–52.
(4) Burke, T. R., Jr.; Yao, Z. J.; Liu, D. G.; Voigt, J.; Gao, Y.
Biopolymers 2001, 60, 32–44.
(1) Darnell, J. E., Jr. Nat. ReV. Cancer 2002, 2, 740–9
.
(2) Yu, H.; Jove, R. Nat. ReV. Cancer 2004, 4, 97–105
.
(5) Burke, T. R., Jr.; Lee, K. Acc. Chem. Res. 2003, 36, 426–33.
10.1021/ol9012662 CCC: $40.75
Published on Web 07/13/2009
 2009 American Chemical Society

Figure 1. Structure of Stat3 inhibitors and their corresponding phosphatase-stable, cell-permeable prodrugs.
destroying the ability of the inhibitor to bind to the SH2
domain.3 Phosphonates, difluoromethylphosphonates, mal-
onates, and heterocycles have been employed as phosphate
surrogates to overcome this deficit.4,5 Although a large
number of inhibitors of the SH2 domains of Grb2, Src, Lck,
p85PI3K, and others have been reported, relatively few have
demonstrated ability to cross cell membranes and inhibit their
targets.3 In several examples, passive diffusion into cells was
accomplished by capping phosphate or phosphonate oxygen
atoms with enzyme-cleavable groups. S-Acyl-2-thioethyl
(SATE),6,7 amino acid phosphoramidate,8,9 nitrofurfuryl
phosphoramidate,10 and pivaloyloxymethyl (POM)11-13 pro-
drug approaches have been reported.
values of 162 and 138 nM, respectively, in a fluorescence
polarization assay, as compared to 290 nM for the starting
peptide (pCinn ) 4-phosphoryloxycinnamate, Haic ) 5-amino-
1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-car-
boxylic acid). Key to increased affinity was replacement of
phosphotyrosine with pCinn. To stabilize against phosphatase
inactivation, the phosphate group was replaced with the
phosphonodifluoromethyl group16 (F2PmCinn). To impart
cell permeability, the POM prodrug method was employed.
This approach required the development of a POM phospho-
nodiester of F2PmCinn. We report here the synthesis of a
F2Pm(POM2)Cinn building block and its use in the prepara-
tion of cell-permeable prodrug analogues of PM-6 (BP-PM6)
and PM-66F (BP-PM279G) (Figure 1). We demonstrate that
they can indeed enter cells and inhibit the phosphorylation
of Stat3.
With the use of conformational constraints, we have
converted our lead peptide inhibitor of Stat3, Ac-pTyr-Leu-
Pro-Gln-Thr-Val-NH2, into high-affinity peptidomimet-
ics.14,15 Of these, pCinn-Haic-Gln-NHBn (PM-6) and pCinn-
Leu-Pro-Gln-NHBn (PM-66F) (Figure 1) exhibited IC50
Stankovic et al.11 synthesized POM prodrugs of phospho-
nodifluoromethylphenylalanine-containing inhibitors of the
Src SH2 domain. It was reported that only one POM group
(6) Liu, W. Q.; Vidal, M.; Mathe, C.; Perigaud, C.; Garbay, C. Bioorg.
Med. Chem. Lett. 2000, 10, 669–72
(7) Liu, W. Q.; Vidal, M.; Olszowy, C.; Million, E.; Lenoir, C.; Dhotel,
H.; Garbay, C. J. Med. Chem. 2004, 47, 1223–33
(8) Gay, B.; Suarez, S.; Caravatti, G.; Furet, P.; Meyer, T.; Schoepfer,
J. Int. J. Cancer 1999, 83, 235–241
(9) Gay, B.; Suarez, S.; Weber, C.; Rahuel, J.; Fabbro, D.; Furet, P.;
Caravatti, G.; Schoepfer, J. J. Biol. Chem. 1999, 274, 23311–23315
.
(12) McKinney, J.; Raimundo, B. C.; Cushing, T. D.; Yoshimura, H.;
Ohuchi, Y.; Hiratate, A.; Fukushima, H.; Xu, F.; Peto, C. Application: WO,
2001, p 85 pp.
.
.
(13) McKinney, J.; Raimundo, B. C.; Cushing, T. D.; Yoshimura, H.;
Ohuchi, Y.; Hiratate, A.; Fukushima, H. Application: US, 2002, p 31 pp.
(14) Mandal, P. K.; Heard, P. A.; Ren, Z.; Chen, X.; McMurray, J. S.
Bioorg. Med. Chem. Lett. 2007, 17, 654–656.
.
(10) Garrido-Hernandez, H.; Moon, K. D.; Geahlen, R. L.; Borch, R. F.
J. Med. Chem. 2006, 49, 3368–76.
(15) Mandal, P. K.; Limbrick, D.; Coleman, D. R.; Dyer, G. A.; Ren,
Z.; Birtwistle, J. S.; Xiong, C.; Chen, X.; Briggs, J. M.; McMurray, J. S.
J. Med. Chem. 2009, 52, 2429–42.
(11) Stankovic, C. J.; Surendran, N.; Lunney, E. A.; Plummer, M. S.;
Para, K. S.; Shahripour, A.; Fergus, J. H.; Marks, J. S.; Herrera, R.; Hubbell,
S. E.; Humblet, C.; Saltiel, A. R.; Stewart, B. H.; Sawyer, T. K. Bioorg.
(16) Burke, T. R., Jr.; Smyth, M. S.; Otaka, A.; Nomizu, M.; Roller,
P. P.; Wolf, G.; Case, R.; Shoelson, S. E. Biochemistry 1994, 33, 6490–4.
Med. Chem. Lett. 1997, 7, 1909–1914
.
Org. Lett., Vol. 11, No. 15, 2009
3395

could be installed on their peptidomimetics. For inhibitors
of the SH2 domains of Stat4 and Stat6, McKinney et al. also
employed F2Pm(POM2)Cinn.12,13 Their synthesis of this unit
employed Arbuzov phosphonylation of 4-iodobenzoate fol-
lowed by DAST treatment to install a diethyl phosphonodi-
fluoromethyl group and subsequent palladium-mediated cross
coupling to add the acryloyl group. After deprotection and
coupling to a peptide mimetic, POM groups were installed
using silver salts of the phosphonate and esterification with
pivaloyloxymethyl iodide (POM-I) in toluene. It is unclear
how successful this approach was as no yield data or
biological testing were reported for the final prodrug.
BP-PM6 was used as the target to develop synthesis
methodology. The first attempts involved esterification of
the phosphonate oxygens of PM6 incorporating F2PmCinn.
F2PmCinn was prepared as an active pentachlorophenyl
carboxyester (5) for use in solid-phase synthesis, as shown
inScheme1.Startingwithiodobenzaldehyde,Horner-Emmons
phate and phosphonate esters,18 we employed the pentachlo-
rophenyl ester to serve as both protection and activation of
the carboxyl group. The tert-butyl ester of 3 was deprotected
with TFA, and the carboxyl group was esterified with
pentachlorophenol and DCC to give intermediate 4. As
expected, the pentachlorophenyl carboxy ester moiety was
stable to TMSI, which was used to cleave the ethyl protecting
groups to yield 5.19
To synthesize the starting mimetic, F2PmCinn-Haic-
Gln(R)-NHBn, Fmoc-Glu-NHBn20 was coupled to Rink
amide resin via the side chain using diisopropylcarbodiimide
and 1-hydroxybenzotriazole (HOBt) (R ) Resin). After
removal of the Fmoc group, Fmoc-Haic-OH was coupled in
the same manner. Tyrosine mimic 5 was then coupled in
the presence of HOBt and diisopropylethylamine (DIEA).
Note that both Fmoc-Haic and 5 were coupled in 1.5-fold
excess, which often required overnight reaction.
The first attempt to prepare BP-PM6 involved POM ester
formation on the solid support. F2PmCinn-Haic-Gln(R)-
NHBn was treated with excess POM-I in the presence of
DIEA in DMF for 24 h. After cleavage from the resin, the
product was a mixture of the mono-POM ester (MP-PM6),
the unprotected phosphonate (NP-PM6), and none of the
POM diester. In a second approach, F2PmCinn-Haic-Gln-
NHBn was cleaved from the resin, purified by HPLC to
>98% purity, and subsequently treated with POM-I/DIEA
in DMF for 24 h at room temperature. The product of this
reaction was also a mixture of unprotected phosphonate and
mono-POM ester. The disilver salt of of F2PmCinn-Haic-
Gln-NHBn was nearly insoluble in toluene, the optimum
solvent for this reaction,21 precluding this method of POM
esterification.
Scheme 1
.
Synthesis of F2PmCinn-OPcp (5) and
F2Pm(POM2)Cinn-OPcp (6)
These unsatisfactory results led to the development of a
preformed POM diester of F2PmCinn-OPcp (6, Scheme 1)
that can be coupled to peptides or peptidomimetics in
solution. To install the POM groups, the phosphonate
oxygens of 5 were neutralized with 2 equiv of NaOH, and
the sodium salt was treated with AgNO3 to give an insoluble
silver salt. After thorough drying and pulverizing, the
phosphonate oxygens were alkylated with 3 equiv of piv-
aloyloxymethyl iodide in toluene (rt, 24 h) to give the
prodrug building block 6.
To synthesize BP-PM6, H-Haic-Gln-NHBn, 7, was syn-
thesized on Rink resin, cleaved with trifluoroacetic acid/
triisopropylsilane/H2O (TFA/TIS/H2O),22 and purified by
reversed-phase HPLC. Active ester 6 was coupled in 1.1-
fold excess to the peptide (Scheme 2). Initial experiments
using HOBt as a catalyst and DIEA as the base in DMF
resulted in 15-25% coupling yields, based on the amount
of starting peptide, after HPLC purification. Addition of a
solution of 1.1 equiv of 6 in CH2Cl2 to the peptide substrate
coupling with tert-butyl (diethylphosphono)acetate gave the
iodocinnamate 2. Copper-cadmium cross coupling of 2 with
diethyl bromodifluoromethylphosphonate17 provided the di-
ethyl phosphonate 3. Since phenolic esters are stable to
trimethylsilyl iodide (TMSI)-mediated dealkylation of phos-
(19) Gordeev, M. F.; Patel, D. V.; Barker, P. L.; Gordon, E. M.
Tetrahedron Lett. 1994, 35, 7585–7588.
(20) Coleman, D. R.t.; Ren, Z.; Mandal, P. K.; Cameron, A. G.; Dyer,
G. A.; Muranjan, S.; Campbell, M.; Chen, X.; McMurray, J. S. J. Med.
Chem. 2005, 48, 6661–70.
(17) Qabar, M. N.; Urban, J.; Kahn, M. Tetrahedron 1997, 53, 11171–
(21) Farquhar, D.; Khan, S.; Srivastva, D. N.; Saunders, P. P. J. Med.
Chem. 1994, 37, 3902–9.
11178.
(18) Blackburn, G. M.; Ingleson, D. J. Chem. Soc., Perkin Trans. 1 1980,
(22) Pearson, D. A.; Blanchette, M.; Baker, M. L.; Guindon, C. A.
Tetrahedron Lett. 1989, 30, 2739–2742.
1150–1153.
3396
Org. Lett., Vol. 11, No. 15, 2009

Scheme 2. Synthesis of Prodrugs
in N-methylpyrrolidone (NMP) in the presence of HOBt and
DIEA increased the yields to 50% (6 was sparingly soluble
in NMP). However, the replacement of HOBt with 0.5 equiv
of dimethylaminopyridine (DMAP) and DIEA with N-
methylmorpholine (NMM) increased the yield to 72% after
HPLC purification. Under these conditions, coupling was
complete in ca. 2 h, as judged by analytical HPLC. In these
experiments, BP-PM6 was purified to >98% purity by
reversed-phase HPLC using gradients of MeCN in H2O.
Note: addition of 0.1% TFA to the HPLC mobile phase
results in premature hydrolysis of one of the POM groups.
The versatility of the active ester approach was demon-
strated in the synthesis of BP-PM279G (Scheme 2). As
above, H-Leu-Pro-Gln-NHBn, 8, was synthesized by solid-
phase methods. In this case, 6 (in CH2Cl2) was added to the
peptide, DMAP, and NMM in MeCN. Based on starting
tripeptide, the yield of BP-PM279G was 75% after HPLC
purification.
BP-PM6 and BP-PM279G were added to cultured MDA-
MB-468 breast tumor cells that possess constitutively phos-
phorylated Stat3. Cells were treated with the inhibitors for
2 h and lysed, and pStat3 was detected by Western blotting.
As shown in Figure 2A, both prodrugs inhibited Stat3
phosphorylation in a dose-dependent manner. BP-PM6, the
less peptidic prodrug, is significantly more potent than BP-
PM279G, which has a higher proportion of natural amino
acids. These results suggest that the prodrugs enter the cells
where the POM groups are cleaved by carboxyesterases,
exposing the aryl difluoromethylphosphonate for binding to
the SH2 domain of Stat3. This breaks up preformed Stat3
dimers exposing the phosphotyrosine to cleavage by phos-
phatases and blocks the binding of Stat3 to receptors leading
to reduced phosphorylation. NP-PM6 and MP-PM6 do not
inhibit Stat3 phosphorylation at 25 µM (Figure 2B). There-
Figure 2. (A) Inhibition of Stat3 phosphorylation in MD-MBA-
468 breast tumor cells by prodrugs BP-PM6 and BP-PM279G. (B)
MP-PM6 (MP) and NP-PM6 (NP) do not inhibit Stat3 phospho-
rylation (BP ) BP-PM6).
fore, two POM groups are required for efficient cell
penetration and inhibitory activity.
In conclusion, we have developed a bis-POM phospho-
nodiester building block that can be used to synthesize
prodrug inhibitors of Stat3. Evaluation of BP-PM6 and BP-
PM279G indicates that the POM groups lead to cell
permeability and that the compounds bind to Stat3 and
prevent its phosphorylation. Full biological evaluation will
be reported under separate cover.
Acknowledgment. We are grateful to the National Cancer
Institute (CA096652) and the M. D. Anderson Cancer Center
University Cancer Fund for support of this work. We also
acknowledge the NCI Cancer Center Support Grant No.
CA016672 for the support of our NMR facility and Chiyi
Zhong of the Department of Experimental Diagnostic Imag-
ing for Mass Spectrometry.
Supporting Information Available: Experimental pro-
cedures for the syntheses in Schemes 1 and 2 and the
evaluation of prodrugs in breast tumor cells. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL9012662
Org. Lett., Vol. 11, No. 15, 2009
3397