Targeting the Src Homology 2 (SH2) Domain of Signal Transducer and Activator of Transcription 6 (STAT6) with Cell-Permeable, Phosphatase-Stable Phosphopeptide Mimics Potently Inhibits Tyr641 Phosphorylation and Transcriptional Activity

Article abstract of DOI:10.1021/acs.jmedchem.5b01321

Signal transducer and activator of transcription 6 (STAT6) transmits signals from cytokines IL-4 and IL-13 and is activated in allergic airway disease. We are developing phosphopeptide mimetics targeting the SH2 domain of STAT6 to block recruitment to phosphotyrosine residues on IL-4 or IL-13 receptors and subsequent Tyr641 phosphorylation to inhibit the expression of genes contributing to asthma. Structure-affinity relationship studies showed that phosphopeptides based on Tyr631 from IL-4Rα bind with weak affinity to STAT6, whereas replacing the pY+3 residue with simple aryl and alkyl amides resulted in affinities in the mid to low nM range. A set of phosphatase-stable, cell-permeable prodrug analogues inhibited cytokine-stimulated STAT6 phosphorylation in both Beas-2B human airway cells and primary mouse T-lymphocytes at concentrations as low as 100 nM. IL-13-stimulated expression of CCL26 (eotaxin-3) was inhibited in a dose-dependent manner, demonstrating that targeting the SH2 domain blocks both phosphorylation and transcriptional activity of STAT6.

Full text of DOI:10.1021/acs.jmedchem.5b01321

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Article
Targeting the Src homology 2 (SH2) domain of signal transducer
and activator of transcription 6 (STAT6) with cell-permeable,
phosphatase-stable phosphopeptide mimics potently
inhibits Tyr641 phosphorylation and transcriptional activity
Pijus K Mandal, Pietro Morlacchi, John Morgan Knight, Todd M Link, Gilbert R. Lee, Roza Nurieva,
Divyendu Singh, Ankur Dhanik, Lydia E. Kavraki, David B. Corry, John E. Ladbury, and John S. McMurray
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01321 • Publication Date (Web): 27 Oct 2015
Downloaded from http://pubs.acs.org on October 31, 2015
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Targeting the Src homology 2 (SH2) domain of signal transducer and activator of
transcription 6 (STAT6) with cellꢀpermeable, phosphataseꢀstable phosphopeptide mimics
potently inhibits Tyr641 phosphorylation and transcriptional activity.
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Pijus K. Mandal, Pietro Morlacchi, J. Morgan Knight, Todd M. Link, Gilbert R. Lee, IV,
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Roza Nurieva, Divyendu Singh, Ankur Dhanik, Lydia Kavraki , David B. Corry, John E.
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Ladbury, John S. McMurray *
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Department of Experimental Therapeutics, Department of Biochemistry and Molecular
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Biology, Center for Biomolecular Structure and Function, Department of Immunology, The
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University of Texas M. D. Anderson Cancer Center, Houston, Texas, The Department of
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Computer Science, Rice University, Houston, Texas, Departments of Medicine and Pathology
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Immunology, The Baylor College of Medicine, Houston, Texas
Current Addresses:
John E. Ladbury
School of Molecular and Cellular Biology
University of Leeds
LC Miall Building
Leeds, LS2 9JT
UK
J.E.Ladbury@leeds.ac.uk
Pietro Morlacchi
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Institute for Applied Cancer Science
The University of Texas M. D. Anderson Cancer Center
901 East Road.
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Houston, Tx 77054
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Todd M. Link
Department of Genomic Medicine
The University of Texas M. D. Anderson Cancer Center
1901 East Road.
Houston, Tx 77054
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Abstract
Signal transducer and activator of transcription 6 (STAT6) transmits signals from cytokines ILꢀ4
and ILꢀ13 and is activated in allergic airway disease. We are developing phosphopeptide
mimetics targeting the SH2 domain of STAT6 to block recruitment to phosphotyrosine residues
on ILꢀ4 or ILꢀ13 receptors and subsequent Tyr641 phosphorylation to inhibit the expression of
genes contributing to asthma. Structureꢀaffinity relationship studies showed that phosphopeptides
based on Tyr631 from ILꢀ4Rα bind with weak affinity to STAT6 whereas replacing the pY+3
residue with simple aryl and alkyl amides resulted in affinities in the mid to low nM range. A set
of phosphataseꢀstable, cellꢀpermeable prodrug analogs inhibited cytokineꢀstimulated STAT6
phosphorylation in both Beasꢀ2B human airway cells and primary mouse Tꢀlymphocytes at
concentrations as low as 100 nM. ILꢀ13ꢀstimulated expression of CCL26 (eotaxinꢀ3) was
inhibited in a doseꢀdependent manner, demonstrating that targeting the SH2 domain blocks both
phosphorylation and transcriptional activity of STAT6.
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Introduction
Signal transducer and activator of transcription 6 (STAT6) transmits signals from cytokines
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ꢀ3
interleukin 4 (ILꢀ4) and ILꢀ13. Under normal conditions, STAT6 is required for helper T cell
type 2 (Th2) development in the context of allergic inflammatory responses and is hypothesized
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to contribute to the polarization of macrophages to the M2 phenotype. Upon receptor binding,
both ILꢀ4 and ILꢀ13 induce phosphorylation of tyrosine residues located within the intracellular
region of ILꢀ4Rα (Figure 1). STAT6, via its SH2 domain, is recruited to these phosphotyrosine
residues and is itself phosphorylated on Tyr641 by associated Janus kinases 1 and 3 (JAK1 and
JAK3). Tyrosine phosphorylated STAT6 (pSTAT6) then dimerizes via reciprocal SH2 domainꢀ
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pTyr interactions, allowing it to translocate to the nucleus, where it participates in the
transcription of allergyꢀrelated genes. The ILꢀ4/ILꢀ13/JAK/STAT6 pathway plays a key role in
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asthma pathogenesis.
Patients have elevated levels of ILꢀ4 and ILꢀ13 in their airways and
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elevated pSTAT6 levels have been found in bronchial epithelium. Stat6 knockout mice do not
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develop airway hyperresponsiveness (AHR) or lung pathology associated with asthma.
Figure 1. Schematic representation of the ILꢀ4/JAK/STAT6 pathway. Note the ILꢀ13 signals in
an analogous manner using the ILꢀ13R1/ILꢀ4Rα coꢀreceptor.
Intervention in the ILꢀ4/ILꢀ13/JAK/STAT6 pathway at several points for the treatment of
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asthma is being actively pursued by the pharmaceutical industry and academic laboratories.
7ꢀ12
These include small molecules without described inhibitory mechanisms,
nonꢀSH2 domain
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directed dimerization inhibitors, JAK inhibitors,
siRNA, decoy oligonucleotides,
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7ꢀ19
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phosphopeptides targeting the SH2 domain of STAT6,
antibodies targeting ILꢀ13 and
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antibodies targeting ILꢀ4Rα.
Our approach is to target the SH2 domain of STAT6 with
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small molecule peptidomimetics to block recruitment to ILꢀ4Rα thereby preventing subsequent
phosphorylation of Tyr641 by JAKs, dimerization, translocation to the nucleus, and transcription
of genes leading to airway inflammation. Proof of principle that steric blockade of the SH2
domain of STAT6 prevents recruitment to ILꢀ4Rα, phosphorylation of Tyr641, and subsequent
transcriptional activity leading to asthma symptoms has been demonstrated using
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phosphopeptides derived from Tyr631 of ILꢀ4Rα, the docking site for STAT6, and Tyr641 of
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STAT6,
with cell penetration peptides attached.
To the best of our knowledge, no systematic structureꢀaffinity or structureꢀactivity studies
of phosphopeptide ligands of the SH2 domain of STAT6 have appeared in the literature. In this
communication we report truncation and amino acid substitutions on a phosphopeptide based on
the sequence surrounding Tyr631 of ILꢀ4Rα to gain an understanding of the determinants for
binding affinity that could be used in the design of STAT6 antagonists. In parallel, a structureꢀ
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affinity study of phosphate analogs of a phosphopeptide mimic reported in the patent literature
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was also conducted. A molecular docking study illustrated key intramolecular interactions that
help explain the higher affinity of the mimics compared to the receptorꢀderived peptide ligand.
BisꢀPOM prodrugs of phosphataseꢀstable phosphonodifluoromethyl analogs of a set of the new
inhibitors provided a structureꢀactivity relationship for inhibition of ILꢀ4 and ILꢀ13 stimulated
phosphorylation of Tyr641in intact human airway epithelial cells. Several inhibitors induced
significant inhibition at 100ꢀ500 nM in this cell line as well as in a human breast cancer cell line
and primary murine T lymphocytes. Expression of the downstream target, CCL26 (eotaxinꢀ3)
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was inhibited, which demonstrates that, in addition to inhibition of Tyr641 phosphorylation, this
class of compounds blocks transcriptional activity of STAT6.
Results
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Affinity of phosphopeptides for STAT6. To understand basic requirements for phosphopeptide
binding to the SH2 domain and to possibly find a lead for peptide mimetic development, we
screened a series of peptides based on the STAT6 docking site of ILꢀ4Rα, Tyr 631. As SH2
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domains typically recognize phosphotyrosine and two to four residues on its Cꢀterminal side,
we prepared a series of Nꢀterminal pTyr phosphopeptides. The fluorescence polarization assay
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of Wu et al. was used to monitor affinity. A construct consisting of an Nꢀterminal His tag, the
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coiledꢀcoil domain, the DNA binding domain, the linker domain, and the SH2 domain of STAT6
was cloned, expressed in E. coli, and purified by nickel IMAC chromatography for use in these
studies. Overall, the peptides displayed rather weak avidity to STAT6 with IC values ranging
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from 2 – 6 ꢀM (Table 1). Peptide length was important as affinities decreased from the
octapeptide 1, (IC = 2 ꢀM) to the tetrapeptide 5, (IC = 6 ꢀM). Substitution of alanine for
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lysine at position pY+1 resulted in a significant reduction in affinity, whilst other amino acids
such as tertiary leucine (Tle) and norleucine (Nle) were equipotent to lysine (7ꢀ9, Table 2). Thus
this position requires at the minimum β substitution and the εꢀamino group of lysine does not
provide interaction energy. A small screen of Nꢀterminal hydrophobic appendages showed no
significant effect on affinity (10ꢀ13, Table 2) and therefore further elaboration of this position
was not pursued. Replacement of pTyr with constrained surrogates 4ꢀphosphoryloxycinnamide
(14) and 7ꢀphosphoryloxyindoleꢀ2ꢀcaboxamide (15), which provided notable improvement in
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affinity for phosphopeptide inhibitors of STAT3 provided no significant increase in binding to
STAT6 in this series of peptides (Table 3). Interestingly, incorporation of 7ꢀ
phosphoryloxybenzofuranꢀ2ꢀcaboxamide (16) led to a substantial decrease in affinity. This
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suggests that the NH group of the indole may be close to a hydrogen bond acceptor such as a
carbonyl oxygen which would repel the oxygen atom of the benzofuran. In summary,
phosphopeptides based on Tyr631 of the ILꢀ4Rα displayed low affinity for STAT6 protein and
were not useful as leads for inhibitor development.
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In the late1990's researchers from Tularik, Inc. recognized the potential of inhibition of
STAT6 for the treatment of allergic airway disease and developed phosphopeptide mimics
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targeting the SH2 domain.
Structureꢀaffinity studies were reported, but only one compound
with the potential to inhibit pSTAT6 in intact cells was described in the patents (17, Figure 2).
Compound 17 is a phosphopeptide mimic in which phosphotyrosine is replaced by the
conformationally constrained cinnamate unit. Stability to phosphatases was designed by
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replacing phosphate with the phosphonodifluoromethyl group. The negatively charged
phosphonate oxygens were blocked with carboxyesteraseꢀlabile pivaloyloxymethyl (POM)
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groups as a means to permeate cells. Importantly, no biological evaluation was given, either in
the patents or in peerꢀreviewed literature; therefore it was not known whether this was a viable
approach for the inhibition of STAT6 phosphorylation. Using our convergent methodology for
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the synthesis of phosphopeptide mimic prodrugs targeting SH2 domains,
we improved the
synthesis of 17 and found that it indeed inhibited both ILꢀ4 and ILꢀ13ꢀstimulated
phosphorylation of STAT6 in Beasꢀ2B cells, an immortalized human bronchial epithelial cell
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line. Significant inhibition of pSTAT6 was observed at concentrations ≥1 ꢀM.
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Figure 2. Lead STAT6 inhibitor (from reference 33).
We embarked on new studies to test structural requirements of phosphateꢀcontaining
analogs of prodrug 17 for affinity for the SH2 domain of STAT6. Interestingly, compound 18a
(
IC = 0.04 ꢀM) was approximately 100ꢀfold more potent than the phosphopeptides based on
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the ILꢀ4Rα docking site (1ꢀ16), which exhibited IC values of 2ꢀ15 ꢀM. Given that 18a has the
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cinnamate group of 14 and the TleꢀPro group of 7, we conclude that placing the aryl groups
closer to the proline in 18a than that of Phe in the peptides is responsible for the increase in
affinity. For STAT3 inhibitors, we found that substitution of a methyl group on the β position of
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the phosphocinnamate pTyr surrogate enhanced affinity 2ꢀ3 fold (pCinn vs βMpCinn). In
keeping with these results, substitution of βMpCinn for pCinn produced a 2ꢀfold increase in
affinity 19a vs 18a (Table 4). 7ꢀPhosphorylꢀindole 2ꢀcarboxylate (20) reduced affinity compared
2
8
to 4ꢀphosphoryloxycinnamate, also consistent with our observations of inhibitors of STAT3.
The literature describing 17 emphasized Cꢀterminal bisꢀaryl and aryl,benzyl amides and
all of the compounds in the reported structureꢀaffinity relationship studies possessed these
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structural features.
To test the requirements of aryl groups, we prepared a series of analogs
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of 18a and 19a with varying Cꢀterminal amide substitution (Table 5). In the cinnamate series,
the diphenylamide (18b) has a 3ꢀfold higher IC than 18a indicating that the iodine contributed
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to affinity. Methylanilide 18c is 4ꢀfold lower in affinity than diphenylamide 18b suggesting that
aromatics are indeed important at the Cꢀterminus. Removal of the methyl group (18d) resulted
in further loss in affinity. Not surprisingly, in the βꢀmethylcinamate series, the same trend in
affinity loss was observed with reduced aryl and methyl substitution on the Cꢀterminal amide
nitrogen (19aꢀd), although the affinities in this series were higher than those of 18aꢀd. No gain
in affinity was achieved by adding one or two methylene unit spacers to anilide 19d, see 19e and
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19f. The cyclohexyl,methyl amide (19g) was twoꢀfold less avid than the phenyl,methyl amide,
19c. The dimethylamide, 19h, is significantly lower in affinity than the phenyl,methyl amide.
Affinity is improved with the larger diethylamide, 19i. These results show that while aryl groups
are important at the Cꢀterminus, reasonable affinity can be achieved with alkyl amides.
Structural requirements of proline were probed (Table 6). For these experiments Nꢀ
methylanilides were employed at the Cꢀterminus. Replacing proline with the acyclic, Nꢀ
methylated amino acid, sarcosine, 21, resulted in approximately 2ꢀfold lower affinity than the
prolineꢀcontaining analogue, 19c, suggesting that relative to sarcosine, the conformational
constraint of proline is important. Interestingly, alanine (22) was equipotent to proline whereas
Nꢀmethylation of the alanine (23) reduced affinity 4ꢀfold. This suggests that the TleꢀPro peptide
bond is likely in the trans conformation when bound to STAT6. Given that XaaꢀYaa peptide
bonds sample significant cis conformations when Yaa is an imino acid, such as Nꢀmethyl
alanine, the lower affinity of 23 relative to 22 may be due to the lower effective concentration of
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the trans conformer in the former.
Contributions of the atoms of the Cꢀterminal amide bond were studied by replacement
with conformationally constrained vinyl analogs (Table 7). Substituting the CONH group of 19d
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with a simple carbonꢀcarbon double bond (24) reduced affinity over 10ꢀfold. Extending the
aliphatic chain by two methylene groups (25) increased affinity to 0.5 ꢀM. Reduction of the
vinyl group of 25 to an alkane (26) resulted in a slight decrease in affinity. Reduction of the
carbonyl group of the methyl,phenyl amide of 19c resulted in a fourꢀfold loss in affinity (27).
Thus the carbonyl and nitrogen atoms of the Cꢀterminal amide contribute to affinity for the SH2
domain of STAT6.
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Computed structure of 18a bound to the SH2 domain of STAT6.
To date our attempts to crystallize STAT6 either alone or in the presence of a
phosphopeptide have not been successful. To elucidate potential interactions between 18a (the
phosphate analog of 17) and STAT6 we employed a molecular modeling strategy that combines
an AutoDockꢀbased incremental protocol of flexible proteinꢀligand docking (DINC) and
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molecular dynamics simulations.
structure of STAT1 (PDB ID 1YVL, chain A), was obtained from ModBase. Using DINC,
8a was docked to the SH2 domain of STAT6. The complex was submitted to molecular
A homology model of STAT6, based on the template
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dynamics simulation in explicit water. We obtained five clusters through kꢀmean clustering
(k=5). The sizes of the clusters were 173, 115, 101, 59, and 52 frames. Throughout the
simulation, RMSD variations of 18a were largely between 1 and 2.5 Å indicating that the peptide
mimetic in the complex was stable. In the computed models, the inhibitor interacts with the
surface of the protein and there do not appear to be deep pockets (Figures 3A and 3B).
Hydrogen bonds are illustrated in the interaction diagram of Figure 3C and the stereo rendering
in Figure 3D. The phosphate participates in ionic interactions with Lys544 and Arg562 and
hydrogen bonding interactions with the main chains of Asp565 and Ser566, which are analogous
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to phosphate interactions with STAT1
and STAT3 observed in xꢀray crystallographic
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structures and computational models of phosphopeptides bound to STAT3.
These hydrogen
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bonds were very stable and were present in greater than 90% of the trajectory frames throughout
the molecular dynamics simulation. The exception was the interaction between O2 of the
phosphate and one of the quanidino NH groups of Arg562 (75% of trajectory frames). The
2
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hydrogen bond between the NH of Tle and the main chain C=O of Asn588 is typical of
phosphopeptide interactions with SH2 domains and was present in 83% of the trajectory frames.
Also noted was a very stable hydrogen bond between the C=O of Tle and the main chain NH of
Gln590 (100% of trajectory frames). Interactions between the C=O of the pY+1 residue and the
NH of Glu638 (analogous to Gln590) were also seen in computed models of phosphopeptides
36, 41
bound to STAT3.
The aryl group of the cinnamate and the Cꢀterminal iodophenyl group
“sandwich” the side chain of Gln590, which extends away from the protein surface (Figures 3A,
3B, and 3D). Not surprisingly, there were several hydrophobic interactions with the SH2 domain
of STAT6, which are depicted as orange surfaces in Figure 3A and which are summarized in the
interaction diagram in Figure 3C. The tertꢀbutyl group of Tle interacts with the side chain
methylene groups of Glu587 and the side chain of His 575. The Cꢀterminal benzene rings
interact with a hydrophobic surface comprised of the side chains of Ile589, Phe592, Leu609,
Leu612, Ile606 and the side chain CH groups of Arg605. This hydrophobic surface does not
2
appear to be able to accommodate the side chain benzene ring of Phe at position pY+3 in the
peptides, which is extended from the proline by the α and β carbons. Thus the peptides exhibit
lower affinity than the mimetics.
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Figure 3. Computed structure of 18a in complex with a homology model of STAT6. A
representative complex from the largest cluster from the final 10 ns of a molecular dynamics
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simulation is shown (see Methods). A. STAT6 (comprised of the linker and SH2 domains) is
shown in surface representation with coloring according to amino acid hydrophobicity on Kyteꢀ
Doolittle scale B. STAT6 is shown in surface representation with coloring according to APBSꢀ
based electrostatic potentials. C. Intermolecular interaction diagram showing interactions
between 18a and amino acids from the SH2 domain of STAT6. Structures in A and B were
drawn using Chimera software, C was drawn using Maestro/Schrödinger software, and D was
rendered in PyMOL.
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Inhibition of Stat6 phosphorylation in intact cells with prodrugs derived from
phosphopeptide mimetics. A set of analogs of 17 (Chart 1) was synthesized to systematically
evaluate the effect of structural modifications on the ability to inhibit STAT6
phosphosphorylation in intact cells (Figure 4). Beasꢀ2B immortalized human airway epithelial
cells were incubated with prodrugs for 2 h and stimulated with ILꢀ4 for 1 h. Levels of pSTAT6
and total STAT6 proteins were evaluated by western blotting of the cell lysates. The first series
was a set of analogs of phosphates 18aꢀd. Starting with 17, possessing a phenyl, 4ꢀiodophenyl
amide, we removed the iodine (28), and then replaced one of the phenyl rings with a methyl
group (29) then a hydrogen (30). As depicted in Figure 4A, removing the iodine from 1 resulted
in a dramatic increase in potency (28). Significant inhibition was observed at 100 nM and
complete inhibition occurred at 500 nM. Substitution of one of the phenyl groups with a methyl
group (29) produced a further increase in potency with significant inhibition at 50 nM and nearly
complete inhibition at 100 nM. Replacing the methyl group with a hydrogen (30), resulted in
approximately 25ꢀfold less potency. Cellular viability (MTS assay, 72 h exposure, Figure 4C)
did not parallel potency for STAT6 inhibition. The trend NPh,PhI < NPh < NMePh < NHPh
2
indicates that smaller amides result in greater cytotoxicity. At concentrations ≤1 ꢀM, diphenyl
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amide 28 and methylanilide 29 did not affect viability although pSTAT6 was completely
inhibited. Thus for these compounds, the reason for the observed cytotoxicity is not STAT6
phosphorylation inhibition. In contrast, for 30, the fraction of viable cells paralleled pSTAT6
inhibition. These results suggest that the Cꢀterminal anilide is engaged in an as yet undefined
offꢀtarget interaction that produces a cytotoxic effect.
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Figure 4. Effect of structure on inhibition of pSTAT6 and cytotoxicity. Beaseꢀ2B immortalized
human epithelial airway cells were treated with the prodrugs for two h and were then stimulate
with ILꢀ4 for 1 h. Cells were lysed and total and phosphorylated STAT6 were estimated by
western blots. For each pair of gels the top is pSTAT6 and the bottom is total STAT6. A.
Prodrugs with cinnamoyl pTyr mimcs. B. Prodrugs with βꢀmethyl pTyr mimics. C. Graphic
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representation the integrals of the bands in A and B. D. Effect of prodrugs on viability of Beasꢀ
2B cells. Cells were treated with the prodrugs for 72 hr and viability was measured with MTT
assays.
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A second set of prodrugs was prepared to test the effect of βꢀmethyl substitution on the
cinnamate on cellular activity, as this substitution produced a small but significant increase in
affinity of the phosphate analogs. Figure 4B shows that 31, the analog of 29, retains potency and
the βꢀmethyl group has no noticeable effect on inhibition of pSTAT6. In 32 a cyclohexyl,methyl
amide group replaced the phenyl,methyl amide of 31. As can be seen this substitution also
resulted in high potency. Cell viabilities of 31 and 32 were nearly identical to that of 29. Thus
the βꢀmethyl group has no effect on cytotoxicity in this cell line.
Interestingly, cellular potency did not parallel affinity for STAT6 of the corresponding
phosphates, 18aꢀd, and 19c, and 19g. Whereas affinity of the phosphate series decreased with
decreasing aromatic character or decreasing methyl substitution, this was not the defining feature
for the prodrug potency. Similar observations were made in the case of our prodrugs targeted to
the SH2 domain of STAT3. For example, changing the Cꢀterminal CONHBn to a simple methyl
group in one series reduced affinity by a factor of two but increased cellular potency of the
3
1
prodrug by 50ꢀfold. In another series, substitution on the ring of a proline had no effect on
42
affinity but increased cellular potencies from 20ꢀ100×. Thus, for this class of phosphonate
prodrugs, biochemical affinity of the ligand for the target SH2 domain is not linearly predictive
of cellular activity.
Other cell lines and other SH2 domains.
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The panel of prodrugs was tested for pSTAT6 inhibition in the triple negative breast cancer cell
line, MDAꢀMBꢀ468. After serum starvation, cells were treated with prodrugs followed by ILꢀ4
stimulation which resulted in pSTAT6 inhibition. Overall the pattern of inhibition paralleled that
of the Beasꢀ2B epithelial airway cells (Supplemental Figure 1). Treatment of serum starved
MDAꢀMBꢀ468 cells with epidermal growth factor (EGF) activates the PI3K pathway leading to
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AKT phosphorylation, a process that is initiated by binding of the SH2 domain of p85, the
4
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regulatory subunit of PI3K, to pTyr residues on the EGF receptor. There was no clear trend for
inhibition of pAkt as a function of structure. Whereas most compounds had no effect, 29 and 31
exhibited mild inhibition at the relatively high concentration of 5 ꢀM. The exception was anilide
30, which potently inhibited pAkt at concentrations as low as 100 nM. MTS assays showed that
for the panel of prodrugs, IC values for this cell line ranged from 5 – 10 ꢀM. It is unclear if the
5
0
decrease in pAkt is due to cross reactivity with the SH2 domains of p85 or due to other nonꢀ
related offꢀtarget effects that might affect this pathway.
The panel of prodrugs was assayed for the ability to inhibit the phosphorylation of
+
STAT1, STAT3, STAT5 and STAT6 in primary mouse CD4 T lymphocytes (Supplementary
Figure 2). Similarly to Beasꢀ2B cells, inhibition of ILꢀ4 stimulated pSTAT6 with 17 was
relatively weak and the others stronger. We further found robust inhibition of ILꢀ2 mediated
STAT5 phosphorylation after treatment with 29, 28, 31 and 32. This is not surprising given the
high homology in the SH2 domains of these two proteins. The hydrophobic surface mentioned
above for STAT6 (Ile589, Phe592, Are605, Ile606, Leu609, and Leu612) is highly homologous
in STAT5 (L643, Phe646, Are661, Leu662, Leu665, and Leu668). The side chain CH groups
2
of Lys644 in STAT5 can also contribute to the hydrophobic binding site in an analogous manner
to that of Gln590 in STAT6. Interestingly, anilide 30 showed selectivity for STAT6 as it was not
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an inhibitor of pSTAT5. None of the prodrugs affected the phosphorylation status of STAT1 or
STAT3.
Inhibition of the expression of CCL26 (eotaxinꢀ3)
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Starting prodrug 17 and analog 31 were tested for their ability to inhibit the expression of the
downstream gene, CCL26 (eotaxinꢀ3), a chemokine involved in the recruitment of eosinophils to
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sites of inflammation.
Beasꢀ2B cells were treated with the inhibitors and after 24 h of
exposure to ILꢀ13, expression of CCL26 mRNA was assessed by quantitative PCR. As can be
seen in Figure 5, addition of ILꢀ13 led to robust expression of CCL26 mRNA (DMSO+). The
expression of CCL26 mRNA was inhibited in a dose dependent manner by both prodrugs. In
both cases, >50% inhibition was achieved at 100 nM. The greater potency of 31 reflects its
greater ability to inhibit STAT6 phosphorylation than 17. Thus steric block of the STAT6 SH2
domain with these prodrugs not only inhibits activation (phosphorylation of Tyr641), it inhibits
subsequent transcriptional activity.
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Figure 5. Prodrugs targeting the SH2 domain of STAT6 inhibit the expression of CCL26
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mRNA. Beas2B cells plated at 4×10 cells/well and incubated overnight in complete media,
serum starved for 2hr, preꢀtreated with drug for 2hr, stimulated with ILꢀ13 overnight in serum
free media, and total CCL26 mRNA was quantitated on cell lysates.
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Chemistry
Phosphopeptide Synthesis. Phosphopeptides in Tables 1 ꢀ 3 were synthesized by manual solid
phase techniques using the Fmoc protection scheme. Cꢀtermini were prepared as amides by the
employment of Rink amide resin. Nꢀtermini were capped with acetyl groups unless otherwise
4
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noted. Phosphotyrosine was introduced as the phosphoroꢀbisꢀdimethylamide.
For the 5ꢀ
phosphoryloxyindole (15) and 5ꢀphosphoryloxybenzofurnanꢀ2ꢀcarboxylates (16), the
corresponding 5ꢀhydroxyꢀ2ꢀcarboxylates were coupled to resinꢀbound peptides and were
phosphorylated using global phosphorylation with N,Nꢀdiisopropylꢀdiꢀtertꢀbutylphosphoramidite
1
in the presence of Hꢀtetrazole. Peptides terminating in 4ꢀphosphoryloxycinnamide and 4ꢀ
phosphoryloxyꢀβꢀmethylcinnamide were capped with the corresponding phosphorylated
2
8
cinnamic acidꢀpentachlorophenyl esters as described previously. Peptides were cleaved from
50
the resin using TFA:TIS:H O. Peptides containing phosphoroꢀbisꢀdimethylamides were left
2
4
7ꢀ49
overnight to deꢀamidate the phosphate group.
All phosphopeptides were purified by reverse
phase HPLC and were the correct molecular weight as measured by electrospray mass
spectrometry.
Synthesis of phosphate analogs of 17. We used convergent synthesis methodology to prepare
phosphates for analysis of affinity for STAT6 protein in which modified dipeptides were
synthesized and coupled to preꢀformed constrained phosphotyrosine surrogates. For inhibitors
18ꢀ20 possessing constrained cinnamate and indoleꢀbased pTyr mimics (Table 4), diphenyl and
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phenyl,4ꢀiodophenyl amines were not amenable to simple coupling to Bocꢀproline (33). In these
cases the first aryl group was installed as the anilide or 4ꢀiodophenylamide of BocꢀProꢀOH using
water soluble EDC as the coupling agent (34a, b) (Scheme 1). The second phenyl group was
added by treatment with triphenylbismuth in the presence of cupric acetate and triethyl amine
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(35a, b).
The Boc group was removed with TFA and the resulting amine was coupled to
Bocꢀtertiaryꢀleucine with HBTU (36a and 36b). Again the Boc group was removed to give
dipeptide amine salts 37a and 37b. Intermediate 37a was capped with pentachlorophenyl 4ꢀ
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phosphoryloxycinnamate (38a) and pentachlorophenyl βꢀmethylꢀ4ꢀphosphoryloxycinnamate
31
(
38b) to give inhibitors 18a and 19a (Table 4), respectively. Amine 37b was acylated with 38a
and 38b to give 18b and 19b, respectively (Table 5). The alternative pTyr surrogate, 5ꢀ
phosphoryloxyindoleꢀ2ꢀcarboxylate was introduced by coupling 37a with 5ꢀdiꢀtertiaryꢀ
butoxyphosphorylꢀindolineꢀ2ꢀcarboxylic acid (Scheme 2) (39) in presence of HBTU and DIPEA.
Treatment with TFA for an hour and purification by HPLC gave 20 (Table 4).
As shown in Scheme 2, 5ꢀdiꢀtertiaryꢀbutoxyphosphorylꢀindolineꢀ2ꢀcarboxylic acid (39)
was prepared by protection of the carboxyl group of 5ꢀhydroxyindoleꢀ2ꢀcarboxylate (40) as a
tertꢀbutyl dimethylsilyl (TBDMS) ester (41) followed by phosphitylation of the hydroxyl group
with N,Nꢀdiethyl diꢀtertꢀbutyl phosphoramidite and subsequent oxidation to the phosphate ester
with tertꢀbutyl hydrogen peroxide (42). Aqueous hydrolysis of the TBDMS ester gave the free
carboxylate, 39 ready for capping 37a (Scheme 1).
Inhibitors in which the Cꢀterminal amide was explored (18cꢀd, 19cꢀi, Table 5) were
prepared by initial routine coupling of amines to Bocꢀproline using EDC (43cꢀi, Scheme 3). The
Boc group was removed with TFA and Bocꢀtertiaryꢀleucine was coupled with HBTU (44cꢀi).
Again the Boc group was removed and the dipeptides were capped with 38a or 38b to give
inhibitors 18cꢀd and 19cꢀi.
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Inhibitors 21 – 23 (Table 6), in which proline was replaced with nonꢀconstrained
sarcosine, alanine, and Nꢀmethylalanine, respectively, were prepared by simple coupling of Nꢀ
methylaniline to the corresponding Bocꢀamino acid and elaborating as in Scheme 3.
Inhibitors 24ꢀ27 (Table 7) were synthesized to establish the importance of the Cꢀterminal
amide bond. As shown in Scheme 4, (S)ꢀ2ꢀstyrylꢀNꢀBocꢀpyrrolidine (46) and (S)ꢀ2ꢀ(3ꢀphenylꢀ
propenyl)ꢀNꢀBocꢀpyrrolidine (48) were synthesized through a Wittig olefination of BocꢀProlinal
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(
45) and the corresponding triphenylphosphonium bromide derivative. Fully reduced (S)ꢀ2ꢀ(4ꢀ
phenylbutyl)ꢀNꢀBocꢀpyrrolidine (50) was obtained by hydrogenation of 48 in presence of
0%PdꢀC. An analog with a reduced amide bond was prepared by consecutive reductiveꢀ
1
amination of Bocꢀprolinal with aniline (52) and then with formaldehyde (40% solution) (53).
Intermediates 46, 48, 50 and 53 were elaborated to dipeptide amines 47, 49, 51 and 54 by TFAꢀ
mediated acidolysis of the Boc protecting group, HBTUꢀmediated coupling with BocꢀTleꢀOH,
and then Boc cleavage with TFA as in Scheme 3. Treatment of the amine salts with 39b gave
inhibitors 24ꢀ27.
Cell permeable prodrugs (Chart 1) were synthesized (Scheme 5) by coupling dipeptide
amines to pentachlorophenyl active esters of bisꢀPOM protected
3
1, 32
phosphonodifloromethylcinnamates 55a or b, that were synthesized as described previously.
Couplings were carried out in NMP in the presence of Nꢀmethylmorpholine with DMAP
catalysis. Products were purified by reverse phase HPLC with gradients of MeCN in H O with
2
no additives in the mobile phase.
Conclusions
The sequence surrounding Tyr631, the docking site of ILꢀ4Rα, binds weakly to STAT6. This
contrasts with STAT3 where a peptide derived from the docking site on gp130 (Tyr904) had low
5
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23, 24
nM affinities.
In the modified dipeptides reported by McKinney et al.
the aryl groups at
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pY+3 were closer to the main chain than the phenyl group of phenylalanine in the
phosphopeptide series. Whereas Cꢀterminal aryl amides produced the highest affinity, low nM
binding constants could also be obtained with Nꢀmethyl anilides and diꢀalkyl amides. The
smaller hydrophobic groups of the latter likely do not penetrate as deeply into the proposed
hydrophobic pocket as do benzene rings. Cellular potency of the bisꢀPOM prodrugs was
dependent on the substituents of the Cꢀterminal amide but did not parallel affinity for STAT6 by
the corresponding phosphates. Cellular activity of a peptidomimetic or any other drug is a
function of many factors that may include 1, the intrinsic affinity of the drug for its target (e.g.
enzyme, transcription factor, miRNA); 2, effectiveness of cell penetration; 3, efflux from the
cell; 4, metabolic stability; 5, partitioning into membranes versus the cytosol, and other possible
interactions. It is not clear which of these factors contributes to the deviation in cellular potency
of the prodrugs from affinity for STAT6 of the corresponding phosphates. Inhibition of cell
viability was inversely proportional to the size of the Cꢀterminal amide with the smaller
methylanilide and anilide analogs being most inhibitory. These apparent offꢀtarget effects can
potentially induce more than one type of biological response in vivo and it will be necessary to
carefully monitor toxicity in animal studies. The prodrugs were quite potent in inhibiting
expression of CCL26, suggesting that this class of compounds will have utility in modulating
immune responses. In the case of asthma, cytotoxicity is detrimental so care will have to be
exercised to achieve the therapeutic window. The inhibitors were selective for STAT6 over
STAT1 and STAT3. The basis of the selectivity can be explained as follows. At the base of the
hydrophobic pocket that accommodates the Cꢀterminal aromatic groups in STAT6 is Phe592. In
the sequences of STAT1, STAT2, STAT3, and STAT4, the corresponding residue is a tyrosine.
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39
In the crystal structure of a phosphopeptide bound to STAT1, and in models calculated for
28, 36, 41
phosphopeptideꢀSTAT3 complexes,
the side chain hydroxide of this tyrosine contacts a
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5
5
5
5
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6
main chain peptide bond in a hydrogen bond which appears to be part of the recognition
determinant for these SH2 domains. Based on these models, this hydroxyl group, and associated
waters of hydration, in STAT1 and STAT3 would repel the Cꢀterminal amide aromatic groups of
the inhibitors, thus leading to selectivity for STAT6 (and STAT5). The observed cross reactivity
to STAT5 in asthma is not likely to be a problem in clinical settings. As STAT5 was reported to
0
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9
0
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5
be required for the asthma phenotype in a STAT6ꢀindependent manner, cross reactivity to
STAT5 may actually contribute to in vivo efficacy. Evaluation of prodrugs in a fungalꢀinduced
murine model of asthma is in progress and will be reported under separate cover.
Methods
a
N ꢀprotected amino acids were purchased from, ChemImpex, or NovaBiochem. HOBt was
from ChemImpex. For the synthesis of phosphopeptides, Rink resin (Advanced Chemtech, Inc.)
with a loading of 0.6 mmol/gm was employed. Anhydrous DMF for amino acid solutions, and
aniline derivatives were 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.
General Procedure for the synthesis of phosphopeptides. Solid phase syntheses were carried
out manually using commercially available Rink resin. Resin, 0.2 gm, was placed in a manual
reactor and swollen and washed with 5 × 10 mL of DMF/CH Cl . Fmoc groups were removed
2
2
with 3 × 6 mL of 20% piperidine/DMF for 5 min each. For coupling, threeꢀfold excesses of
Fmocꢀamino acids, DIC, and HOBt were used in 8ꢀ10 mL of DMF/CH Cl and were allowed to
2
2
proceed until resin samples tested negative with ninhydrin tests. After coupling and deprotection
steps, resins were washed with 5 ×10 mL of DMF/CH Cl . On completion of the peptide chain,
2
2
resins were washed with CH Cl (3 × 10 mL) and were treated with TFA: TIS: H O (95:2.5:2.5)
2
2
2
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(
3 × 5 mL) for 15 min each. The combined filtrates volumes were reduced in vacuo. Peptides
were precipitated in Et O and collected by centrifugation. After drying, peptides were purified by
2
reverse phase HPLC on a Varian Dynamax HPLC using a Phenomenex Luna C18 (2) 10 ꢀM, 2.1
10
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43
44
45
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47
48
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53
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56
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58
59
60
x 25 cm column. Gradients of 0.1% TFAꢀMeCN in 0.1% TFAꢀH O with a flow rate of 20
2
mL/min were employed. For prodrugs, no TFA was used in the mobile phase. Peptides were
tested for purity by reverse phase HPLC on a Waters Alliance HPLC using a Phenomenex Luna
C18 (2) 5ꢀM 2.1 x 250 mm column at a flow rate of 0.4 mL/min. A gradient of 0ꢀ40%
MeCN/30 min was used for phosphopeptides and peptide intermediates. For prodrugs the
gradient was 10ꢀ80% MeCN/30 min. Phosphopeptides and prodrug intermediates were dried in
5
3
vacuo over P O at 37° for 24 h prior to use. All compounds were >95% pure (HPLC) before
2
5
evaluation. Purities, yields, and mass spectral characteristics of phosphopeptides and prodrugs
are provided in the supporting information.
Synthesis of 4ꢀ(diꢀtertꢀbutoxyphosphoryloxy)ꢀIndoleꢀ2ꢀcarboxylic acid, 40: To a
suspension of 5ꢀhydroxyindoleꢀ2ꢀcarboxylic acid (1.0 g, 5.64 mmol) and tertꢀbutyldimethylsilyl
chloride (0.85g, 5.64 mmol) in 10 mL of dry THF, Nꢀmethylmorpholine (0.62 mL, 5.64 mmol)
was added at room temperature. After 15 min, 1Hꢀtetrazole (1.6 g, 22.56 mmol) was added in
one portion followed by the addition of diꢀtertꢀbutyl N,Nꢀdiethylphosphoramidite (3.9 g, 11.3
mmol) in 15 mL of dry THF. After stirring for 2 h, 70% aq. tertꢀbutylhydroperoxide (3.0 mL,
16.9 mmol) was added at 0°C and the mixture was stirred for 2 h more. The reaction was
quenched with slow addition of an aqueous solution of 10% Na S O and stirred for 1.0 h. It was
2
2
5
then diluted with ethyl acetate (30 mL) and extracted with an additional amount of ethyl acetate
(2 × 20 mL). The combined organic parts were washed with water followed by brine and dried
(
MgSO ). Solvent was removed and the crude product was triturated with hexaneꢀether. The
4
product was collected by filtration and dried to give 1.5 g (71%) of a white solid, which was used
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1
without further purification. H NMR (DMSOꢀd , 300 MHz) δ1.26 (s, 18H), 6.88ꢀ6.94 (m, 2H),
6
1
3
7.21ꢀ7.26 (m, 2H), 11.62 (s, 1H). C NMR (DMSOꢀd , 75 MHz) δ 29.9, 83.1, 83.2, 107.8,
6
+
1
12.0, 113.6, 118.5, 127.3, 130.1, 134.8, 145.2, 145.3, 163.1. MS (ESI) m/z : (M+1) calcd:
70.3; found 370.4.
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3
General procedure for Bocꢀprolinamide formation:
A solution of Bocꢀproline (1.0 g, 4.64 mmol), the appropriate amine (1.0 equiv) and EDC (1.2
equiv) in 60 mL of dry dichloromethane was stirred for overnight. It was then transferred to a
separatory funnel with an additional 20 mL of CH Cl and washed with 5% HCl (2 × 30 mL)
2
2
followed by 10% NaHCO (2 × 30 mL) and brine (1 × 20 mL). The organic layer was dried
3
(MgSO ) and concentrated under reduced pressure. The residue was used as such for the next
4
step or purified by flash chromatography with gradients of EtOAc in hexanes.
3
3
BocꢀProꢀNHPhI, 34a
1
BocꢀProꢀNHPh, 34b: Yield 1.2 gm, 90%, starting from 4.64 mmol of BocꢀProline. HꢀNMR
(
300 MHz, CDCl ) δ1.5 (s, 9H), 1.91ꢀ1.95 (m, 3H), 2.52 (m, 1H) 3.44 (m, 2H), 4.44(m, 1H), 7.1
3
+
(m, 1H), 7.3 (t, J = 7.6 Hz, 2H), 7.5 (d, J = 8.0 Hz, 2H), 9.5 (brs, 1H). MS (ESI) m/z : (M+23)
calcd: 313.2; found 313.3
1
BocꢀProꢀN(Me)Ph, 43c: Yield 1.1 gm, 78%, starting from 4.64 mmol of BocꢀProline. HꢀNMR
(300 MHz, CDCl ) δ 1.45 (s, 9H), 1.48(isomer, s, 9H), 1.65ꢀ2.0 (m, 4H), 3.28 (s, 3H), 3.3ꢀ3.47
3
(
m, 1H), 3.5ꢀ3.6 (m, 1H), 4.14 (m, 1H), 4.3 (isomer, m, 1H), 7.2ꢀ7.47 (m, 5H). MS (ESI) m/z :
+
(
M+23) calcd: 327.2; found 327.3.
1
BocꢀProꢀNHCH Ph, 43e: Yield 1.3 gm, 92%, starting from 4.64 mmol of BocꢀProline. Hꢀ
2
NMR (300 MHz, CDCl ) δ 1.4 (s, 9H), 1.82ꢀ1.98 (m, 2H), 2.1ꢀ2.43 (m, 2H), 3.4 (m, 2H), 4.2ꢀ
3
+
4
.65 (m, 3H), 6.4 (brs, 1H), 7.24ꢀ7.34 (m, 5H). MS (ESI) m/z : (M+23) calcd: 327.2; found
327.3.
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BocꢀProꢀNHCH CH Ph, 43f: Yield 1.2 gm, 85%, starting from 4.64 mmol of BocꢀProline. Hꢀ
2
2
NMR (300 MHz, CDCl ) δ 1.42 (s, 9H), 1.74ꢀ1.9 (m, 2H), 1.95ꢀ2.4 (m, 2H), 2.7ꢀ2.9 (m, 2H),
3
+
3
.27ꢀ3.4 (m, 2H), 3.4ꢀ3.6 (m, 2H), 4.22 (m, 1H), 7.2ꢀ7.33 (m, 6H). MS (ESI) m/z : (M+23)
calcd: 341.2; found 341.3.
BocꢀProꢀN(Me)C H , 43g: Yield 1.1 gm, 76%, starting from 4.64 mmol of BocꢀProline. Hꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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28
29
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37
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43
44
45
46
47
48
49
50
51
52
53
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55
56
57
58
59
60
1
6
11
NMR (300 MHz, CDCl ) δ1.05ꢀ1.2 (m, 1H), 1.25ꢀ1.48 (m, 14H), 1.5ꢀ1.73 (m, 4H), 1.74ꢀ2.25
3
(m, 6H), 2.8 (s, 1.5H), 2.9 (s, 1H), 2.92 (s, 1H), 3.4ꢀ3.7(m, 2H), 4.4 (m, 0.5H), 4.5(m, 0.5H),
+
4.65 (m, 0.5H). MS (ESI) m/z : (M+23) calcd: 333.2; found 333.3
1
BocꢀProꢀNMe , 43h: Yield 1.0 gm, 79%, starting from 4.64 mmol of BocꢀProline. HꢀNMR
2
(
300 MHz, CDCl ) δ 1.4 (s, 9H), 1.46 (isomer, s, 9H), 1.78ꢀ1.92 (m, 4H), 1.96ꢀ2.23 (isomer, m,
3
4H), 2.96 (s, 3H), 2.97 (isomer, s, 3H), 3.06 (s, 3H), 3.1 (isomer, s, 3H), 3.4ꢀ3.5 (m, 2H), 3.53ꢀ
+
3
.66 (isomer,m, 2H). 4.55 (m, 1H), 4.67 (m, 1H). MS (ESI) m/z : (M+23) calcd: 265.2; found
265.3
1
BocꢀProꢀNEt , 43i: Yield 1.1 gm, 87%, starting from 4.64 mmol of BocꢀProline. HꢀNMR (300
2
MHz, CDCl ) δ 1.08ꢀ1.18 (m, 6H), 1.2ꢀ1.28 (isomer, m, 6H), 1.4 (s, 9H), 1.45 (s, 9H), 1.76ꢀ1.92
3
(m, 4H), 1.97ꢀ2.23 (isomer, m, 4H), 3.2ꢀ3.7 (m, 12H, including isomer), 4.45 (m, 1H), 4.6 (m,
+
1
H). MS (ESI) m/z : (M+23) calcd: 293.2; found 293.3
5
1, 52
General procedure for arylation of amides:
To a stirred solution of amide 34a or b (5.0 mmol) in dry CH Cl (50 mL), was added
2
2
triphenylbismuth (2.0 equiv), Cu(OAc) (2.0 equiv) and dry triethylamine (3.0 equiv). After
2
completion of the reaction (as monitored by HPLC), the solvent was evaporated and the residue
was diluted with ether (150 mL) and filtered through celite. The filtrate was then washed with
5
% HCl (2 × 30 mL) followed by brine and was dried over MgSO . Concentration under reduced
4
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pressure followed by purification by silica gel chromatography using 10% ethylacetateꢀhexane
mixture afforded the title compounds.
3
3
BocꢀProꢀN(Ph)PhI, 35a
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BocꢀProꢀNPh , 35b Yield 0.9 gm, 71%, starting from 3.44 mmol of 35b. HꢀNMR (600 MHz,
2
CDCl ) δ 1.46 (s, 9H), 1.52 (s, 9H, isomer), 1.72ꢀ1.8 (m, 2H), 1.87ꢀ1.94 (m, 1H), 1.96ꢀ2.1(m,
3
5H), 3.36ꢀ3.4 (m, 1H), 3.46ꢀ3.5 (m, 1H), 3.55ꢀ3.64 (m, 2H), 4.3 (m, 1H), 4.44 (m, 1H), 7.1ꢀ7.5
+
(
m, 20H, including isomer). MS (ESI) m/z : (M+23) calcd: 389.2; found 389.3
General procedure for dipeptide formation:
A solution of the appropriate Bocꢀprolinamide (2.0 g) in 10 mL of 95%TFA/CH Cl was stirred
2
2
for 1 h. The solution was concentrated under vacuum. Residual TFA was stripped off with by the
addition and evaporation of CCl (2 ×10 mL). The residue was treated with BocꢀTleꢀOH (1.2
4
equiv), HBTU (1.2 equiv), and DIEA (2.5 equiv) in 50 mL of dry CH Cl for overnight. The
2
2
organic layer then diluted with an additional 50 mL of CH Cl and washed with 5% HCl (3 × 30
2
2
mL) followed by 10% NaHCO (2 × 30 mL) and brine. After drying (MgSO ) and concentration
3
4
under vacuum, crude products were sufficiently pure and were used as such for the next step.
3
3
BocꢀTleꢀProꢀN(Ph)PhI, 36a
1
BocꢀTleꢀProꢀNPh , 36b: Yield 0.7 gm, 77%, starting from 1.9 mmol of HꢀProꢀNPh . HꢀNMR
2
2
(
600 MHz, CDCl ) δ 1.06 (s, 9H), 1.40 (s, 9H), 1.83 (m, 1H), 1.97ꢀ2.06 (m, 3H), 3.73 (m, 1H),
3
3.83 (m, 1H), 4.30 (d, J = 9.6 Hz, 1H), 4.55 (m, 1H), 5.38 (d, J = 9.6 Hz, 1H), 7.15ꢀ7.52 (m,
+
1
0H). MS (ESI) m/z : (M+23) calcd: 502.3; found 502.5
BocꢀTleꢀProꢀN(Me)Ph, 44c Yield 0.69 gm, 68%, starting from 2.44 mmol of HꢀProꢀN(Me)Ph.
1
HꢀNMR (600 MHz, CDCl ) δ 1.00 ( s, 9H), 1.32 (s, 9H), 1.64ꢀ1.72 (m, 1H), 1.78 (m, 2H), 1.96
3
(m, 1H), 3.2 (s, 3H), 3.62 (m, 1H), 3.72 (m, 1H), 4.22 (d, J = 9.6 Hz, 1H), 4.34 (t, J = 7.4 Hz,
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H), 5.28 (d, J = 9.6 Hz, 1H), 7.26ꢀ7.38 (m, 5H). MS (ESI) m/z : (M+23) calcd: 440.3; found
440.5
1
BocꢀTleꢀProꢀNHPh, 44d: Yield 0.83 gm, 79%, starting from 2.63 mmol of HꢀProꢀNHPh. Hꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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28
29
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31
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34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
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53
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60
NMR (600 MHz, CDCl ) δ1.00 (s, 9H), 1.44 (s, 9H), 1.9 (m, 1H), 2.03 (m, 1H), 2.16 (m, 1H),
3
2.55 (m, 1H), 3.66 (m, 1H), 3.82 (m, 1H), 4.35 (d, J = 9.7 Hz, 1H), 4.83 (m, 1H), 5.26 (d, J = 9.6
+
Hz, 1H), 7.05 (m, 1H), 7.27 (m, 2H), 7.47 (m, 2H), 9.37 (brs, 1H). MS (ESI) m/z : (M+23)
calcd: 426.3; found 426.4
BocꢀTleꢀProꢀNHCH Ph, 44e: Yield 0.72 gm, 71%, starting from 2.45 mmol of HꢀProꢀ
2
1
NHCH Ph. HꢀNMR (600 MHz, CDCl ) δ 0.88 (s, 9H), 1.42 (s, 9H), 1.85ꢀ1.9 (m, 1H), 1.92ꢀ2.0
2
3
(m, 1H), 2.14 (m, 1H), 2.41ꢀ2.45 (m, 1H), 3.61 (m, 1H), 3.75 (m, 1H), 4.26 (d, J = 9.6 Hz, 1H),
4.32 (dd, J = 5.4 and 15.0 Hz, 1H), 4.46 (dd, J = 6.6 and 15.0 Hz, 1H), 4.63 (dd, J = 2.4 and 7.8
+
Hz, 1H), 5.2 (d, J = 9.6 Hz, 1H), 7.23ꢀ7.30 (m, 5H). MS (ESI) m/z : (M+23) calcd: 440.3;
found 440.4
BocꢀTleꢀProꢀNHCH CH Ph, 44f: Yield 0.53 gm, 66%, starting from 1.83 mmol of Hꢀ Proꢀ
2
2
1
NHCH CH Ph. HꢀNMR (600 MHz, CDCl ) δ 0.96 (s, 9H), 1.42 (s, 9H), 1.88ꢀ1.96 (m, 2H),
2
2
3
2.06ꢀ2.09 (m, 1H), 2.28 (m, 1H), 2.74ꢀ2.81 (m, 2H), 3.47 (m, 2H), 3.61 (m, 1H), 3.77(m, 1H),
4.3(d, J = 9.6 Hz, 1H), 4.53 (m, 1H), 5.28 (d, J = 9.6 Hz, 1H), 6.9 (m, 1H), 7.16ꢀ7.21 (m, 3H),
+
7
.27ꢀ7.3 (m, 2H). MS (ESI) m/z : (M+23) calcd: 454.3; found 454.5
BocꢀTleꢀProꢀN(Me)C H , 44g Yield 0.62 gm, 61%, starting from 2.38 mmol of HꢀProꢀ
6
11
1
N(Me)C H . HꢀNMR (600 MHz, CDCl ) δ1.04 (s, 6H), 1.05 (s, 3H), 1.3ꢀ1.4 (m, 4H), 1.4ꢀ1.44
6
11
3
(m, 11H), 1.53ꢀ1.61 (m, 1H), 1.62ꢀ1.7 (m, 2H), 1.74ꢀ1.8 (m, 1H), 1.81ꢀ1.9 (m, 2H), 1.9ꢀ2.00 (m,
1
4
3
H), 2.06ꢀ2.24 (m, 2H), 2.8 (s, 1H), 2.95 (s, 2H), 3.6 (m, 0.5H), 3.73 (m, 1H), 3.86 (m, 1H), 4.3ꢀ
+
.4 (m, 1.5H), 4.83 (m, 0.5H), 4.9 (m, 0.5H), 5.35 (m, 1H). MS (ESI) m/z : (M+23) calcd:
89.2; found 389.3
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BocꢀTleꢀProꢀNMe , 44h Yield 1.1 gm, 60%, starting from 4.92 mmol of HꢀProꢀNMe . HꢀNMR
2
2
(
600 MHz, CDCl ) δ 1.04 (s, 9H), 1.42 (s, 9H), 1.86 (m, 1H), 1.95 (m, 1H), 2.1 (m, 1H), 2.18
3
(
m, 1H), 2.94 (s, 3H), 3.11 (s, 3H), 3.74 (m, 1H), 3.86 (m, 1H), 4.33 (d, J = 9.6 Hz, 1H), 4.87 (m,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
H), 5.29 (d, J = 9.6 Hz, 1H). MS (ESI) m/z : (M+23) calcd: 378.3; found 378.4
1
BocꢀTleꢀProꢀNEt , 44i Yield 1.4 gm, 64%, starting from 5.9 mmol of HꢀProꢀNEt . HꢀNMR
2
2
(
600 MHz, CDCl ) δ 1.03 (s, 9H), 1.1 (t, J = 7.2 Hz, 3H), 1.27 (t, J = 7.2 Hz, 3H), 1.42 (s, 9H),
3
1
.86ꢀ1.96 (m, 2H), 2.12ꢀ2.18 (m, 2H), 3.28ꢀ3.42 (m, 3H), 3.53 (m, 1H), 3.73 (m, 1H), 3.87 (m,
+
1H), 4.32 (d, J = 10.2 Hz, 1H), 4.76 (m, 1H), 5.34 (d, J = 9.6 Hz, 1H). MS (ESI) m/z : (M+23)
calcd: 406.3; found 406.5
5
6
Synthesis of (S)ꢀ2ꢀstyrylꢀNꢀBocꢀpyrrolidine, 46: nꢀBuLi (1.0 mL, 2.5 mmol) was added
dropwise via syringe over 10 minutes to a solution of benzylꢀtriphenylphosphonium bromide
(
1.0 g, 2.3 mmol) in 20 mL of dry THF under argon at ꢀ78°C. The mixture was stirred for 1 h,
then BocꢀLꢀprolinal (0.46g, 2.3 mmol) in 20 mL THF was added dropwise. The reaction was
allowed to stir for 24h at room temperature. The reaction mixture then quenched with 30 mL of
saturated NH Cl, diluted with ethyl acetate, washed with brine (2 × 25 mL), dried over sodium
4
sulfate, and concentrated in vacuo. Silica gel chromatography (5:1 hexanes/EtOAc) yielded
1
0
.480 g (76%). H NMR (CDCl , 300 MHz) δ 1.42 (s, 9H), 1.8ꢀ2.2 (m, 4H), 3.45 (brs, 1H), 4.3ꢀ
3
4.8 (m, 1H), 5.58ꢀ6.15 (m, 1H), 6.35ꢀ6.45 (m, 1H), 7.2ꢀ7.4 (m, 5H).
Synthesis of (S)ꢀ2ꢀ(4ꢀphenylꢀbutꢀ1ꢀenyl)ꢀNꢀBocꢀpyrrolidine, 48: The same procedure as
described for the synthesis of 46 was followed starting with 1.0 g of (3ꢀphenylpropyl)ꢀ
triphenylphosphonium bromide (2.2 mmol) and 0.483 g of Bocꢀprolinal (2.2 mol). Yield: 0.452 g
1
(
1.5 mmol, 68%). H NMR (CDCl3, 300 MHz) δ 1.43 (s, 9H), 1.68ꢀ1.92 (m, 4H), 2.37ꢀ2.52 (m,
2
H), 2.56ꢀ2.81 (m, 2H), 3.3ꢀ3.42 (m, 2H), 4.45 (brs, 1H), 5.27ꢀ5.45 (m, 2H), 7.14ꢀ7.31 (m, 5H).
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Journal of Medicinal Chemistry
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(
S)ꢀ4ꢀphenbutylꢀNꢀBocꢀpyrrolidine, 50: A 50ꢀmL roundꢀbottom flask fitted with a 3ꢀway tap
and balloon was charged with 400 mg of 10% PdꢀC. The syringe and stirred for several minutes.
Alkene 49 (405 mg) in 5 mL EtOH was added via syringe and stirred for 2 h. Hydrogen gas was
added as the balloon deflated. Upon complete reaction (TLC) the catalyst was removed by
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filtration. The product was concentrated in vacuo to yield. Colorless oil, yield 376 mg (93%). H
NMR (CDCl , 300 MHz) δ 7.15ꢀ7.31 (m, 5H), 3.70ꢀ3.76 (br m, 1H), 3.26ꢀ3.41 (m, 2H), 2.58ꢀ
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2.64 (m, 2H), 1.59ꢀ1.93 (m, 7H), 1.49 (s, 9H), 1.30ꢀ1.35 (m, 3H).
Synthesis of NꢀBoc (S)ꢀ2ꢀ((methyl(phenyl)amino)methyl)pyrrolidine, 53. To a stirred
solution of 45 (1.00 g, 5.02 mmol) and aniline (0.78 g, 6.02mmol) in MeOH:acetic acid (9:1) (10
mL) was added NaCNBH (0.63 g, 10.04mmol) at room temperature. The mixture was stirred for
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1h, evaporated to dryness and the residue extracted with AcOEt (2 x 30 mL). The organic phase
was washed with 5% NaHCO , brine, dried (MgSO ) and evaporated. The product 52 (1.0 g, 3.2
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mmol) was then dissolved in 10 mL of MeCN: acetic acid (9:1), and 37% formalin (10 mL) and
sodium cyanoborohydride (0.6g, 9.6 mmol) were added. The mixture was stirred for 4 h at room
temperature. The solution was evaporated in vacuo, the residue was dissolved in AcOEt (50 mL)
and was washed with 1N KOH followed by brine. The organic phase was dried (MgSO4),
evaporated, and the crude product was purified by silica gel chromatography using 30% EtOAcꢀ
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hexane mixture yielding 0.640 gm (2.2 mmol) of 53 (61%). H NMR (CDCl , 300 MHz) δ1.52
3
(s, 9H), 1.8ꢀ1.95 (m, 4H), 2.98 (s, 3H), 3.0ꢀ3.21 (m, 1H), 3.28ꢀ3.48 (m, 2H), 3.56ꢀ3.72 (m,
+
1
H),4.15 (m, 1H), 6.68 (m, 1H), 6.74 ꢀ6.84 (m, 2H), 7.18ꢀ7.26 (m, 2H). MS (ESI) m/z : (M+H)
calcd: 291.2; found 291.3.
General procedure for phosphopeptide/phosphopeptidomimetic formation: Bocꢀprotected
dipeptide (0.5 g) was treated with 5.0 mL of 95% TFAꢀCH Cl for 1h. After concentration under
2
2
vacuum the residue was diluted with 10 mL of water, neutralized with conc. NH OH and
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