Novel tetrahydropyran-based peptidomimetics from a bioisosteric transformation of a tripeptide. Evidence of their activity at melanocortin receptors

Article abstract of DOI:10.1016/S0968-0896(03)00274-8

We have prepared novel peptidomimetics based on a 2,4,6-trisubstituted tetrahydropyran. This scaffold was constructed in an isosteric transformation using conceptual constraints imposed on a tripeptide moiety involving O_i′-C_i+1^γ

Full text of DOI:10.1016/S0968-0896(03)00274-8

Bioorganic& Medicinal Chemistry 11 (2003) 3053–3063
Novel Tetrahydropyran-Based Peptidomimetics from a
Bioisosteric Transformation of a Tripeptide. Evidence of Their
Activity at Melanocortin Receptors
Adam W. Mazur,* Anna Kulesza, Rajesh K. Mishra, Doreen Cross-Doersen,
Anne F. Russell and Frank H. Ebetino
Procter & Gamble Pharmaceuticals, Health Care Research Center, Mason, OH 45040, USA
Received 23 January 2003; revised 14 April 2003; accepted 16 April 2003
Abstract—We have prepared novel peptidomimetics based on a 2,4,6-trisubstituted tetrahydropyran. This scaffold was constructed
in an isosteric transformation using conceptual constraints imposed on a tripeptide moiety involving Oi0–Cig+1 and Oi0–Ni+2 formal
cyclization modes. A series of regioselective transformations commencing with a substituted dihydropyran-4-one readily provided
the required analogues. Specific tetrahydropyrane analogues modeled on PheArgTrp as a truncated version of the melanocortin
receptor message sequence, showed activity at the melanocortin receptors MC4R and MC1R. Thus, the 2,4,6-trisubstituted tetra-
hydropyran scaffold has provided a potentially useful peptidomimetic lead, and conceptual cyclization of peptide moieties can offer
a valuable design strategy in peptidomimeticresearch.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
While nitrogen-containing saturated ring systems con-
stitute fundamental peptidomimetic scaffolds, the cor-
responding oxacycles are less frequently used for this
purpose. Hirschmann1 reported the first glucose-based
Scheme 1. Examples of frequently used modes of conceptual bridging
atoms in dipeptide moieties leading to cyclic peptidomimetics. Modes
peptidomimeticsomatostatin agonist and several efforts
have subsequently been reported for utilization of the
pyranose structure to mimick peptide motifs.2À10 How-
ever, these approaches involved mainly transformations
of carbohydrate structures, in particular including cap-
ping the hydroxyl groups with alkyl or acyl groups. This
methodology leaves excess oxygen atoms in the mol-
ecule because their removal would usually involve more
elaborate transformations. Consequently, the pyranose
structure merely serves as an attachment center for spa-
tial display of a few or several substituents, but the
structures do not have a clear topological relationship
to their peptide counterpart. On the other hand, more
direct translations have been accomplished with many
nitrogen-containing heterocycles designed by con-
ceptual bridging of the peptide backbone as illustrated
in Scheme 1.
a
of bridging: a. NiÀNi+1
;
b. NiÀCi+10NiÀCia;
c
.
CÀNi+1
; d.
i
Cai ÀCai+1
.
In this report, we want to present two aspects of our
petidomimetic research. One, concerns the development
of oxacyclic peptidomimetics. These structures differ from
the earlier pyranose-based systems by having closer topo-
logical relationships to the constrained peptide structure
and by the fact that they are derived from amino acids
not carbohydrates. The other aspect of our report is the
demonstration of conceptual bridging of peptide struc-
tures as a low-cost technique in the arsenal of drug dis-
covery tools. This methodology can rapidly produce early
peptidomimeticleads from the initial peptide structure,
without prior knowledge of the active conformation.
The bridging operations, presented in Scheme 1 have
generated many heterocyclic structures including
*Corresponding author. Tel.: +1-513-573-4636; e-mail: amazur@
cinci.rr.com
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00274-8

3054
A. W. Mazur et al. / Bioorg. Med. Chem. 11 (2003) 3053–3063
oxopiperazines and diazepines (mode a, b),11 Freidinger
lactams12,13 (mode c), piperidines (mode c, d), and so
on. Toniolo14 reviewed early work regarding the design
of peptidomimetics using 18 modes of constraints within
the tripeptide moiety. In most cases, these various
modes of cyclization have been used to design peptido-
mimetics addressing specific secondary structures of the
parent peptides, mostly the b- and g-turn motifs.15
Nonetheless, a large number of possible cyclization
modes, as well as a multitude and diversity of associated
chemical structures, brought us to the use of this
method, as a conceptual and mnemonic device in the
discovery of peptidomimetics, without a prior detailed
elucidation of the exact secondary structures of the cor-
responding peptides.
bridging function becomes either oxygen in (C) or a
methylene group in (D).
In the second case (Chart 2), the bridging in (B) con-
nects the backbone Ci0¼Oi0 and Ni+1. The centers Oi0
and Ni+1 become either O in (E) or CH2 in (F) while the
amide bond C’i+1–Ni+2 is replaced by CH2–CH in both
constructs. Structures (E-1) and (F-1) represent further
divergence from peptide moieties as well as a structural
simplification to facilitate synthesis.
In order to determine if the described transformations
of the tripeptide can actually produce bioisosteric
structures, we have applied this concept to finding non-
peptide ligands for the melanocortin receptors (MCR),
as a part of our ongoing efforts in this area.16À18 Among
five subtypes of these receptors, MC1R has been linked
to skin pigmentation19 while MC4R has been associated
with feeding behavior20,21 and modifying sexual activity
in humans.22
Results
Design of oxacylic peptidomimetics
Herein, we present a design and an initial evaluation of
2,4,6-trisubstituted tetrahydropyrans based on the
conceptual bridging of the carbonyl group Ci0 in a tri-
peptide. Our design strategy is shown in Charts 1 and 2.
It involves topological transformation of the tripeptide
moiety into the tetrahydropyran system that is isosteric
to the parent peptide. In addition, the designed tetra-
hydropyran analogues can also be viewed as isosteres of
a constrained amide bond.
As a model tripeptide for (A) and (B), we have selected
AcDPhe-Arg-TrpNH2 (1), a truncated version of the
message sequence His-Phe-Arg-Trp of peptide agonists
at the melanocortin receptors.23 The peptide (1) is
known to demonstrate weak but significant activity at
MC4R. We also chose 2-naphthyl moiety to be an iso-
stericreplacement for indole in Trp as a synthetiaclly
more accessible starting benchmark for peptidomimetic
synthesis.24
In the first case (Chart 1), the tripeptide (A) was con-
ceptually bridged by connecting the backbone Ci0¼Oi0
group to Cgi+1 of the side chain. The tetrahydropyran
structures (C) and (D) are obtained from (A) by repla-
cing Ni+1 with carbon and oxygen, respectively. The
The specific compounds that were made and tested in
our in vitro assays are listed in Chart 3. Analogues 10
and 13 correspond to the construct D from Chart 1.
Structurally, these two peptidomimetics differ from each
other by a number of CH2 groups in the arginine-related
side chain. The three remaining structures 18, 23, and 24
Chart 1. Design of tetrahydropyran analogues using Ci0¼Oi0ꢀ ꢀ ꢀCig+1
mode of peptide bridging.
Chart 2. Design of tetrahydropyran analogues using Ci0¼Oi0ꢀ ꢀ ꢀNi+2
Chart 3. Compounds tested at the melanocortin receptors MC4R and
MC1R.
mode of peptide bridging.

A. W. Mazur et al. / Bioorg. Med. Chem. 11 (2003) 3053–3063
3055
originated from the design illustrated in Chart 2. The
structure 18 corresponds to F-1 while 23 and 24 have
been derived from E-1. As compared to the original
constructs, F and E, all three synthesized compounds
are lacking the carboxamide terminus and 18 is a mix-
ture of diatereoisomers at C-4. Also, analogues 23 and
24 contain the chain corresponding to the arginine side
chain in B shortened by one CH2. They also represent
separate isomers that would relate respectively to l- and
d-isomers of arginine in B.
structures. Prior to the hydrolysis of CN group, the 4-
keto group in 4a and 4b had to be protected in the form
of a dimethyl acetal (step a, i) to prevent decomposition
of the material. The acetal was subsequently hydrolyzed
with acetic acid (step a, iii) and the free carboxyl group
was esterified with TMS diazomethane (step a, iv).
The next stage in the conversions of 6a and 6b involved
installation of the cyano and cyanomethylene groups at
C-4 as precursors of the guanidine function relating to
the tripetide arginine site.
Synthesis
The key intermediates for the preparation of all descri-
bed 2,4,6-trisubstituted tetrahydropyrane analogues
have been 3,4-dihydro-2H-pyran-4-ones 3a and 3b.
These are derived from a convenient hetero Diels–Alder
cyclization approach.25 Amino acid aldehydes used for
this reaction ascertained the first substitution in the
pyran ring. Two remaining substituents were introduced
sequentially utilizing properties of the conjugated
ketone system in 3a and 3b. The reaction sequence
started with 1,4-additions of the nucleophiles such as
the cyano group26 (step b) and alkynes27 (step c) result-
ing in analogues 4a, 4b and 5a, 5b. All these reactions
took place with high diastereoselectivity.
While 5a and 5b contained the required stereochemistry
at C-6, the configuration of this center in 4a and 4b had
to be reversed. We discovered that basic hydrolysis of
the cyano group (Scheme 3) occurs with epimerization
thus furnishing the correct diastereoisomers of 6a and
6b. The configurations of the center at C-6 for 4b and 6b
were determined by difference Nuclear Overhauser
Effect (NOE) NMR spectroscopy at 500 MHz. In the
case of 4b, irradiation of the H-2 resonance showed a
significant NOE to a proton at the carbon adjacent to
the NH Bocgroup. Additionally, irradiation of H-6
showed NOEs to both protons at position 5, but no
significant NOE was detected to H-2. For 6b, irradia-
tion of H-2 showed a strong NOE to H-6 indicating that
these two protons are in close proximity on the same
side of the ring. These data confirmed the proposed
Scheme 3. Reagents and conditions: (a) (i) HC(OMe)3, TsOH, MeOH;
(ii) KOH, EtOH, H2O; (iii) AcOH, THF; (iv) TMSCH2N2, MeOH; (b)
(EtO)2OPCN, NaCN, THF, rt; (c) (EtO)2OPCH2CN, NaH, THF,
À40 ꢁC; (d) SmI2, t-BuOH, THF, rt; (e) (i) LiOH, H2O, THF, rt; (ii) 2-
Nal-CONHMe, HOBt, NMM, EDCI, DMF, rt; (f) H2, Ni/Ra,
MeOH/NH4OH (1:4); (g) H2, NaBH4, PdCl2, MeOH; (h) (i)
BocNHC(¼NBoc)SMe, HgCl2, Et3N, DMF, 0 ꢁC; (ii) TFA, DCM.
Scheme
2. Reagents
and
conditions:
(a)
CH2¼C(OSi-
C(OSiMe3)CH¼CHOMe, LiClO4, Et2O, rt; (b) TMSCN, BF3Et2O,
DCM, 0 ꢁC; (c) R-CꢂCH, BuLi, TMSI, CuI*DMS, THF.

3056
A. W. Mazur et al. / Bioorg. Med. Chem. 11 (2003) 3053–3063
Thus, the 4-keto group in 6a was converted to the cyano
intermediate 8, constituting a mixture of isomers, by
means of cyanohydrin phosphate 7 (Scheme 3, step b)
followed by reduction of the latter with samarium ido-
die.28 The ester function in 8 was replaced with l-naph-
thylalanine methylcarboxamide to produce 9 using
standard methodologies of ester hydrolysis and amide
bond formation. The cyano group in 9 was reduced to the
corresponding amine with Raney nickel and subsequently
converted to 10 by a typical guanidinylation technique29
followed by TFA cleavage of all Boc protections.
obtained a single compound 24 but 23 was not com-
pletely isolated from the other isomer and contained
about 20% of 24.
Biological testing
Table 1 shows the biological activity of 2,4,6-trisub-
stituted tetrahydropyran peptidomimetics. The com-
pounds were evaluated in the binding and functional
cell-based assays. Analogues 10 and 13, corresponding
to the construct D, have binding affinity and agonist
potency at MC4R similar to those of 1. On the other
hand, Emax values of 10 and 13 indicate that they may
be only partial agonists at this receptor. One particular
difference between these two peptidomimetics is the
emergence of MC1R binding in 13. Over all, the pepti-
domimetics 10 and 13 appear to be bioisostericwith
respect to the model tripeptide 1.
The cyanomethylene group was introduced to 6b using
a Horner–Emmons reaction30 (Scheme 3, step c) result-
ing in 11. A transformation of 11 into 13 essentially
followed the same methodology as the one applied for
making 10 from 8 with one additional step (Scheme 3,
step g) involving reduction of the conjugated double bond
with NaBH4/PdCl2.31 This hydrogenation method was
used for a chemoselective reduction of carbon–carbon
double bond in a,b-unsaturated carbonyl compounds.32
The analogues 17, related to F, as well as 23, and 24,
related to the construct E do not show the agonist
The reactions listed in Scheme 4 represent the conversion
of 5a to 18. En route, reduction of ketone 14 produced a
mixture of diasteroisomericalcohols in the ratio of about
2:1. This mixture was carried to the final product 18.
Table 1. Affinity and functional activity of investigated analogues at
the melanocortin receptors MC4R and MC1R
MC4R
MC1R
Ki (nM) EC50 (nM) (Emax%) Ki (nM) EC50 (nM) (Emax%)
Analogues relating to the construct E-1 in Chart 2, were
derived from ketone 5b (Scheme 5). A Horner–Emmons
reaction of 5b followed by hydrogenation of the double
bond in 18 produced two diastereoisomers 19a and 19b
in a 2:1 ratio. Using NOESY and NOE techniques, we
assigned configuration 19b to the major and 19a to the
minor isomer. We carried out the subsequent steps c
and d with diastereoisomericmixtures of starting mate-
rials and separated the products at each step to confirm
the structure of each isomer. In the final purification, we
1
1188
3395
1163
797
1701
5472
2803 (49)
2463 (45)
4269 (52)
>100000
>100000
>100000
>5000
>5000
1445
2858
3310
10
13
18
23
24
>25 000
1636 (69)
1498 (64)
>5000
Scheme 4. Reagents and conditions: (a) H2, Pd/C 10%, AcOEt; (b) l-
Selectride, THF, À78 ꢁC; (c) (i) 2-(bromomethyl)naphthalene, NaH,
NaI, THF; (ii) TBAF, THF (d) BocHNC(¼NH)NHBoc, DEAD,
PPh3, THF, 0 ꢁC; (e) TFA, DCM.
Scheme 5. Reagents and conditions: (a) (EtO)2OPCH2CN, NaH,
THF, À40 ꢁC; (b) H2, 40PSI, Wilkinson cat., toluene; (c) (i) H2, Ni/
Ra, MeOH/NH4OH (1:4); (ii) BocNHC(¼NBoc)SMe, HgCl2, Et3N,
DMF, 0 ꢁC; (d) TFA, DCM.

A. W. Mazur et al. / Bioorg. Med. Chem. 11 (2003) 3053–3063
3057
activity at MC4R. Compounds 18 and 23 have similar
binding affinity at MC4R to that of 1 with 18 appearing
somewhat more potent. The key difference in the
measured biological activity of 18 and 23 versus 10 and
13 is an agonist effect at MC1R of the two former
compounds.
three hMCRs using a cell-based assay that is specific for
each subtype of MCRs (MC1R, MC3R, MC4R). Each
subtype was stably transfected into HEK293 cells see-
ded in 96 well poly-l-lysine coated plates (VWR) with
DMEM culture media containing 10% FBS, 1% Mini-
mum Essential Amino Acids, 0.1% Penn/Strep and
0.1% l-glutamine (Gibco). NDP-MSH-Eu was dis-
solved in MEM (Gibco)+10% Seablock (Pierce)+1%
DMSO (Sigma ) mixture to attain a final concentration
of 25 nM. This is to be added fresh each assay.
Discussion and Conclusions
Even though compared to other recently published
results33 we have obtained only low activity at the mel-
anocortin receptors, this work does illustrate that 2,4,6-
trisubstituted tetrahydropyrans designed by conceptual
bridging the tripeptide moiety between Oi0 and Cig+1 or
Ni+2 are ligands of the melanocortin receptors MC1R
and MC4R. Therefore, we can consider this transfor-
mation as being bioisostericwith respect to the model
peptide. It should be noted that our design hasn’t been
based on attempts to mimic a particular secondary
structure of the peptide ligand, for example, b-turn but
only on a topological relationship between the peptide
and 2,4,6-trisubstituted tetrahydropyrans. Whether the
reported structures correspond to any potential second-
ary peptide structures can possibly be determined by
conformational analysis of 10, 13, 18, 23 and 24. This
analysis could also aid determination of conformational
characteristics of ligands required for the activity at the
melanocortin receptors.
Binding activity (calculated as IC50 and Ki values) was
determined in these cell lines by measuring the dis-
placement of a constant concentration of europium
labeled NDP-a-MSH with competing unlabeled ligands.
Europium activity was detected using time resolve
fluorometry on a Wallacplate reader under the Bio-
works Europium program.
In vitro functional assay for the MCRs. The agonist
activity of MCR ligands was evaluated at three hMCRs
using the above MCR expressing HEK293 cells which
were stably transfected with a reporter system consisting
of a c-AMP responsive element (CRE) coupled to a
luciferase reporter gene. Agonist activity was deter-
mined by assaying cells for luciferase activity in Packard
96-well view plate using luciferase assay buffer contain-
ing tricine (20 mM), MgCOꢃ3Mg(OH)2ꢃ 5H2O (1.07 mM),
MgSOꢃ47H2O (2.67 mM), EDTA (0.1 mM) and lucifer-
ase substrate containing: DTT (5 mg/mL), ATP (0.292
mg/mL), co-enzyme A (0.208 mg/mL), d-luciferin
(0.142 mg/mL). Responses were compared to the effect
of NDP-MSH (MT-I) and expressed as a % of max-
imum activity of MT-I (Emax) which is considered to be
a full agonist at each of the three MCRs.
While it will remain important to analyze peptide sec-
ondary structures and continue designing non-peptide
mimetics using conformational information from pep-
tide leads, a systematicreview of possible peptide con-
straint modes and structures associated with them may
also provide a fertile ground for the design of diverse
non-peptide analogues. As a result of this formal ana-
lysis, one can envision initiation of a peptidomimetic
strategy through the systematicdesign of a library of
surrogates for constraint modes of peptide ligands.
When screened against biological targets, these libraries
may provide access to initial hits. Thus, such an
approach can be particularly effective in early discovery
of non-peptide ligands from peptide leads. It can sup-
plement other methods such as computer modeling and
high throughput screening.
Chemistry
General information
1H and 13C NMR spectra were recorded with Varian
Inova 300 and 500 spectrometers; the data are reported
as follows: chemical shift in ppm from Me4Si line as
external standard, multiplicity (b=broad, s=singlet,
d=doubled, t=triplet, q=quartet, m=multiplet) and
coupling constants. Normal resolution mass spectra and
LC–MS were performed on a Micromass ZMD-400
spectrometer. High resolution mass spectra were
obtained with AutoSpecSpectrometer at the Nebraska
Center for Mass Spectrometry, University of Nebraska.
Flash chromatography was carried out on a Biotage
system using Flash 40 silica cartridges. Analytical
HPLC was performed using a ThermoQuest system
equipped with MetaChem, Polaris, C18 3m 4.6ꢄ250 mm
column, using linear gradient elution starting from
CH3CN/0.1% phosphoricacid in water (20:80) to 100%
CH3CN over 20 min (1 mL/min). Preparative HPLC
was done on Rainin Dynamax and Varian ProStar sys-
tems equipped with MetaChem, Polaris, C18 10m col-
umn 50ꢄ250 mm using gradient elution starting from
CH3CN/0.1% trifluoroacetic acid in water to 100%
CH3CN over 50 min. All reactions were carried out
In summary, a conceptual bridging of the tripeptide
moiety led to the design of novel 2,4,6-tetrahydropyran
compounds. A sound synthetic methodology was
subsequently developed to make these analogues. Test-
ing on the melanocortin receptors confirmed the
hypothesis that this bridging approach could produce
bioisostericpeptidomimetisc based on the tetrahydro-
pyran moiety.
Experimental
Biology
In vitro assays. In vitro binding assay for the MCRs.
The binding activity of MCR ligands was evaluated at

3058
A. W. Mazur et al. / Bioorg. Med. Chem. 11 (2003) 3053–3063
under argon atmosphere. Anhydrous solvents and
reagents were purchased from commercial sources.
Reactions were monitored by analytical HPLC and
thin-layer chromatography using Baker-flex silica plates
1B2-F. In addition to characterization data provided
below, we obtained for each compound normal resolu-
tion MS and LC–MS that were in agreement with the
proposed structures.
for 1 h. The reaction was quenched by addition of
saturated NH4Cl at À30 ꢁC and the mixture was stirred
for 30 min at room temperature. The layers were sepa-
rated and the aqueous phase was extracted with Et2O.
The combined ether layer was washed with 1M
Na2S2O3, brine and dried over MgSO4. Evaporation of
the solvents gave product that was purified on a silica
column (hexane:EtOAc, 8:2). This procedure gave pro-
duct with the yields 60–80%.
A general method for the cyano group addition to 3a and
3b (Scheme 2b). To a stirred solution of TMSCN (3.6
mL, 26.6 mmol) and ketone (8.7 mmol) in DCM (125
mL) was added BF3Et2O (0.12 mL, 0.87 mmol) at 0 ꢁC.
The reaction mixture was stirred for 2 h at 0 ꢁC, quen-
ched with NH4Cl and extracted with DCM. Combined
(1R-{6R-[3-(tert-Butyl-dimethyl-silanyloxy)-prop-1-ynyl]-
4-oxo-tetrahydro-pyran-2R-yl}-2-phenyl-ethyl)-carbamic
acid tert-butyl ester 5a. HPLC Rt. 22.22, 1H NMR
(300 MHz; CDCl3) d 7.36–7.18 (m, 5H), 5.19 (d, 1H,
J=6.6 Hz), 4.97 (d, 1H, J=10.2 Hz), 4.23 (s, 2H), 4.23–
4.04 (m, 1H), 3.88–3.72 (m, 1H), 3.02–2.82 (m, 2H),
2.80–2.70 (m, 1H), 2.61–2.38 (m, 2H), 2.24–2.12 (m,
1H), 1.4 (s, 9H), 0.92 (s, 9H), 0.11 (s, 6H); 13C NMR
(300 MHz, CDCl3) d 204.9, 156.0, 137.8, 130.0, 128.6,
126.7, 88.3, 80.7, 80.0, 71.0, 65.6, 55.2, 51.7, 46.7, 45.0,
39.0, 28.6, 26.0, 18.3, À5.0; HRFAB (positive) m/e
488.2832 calculated for C27H41NO5Si (M+H)+, found
488.2829.
organicextracts were washed with NaHCO dried over
3
MgSO4 and concentrated under reduced pressure. The
residue containing TMS enol ether was dissolved in
THF and treated with 1 M HCl (1:1 mixture) then
extracted and dried over MgSO4. Purification by flash
chromatography (hexanes/EtOAc, 6:4) afforded the
product.
[1-(6R-Cyano-4-oxo-tetrahydro-pyran-2R-yl)-2R-phenyl-
1
ethyl]-carbamic acid tert-butyl ester 4a. (2.4 g, 79%). H
[1-(6R-Naphthalen-2-ylethynyl-4-oxo-tetrahydro-pyran-
2R-yl)-2-phenyl-ethyl]-carbamic acid tert-butyl ester 5b.
HPLC Rt. 21.32 H NMR (300 MHz; CDCl3) d 7.96–
NMR (300 MHz; CDCl3) d 7.38–7.22 (m, H), 5.29 (d,
J=6.9 Hz, 1H), 4.85 (d, 10.2 Hz, 1H), 4.20–4.10 (m,
1H), 3.97–3.86 (m, 1H), 3.00–2.81 (m, 3H), 2.70–2.58
(m, 2H), 2.42–2.35 (m, 1H), 1.4 (s, 9H); 13C NMR
(300 MHz, CDCl3) d 201.4, 155.8, 137.1, 129.6, 129.0,
127.1, 116.2, 80.2, 74.4, 64.5, 54.9, 44.5, 43.5, 38.8, 28.5.
1
7.78(m, 3H), 7.61–7.51 (m, 2H), 7.45–7.39 (m, 1H),
7.38–7.17 (m, 3H), 7.17–7.04 (m, 3H), 5.45 (d, 1H,
J=6.6 Hz), 5.09 (d, 1H, J=10.2 Hz), 4.45–4.35 (m, 1H),
3.96–3.98 (m, 1H), 3.12–2.96 (m, 2H), 2.95–2.82 (m,
1H0, 2.81–2.46 (m, 2H), 2.38–2.18 (m, 1H), 1.5 (s, 9H);
13C NMR (300 MHz, CDCl3) d 205.0, 156.0, 137.9,
133.3, 133.0, 12.5, 129.9, 128.8, 128.6, 128.2, 128.1,
127.3, 127.0, 126.7, 119.1, 89.8, 85.3, 80.0, 71.0, 66.3,
55.5, 47.1, 45.2, 39.0, 28.7; HRFAB (positive) m/e
470.2331 calculated for C30H32NO4 (M+H)+, found
470.2329.
[1R-(6R-Cyano-4-oxo-tetrahydro - pyran - 2R - yl) - 2 - (4 -
fluoro-phenyl)-ethyl]-carbamic acid tert-butyl ester 4b.
1H NMR (300 MHz; CDCl3) d 7.28–7.10 (m, 2H),
7.05–6.90 (m, 2H), 5.27 (d, J=7.5 Hz), 4.90 (d, J=10.2
Hz, 1H), 4.18–4.04 (m, 1H), 3.92–3.78 (m, 1H), 2.96–
2.78 (m, 3H), 2.72–2.50 (m, 2H), 2.42–2.28 (m, 1H), 1.4
(s, 9H); 13C NMR (300 MHz, CDCl3) d 201.4, 163.7,
155.9, 132.9, 131.2, 116.3, 115.9, 115.6, 80.3, 74.5, 64.6,
54.9, 44.5, 43.5, 38.0, 28.5; HRFAB (positive) m/e
363.1720 calculated for C19H24N2O4F (M+H)+, found
363.1718.
A general procedure for a transformation of the cyano
group in 6a and 6b to the methyl ester. Formation of di-
methylacetal and hydrolysis of cyano group (Scheme 3a,
i and ii). A solution of ketone (6.7 mmol) in MeOH (70
mL), trimethylortoformate (20 mL) and p-toluene-
sulfonicacid (0.2 g) was heated for 12 h at 80 ꢁC. After
cooling to room temperature, the reaction mixture was
neutralized with 1 M KOH and evaporated under
reduced pressure. The residue was refluxed for 1 h in a
mixture of EtOH (30 mL) and 1 M KOH (30 mL, 30
mmol), the reaction mixture was diluted with water and
extracted with ether. Then water phase, containing the
product, was acidified with 10% HCl and extracted with
EtOAc. The organic phase was dried over MgSO4 and
concentrated under reduced pressure. The crude pro-
duct was dissolved (Scheme 3a, ii) in a solution of THF
(50 mL) and 2.5 M acetic acid (50 mL). The reaction
mixture was heated at 60 ꢁC for 12 h, extracted with
ether to remove impurities, and the aqueous phase was
acidified to pH 2–3. The product was extracted with
EtOAc, the organic phase was dried over MgSO4 and
concentrated under reduced pressure A solution of this
crude product in MeOH (30 mL) (Scheme 3a, iv) was
exposed to five cycles of esterifications. Each cycle con-
A general method for addition of acetylenes to enone 3a
(Scheme 2c.). Preparation of a copper iodide-dimethyl-
sulfide complex. Copper(I) iodide was dissolved in
Et2O/DMS mixture and filtered. The filtrate was diluted
with hexanes to precipitate CuI*1.5DMS complex as
white crystals. This complex was dried at room temp.
under vacuum for 1–2 h to give CuI*0.75DMS as white
powder.
Typical procedure for conjugate addition of alkynylcop-
per reagents. n-Butyllithium (1.5 mL, 2.4 mmol, 1.6 M)
was added at À10C to a stirred solution of an acetylene
derivative (2.4 mmol) in THF (5 mL). After 20 min,
CuI*0.75DMS complex (0.63 g, 2.7 mmol) was added in
one batch. The stirring was continued for 45 min at
À10 ꢁC, the temperature was lowered to À78 ꢁC and
TMSI (0.335 mL, 2.4 mmol) was added. After 5 min, a
solution of enone 3a (1.6 mmol) in THF (5 mL) was
added and the reaction mixture was stirred at À30 ꢁC

A. W. Mazur et al. / Bioorg. Med. Chem. 11 (2003) 3053–3063
3059
sisted of cooling the reaction mixture to 0 ꢁC, dropwise
addition of TMSCH2N2 (1.8 mL) and stirring at room
temperature for 1 h. After the last cycle, the excess of
TMSCH2N2 was decomposed by careful treatment with
acetic acid and solvents were removed under reduced
pressure. The residue was dissolved in ether, washed
with saturated aqueous NaHCO3 and dried over
MgSO4. Purification on a silica column (hexanes/EtOAc
7:3) afforded products with the overall yields of 45–
55%.
6R-(1R-tert-Butoxycarbonylamino-2-phenyl - ethyl) - 4 -
cyano-tetrahydro-pyran-2S-carboxylic acid methyl ester
8 (Scheme 3d). To a solution of crude 7 (0.5 g, 0.93
mmol) in THF (7 mL) and t-BuOH (0.09 mL, 0.93
mmol) was degassed with argon and solution of SmI2
(28 mL, 2.8 mmol, 0.1 M in THF) was added at room
temperature. Progress of the reaction was followed by
HPLC and SmI2 was added. After 4 h, reaction mixture
was quenched with 10% HCl and extracted with ethyl
ether. Extracts were washed with 5% Na2S2O3, H2O
and brine and dried over MgSO4. After removal of sol-
vent under reduced pressure, the crude product of was
purified by flash chromatography (hexane/EtOAc, 2:1)
to give 0.25 g, (69%) of 6R-(1R-tert-butoxycarbonyl-
amino-2-phenyl-ethyl)-4-cyano-tetrahydro-pyran-2S-car-
boxylic acid methyl ester 8. 1H NMR (300 MHz; CDCl3)
d 7.41–7.07 (m, 5H), 5.05–4.80 (m, 1H), 4.4–4.20 (m,
1H), 4.2–4.05 (m, 1H), 4.98–4.80 (m, 1H), 3.81 (s, 3H),
3.68–3.60 (m, 1H), 3.30–3.3.08 (m, 1H), 3.00–2.87 (m,
2H), 2.40–2.08 (m, 1H), 1.9–1.60 (m, 2H), 1.4 (s, 9H);
13C NMR (300 MHz, CDCl3) d 170.5, 156.0, 138.2,
129.7, 128.8, 126.6, 120.6, 79.9, 75.0, 73.0, 55.1, 52.7,
38.8, 31.1, 30.7, 28.6.
6R-(1R-tert-Butoxycarbonylamino-2-phenyl-ethyl)-4-oxo-
1
tetrahydro-pyran-2S-carboxylic acid methyl ester 6a. H
NMR (300 MHz; CDCl3) d 7.33–7.17 (m, 5H), 5.15 (d,
J=10.2 Hz, 1H), 4.14 (dd, J1=4.5 Hz, J2=10.8, 1H),
3.99–43.90 (m, 1H), 3.85 (s, 3H), 3.63–3.54 (m, 1H),
3.04–2.88 (m, 2H), 2.70–2.52 (m, 2H), 2.28–2.00 (m,
1H), 2.00–1.80 (m, 1H), 1.42 (s, 9H); 13C NMR
(300 MHz, CDCl3) d 204.8, 170.0, 156.0, 137.9, 129.6,
129.5, 128.9, 126.9, 79.9, 75.8, 75.0, 54.8, 52.9, 44.8,
43.8, 38.8, 28.6; HRFAB (positive) m/e 400.1736
calculated for C20H27NO6Na (M+Na)+, found
400.1729.
6R-[1R-tert-Butoxycarbonylamino-2-(4-fluoro-phenyl)-
ethyl]-4-oxo-tetrahydro-pyran-2S-carboxylic acid methyl
ester 6b. H NMR (300 MHz; CDCl3) d 7.24–7.15 (m,
{1R-[4-Cyano-6S-(1R-methylcarbamoyl-2-naphthalen-2-
yl - ethylcarbamoyl) - tetrahydro-pyran-2R-yl]-2-phenyl-
ethyl}-carbamic acid tert-butyl ester 9 (Scheme 3e, i).
Methyl ester 8 (0.1 g, 0.26 mmol) was dissolved in
THF/H2O (2 mL/0.6 mL) and water solution of LiOH
(0.014 g, 0.6 mmol) was added. The reaction was stirred
for 2 h at room temperature, concentrated under
reduced pressure to about 1/3 volume and treated with
1 N HCl to pH 2–3. The product (acid) was extracted
with ethyl acetate. The organic phase was washed with
water until pH 4 and concentrated (Scheme 3e, ii). To a
solution of crude acid in DMF (1 mL) were added
HOBt (0.07 g, 0.52 mmol), NMM (0.23 mL, 2.1 mmol)
and l-Nal-NH2 (0.093 g, 0.28 mmol, TFA salt) follow-
ing by addition of EDCI (0.061 g, 0.32 mmol). The
reaction mixture was stirred overnight at room tem-
perature, diluted with water, extracted with ethyl ace-
tate. Purification on a preparative HPLC afforded 0.14
g (92%) of {1R-[4-Cyano-6S-(1R-methylcarbamoyl-2-
naphthalen-2-yl-ethylcarbamoyl)-tetrahydro-pyran-2r-yl]-
2-phenyl-ethyl} - carbamic acid tert-butyl ester 9 as a
mixture of two diastereoisomers in a ratio 1:1 with
HPLC retention times 16.73 and 16.94 min, respectively.
1H NMR (300 MHz; CDCl3) d 7.85–7.80 (m, 2H), 7.73
(s, 1H), 7.53–7.35 (m, 3H), 7.34–7.20 (m, 3H), 7.17–7.12
(m, 2H), 5.01 (d, J=9.9 Hz, 1H), 4.80–4.4.68 (m, 2H),
4.16–4.06 (m, 1H), 4.00–3.89 (m, 1H), 3.82–3.66 (m,
1H), 3.34–3.26 (m, 2H), 3.00 (s, 1H), 2.84–2.74 (m, 3H),
2.19–2.08 (m, 1H), 1.80–1.58 (m, 2H), 1.4 (s, 9H), 1.20–
1.00 (m, 1H); 13C NMR (300 MHz, CDCl3) d 171.8,
170.6, 156.1, 137.5, 134.3, 133.7, 132.7, 129.3, 128.8,
128.7, 128.5, 128.0, 127.8, 127.5, 127.0, 126.7, 126.2,
120.4, 80.0, 74.8, 73.7, 54.7, 54.5, 38.6, 38.5, 30.2, 29.8,
28.7, 26.7, 24.8.
1
2H), 7.06–6.95 (m, 2H), 5.04 (d, J=9.9 Hz, 1H), 4.16
(dd, J1=4.5 Hz, J2=10.8 Hz, 1H), 3.87 (s, 3H), 3.86–
3.74 (m, 1H), 3.68–3.52 (m, 1H), 3.04–2.88 (m, 2H),
2.70–2.50 (m, 3H), 2.34–2.20 (m, 1H), 1.4 (s, 9H); 13C
NMR (300 MHz, CDCl3) d 204.6, 170.8, 169.9, 163.5,
160.3, 156.0, 133.7, 131.2, 130.9, 115.7, 115.4, 79.9, 77.4,
76.0, 75.0, 54.7, 52.8, 44.2, 43.7, 38.0, 28.5; HRFAB
(positive) m/e 396.1822 calculated for C20H27NO6F
(M+H)+, found 396.1827.
6R-(1R-tert-Butoxycarbonylamino - 2 - phenyl - ethyl) - 4 -
cyano-4-(diethoxy-phosphoryloxy)-tetrahydro-pyran-2S-
carboxylic acid methyl ester 7 (Scheme 3b). To a solu-
tion of ketone 6a (0.66 g 1.8 mM) and diethylcyano-
phosphate (0.88 g 5.4 mM) in dry THF (30 mL) was
added 0.5 M solution of NaCN in DMF (10.8 mL) and
the mixture was stirred at room temperature for 2 h.
The reaction mixture was quenched with water and
extracted with a mixture of hexane and ethyl acetate
(1:1). The organiclayer was washed with brine several
times, dried over MgSO4 and concentrated under
reduced pressure affording crude product that was sub-
jected to flash chromatography on a silica column (hex-
ane/EtOAc, 2:1). Yield 0.45. (90%) of 6R-(1R-tert-
butoxycarbonylamino-2-phenyl-ethyl)-4-cyano-4-(diethoxy-
phosphoryloxy) - tetrahydro - pyran - 2S- carboxylic acid
methyl ester 7. 1H NMR (300 MHz; CDCl3) d 7.40–7.10
(m, 5H), 4.95 (d, J=9.9 Hz, 1H), 4.17(q, J=7.2 Hz,
4H), 4.14–1.05 (m, 1H), 3.98–3.86 (m, 1H), 3.83 (s, 3H),
3.68–3.54 (m, 1H), 2.98.286 (m, 2H), 2.84–2.74 (m, 1H),
2.34–2.24 (m, 1H), 2.12–1.90 (m, 2H), 1.4 (s, 9H), 1.36
(s, 6H); 13C NMR (300 MHz, CDCl3) d 169.3, 155.8,
137.6, 129.7, 128.8, 126.9, 117.4, 79.9, 74.2, 73.2, 65.0,
54.6, 52.9, 39.0, 28.5, 16.3; HRFAB (positive) m/e
563.2134 calculated for C25H37N2O9PNa (M+Na)+,
found 563.2159.
6R-(1R-Amino-2-phenyl-ethyl)-4-guanidinomethyl-tetra-
hydro-pyran-2S-carboxylic acid (1R-methylcarbamoyl-2-
naphthalen-2-yl-ethyl)-amide 10 (Scheme 3f). A solution
of 9 (0.034 g, 0.06 mmol) in MeOH/NH4OH (4:1) (10

3060
A. W. Mazur et al. / Bioorg. Med. Chem. 11 (2003) 3053–3063
mL) was hydrogenated overnight in the presence of
Raney nickel at 40 psi, room temperature. The catalyst
was removed by filtration; the filtrate was concentrated
to an oily residue (Scheme 3h). A solution of crude
amine, 1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseu-
dourea (CAS# 25508-20-7) (0.24 g, 0.08 mmol) and
triethylamine (0.015 mL) in DMF (1 mL) was treated
with HgCl2 (0.021 g, 0.08 mmol) at 0 ꢁC and stirred at
this temperature for 2 h. The reaction mixture was
diluted with ethyl acetate (15 mL), the solids were
removed by filtration, the filtrate evaporated and the
residue was purified on a silica column using hexanes/
ethyl acetate 1/1 affording 0.03 g of the product which
was subsequently treated with a solution of dichloro-
methane, trifluoroacetic acid and water in 2:1:0.1 ratio
(1 mL) for 3 h at room temperature. The reaction mix-
ture was diluted with 1,2-dichloroethane and repeatedly
evaporated with fresh portions of this solvent to remove
residual trifluoroacetic acid. The final residue was pur-
ified by a preparative HPLC affording 0.022 g (70%) of
6R-(1R-Amino-2-phenyl-ethyl)-4-guanidinomethyl-tetra-
hydro-pyran-2S-carboxylic acid (1R-methylcarbamoyl-2-
naphthalen-2-yl-ethyl)-amide 10. 1H NMR (300 MHz;
CD3OD) d 7.88–7.70 (m, 3H), 7.50–7.26 (m, 8H), 7.14–
7.08 (m, 1H), 4.08–3.98 (m, 1H), 3.74–3.44 (m, 2H),
3.42–3.38 (m, 1H), 3.33–3.20 (m, 2H), 3.29–2.86 (m,
3H), 2.75 (s, 3H), 2.30–2.06 (m, 1H), 1.90–1.50 (m, 4H),
1.38–1.22 (m, 1H); 13C NMR (300 MHz, CDCl3) d
174.4, 173.4, 159.2, 136.7, 136.0, 134.4, 130.9, 130.8,
130.7, 130.5, 129.7, 129.4, 129.3129.1128.9, 128.8, 128.7,
127.7, 127.2, 74.5, 71.8, 57.4, 43.0, 39.9, 37.3, 36.9, 31.9,
31.0, 30.2, 29.9, 26.8; HRFAB (positive) m/e 531.3084
calculated for C30H39N6O3 (M+H)+, found 531.3078.
yl]-2-(4-fluoro-phenyl)-ethyl]-carbamic acid tert-butyl es-
ter 12. This was obtained from 11 (0.15 g) analogously
to 9 from 8. H NMR (300 MHz; CDCl3) d 7.90–7.64
1
(m, 3H), 7.62–7.34 (m, 4H), 7.12–7.88 (m, 4H), 5.32 (s,
1H), 5.15 (d, J=9.0 Hz, 1H), 4.84–4.64 (m, 1H), 4.02–
3.83 (m, 1H), 3.82–3.64 (m, 1H), 3.48–3.10 (m, 3H),
2.96–2.54 (m, 7H), 2.43–2.02 (m, 2H), 1.91–1.70 (m,
1H), 1.4 (s, 9H); 13C NMR (300 MHz, CDCl3) d 171.9,
170.0, 163.6, 159.8, 156.1, 134.2, 133.7, 133.3, 132.7,
130.8, 128.7, 128.4, 128.0, 127.7, 127.5, 126.7, 126.3,
115.8, 115.6, 96.2, 80.24, 77.5, 76.9, 76.6, 54.9, 54.5,
53.7, 38.6, 37.9, 37.6, 37.2, 35.2, 28.7, 26.7; HRFAB
(positive) m/e 639.2959 calculated for C35H41N4O5F Na
(M+Na)+, found 639.2986.
6R-[1R-Amino-2-(4-fluoro-phenyl)-ethyl]-4-(2-guanidino-
ethyl)-tetrahydro-pyran-2S-carboxylic acid (1R-methyl-
carbamoyl-2-naphthalen-2-yl-ethyl)-amide 13 (Scheme
3g). Sodium borohydride (0.02 g, 0.52 mmol) was
added to a mixture of PdCl2 (0.04 g, 0.22 mmol) in
methanol (1 mL). Subsequently, 12 (0.5 g, 0.8 mmol)
was added and the mixture was stirred under normal
hydrogen pressure for 3 h. After filtration of solids, the
filtrate was concentrated under reduced pressure and
the residue was converted to 13 with procedures
(Scheme 3f, h) analogues to those used for making 10
from 9, including final purification on a preparative
HPLC affording 0.27 g (60%) of 6R-[1R-amino-2-(4-
fluoro-phenyl)-ethyl]-4-(2-guanidino-ethyl)-tetrahydro-
pyran-2S-carboxylic acid (1R-methylcarbamoyl-2-naph-
thalen-2-yl-ethyl)-amide 13. HPLC retention time. 7.71
1
min; H NMR (300 MHz; CDCl3) d 7.90–7.76 (m, 3H),
7.72 (s, 1H), 7.54–7.38 (m, 3H), 7.34–7.25 (m, 2H),
7.16–7.07 (m, 2H), 5.00–4.84 (m, 1H), 3.84–3.76 (m,
1H), 3.58–3.34 (m, 3H), 3.22–2.98 (m, 3H), 2.96–2.85
(m, 2H), 2.82–2.70 (m, 3H), 1.83–1.50 (m, 4H), 1.50–
1.20 (m, 2H), 1.03–0.90 (m, 1H), 1.57–1.28 (m, 1H);
HRFAB (positive) m/e 563.3136 calculated for
C31H39FN6O3Na (M+H)+, found 563.3146.
6R-[1R-tert-Butoxycarbonylamino-2-(4-fluoro-phenyl)-
ethyl]-4-cyanomethylene-tetrahydro-pyran-2S-carboxylic
acid methyl ester 11 (Scheme 3c). Diethylcyanomethyl-
phosphate (0.1 mL, 0.61 mmol) was added dropwise
into a mixture of NaH (0.03 g, 0.61 mmol, 60% in
mineral oil) in THF (5 mL) (mineral oil was removed
with hexane prior to addition of THF) at 0 ꢁC and the
reaction mixture was stirred for 1 h. The mixture was
cooled to À40 ꢁC, and ketone 6b (0.2 g, 0.51 mmol) in
THF (0.5 mL) was added. After stirring for 3 h, the
reaction mixture was quenched with saturated NH4Cl.
The product was extracted with Et2O and purified on a
silica column (hexanes/EtOAc, 2:1) affording (0.2 g,
(1R-{6S-[3-(tert-Butyl-dimethyl - silanyloxy) - propyl] - 4 -
oxo-tetrahydro-pyran -2R-yl}-2-phenyl-ethyl)-carbamic
acid tert-butyl ester 14 (Scheme 4a). Raney nickiel was
added to a solution of 5a (0.15 g, 0.31 mmol) in EtOAc
and the mixture was stirred at 50 ꢁC for 0.5 h to deacti-
vate possible traces of sulfur contamination left from
the Michael addition. The solids were removed by fil-
tration and the filtrate was hydrogenated using Pd/C
10% at normal conditions for 1 h. After filtration, the
solvent was evaporated under reduced pressure and the
residue was chromatographed (hexane/EtOAc, 7:3) to
give 0.14 g (92%) (1R-{6S-[3-(tert-Butyl-dimethyl-sila-
nyloxy)-propyl]-4-oxo-tetrahydro-pyran-2R-yl}-2-phenyl-
ethyl)-carbamic acid tert-butyl ester 14. 1H NMR
(300 MHz; CDCl3) d 7.40–7.15 (M, 5H), 4.97 (d, 1H,
J=9.6 Hz), 4.44–4.26 (m, 1H), 3.99–3.90 (m, 1H), 3.89–
3.80 (m, 1H), 3.74–3.56 (m, 2H), 3.03–2.82 (m, 2H),
2.79–2.49 (m, 2H), 2.36–2.16 (m, 2H), 1.72–1.55 (m,
4H), 1.4 (s, 9H), 0.92 (s, 9H), 0.85 (m, 6H); 13C NMR
(300 MHz, CDCl3) d 207.6, 156.0, 138.1, 129.5, 128.7,
126.7, 79.8, 74.1, 70.0, 62.7, 55.1, 46.0, 44.8, 39.1, 28.6,
26.2, 18.6,-5.1; HRFAB (positive) m/e 492.3145 calcu-
lated for C27H46NO5Si (M+H)+, found 492.3137.
94%),
6R-[1R-tert-butoxycarbonylamino-2-(4-fluoro-
phenyl)-ethyl]-4-cyanomethylene-tetrahydro-pyran-2S-
carboxylic acid methyl ester 11. HPLC retention time.
16.27 min. 1H NMR (300 MHz; CDCl3) d 7.25–7.10 (m,
2H), 7.06–6.94 (m, 2H), 5.32–5.18 (m, 1H), 5.10–4.94
(m, 1H), 3.87–3.81 (m, 5H), 3.4–3.22 (m, 1H), 3.20–2.82
(m, 2H), 2.78–2.56 (m, 1H), 2.54–2.28 (m, 2H), 2.26–
2.10 (m, 1H), 1.4 (s, 9H); 13C NMR (300 MHz, CDCl3)
d 169.9, 163.6, 160.2, 156.0, 155.8, 133.7, 131.0, 130.9,
116.0, 115.8, 115.5, 96.1, 80.1, 76.9, 76.8, 76.7, 76.0,
75.8, 54.8, 52.8, 38.2, 38.0, 37.5, 37.3, 35.2, 35.1, 28.6;
HRFAB (positive) m/e 441.1802 calculated for
C22H27N2O5FNa (M+Na)+, found 441.1787.
[1R-[4-Cyanomethylene-6S-(1R-methylcarbamoyl - 2 -
naphthalen-2-yl-ethylcarbamoyl)-tetrahydro-pyran-2R-

A. W. Mazur et al. / Bioorg. Med. Chem. 11 (2003) 3053–3063
3061
(1R-{6S-[3-(tert-Butyl-dimethyl-silanyloxy) - propyl] - 4 -
hydroxy-tetrahydro-pyran-2R-yl}-2-phenyl-ethyl)-car-
bamic acid tert-butyl ester 15 (Scheme 4b). A solution
of ketone 14 (0.2 g, 0.41 mmol) in THF at À78 ꢁC was
treated dropwise with l-Selectride stirred 0.5 h. The
reaction was quenched with 10% aq NH4Cl, the pro-
duct extracted with ether, dried with MgSO4 and the
extract was concentrated under reduced pressure. The
residue was purified by silica gel chromatography (hex-
ane/EtOAc8:2) giving 0.16 g (yield 80%) of 15, two
diastereoisomers. HPLC retention times 21.92 and 22.58
azodicaboxylate (0.04 mmol) was added dropwise at
0 ꢁC to a solution of alcohol 16 (0.015 g, 0.03 mmol),
1,3-bis(tert-butoxycarbonyl)-guanidine (0.012 g, 0.045
mmol) and triphenylphosphine (0.011 g, 0.04 mmol) in
anhydrous THF (0.5 mL) over 2 h. The reaction mix-
ture was concentrated under reduced pressure. The
residue was purified on a silica column (hexane/EtOAc
8:2) to give 0.017 g (yield 75%) of 17. 1H NMR
(300 MHz; CDCl3) d 7.94–7.72 (m, 3H), 7.56–7.44 (m,
2H), 7.38–7.19 (m, 7H), 5.10–4.96 (m, 1H), 4.84–4.52
(m, 2H), 4.26–4.10 (m, 1H), 4.09–3.68 (m, 4H), 3.64–
3.49 (m, 1H), 2.10–1.56 (m, 8H), 1.5 (bs, 27H); 13C
NMR (300 MHz, CDCl3) d 161.1, 158.6, 156.1, 137.4,
136.3, 134.1, 134.2, 129.7, 128.6, 128.1, 128.0, 126.4,
126.1, 125.9, 84.0, 81.7, 79.3, 72.9, 71.2, 53.4, 44.5, 39.2,
35.5, 30.0, 28.6, 28.4, 25.8.
1
min with the ratio 2.2:1. H NMR (300 MHz; CDCl3) d
7.35–7.17 (m, 5H), 5.01 (d, 0.31H, J=9.6 Hz), 4.81 (d,
0.69H, J=8.4 Hz), 4.28–4.02 (m, 1H), 4.01–3.79 (m,
2H), 3.78–3.48 (m, 3H), 3.04–2.72 (m, 2H), 2.04–1.42
(m, 8H), 1.4 (s, 9H), 0.93 (s, 9H), 0.08 (s, 6H); 13C
NMR (300 MHz, CDCl3) d 156.0, 138.8, 138.2, 129.8,
129.6, 128.6, 126.5, 79.4, 73.5, 71.3, 68.6, 67.8, 64.7,
64.4, 63.7, 63.0, 55.6, 53.2, 38.7, 38.1, 35.5, 31.4, 29.9,
28.6, 26.2, 18.6, À5.0; HRFAB (positive) m/e 494.3302
calculated for C27H48NO5Si (M+H)+, found 494.3298.
N-{3-[6S-(1R-Amino-2-phenyl-ethyl)-4-(naphthalen-2-yl-
methoxy)-tetrahydro-pyran-2R-yl]-propyl}-guanidine 18.
This was obtained from 17 by a standard Boccleavage
using trifluoroacetic/dichlomethane/water 1/2/0.1 at
room temperature and purification on a preparative
HPLC. HPLC retention time time 8.77 min; HRFAB
(positive) m/e 461.2917 calculated for C28H37N4O2
(M+H)+, found 461.2912.
{1R-[6S-(3-Hydroxy-propyl)-4-(naphthalen-2-ylmethoxy)
-tetrahydro-pyran-2R-yl]-2-phenyl-ethyl}-carbamic acid
tert-butyl ester 16 (Scheme 4c, i). Sodium hydride
(0.032 g, 0.82 mmol) was placed in the dried flask under
argon and washed with hexane to remove mineral oil,
the THF (1.0 mL) was added followed by dry sodium
iodide and the solution was cooled to 0 ꢁC. To this
mixture, alcohol 15 (0.12 g, 0.41 mmol) dissolved in
THF (0.5 mL) was added and the mixture was stirred at
0 ꢁC for 0.5 h. Then bromide (0.09 g, 0.43 mmol) dis-
solved in DMF (0.2 mL) was added dropwise and the
reaction mixture was stirred at room temperature for 12
h. The work-up involved addition of 10% aq NH4Cl,
extracted of the product with ether. Purification on a
silica column (hexane/EtOAc: 95:5) furnished (0.08 g,
52%) of {1R-[6S-[3-(tert-Butyl-dimethyl-silanyloxy)-
propyl]-4-(naphthalen-2-ylmethoxy)-tetrahydro-pyran-2R-
yl]-2-phenyl-ethyl}-carbamic acid tert-butyl ester that
was dissolved in a mixture of acetic acid (Scheme 4c, ii),
water and THF in ratio 3:1:1. After overnight stirring
the reaction mixture wa evaporated and chromato-
graphed on a silica column (hexane/EtOAc, 8:2) produ-
cing 0.04 g, (32%) in two steps of {1R-[6S-(3-Hydroxy-
propyl)-4-(naphthalen-2-ylmethoxy)-tetrahydro-pyran-2R-
yl]-2-phenyl-ethyl}-carbamic acid tert-butyl ester 16.
[1R-(4-Cyanomethylene-6R-naphthalen-2-ylethynyl-tetra-
hydro-pyran-2R-yl)-2-phenyl-ethyl]-carbamic acid tert-
butyl ester 19 (Scheme 5a). Diethylcyanomethylpho-
sphate (0.028 mL, 0.17 mmol) was added dropwise into
a mixture of NaH (0.007 g, 0.17 mmol, 60% in mineral
oil) in THF (1 mL) (mineral oil was removed with hex-
ane) at 0 ꢁC and the reaction mixture was stirred for 1 h.
Then mixture was cooled to À40 ꢁC and ketone 5b
(0.064 g, 0.14 mmol) dissolved in THF (1 mL) was
added. After stirring for 1 h, the reaction was quenched
with 10% aq NH4Cl, extracted with Et2O. Purification
on a silica column (hexanes/EtOAc 2:1) afforded 19
(0.06 g, 87%). HPLC retention time 21.59 min; 1H
NMR (300 MHz; CDCl3) d 7.98–7.79 (m, 4H), 7.62–
7.52 (m, 2H), 7.50–7.23 (m, 3H), 7.20–7.06 (m, 3H),
5.38–5.22 (m, 2H), 5.06–4.91 (m, 1H), 4.14–4.01 (m,
1H0, 3.99–3.78 (m, 1H), 3.12 (d, 0.59H, J=13.8 Hz),
3.00–2.88 (m, 2H), 2.85–2.67 (m, 1.41H), 2.58–2.40 (m,
1H), 2.26–2.16 (m, 1H), 1.4 (s, 9H); 13C NMR
(300 MHz, CDCl3) d 159.9, 156.1, 138.0, 133.3, 133.1,
132.3, 130.0, 128.7, 128.3, 128.1, 127.2, 127.0, 126.9,
126.7, 119.4, 116.5, 96.7, 96.5, 89.4, 85.3, 85.2, 80.0,
71.3, 70.9, 66.5, 55.4, 40.7, 39.0, 38.7, 38.2, 35.8, 28.7;
HRFAB (positive) m/e 493.2491 calculated for
C32H33N2O3 (M+H)+, found 493.2490.
1
HPLC retention time. 18.85 min; H NMR (300 MHz;
CDCl3) d 7.91–7.81 (m, 3H), 7.78 (s, 0.57H), 7.75 (s,
0.43H), 7.56–7.38 (m, 3H), 7.34–7.12 (m, 5H), 4.98 (d,
1H, J=9 Hz), 4.80–4.58 (m, 2H), 4.22–3.72 (m, 3H),
3.71–3.62 (m, 2H), 3.62–3.50 (m, 1H), 2.96–2.72 (m,
2H), 2.08–1.84 (m, 2H), 1.83–1.50 (m, 6H), 1.4 (s, 9H);
13C NMR (300 MHz, CDCl3) d 156.0, 133.5, 133.2,
129.6, 128.6, 128.5, 128.1, 127.9, 126.3, 126.1, 125.9,
79.4, 73.1, 71.4, 71.1, 70.4, 70.1, 69.5, 68.2, 63.1, 62.8,
55.4, 53.2, 39.0, 35.7, 35.2, 32.7, 29.7, 28.7; HRFAB
(positive) m/e 520.3063 calculated for C32H42NO5
(M+H)+, found 520.3056.
{1R-[4-Cyanomethyl-6S-(2-naphthalen-2-yl-ethyl)-tetra-
hydro-pyran-2R-yl]-2-phenyl-ethyl}-carbamic acid tert-
butyl ester 20 (Scheme 5b). Nitrile 19 (0.05 g, 0.1 mmol)
was hydrogenated in toluene (4 mL) in a presence of
Wilkinson catalyst at 40PSI for 2 days. The reaction
mixture was filtered, evaporated and chromatographed
on a silica column (hexane/EtOAc, 8:1), affording 0.04 g
(yield 80%) of 20. (two diastereoisomers in ratio 4.5:1)
1H NMR (300 MHz; CDCl3) d 7.9–7.75 (m, 3H), 7.63 (s,
1H), 7.54–7.40 (m, 2H), 7.39–7.19 (m, 6H), 5.02 (d,
0.18H, J=9.6 Hz), 4.75 (d, 0.72H, J=8.7 Hz), 4.38–
{1R-[6S-(3-(bis-Boc)Guanidino-propyl)-4-(naphthalen-2-
ylmethoxy)-tetrahydro-pyran-2R-yl]-2-phenyl-ethyl}-car-
bamic acid tert-butyl ester 17 (Scheme 4d). Diisopropyl-

3062
A. W. Mazur et al. / Bioorg. Med. Chem. 11 (2003) 3053–3063
4.06 (m, 1H), 3.95–3.62 (m, 2H), 3.10–2.91 (m, 2H),
2.90–2.68 (m, 2H), 2.36–2.19 (m, 2H), 2.18–2.01 (m,
1H), 2.00–1.74 (m, 4H), 1.73–1.49 (m, 2H), 1.4 (m, 9H);
13C NMR (300 MHz, CDCl3) d 156.0, 139.9, 137.7,
134.0, 133.9, 132.2, 132.3, 129.8, 129.6, 128.7, 128.3,
127.9, 127.5, 126.8, 126.4, 126.2, 125.4, 118.1, 79.6, 72.5,
72.3, 70.7, 67.8, 55.6, 50.9, 39.2, 38.8, 38.2, 36.6, 34.7,
34.6, 32.8, 32.6, 32.1, 31.8, 29.1, 28.7, 28.2, 27.9, 24.8,
24.6; HRFAB (positive) m/e 499.2961 calculated for
C32H39N2O3 (M+H)+, found 499.2980.
2H), 7.40–7.08 (m, 6H), 5.03 (d, 1H, J=9.6 Hz), 4.15–
4.02 (m, 1H), 3.93–3.79 (m, 1H), 3.78–3.57 (m, 1H),
3.53–3.31(m, 2H), 3.08–2.67 (m, 4H), 1.98–1.62 (m, 9H),
1.53 (s, 9H), 1.46 (bs, 18H).
13C NMR (300 MHz, CDCl3) d 162.9, 1565, 156.1,
153.5, 139.6, 139.0, 133.9, 132.3, 130.0, 129.7, 128.7,
128.2, 127.9, 127.7, 127.6, 126.7, 126.5, 126.2, 125.4,
83.8, 80.8, 79.3, 72.7, 68.2, 55.8, 39.0, 36.4, 35.6, 35.2,
32.9, 32.3, 30.0, 28.7, 28.5, 28.2, 27.7.
The mixture of diastereoisomers 20 was separated on a
preparative HPLC.
N-{2-[2R-(1R-Amino-2-phenyl-ethyl)-6S-(2-naphthalen-2-
yl-ethyl)-tetrahydro-pyran-4S-yl]-ethyl}-guanidine 23 and
N-{2-[2R-(1R-Amino-2-phenyl-ethyl)-6S-(2-naphthalen-
2-yl-ethyl)-tetrahydro-pyran-4R-yl]-ethyl}-guanidine 24
(Scheme 5d). This was obtained by cleavage of the Boc
groups from 21 and 22 (diastereoisomers mixture) with
a solution of dichloromethane, triflouroacetic acid and
water 2:1:0.1 for 3 h at room temperature. The diaster-
eoisomers 23 and 24 were separated on a preparative
Major isomer of 20 HPLC retention time 20.74 min. 1H
NMR (300 MHz; CDCl3) d 7.87–7.76 (m, 3H), 7.64 (s,
1H), 7.52–741 (m, 2H), 7.38–7.22 (m, 6H), 4.76 (d,
J=7.5 Hz, 1H), 4.36–4.17 (m, 1H), 3.86–3.69 (m, 2H),
3.09–2.93 (m, 2H), 2.86–2.70 (m, 2H), 2.32–2.22 (m,
2H), 2.20–2.01 (m, 1H), 2.00–1.74 (m, 4H), 1.54 (td,
J1=6 Hz, J2=12.9 Hz, 1H), 1.44 (s, 9H), 1.25–1.10 (m,
1H); 13C NMR (300 MHz, CDCl3) d 156.0, 140.0, 137.7,
133.9, 132.2, 129.8, 128.7, 128.2, 127.9, 127.7, 127.5,
126.8, 126.4, 126.2, 124.3, 118.1, 79.6, 75.5, 70.7, 50.9,
38.8, 38.2, 36.6, 32.6, 31.8, 28.7, 28.2, 24.6.
1
HPLC. Major isomer 24 retention time 8.91 min; H
NMR (300 MHz; CDCl3) d 7.86–7.76 (m, 3H), 7.68 (s,
1H), 7.50–7.30 (m, 8H), 4.20–3.95 (m, 2H), 3.88–3.70
(m, 1H), 3.11–3.00 (m, 3H), 2.95–2.83 (m, 3H), 2.14–
2.00 (m, 1H), 1.97–1.80 (m, 3H), 1.75–1.58 (m, 1H),
1.55–1.38 (m, 3H), 1.17–1.05 (m, 1H); 13C NMR
(300 MHz, CDCl3) d 157.4, 139.9, 135.7, 134.0, 132.4,
129.2, 129.0, 127.8, 127.4, 127.2, 127.0, 126.0, 125.8,
125.1, 73.1, 71.0, 51.9, 38.5, 37.6, 36.3, 35.9, 35.3, 31.4,
31.3, 27.5; HRFAB (positive) m/e 445.2967 calculated
for C28H37N4O1 (M+H)+, found 445.297.
Minor isomer of 20 HPLC retention time 21.75 min; 1H
NMR (300 MHz; CDCl3) d: 7.88–7.76 (m, 3H), 7.64 (s,
1H), 7.55–7.44 (m, 2H), 7.39–7.22 (m, 6H), 5.02 (d,
J=9.6 Hz, 1H), 4.20–4.04 (m, 1H), 3.94–3.80 (m, 1H),
3.68 (d, J=11.4 Hz, 1H), 3.08–2.75 (m, 4H), 2.36–2.00
(m, 4H), 1.78–1.50 (m, 4H), 1.4 (s, 9H), 1.38–1.23 (m,
1H); 13C NMR (300 MHz, CDCl3) d 156.1, 139.2, 138.7,
133.9, 132.3, 1129.6, 128.8, 128.3, 128.0, 127.6, 127.5,
126.7, 126.3, 125.6, 118.1, 79.6, 72.3, 67.8, 55.6, 39.2,
34.8, 34.6, 32.7, 32.1, 28.7, 27.9, 24.8.
Minor isomer 23 retention time 9.91 min; 1H NMR
(300 MHz; CDCl3) d 7.86–7.77 (m, 3H0, 7.68 (s, 1H),
7.52–7.30 (m, 8H), 4.27–4.16 (m, 2H), 3.79 (d, 1H,
J=9.6 Hz), 3.52–3.42 (m, 1H), 3.27–3.12 (m, 2H), 3.10–
2.82 (m, 4H), 1.96–1.80 (m, 2H), 1.79–1.66 (m, 2H),
1.60–1.25 (m, 3H), 1.00–0.91 (m, 1H); 13C NMR
(300 MHz, CDCl3) d 168.2, 157.5, 139.4, 135.9, 134.0,
132.5, 131.2, 129.2, 129.0, 128.7, 127.9, 127.4, 127.2,
126.2, 125.9, 125.1, 73.67.9, 67.2, 56.8, 39.0, 38.5, 35.8,
34.7, 32.5, 32.0, 30.5, 29.0, 27.0.
N-{2-[2R-(1R-BocAmino-2-phenyl-ethyl)-6S-(2-naphtha-
len-2-yl-ethyl)-tetrahydro-pyran-4S-yl]-ethyl}-bis(Boc)-
guanidine 21 and N-{2-[2R-(1R-Bocamino-2-phenyl-
ethyl)-6S-(2-naphthalen-2-yl-ethyl)-tetrahydro-pyran-4R-
yl]-ethyl}-bis(Boc)guanidine 22 (Scheme 5c). Nitrile 20
(0.06 g, 0.07 mmol) was converted to 21 and 22 by
hydrogenation and guanidylation using the same pro-
cedure as those applied for transformations of 10
affording 0.06 g (87%) of diastereoisomers 21 and 22 in
a ratio 2.2:1 which were separated on a preparative
HPLC. Major isomer 22: HPLC retention time 23.58
min; 1H NMR (300 MHz; CDCl3) d 11.5 (s, 1H), 8.36 (t,
1H, J=9.6 Hz), 7.90–7.70 (m, 3H), 7.63 (s, 1H), 7.54–
7.37 (m, 2H), 7.36–7.15 (m, 6H), 4.76 (d, 1H, J=7.2
Hz), 4.46–4.19 (m, 1H), 3.86–3.57 (m, 2H), 3.56–3.28
(m, 1H), 3.20–2.88 (m, 2H), 2.87–2.60 (m, 2H), 1.98–
1.62 (m, 8H), 1.54 (s, 9H), 1.51 (s, 9H), 1.43 (s, 9H),
1.10–0.90 (m, 1H); 13C NMR (300 MHz, CDCl3) d
163.5, 156.5, 156.0, 153.6, 140.3, 137.9, 133.9, 132.2,
130.0, 128.6, 128.1, 127.8, 127.7, 127.6, 126.7, 126.4,
126.1, 125.3, 83.6, 80.0, 79.4, 73.0, 70.8, 50.5, 38.6, 37.8,
36.6, 32.7, 32.3, 28.7, 28.5, 28.3.
References and Notes
1. Hirschmann, R.; Nicolaou, K. C.; Pietranico, S.; Salvino,
J.; Leahy, E. M.; Sprengeler, P. A.; Furst, G.; Strader, C. D.;
Smith, A. B., III J. Am. Chem. Soc. 1992, 114, 9217.
2. Recent comprehensive review on the application of carbo-
hydrate-based scaffolds in drug design: Gruner, S. A. W.;
Locardi, E.; Kessler, H. Chem. Rev. 2002, 102, 491.
3. Review of peptidomimetics based on glucose and 5-meme-
bered fused oxacyclic scaffolds: Peri, F.; Cipolla, L.; Forni, E.;
Nicotra, F. Monatsh. Chem. 2002, 133, 369.
4. Review on the application of glycosamino acids as building
blocks for combinatorial synthesis of peptidomimetics:
Schweizer, F. Angew. Chem. 2002, 41, 230.
5. Chakraborty, T. K.; Jayaprakash, S.; Ghosh, S. Comb.
Chem. High T. Scr. 2002, 5, 373.
6. Review on the application of sugar amino acids in design-
ing bioactive molecules: Chakraborty, T. K.; Ghosh, S.; Jaya-
prakash, S. Curr. Med. Chem. 2002, 9, 421.
1
Minor isomer 21 HPLC retention time 24.34 min; H
NMR (300 MHz; CDCl3) d 11.41 (s, 1H), 8.48 (bs, 1H),
7.91–7.72 (M, 3H), 7.69–7.59 (m, 1H), 7.55–7.41 (m,
7. Recent application of glucal cycloadducts for NK-2

A. W. Mazur et al. / Bioorg. Med. Chem. 11 (2003) 3053–3063
3063
peptidomimeticligands: Capozzi, G.; Giannini, S.; Menichetti,
S.; Nativi, C.; Giolitti, A.; Patacchini, R.; Perrotta, E.; Alta-
mura, M.; Alberto Maggi, C. Bioorg. Med. Chem. Lett. 2002,
12, 2263.
8. Review on the design and activity of sugar amino acids
based peptidomimetics: Lohof, E.; Burkhart, F.; Born, M. A.;
Planker, E.; Kessler, H. Advances in Amino Acid Mimetics and
Peptidomimetics 1999, 2, 263.
9. Synthetic protocols concerning azasugars: Le Merrer, Y.;
Poitout, L.; Depezay, J. C. Method. Mol. Med. 1999, 23, 227.
10. Nicolaou, K. C.; Trujillo, J. I.; Chibale, K. Tetrahedron
1997, 53, 8751.
11. Han, Y.; Mierke, D. F.; Chorev, M. Biopolymers 2002, 64,
1.
12. Freidinger, R. M.; Perlow, D. S.; Veber, D. F. J. Org.
Chem. 1982, 47, 104.
13. Aube, J. Adv. Amino Acid Mimetics and Peptidomimetics
1997, 1, 193.
14. Toniolo, C. Int. J. Pept. Protein Res. 1990, 35, 287.
15. Hruby, V. J.; Balse, P. M. Curr. Med. Chem. 2000, 7, 945.
16. Mazur, A. W.; Tian, X.; Hu, X. E.; Ebetino, F. H. WO
0285925, 2002.
Kost, T.; Luther, M.; Chen, W.; Woychik, R. P.; Wilkison,
W. O. Nature 1994, 371, 799.
22. Wessells, H.; Levine, N.; Hadley, M. E.; Dorr, R.; Hruby,
V. Int. J. Impot. Res. 2000, 12 (Suppl. 4), S74.
23. Haskell-Luevano, C.; Hendrata, S.; North, C.; Sawyer,
T. K.; Hadley, M. E.; Hruby, V. J.; Dickinson, C.; Gantz, I. J.
Med. Chem. 1997, 40, 2133.
24. Holder, J. R.; Xiang, Z.; Bauzo, R. M.; Haskell-Luevano,
C. J. Med. Chem. 2002, 45, 5736.
25. Grieco, P. A.; Moher, E. D. Tetrahedron Lett. 1993, 34, 5567.
26. Willson, T. M.; Kocienski, P.; Jarowicki, K.; Isaac, K.;
Hitchcock, P. M.; Faller, A.; Campbell, S. F. Tetrahedron
1990, 46, 1767.
27. Eriksson, M.; Iliefski, T.; Nilsson, M.; Olsson, T. J. Org.
Chem. 1997, 62, 182.
28. Konieczny, M. T.; Toma, P. H.; Cushman, M. J. Org.
Chem. 1993, 58, 4619.
29. Kim, K. S.; Qian, L. Tetrahedron Lett. 1993, 34, 7677.
30. Wadsworth, W. S., Jr. Org. React. 1977, 25, 73.
31. Hudlicky, M. Reductions in Organic Chemistry (ACS
monograph 188); American Chemical Society: Washington,
DC. 1996; p 298.
17. Ebetino, F. H.; Mazur, A. W.; Hayes, J. C.; Wang, F.;
Solinsky, M. G.; Colson, A. O.; Lin, Q. WO 0226774, 2001.
18. Mazur, A. W.; Wang, F.; Sheldon, R. J.; Ebetino, F. H.
WO 0058361, 2000.
19. Abdel-Malek, Z.; Suzuki, I.; Tada, A.; Im, S.; Akcali, C.
Ann. N. Y. Acad. Sci. 885, 117.
20. Ollmann, M. M.; Wilson, B. D.; Yang, Y. K.; Kerns, J. A.;
Chen, Y.; Gantz, I.; Barsh, G. S. Science 1997, 278, 135.
21. Lu, D.; Willard, D.; Patel, I. R.; Kadwell, S.; Overton, L.;
32. Russell, T. W.; Duncan, D. M.; Hansen, S. C. J. Org.
Chem. 1976, 42, 551.
33. Sebhat, I. K.; Martin, W. J.; Ye, Z.; Barakat, K.; Mosley,
R. T.; Johnston, D. B. R.; Bakshi, R.; Palucki, B.; Weinberg,
D. H.; MacNeil, T.; Kalyani, R. N.; Tang, R.; Stearns, R. A.;
Miller, R. R.; Tamvakopoulos, C.; Strack, A. M.; McGowan,
E.; Cashen, D. E.; Drisko, J. E.; Hom, G. J.; Howard, A. D.;
MacIntyre, D. E.; van der Ploeg, L. H. T.; Patchett, A. A.;
Nargund, R. P. J. Med. Chem. 2002, 45, 4589.