Design, synthesis, and in vitro activity of peptidomimetic inhibitors of myeloid differentiation factor 88

Article abstract of DOI:10.1021/jm070723u

We describe the design and synthesis of a peptidomimetic library derived from the heptapeptide AC-RDVLPGT-NH₂, belonging to the Toll/IL-1 receptor (TIR) domain of the adaptor protein MyD88 and effective in inhibiting its homodimerization. The a

Full text of DOI:10.1021/jm070723u

J. Med. Chem. 2008, 51, 1189–1202
1189
Design, Synthesis, and In Vitro Activity of Peptidomimetic Inhibitors of Myeloid Differentiation
Factor 88
Nicola Fantò,*,† Grazia Gallo,† Andrea Ciacci,† Mauro Semproni,† Davide Vignola,† Marco Quaglia,† Valentina Bombardi,‡
Domenico Mastroianni,‡ M. Pia Zibella,‡ Giancarlo Basile,‡ Marica Sassano,‡ Vito Ruggiero,† Rita De Santis,† and
Paolo Carminati†
Research and DeVelopment, Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Pomezia (RM), Italy, and Tecnogen S.p.A.,
81015 Piana di Monte Verna (CE), Italy
ReceiVed June 21, 2007
We describe the design and synthesis of a peptidomimetic library derived from the heptapeptide
Ac-RDVLPGT-NH2, belonging to the Toll/IL-1 receptor (TIR) domain of the adaptor protein MyD88 and
effective in inhibiting its homodimerization. The ability of the peptidomimetics to inhibit protein–protein
interaction was assessed by yeast 2-hybrid assay and further validated in a mammalian cell system by
evaluating the inhibition of NF-κB activation, a transcription factor downstream of MyD88 signaling pathway
that allows production of essential effector molecules for immune and inflammatory responses.
Introduction
Among the transcription factors, nuclear factor-κB (NF-κBa) is
one of the most intensively investigated owing to its involvement
in inflammation, immunity, cell proliferation, and apoptosis. NF-
κB recognizes consensus sequences for the enhancers of various
genes coding for proinflammatory cytokines (TNF, IL-1, IL-2,
IL-6, IL-11, IL-17, GM-CSF), chemokines (IL-8, RANTES,
MIP-1R, MCP-2), adhesive molecules (ICAM-1, VCAM-1,
E-selectin), and enzymes producing inflammatory mediators
(iNOS and COX2).1
Figure 1. Schematic representation of the three different portions that
characterize the consensus peptide (yellow ) ꢀ-turn; grey ) hydro-
phobic spacer; blue ) charged portion).
In response to various damaging stimuli, NF-κB activation
is observed in almost all cells involved in the immune
response: neutrophils, macrophages, lymphocytes, and en-
IL-1R/TLR and MyD88 receptors, one of the conserved zones
dothelial, epithelial, and mesenchymal cells. Therefore, the
consists of a loop between the second ꢀ-strand and the second
immediate, transitory activation of NF-κB constitutes a
R-helix (BB loop) whose consensus sequence is RDXΦ1Φ2GX,
characteristic of primary importance in the functioning of
where X is any amino acid and Φ1Φ2 are two hydrophobic
the normal physiological response to pathogenic damage.
ones. We were the first to demonstrate that the heptapeptide,
Nevertheless, dysregulation of this finely tuned mechanism,
Ac-RDVLPGT-NH2, derived from the TIR domain of MyD88,
occurring as an excessive, persistent activation, is closely
was effective in inhibiting its homodimerization.4 Subsequent
associated with chronic inflammatory diseases.2
studies carried out by different groups corroborated our initial
Recent research lends support to the hypothesis concerning
results in additional experimental settings.5,6
the existence of a common transduction mechanism in the
Protein–protein interaction as target of small molecule
IL-1R/TLR superfamily3 context. In particular, the adaptor
inhibitors still represents a challenging task.7 The recent
protein, MyD88, seems to play a crucial role in the
identification of a crucial subregion of MyD88 TIR domain4
transduction events triggered by all TLR receptors, (except
TLR3) as well as IL-1 and IL-18 receptors.
provides a good starting point to mimic, though it must be
emphasized that the intracellular location of the target hinders
Therefore, the inhibition of an adaptor protein, such as
the use of peptides and represents an additional barrier to the
MyD88, involved in the activation of NF-κB, triggered by
druggability of candidate structures.
signals from receptors that recognize distinct ligands but share
The present study aimed at identifying mimetics of this
the same transduction pathway, is expected to be more effective,
particular portion of MyD88, which might interfere with its
in principle, than inhibition of individual ligand activities. On
homodimerization. This would make it possible to prevent
alignment of the primary sequences of the TIR domains of the
recruitment of MyD88 to each of the IL-1R/TLR receptors, thus
interfering with their signaling and proving the effectiveness
of this approach.
* To whom correspondence should be addressed. Phone: 39-6-9139-3445.
Fax: 39-6-9139-3638. E-mail: nicola.fanto@sigma-tau.it.
†
Sigma-Tau Industrie Farmaceutiche Riunite S.p.A.
Tecnogen S.p.A.
Results and Discussion
‡
a Abbreviations: TIR, Toll/IL-1 receptor; MyD88, myeloid differentiation
factor 88; NF-κB, nuclear factor-κB; IL, interleukin; IL-1R, interleukin-1
receptor; TLR, toll-like receptor; 2HyS, 2-hybrid system; AD, activation
domain; BD, binding domain; UAS, upstream activation sequence; COSY,
correlation spectroscopy; HSQC, heteronuclear single-quantum correlation;
HMBC, heteronuclear multiple-bond coherence.
Library Enumeration. Figure 1 illustrates the strategy for
constructing mimetics of the consensus peptide of MyD88 TIR
domain; the structure of the consensus peptide is subdivided
into three distinct portions: (a) a charged portion consisting of
the Arg-Asp amino acids, (b) a hydrophobic portion consisting
10.1021/jm070723u CCC: $40.75
2008 American Chemical Society
Published on Web 02/15/2008

1190 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Fantò et al.
Table 1. Selection of 18 Commercially Available Arginine Mimetic Groups
of the Val-Leu amino acids, and (c) a ꢀ-turn portion consisting
of the Leu-Pro-Gly-Thr amino acids.
For each of these portions, a certain type of mimetic was
chosen to be substituted alternatively or simultaneously in the
consensus peptide sequence, maintaining the amide bond as the
functional link between the three groups.
Table 1 shows the 18 commercially available arginine
mimetic groups used, where a hydrophobic structure contains
a functional group that modulates basicity from that of arginine
to zero. There is a hydrophobic spacer covering the distance
between arginine and proline, with a limited number of rotational
degrees of freedom and an aromatic linker ring differently
substituted and functionalized, where only one carboxylic acid
group and one primary amine group is present and engaged in
an amide bond. Table 2 shows the 39 commercially available
spacers used. Table 3 shows that the central portion of the ꢀ-turn
(Pro-Gly) is substituted with 10 different ꢀ-turn mimetics.8–11
When combining all the building blocks, 4368 direct and
234 retroinverse peptidomimetics may be obtained. Therefore,
we chose to restrict the number of compounds and synthesize
a diverse but representative set. A virtual library was then
enumerated, and molecules possessing more than one viola-
tion of the “rule of five”12 were removed. Building blocks
showing bad superposition on the 3D structure of the BB-
loop, poor reactive groups, and high toxicity groups were
also removed.
The final scaffold selection on the remaining 1946
molecules was then realized by an experimental design
(Onion/D-optimal design, Figure 2),13 where the EVA
structural descriptors,14 based on calculated fundamental
molecular vibrational frequencies, were used.
Chemistry. A diverse library of 83 molecules was selected
(Table I, Supporting Information) and then synthesized by
solid-phase synthesis. Chart 1 shows the structures of a

Inhibitors of Myeloid Differentiation Factor 88
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1191
Table 2. Selection of 39 Commercially Available Hydrophobic Spacers
reference compound 115 and molecules having the best
biological results. Schemes 1-3 condense the synthetic
strategies to prepare all molecules. Schemes 2 and 3 show
synthesis examples of direct 2 and retro 3 peptidomimetics.
Each peptidomimetic sequence was synthesized on polymer-
supported (aminomethyl)polystyrene (Rink amide) using
standard Fmoc protocols.
The building blocks, useful for the synthesis of peptidomi-
metic compounds containing the ꢀ-turn mimetics, were syn-
thesized using methods described in the literature8–11 and

1192 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Table 3. Selection of 10 ꢀ-Turn Mimetics
Fantò et al.
Figure 2. Selection of 83 compounds (red points) by an onion/D-
optimal design. A total of 3 of 20 principal components are represented
for clarity.
The 2HyS is based on an ability to reconstitute the GAL4
transcription factor, in vitro, which can be divided into two
functional domains: the activation domain (AD) and the binding
domain (BD).16 When these two domains are fused with two
proteins capable of interacting, the result is a functional
reconstitution of GAL4 which, in turn, activates transcription
of a number of reporter genes under the control of its own
upstream activation sequence (UAS). Transcription of the
reporter genes under control of the UAS of GAL4 permits the
synthesis of enzymes important for growth in selective medium.
The assay is carried out by using 384-well plates, where the
wells are coated with a silicone matrix incorporating a fluores-
cent substance, the emission of which is sensitive to oxygen
levels.17 When an interaction occurs between the two proteins
fused with the GAL4 BD and AD domains, respectively, yeast
is able to grow in the selective medium, consuming oxygen.
Hence, the emitted fluorescence, which increases proportionately
over time, can be detected by using a suitable fluorescence
reader. If the yeast grows in the presence of molecules capable
of inhibiting this interaction, transcription of the reporter genes
abates, slowing down the ability to grow in minimal medium
with consequent reduction of the fluorescence signal. The
fluorescence growth curves generated by the 2HyS were
analyzed by means of a system specifically elaborated for this
type of investigation (Chrysallis s.a.s.). The software provides
quantitative parameters capable of describing the characteris-
tics of the individual curves. In particular, seven different
descriptors were used: plateau slope and height, plateau range,
growth time, time to grow to 50% of plateau height, “hump”
factor, and sigmoidal τ. Through these quantitative parameters,
it is possible to obtain a multidimensional representation of the
set of experimental curves, and after using extraction techniques
by principal component analysis, a two-dimensional representa-
tion of the data is constructed to highlight, as clearly as possible,
the differences between the curves. This type of representation
allows identification of clusters of curves with similar behavior
and a clear indication of which curves are generated by
compounds influencing oxygen consumption and, thus, yeast
growth.
modified using the methods described below, as shown in
Schemes 4-6.
Biological Results. The primary screening was first per-
formed by using a 2-hybrid system (2HyS) in yeast Saccharo-
myces cereVisiae to evaluate the inhibition of MyD88 ho-
modimerization and then, by means of a secondary functional
test, to evaluate the inhibition of NF-κB in HeLa cells.
Activation of NF-κB is an event that takes place downstream
of MyD88 homodimerization and recruitment, through its TIR
domain, to activated receptor complexes. The ability of com-
pounds that inhibited activation of NF-κB downstream of the
signaling cascade triggered by IL-1ꢀ was investigated. All the

Inhibitors of Myeloid Differentiation Factor 88
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1193
Chart 1. Structures of Reference Compound 1 and Molecules Having the Best Biological Results after Primary Screening (2-6)
Scheme 1. General Scheme of Parallel SPPSa
a Reagents and conditions: (a) Typical deprotection procedure according to the literature: DMF/CH2Cl2, 25% piperidine in DMF, 20 min, rt; (b) Typical
procedure for the coupling of ꢀ-turn mimetic to the amine group: ꢀ-turn mimetic, HOBt/TBTU, DIPEA, DMF, 120 min, rt; (c) Typical procedure for the
coupling of a spacer: Fmoc-spacer, HOBt/TBTU, DIPEA, DMF, 120 min, rt; (d) Typical procedure for the coupling of an arginine mimetic group: arginine
mimetic, HOBt/TBTU, DIPEA, DMF, 120 min, rt; (e) Typical cleavage procedure: TFA-Tis-H2O [95:2.5:2.5], 120 min, rt; (f) Typical procedure for the
coupling of an arginine mimetic with formation of the guanidine group: Fmoc-isothiocyanate, DMF, 12 h, rt.
Scheme 2. Synthesis of 2a
a Reagents and conditions: (a) DMF/CH2Cl2, 25% piperidine in DMF, 20 min, rt; (b) 18, HOBt/TBTU, DIPEA, DMF, 120 min, rt; (c) Fmoc-SP19-OH,
HOBt/TBTU, DIPEA, DMF, 120 min, rt; (d) PAM4, HOBt/TBTU, DIPEA, DMF, 120 min, rt; (e) TFA-Tis-H2O [95:2.5:2.5], 120 min, rt.
molecules were concomitantly assayed for cell viability to rule
out toxic effects.
Because known inhibitors of MyD88 homodimerization are
not yet available, it is not possible to predict how effectively

1194 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Scheme 3. Synthesis of 3a
Fantò et al.
a Reagents and conditions: (a) DMF, 12 h, rt; (b) CH3I/DIPEA, DMF, 2 h, rt; (c) 1,4 diaminobutane, DMF, 12 h, rt; (d) Fmoc-SP38-OH, HOBt/TBTU,
DIPEA, DMF, 120 min, rt; (e) TBDPS-O-BETA6-Cl, sym-collidine, THF, 70 °C; (f) Bu4N+F-, THF, 25 °C; (g) TFA-Tis-H2O [95:2.5:2.5], 120 min, rt.
the inhibition of MyD88 dimerization translates into the
inhibition of IL-1ꢀ-induced activation of NF-κB. However, as
reference compound in the NF-κB assay, we used a cell-
permeable, end-protected L-valyl derivative modeled on the
MyD88-binding sequence of the TIR domain BB-loop of IL-
1RI; this compound 1 (i.e., hydrocinnamoyl-L-valyl pyrroli-
dine)15 was able to specifically inhibit TIR domain-mediated
MyD88/IL-1RI interaction, successfully reducing IL-1ꢀ-induced
fever in mice in vivo and attenuating IL-1ꢀ-induced, but not
LPS-induced, activation of MAP kinase signaling cascades in
murine lymphocytes and EL4 thymoma cells in vitro.
BAY 11-7085, a known pharmacologic inhibitor of IκBR
phosphorylation,18 was used as an additional reference com-
pound in the NF-κB assay. The maximal inhibitory activity
observed in our experimental setting was 45% at 10 µM (Table
II in Supporting Information).
Chart 1 and Table 4 summarize the structures and positive
results in both 2HyS screening and NF-κB assay (expressed as
percentage inhibition at 100 µM) obtained with the molecules
under investigation and reference compound 1. In our experi-
mental setting, the inhibitory activities scored by our screened
compounds ranged from 17 to 26% (Table 4). Interestingly, the
highest activity was shown by 2 (26% inhibition) and was very
close to the activity exhibited by Bartfai’s compound 1 (31%
inhibition at 100 µM).
scribed in the present investigation interfere with MyD88
dimerization, thereby anticipating that they may interfere with
signaling from additional TLRs. In fact, we found that 2
effectively inhibited signaling even from ligands operating in
an intracellular compartment such as the CpG, that is, the ligand
of TLR9, a receptor that depends solely on MyD88 for signal
transduction.19
The remarkable progress in the elucidation of TLR signaling
has elicited great interest from a number of pharmaceutical
companies20 inasmuch as it may lead, in the future, to an entirely
new class of therapeutic agents. Indeed, both agonists and
antagonists of IL-1R/TLR signaling can find therapeutic ap-
plication, depending on the clinical setting. Activators may have
adjuvant roles, thus improving vaccine efficacy and reinforcing
the immune response to cancer,21 while inhibitors may help
dampen inflammation in disorders such as allergy, atheroscle-
rosis, and autoimmune diseases.22
SAR Analysis. 2HyS experiments were carried out to assess
the ability of the active molecules to specifically interfere with
MyD88 homodimerization, while NF-κB assays aimed at testing
whether inhibition of MyD88 dimerization translated into
inhibition of IL-1ꢀ-induced activation of NF-κB. Since both tests
are carried out on cell cultures (yeast and mammalian cells),
cell membrane/wall permeation, and intrinsic toxicity are strong
limiting factors in the selection of positive candidates and in
the subsequent SAR analysis.
Nevertheless, some information can be drawn from the
present findings on the structure of tested compounds: generally
a noncharged arginine mimetic group, a spiro ꢀ-turn and a
flexible spacer are preferred. The most represented building
block is BETA8 (contained in 2, 4, and 6); the characteristic of
this ꢀ-turn mimetic is a bulky S-containing bicyclic ring linked
to natural proline. Activity is enhanced when BETA8 is merged
to a para-disubstituted six-membered ring. Figure 3 shows the
superimposition of compounds in Table 4 and the MyD88
consensus peptide. Noted is a good alignment of the ꢀ-turn
mimetic skeleton, while unexpectedly bulky hydrophobic groups
Moreover, it is worth mentioning that using a different
methodology, based on an in-house chemiluminescent NF-κB
reporter gene assay, not only were we able to confirm the
activity of 2 in vitro, but we further showed that 2 is indeed
able to counteract IL-1ꢀ signaling in vivo by significantly
decreasing IL-1ꢀ-dependent production of IL-6 in mice.19
Interestingly, the doses of 2 utilized in our in vivo study, ranging
from 50 to 200 mg/kg, are in the same dose range as those
used by Bartfai’s compound 1 (namely, 100 mg/kg) in coun-
teracting IL-1ꢀ-induced fever in mice.
At variance with Bartfai’s inhibitor,15 which only blocks the
interaction between IL-1R and MyD88, the compounds de-

Inhibitors of Myeloid Differentiation Factor 88
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1195
Scheme 4. Synthesis of 18a
a Reagents and conditions: (a) t-Bu-CHO, cat. CF3COOH, n-pentane, reflux, 6 d; (b) (1) LDA, THF, –78 °C, 30 min; (2) CH2dCH-CH2-Br, 0 °C, 3 h;
(c) silica gel, MeOH/H2O, rt, 72 h; (d) (Boc)2O, (CH3)4N+OH- ·5H2O, CH3CN, rt, 72 h; (e) CH3I, KHCO3, rt, 20 h; (f) OsO4, NaIO4, MeOH/H2O, rt, 20 h;
(g) D-homocys-OH·HCl, NaOH, H2O/EtOH, rt, 40 h,; (h) (1) NEt3, DMF, 70 °C, 3 d; (2) CH3I, KHCO3, DMF, rt, 24 h; (i) (1) K2CO3, MeOH/H2O, 20 h,
rt; (2) pH 5; (l) TFA, rt, 3 h; (m) (1) Fmoc-OSu, NaHCO3, H2O/(CH3)2CO, rt, 20 h; (2) HCl 2N, pH 3.
Table 4. Primary Screening Results
cmpd
composition
2HyS % NF-κB inhibition
1
2
3
4
5
6
hydrocinnamoyl-L-valyl pyrrolidine n.d.a
31
26
24
23
18
17
PAM4-SP19-BETA8-NH2
AM8-SP38-BETA6
Y
Y
Y
Y
Y
PAM6-SP20-BETA8-NH2
PAM8-SP20-BETA3-NH2
PAM3-SP33-BETA8-NH2
a Not determined.
The listed compounds, with their primary screening results,
represent hits that may be further evaluated by investigating
their activity in additional and more sensitive functional assays,
addressing their effects on cells of the immune system. The
most interesting ones may be used as a starting point to generate
homologous series aimed at obtaining derivatives with increased
inhibitory activity.
Figure 3. Superimposition of active molecules (tube; 2 (cyan), 3
(orange), 4 (pink), 5 (green), and 6 (goldenrod)) onto MyD88 consensus
peptide (ball and stick; yellow, grey, and blue).
Experimental Section
Computational Methods. A virtual library of 4368 direct and
234 retroinverse peptidomimetics was enumerated using TSAR
software.23 The rule of five properties (logP < 5, HBA < 10, HBD
< 5, MW < 600, and rotatable bonds < 8) were calculated using
TSAR software. EVA descriptors14 were used to describe the
occupy the Gly CR space; also observed is a preferential
agreement on the position of the second amide bond and Asp-
Val junction; the arginine mimetic hydrophobic fragment is
located on the methylene portion of the Arg side chain.

1196 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Scheme 5. Synthesis of 24a
Fantò et al.
a Reagents and conditions: (a) DMSO, 120 °C, 5 h; (b) TBDPSCl, imidazole, DMF, rt, 3.5 h; (c) (1) NaH, –40 °C, 45 min; (2) BrCH2COOtBu, rt, 2 h;
(d) CH2Cl2/TFA, rt, 45 min.
structures in the experimental design. Principal component analysis
was performed on the molecular descriptors matrix using SIMCA-P
software.24 Extracted principal components were processed into
MODDE software25 to perform an onion/D-optimal design.13
Chemistry. General Methods. Target compounds described in
Chart 1 and Table 4 and all final compounds described in Table 1
of Supporting Information were synthesized on polymer-supported
(aminomethyl)polystyrene (Rink amide MBHA, 0.74 mmol/g) with
standard 9-fluorenylmethyloxycarbonyl (Fmoc) protocols, using
parallel solid-phase peptide synthesis (SPPS) procedures, according
to Scheme 1. Single coupling reactions were normally carried out
in dimethylformamide (DMF) and 1-hydroxy-1-H-benzotriazole/
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium tetrafluorobo-
rate (HOBt-TBTU) was used for chemical activation of the
carboxylic component of Fmoc-protected building blocks, while
diisopropylethylamine (DIPEA) was used as a proton scavenger.
Poor nucleophilic amino peptidyl resins were efficiently coupled,
using acid chlorides of corresponding building blocks at 70 °C for
2 h in tetrahydrofuran (THF) in the presence of sym-collidine. Fmoc-
protected amino acids, solvents, and reagents were purchased from
Chem-Impex, while Rink amide MBHA resin and unprotected
amino benzoic derivatives (spacers) were supplied by Sigma-
Aldrich. The amine function of the benzoic derivatives was
protected by Fmoc according to methods described in the
literature.26a Target compounds were characterized by mass spec-
trometry and purity was determined by analytical HPLC on
Thermofinnigan LCQ-Duo with adjustable API-ESI ion source,
using a gradient system composed of solvent (A) H2O + 0.1% TFA
and (B) MeCN + 0.1% TFA from 95 to 40% solvent (A) in 13
min; Phenomenex Luna RP-C18 column, 3 µm, 50 × 2 mm; flow
rate 0.5 mL/min. Semipreparative HPLC purifications of target
compounds were performed on Labservice Analitica Labflow 2000
and 3000 with a Knauer K-2501 UV detector and with a Phenom-
enex Jupiter-Proteo RP-C12 column, 250 × 21.2 mm, 10 µm, 90
Å; using a gradient system composed of solvent (A) H2O/MeCN/
TFA 97/3/0.1 and solvent (B) MeCN/H2O/TFA 80/20/0.1 from 95
to 40% solvent (A) in 30 min; flow rate 15 mL/min. NMR spectra
of intermediate compounds were obtained on a Varian VXR 300
and spectra elaboration was performed by standard Varian software
(VNMR, 6.1C release); chemical shifts are reported in parts per
million (ppm) using the middle resonance of D2O, CDCl3, or
MeOH-d4 as an internal standard. NMR spectra of target compounds
were obtained at 27 °C on a Varian Inova-500 equipped with a
triple resonance indirect probe operating at 500.0 MHz for 1H and
125.67 MHz for 13C (Varian). Data acquisition, Fourier transforma-
tion, and spectra elaboration were performed by standard Varian
software (VNMR, 6.1C release); chemical shifts are reported in
parts per million (ppm) using the middle resonance of DMSO-d6
or MeOH-d4 as an internal standard. About 5 mg of all samples
were dissolved in DMSO-d6 or MeOH-d4 (Sigma-Aldrich) and
extensively analyzed by means of 2D-NMR spectroscopy (1H-1H
COSY, 1H-13C gs-HSQC, 1H-13C gs-HMBC). The following
abbreviations are used to describe peak patterns when appropriate:
b ) broad, s ) singlet, d ) doublet, dd ) double–doublet, dt )
double triplet, t ) triplet, q ) quartet, m ) multiplet. Intermediate
compounds were characterized by mass spectrometry on Single
Quadrupole Micromass Waters ZQ2000 mass spectrometer with
ESI ion source. Melting points were obtained in open capillary tubes
and are uncorrected. Thin layer chromatography (TLC) was carried
out on Merck precoated plates (Kieselgel 60F254), and compounds
were visualized using a UV lamp at 254 and 366 nm. Elemental
analyses were performed on a Thermo EA1110 CHNS-O instru-
ment. Reference compound 1 (i.e., hydrocinnamoyl-L-valyl pyrro-
lidine) was prepared according to the literature.15
Synthesis of (2R,4′R,8a′R)-1-[(9H-Fluoren-9-yl-methoxy)car-
bonyl]-6′-oxotetrahydro-2′H-spiro[pyrrolidine-2,7′-pyrrole-[2,1-
b][1,3]thiazine]-4′-carboxylic Acid, Fmoc-BETA8-OH (18):
(3S,7aS)-3-tert-Butyltetrahydro-1H-pyrrolo[1,2-c][1,3]oxazol-1-
one (8). Compound 8 was prepared according to the literature (yield
80%).11a Bp 84–85 °C (0.05 mm). 1H NMR (300 MHz, CDCl3): δ
0.92 (s, 9H), 1.60–1.92 (m, 2H), 1.94–2.30 (m, 2H), 2.64–2.98 (m,
1H), 3.05–3.35 (m, 1H), 3.80 (dd, 1H), 4.50 (s, 1H).
(3S,7aR)-7a-Allyl-3-tert-butyltetrahydro-1H-pyrrolo[1,2-
c][1,3]oxazol-1-one (9). Compound 9 was prepared according to
the literature (yield 75%).11a Bp 80–82 °C (0.01 mm). TLC solvents:
n-hexane/AcOEt 8/2, Rf 0.72. 1H NMR (300 MHz, CDCl3): δ 0.87
(s, 9H), 2.14–1.34 (m, 4H), 2.35 (d, 2H), 3.05–2.70 (m, 2H), 4.20
(s, 1H), 5.24–4.90, 6.14–5.60 (m, 3H).
(R)-2-Allyl-proline (10). Compound 10 was prepared according
1
to the literature (yield 70%).11b [R]20 ) –46.3 (c 0.5, H2O). H
D
NMR (300 MHz, D2O): δ 2.00 (m, 3H), 2.04 (m, 1H), 2.58 (dd,
1H), 2.90 (dd, 1H), 3.45 (m, 2H), 5.25 (s, 1H), 5.32 (d, 1H),
5.65–5.90 (m, 1H). ESIMS m/z 156.2 (M + H)+.

Inhibitors of Myeloid Differentiation Factor 88
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1197
Scheme 6. Synthesis of 33a
a Reagents and conditions: (a) t-Bu-CHO, cat. TFA, n-pentane, reflux, 6 d; (b) (1) LDA, THF/DMPU, –78 °C, 1 h; (2) CH2dCH-CH2-CH2-Br, –78 °C,
20 h; (c) silica gel, MeOH/H2O, rt, 72 h; (d) (Boc)2O, (CH3)4N+OH- ·5H2O, rt, 72 h; (e) Gly OMe·HCl, DCC, HOBt, NEt3, CHCl3, N2, rt, 20 h; (f) OsO4,
N2, NaIO4, MeOH/H2O, rt, 24 h; (g) NaBH3CN, THF/TFA, N2, rt, 20 h; (h) (1) K2CO3, MeOH/H2O, rt, 20 h; (2) HCl, pH 2-3; (i) TFA/CH2Cl2, rt, 1 h;
(l) (1) Fmoc-OSu, NaHCO3, H2O/(CH3)2CO, rt, 24 h; (2) HCl 2 N, pH 2–3.
(R)-2-Allyl-N-(tert-butoxycarbonyl)-proline (11). Compound
(2R,4′R,8a′R)-6′-Oxotetrahydro-1H,2′H-spiro[pyrrolidine-
2,7′-pyrrolo[2,1-b][1,3]thiazine]-1,4′-dicarboxylic acid 1-tert-
Butyl 4′-Methyl Ester (15). Compound 15 was prepared according
to the literature (yield 40%).11c Mp 152–154 °C (lit.11c mp 156–158
°C). [R]20D ) +129.6 (c 1.2, CHCl3). 1H NMR (300 MHz, CDCl3):
δ 1.39, 1.42 (s, 9H), 1.70–2.10 (m, 4H), 2.10–2.40 (m, 3H), 2.62
(dt, 1H), 2.82 (dd, 1H), 2.90–3.10 (m, 1H), 3.40–3.60 (m, 2H),
3.76, 3.79 (s, 3H), 5.05–5.18 (m, 1H), 5.32, 5.43 (d, 1H). ESIMS
m/z 393.3 (M + Na)+.
(2R,4′R,8a′R)-1-(tert-Butoxy-carbonyl)-6′-oxotetrahydro-2′H-
spiro[pyrrolidine-2,7′-pyrrole[2,1-b]-[1,3]-thiazine]-4′-carboxy-
lic Acid (16). (2R,4′R,8a′R)-6′-Oxotetrahydro-1H,2′H-spiro[pyrro-
lidine-2,7′-pyrrole[2,1-b][1,3]thiazine]-1,4′-dicarboxylic acid 1-tert-
butyl 4′-methyl ester 15 (2.55 g, 6.9 mmol) was dissolved in 45
mL of MeOH and 45 mL of H2O; K2CO3 (1.9 g, 13.8 mmol) was
added to the solution and kept under stirring at room temperature
for 20 h, then treated by lowering the pH to 5, bringing to dryness
under reduced pressure. The residue obtained was collected with
H2O, brought to pH 2–3 and extracted with CHCl3. The organic
phase was dried over anhydrous Na2SO4 and the solvent was
evaporated. The pitchy mass was crystallized with ethyl ether,
thereby obtaining a filterable white solid (2.4 g, 97%). TLC solvents:
CHCl3/MeOH/CH3COOH 7.95/1.95/0.1, Rf 0.65. 1H NMR (300
MHz, CDCl3)δ 1.40, 1.45 (2d, 9H), 2.70–2.05 (m, 4H), 2.00–2.30
(m, 1H), 2.25–2.55 (m, 1H), 2.55–2.80 (m, 1H), 2.75–3.15 (m, 1H),
3.50 (m, 2H), 3.70 (t, 2H), 4.98 (dd, 1H), 5.10 (d, 1H). ESIMS
m/z 379.3 (M + Na)+.
11 was prepared according to the literature (yield 85%).11c [R]20
D
1
) +58.4 (c 1.2, MeOH). H NMR (300 MHz, CDCl3): δ 1.40,
1.45 (s, 9H), 1.75–1.94 (m, 2H), 1.98–2.02, 2.35–2.40 (m, 1H),
2.12–2.16 (m, 1H), 2.58 (dd, 1H), 2.95 (dd, 1H), 3.28–3.40 (m,
1H), 3.50–3.70 (m, 1H), 5.10–5.18 (m, 2H), 5.62–5.82 (m, 1H),
10.90 (bs, 1H). ESIMS m/z 256.3 (M + H)+.
(R)-2-Allyl-N-(tert-butoxycarbonyl)-proline Methyl Ester (12).
Compound 12 was prepared according to the literature (yield
95%).11c [R]20 ) +49.6 (c 0.9, MeOH). 1H NMR (300 MHz,
D
CDCl3): δ 1.40, 1.45 (s, 9H), 1.76–1.93 (m, 2H), 2.02–2.16 (m,
2H), 2.58 (dd, 1H), 2.95, 3.10 (dd, 1H), 3.30–3.40 (m, 1H),
3.55–3.70 (m, 1H), 3.72 (s, 3H), 5.10–5.15 (m, 2H), 5.72–5.78 (m,
1H). ESIMS m/z 292.4 (M + Na)+.
(2R)-2-(2-Oxoethyl)pyrrolidine-1,2-dicarboxylic Acid 1-tert-
Butyl 2-Methyl Ester (13). Compound 13 was prepared according
to the literature (yield 80%).11c [R]20 ) +136.7 (c 1.0, CHCl3).
D
1H NMR (300 MHz, CDCl3): δ 1.38, 1.40 (s, 9H), 1.80–2.00 (m,
2H), 2.14–2.28 (m, 2H), 2.70–3.09 (m, 2H), 3.42–3.78 (m, 2H),
3.70, 3.72 (s, 3H), 9.77, 9.78 (s, 1H). ESIMS m/z 294.4 (M + Na)+.
(4R)-2-{[(2R)-1-(tert-Butoxycarbonyl)-2-(methoxycarbonyl)pyr-
rolidin-2-yl]methyl}-1,3-thiazinane-4-carboxylic Acid (14). Com-
pound 14 was prepared according to the literature (yield 65%).11c
Mp 90–95 °C (lit.11c mp 94–97 °C). 1H NMR (300 MHz, CDCl3):
δ 1.46 (s, 9H), 1.75–2.20 (m, 4H), 2.15–2.82 (m, 4H), 2.82–3.15
(m, 2H), 3.18–4.62 (m, 3H), 4.78 (s, 3H), 4.20, 4.34 (s, 1H). ESIMS
m/z 389.5 (M + H)+.

1198 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Fantò et al.
(2R,4′R,8a′R)-4′-Carboxy-6′-oxotetrahydro-2′H-spiro[pyrro-
lidine-2,7′-pyrrole[2,1-b][1,3]thiazine]Trifluoroacetate(17).(2R,4′R,8a′R)-
1-(tert-Butoxycarbonyl)-6′-oxotetrahydro-2′H-spiro[pyrrolidine-2,7′-
pyrrole[2,1-b][1,3]thiazine]-4′-carboxylic acid 16 (2.1 g, 5.8 mmol)
was dissolved in 45 mL of trifluoroacetic acid. The mixture was
kept under stirring at room temperature for 3 h and evaporated to
dryness at 30 °C under reduced pressure. The residue obtained was
solubilized in a small amount of CHCl3, precipitated with ethyl
ether, and the solid formed was rapidly filtered. This step was
performed twice, obtaining a very hygroscopic white solid (1.55
g, 74%). TLC solvents: CHCl3/isoPrOH/MeOH/H2O/AcOH 4.2/
0.7/2.8/1.05/1.05, Rf 0.56. 1H NMR (300 MHz, D2O): δ 1.75–2.00
(m, 1H), 2.00–2.38 (m, 5H), 2.45 (dd, 1H), 2.70 (dt, 1H), 2.77–3.05
(m, 2H), 3.25–3.55 (m 2H), 3.96 (d, 1H), 5.18 (d, 1H). ESIMS
m/z 258.3 (M + H)+.
Step 4: Cleavage of Sequence from the Resin. Typical
Procedure. The dry resin from the previous step was treated with
a mixture of TFA/triisopropylsilane/H2O (TFA-Tis-H2O) 95:2,5:
2.5 (1.0 mL). The reaction was shaken for 2 h and then filtered
off. The solution, evaporated under reduced pressure, yielded a
residue which, after RP-HPLC purification, provided pure 2 (9.3
mg, 29%); analytical HPLC showed a peak related to the product
at 12.28 min, purity > 93%. Mass value in agreement with
theoretical value: ESIMS m/z 591.04 (M + H)+, 613.18 (M + Na)+.
1H NMR (Figure I of the Supporting Information reports the
numbering system used for the attribution of main signals, 500
MHz, DMSO-d6): δ 1.52–1.64 (m, 1H, 3-CH″), 1.86–1.98 (m, 3H,
7-CH″,11-CH″, 12-CH″), 1.98–2.13 (m, 2H, 11-CH”, 12-CH”),
2.53–2.57 (m, 1H, 3-CH”), 2.61–2.83 (m, 3H, 2-CH2, 7-CH”),
3.41–3.53 (m, 1H, 10-CH″), 3.64 (s, 2H, 20-CH2), 3.66 (d, J )
7.9 Hz, 1H, 10-CH”), 4.61 (d, J ) 4.6 Hz, 1H, 4-CH), 4.85 (s, 2H,
30-CH2), 4.97 (dd, J ) 7.6, 1.6 Hz, 1H, 6-CH), 7.11 (d, J ) 8.9
Hz, 1 H, 37-CH), 7.17 (d, J ) 8.2 Hz, 2H, 22-CH, 26-CH), 7.21
(bs, 1H, 19-NH′), 7.38 (dd, J ) 8.9, 1.8 Hz, 1H, 36-CH), 7.50 (bs,
1H, 19-NH”), 7.53 (d, J ) 8.2 Hz, 2H, 23-CH, 25-CH), 7.61 (d, J
) 1.8 Hz, 1H, 34-CH), 10.14 (s, 1H, 27-NH); 13C NMR (125.67
MHz, DMSO-d6): δ 24.48 (11-C), 25.59 (3-C), 26.02 (2-C), 37.91
(7-C), 38.46 (12-C), 41.20 (20-C), 48.63 (10-C), 52.07 (4-C), 55.04
(6-C), 66.39 (8-C), 68.60 (30-C), 116.04 (37-C), 120.20 (23-C, 25-
C), 123.21 (33-C), 125.75 (35-C), 128.74 (36-C), 130.05 (22-C,
26-C), 130.09 (34-C), 130.81 (21-C), 137.49 (24-C), 153.37 (32-
C), 166.26 (28-C), 169.84 (16-C), 170.80 (14-C), 172.61 (13-C).
Synthesis of [(2R,11aS)-2-{[tert-Butyl(diphenyl)silyl]oxy}-5,11-
dioxo-2,3,11,11a-tetrahydro-1H-pyrrole[2,1-c][1,4]benzodiazepine-
10(5H)-yl]-acetic Acid, TBDPS-O-BETA6 (24): (2R,11aS)-2-
Hydroxy-2,3-dihydro-1H-pyrrole[2,1-c][1,4]benzodiazepine-
5,11(10-H,11aH)-dione (21). Compound 21 was prepared according
to the literature (yield 80%).10 TLC solvent AcOEt Rf 0.3, mp
(2R,4′R,8a′R)-1-[(9H-Fluoren-9-yl-methoxy)carbonyl]-6′-ox-
otetrahydro-2′H-spiro[pyrrolidine-2,7′-pyrrole-[2,1-b][1,3]thiazine]-
4′-carboxylic Acid (18). (2R,4′R,8a′R)-4′-Carboxy-6′-oxotetrahydro-
2′H-spiro[pyrollidine-2,7′-pyrrole[2,1-b]-[1,3]thiazine] Trifluoroacetate
17 (1.48 g, 4.0 mmol) was dissolved in 50 mL of H2O. NaHCO3
(0.67 g, 8.0 mol) was added to the solution followed by 1.42 g
(4.2 mmol) of Fmoc-N-OSu dissolved in 75 mL of acetone. The
reaction solution was left under stirring at room temperature for
20 h, eliminating the acetone under reduced pressure. H2O was
added and the solution was washed with Et2O. The aqueous solution
was brought to pH 3 with HCl 2N and extracted with CHCl3. The
organic phase was dried over anhydrous Na2SO4 and the solvent
was evaporated, obtaining a glassy white solid (1.9 g, 98%). TLC
solvents: CHCl3/MeOH/AcOH 7.95/1.95/0.1, Rf 0.74. Mp 108–
115 °C. [R]20 ) +70.1 (c 0.5, MeOH). 1H NMR (300 MHz,
D
CDCl3): δ 1.85 (m, 1H), 1.94 (dd, 1H), 2.05 (m, 1H), 2.15 (m,
1H), 2.42 (m, 1H), 2.67 (d, 1H), 2.82 (m, 2H), 3.00 (m, 1H), 3.58
(dd, 2H), 4.20 (t, 1H). HPLC: column, Symmetry C18 (5µ) 3.9 ×
150 mm; mobile phase, KH2PO4 50 mM/CH3CN 65/35; flow rate,
1.0 mL/min; RT, 12.19 min. ESIMS m/z 501.3 (M + Na)+. Anal.
(C26H26N2O5S·2H2O) C, H, N. C: calcd, 60.70; found, 56.77. H:
calcd, 5.84; found, 4.85. N: calcd, 5.45; found, 5.06. S: calcd, 6.23;
found, 5.26.
200–202 °C (lit.10 mp 198–200 °C). [R]20 ) +410.2 (c 0.02,
D
1
MeOH). H NMR (300 MHz, DMSO-d6): δ 1.92 (m, 1H), 2.61
(m, 1H), 3.50 (m, 1H), 3.62 (dd, 1H), 4.22 (dd, 1H), 4.32 (m, 1H),
5.23 (d, 1H), 7.16 (d, 1H), 7.23 (t, 1H), 7.53 (m, 1H), 7.82 (dd,
1H), 10.55 (s, 1H). ESIMS m/z 255.2 (M + Na)+. Anal.
(C12H12N2O3) C, H, N. C: calcd, 62.06; found, 61.90; H: calcd,
5.21; found, 5.18; N: calcd, 12.06; found, 11.98.
SPPS Synthesis of (2R,4′R,8a′R)-1-[(4-{[(2,4-Dichlorophenoxy)-
acetyl]amino}phenyl) acetyl]-6′-oxotetrahydro-2′H-spiro[pyr-
rolidine-2,7′-pyrrolo[2,1-b][1,3]thiazine]-4′-carboxamide, AM4-
(2R,11aS)-2-{[tert-Butyl(diphenyl)-silyl]oxy}-2,3-dihydro-1H-
pyrrole[2,1-c][1,4]benzo diazepine-5,11(10-H,11aH)-dione (22).
(2R,11aS)-2-Hydroxy-2,3-dihydro-1H-pyrrole[2,1-c][1,4]benzodiazepine-
5,11(10H,11aH)-dione 21 (2.3 g, 10.0 mmol) was dissolved in 30 mL
of DMF; then imidazole (3.4 g, 50.0 mmol) and also 5.6 mL (22.0
mmol) of tert-butyl-diphenyl-silylchloride were added to this solution
dropwise under stirring. The solution was kept under stirring at room
temperature for 3.5 h, and then 100 mL of H2O were added; for
extraction, 100 mL of CH2Cl2 were used, washing the organic phase
with H2O. The organic phase was dried over anhydrous Na2SO4, and
the solvent was evaporated under reduced pressure using a mechanical
pump, obtaining a dense oil that was solubilized hot in n-hexane/AcOEt
and reprecipitated several times. After drying with the mechanical
pump, an amorphous solid was obtained (3.9 g, 83%). TLC solvents:
n-hexane/AcOEt 6/4, Rf 0.5. 1H NMR (300 MHz, CDCl3): δ 1.03 (s,
9H), 2.20 (m, 1H), 2.78 (m, 1H), 3.55, 3.60 (2d, 1H), 3.85, 3.90 (2d,
1H), 4.25 (m, 1H), 4.55 (m, 1H), 7.00 (d, 1H), 7.25–7.55 (m, 8H),
7.60–7.80 (m, 4H), 8.02 (d, 1H), 8.65 (s, 1H). ESIMS m/z 493.6 (M
+ Na)+. Anal. (C28H30N2O3Si) C, H, N. C: calcd, 71.45; found, 70.47;
H: calcd, 6.42; found, 6.67; N: calcd, 5.95; found, 5.53.
[(2R,11aS)-2-{[tert-Butyl-(diphenyl)silyl]oxy}-5,11-dioxo-
2,3,11,11a-tetrahydro-1H-pyrrole-[2,1-c]-[1,4]benzodiazepine-
10(5H)-yl)-acetic Acid tert-Butyl Ester (23). NaH 60% (330 mg,
8.0 mmol) was suspended in 6 mL of anhydrous THF after washing
several times with THF. The suspension was cooled to –40 °C and
(2R,11aS)-2-{[tert-butyl(diphenyl)-silyl]oxy}-2,3-dihydro-1H-pyr-
role[2,1-c][1,4]benzodiazepine-5,11(10H, 11aH)-dione 22 (3.45 g,
7.3 mmol), dissolved in 18 mL of THF, was added dropwise,
maintaining the solution under stirring. After 45 min, tert-butyl-
bromoacetate (1.2 mL, 8.0 mmol) was added dropwise, and the
mixture was left to react under stirring for 2 h, allowing the
SP19-BETA8-NH2 (2). Step 1: Attachment of ꢀ-Turn Mimetic
14 to Rink Amide MBHA. Typical Procedure. Compound 18
(250 µmol, 116 mg), prepared according to Scheme 4, was dissolved
in a 1.0 M solution of DIPEA in DMF (0.5 mL) and a 0.5 M
solution of HOBt-TBTU in DMF (0.5 mL). The mixture was added
to Rink amide MBHA resin, previously treated for Fmoc removal
(66 mg, ∼50 µmol). The suspension was shaken for 2 h, filtered
off, and, after washing with DMF, the resin was allowed to react
for 20 min with a 25% piperidine solution in DMF (1 mL) for
Fmoc removal. After washing, the resin was used for the subsequent
step.
Step 2: Attachment of Spacer (Fmoc-SP19-OH) to H-ꢀ-
Turn Mimetic-Rink Amide MBHA Resin (H-BETA8-Rink
Amide Resin). Typical Procedure. Fmoc-SP19-OH (250 µmol,
94 mg), dissolved in a 1.0 M solution of DIPEA in DMF (0.5 mL)
and a 0.5 M solution of HOBt-TBTU in DMF (0.5 mL), was added
to BETA8-Rink amide MBHA resin. The suspension was gently
shaken for 2 h, filtered off, washed, and then the resin was allowed
to react for Fmoc removal, as described in Step 1. Then the resin
was dried with a nitrogen flow and used for the subsequent
step.
Step 3: Attachment of Arginine Mimetic (PAM4) to H-Spacer-
ꢀ-Turn Mimetic-Rink Amide MBHA Resin (H-SP19-BETA8-
Rink Amide Resin). Typical Procedure. To the pseudoeptidyl
resin from Step 2, suspended in THF (1.0 mL) in a glass reactor
with a screw cap, was added sym-collidine (500 µL) and acid
chloride of PAM4 (250 µmol), then shaking for 2 h at 70 °C.
Subsequently, the resin was filtered off, washed, dried, and allowed
to react to get cleavage of the peptidomimetic sequence.

Inhibitors of Myeloid Differentiation Factor 88
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1199
temperature to rise to room temperature. The suspension was then
poured into 50 mL of H2O and extracted twice with 30 mL of
CH2Cl2, washing the pooled organic phases several times with brine
and dried over anhydrous Na2SO4. The solution was evaporated
under reduced pressure, obtaining an oil that, after repeated washing
with petroleum ether and drying with a mechanical pump, gave an
amorphous solid (3.6 g, 84%). TLC solvents: n-hexane/AcOEt 7/3,
(d, J ) 16.7 Hz, 1H, 17-CH”), 7.43 (t, J ) 7.6 Hz, 1H, 13-CH),
7.54 (d, J ) 8.4 Hz, 1H, 11-CH), 7.68 (t, 1H, 12-CH), 7.69 (d,
1H, 23-CH), 7.84–7.92 (m, 3H, 14-CH, 28-CH, 29-CH), 7.96 (d,
J ) 9.1 Hz, 1H, 24-CH), 8.34 (bs, 2H, 26-CH, 31-CH); 13C NMR
(125.67 MHz, MeOH-d4): δ 26.06 (37-C), 26.71 (36-C), 34.61 (4-
C), 39.09 (35-C), 41.00 (38-C), 52.93 (17-C), 53.66 (2-C), 56.27
(5-C), 68.63 (3-C), 116.10 (31-C), 120.75 (23-C), 122.87 (11-C),
124.25 (29-C), 126.17 (13-C), 127.33 (26-C), 127.79 (28-C), 129.26
(9-C), 129.67 (24-C), 129.74 (14-C), 129.90 (25-C), 130.72 (30-
C), 132.76 (12-C), 135.65 (27-C), 137.81 (22-C), 140.38 (8-C),
157.51 (40-C), 166.80 (10-C), 167.78 (18-C), 169.19 (32-C), 169.88
(6-C).
SPPS Synthesis of (2R,4′R,8a′R)-1-[N-(4-{[(5-Methylpyrazin-
2-yl)carbonyl]amino}benzoyl)-ꢀ-alanyl]-6′-oxotetrahydro-2′H-
spiro[pyrrolidine-2,7′-pyrrolo[2,1-b][1,3]thiazine]-4′-carboxa-
mide, PAM6-SP20-BETA8-NH2 (4). The peptidomimetic sequence
was synthesized on Rink amide MBHA resin (66 mg, ∼50 µmol),
as described in typical procedures (Steps 1–4 of SPPS synthesis of
2). Cleavage and purification of the crude product by RP-HPLC
gave 4 (5.5 mg, 17.5%); analytical HPLC showed a peak related
to the product at 8.87 min, purity > 95%. Mass value agrees with
theoretical value: ESIMS m/z 566.23 (M + H)+, 588.35 (M + Na)+.
1H NMR (Figure I in Supporting Information reports numbering
system used for the attribution of main signals, 500 MHz, DMSO-
d6): δ 1.55–1.66 (m, 1H, 3-CH″), 1.84–2.00 (m, 3H, 9-CH″, 10-
CH″, 6-CH″), 1.99–2.12 (m, 2H, 9-CH”, 10-CH”), 2.52–2.58 (m,
2H, 3-CH”, 13-CH″), 2.65 (s, 3H, 25-CH3), 2.66–2.85 (m, 4H,
4-CH2, 6-CH”, 13-CH”), 3.35–3.55 (m, 3H, 11-CH″, 14-CH2), 3.66
(t, J ) 8.1 Hz, 1H, 11-CH”), 4.63 (d, J ) 4.7 Hz, 1H, 2-CH), 5.00
(dd, J ) 9.0, 2.6 Hz, 1H, 5-CH), 7.32 (s, 1H, 28-NH′), 7.55 (s,
1H, 28-NH”), 7.84 (d, J ) 8.5 Hz, 2H, 17-CH, 17’CH), 8.01 (d, J
) 8.5 Hz, 2H, 18-CH, 18′-CH), 8.42 (t, J ) 5.5 Hz, 1H, 27-NH),
8.72 (s, 1H, 24-CH), 9.18 (s, 1H, 22-CH), 10.85 (s, 1H, 26-NH);
13C NMR (125.67 MHz, DMSO-d6): δ 22.18 (25-C), 24.45 (10-
C), 25.68 (3-C), 25.99 (4-C), 34.89 (13-C), 36.16 (14-C), 37.88
(6-C), 38.51 (9-C), 48.37 (11-C), 52.10 (2-C), 55.08 (5-C), 66.25
(7-C), 120.42 (18-C, 18′-C), 128.53 (17-C, 17′-C), 130.43 (16-C),
141.54 (19-C), 142.86 (21-C), 143.49 (24-C), 143.84 (22-C), 158.12
(23-C), 162.86 (20-C), 166.45 (15-C), 170.10 (12-C), 170.86 (1-
C), 172.73 (8-C).
1
Rf 0.5. H NMR (300 MHz, CDCl3): δ 1.03 (s, 9H), 1.45 (s, 9H),
2.15 (m, 1H), 2.80 (m, 1H), 3.55, 3.60 (2d, 1H), 3.75, 3.82 (2d,
1H), 4.30 (m, 1H), 4.05–4.60 (dd, 2H), 4.62 (m, 1H), 7.20 (d, 1H),
7.30–7.60 (m, 8H), 7.60–7.80 (m, 4H), 8.00 (d, 1H); ESIMS m/z
607.5 (M + Na)+. Anal. (C34H40N2O5Si) C, H, N. C: calcd, 69.83;
found, 68.87; H: calcd, 6.89; found, 7.37; N: calcd, 4.79; found,
4.01.
[(2R,11aS)-2-{[tert-Butyl(diphenyl)silyl]oxy}-5,11-dioxo-
2,3,11,11a-tetrahydro-1H-pyrrole[2,1-c][1,4]benzodiazepine-
10(5H)-yl]-acetic Acid (24). [(2R,11aS)-2-{[tert-butyl-(diphenyl
)silyl]oxy}-5,11-dioxo-2,3,11,11a-tetrahydro-1H-pyrrole[2,1-c]-
[1,4]benzo diazepine-10(5H)-yl)-acetic acid tert-butyl ester 23 (3.6
g, 6.1 mol) was suspended in 30 mL of CH2Cl2, and 25 mL of
trifluoroacetic acid were added dropwise, under stirring, at 15 °C.
The temperature was allowed to rise to room temperature, and the
solution was left under stirring for 45 min. The reaction solution
was brought to dryness under reduced pressure with a mechanical
pump, and the residue was solubilized in ethyl ether from which it
was precipitated by cooling, yielding a white solid (2.6 g, 81%).
TLC solvents: CHCl3/MeOH 7/3, Rf 0.7; mp 191–193 °C. [R]20
D
) +246.2 (c 1.0, CHCl3). HPLC: Inertsil ODS 3 column (5µ), 4.6
× 250 mm; mobile phase, CH3CN/KH2PO4 50 mM 70/30; pH 3
(H2PO4 85%); flow rate, 1.0 mL/min, t ) 30 °C; RT, 10.79 min.
1H NMR (300 MHz, CDCl3): δ 1.03 (s, 9H), 2.05 (m, 1H), 2.75
(m, 1H), 3.50, 3.55 (2d, 1H), 3.75, 3.78 (2d, 1H), 4.25 (m, 1H),
4.20–4.57 (dd, 2H), 4.60 (m, 1H), 7.10 (d, 1H), 7.20–7.50 (m, 8H),
7.60 (m, 4H), 7.90 (d, 1H). ESIMS m/z 551.2 (M + Na)+. Anal.
(C30H32N2O5Si) C, H, N. C: calcd, 68.16; found, 67.12; H: calcd,
6.10; found, 6.09; N: calcd, 5.30; found, 5.28.
SPPS Synthesis of N-(4-{[Amino(imino)methyl]amino}butyl)-
6-({[(2R,11aS)-2-hydroxy-5,11-dioxo-2,3,11,11a-tetrahydro-1H-
pyrrolo[2,1-c][1,4]benzodiazepin-10(5H)-yl]acetyl}amino)-2-
naphthamide, AM8-SP38-BETA6 (3). Typical Procedure for
the Coupling of an Arginine Mimetic with Formation of the
Guanidine Group. Fmoc-isothiocyanate (0.5 mmol) in DMF (2.0
mL) was added to Rink amide MBHA resin (66 mg, ∼50 µmol)
free of Fmoc protecting group. The suspension was shaken for 12 h,
filtered off, washed with DMF, and reacted with a solution of CH3I
(10 equiv relative to the resin) and DIPEA (30 equiv relative to
the resin) in DMF (3.0 mL). The mixture was shaken for 2 h at
room temperature, filtered off, and washed with DMF. Then
2-methyl-isothioureyl-resin was added, recovered from the previous
reaction, to a solution of 1,4-diaminobutane (10 equiv relative to
the resin) in DMF (2.0 mL) and shaken for 12 h at room
temperature. Afterward, the resin was filtered off and washed with
DMF.
Synthesis was then accomplished following the typical proce-
dures described in Steps 1 and 3 of SPPS synthesis of 2, using
Fmoc-SP38-OH (250 µmol) and tert-butyldiphenylsilyl-O-BETA6
acid chloride (TBDPS-O-BETA6-Cl; 250 µmol), prepared using
24 (Scheme 5) by treatment with thionyl chloride and cleavage of
the TBDS protective group with Bu4N+F–, as previously
described.26b Cleavage from the resin, as described in Step 4 of
the SPPS synthesis of 2, and RP-HPLC purification of crude product
gave 3 (0.9 mg, 2.85%); analytical HPLC showed a peak related
to the product at 6.78 min, purity > 90%. Mass value agrees with
theoretical value: ESIMS m/z 572.33 (M + H)+. 1H NMR (Figure
I of Supporting Information reports numbering system used for the
attribution of main signals, 500 MHz, MeOH-d4): δ 1.64–1.80 (m,
4 H, 36-CH2, 37-CH2), 2.08–2.17 (m, 1 H, 4-CH″), 2.86–2.96 (m,
1H, 4-CH”), 3.28 (t, J ) 6.9 Hz, 2H, 38-CH2), 3.50 (t, J ) 6.0 Hz,
2H, 35-CH2), 3.69 (dd, J ) 12.2, 4.7 Hz, 1H, 2-CH″), 3.77 (dd, J
) 12.2, 2.7 Hz, 1H, 2-CH”), 4.45 (t, J ) 6.0 Hz, 1H, 5-CH),
4.55–4.60 (m, 1H, 3-CH), 4.64 (d, J ) 16.7 Hz, 1H, 17-CH″), 4.84
Synthesis of {(5S)-1-[(9H-Fluoren-9-yl-methyloxy)-carbonyl]-
6-oxo-1,7-diazaspiro[4.5]dec-7-yl}-acetic Acid, Fmoc-BETA3-
OH (33). (3R,7aS)-7a-But-3-en-1-yl-3-tert-butyltetrahydro-1H-
pyrrolo[1,2-c][1,3]oxazol-1-one (25). Compound 25 was prepared
according to the literature (yield 50%).11c TLC solvents: n-hexane/
1
AcOEt 8/2, Rf 0.62. H NMR (300 MHz, CDCl3): δ 0.88 (s, 9H),
1.55–2.02 (m, 5H), 2.02–2.35 (m, 3H), 2.82–3.05 (m, 2H), 4.25
(s, 1H), 4.92–5.12 (m, 2H), 5.72–5.95 (m, 1H).
(S)-2-But-3-en-1-yl-proline (26). Compound 26 was prepared
according to the literature (yield 60%).11c Mp 276–277 °C (lit.11c
1
mp 281–282 °C dec). [R]20 ) –70.2 (c 1.0, MeOH). H NMR:
D
(300 MHz, DMSO-d6): δ 1.52–2.10 (m, 7H), 2.19–2.23 (m, 1H),
2.94–3.15 (m, 1H), 3.16–3.22 (m, 1H), 4.88–4.99 (m, 2H), 5.67–
5.82 (m, 1H), 8.55 (bs, 1H). ESIMS m/z 192.2 (M + Na)+.
(S)-2-But-3-en-1-yl-N-(tert-butoxycarbonyl)-proline (27). Com-
pound 27 was prepared according to the literature (yield 93%).11c
mp 86–88 °C (lit.11c mp 90–91 °C). [R]20D ) +36.8 (c 2.0, MeOH).
1H NMR (300 MHz, CDCl3): δ 1.48, 1.53 (s, 9H), 1.70–1.92 (m,
2H), 1.92–2.20 (m, 4H), 2.20–2.40 (m, 1H), 2.75–2.85 (m, 1H),
3.25–3.45 (m, 1H), 3.45–3.62 (m, 1H), 4.94–5.14 (m, 2H),
5.71–5.94 (m, 1H), 10.95 (bs, 1H). ESIMS m/z 292.3 (M + Na)+.
(S)-2-But-3-en-1-yl-N-(tert-butoxy-carbonyl)-prolylglycine
Methyl Ester (28). (S)-2-But-3-en-1-yl-N-(tert-butoxycarbonyl)-
proline 27 (8.0 g, 29.7 mmol), glycine methylester hydrochloride
(3.7 g, 29.7 mmol), and hydroxybenzotriazole (4.0 g, 29.7 mmol)
were solubilized in 100 mL of anhydrous CHCl3. TEA (4.1 mL,
29.7 mmol) and then DCC (6.1 g, 29.7 mmol), dissolved in 100
mL of anhydrous CHCl3, were added to the solution. The reaction
mixture was left under stirring overnight at room temperature in
an N2 atmosphere. The dicyclohexylurea (DCU) formed was
filtered, and the filtrate was then brought to dryness at reduced

1200 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Fantò et al.
pressure. The residue obtained was then shaken with Et2O and
filtered to eliminate any DCU; the liquid phase was washed with
NaHCO3 1M, brine, 10% citric acid and then again with brine.
The organic phase was dried over anhydrous Na2SO4, and the
solvent was evaporated under reduced pressure, obtaining 13 g of
a yellow oil that was purified using a silica gel chromatography
column, eluting with n-hexane/AcOEt 2/1, Rf 0.65. Purification
dissolved in a solution of 60 mL of CH2Cl2 and 60 mL of
trifluoroacetic acid, and the reaction solution was left stirring for
1 h at room temperature. Next, it was brought to dryness under
reduced pressure at 30 °C, and the residue was collected with H2O
and brought to dryness again under reduced pressure, drying
thoroughly with an oil pump, to obtain product 28 as a light-colored
oil (3.1 g, 95%). TLC solvents: CHCl3/isoPrOH/MeOH/H2O/AcOH
4.2/0.7/2.8/1.05/1.05, Rf 0.4. 1H NMR (300 MHz, D2O): δ
1.85–2.30 (m, 8H), 3.20–3.60 (m, 4H), 4.05 (d, 2H). ESIMS m/z
214.2 (M + H)+.
yielded 24 as a white solid (9.4 g, 93%). TLC solvents: n-hexane/
1
AcOEt 2/1. [R]20 ) –12.8 (c 0.7, MeOH). H NMR (300 MHz,
D
CDCl3): δ 1.54, 1.60 (2s, 9H), 1.75 (m, 3H), 2.10 (m, 4H), 2.75
(m, 1H), 3.35 (m, 1H), 3.58 (m, 1H), 3.75 (s, 3H), 4.05 (m, 2H),
5.00 (m, 2H), 5.82 (m, 1H), 6.50, 8.32 (2sa, 1H). ESIMS m/z 363.4
(M + Na)+.
{(5S)-1-[(9H-Fluoren-9-yl-methyloxy)-carbonyl]-6-oxo-1,7-
diazaspiro[4.5]dec-7-yl}-acetic Acid (33). (5R)-7-(Carboxym-
ethyl)-6-oxo-7-aza-1-azoniaspiro-[4.5]decano trifluoroacetate 32 (3.2
g, 10.0 mmol) was dissolved in 100 mL of H2O. NaHCO3 (1.7 g,
20.0 mmol) and then Fmoc-N-OSu (3.7 g, 11.0 mmol), dissolved
in 150 mL of acetone, was added to the solution. The reaction
mixture was left stirring at room temperature for 24 h, and after
evaporating the acetone under reduced pressure, it was diluted with
H2O and washed with Et2O. The aqueous phase was brought to
pH 2–3 with HCl 2 N and extracted with CHCl3; the organic phase
was dried over anhydrous Na2SO4, and the solvent was evaporated
under reduced pressure. The product obtained was crystallized with
CH2Cl2 and Et2O, thus obtaining 33 as a white solid (1.4 g, 32%).
TLC solvents: CHCl3/MeOH/AcOH 7.95/1.95/0.1, Rf 0.8.; mp
(5S)-7-(2-Methoxy-2-oxoethyl)-6-oxo-1,7-diazaspiro[4.5]decano-
1-carboxylic Acid tert-Butyl Ester (30). (S)-2-But-3-en-1-yl-N-
(tert-butoxycarbonyl)-prolylglycine methyl ester 28 (9.15 g, 27.0
mmol) was dissolved in 300 mL of MeOH/H2O 2/1. OsO4 (0.33 g,
1.3 mmol) was added to the solution and left stirring for 10 min,
after bubbling of N2, while adding NaIO4 (17.1 g, 80.0 mmol)
portionwise. A white precipitate formed from the dark-colored
solution, and it was left stirring at room temperature for 24 h. H2O
was added to the reaction mixture until a solution was obtained
and then it was extracted several times with AcOEt. The pooled
organic phases were washed with H2O, dried over anhydrous
Na2SO4, and the solvent was evaporated under reduced pressure,
obtaining 9.2 g of a mixture of the diastereoisomers of (5R)-8-
hydroxy-7-(2-methoxy-2-oxoethyl)-6-oxo-1,7-diazaspiro[4.5]-decano-
1-carboxylic acid tert-butyl ester 29 as a dark-colored oil. TLC
solvent: AcOEt; Rf 0.39 and 0.52 (diastereoisomers). 1H NMR (300
MHz, CDCl3): δ 1.40, 1.48 (s, 9H), 1.60–1.65 (m, 1H), 1.65–2.00
(m, 4H), 2.08 (m, 1H), 2.10–2.80 (m, 2H), 3.25–3.40 (m, 1H),
3.50–, 3.70 (m, 2H), 3.75 (s, 3H), 4.05 (m, 2H), 8.20 (bs, 1H).
This product was allowed to react without any further purification.
(5R)-8-Hydroxy-7-(2-methoxy-2-oxoethyl)-6-oxo-1,7-
diazaspiro[4.5]decano-1-carboxylic acid tert-butyl ester 29 (9.0 g, 26.0
mmol) was dissolved in 180 mL of anhydrous THF. Trifluoroacetic
acid (18 mL) was added to the solution, which was cooled in an
ice bath before adding NaBH3CN (4.68 g, 74.0 mmol). The reaction
mixture was left stirring for 20 h at room temperature under a
nitrogen atmosphere and then alkalinized with K2CO3. The solution
was separated from the residue by filtration, and the filtrate was
then brought to dryness under reduced pressure. The amorphous
mass obtained was dissolved in H2O and extracted several times
with CH2Cl2. The organic phase was dried over anhydrous Na2SO4,
and the solvent was evaporated under reduced pressure. A total of
9 g of 30 was obtained as a dark-colored oil that was purified by
silica column chromatography, eluting with n-hexane/AcOEt 1/3
to obtain 30 as a light-colored oil (4.5 g, 53%). TLC solvent: AcOEt,
122–123 °C. [R]20 ) –26.4 (c 0.5, MeOH). HPLC: Symmetry
D
C18 (5µ) column, 3.9 × 150 mm; mobile phase, KH2PO4 50 mM
pH 3/CH3CN 65/35; flow rate, 1.0 mL/min, RT, 5.2 min. 1H NMR
(300 MHz, CDCl3): δ 1.20, 1.38, 1.62 (3m, 1H), 1.78–2.20 (m,
5H), 2.22–2.50 (m, 2H), 2.80, 3.45 (2m, 1H), 3.25 (m, 1H), 3.60
(dd, 1H), 3.65 (m, 1H), 3.80, 3.85 (2d, 1H), 4.15, 5.2 (2m, 1H),
4.2–4.35 (m, 1H), 4.40 (m, 1H), 4.60 (m, 1H), 7.20–7.45 (m, 8H),
7.58 (m, 2H), 7.75 (d, 2H). ESIMS m/z 457.4 (M + Na)+. Anal.
(C25H26N2O5) C, H, N. C: calcd, 69.10; found, 67.81; H: calcd,
6.03; found, 5.90; N: calcd, 6.44; found, 6.31.
SPPS Synthesis of N-{3-[(5R)-7-(2-Amino-2-oxoethyl)-6-oxo-
1,7-diazaspiro[4.5]dec-1-yl]-3-oxopropyl}-4-[(1-naphthylace-
tyl)amino]benzamide, PAM8-SP20-BETA3-NH2 (5). The pep-
tidomimetic sequence was synthesized on Rink amide MBHA resin
(66 mg, ∼50 µmol), as described in typical procedures (Steps 1–4
of SPPS synthesis of 2), using 33, prepared according to Scheme
6. Cleavage and purification of the crude product by RP-HPLC
gave 5 (3.5 mg, 11.6%); analytical HPLC showed a peak related
to the product at 10.02 min, purity > 94%. Mass value agrees with
theoretical value: ESIMS m/z 570.17 (M + H)+, 592.36 (M + Na)+.
1H NMR (Figure I in Supporting Information reports numbering
system used for the attribution of main signals, 500 MHz, DMSO-
d6): δ 1.65–2.06 (m, 7H, 5-CH″, 6-CH2, 9-CH2, 13-CH2), 2.25 (td,
J ) 12.7, 4.4 Hz, 1H, 5-CH”), 2.39–2.48 (m, 1H, 8-CH″), 2.54–2.66
(m, 1H, 8-CH”), 3.21–3.28 (m, 1H, 10-CH″), 3.34 (d, J ) 16.6
Hz, 1H, 2-CH″), 3.36–3.54 (m, 4H, 12-CH2, 10-CH”, 7-CH″),
3.63–3.70 (m, 1H, 7-CH”), 4.18 (s, 2H, 20-CH2), 4.37 (d, J ) 16.6
Hz, 1H, 2-CH”), 7.20 (s, 1H, 33-NH′), 7.29 (s, 1H, 33-NH”),
7.46–7.60 (m, 4H, 29-CH, 30-CH, 23-CH, 28-CH), 7.68 (d, J )
8.8 Hz, 2H, 17-CH, 17′-CH), 7.79 (d, J ) 8.7 Hz, 2H, 16-CH,
16′-CH), 7.86 (dd, J ) 7.6, 1.3 Hz, 1H, 24-CH), 7.95 (dd, J ) 8.6,
1.3 Hz, 1H, 22-CH), 8.13 (d, J ) 8.3 Hz, 1H, 27-CH), 8.27 (t, J )
5.7 Hz, 1H, 32-NH), 10.54 (s, 1H, 31NH); 13C NMR (125.67 MHz,
DMSO-d6): δ 21.23 (6-C), 24.02 (9-C), 31.93 (5-C), 35.03 (8-C),
36.15 (12-C), 37.29 (13-C), 41.36 (20-C), 48.92 (7-C), 49.26 (10-
C), 52.09 (2-C); 66.43 (4-C), 118.97 (17-C, 17′-C), 124.89 (27-
C), 126.25 (29-C), 126.41 (23-C), 126.85 (28-C), 128.02 (24-C),
128.62 (30-C), 128.70 (16-C, 16′-C), 129.15 (22-C), 129.58 (15-
C), 132.71 (25-C), 132.92 (26-C), 134.09 (21-C), 142.47 (18-C),
166.31 (14-C), 169.76 (11-C), 170.11 (19-C), 170.81 (3-C), 171.78
(1-C).
Rf 0.58. [R]20 ) –58.6 (c 1.3, MeOH). 1H NMR (300 MHz,
D
CDCl3): δ 1.40, 1.60 (s, 9H), 1.60–2.15 (m, 6H), 2.15–3.80 (m,
2H), 3.30 (m, 1H), 3.30–3.80 (m, 4H), 3.75, 3.76 (2s, 3H), 4.70,
4.78 (2d, 1H). ESIMS m/z 349.2 (M + Na)+.
[(5S)-1-(tert-Butoxycarbonyl)-6-oxo-1,7-diazaspiro[4.5]dec-
7-yl]-acetic Acid (31). (5S)-7-(2-Methoxy-2-oxoethyl)-6-oxo-1,7-
diazaspiro[4.5]decano-1-carboxylic acid tert-butyl ester 30 (4.0 g,
12.0 mmol) was dissolved in a solution of 60 mL of MeOH and
60 mL of H2O, and then K2CO3 (3.32 g, 24.0 mmol) was added.
The reaction mixture was kept at room temperature under stirring
for 20 h, then brought to pH 5 with HCl 2 N, and evaporated under
reduced pressure. The residue obtained was dissolved in H2O,
brought to pH 2–3, and extracted several times with CH2Cl2. The
organic phase was dried over anhydrous Na2SO4, and the solvent
was evaporated under reduced pressure, obtaining 31 as a white
solid (3.2 g 86%). TLC solvents: CHCl3/MeOH/AcOH 7.95/1.95/
1
0.1, Rf 0.65. [R]20 ) –58.4 (c 0.5, MeOH). H NMR (300 MHz,
D
CDCl3): δ 1.43 (s, 9H), 1.60–2.15 (m, 6H), 2.15–2.50 (m, 2H),
3.25 (m, 1H), 3.30–3.80 (m, 4H), 4.43, 4.98 (2d, 1H). ESIMS m/z
335.3 (M + Na)+.
(5R)-7-(Carboxymethyl)-6-oxo-7-aza-1-azoniaspiro[4.5]decano
Trifluoroacetate (32). [(5S)-1-(tert-Butoxycarbonyl)-6-oxo-1,7-di-
azaspiro-[4.5]dec-7-yl]-acetic Acid 31 (3.1 g, 10.0 mmol) was
SPPS Synthesis of (2R,4′R,8a′R)-1-[(3-{[(4-Chlorophenoxy)-
acetyl]amino}-1H-1,2,4-triazol-5-yl)carbonyl]-6′-oxotetrahydro-
2′H-spiro[pyrrolidine-2,7′-pyrrolo[2,1-b][1,3]thiazine]-4′-car-
boxamide, PAM8-SP33-BETA8-NH2 (6). The peptidomimetic
sequence was synthesized on Rink amide MBHA resin (66 mg,
∼50 µmol), as described in typical procedures (Steps 1–4 of SPPS

Inhibitors of Myeloid Differentiation Factor 88
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1201
synthesis of 2). Cleavage and purification of the crude product by
RP-HPLC gave 6 (1.3 mg, 4.53%); analytical HPLC showed a peak
related to the product at 10.08 min, purity > 93%. Mass value
agrees with theoretical value: ESIMS m/z 534.17 (M + H)+, 556.27
(M + Na)+. 1H NMR (Figure I in Supporting Information reports
numbering system used for the attribution of main signals, 500
MHz, DMSO-d6): δ 1.90–2.08 (m, 5H, 9-CH2, 10-CH2, 6-CH″),
2.53–2.57 (m, 2H, 3-CH2), 2.67–2.83 (m, 3H, 6-CH”, 4-CH2),
3.50–3.58 (m, 1H, 11-CH″), 3.67–3.74 (m, 1H, 11-CH”), 4.62 (dd,
J ) 5.8, 1.70 Hz, 1H, 2-CH), 4.76 (d, J ) 15.6 Hz, 1H, 16-CH″),
4.82 (d, J ) 15.6 Hz, 1H, 16-CH”), 5.00 (dd, J ) 9.4, 2.8 Hz, 1H,
5-CH), 6.94 (d, J ) 9.2 Hz, 2H, 18-CH, 18′-CH), 7.09 (bs, 1H,
22-NH), 7.13 (bs, 1H, 19-NH′), 7.23 (bs, 1H, 19-NH”), 7.32 (d, J
) 9.2 Hz, 2H, 19-CH, 19′-CH), 7.51 (bs, 1H, 21-NH); 13C NMR
(125.67 MHz, DMSO-d6): δ 24.88 (10-C), 25.94 (3-C), 26.18 (4-
C), 37.88 (6-C), 38.28 (9-C), 47.29 (11-C), 52.31 (2-C), 55.22 (5-
C), 67.01 (7-C), 67.27 (16-C), 117.43 (18-C, 18′-C), 125.66 (20-
C), 130.10 (19-C, 19′-C), 157.78 (17-C), 166.90 (15-C), 168.92
(12-C), 171.04 (1-C), 172.69 (8-C).
Biology. Yeast 2-Hybrid Assay. The vectors (pGBKT7 and
pGADT7) used for the expression of MyD88 fused with the AD
and BD domains were supplied by Clontech, as was the yeast strain
AH109 (MATa, trp1-901, leu2-3 112, ura3-52, his3-200, gal4∆,
gal80∆) and the minimal media SGd/-Leu/-Trp e SGd/-Ade/-His/-
Leu/-Trp. The 384-well plates used for the assay were supplied by
BD Biosciences (Oxygen BioSensor Plates), and the instrument for
fluorescence measurements was the Fusion fluorescence detector
(PerkinElmer). The AH109 yeast strain, cotransformed with the
two gene fusions (AD-MyD88 and BD-MyD88), was preinoculated
in 2 mL of SGd/-Leu/-Trp and incubated overnight at 30 °C under
stirring at 200 rpm; the preinoculum was then diluted (1/20) in
100 mL of SGd/-Ade/-His/-Leu/-Trp for each well of the 384-well
plates in the presence of the molecule to be assayed (final
concentration: 100 µM). The plate was incubated in the Fusion
instrument at 30 °C and the fluorescence emitted by each individual
well was measured using an excitation wavelength of 485 nm,
reading the emission at 630 nm from the bottom of the plate every
90 min for a total of 25 readings. Fluorescence intensity is an
arbitrary unit, therefore, normalization is necessary: the fluorescence
intensity of a well at a given time t must be divided by the initial
fluorescence intensity of the same well.
The compounds considered positive in the NF-κB inhibition assay
were those showing a percentage inhibition g15%. The toxicity of
positive compounds was evaluated by MTT assay.28
Acknowledgment. The authors wish to thank Mr. Augusto
Liberati and Dr. Silvia Vincenti for their valuable contribution
and Ms. Marlene Deutsch for editing the manuscript.
Supporting Information Available: Elemental analysis data,
complete peptidomimetic sequences analytical data, 1H NMR and
13C NMR spectra, HPLC and ESIMS tracings of target compounds,
and activity of reference compound BAY 11-7085. This information
is available free of charge via the Internet at http://pubs.acs.org.
References
(1) Ghosh, S.; May, M. J.; Kopp, E. B. NF-kappa B and Rel proteins:
Evolutionarily conserved mediators of immune responses. Annu. ReV.
Immunol. 1998, 16, 225–260.
(2) Barnes, P. J.; Karin, M. Nuclear factor-kappaB: A pivotal transcription
factor in chronic inflammatory diseases. New Engl. J. Med. 1997, 336,
1066–1071.
(3) O’Neill, L. A. J.; Dinarello, C. A. The IL-1 receptor/toll-like receptor
superfamily: Crucial receptors for inflammation and host defense.
Immunol. Today 2000, 21, 206–209.
(4) Loiarro, M.; Sette, C.; Gallo, G.; Ciacci, A.; Fantò, N.; Mastroianni,
D.; Carminati, P.; Ruggiero, V. Peptide-mediated interference of TIR
domain in MyD88 inhibits interleukin-1-dependent activation of NF-
κB. J. Biol. Chem. 2005, 280, 15809–15814.
(5) Toshchakov, V. U.; Basu, S.; Fenton, M. J.; Vogel, S. N. Differential
involvement of BB loops of Toll-IL-1 resistance (TIR) domain-
containing adapter proteins in TLR4- versus TLR2-mediated signal
transduction. J. Immunol. 2005, 175, 494–500.
(6) Li, C.; Zienkiewicz, J.; Hawiger, J. Interactive sites in the MyD88
Toll/Interleukin (IL) 1 receptor domain responsible for coupling to
the IL1ꢀ signaling pathway. J. Biol. Chem. 2005, 280, 26152–26159.
(7) Fry, D. Protein-protein interactions as targets for small molecule drug
discovery. Biopolymers 2006, 84, 535–552.
(8) Genin, M. J.; Ojala, W. H.; Gleason, W. B.; Johnson, R. L. Synthesis
and crystal structure of a peptidomimetic containing the (R)-4.4-spiro
lactam type-II ꢀ-turn mimic. J. Org. Chem. 1993, 58, 2334–2337.
(9) Long, R. D.; Moeller, K. D. Conformationally constrained peptide
mimetics: The use of a small lactam ring as an HIV-1 antigen
constraint. J. Am. Chem. Soc. 1997, 119, 12394–12395.
(10) Peña, M. R.; Stille, J. K. A total synthesis of anthramicin: Application
of palladium-catalyzed coupling reactions for the attachment of the
acrylic side chain. J. Am. Chem. Soc. 1989, 111, 5417–5424.
(11) (a) Seebach, D.; Boes, M.; Naef, R.; Schweizer, B. Alkylation of amino
acids without loss of the optical activity: Preparation of R-substituted
proline derivates. A case of self-reproduction of chirality. J. Am. Chem.
Soc. 1983, 105, 5390–5398. (b) Genin, M. J.; Baures, P. W.; Johnson,
R. L. An improved method of oxazolidinone hydrolysis in the
asymmetric synthesis of R-alkylprolines. Tetrahedron Lett. 1994, 23
(35), 4967–4968. (c) Khalil, E. M.; Ojala, W. H.; Pradhan, A.; Nair,
V. D.; Gleason, W. B.; Mishra, R. K.; Johnson, R. L. Design, synthesis,
and dopamine receptor modulating activity of spiro bicyclic peptido-
mimetics of L-prolyl-L-leucyl-glycinamide. J. Med. Chem. 1999, 42,
628–637.
Biology. NF-KB Inhibition Assay. HeLa (ECACC nr 93021013)
cells were cultured in EMEM medium supplemented with 2 mM
glutamine, 1% nonessential amino acids, 7.5% FBS (Fetal Bovine
Serum) and 10 mL/L of a solution of penicillin/streptomycin (10000
units/mL penicillin and 10 mg/mL streptomycin). All reagents
described were provided by Sigma-Aldrich. Cells were seeded in
six-well plates at a density of 3 × 105 cells/well in complete
medium and incubated overnight at 37 °C, 5% CO2. After 18 h,
cells were washed twice with PBS and then resuspended with 1
mL/well of EMEM without serum.
The compounds, dissolved in DMSO, were subsequently added
to the cells at a final concentration of 100 µM, with the cells
incubated for 6 h at 37 °C, 5% CO2. The cells were stimulated
with 5 ng/mL of recombinant human IL-1R (Sigma-Aldrich) for
30 min at 37 °C, 5% CO2. After stimulation with IL-1R, the cells
were washed twice with PBS, collected by cell scraping, and
centrifuged at 800 rpm at 4 °C for 10 min. Activation/inhibition of
NF-κB was evaluated by using the TransAM NF-κB p65 Kit (Active
Motif) that allows detection of activated NF-κB through a colori-
metric reaction.27 Pellets were resuspended in lysis buffer (provided
by kit), incubated for 10 min at 4 °C, and centrifuged at maximum
speed at 4 °C. About 10 µg of total proteins, measured by Bradford
Assay (BioRad), were utilized to perform these experiments.
Percentage of inhibition is calculated by processing the values
obtained in the spectrophotometric readings as follows: 100 - (IL*
- C*/IL - C) ) % inhibition of NF-κB, where IL* ) A450 of
cells stimulated and treated with the molecules under investigation;
C* ) A450 of cells not stimulated and treated with the molecules;
IL ) A450 of cells stimulated with IL-1R and not treated with the
molecules; and C ) A450 of cells neither stimulated nor treated.
(12) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Experimental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. AdV. Drug
DeliVery ReV. 1997, 23, 3–25.
(13) Olsson, I.-M.; Gottfries, J.; Wold, S. D-Optimal onion designs in
statistical molecular design. Chemom. Intell. Lab. Syst. 2004, 73, 37–
46.
(14) Ferguson, A. M.; Heritage, T.; Jonathon, P.; Pack, S. E.; Phillips, L.;
Rogan, J.; Snaith, P. J. EVA: A new theoretically based molecular
descriptor for use in QSAR/QSPR analysis. J Comput.-Aided Mol.
Des. 1997, 11, 143–152.
(15) Bartfai, T.; Behrens, M. M.; Gaidarova, S.; Pemberton, J.; Shivanyuk,
A.; Rebek, J., Jr. A low molecular weight mimic of the Toll/IL-1
receptor/resistance domain inhibits IL-1 receptor-mediated responses.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7971–7976.
(16) (a) Fields, S.; Song, O. A novel genetic system to detect protein-protein
interactions. Nature 1989, 340, 245–246. (b) Chien, C. T.; Bartel, P. L.;
Sternglanz, R.; Fields, S. The two-hybrid system: a method to identify
and clone genes for proteins that interact with a protein of interest.
Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 9578–9582.
(17) Wodnicka, M.; Guarino, R. D; Hemperly, J. J; Timmins, M. R; Stitt,
D.; Pitner, J. B. Novel fluorescent technology platform for high

1202 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Fantò et al.
throughput cytotoxicity and proliferation assays. J. Biomol. Screen.
2000, 5, 141–152.
(18) Pierce, J. W.; Schoenleber, R.; Jesmok, G.; Best, J.; Moore, S. A.;
Collins, T. E.; Gerritsen, M. E. Novel inhibitors of cytokine induced
IκBR phosphorylation and endothelial cell adhesion molecule expres-
sion show anti-inflammatory effects in vivo. J. Biol. Chem. 1997, 272,
21096–21103.
(19) Loiarro, M.; Capolunghi, F.; Fantò, N.; Gallo, G.; Campo, S.; Arseni,
B.; Carsetti, R.; Carminati, P.; De Santis, R.; Ruggiero, V.; Sette, C.
Inhibition of MyD88 dimerization and recruitment of IRAK1 and
IRAK4 by a novel peptidomimetic compound. J. Leukocyte Biol. 2007,
82, 801–810.
(20) (a) Hoffman, E. S.; Smith, R. E.; Renaud, R. C., Jr. TLR-targeted
therapeutics. Nat. ReV. 2005, 4, 879–880. (b) Wickelgren, I. Targeting
the tolls. Science 2006, 312, 184–187. (c) Gearing, A. J. H. Targeting
toll-like receptors for drug development: A summary of commercial
approaches. Immunol. Cell Biol. 2007, 85, 490–494.
(22) Ulevitch, R. J. Therapeutics targeting the innate immune system. Nat.
ReV. Immunol. 2004, 4, 512–520.
(23) TSAR3D, v. 3.3; Accelrys Inc.: 9685 Scranton Road, San Diego, CA
92121–3752.
(24) SIMCA-P, v. 10.0; Umetrics AB: Box 7960, SE-90719 Umeå, Sweden.
(25) MODDE, v. 7.0; Umetrics AB: Box 7960, SE-90719 Umeå, Sweden.
(26) (a) Atherton, E.; Sheppard, R. C. The Fluorenylmethoxycarbonyl
Amino Protective Group. In The Peptides; Udenfriend, S., Meienhofer,
J., Eds.; Academic Press: New York, 1987; Vol. 9, pp 1–39. (b)
Hanessian, S.; Lavalle, P. The preparation and synthetic utility of tert-
butyldiphenylsilyl ethers. Can. J. Chem. 1975, 53, 2975–2977.
(27) Renard, P.; Ernest, I.; Houbion, A.; Art, M.; Le Calvez, H.; Raes, M.;
Remacle, J. Development of a sensitive multi-well colorimetric assay
for active NFkappaB. Nucleic Acids Res. 2001, 29, e21.
(28) Mosmann, T. J. Rapid colorimetric assay for cellular growth and
survival: Application to proliferation and cytotoxicity assays. J. Im-
munol. Methods 1983, 65, 55–63.
(21) Krieg, A. M. Therapeutic potential of Toll-like receptor 9 activation.
Nat. ReV. Drug DiscoVery 2006, 5, 471–484.
JM070723U