Conformationally constrained peptidomimetic inhibitors of Signal transducer and activator of transcription 3: Evaluation and molecular modeling

Article abstract of DOI:10.1021/jm801491w

Signal transducer and activator of transcription 3 (Stat3) is involved in aberrant growth and survival signals in malignant tumor cells and is a validated target for anticancer drug design. We are targeting its SH2 domain to prevent docking to cytokine an

Full text of DOI:10.1021/jm801491w

J. Med. Chem. 2009, 52, 2429–2442
2429
Conformationally Constrained Peptidomimetic Inhibitors of Signal Transducer and Activator of
Transcription 3: Evaluation and Molecular Modeling
Pijus K. Mandal,†,‡ Donald Limbrick,§,‡ David R. Coleman, IV,†,| Garrett A. Dyer,† Zhiyong Ren, J. Sanderson Birtwistle,†
Chiyi Xiong,# Xiaomin Chen, James M. Briggs,§ and John S. McMurray*,†
Department of Experimental Therapeutics, Department of Biochemistry and Molecular Biology, and Department of Experimental Diagnostic
Imaging, The UniVersity of Texas M. D. Anderson Cancer Center, 1515 Holcombe BouleVard, Houston, Texas 77030, and Department of
Biology and Biochemistry, UniVersity of Houston, 4800 Calhoun Road, Houston, Texas 77204-5001
ReceiVed NoVember 26, 2008
Signal transducer and activator of transcription 3 (Stat3) is involved in aberrant growth and survival signals
in malignant tumor cells and is a validated target for anticancer drug design. We are targeting its SH2
domain to prevent docking to cytokine and growth factor receptors and subsequent signaling. The amino
acids of our lead phosphopeptide, Ac-pTyr-Leu-Pro-Gln-Thr-Val-NH2, were replaced with conformationally
constrained mimics. Structure-affinity studies led to the peptidomimetic, pCinn-Haic-Gln-NHBn (21), which
had an IC50 of 162 nM (fluorescence polarization), compared to 290 nM for the lead phosphopeptide (pCinn
) 4-phosphoryloxycinnamate, Haic ) (2S,5S)-5-amino-1,2,4,5,6,7-hexahydro-4-oxo-azepino[3,2,1-hi]indole-
2-carboxylic acid). pCinn-Haic-Gln-OH was docked to the SH2 domain (AUTODOCK), and the two highest
populated clusters were subjected to molecular dynamics simulations. Both converged to a common peptide
conformation. The complex exhibits unique hydrogen bonding between Haic and Gln and Stat3 as well as
hydrophobic interactions between the protein and pCinn and Haic.
Introduction
Tyr705 and subsequent dimer formation. Therefore, transloca-
tion to the nucleus and binding to Stat3 response elements on
the promotors of the above-mentioned cell cycling, survival,
and angiogenesis genes will be inhibited, resulting in reduced
tumor growth. Peptidomimetic development requires information
gained from structure activity relationship (SARa) and structural
studies to design or otherwise find nonpeptidic scaffolds to
present the pharmacophores of a lead peptide in the correct
spatial arrangement for optimal binding to the target. To find a
lead peptide for inhibitor development, we screened several
receptor docking sites for Stat3 and found that the sequence
from gp130 904-909, Ac-pTyr-Leu-Pro-Gln-Thr-Val-NH2 (1),
is a high affinity ligand for the Stat3 SH2 domain.15 We recently
reported SAR studies on the peptide that showed that the pY -
1 acetyl group could be replaced by a hydrocinnamoyl group,
the optimum residue at pY + 1 is Leu, cis-3,4-methanoproline
at pY + 2 results in enhanced interaction, Gln is optimal at pY
+ 3, the side chain CONH2 is involved in important hydrogen
bonds, and the C-terminal Thr-Val dipeptide can be replaced
with a benzyl group.16 This study provided important informa-
tion on the pharmacophores of the lead peptide but gave little
information on the conformation of the bound peptide.
Signal transducer and activator of transcription 3 (Stat3) is a
latent, cytosolic transcription factor that transmits signals directly
from cell surface receptors to the nucleus (reviewed in refs 1-3).
On stimulation by IL-6 family of cytokines, or growth factors
such as EGF or PDGF, Stat3 is recruited to phosphotyrosine
residues on the cell surface receptor via its SH2 domain and
becomes phosphorylated on Tyr705. The protein then forms a
dimerized complex in which the SH2 domain of one protein
binds to the phosphotyrosine of the other and vice versa. The
Stat3-dimer then translocates to the nucleus where it initiates
transcription of cell cycling genes such as cyclin D1 and p21waf
,
antiapoptotic genes such as survivin and Bcl-xL, and angio-
genesis factors such as vascular endothelial growth factor. Stat3
is constitutively activated in cancers of the breast, prostate, lung,
head and neck, multiple myeloma, leukemia, and others
(reviewed in refs 4-8). Studies on cell lines have showed that
inhibiting Stat3 activity with the use of antisense oligonucle-
otides, dominant negative constructs,4-8 and decoy oligonucleo-
tides9-14 reduces the growth of cancer cells and induces
apoptosis. Thus, Stat3 is a target for antitumor drug design.
We are developing Stat3 inhibitors by targeting the SH2
domain with peptidomimetics derived from phosphopeptides.
Such compounds are expected to prevent binding to cytokine
or growth factor receptors, thus preventing phosphorylation of
NMR or X-ray crystal structures of phosphopeptides bound
to SH2 domains have provided the basis for the development
of inhibitors of Grb2 and Src-family kinases (reviewed in refs
17-21). Information on intermolecular interaction and on the
bioactive conformation has led to the development of highly
potent, non-peptide inhibitors of Src,22,23 Lck,24 and Grb2.25-28
The crystal structure of Stat3ꢀ dimer in complex with DNA29
reveals the interactions between the phosphopeptide segment,
* To whom correspondence should be addressed. Phone: 713-745-3763.
Fax: 713-792-1204. E-mail: jmcmurra@mdanderson.org.
†
Department of Experimental Therapeutics, The University of Texas
M. D. Anderson Cancer Center.
‡
Both authors contributed equally to this work.
University of Houston.
Current address: Department of Chemical and Biological Engineering,
§
a Abbreviations: ABN, azabicyclo[4.3.0]nonane-9-carboxylate; DIC,
diisopropylcarbodiimide; Gaba, γ-aminobutyric acid; GB, generalized Borne;
Haic, 5-amino-1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-car-
boxylic acid; ∆Haic, “dehydro-Haic” 5-amino-1,2,4,5-tetrahydro-4-oxoaze-
pino[3,2,1-hi]indole-2-carboxylic acid; Met(O), methionine sulfoxide; Me-
t(O2), methionine sulfone; pCinn, 4-phosphoryloxycinnamic acid; pInd,
5-phosphoryloxyindole-2-carboxylate; SAR, structure-activity relationship;
pTic, 4-phosphoryloxy-1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid.
|
South Dakota School of Mines and Technology, 501 E. St. Joseph, Rapid
City, South Dakota, 57701.
Department of Biochemistry and Molecular Biology, The University
of Texas M. D. Anderson Cancer Center.
#
Department of Experimental Diagnostic Imaging, The University of
Texas M. D. Anderson Cancer Center.
10.1021/jm801491w CCC: $40.75
2009 American Chemical Society
Published on Web 03/31/2009

2430 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Mandal et al.
Scheme 1. Synthesis of 4-(Di-tert-butylphosphoryloxy)cinnamic Acid
Tyr(PO3H2)705-Leu-Lys-Thr-Lys-Phe, of one protein molecule
and the SH2 domain of the other. The pTyr-Leu dipeptide makes
contacts in an analogous manner to the pY and pY + 1 residues
in other SH2-phosphopeptide complexes.26,30-38 However,
because of the structural difference between Lys-Thr-Lys-Phe
of Stat3 and Pro-Gln-Thr-Val of our lead peptide, no direct
information on the conformation or intermolecular interaction
of the (pY + 2)-(pY + 4) residues of the latter can be obtained
from this model. In this communication, we describe the
incorporation of conformational constraints into our lead peptide
that provided information on the bound conformation. The result
was a high affinity, peptidomimetic inhibitor with enhanced
affinity for Stat3, compound 21.
Having found conformational constraints that improve the
affinity of our lead peptide, we studied potential inhibitor-protein
interactions using AUTODOCK39 and selected two complexes
for further study. Extended molecular dynamics simulations
resulted in a converged conformation of the inhibitor and
provided insight into the basis of affinity of the peptidomimetic.
2-carboxylic acid (pInd), N-acetyl-4-phosphoryloxy-1,2,3,4-
tetrahydro-3-isoquinolinecarboxylic acid (Ac-Tic), 2-phospho-
ryloxy-7-carboxylnaphthalene, and 6-phosphoryloxyindole-3-
acetic acid, were prepared by global phosphorylation of the
corresponding hydroxy compounds on solid supports using
dibenzyl-N,N-diisopropyl phosphoramidite/tetrazole and oxida-
tion with tert-butyl hydroperoxide after coupling to their peptide
chains (reviewed in ref 43). Peptides were cleaved and purified
as above.
Synthesis of Analogues of Fmoc-Haic, (2S,5S)-5-(9-Fluo-
renyloxycarbonyl)amino-1,2,4,5,6,7-hexahydro-4-oxoazepi-
no[3,2,1-hi]indole-2-carboxylic Acid. Fmoc-Haic-OH (44) was
obtained commercially or was synthesized by the method of
De Lombaert et al.44,45 (Scheme 2). Friedel-Crafts cyclization
of 41 results in approximately 10% of the 2R,5S diastereoisomer
(43), which was isolated by fractional crystallization of the crude
material in Et2O. It can be converted to the 2S,5S ketone 42 by
treatment with DBU and taken on to 44. To prepare the 2R,5S
diastereoisomer for use in solid phase synthesis (45), compound
43 was hydrogenated and deprotected with LiOH and the amino
group capped with an Fmoc group. To prepare 6,7-dehydro-
Haic, partial reduction of ketone 42 was accomplished by
hydrogenation in THF to give alcohol 46 which was dehydrated
using trifluoracetic anhydride and TFA. The amino and carboxyl
protecting groups were removed with LiOH, and Fmoc protec-
tion was applied to the amino group providing compound 47.
Stereoisomers of Fmoc-2-aminoazabicyclo[4.3.0]nonane-9-car-
boxylate (ABN) were synthesized as described.46
Synthesis of Inhibitors Containing Glutamine Ana-
logues. Inhibitors 27, 29, and 30 (Table 5) were prepared by
coupling Fmoc-Glu-OMe (50a), Fmoc-Glu-NHMe (50b), and
Fmoc-Glu-NHiPr (50c), respectively, to Rink resin via their side
chains using DIPCDI/HOBt followed by assembly of the
remainder of the compound using standard peptide synthesis
protocols. Glutamine analogues 50a-c were synthesized by
derivatization of Fmoc-Glu(OtBu)-OH (48) with MeOH or the
appropriate amine with PyBOP/DIPEA followed by cleavage
of the side chain tert-butyl group with TFA (Scheme 3). Fmoc-
pyrrolidine-3-acetic acid, used in the synthesis of 25, was
prepared as described by Coleman et al.16
Chemistry
Peptide Synthesis. Peptides were synthesized using manual
solid phase techniques employing the Fmoc protection scheme
and either Rink resin or Wang resin. Couplings were mediated
with either diisopropylcarbodiimide (DIC)/1-hydroxybenzotria-
zole (HOBt) or PyBOP/HOBt/DIEA. Fmoc removal was ac-
complished with two treatments of 20% piperidine in DMF for
5 min each. Peptides incorporating a C-terminal glutamine
R-benzylamide were synthesized by coupling Fmoc-Glu-
NHBn,16 via the side chain, to Rink resin. Peptides were cleaved
from the solid support with TFA/triethylsilane/H2O (95:2.5:
2.5)40 and were purified by reverse phase HPLC.
Synthesis of Inhibitors Incorporating Constrained
Tyrosine Mimics. 4-Phosphoryloxycinnamic acid (pCinn) was
introduced onto peptide chains using the protected building
block, 4-(di-tert-butoxyphosphoryloxy)cinnamic acid (39, Scheme
1). This was prepared by methodology initially described by J.
Perich et al. for the synthesis of phosphotyrosine derivatives.41,42
The carboxyl group of 4-hydroxycinnamic acid (36) was
selectively protected as a TBDMS ester, and the hydroxyl group
was phosphitylated with di-tert-butyl-N,N-diisopropyl phos-
phoramidite in the presence of tetrazole. The phosphorus was
oxidized with tert-butyl hydroperoxide, and the TBDMS ester
was removed with aqueous bicarbonate to afford 4-(di-tert-
butoxyphosphoryloxy)cinnamic acid (39). This agent was
coupled to peptides using PyBOP/HOBt/DIPEA. The other
constrained phosphotyrosine mimics, 5-phosphoryloxyindole-
Results and Discussion
In a previous study the lead peptide, 1 was truncated to
pentapeptide 2 and tetrapeptide 3.16 The last two peptides
showed reduced affinity for Stat3, suggesting that hydrophobic
contacts exist between the C-terminal Val and Stat3 (Table 1).
Replacement of the Thr-Val dipeptide unit with a benzyl group

Peptidomimetic Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2431
Scheme 2. Synthesis of Fmoc-Haic-OH and Its Analogues
Scheme 3. Synthesis of R-Carboxy Analogues of Fmoc-glutamine
Table 1. IC50 Values of Peptide Inhibitors of Stat3a
peptide
IC50 (nM)
Table 2. Effect of pTyr Mimics on the Affinity of
XXX-Leu-Pro-Gln-Thr-NH2
1
2
3
4
Ac-Tyr(PO3H2)-Leu-Pro-Gln-Thr-Val-NH2
Ac-Tyr(PO3H2)-Leu-Pro-Gln-Thr-NH2
Ac-Tyr(PO3H2)-Leu-Pro-Gln-NH2
290 ( 63
739 ( 31
856 ( 41
409 ( 15
Ac-Tyr(PO3H2)-Leu-Pro-Gln-NHBn
a IC50 values from Coleman et al.16
brought the IC50 to 409 nM, thus confirming the presence of
these contacts.16 Peptides 2-4 served as leads to which the
effects of the constrained amino acid substitutions described
below were compared.
Phosphotyrosine Constraints. The X-ray crystal structure
of dimerized Stat3ꢀ bound to DNA29 shows interactions between
the phosphate group of pTyr705 and the opposing SH2 domain
which are analogous to those observed in NMR and crystal
structures of Src,30-33 Grb2,26,34,35 Lck,36 and p85 PI3K.37,38
In the Stat3 structure,29 the dihedral angle of the C, CR, Cꢀ,
and Cγ atoms of pTyr is 174°, also typical of those found in
phosphopeptide ligands of other SH2 domains26,30-38 4-Phos-
phoryloxycinnamate (pCinn)47 and 5-phosphoryloxyindole-2-
carboxylate (pInd)48 both constrain this dihedral angle to 180°.
Substitution of the pTyr of peptide 2 with these two mimics
enhanced the activity of our peptide 4- to 5-fold, decreasing
the IC50 from 739 to 136 and 186 nM, respectively (5 and 6,
Table 2). The use of pCinn as a phosphotyrosine replacement
in inhibitors of the Src SH2 domain was reported by Shahripour
et al.47 In this paper the activity of the lead was reduced 11-
fold by replacing pTyr with 4-phosphoryloxycinnamide. Re-
searchers from Tularik, Inc. used pCinn in inhibitors of
Stat6,49,50 although no comparison with phosphotyrosine was
provided in their patents. Vu et al.48 reported that the use of
pInd in development of inhibitors of the SH2 domain of Zap70
reduced activity of their lead 7-fold. In the case of Stat3,
constraining the C-CR-Cꢀ-Cγ dihedral angle to 180° appears

2432 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Mandal et al.
Table 4. Affinities of Derivatives of 4, Ac-pTyr-Leu-Pro-Gln-NHBn
Table 3. Effect of Leu-Pro Mimics on the Affinity of
Ac-pTyr-XXX-Gln-Thr-NH2
compd
IC50 (nM)
17
18
19
20
21
22
23
24
25
pCinn-Leu-Pro-Gln-NHBn
pCinn-(3S,6S,9S)-ABN-Gln-NHBn
pCinn-(3S,6R,9S)-ABN-Gln-NHBn
pCinn-(3S,6R,9R)-ABN-Gln-NHBn
pCinn-Haic-Gln-NHBn
pCinn-∆Haic-Gln-NHBn
pInd-Haic-Gln-NHBn
pCinn-Haic-Gaba
pCinn-Haic-pyrrolidine acetamide
138 ( 8a
604 ( 68
45700 ( 7710
59800 ( 7580
162 ( 19
195 ( 20
190b
815 ( 84
595 ( 92
a From ref 69. b A single assay was run.
an approximately 5-fold loss in affinity (11, Table 3). Incorpora-
tion of the (3S,6R,9R)-isomer was even more deleterious
(compound 12, IC50 ) 24 700 nM). In compound 12 the
configuration of CR of the five-membered ring (C9) is R,
analogous to D-Pro, which we reported to greatly reduce affinity
for Stat3.16
Replacing Leu-Pro with Haic (5-[(S)-amino]-1,2,4,5,6,7-
hexahydroazepino[3,2,1-hi]indole-4-one-2-(S)-carboxylate) pro-
duced an inhibitor with IC50 of 231 nM (compound 13, Table
3). The synthesis of Haic by the method of De Lombaert et
al.44,45 produces roughly 90% of the 2S,5S diastereomer and
10% of the 2S,5R diastereomer during the annelation of the
seven-membered ring. We completed the synthesis of Fmoc-
(2S,5R)-Haic (45) and used this to prepare 14. The IC50 of the
peptide containing this diastereomer was 1650 nM, a 7-fold
reduction in activity compared to the (2S,5S)-stereoisomer.
Compound 14 was only 2-fold lower in affinity than the parent
peptide 2. The seven-membered ring of Haic displays noticeable
flexibility.44 In spite of the opposite configuration at C5, it
appears that the (2S,5R)-stereoisomer can indeed fold to present
the phenylphosphate and carboxamide pharmacophores for
interaction with the SH2 domain. The tricyclic structure of Haic
is important. Uncoupling of the azepine ring of Haic by
substituting with leucinyl-(S)-imidazoline-2-carboxylate caused
a 20-fold loss in affinity (15, IC50 ) 4640 nM). Replacing pTyr
of compound 13 with the 4-phosphoryloxydihydrocinnamate
resulted in a 14-fold loss in affinity (16, IC50 ) 3240 nM). The
acetamido group appears to add to the affinity of 13.
In a previous publication16 Gln was substituted with a wide
variety of flexible and constrained ω-amino acid amides. None
were as effective as glutamine. We also found that substituting
a benzylamide on the C-terminus (4) produced a slight enhance-
ment in affinity over the Thr-NH2 in peptide 2. To create
peptidomimetics, pCinn was added to the ABN and Haic units
and the Thr-NH2 was replaced with a benzylamide. Again, the
ABN unit was not as favorable as Haic (Table 4). Incorporation
of (3S,6S,9S)-ABN (18) resulted in an IC50 of 604 nM, 4-fold
lower affinity than that of the Leu-Pro analogue 17 (IC50 ) 138
nM). The (3S,6R,9S)- and (3S,6R,9R)-diastereomers of ABN
caused large decreases in affinity (compounds 19 and 20, IC50
of 45 700 and 59 800 nM, respectively). Incorporation of Haic
resulted in an IC50 value of 162 nM (21), nearly identical to
that of the Leu-Pro analogue, 17. In an attempt to constrain the
conformation of the seven-membered ring of the Haic unit, we
synthesized Fmoc-6,7-dehydro-Haic (47) and incorporated it into
the mimetic to give compound 22. The IC50 of 195 nM is
somewhat lower than that of the saturated compound 21 (162
nM). Incorporating pInd onto the Haic-Gln-NHBn framework
(22) resulted in an IC50 of 190 nM. This slight reduction
compared to pCinn (20) parallels that found in the more peptidic
compounds, 6 vs 5.
to have locked the aromatic phosphate in a favorable orientation
for contact with the protein which likely reduced the entropy
penalty on binding. Substitution of pTyr with Ac-pTic, 2-phos-
phoryloxy-7-carboxylnaphthalene or 6-phosphoryoxyindole-3-
acetic acid resulted in inactive compounds (7-9). Movement
of the phosphate group to position 3 of the aromatic ring of
cinnamic acid also abrogated activity (peptide 10).
Replacement of (pY + 1)-(pY + 2) Dipeptide Unit. We
recently reported that substitution of proline of peptide 2 with
the pseudo-proline (ΨPro) analogues 2,2-dimethyl-1,3-oxazo-
lidine-5-carboxylate and 2,2,4-trimethyl-1,3-oxazolidine-5-car-
boxylate resulted in reduced affinity for Stat3.51 That the lower
activity correlated with the increased extent of cis isomerization
of the Leu-ΨPro peptide bond suggests that the corresponding
peptidebondistransinourleadpeptides.51 Azabicyclo[4.3.0]nonane-
9-carboxylate (ABN) has been studied in the context of ꢀ-turn
mimetics and has been used to good effect as a conformational
constraint.52-59 Replacement of the Leu-Pro unit with ABN
would hold the peptide bond in the trans conformation and
would also constrain the φ dihedral angle of Leu, thus reducing
the amount of conformational space available to the peptide
and the entropy penalty on binding. Incorporation of (3S,6S,9S)-
ABN into peptide 2 increased the IC50 from 740 to 3530 nM,
Haic is a known dipeptide mimetic that has been used in
protease inhibitor development,60,61 bradykinin antagonists,62

Peptidomimetic Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2433
Table 5. Contributions of Gln-X to the Binding of pCinn-Haic-Based
Peptidomimetics to Stat3
the trajectories. In the discussion below, amino acids from the
protein will be designated by the one-letter code followed by
the residue number, e.g., E638. Features from the inhibitor will
be designated by fragment, e.g., Gln side chain. Rigid-protein/
flexible-ligand docking of 25 to Stat3 was modeled in an
automated manner using AUTODOCK 4.0.39 Two poses, in
which 25 was in an extended conformation (pose A, Figure 1A)
and a bent conformation (pose B, Figure 1B), were selected
from the docking study for subsequent molecular modeling
explorations. Pose A was chosen because it was a member of
the largest cluster of docking poses. Pose B was selected because
of proximity of the glutamine side chain amide to residues E638,
P639, and Y640 of Stat3, which were hypothesized to be close
in peptide complexes with the SH2 domain of Stat3.65
compd
IC50 (nM)
26
27
28
29
30
31
32
33
34
35
pCinn-Haic-Gln-OH
pCinn-Haic-Gln-OMe
pCinn-Haic-Gln-NH2
pCinn-Haic-Gln-NHMe
pCinn-Haic-Gln-NHiPr
pCinn-Haic-NH2
pCinn-Haic-Ala-NH2
pCinn-Haic-Nle-NH2
pCinn-Haic-Met(O)-NH2
pCinn-Haic-Met(O2)-NH2
359 ( 30
141 ( 14
233 ( 27
269 ( 22
305 ( 46
35600 ( 5010
25060 ( 2750
19200 ( 1300
7600 ( 10
7030 ( 1300
major histocompatibility complex antagonist studies,63 and
opioid receptor agonists.64 This is the first reported use in an
SH2 domain inhibitor.
After extensive energy minimization with implicit solvent
followed by explicit solvent, the two poses were separately
heated, equilibrated, and subjected to 3 ns of molecular dynamics
simulations containing explicit water. After about 1.5 ns both
simulations exhibited a decrease in main-chain rmsd of both
ligand and protein and convergence to a similar inhibitor
conformation and intermolecular hydrogen bonding features.
Both trajectories were separately clustered on the basis of
similarity of the rmsd of the inhibitor.
Incorporation of Glutamine Mimics. Glutamine is es-
sentially 4-carboxy-4-aminobutyramide, an analogue of Gaba.
Replacement of Gln-NHBn of compound 21 with Gaba (24)
resulted in an IC50 value of 815 nM, a 5-fold loss in affinity
(Table 4). Constraining the conformation of Gaba by tethering
the nitrogen with C3, i.e., using pyrrolidine-3-acetamide (com-
pound 25, IC50 ) 815 nM), did not have an appreciable effect
on affinity. Thus, Gln-NHBn produced the greatest affinity,
which is in keeping with earlier work in which Gaba and
pyrrolidine-3-acetamide provided less affinity than Gln-NHBn
in the peptide Ac-pTyr-Leu-Pro-XXX.16
To ascertain the contribution of the aromatic ring of the
C-terminal R-benzylamide in the pCinn-Haic-Gln-NHBn, a
series of substitutions was made on the main chain carboxyl
group of Gln of compound 21. Thus, carboxyl, methyl ester,
and a set of amides were assayed (compounds 26-30, Table
5). The difference in affinity of a negatively charged carboxyl
The negatively charged phosphate of pCinn is located in the
electropositive phosphate binding pocket formed partly by K591
and R609 throughout the entirety of both simulations. These
ionic interactions are analogous to those observed in the crystal
structure of the Stat3 dimer29 and are typical of phosphopeptide-
SH2 domain complexes.26,30-38 The phosphate-R609 hydrogen
bond exists for 100.0% and 99.9% of the total 3 ns MD
trajectories of poses A and B, respectively. Similarly the
phosphate-K591 hydrogen bond exists for 98.0% and 97.9%
of the 3 ns MD trajectories of poses A and B, respectively.
Other hydrogen bonds observed between Stat3 and the phos-
phopeptide phosphate in both the Stat3 crystal structure and
the molecular dynamics simulations are as follows. The interac-
tion between the backbone nitrogen of E612 and one of the
phosphate oxygens exists for 93.0% and 84.9% of the 3 ns
trajectories of poses A and B, respectively, and the hydrogen
bond between the side chain hydroxyl group of Stat3 S613 and
one of the phosphate oxygens exists for 62.4% and 10.3% in
the trajectories of poses A and B, respectively, which differs
from the crystal structure of Stat3.29 The placement of the
phosphate moiety in this pocket is consistent with site-directed
mutations which demonstrated that K591 and R609 are essential
for binding four to six residue phosphopeptides with pYXXQ
consensus sequences.66 On the basis of fidelity of the hydrogen
bonds observed in the Stat3 crystal structure29 and consistency
with experimental evidence, it can be concluded that the
molecular dynamics simulations reasonably predict the interac-
tion between Stat3 and the pCinn unit of 26.
Hydrogen bonds between the pY + 1 main chain amide
nitrogen and the backbone carbonyl oxygen of the ꢀD4 residue
exist in most known complexes between SH2 domains and
phosphopeptide ligands,26,30-38 and the STATs are no excep-
tion.29,67,68 In crystal structures of Stat3 and Stat1 the backbone
oxygen of Stat3, S636 (Stat1:A630) makes a hydrogen bond
with the pY + 1 backbone nitrogen of the opposing protein29,67
or a phosphopeptide ligand.68 A structure-activity relationship
study demonstrated through leucine N-methylation that this
hydrogen bond is essential for high affinity recognition of
phosphopeptides by Stat3.16 However, hydrogen bonds between
the analogous exocyclic amide nitrogen of Haic and the
backbone oxygen of S636 were observed for only 22-25% of
group (26, IC50 ) 359 nM and the benzyl group (21, IC50
)
162 nM) is only 2-fold, and the other substitutions fall within
that range. Thus, the contribution of the C-terminal benzene
ring of 21 is relatively small. However, the contribution of the
alkylcarboxamide side chain is large. pCinn-Haic-NH2 (31) has
an IC50 value of 35 µM, 42-fold weaker than Gaba containing
compound 24 (819 nM, Table 2) and 150-fold weaker than
compound 28 containing Gln-NH2 (IC50 ) 233 nM). Replace-
ment of glutamine in 28 with alanine (32) or norleucine (33),
neither of which possesses a side chain CONH2 group, also
resulted in compounds with IC50 values in the micromolar range.
Docking of pCinn-Haic-Gln-OH to the SH2 domain of
Stat3. To determine possible inhibitor conformations and to
study intermolecular interactions, pCinn-Haic-Gln-OH, 26, was
docked to the SH2 domain of Stat3 and then subjected to
minimization and molecular dynamics simulations. Stat3, like
all STATs, is a multidomain protein consisting of an amino
terminal domain, a coiled coil domain, a DNA binding domain,
a linker domain, an SH2 domain, and the C-terminal transac-
tivation domain. The Stat3ꢀ isoform lacks the amino terminal
domain and the 55 residues of the transactivation domain, which
are replaced by seven unique amino acids (Schaefer et al.,
1995).94 The coordinates from the crystal structure of Stat3ꢀ,29
PDB code 1BG1, were used for the simulations. For the initial
docking experiments, residues 136-688 were used and the
ligand was docked into the SH2 domain. This fragment of Stat3
does not contain amino acids C-terminal to the SH2 domain.
For the molecular dynamics simulations, residues 585-688,
consisting of the linker and SH2 domains, were employed. The
smaller protein fragment was used to reduce computation time,
and inclusion of the linker domain ensured that the conformation
of the distal side of the SH2 domain would not change during

2434 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Mandal et al.
Figure 1. Structures of the poses of docking compound 26 to the SH2 domain of Stat3 from the initial AUTODOCK study: (A) pose A, a
representative from the most populated cluster from the AUTODOCK study exhibiting the extended conformation; (B) pose B, a representative
from the cluster possessing the bent conformation. These figures represent complexes that were energy-minimized with implicit solvent followed
by minimization with explicit solvent. Water molecules were removed for clarity. Compound 26 is depicted as sticks: green, carbon; blue, nitrogen;
red, oxygen. Hydrogens were removed for clarity. Stat3 is represented as a surface rendition. The following residues are highlighted: phosphate
binding pocket (blue); S636 and P639 (purple); E638 (red); Y640 (yellow); Y657 (orange) M660 (brown). The images were generated with PyMol.70
least 74% of the time for both conformations with only a 4.8%
difference between the two simulations. The persistence of
hydrogen bonds between 25 and the side chains of Y640 and
Y657 in both simulations suggests that these interactions are
important for stabilizing the inhibitor-protein complex. An
analogous hydrogen bond between the main chain CdO of the
Pro of phosphopeptide ligand Ac-pTyr-Asp-Lys-Pro-His-NH2
and the side chain hydroxyl group of Y651 (analogous to Y657
of Stat3) was observed in the recently reported crystal structure
of Stat1.68
In both MD trajectories, at least one water molecule bridges
the side chain NH2 of Gln to the main chain carbonyl oxygens
of either E638 or P639. For example, in the most populated
cluster of the pose A simulation (61% of the snapshots) a water
molecule coordinates a hydrogen bond between the backbone
oxygen of E638 and the side chain amide nitrogen of Gln. In
the second cluster of the pose A simulation (26% of the
snapshots), two water molecules are involved in a hydrogen
bond network between the following atoms: the backbone
oxygens of E638 and P639, the side chain oxygen of E638,
and the side chain amide nitrogen of 26.
Figure 2. Percentage of time that specific hydrogen bonds exist
between 26 and Stat3 in the final 1.5 ns of the MD simulations starting
Methionine sulfoxide, Met(O), is a glutamine isostere in
which the side chain carbonyl group is replaced by a sulfoxide
and the NH2 is replaced by a methyl group. The sulfonyl group
could serve as a hydrogen bond acceptor from Y640, but the
terminal methyl group is not a hydrogen bond donor. To test
for the presence of hydrogen bonding between the side chain
NH2 of Gln and Stat3, Met(O) and the dioxygenated analogue
methionine sulfone (Met(O2)) were incorporated into compound
28 (chosen for the ease of synthesis). The modified analogues
34 (containing Met(O)) and 35 (containing Met(O2)) resulted
in about a 10-fold loss in affinity, which is consistent with loss
of hydrogen bonding between Gln and Stat3.
In addition to the similarity of intermolecular hydrogen
bonding behavior during the final 1.5 ns of both MD trajectories,
the contributions to the binding free energies of each residue
of Stat3 are identical within standard deviation with the
exception of M660. In the complex starting with pose A, this
residue contributes -3.7 kcal/mol to the total molecular
with pose A and pose B.
the time in the two trajectories (Figure 2). A hydrogen bond
between the endocyclic carbonyl oxygen of Haic and the
backbone nitrogen of E638 exists for 60% in the MD trajectory
of pose A compared to only 23% in that of pose B (Figure 2).
Compound 26 makes a hydrogen bond with either S636 or E638
for 80% of the trajectory starting with pose A and 50% of that
starting with pose B. The data suggest that the inhibitor may
toggle back and forth in position between these two interaction
sites.
A hydrogen bond exists between the side chain amide oxygen
of Gln and the side chain hydroxyl group of Y640 approximately
70% of the final 1.5 ns of both MD simulations (Figure 2). The
difference in the percent of the simulation of this hydrogen bond
between the two conformations is only 1.9%. A hydrogen bond
also exists between the exocyclic carbonyl oxygen of Haic and
the phenolic hydroxyl group of Y657 side chain hydroxyl at

Peptidomimetic Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2435
Figure 3. Proposed model for pCinn-Haic-Gln-OH bound to the SH2 domain of Stat3. The central representative structure from the largest cluster
of the MD simulation starting with pose A is shown. (A) Compound 26 is depicted as sticks: green, carbon; blue, nitrogen; red, oxygen. Hydrogens
were removed for clarity. Stat3 is shown as a surface representation with the following residues highlighted: phosphate binding pocket (blue); S636
and P639 (purple); E638 (red); Y640 (yellow); Y657 (orange); M660 (brown). (B) Hydrogen bonds between 26 and Stat3. The side chain of E638
was removed for clarity. Stat3 amino acids are depicted in the light-blue coloring scheme: light-blue, carbon; blue, nitrogen; red, oxygen. The
images were generated with PyMol.70
mechanical/generalized Born (GB) binding free energy. Interest-
ingly, there is negligible contribution in the structure resulting
from pose B. Decomposition of the contribution of M660 into
its individual energetic components reveals a significant van
der Waals interaction (-3.2 kcal/mol) and hydrophobic interac-
tion (-0.4 kcal/mol) in the complex starting with pose A.
The discrepancy in the contribution of M660 to the binding
free energy between the two simulations correlates with the
distance between this amino acid and the hydrophobic ring
system of Haic. In the MD simulation starting with pose B, the
distance between M660 Cꢀ and the fused-ring system of Haic
was 5.1-12.8 Å in the final 1.5 ns of the trajectory. The closest
distance of 5.1 Å occurs in only one of the 1500 snapshots,
and in this instance there are three water molecules between
M660 Cꢀ and rings of Haic, which would preclude hydrophobic
interactions. The existence of water molecules between M660
and Haic persisted throughout the entire final 1.5 ns of the 3 ns
trajectory.
In contrast, in the MD trajectory of pose A, the distance
between M660 Cꢀ and Haic ranged from 3.3 to 6.3 Å
throughout the final 1.5 ns. Moreover, it is within 5.1 Å for
1433 ps out of 1500 ps (95.5%). There are no water molecules
between the M660 Cꢀ and Haic in this simulation, which, in
conjunction with the GB free energy calculations, suggests
significant hydrophobic interactions.
In the simulation of pose A, a hydrogen bond exists between
the main chain NH of M660 and the side chain hydroxyl group
of Y657 6% of the final 1.5 ns. This pair is <3.5 Å in the
remaining simulation, indicating high potential for hydrogen
bonding.
remains in place relative to the remainder of the protein. This
is likely due to water molecules that packed the space between
the loop and the inhibitor when we hydrated the molecular
system. In trajectory A the loop does not retain its intraloop
hydrogen bonds and it moves relative to the remainder of the
protein. No waters were able to fit between this loop and the
inhibitor because in the starting complex of pose A the Gln
side chain covered the loop during the solvent soaking step prior
to the molecular dynamics simulation. The lack of water
molecules allowed movement of the loop, resulting in hydro-
phobic contact between M660 and Haic and hydrogen bonding
to Y657.
It is unknown whether interaction with M660 contributes to
the binding of compound 15, in which Haic has been “split”
into leucine and the bicyclic proline analogue 2-carboxyindoline.
The reduction in affinity may be due to increased entropy
penalty resulting from rotation of the Leu ψ bond which is not
present in Haic. Alternatively, collisions between the side chain
of Leu and the closest aromatic proton of 2-carboxyindoline
may result in conformations of peptide 15 that are unfavorable
for binding.
Both E638 and P639 contribute to the free energy of binding
in the MD simulations of both poses, through van der Waals
and hydrophobic interactions. The side chains of these residues
contact the ꢀ and aromatic carbons of pCinn. These interactions
were observed in the crystal structure of the Stat3 dimer29 and
the model of Ac-pTyr-Leu-Pro-Gln-NHBn bound to the SH2
domain of Stat3.65
Integration of the experimental evidence with the molecular
dynamics data is consistent with the predicted central/
representative structure of the largest cluster (61% of the
snapshots) from the extended-conformation MD trajectory
(Figure 3). The hydrogen bonds of all but the phosphate are
depicted in Figure 3B.
M660 resides in a loop of about 10 residues from K658 to
S668. The rmsd analysis of the backbone atoms of residues
within this loop reveals significantly more deviation in the pose
A trajectory compared to that of pose B. The data seem
counterintuitive considering that M660 is “tethered” to Haic by
hydrophobic and indirect hydrogen bond interactions in trajec-
tory A but exhibits no interaction at all in trajectory B. Visual
analysis of the latter trajectory shows that the loop maintains
its ꢀ-sheet secondary structure throughout the simulation and
Conclusions
Compared to the original lead hexapeptide 1, we have
significantly reduced peptide character and by judicious incor-

2436 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Mandal et al.
mixture was stirred for 1 h more. The reaction was quenched with
slow addition of an aqueous solution of 10% Na2S2O5 and stirred
for 20 min. It was then diluted with ether (50 mL) and extracted
with an additional amount of ether (2 × 50 mL). The combined
organic parts were washed with water followed by brine and dried
(MgSO4). Solvent was removed, and the crude product was
triturated with hexane-ether. The product was collected by filtration
and dried to give 0.65 g of a white solid. To obtain more product,
the filtrate was concentrated and extracted with 5% NaHCO3 (3 ×
10 mL). The combined extracts were carefully acidified to pH
3.5-4.0 with slow addition of solid citric acid and were extracted
with DCM (3 × 25 mL). The combined organic parts were washed
with brine, dried (MgSO4), and evaporated to dryness to give the
product as white foam (0.21 g). Compound 39 was used in solid
phase synthesis with no further purification. 1H NMR (CDCl3, 500
MHz) δ 1.5 (s, 18H), 6.36 (d, J ) 16.0 Hz, 1H), 7.25 (d, J ) 8.0
Hz, 2H), 7.5 (d, J ) 8.0 Hz, 2H), 7.7 (d, J ) 16.0 Hz, 1H).
Synthesis of Fmoc-(5-[(R)-amino]-1,2,4,5,6,7-hexahydroaze-
pino[3,2,1-hi]indole-4-one-2-(S)-carboxylate), 45. A solution of
(R)- 4,7-dioxo-5-(2,2,2-trifluoroacetylamino)-1,2,4,5,6,7-hexahy-
droazepino[3,2,1-hi]indole-2-carboxylic acid methyl ester (43)44,45
(850 mg, 2.3 mmol) was suspended in AcOH (65 mL) and
hydrogenated with 10% Pd/C (850 mg) at 50 psi and ambient
temperature for 24 h. The catalyst was removed by filtration through
Celite and the solvent removed under reduced pressure. The residue
was dissolved in EtOAc (50 mL) and the solution washed with
saturated NaHCO3 (2 × 50 mL) and saturated brine (50 mL) before
drying (MgSO4). Filtering and removal of the solvent under reduced
pressure gave a white solid (744 mg, 87%) which was used without
further purification. 1H NMR (CDCl3, 300 MHz) δ 2.0 (m,1H), 2.5
(m,1H), 3.1 (m,2H), 3.5 (m,2H), 3.8 (s,3H), 4.5 (dd,1H), 5.1 (m,1H),
7.1 (m,3H), 7.8 (s,1H).
poration of conformational constraints have increased the affinity
nearly 2-fold. Constraining the C-CR-Cꢀ-Cγ dihedral angle
of the phosphotyrosine to 180° led to a major increase in affinity,
likely the result of reduced entropy penalty of binding. The
affinities of compounds incorporating Haic provide further
evidence that the Leu-Pro peptide bond in peptide inhibitors
1-4 is in the trans conformation. Modeling of pCinn-Haic-Gln-
OH suggests that the basis of affinity of the Haic molecules is,
in addition to the ionic interactions with the phosphate, mediated
by hydrogen bonds between the inhibitor and various groups
on the protein and by hydrophobic interactions with the aromatic
ring of the dipeptide mimic. Loop 658-668 of Stat3 moved in
relation to the bulk of the protein. This movement placed M660
in proximity to the inhibitor, allowing a hydrophobic contact
with the aromatic groups of Haic, which may be one reason
why this Leu-Pro mimic was more effective than the ABN
groups. Compound 21, pCinn-Haic-Gln-NHBn, is a peptido-
mimetic containing only one natural amino acid, Gln.
Currently two models have been published on the nature of
phosphopeptide binding to the SH2 domain of Stat3. Shao et
al.66 proposed that on docking to the protein, peptides form
ꢀ-turns and that, in addition to the phosphotyrosine interactions,
the side chain carbonyl group of Gln at position pY + 3
participates in hydrogen bonding with the main chain NH of
E638. McMurray65 hypothesized in a model of peptide 4 that
the side chain of Gln participates in an extensive hydrogen
bonding network in which the NH2 interacts with the CdO’s
of E638 and P639 and carbonyl oxygen participates in a
hydrogen bond with the side chain of Q644 and a water
mediated hydrogen bond with the side chain of E638. The
models generated here suggest that the side chain of Gln does
indeed participate in hydrogen bonds but to residues that are
different from the previous models. It seems reasonable that
the constrained nature of Haic does not allow the Gln to insert
itself as deeply into the pocket as does the more flexible Leu-
Pro central dipeptide in McMurray’s model. However, Haic
provides interactions with M660 that the peptide does not. These,
in addition to the entropic gain of restricted rotation of the pCinn
and Leu ψ dihedral angle, provide binding energy that peptides
such as 4 do not exhibit. Thus, the IC50 of the Haic peptides is
low. The interactions suggested by the modeling have generated
ideas for modification of Haic and Gln, which will be reported
on completion of those studies.
The intermediate (520 mg, 1.46 mmol) was dissolved in THF
(15 mL), and LiOH (210 mg, 8.76 mmol) in H2O (7.5 mL) was
added. The mixture was stirred vigorously for 2 h and acidified
with 2 N HCl (6 mL), and the solvents were removed under reduced
pressure. Sodium carbonate (950 mg, 8.93 mmol) dissolved in water
(18 mL) was added to the solid residue and the mixture stirred for
5 min. Fmoc-OSu (492 mg, 1.46 mmol) dissolved in dioxane (18
mL) was added, and the stirring continued for 20 h. The solvent
was removed under reduced pressure, and water (150 mL) was
added to the solid residue and the mixture acidified with 2 N HCl
(12 mL). The aqueous suspension was extracted with EtOAc (2 ×
100 mL). The extract was washed with water (3 × 100 mL) and
saturated brine (100 mL) before drying over anhydrous magnesium
sulfate. Removal of the solvent gave a solid residue (649 mg) of
which an amount of 600 mg was purified by silica gel chromatog-
raphy: EtOAc (300 mL), EtOAc/AcOH 99.7:0.3 (650 mL). Yield:
Several authors have reported the development of Stat3
inhibitors that incorporate phopshotyrosine or phosphotyrosine
mimic.71-76 These compounds did not contain glutamine or a
mimic and displayed low affinity for the target, as judged by
electrophoretic mobility shift assays or fluorescence polarization.
Modifications to the structure of glutamine reported previ-
ously15,16,69 and here point to the importance of the alkylcar-
boxamide group for affinity. Our previous model65 and the
models generated here suggest that the side chain amide group
participates in hydrogen bonding interactions with Stat3 that
have a major impact on binding energy.
1
353 mg, 56%. HRMS (M + H), 469.1742 (theory, 469.1763). H
NMR (DMSO-d6, 300 MHz) δ 2.0 (m, 2H), 3.0 (m, 3H), 3.4 (m,
1H), 4.2 (m, 4H), 4.9 (dd, 1H), 7.0 (m, 3H), 7.3 (m, 4H), 7.8 (m,
5H), 12.7 (s, 1H). 13C NMR (DMSO- d6) δ 27.6, 30.8, 31.1, 47.2,
56.6, 61.1, 66.1, 120.6, 123.8, 125.7, 126.1, 127.6, 128.1, 129.7,
131.7, 138.9, 141.2, 144.3, 144.4, 156.3, 169.4, 172.9.
Synthesis of (S)-7-Hydroxy-4-oxo-5-(2,2,2-trifluoroacetylamino)-
1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxylic Acid
Methyl Ester, 46. A solution of 4244,45 (500 mg, 1.35 mmol) and
AcOH (1 mL) in THF (10 mL) was hydrogenated with 10% Pd/C
(21 mg) at 49 psi and ambient temperature for 4 h. The catalyst
was removed by filtration through Celite and the filtrate washed
with saturated aqueous NaHCO3 (2 × 30 mL) and saturated brine
(20 mL) before drying (MgSO4). Filtration and removal of the
solvent under reduced pressure gave a white solid (470 mg).
Purification was by silica gel chromatography: EtOAc/hexane 1:4
(400 mL), EtOAc/hexane 3:7 (180 mL), 30 mg (1), EtOAc/hexane
3:7 (680 mL), 200 mg (3), EtOAc/hexane 4:6 (410 mL) gave 430
Experimental Procedures
Synthesis of 4-(Di-tert-butoxyphosphoryloxy)cinnamic Acid,
39. To a suspension of 4-hydroxycinnamic acid (1.0 g, 6.1 mmol)
and tert-butyldimethylsilyl chloride (0.92 g, 6.1mmol) in 10 mL
of dry THF, N-ethylmorpholine (0.6 mL, 5.5 mmol) was added at
room temperature. After 10 min, 1H-tetrazole (1.5 g, 21.3 mmol)
was added in one portion followed by the addition of di-tert-butyl
N,N-diethylphosphoramidite (4.2 g, 15.2 mmol) in 10 mL of dry
THF. After the mixture was stirred for 2 h, 70% aqueous tert-butyl
hydroperoxide (2.5 mL, 18.3 mmol) was added at 0 °C and the
1
mg of 46 (86%): H NMR (CDCl3, 300 MHz) δ 2.4 (m,1H), 2.7
(m,1H), 3.2 (dd,1H), 3.5 (m,1H), 3.8 (s,3H), 4.5 (m,1H), 5.25
(m,1H), 5.35 (m,1H), 7.2 (m,2H), 7.5 (m,1H), 7.8 (d,1H) ppm.

Peptidomimetic Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2437
Synthesis of Fmoc-(5-[(S)-Amino]-1,2,4,5-tetrahydroazepi-
no[3,2,1-hi]indole-4-one-2-(S)-carboxylate), 47. Compound 46
(780 mg, 2.1 mmol) was dissolved in trifluoroacetic acid (10 mL)
and stirred at room temperature for 5 h. The solvent was removed
under reduced pressure and the residue dissolved in EtOAc (100
mL). This solution was washed with 5% NaHCO3 (2 × 75 mL)
and saturated brine (50 mL) before drying over MgSO4. Filtration
and removal of the solvent gave a solid (760 mg) which was purified
by silica gel chromatography: EtOAc/hexane 1:4 (400 mL), 680
mg, 92% (4), mp 120-124 °C. 1H NMR (CDCl3, 300 MHz) δ 3.3
(dd,1H), 3.65 (m,1H), 3.7 (s,3H), 4.7 (m,1H), 5.4 (dd,1H), 5.7
(dd,1H),6.8 (dd,1H), 7.2 (m,3H), 7.9 (s,1H).
2H), 4.32 (m, 1H), 7.33 (t, J ) 7.0 Hz, 2H), 7.42 (t, J ) 7.0 Hz,
2H), 7.5 (d, J ) 8.0 Hz, 1H), 7.74 (t, J ) 7.0 Hz, 2H), 7.8 (d, J )
4.5 Hz), 7.9 (d, J ) 7.5 Hz, 2H). 13C NMR (DMSO-d6, 125.0 MHz)
δ 26.1, 27.7, 28.2, 31.9, 47.2, 54.4, 66.1, 80.1, 120.6, 125.8, 127.5,
128.1, 141.2, 144.2, 144.4, 156.4, 172.1. Anal. (C25H30N2O5) Calcd:
C, 68.47; H, 6.90; N, 6.39. Found: C, 68.45; H, 6.90; N, 6.37.
Synthesis of Fmoc-Glu-NHMe, 50b. 50b was prepared from
49b (0.50 g) using identical conditions as for 50a. Yield: 0.38 g of
1
a white powder. H NMR (DMSO-d6, 500 MHz) δ 1.77 (m, 1H),
1.95 (m, 1H), 2.25 (m, 2H), 2.6 (s, 3H), 4.0 (m, 1H), 4.2-4.33 (m,
3H), 7.33 (t, J ) 7.0 Hz, 2H), 7.42 (t, J ) 7.0 Hz, 2H), 7.5 (d, J
) 8.0 Hz, 1H), 7.74 (m, 2H), 7.8 (d, J ) 4.5 Hz, 1H), 7.9 (d, J )
7.5 Hz, 2H), 12.1 (s, 1H). 13C NMR (DMSO-d6, 125.0 MHz) δ
26.1, 27.7, 30.7, 47.2, 54.5 66.1, 120.6, 125.8, 127.5, 128.1, 141.2,
144.2, 144.4, 156.4, 172.2, 174.3. Anal. (C21H22N2O5) Calcd: C,
65.96; H, 5.80; N, 7.33. Found: C, 66.11; H, 5.95; N, 7.23.
Synthesis of Fmoc-Glu (OtBu)-NHiPr, 49c. 49c was prepared
from Fmoc-Glu(OtBu)-OH (1 g, 2.35 mmol) using identical
conditions as for 49a except that isopropylamine (0.2 mL, 2.6
mmol) in 5 mL of dry CH2Cl2 was used instead of MeOH. Yield:
The unsaturated intermediate (520 mg, 1.49 mmol) was dissolved
in THF (18 mL), and LiOH (212 mg, 8.80 mmol) in H2O (9 mL)
was added. The mixture was stirred vigorously for 2 h and acidified
with HCl, and the solvent was removed under reduced pressure.
Sodium carbonate (933 mg, 8.80 mmol) dissolved in water (15 mL)
was added to the solid residue and the mixture stirred for 5 min.
Fmoc-OSu (562 mg, 1.49 mmol) dissolved in dioxane (15 mL)
was added, and the stirring continued for 20 h. The solvent was
removed under reduced pressure, water (70 mL) was added to the
solid residue, and the mixture was acidified with 2 N HCl (6 mL).
The aqueous suspension was extracted with EtOAc (3 × 70 mL).
The extract was washed with water (4 × 70 mL) and saturated
brine (70 mL) before drying (MgSO4). Removal of the solvent gave
a solid residue (734 mg) which was purified by silica gel
chromatography: EtOAc/hexane 1:1 (250 mL), EtOAc/AcOH 99.7:
0.3 (410 mL). Yield, 317 mg, 46%. HRMS, 467.1587 (theory,
467.1607). 1H NMR (DMSO, 300 MHz) δ 3.1 (d,1H), 3.7 (m,1H),
4.3 (m,4H), 5.1 (d,1H), 5.7 (dd,1H), 6.8 (dd,1H), 7.1 (t,1H), 7.2
(d,1H), 7.3 (m,3H), 7.4 (t,2H), 7.7 (t,2H), 7.9 (d,2H), 8.1 (d,1H),
12.9 (s,1H) ppm. 13C NMR (DMSO, 300 MHz) δ 32.6, 47.1, 54.6,
61.8, 66.3, 120.6, 124.0, 125.1, 125.7, 125.8, 126.3, 127.6, 128.1,
128.3, 132.8, 139.2, 141.2, 144.2, 144.3, 156.5, 166.4, 172.5.
Synthesis of Fmoc-Glu(OtBu)-OMe, 49a. To a stirred solution
of Fmoc-Glu(OtBu)-OH (1.0 g, 2.35 mmol), DIPEA (0.9 mL, 4.7
mmol) in 10 mL of dry CH2Cl2 and 2 mL of dry MeOH, at 0 °C,
a solution of PyBOP (1.4 g, 2.6mmol) in 5 mL of dry CH2Cl2 was
added dropwise. The mixture was stirred overnight and was
transferred to a separatory funnel, with an additional amount of
CH2Cl2 (30 mL). The organic layer then washed with 10% HCl,
10% NaHCO3, brine and dried (MgSO4). Solvent was removed,
the crude then purified by silica gel column chromatography elution
with 25% EtOAc-hexane to yield yielded 0.90 g of pure Fmoc-
1
0.90 g of pure Fmoc-Glu(OtBu)-NHCH(Me)2. H NMR (DMSO-
d6, 500 MHz) δ 1.07 (m, 6H), 1.4 (s, 9H), 1.78 (m, 1H), 1.9 (m,
1H), 2.23 (m, 2H), 3.86 (m, 1H), 4.0 (m, 1H), 4.22-4.3 (m, 3H),
7.33 (m, 2H), 7.40-7.46 (m, 3H), 7.65-7.8 (m, 3H), 7.9 (d, J )
7.0 Hz, 2H). 13C NMR (DMSO-d6, 125.0 MHz) δ 22.7, 22.8, 28.2,
31.9, 47.2, 54.4, 66.1, 80.1, 120.5, 125.8, 127.5, 128.1, 141.2, 144.2,
144.4, 156.3, 170.7, 172.1. Anal. (C27H34N2O5) Calcd: C, 69.50;
H, 7.35; N, 6.00. Found: C, 69.11; H, 7.32; N, 5.91.
Synthesis of Fmoc-Glu-NHiPr, 50c. 50c was prepared from 49c
(0.50 g) using identical conditions as for 50a. Yield: 0.40 g of a
1
white powder. H NMR (DMSO-d6, 500 MHz) δ 1.06 (m, 6H),
1.75 (m, 1H), 1.88 (m, 1H), 2.26 (m, 2H), 3.83 (m, 1H), 4.0 (m,
1H), 4.2-4.3 (m, 3H), 7.33 (m, 2H), 7.4-7.46 (m, 3H), 7.74 (m,
3H), 7.9 (d, J ) 7.5 Hz, 2H), 12.1 (s, 1H). 13C NMR (DMSO-d6,
125.0 MHz) δ 22.7, 22.8, 28.1, 30.8, 47.2, 54.4, 66.1, 120.5, 125.8,
127.5, 128.1, 141.2, 144.2, 144.4, 156.3, 170.8, 174.4. Anal.
(C23H26N2O5) Calcd: C, 67.30; H, 6.38; N, 6.82. Found: C, 66.38;
H, 6.39; N, 6.68.
Molecular Modeling of the Interactions of 26 and the SH2
domain of Stat3. The coordinates for Stat3ꢀ (PDB code 1bg1)29
were obtained from the Research Collaboratory for Structural
Bioinformatics (RCSB) Brookhaven Protein Data Bank (PDB,
http://www.rcsb.org/pdb).77 Compound 26 and fragments thereof
were computationally constructed using the Builder module of
InsightII 98.0.78 The coordinates of Haic from a peptidomimetic
inhibitor bound to the MHC class II HLA-DR (PDB code 1d5x)79
were used as a template for this dipeptide mimic.
1
Glu(OtBu)-OMe. H NMR (DMSO-d6, 500 MHz) δ 1.4 (s, 9H),
1.8 (m, 1H), 1.95 (m, 1H), 2.3 (m, 2H), 3.64 (s, 3H), 4.1 (m, 1H),
4.24 (m, 1H), 4.32 (d, J ) 7.0 Hz, 2H), 7.34 (t, J ) 7.0 Hz, 2H),
7.43 (t, J) 7.0 Hz, 2H), 7.72 (m, 2H), 7.78 (d, J ) 8.0 Hz, 1H),
7.91 (d, J ) 7.5 Hz, 2H). 13C NMR (DMSO-d6, 125.0 MHz) δ
26.5, 28.2, 31.5, 47.1, 52.4, 53.4, 66.1, 80.3, 120.6, 125.7, 127.5,
128.1, 141.2, 144.3, 156.5, 171.9, 173.0. Anal. (C25H29NO6) Calcd:
C, 68.32; H, 6.65; N, 3.19. Found: C, 68.50; H, 6.70; N, 3.24.
Synthesis of Fmoc-Glu-OMe, 50a. Fmoc-Glu(OtBu)-OMe (0.50
g) was treated with neat TFA (5 mL) for 1 h. The TFA was removed
under vacuum, and residual acid was removed by adding and
evaporating tolune (2 × 5 mL). The product was recrystallized from
ether-hexane to yield 0.40 g of a white powder. 1H NMR (DMSO-
d6, 500 MHz) δ 1.82 (m, 1H), 1.98 (m, 1H), 2.34 (m, 2H), 3.64 (s,
3H), 4.1 (m, 1H), 4.24 (m, 1H), 4.32 (m, 2H), 7.35 (t, J ) 7.5 Hz,
2H), 7.43 (t, J ) 7.5 Hz, 2H), 7.73 (d, J ) 7.5 Hz, 2H), 7.8 (d, J
) 8.0 Hz, 1H), 7.9 (d, J ) 7.5 Hz, 2H), 12.2 (s, 1H). 13C NMR
(DMSO-d6, 125.0 MHz) δ 26.4, 30.4, 47.1, 52.4, 53.5, 66.1, 120.6,
125.7, 127.5, 128.1, 141.2, 144.2, 156.6, 173.0, 174.1. Anal.
(C21H21NO6) Calcd: C, 65.79; H, 5.52; N, 3.65. Found: C, 64.99;
H, 5.53; N, 3.66.
Preparation of Ligands. Fragments pCinn-NH2 and H-Haic-
Gln-OH were assigned consistent valence force-field (CVFF) partial
charges with InsightII 98.0/FDiscover 2.98.78 Two of the oxygen
atoms of the pCinn-NH2 phosphate group were manually assigned
a formal charge of -1, bringing the total formal charge of the
fragment to -2. One of the oxygen atoms of the carboxyl group
of H-Haic-Gln-OH was manually assigned a formal charge of -1,
bringing the total formal charge of the fragment to -1. Formal
charges were initially assigned with the CVFF in InsightII 98.0 so
that the bond order in the output structure would be correctly set
for subsequent processing in the Antechamber module of the
Assisted Model Building with Energy Refinement 8 (Amber 8)
molecular simulation programs.80 pCinn-NH2 and H-Haic-Gln-OH
were processed separately because the complete compound 26 (68
atoms) was too large for the Antechamber module. Atomic partial
charges, originally assigned through CVFF, were adjusted using
the generalized Amber force field (GAFF). Partial atomic charges
were assigned using the AM1-bond charge correction (AM1-BCC)
method.81 GAFF was also used to assign bond and angle force
field potentials.82 To create the complete compound 26, the NH2
group of pCinn-NH2 was deleted in InsightII 98.078 and the acyl
group was appended to the exocyclic amino group of N-Haic-Gln-
OH in the Leap module of Amber 8. To compensate for the abrupt
change in charge distribution caused by joining of these fragments,
Synthesis of Fmoc-Glu(OtBu)-NHMe, 49b. 49b was prepared
from Fmoc-Glu(OtBu)-OH (1 g, 2.35 mmol) using identical
conditions as for 49a except that MeNH2 ·HCl (0.16 g, 2.35 mmol)
1
was used in place of the MeOH. Yield, 0.93 g of 49b. H NMR
(DMSO-d6, 500 MHz) δ 1.4 (s, 9H), 1.75 (m, 1H), 1.9 (m, 1H),
2.2 (m, 2H), 2.6 (d, J ) 4.5 Hz, 3H), 3.96 (m, 1H), 4.2-4.3 (m,

2438 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Mandal et al.
a third fragment comprising pCinn, up to but excluding the
phosphate and the phenyl groups, and Haic was generated and
processed in Antechamber. The overlapping charges assigned to
the third fragment were used to replace the corresponding charges
in the complete 26 construct to better approximate a true charge
distribution.
pCinn-Haic docking conditions were used for 26 except that 42,
40, and 42 grid points in the X, Y, and Z directions, respectively,
were calculated to create the final affinity grid and only three of
six torsdof were used in the FEB calculation. After 26 was docked
to the Stat3 SH2 domain, one structure was selected as a precursor
to another starting structure. A second docking experiment was
executed with parameters identical to those of the first 26. Two
structures from the second docking experiment were selected as
final docking poses, one of which was a member of the largest
docking cluster (extended conformation, pose A) and the other
belonging to a cluster that placed the glutamine side chain in the
pocket formed by Stat3 residues E638, P639, and Y640 (bent
conformation, pose B).
Implicit-Solvent Energy Minimization. The two poses were
assigned Amber99 and GAFF force field parameters77 in InsightII
98.0/CDiscover 3,78 respectively. The energy landscapes of both
systems were searched for local energy minima to remove high
energy atomic “clashes” that may have arisen between Stat3 and
26. Energy minimizations were executed in a stepwise manner using
a distance-dependent dielectric constant to implicitly represent
continuum solvent and without nonbonded (van der Waals and
electrostatic) cutoffs. Each minimization was terminated when the
change in total energy of the complex was negligible and the
derivative of the gradient was less than 0.01.
Pose A. The Stat3/26 complex of pose A was subjected to the
following 24 500 step energy minimization procedure: (1) 1000 SD
followed by 1500 CG on compound 26 hydrogens; (2) 1000
SD followed by 1000 CG on 26 and Stat3 hydrogens; (3) 2000 SD
followed by 3000 CG on the 26 hydrogens and the Stat3 side chains;
(4) 2000 SD followed by 3000 CG on 26, and the Stat3 side chains
including the R carbons; (5) 2000 SD followed by 8000 CG on all
atoms of both 26 and Stat3.
Pose B. The Stat3/26 complex of pose B was subjected to the
following 13 700 step energy minimization procedure: (1) 600 SD
followed by 600 CG on compound 26 hydrogens; (2) 500 CG on
the Stat3 hydrogens; (3) 500 SD followed by 500 CG on the Stat3
side chains excluding the R carbons; (4) 1000 SD followed by 1000
CG on 26 and the Stat3 side chains excluding the R carbons; 5)
1000 SD followed by 8000 CG on all atoms of both 26 and Stat3.
Explicit-Solvent Molecular Dynamics. Both docking poses
were solvated in a 15 Å truncated octahedral box with explicit water
molecules using the xleap module of Amber 8. The total charge
on the Stat3 protein, which was protonated with the CVFF at pH
7.4, was +1, and the total charge on 26 was -3, bringing the total
charge on the poses to -2. The charges on each simulation system
were neutralized by replacing two of the explicit waters in the
truncated octahedral boxes with two sodium ions (Na+) at a distance
of greater than 3.5 Å from the protein-ligand complexes. This
rendered the net charge on the simulation system zero, which is a
requirement of the method that we used for treating long-range
electrostatic effects (see below). Water molecules were modeled
using the TIP3P force field parameters.87
Energy Minimization. The Amber99 force field83 was converted
by the AmberFFC 1.3 program84 to a format which could be read
by InsightII 98.0/CDiscover 3.78 To reduce the number of torsions
during the automated rigid-protein/flexible-ligand docking, 26 was
separated in InsightII 98.078 into pCinn-NH2 and pCinn-Haic-OH
fragments. The pCinn-NH2 atoms, excluding the phosphophenyl
group, were subjected to 40 steps of steepest-descent (SD) followed
by 200 steps of conjugate-gradient (CG) energy minimizations in
InsightII 98.0/CDiscover 3,78 at which point the change in total
energy (nonbonded and internal) was negligible and the derivative
of the gradient was less than 0.01. Energy minimizations were
executed using a distance-dependent dielectric constant to implicitly
represent continuum solvent and without nonbonded (electrostatic
and van der Waals) cutoffs. The coordinates for the atoms of the
phosphophenyl group were held fixed during the minimizations.
Automated Rigid-Protein/Flexible-Ligand Docking. Hydrogen
atoms were added to the Stat3 crystal structure at pH 7.4 in InsightII
98.0.78 The charges and Lennard-Jones potentials for all the atoms,
including the added hydrogens, were subsequently adjusted with
Amber99 force field parameters.83 All ligands (26 and its fragments)
were docked to Stat3 with Autodock 4.039 in an automated rigid-
protein/flexible-ligand manner, using a free-energy and charge-based
desolvation scoring function.85 Stat3 (residues 136-688) served
as the rigid protein, and all atomic partial charges assigned by the
Amber99 force field83 in InsightII 98.078 were accepted. Atomic
partial charges assigned by Antechamber to 26 and its fragments
were accepted. All ligands were docked to the Stat3 SH2 domain
using the Lamarckian genetic algorithm (LGA) method86 using a
distance-dependent dielectric. A maximum of 256 LGA runs were
executed for each docking experiment. The torsional free energies
(internal 1-4 interactions) and internal electrostatics of all ligands
were included in the free energy calculations. The ligand structures
resulting from each docking study were clustered with an rmsd of
1.0 Å. All dockings utilized ligand-centered grid maps of 0.375 Å
spacing for each atom type of the ligand and for the electrostatic
(e) and desolvation (d) maps.
Docking of pCinn-NH2. The pCinn-NH2 fragment was docked
to the Stat3 SH2 domain first. The C, N, P, A, OA, HD, d, and e
centered grid maps, with 36, 30, and 32 grid points in the X, Y,
and Z directions, respectively, were calculated to create the final
affinity grid. LGA was used to generate an initial population of
300 different structures. The structure with the lowest (most
favorable) free energy of binding (FEB) survived to seed the next
population of 300 structures. A maximum of 27 000 generations
of populations of 300 structures were seeded, and a maximum of
25 million energy calculations were evaluated. Autodock 4.0
identified four torsional degrees of freedom (torsdof) in pCinn-
NH2 to be used in the FEB calculation, and all four torsions (ndihe)
were active and allowed to rotate in 30° increments during the
docking. The pCinn-NH2 fragment was allowed to translate in 1 Å
increments and to rotate about its axis in 30° increments. All other
parameters, including the LGA parameters, were assigned the
Autodock 4.0 default values.
Docking of pCinn-Haic-OH. After the pCinn-NH2 fragment was
docked to the Stat3 SH2 domain, one structure was selected as a
precursor to the pCinn-Haic-OH starting structure. The same set
of docking conditions used for pCinn-NH2 was used for pCinn-
Haic-OH except that 40, 40, and 54 grid points in the X, Y, and Z
directions, respectively, were calculated to create the final affinity
grid. Also, a maximum of 2.5 million energy calculations were
evaluated, and only three of six torsdof were used in the FEB
calculation.
Systems Setup. The heating, equilibration, and production phases
of all molecular dynamics (MD) simulations were executed in
NAMD using a fixed number of particles, fixed pressure, and fixed
temperature (NPT) ensemble. The pressures of the systems were
fixed to 1.013 25 bar by coupling to a Berendsen pressure bath88
and were assigned a compressibility of 4.57 × 10-5 bar. After the
systems were heated and during equilibration, the temperatures of
the systems were fixed to 310 K by coupling to a temperature bath
with temperature reassignment every 1 ps. Atomic positions,
trajectories, forces, and other system parameters were sampled in
2 fs time intervals by fixing hydrogen covalent bond lengths using
the SHAKE algorithm89 with a 1.0 × 10-8 Å tolerance. A 12 Å
interaction cutoff, which gradually approached zero beginning at a
distance of 8 Å, was used to truncate the nonbonded and long-
range electrostatics of the systems. Any atom pairs that vacillated
beyond and within the 12 Å cutoff but did not exceed 13 Å were
included in the calculations. The particle mesh Ewald (PME)
summation method90 was employed to correct for the cutoffs of
all long-range electrostatic interactions. The octahedral solvent
Docking of pCinn-Haic-Gln-OH, 26. One structure from the
pCinn-Haic docking study was selected as a precursor for the
starting structure for the complete inhibitor 26. The same set of

Peptidomimetic Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2439
boxes of the systems were replicated in all dimensions using
periodic boundary conditions. The periodic cells of both systems
were periodically adjusted as the size (volume) of the systems
changed during the equilibration phases of the MD simulations.
Each unit of the cell was allowed to adjust its size (volume) in
response to changes that may have arisen during NPT sampling.
The Amber9982 force field was used by NAMD to assign all atomic
parameters and implement all force field equations.
Energy Minimization. The energy landscapes of both systems
were searched for local energy minima to remove high energy
atomic “clashes” that may have arisen between the added waters,
the added ions, and the poses. Energy minimizations were executed
in a stepwise manner in explicit solvent with a 12 Å nonbonded
cutoff. Each minimization step was terminated when the change in
total energy of the docking pose and the change in the derivative
of the gradient was negligible. SD minimizations were executed
with the Sander module of Amber 8, and CG minimizations were
executed with NAMD 2.61B.91
Heating. Atomic velocities were reassigned at higher kinetic
energies until the average kinetic energies of the systems were
slowly, over the span of 31 ps (ps) and with temperature
reassignment every 1 ps, brought to 310 K. After the average kinetic
energies of the systems reached 310 K, the average kinetic energies
were held at 310 K for 19 ps by temperature reassignment every 1
ps, as necessary.
Equilibration. After the systems were heated, velocities were
reassigned in both systems every 1 ps to keep the average kinetic
energy at 310 K until the change in total energy as a function of
time became negligible.
the Stat3 backbone atoms were least-squares-fitted and a 1 Å cutoff
was used. The representative structure from each cluster was the
structure with the smallest average distance to all other structures
in the cluster.
Poisson-Boltzmann and Generalized Born Electrostatics. For
both systems, Amber 880 was used to separately calculate the
solvation energy of the isolated implicitly solvated Stat3, the isolated
implicitly solvated 26, and the isolated implicitly solvated docking
pose. The polar (electrostatic/Coulombic) component of the sol-
vation energy was calculated using finite-difference methods to
solve the linearized Poisson-Boltzmann (PB) equation. The
generalized Born (GB) method was also used to approximate the
linearized PB equation and calculate electrostatic and nonpolar
solvation energies. The nonpolar component of the solvation energy
was calculated by measuring the solvent-accessible surface area
(SASA) with a 1.4 Å water probe and subsequently multiplying
the SASA value with the empirical constant 0.0072 (kcal/mol)/Å2.
The total solvation energy was determined by summing the nonpolar
and polar components. The total solvation contribution to the free
energy of binding (FEB) of 26 to Stat3 was determined by
subtracting the total solvation energy of the isolated implicitly
solvated Stat3 and the total solvation energy of the isolated
implicitly solvated Stat3 from the total solvation energy of the
isolated implicitly solvated 26. For the electrostatics calculations,
the solute dielectric was set to 2, the solvent dielectric constant
was set to 80, the ionic strength set to 0, and the grid spacing set
to 0.25 Å. For the gas phase (vacuum) molecular mechanics
energies (Coulombic/electrostatic, van der Waals, internal), the
external dielectric was set to 1. MM-PBSA and MM-GBSA
calculations were performed on 150 structures from every 100 ps
of the final 1.5 ns of a 3 ns trajectory.
Production. After equilibration, both systems were allowed to
evolve without temperature reassignment. The size of the periodic
cell remained constant during NPT sampling. A 3 ns MD trajectory
was sampled for both systems.
Acknowledgment. We are grateful to the National Cancer
Institute (Grant 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
CA016672 for the support of our NMR facility and both the
M. D. Anderson Cancer Center Proteomics Facility and Ray-
mond Jacobson of the Structural Biology Program for mass
spectrometry. J.M.B. and D.L. acknowledge support from the
NASA University Research Center through Texas Southern
University. Funding as an Odyssey Fellow (Z.R.) was supported
by the Odyssey Program and the Cockrell Foundation Award
for Scientific Achievement at University of Texas M. D.
Anderson Cancer Center.
Pose A. The solvated docking-pose A contained a total of 46 396
atoms, which included 194 Stat3 residues (3111 atoms), 68
compound 26 atoms, 14 405 explicit waters (43 215 atoms), and
two Na+ ions. The system was subjected to the following
minimization procedure: (1) 7000 SD followed by 46 000 CG steps
on the solvent; (2) 31 ps (15 500 time steps) of solvent heating
followed by 19 ps (9500 time steps) of temperature reassignment
to 310 K; (3) 493 ps (246 500 time steps) of solvent equilibration
with temperature reassignment to 310 K; (4) 2000 SD followed by
6000 CG steps on the docking pose; (5) 1000 CG energy
minimization on the system; (6) 31 ps (15 500 time steps) of system
heating followed by 19 ps (9500 time steps) of temperature
reassignment to 310 K; (7) 200 ps (100 000 time steps) of system
equilibration with temperature reassignment to 310K; (8) 3 ns
system sampling without temperature reassignment.
Pose B. The solvated docking-pose B contained a total of 44 944
atoms;, which included 194 Stat3 residues (3111 atoms), 68
compound 26 atoms, 13 921 explicit waters (41 763 atoms), and
two Na+ atoms. The system was subjected to the following
minimization procedure: (1) 7000 SD steps on the solvent; (2) 6000
CG energy minimization on the solvent hydrogens; (3) 12 000 CG
steps on the solvent; (4) 31 ps (15 500 time steps) of solvent heating
followed by 19 ps (9500 time steps) of temperature reassignment
to 310 K; (5) 810 ps (405 000 time steps) of solvent equilibration
with temperature reassignment to 310 K; (6) 1000 SD followed by
6000 CG energy minimizations on the docking pose; (7) 31 ps
(15 500 time steps) of heating with temperature reassignment to
310 K (37 °C); (8) 100 ps (50 000 time steps) of equilibration with
temperature reassignment to 310 K; (9) 200 ps (100 000 time steps)
of system equilibration temperature reassignment to 310K; (10) 3
ns system sampling without temperature reassignment.
Supporting Information Available: General synthesis proce-
dures, general HPLC procedures, a table of mass spectral data of
compounds 5-34, HPLC chromatograms of compounds 5-34, and
NMR spectra of compounds 45 and 47. This material is available
free of charge via the Internet at http://pubs.acs.org.
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