Design, synthesis, and conformational analysis of eight-membered cyclic peptidomimetics prepared using ring closing metathesis

Article abstract of DOI:10.1016/j.bmc.2004.06.015

As part of a program to identify novel scaffolds that adopt defined secondary structure when incorporated into peptides, we have designed and prepared a library of constrained eight-membered ring lactams based upon 7-amino-8-oxo-1,2,3,6,7-pentahydroazocin

Full text of DOI:10.1016/j.bmc.2004.06.015

Bioorganic & Medicinal Chemistry 12 (2004) 4375–4385
Design, synthesis, and conformational analysis of eight-membered
cyclic peptidomimetics prepared using ring closing metathesis
Christopher J. Creighton,* Gregory C. Leo, Yanming Du and Allen B. Reitz*
Drug Discovery Division, PO Box 776, Welsh and McKean Rds.,
Johnson & Johnson Pharmaceutical Research and Development, LLC, Spring House, PA 19477-0777, USA
Received 14 April 2004; revised 10 June 2004; accepted 11 June 2004
Available online 2 July 2004
The authors wish to dedicate this manuscript to the memory of Professor Murray Goodman with respect and gratitude
Abstract—As part of a program to identify novel scaffolds that adopt defined secondary structure when incorporated into peptides,
we have designed and prepared a library of constrained eight-membered ring lactams based upon 7-amino-8-oxo-1,2,3,6,7-penta-
hydroazocine-2-carboxylic acid. Ring closing metathesis (RCM) was employed as the key step, proceeding in high yields to afford
the Z olefin. In this reaction sequence, the first generation benzylidene ruthenium RCM catalyst was superior to the second-gen-
eration imidazoline catalyst, which gave extensive oligomerization at higher concentrations. Conformational analysis of the 2S,7S
and 2R,7S stereoisomers revealed that the 2R,7S isomer is a Type VIa b-turn in the solid state (X-ray crystal structure) and in water
(NMR analysis). The Type VIa b-turn is relatively rare, typically bearing the cis amide bond found in proline-containing sequences.
The 2S,7S diastereomer has an extended geometry of the pendent amide chains. The corresponding saturated derivatives (7-amino-
8-oxoazocane-2-carboxylic acid) were also synthesized and investigated. The 2S,7S azocane bears an extended geometry and mimics
the Cþ conformer of ox-[Cys-Cys], found in a variety of naturally occurring peptides. The scaffolds described here are useful for the
design of constrained peptidomimics with defined secondary structure.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Table 1. b-Turns and their prevalence.3
1. Introduction
ψ1
R
2 φ2
ω
Understanding and controlling the secondary structure
of natural and synthetic peptides and proteins is essen-
tial for mediating the overall topology required to
establish proper biological function.1 Among the com-
mon structural elements found in nature, the b-turn is
the primary reverse turn and has been extensively
studied. The b-turn consists of four amino acids ori-
ented in such a way that the peptide backbone changes
direction by ca. 180ꢁ (Table 1).2 A 10-membered ring
hydrogen bond is usually formed between the carbonyl
oxygen of the first or i residue, and the hydrogen on the
amide of the fourth or i þ 3 residue. Also, the distance
between the a-carbons of the i and i þ 3 residues should
O
R
ψ2
φ1
N
O
i+2
i+1
H
HN
N
R
O
H
i
i+3
R
b-Turn Position Position Position Position Abundance
type
i þ 1: /1 i þ 1: w1 i þ 2: /2 i þ 2: w2 (%)
I
I0
)60
60
)30
30
)90
90
0
34
4
0
II
II0
)60
60
120
)120
120
120
10
80
0
13
4
)80
)90
)60
)50
)120
0
VIa
VIb
IV
VIII
)60
)120
)60
)60
0
<1
1
0
ꢀ
vary between 4 and 7 A. The characterization and clas-
20
120
35
9
sification of the dihedral angles of the backbone atoms
)30
Keywords: Type VI b-turns; Ring closing metathesis; Conformational
analysis; Peptidomimetics.
are used to categorize different types of b-turns with at
most a 30ꢁ difference from standard angles, except that
one angle can deviate by as much as 45ꢁ. A recent study
looking at structures deposited in the Protein Data Bank
* Corresponding authors. Tel.: +1-215-628-5348; fax: +1-215-628-4985
(C.J.C.); tel.: +1-215-628-5615; fax: +1-215-540-4776 (A.B.R.);
e-mail addresses: ccreight@prdus.jnj.com; areitz@prdus.jnj.com
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.06.015

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C. J. Creighton et al. / Bioorg. Med. Chem. 12 (2004) 4375–4385
(http://www.rcsb.org/pdb/) has provided insight as to
which b-turns are found in nature in the solid state.3 The
Type I, II, and IV b-turns are the most prevalent, with
the I0 and II0 designations referring to the exact opposite
value for each of the dihedral angles. Type VI b-turns
are unique in that they bear a cis-amide bond between
the i þ 1 and i þ 2 residues, and thus typically have a
proline at the i þ 3 residue. Peptidomimetics are often
designed and prepared in order to restrict the backbone
dihedral angles to the values for specific b-turns, with
the best mimics being those in which all of the dihedral
angles fall within the appropriate ranges. The Type VIa
mimics that have been described fall into two broad
structural classes: modified proline derivatives4 and rigid
cyclic structures.5 The Type VI b-turn has been impli-
cated in several important biological functions such as
the thrombin-catalyzed cleavage of the V3 loop of HIV
gp1206 and substrate recognition of peptidyl prolyl
isomerases.7 We here describe the use of the ring closing
metathesis reaction to prepare a series of eight-mem-
bered ring, constrained dipeptide derivatives, one of
which is a Type VIa b-turn both in aqueous solution and
the solid state.
O
O
O
O
O
O
H
N
NH
S
NH
H2N
N
H
OH
O
S
1
2
O
O
O
O
H
H
N
NH
NH
N
Me
Me
Me
Me
Me
Me
N
N
H
H
O
O
4
3
5
O
O
H
H
NH
NH
N
N
Me
Me
N
N
H
H
O
O
6
Figure 1. Constrained eight-membered ring dipeptides.
work, we have fully characterized 5 as a Type VIa b-turn
mimic in both water and the solid state.
We have been interested in the conformational prefer-
ences of ox-[Cys-Cys] 1,8 an unusual dipeptide found in
the catalytic domain of mercuric ion reductase,9 mal-
formin10 and in the N-terminal extracellular domain of
most nicotinic acetylcholine receptor (nAChR) protein
subunits (Fig. 1).11 We have designed mimetics of 1
based upon the 7-amino-8-oxoazocane-2-carboxylic acid
scaffold 2, such as 3–6.12
2. Results and discussion
We employed ring closing metathesis (RCM)17 as the
key step for the preparation of the required eight-
membered ring starting from two adjacent allylglycine
(Agy) residues. It had been reported that Agy-Agy did
not undergo intramolecular RCM presumably because
the thermodynamically stable s-trans conformation of
the amide bond did not allow for the necessary orien-
tation of the pendant olefins.18 Therefore, we added the
acid labile 2,4-dimethoxybenzyl group (DMB) onto the
amide nitrogen to facilitate the formation of the re-
quired s-cis form (Scheme 1). The DMB group was
chosen because it can be readily removed under acidic
conditions, and the chemistry could be applied to a
dialkoxybenzaldehyde resin for immobilization onto a
In addition to being closely related to ox-[Cys-Cys],
compounds derived from scaffold 2 may be useful for
studying the structure activity relationships of a variety
of biologically relevant peptides including somato-
statin,13 malformin, Arg-Gly-Asp (RGD) peptides,14
and enkephalins.15 Also, 2 is an attractive building block
for the design and synthesis of polymeric, supermole-
cular structures that may have interesting physical
properties.16 We here provide a full report on the design
and synthesis of 3–6, as well as a thorough conforma-
tional analysis using both X-ray crystallography and
NMR spectroscopy in water. During the course of this
solid support.
tively alkylated with 2,4-dimethoxybenzaldehyde to give
8, which was coupled with N-(Boc)- -allylglycine using
L-Allylglycine methyl ester 7 was reduc-
L
HATU and HOAt to afford dipeptide 9.
DMB
N
O
O
O
O
DMB
N
O
O
H2N
a
DMBNH
b
c
BocHN
OMe
OMe
BocNH
AcHN
OMe
OMe
75%
50%
80%
7
8
11
9
O
O
O
DMB
O
H
N
H
O
O
N
N
AcHN
BocNH
g
e,f
d
60%
NHMe
NHMe
NHMe
95%
88%
3
12
4
Scheme 1. DMB ¼ 2,4-(MeO)2PhCH2. Reagents and conditions: (a) 2,4-dimethoxybenzaldehyde, Na(AcO)3BH; (b) Boc-
L-allylglycine, HOAt/
HATU, N-ethyl morpholine; (c) Cl2Ru(PCy3)2 ¼ CHPh (10), DCM, reflux (0.003 M substrate), 1 day; (d) 2 M MeNH2/MeOH; (e) TFA, Et3SiH;
(f) acetic anhydride; (g) H2, Pd/C.

C. J. Creighton et al. / Bioorg. Med. Chem. 12 (2004) 4375–4385
4377
incorporation into peptides for biological evaluation,
which will be described in due course.
First Generation 3 mM
Substrate
100
50
0
Use of the second-generation imidazoline ruthenium
benzylidene complex 1319 for the conversion of 9 to 11
proved to be much less satisfactory than what we
had seen for 10, as a time-dependent oligomerization
was observed. We investigated the RCM reaction of
9 with catalysts 10 and 13 in more detail because
we required >10 g quantities of 3 and 4 and wanted
to increase the concentration. Under certain condi-
tions, we observed the formation of acyclic and
cyclic dimers 14 and 15, respectively, as mixtures of
six or eight possible geometric and regioisomers
(Scheme 2).
Starting Material 9
Product 11
Acyclic Dimer 14
Cyclic Dimer 15
0
25
50
75
100
Time (h)
The RCM reaction of 9 was conducted at concentra-
tions of 12, 50, and 100 mM with 10 mol % of either 10
or 13, and monitored over the period of ca. 1 day in
methylene chloride at ambient temperature (Fig. 3). The
reaction of 9 at 12 mM with first-generation catalyst 10
was similar to that observed at 3 mM except that open
dimer 14 was formed to a substantial extent (ca. 10%)
after 23 h. Furthermore, more 14 was produced at con-
centrations of 50 and 100 mM, being found at the same
level as product 11 at 100 mM after 23 h. The use of
second-generation catalyst 13 also afforded a substantial
amount of 14, but dimer 15 was the predominant
product after 22 h at all concentrations. In these exper-
iments, using either catalyst, the expected monocyclic
product 11 appeared rapidly during the first five hours,
and then the levels did not increase further. We propose
that the reaction manifold may initially start with for-
mation of 11, which then undergoes an additional
metathesis with another molecule of 11 to afford 14. For
the more powerful catalyst 13, the reaction proceeds
further to give closed dimer 15 as the terminal and
prevalent product.
Figure 2. Ring closing metathesis of 9 to give primarily 11 using the
first generation Grubbs catalyst 10.
Ring closing metathesis of 9 (3 mM) using first-genera-
tion Grubbs catalyst 10 reproducibly provided protected
olefin 11 in high yield (80%). This reaction was moni-
tored with time at ambient temperature using a 3 mM
concentration of 9, which revealed that a high conver-
sion to 11 was achieved over the course of 2–3 days and
that very few other products were observed (Fig. 2).
Mes
N
N Mes
CHPh
Cl
Cl
P(c-C6H11 3
)
Ru
Cl
Cl
CHPh
Ru
P(c-C6H11 3
P(c-C6H11 3
)
)
Mes = 2,4,6-Me3Ph
10
13
To complete the synthesis, methyl ester 11 was treated
with methylamine to form amide 12. Acidolysis of the
Boc and 2,4-dimethoxybenzyl groups followed by
treatment of the resultant TFA salt with acetic anhy-
dride and base provided target 3, and hydrogenation of
3 (Pd/C) afforded the corresponding saturated derivative
An X-ray crystal structure for 3 was obtained revealing
cis olefin and amide geometry, and the eight-membered
ring is in a folded twist-boat–boat conformation similar
to a low energy form of cyclooctadiene (Fig. 4).20 The
peptide backbone is in an extended conformation, with
the dihedral angles of the backbone residues listed in
Table 2.
4. A similar reaction sequence starting with
cine methyl ester instead of 7 gave 5 and 6. Also, we
D-allylgly-
have prepared the
D,D- and D,L-variants of 3 for
DMB
N
DMB
N
O
O
O
O
BocHN
BocHN
OMe
OMe
Ru Catalyst
9
11
+
+
10 or 13
MeO
O
MeO
NHBoc
NHBoc
N
DMB
N
O
O
DMB O
14: Open Dimer
15: Closed Dimer
Scheme 2.

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C. J. Creighton et al. / Bioorg. Med. Chem. 12 (2004) 4375–4385
12 mM
100 mM
50 mM
100
75
50
25
0
100
75
50
25
0
100
75
50
25
0
Catalyst
10
0
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
20
25
100
75
50
25
0
100
75
100
75
50
25
0
Catalyst
50
13
25
0
0
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
20
25
Time (h)
Figure 3. Conversion of 9 (M) to 11 (j), 14 (}), and 15 (.) using either the first generation Grubbs catalyst 10 (top) or the second-generation
catalyst 12 (bottom) at concentrations of 12, 50, or 100 mM in methylene chloride at ambient temperature varying over time.
The X-ray crystal structure of saturated-derivative 4
(crystals prepared in ethyl ether) revealed an s-cis amide
bond, but the eight-membered ring was no longer folded
upon itself and the orientation of the two pendant
groups of the peptide backbone was relatively more
compact than for compound 3 (Fig. 5). The dihedral
angles for 4 mimic very closely those found for the Cþ
conformer of ox-[Cys-Cys] 1.8 Therefore, compounds in
which 4 is incorporated would be expected to serve as
close surrogates for 1, which suffers the limitations im-
parted by the disulfide bond such as the possibility of
reduction to the corresponding ring-opened Cys-Cys
dipeptide unit.
The X-ray crystal structure for diastereomer 5 was also
obtained (Fig. 6). This compound contains an s-cis
Figure 4. Crystal structure of 3 showing the cis olefin, s-cis amide
bond, and extended orientation of the peptide backbone.
Table 2. Dihedral angles for 3–5 and those expected for a Type VIa b-turn
χ21
χ11
φ1
ψ
2
NHMe
AcHN
N
HφO2
O
ψ1
ω1
Dihedral
3 (X-ray)
4 (X-ray)
5 (X-ray)
5 (1H NMR)
)63
Type V1a b-turn3
/
)93
)148
177
)70
)148
152
)64
)94
146
19
)60
)90
120
0
1
/
)85
2
a
w1
w2
x1
v11
v21
a
90
)12
)101.5
45.3
166
)7
175.3
4
0
162
43
0
169
42
––
––
)66.9
a These dihedrals cannot be determined by NMR without 15N enrichment.

C. J. Creighton et al. / Bioorg. Med. Chem. 12 (2004) 4375–4385
4379
Figure 5. Left: ORTEP representation of the X-ray crystal structure of 4. Right: superimposition of the Cþ conformer of ox-[Cys-Cys]8 and 4 (yellow
atoms are the sulfurs in ox-[Cys-Cys] 1).
Figure 6. Left: ORTEP representation of the X-ray crystal structure of 5. Right: compound 5 has been rotated to highlight the 10-membered b-turn
hydrogen bond.
R
ꢀ
amide and cis olefin, and the eight-membered ring is in a
folded boat–boat conformation. Unlike 3 and 4, the
pendant substituents on 5 are in a compact, turn ori-
water, with the exception of the 1.6 A value for the H6
and H6S protons, which cannot be pulsed in the NOE
experiment due to the proximity of their chemical shift
(Table 3). In addition, the proton on C5 exhibited a
relatively weak NOE to the H1N and HC7 protons,
indicating that the folded boat–boat conformation of
the eight-membered ring is also present in solution. A
strong NOE was observed between the H2N and pro R
HC3 protons, a weak NOE between the H2N and HC2
protons, and a weak, ambiguous NOE between the H2N
and pro S HC3 protons. These data indicate that atoms
N2, C10, O3, and C11 are in a similar orientation as that
shown by the ORTEP diagram in Figure 6. Finally a
relatively strong NOE between the H3N and HC2 pro-
tons was detected whereas H3N exhibited weak NOE’s
to the H1N and HC7 protons. These observations sup-
port the same alignment of the C-terminal portion of 5
as seen in the crystal structure.
ꢀ
entation, and the distance between C9 and C11 is 5.9 A,
consistent with the b-turn designation.21 The carbonyl
oxygen O3 of the acetyl moiety is involved in a hydrogen
bond to the NH of the terminal N-methyl amide as part
of a 10-membered ring.
1
1
To complement the X-ray analysis, H–1H and H–13C
coupling constants were determined for 5 in water, and
the dihedral angles /1, /2, x2, v11, and v21 were calcu-
lated based upon the Karplus equation (Table 2).22
Extensive 2-D NMR analysis was performed using
HMQC and ROESY techniques, to assign atom con-
nectivity and coupling constants as appropriate. The /,
w, and x dihedral angles for 5 from both the solid state
and in water indicate that it is a Type VIa b-turn. The w1
and w2 angles for 5 could not be derived using NMR
techniques, so it is important to note that the v11 and v21
values from both methods are also very similar. The
dihedral angles obtained from X-ray and NMR are all
within 10ꢁ of each other, showing high concordance
between the solid state and solution conformations.
We reduced 5 to 6 using standard hydrogenation con-
ditions. During the course of our work, 6 was inde-
pendently described by Holmes and co-workers,
prepared via a ca. 10-step procedure.5b Because of the
route employed, these authors did not have access to
olefin 5, but characterized 6 as a Type VIb b-turn by 2-D
NMR analysis in solution, and were able to obtain an
X-ray crystal structure of a synthetic precursor earlier in
the pathway, which supported the Type VI b-turn con-
formation at that stage. Consistent with our observa-
tions, they were not able to prepare crystals of 6
suitable for X-ray diffraction. In an accompanying
Nuclear Overhauser enhancements (NOE) were also
determined in aqueous solution, and were consistent
with the interatomic H–H distances observed in the
crystal structure for 5. In all cases in which the inter-
atomic H–H distances obtained from the crystal struc-
ꢀ
ture were below 3.0 A an NOE was also observed in

4380
C. J. Creighton et al. / Bioorg. Med. Chem. 12 (2004) 4375–4385
Table 3. Interatomic H–H distances from the crystal structure of 5, and NOE effects observed (shaded boxes)
HC3R HC3S
HC6R HC6S
HC11 H2N HC2
2.5 4.1 4.7
2.8 2.3
2.9
HC4 H1N HC7
HC5 H3N HC9
HC11
H2N
5.0
2.7
2.3
1.6
6.8 5.7 7.1 6.0
4.4 4.7 5.9 4.6
3.8 3.5 3.7 1.9
2.3 4.4 5.1 3.9
7.5
6.0
3.4
4.9
7.8 4.6 5.9
5.7 4.5 6.2
4.0 2.6 4.2
5.0 5.1 6.9
2.5
4.1
4.7
HC2
2.8
HC3R
2.3 2.9
HC3S
HC4
H1N
HC7
5.0
2.7 2.3 1.6
2.6 4.9 5.1 3.0
4.3
3.8 4.9 6.5
6.8
5.7
7.1
6.0
4.4 3.8 2.3
4.7 3.5 4.4
5.9 3.7 5.1
4.6 1.9 3.9
2.6
4.9
5.1
3.0
4.5 4.5 3.6
4.0
4.0
2.3
1.6
2.2 5.8 7.5
4.0 2.9 4.6
3.0 3.3 4.4
2.8 3.2 4.4
4.5
4.5 2.2
2.2 3.8
2.9
HC6R
3.6 3.8 2.9
HC6S
HC5
H3N
HC9
7.5
6.0 3.4 4.9
4.3
4.0 4.0 2.3 1.6
2.3 4.1 4.9
5.3 6.7
7.8
4.6
5.9
5.7 4.0 5.0
4.5 2.6 5.1
6.2 4.2 6.9
3.8
4.9
6.5
2.2 4.0 3.0 2.8
5.8 2.9 3.3 3.2
7.5 4.6 4.4 4.4
2.3
4.1
4.9
5.3
2.0
6.7 2.0
Red color indicates protons that overlap with each other.
Of the 23 observed NOE's, 8 are ambiguous.
Distances to methyl protons are measured from the carbon atom.
paper, Holmes and co-workers described the synthesis
of the enantiomer of 4 using a 12-step procedure with an
overall yield of 22–27% from a serine aldehyde deriva-
tive.5a They obtained crystals of ent-4 from chloroform–
hexane and found the pendant groups to be in an ex-
tended orientation consistent with our findings, but their
unit cell included two slightly different conformers. We
observed a single conformer in the structure of 4, which
could be due to polymorphic forms of 4 and ent-4
caused by different solvents and conditions in the crys-
tallization.
The olefin found in 3 and 5 also offers an attractive
synthetic handle for further elaboration. Intermediate 11
bearing latent amino and carboxylic acid functionality
has been converted to diketopiperazines, which have
interesting conformational properties and a tape crystal
packing orientation in the solid state.23
Conformational analysis of the target structures by
X-ray crystallography and 2-D NMR techniques has
proved to be particularly informative. Compounds 3
and 4 adopt an extended orientation of the pendant
substitution. For example, 4 mimics the Cþ conformer
of ox-[Cys-Cys]8 and may be useful in the design of
small molecules that interfere with the binding of ligands
such as bungarotoxin and acetylcholine to nicotinic
acetylcholine receptors. Our serendipitous discovery of
2S,7R isomer 5 as a Type VIa b-turn based on both
X-ray in the solid state and 2-D NMR in aqueous
solution complements the structure determined for 6 as
a Type VIb b-turn based upon NMR analysis.5b Given
the importance of b-turn secondary structures in bio-
organic chemistry, the identification of this unique Type
VIa b-turn provides another useful tool for the design of
novel, constrained peptidomimetics.
Halab and Lubell have described a series of 5-t-BuPro
derivatives that are Type VIa b-turns.4a–c High (>80%)
proportions of the cis-Pro rotamer with this conforma-
tion were seen in solution by NMR and CD techniques,
if the Pro was appropriately N- and C-substituted.4a
X-ray structures were determined for dipeptides 18 and
19, and both fit the criteria for Type VIa b-turn form.
O
t-Bu
N
R
NHMe
18 R = Ac-L-Tyr
19 R = Ac-L-Leu
4. Experimental section
4.1. General methods
3. Conclusion
1H NMR spectra were collected on Bruker 300-MHz
NMR spectrometer unless otherwise specified. Chemical
shifts are reported relative to the resonance of tetra-
methylsilane (TMS) or the corresponding solvent peak.
During the conformational studies, NMR experiments
were carried out at 14.1 T using approximately 10 mg of
test compound dissolved in 0.5 mL of water with
0.05 mL D2O for lock stabilization. The pH was ad-
justed to 6.5, and the data was acquired at 1 ꢁC. Low
temperature was used to shift the water resonance to
lower field to avoid overlap with one of the alpha pro-
tons and also to sharpen the amide signals. Proton and
carbon spectra were referenced to TSP. The 600-MHz
We prepared a series of compounds based upon core
scaffold 7-amino-8-oxo-1,2,3,6,7-pentahydroazocine-2-
carboxylic acid 2. The key step in the synthesis was the
ring closing metathesis of two sequential allylglycine
residues bearing N-(2,4-dimethoxy)benzyl amide sub-
stitution, which allowed for the pendent olefins to be in
close proximity. After removing the protecting groups,
an acetyl and a methyl amide at the N- and C-termini of
each scaffold were incorporated, respectively. The syn-
thetic approach we have described is compact and ver-
satile, providing 4 in ca. 17% overall yield after only six
synthetic steps starting from L-allylglycine methyl ester.

C. J. Creighton et al. / Bioorg. Med. Chem. 12 (2004) 4375–4385
4381
1H NMR spectrometer that was employed as indicated
was used with a triple resonance (H, C, N) and triple
axis gradient probe. The gradient amplifier delivered 10
amps per channel. Standard experiments were used to
assign the proton and carbon resonances, and mea-
surement of the heteronuclear couplings was done as
previously described.6 Mass spectra were obtained with
a single quadrapole and fitted with an electrospray
source. Purifications were conducted by preparative thin
layer chromatography using 20 · 20 cm tapered silica gel
plates or by flash column chromatography on silica gel
(230–400 mesh). Reagents and solvents were purchased
and used without further purification. Crystallographic
data (excluding structure factors) for the compounds
reported in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supple-
mentary publications CCDC-176870, CCDC-187971,
and CCDC 189564. Copies of the data can be obtained
free of charge on application to CCDC, 12 Union Road,
Cambridge CB21EZ, UK (fax: +44-1223-336-033;
deposite@ccdc.cam.ac.uk).
27.9 mmol) was dissolved in methylene chloride (90 mL),
and Boc- -allylglycine (6.0 g, 27.9 mmol), HOAt (3.8 g,
L
27.9 mmol), HATU (10.6 g, 27.9 mmol), and N-ethyl-
morpholine (3.6 mL, 27.9 mmol) were added to the
solution. After 12 h at ambient temperature, the solvent
was removed, and the crude mixture was suspended in
diethyl ether and then filtered through a pad of Celite.
The filtrate was poured into a separatory funnel and
washed 3X with 10% Na2CO3 in water, 1X with satu-
rated brine solution, 3X with 1 N HCl and 1X with
saturated brine. The organic layer was dried with
anhydrous MgSO4 and the solvent was removed under
reduced pressure, and purification was accomplished by
flash chromatography (3:1 hexanes/ethyl acetate) to
1
provide 9 as a clear, colorless oil (6.5 g, 50%). H NMR
(CDCl3) d 7.12 (d, 1H, 9 Hz), 6.45, (m, 2H), 5.7 (m, 1H),
5.35 (d, 1H, 9 Hz), 5.0 (m, 5H); 4.65 (d, 1H, 15 Hz), 4.35
(d, 1H, 15 Hz), 4.05 (m, 1H), 3.75 (m, 6H), 3.55 (s, 3H),
2.7 (m, 1H); 2.55 (m, 2H), 2.35 (m, 1H), 1.5 (s, 9H). MS
m/z (% relative intensity, ion): 477 (30, M+Hþ), 499 (30,
M+Naþ) 151 (100, M+ of DMB).
4.1.1. N-2,4-Dimethoxybenzyl
L
L
-allylglycine methyl ester
(8). A suspension of -allylglycine (15 gm, 130 mmol) in
4.1.3. (2S,7S)-7-N-(Boc)amino-1-(2,4-dimethoxybenzyl)-
8-oxo-1,2,3,6,7-pentahydroazocine-2-carboxylic
acid
methanol (260 mL) was cooled with an ice bath to 0 ꢁC
and thionyl chloride (14.26 mL, 195 mmol) was added
dropwise via an addition funnel. Once the thionyl
chloride addition was completed, the ice bath was re-
moved and the reaction allowed to proceed overnight.
The next morning the solvent was removed under re-
duced pressure. The corresponding oil was triturated for
4 h in an 95:5 ethyl ether/ethyl acetate mixture. The
resulting solid (7) was collected on a glass-fritted funnel
(9.5 g, 57.54 mmol), and placed into a separatory funnel
and treated with excess 10% Na2CO3 in water. The free
base was extracted from the aqueous layer with ethyl
ether. The organics were dried with anhydrous MgSO4,
and the ethyl ether was removed under reduced pressure.
The residue (7.27 g, 56.32 mmol) was dissolved in 1,2-
dichloroethane (200 mL), and an equimolar amount of
methyl ester (11). Intermediate 9 (3.0 g, 6.25 mmol) was
dissolved in methylene chloride (200 mL) and added to a
5 L flask equipped with a condenser. An additional 1.8 L
of methylene chloride was added to the reaction fol-
lowed by the addition of ruthenium catalyst 10 (0.514 g,
0.625 mmol). The reaction was heated to reflux for 60 h
under a stream of dry nitrogen. After the formation of
compound 11 was complete as determined by LC/MS,
the solvent was removed and the crude product purified
by flash chromatography (3:1 hexanes/ethyl acetate) to
provide 2.25 g of a clear, light brown oil of 11 (80%): 1H
NMR (CDCl3) d 7.09 (d, 1H, J ¼ 8 Hz), 6.40 (m, 2H),
6.20 (d, 1H, J ¼ 5 Hz), 5.72 (m, 1H), 5.45 (m, 1H), 4.95
(m, 2H), 4.60 (d, 1H, J ¼ 15 Hz), 4.28 (d, 1H,
J ¼ 15 Hz), 3.80 (s, 3H), 3.80 (s, 3H), 3.44 (s, 3H), 3.00
(m, 1H), 2.70 (m, 2H), 2.35 (ddd, 1H, J ¼ 16, 8, 3 Hz),
1.45 (s, 9H). HPLC Rt ¼ 4.06 min. MS m=z (% relative
intensity, ion): 449 (20, M+Hþ), 471 (20, M+Naþ) 151
(100, M+ of DMB). Anal. Calcd for C23H32N2O7: C,
61.59; H, 7.19; N, 6.25. Found: C, 61.82; H, 7.20; N,
5.96.
2,4-dimethoxybenzaldehyde
was
added
(9.36 g,
56.32 mmol) followed by sodium triacetoxyborohydride
(16.71 g, 78.85 mmol). The reaction was allowed to
proceed for 12 h at ambient temperature, and the excess
borohydride was quenched with 10% Na2CO3 in water
followed by removal of DCE under reduced pressure.
Ethyl ether was added to the reaction and the organic
and aqueous layers were poured into a separatory fun-
nel. The ether layer was washed 3X with 10% aqueous
Na2CO3 and dried with anhydrous MgSO4, and ethyl
ether was removed under reduced pressure to provide a
clear yellow oil. The desired compound was purified
using flash chromatography (65:35 hexanes/ethyl ace-
tate) yielding 11.8 g of a clear, colorless, oil of 8 (75%),
which is best kept for long-term storage as the HCl salt.
1H NMR, free base: (CDCl3) d 7.1 (d, 1H, J ¼ 8:6 Hz),
6.4 (m, 2H), 5.7 (m, 1H), 5.1 (m, 2H), 3.5–3.9 (m, 10H),
2.4 (m, 2H), 2.0 (1H, broad).
4.1.4. (2S,7S)-7-N-(Boc)amino-1-(2,4-dimethoxybenzyl)-
8-oxo-1,2,3,6,7-pentahydroazocine-2-carboxylic
acid
methyl amide (12). Lactam 11 (0.5 g 1.11 mmol) was
dissolved in a 2 M solution of methylamine in MeOH
(20 mL) and the reaction vessel was sealed with a rubber
septum. After 48 h, the solvent was then removed under
reduced pressure and the resulting oil purified by flash
chromatography (3:1 ethyl acetate/hexanes) to yield
1
0.3 g of 12 a white solid (60%). H NMR (DMSO-d6) d
8.35 (m, 1H), 6.85 (d, 1H, J ¼ 8), 6.58 (d, 1H J ¼ 6),
6.45 (d, 1H, J ¼ 2), 6.35 (dd, 1H, Ja ¼ 6, Jb ¼ 2), 5.7 (m,
1H), 5.55 (m, 1H), 5.15 (m, 1H), 4.85 (m, 1H), 4.31 (d,
1H, J ¼ 15), 4.03 (d, 1H, J ¼ 15), 3.75 (s, 3H), 3.70 (s,
3H), 3.32 (s, 3H), 3.10 (m, 1H), 2.68 (m, 1H), 2.35 (m,
1H), 2.30 (d, 3H, J ¼ 4), 2.05 (m, 1H), 1.40 (s, 9H). MS
4.1.2.
allylglycine methylester (9). Compound
Boc-
L
-allylglycine-N-(2,4-dimethoxybenzyl)-
8
L-
(7.79 g,

4382
C. J. Creighton et al. / Bioorg. Med. Chem. 12 (2004) 4375–4385
(m/z, % relative intensity, ion) 470 (10%, M+Naþ) 448
(10, M+Hþ), 151 (100, M+ of DMB).
ꢀ
C–H bond lengths of 0.95–1.00 A) ‘riding’ on their
respective carbon atoms. The two methyl groups (C9,
C11 and their hydrogens) were refined as rigid rotors
(using idealized sp3-hybridized geometry and a C–H
ꢀ
4.1.5. (2S,7S)-7-Acetylamino-8-oxo-1,2,3,6,7-pentahydro-
azocine-2-carboxylic acid methyl amide (3). Compound
12 (0.04 g, 0.08 mmol) was treated with a solution of
triethylsilane (0.020 mL) and water (0.010 mL) in TFA
(0.200 mL). The reaction was allowed to proceed for
16 h after which the solvent was removed and the crude
oil was treated with excess ethyl ether. The product was
triturated in ether for 4 h and then collected on a glass-
fritted funnel (0.025 g, >95%). This solid (0.025 g,
0.080 mmol) was then suspended in methylene chloride
(0.240 mL) and treated with triethylamine (0.022 mL
bond length of 0.96 A) with three rotational parameters
for each in least-squares cycles. The refined values of
these rotational parameters gave N–C–H and C–C–H
angles, which ranged from 108ꢁ to 112ꢁ. The isotropic
thermal parameters for H1N, H2N, and H3N refined to
2
ꢀ
final Uiso values of 0.02(1), 0.03(1), and 0.05(2) A ,
respectively. The isotropic thermal parameters of the
remaining hydrogen atoms were fixed at values 1.2
(nonmethyl) or 1.5 (methyl) times the equivalent iso-
tropic thermal parameters of the carbon atoms to which
they are covalently bonded.
0.160 mmol)
and
acetic
anhydride
(0.008 mL
0.080 mmol). After 3 h, the solvent was removed by re-
duced pressure and the compound purified by flash
chromatography (95:5 chloroform/methanol) yielding
4.1.7. (2S,7S)-7-Acetylamino-8-oxoheptahydroazocine-2-
carboxylic acid methyl amide (4). Compound 3 (0.040 g,
0.1 mmol) was dissolved in a mixture of methanol
(3.0 mL) and water (1.0 mL). To this solution was added
approximately 10 mg of 10% Pd/C and the reaction
mixture was treated with 1 atm of hydrogen and allowed
to react for 4 h at room temperature. After 4 h, the
reaction mixture was filtered over a pad of Celite and
washed 3X with methanol. The organics were combined
and the solvent removed under reduced pressure to yield
1
0.017 g of 3 a white solid (88%). H NMR (600 MHz,
H2O) d 8.47 (d, 1H, J ¼ 6:6), 8.18 (q, 1H, 4.2), 7.50 (d,
1H, J ¼ 7:8), 5.7 (m, 1H), 5.5 (1H, m), 4.9 (m, 1H), 4.70
(m, 1H), 2.90 (m, 2H), 2.68 (d, 3H, J ¼ 4:2), 2.30 (m,
1H), 2.13 (m, 1H), 1.98 (s, 3H). 13C NMR (151 MHz,
H2O) d 175.6, 173.6, 173.0, 127.2, 127.1, 53.5, 51.2, 32.1,
30.8, 26.1 (2C).
1
4 as a white solid (0.040 g, >95%). H NMR (600 MHz,
H2O) d 8.1 (d, 1H, J ¼ 5:4), 7.95 (m, 1H), 7.43 (d, 1H,
J ¼ 6:6), 4.42 (m, 1H), 4.36 (m, 1H), 2.58 (d, 3H,
J ¼ 4:8), 1.85 (s, 3H), 1.85 (m, 3H), 1.52 (m, 6H), 1.30
(m, 1H). 13C NMR (151 MHz, H2O) d 179.1, 176.4,
176.1, 57.4, 53.3, 37.8, 36.4, 28.9, 26.4, 25.5, 24.3.
4.1.6. X-ray diffraction of lactam 3. Single crystals of
C11H17N3O3 are, at )80 2 ꢁC, orthorhombic, space
ꢀ
group P212121-D (No. 19) with a ¼ 7:538ð1Þ A,
3
ꢀ
ꢀ
ꢀ
b ¼ 11:511ð2Þ A, c ¼ 13:981ð2Þ A, V ¼ 1213:2ð3Þ A and
Z ¼ 4 molecules dcalcd ¼ 1:310 g cmꢀ3
;
la ðMoKÞ ¼
0:097 mmꢀ1. A full hemisphere of diffracted intensities
(omega scan width of 0.30ꢁ) was measured using
graphite-monochromated MoKa radiation on a Sie-
mens/Bruker SMART CCD Single Crystal Diffraction
System. X-rays were provided by a normal-focus sealed
X-ray tube operated at 50 kV and 40 mA. Lattice con-
stants were determined with the Siemens/Bruker SAINT
software package using peak centers for 855 reflections.
A total of 5535 integrated reflection intensities having
2HðMoKÞ < 46:50ꢁ were produced using the Siemens/
Bruker program SAINT. A total of 1737 of these were
independent and gave Rint ¼ 0:110. The Siemens/Bruker
SHELXTL-PC software package was used to solve the
structure using ‘direct methods’ techniques. All stages of
weighted full-matrix least-squares refinement were con-
ducted using Fo2 data with the SHELXTL-PC Version 5
software package. Final agreement factors at conver-
gence are: R1 (unweighted, based on F ) ¼ 0.049 for 1013
independent reflections having 2HðMoKÞ < 46:50ꢁ and
I > 2rðIÞ; R1 (unweighted, based on F ) ¼ 0.120 and wR2
(weighted, based on F 2) ¼ 0.069 for all 1737 independent
reflections having 2HðMoKÞ < 46:50ꢁ. The structural
model incorporated anisotropic thermal parameters for
all nonhydrogen atoms and isotropic thermal parame-
ters for all hydrogen atoms. Amine hydrogen atoms
H1N, H2N and H3N were located from a difference Fou-
rier map and refined as independent isotropic atoms.
The remaining hydrogen atoms were included in the
structure factor calculations as idealized atoms (assum-
ing sp2- or sp3-hybridization of the carbon atoms and
In a similar manner as for 11, 12, 3, and 4, compounds
16, 17, 5, and 6 were also prepared.
DMB
N
O
O
DMB
N
O
O
RCM
BocHN
BocHN
5,6
OMe
OMe
17
16
4.1.8. Boc-
L
-allylglycine-N-2,4-dimethoxybenzyl-
D
-allyl-
glycine methylester (16). 50–80% yield. 1H NMR
(CDCl3) d 7.15 (d, 9.3), 6.42 (m, 2H), 5.75 (m, 1 H), 5.5
(m, 1H), 5.40 (d, 8.7), 5.12 (m, 2 H), 5.04 (m, 1H), 4.90
(m, 2H), 4.68 (d, 1H, J ¼ 1:6), 4.30 (d, 1H, J ¼ 15:4),
4.11 (m, 2H), 3.80 (s, 3H), 3.79 (s, 3H), 3.55 (s, 3H), 2.78
(m, 1H), 2.42 (m, 3H), 1.44 (s, 9H). Crystals were
obtained from diethyl ether for X-ray diffraction
studies.
4.1.9. (2R,7S)-7-N-(Boc)amino-1-(2,4-dimethoxybenzyl)-
8-oxo-1,2,3,6,7-pentahydroazocine-2-carboxylic
acid
1
methyl ester (17). 70–80% yield. H NMR (CDCl3) d
7.22 (d, 1H, J ¼ 8:1), 6.40 (m, 2H), 5.64 (m, 2H), 5.30
(m. 1H), 4.90 (m, 1H), 4.78 (d, 1H, J ¼ 14:1), 4.36 (d,
1H, J ¼ 14), 3.79 (s, 3H), 3.77 (s, 3H), 3.69 (s, 3H), 2.85
(m, 2H), 2.65 (m, 1H), 2.10 (m, 1H), 1.43 (s, 9H).

C. J. Creighton et al. / Bioorg. Med. Chem. 12 (2004) 4375–4385
4383
Crystals were obtained from diethyl ether for X-ray
diffraction studies.
parameters of the carbon atoms to which they are
covalently bonded.
4.1.10. (2R,7S)-7-Acetylamino-8-oxo-1,2,3,6,7-pentahyd-
roazocine-2-carboxylic acid methyl amide (5). 50–70%
overall yield. H NMR (600 MHz, H2O) d 8.47 (d, 1H
J ¼ 3:6), 8.40 (m, 1H), 8.02 (d, 1H, J ¼ 7:8), 5.77 (m,
1H), 5.70 (m, 1H), 4.86 (m, 1H), 4.02 (m, 1H), 2.78 (m,
3H), 2.76 (d, 3H, J ¼ 4:8), 2.2 (m, 1H), 2.0 (s, 3H). 13C
NMR (151 MHz, H2O) d 180.5, 176.9, 176.6, 132.0,
129.6, 57.5, 54.4, 33.0, 32.7, 29.1, 23.9. Crystals were
obtained from diethyl ether for X-ray diffraction studies.
4.1.12. (2R,7S)-7-Acetylamino-8-oxoheptahydroazocine-
2-carboxylic acid methyl amide (6). >95% yield. 1H
NMR (600 MHz, H2O 500 lL, D2O 50 rmuL, 7 lL 1 N
HCl, pH 6.2, T ¼ 285 K) d 7.89 (d, 1H, 5.41 Hz), 7.75
(m, 1H), 7.5 (m, 1H), 4.22 (d, 1H, 9.75 Hz), 4.21, (m,
1H), 2.37 (d, 3H, 4.76 Hz), 1.61 (s, 3H), 1.60 (m, 2H),
1.30 (m, 4H), 1.10 (m, 2H). This spectrum was similar to
that previously reported.5b
1
4.1.13. X-ray diffraction of lactam 6. Single crystals of
C11H19N3O3 are, at 20 2 ꢁC, orthorhombic, space
4.1.11. X-ray diffraction of lactam 5. Single crystals of
C11H17N3O3 are, at )80 2 ꢁC, orthorhombic, space
ꢀ
group P212121-D (No. 19) with a ¼ 9:661ð1Þ A,
3
ꢀ
ꢀ
ꢀ
ꢀ
group P212121-D (No. 19) with a ¼ 6:992ð2Þ A,
b ¼ 14:836ð1Þ A, c ¼ 18:431ð3Þ A, V ¼ 2641:7ð5Þ A and
3
ꢀ
ꢀ
ꢀ
b ¼ 9:099ð3Þ A, c ¼ 17:814ð6Þ A, V ¼ 1133:3ð7Þ A and
Z ¼ 8 formula units {dcalcd ¼ 1:213 g cmꢀ3; laðMoKÞ ¼
Z ¼ 4 molecules {dcalcd ¼ 1:402 g cmꢀ3
;
laðMoKÞ ¼
0:089 mmꢀ1}.
A total of 2735 reflections having
0:104 mmꢀ1}. A full hemisphere of diffracted intensities
(omega scan width of 0.30ꢁ) was measured using
graphite-monochromated MoKa radiation on a Bruker
SMART CCD Single Crystal Diffraction System. X-rays
were provided by a normal-focus sealed X-ray tube
operated at 50 kV and 40 mA. Lattice constants were
determined with the Bruker SAINT software package
using peak centers for 550 reflections. A total of 4959
integrated reflection intensities having 2HðMoKÞ <
46:52ꢁ were produced using the Bruker program
SAINT. A total of 1628 of these were independent and
gave Rint ¼ 0:123. The Bruker SHELXTL-PC software
package was used to solve the structure using ‘direct
methods’ techniques. All stages of weighted full-matrix
least-squares refinement were conducted using Fo2 data
with the SHELXTL-PC Version 5 software package. Final
agreement factors at convergence are: R1 (unweighted,
based on F ) ¼ 0.051 for 1055 independent reflections
having 2HðMoKÞ < 46:52ꢁ and I > 2rðIÞ; R1 (un-
weighted, based on F ) ¼ 0.099 and wR2 (weighted, based
on F 2) ¼ 0.112 for all 1628 independent reflections
having 2HðMoKÞ < 46:52ꢁ. The structural model
incorporated anisotropic thermal parameters for all
nonhydrogen atoms and isotropic thermal parameters
2HðMoKÞ < 45:8ꢁ (the equivalent of 0.6 limiting CuK
sphere) were collected on a computer-controlled Bruker
P4 Single Crystal Diffractometer using 0.90ꢁ-wide x
scans and graphite-monochromated MoK radiation;
2551 of these reflections were independent. X-rays were
provided by a normal-focus sealed X-ray tube operated
at 50 kV and 40 mA. Lattice constants were determined
with the Bruker XSCANS software package using 54
centered reflections. The Bruker SHELXTL-PC (version
5) software package was used to solve and refine the
structure. ‘Direct methods’ techniques were used to
solve the structure and the resulting structural parame-
ters have been refined with F 2 data to convergence using
counter-weighted full-matrix least-squares techniques
and a structural model, which incorporated anisotropic
thermal parameters for all nonhydrogen atoms and
isotropic thermal parameters for all hydrogen atoms.
Final agreement factors are: R1 (unweighted, based on
F ) ¼ 0.057 for 1729 independent reflections having
2HðMoKÞ < 45:8ꢁ and I > 2rðIÞ; R1 (unweighted, based
on F ) ¼ 0.096 and wR2 (weighted, based on F 2) ¼ 0.148
for all 2551 independent reflections having
2HðMoKÞ < 45:8ꢁ. Amide hydrogen atoms H1N, H2N
,
H3N, H21N, H22N, and H23N were located from a differ-
ence Fourier map and refined as independent isotropic
atoms. The four methyl groups (C9, C11, C29, C31 and
their hydrogens) were refined as rigid rotors (using ide-
alized sp3-hybridized geometry and a C–H bond length
for all hydrogen atoms. Amide hydrogen atoms H1N
,
H2N and H3N were located from a difference Fourier
map and refined as independent isotropic atoms. The
remaining hydrogen atoms were included in the struc-
ture factor calculations as idealized atoms (assuming
sp2- or sp3-hybridization of the carbon atoms and C–H
ꢀ
of 0.96 A), which were allowed to rotate about their N–
C or C–C bonds in least-squares cycles. The remaining
hydrogen atoms were included in the structure factor
calculations as idealized atoms (assuming sp3-hybrid-
ization of the carbon atoms and C–H bond lengths of
ꢀ
bond lengths of 0.95–1.00 A) ‘riding’ on their respective
carbon atoms. The two methyl groups (C9, C11 and their
hydrogens) were refined as rigid rotors (using idealized
sp3-hybridized geometry and a C–H bond length of
ꢀ
0.97 or 0.98 A) ‘riding’ on their respective carbon atoms.
The isotropic thermal parameters for H1N, H2N, H3N
ꢀ
0.96 A) with three rotational parameters for each in
,
least-squares cycles. The refined values of these rota-
tional parameters gave N–C–H and C–C–H angles,
which ranged from 104ꢁ to 115ꢁ. The isotropic thermal
parameters for H1N, H2N, and H3N refined to final Uiso
values of 0.04(2), 0.05(1), and 0.06(2) A , respectively.
The isotropic thermal parameters of the remaining
hydrogen atoms were fixed at values 1.2 (nonmethyl) or
1.5 (methyl) times the equivalent isotropic thermal
H21N, H22N, and H23N refined to final Uiso values of
2
ꢀ
0.07(2), 0.05(2), 0.08(3), 0.10(2), 0.04(2), and 0.03(2) A ,
respectively. The isotropic thermal parameters of the
remaining hydrogen atoms were fixed at values 1.2
(nonmethyl) or 1.5 (methyl) times the equivalent iso-
tropic thermal parameters of the carbon atoms to which
they are covalently bonded. The second crystallo-
graphically independent molecule in the asymmetric unit
2
ꢀ

4384
C. J. Creighton et al. / Bioorg. Med. Chem. 12 (2004) 4375–4385
Manasse, H. R., Jr. Eds.; Chapman & Hall: New York,
1993, pp 366–378; Hirschmann, R.; Nicolaou, K. C.;
Pietranico, S.; Salvino, J.; Leahy, E. M.; Sprengeler, P. A.;
Furst, G.; Strader, C. D.; Smith, A. B., III; Strader, C. D.;
Cascieri, M. A.; Candelore, M. R.; Donaldson, C.; Vale,
W.; Maechler, L. J. Am. Chem. Soc. 1992, 114, 9217–9218;
(f) Flynn, G. A.; Giroux, E. L.; Dage, R. C. J. Am. Chem.
Soc. 1987, 109, 7914–7915.
appears to be slightly disordered in the lattice with
carbon atoms C24 and C25 having substantially larger
equivalent isotropic thermal parameters than the
remaining nonhydrogen atoms in the asymmetric unit.
This disorder is presumably responsible for the short
C24–C25 bond length of 1.288(11) A. Analytical and
spectroscopic data for the compound indicate the pres-
ence of a single species with no C@C double bonds.
ꢀ
3. (a) Guruprasad, K.; Raykumar, S. J. Biosci. 2000, 25,
143–156; See also: (b) Hutchinson, E. G.; Thornton, J. M.
€
Protein Sci. 1994, 3, 2207–2216; See also: (c) Muller, G.;
Gurrath, M.; Kurz, M.; Kessler, H. Proteins: Struct.,
Funct. Genet. 1993, 15, 235.
4.2. Concentration studies of the ring closing metathesis
reaction
4. (a) Halab, L.; Lubell, W. D. J. Am. Chem. Soc. 2002, 124,
2474–2484; (b) Halab, L.; Lubell, W. D. J. Org. Chem.
1999, 64, 3312–3321; (c) Halab, L.; Becker, J. A. J.;
Compound 9 was added to four 10 mL round bottom
flasks (0.025 g, 0.052 mmol), and treated with an
increasing amount (0.55, 1.1, 2.2, and 4.3 mL) of di-
chloromethane corresponding to 100, 50, 24, and 12 mM
solutions. To each reaction vessel first-generation
Grubbs catalyst 10 was added (0.004 g, 0.0052 mmol),
and this time point was taken to be t0. Each vessel was
equipped with a rubber septum and purged with N2.
Periodically, a 20 lL aliquot of each reaction was re-
moved via syringe and diluted with 20 lL of methanol.
From this mixture, 2 lL was injected on an LC-MS
instrument (MS for 14: C48H68N4O14, m=e 925.1, M+1;
for 15: C46H64N4O14, m=e 897.0, M+1). The same pro-
cedure was used for studies involving second-generation
catalyst 13 (0.004 g, 0.0052 mmol).
ꢁ
Darula, Z.; Tourwe, D.; Kieffer, B. L. J. Med. Chem. 2002,
45, 5353–5357; (d) Boruah, A.; Rao, I. N.; Nandy, J. P.;
Kumar, S. K.; Kunwar, A. C.; Iqbal, J. J. Org. Chem.
2003, 68, 5006–5008; (e) Zhang, W.-J.; Berglund, A.; Kao,
J. L.-F.; Couty, J.-P.; Gershengorn, M. C.; Marshall, G.
R. J. Am. Chem. Soc. 2003, 125, 1221–1235.
5. (a) Derrer, S.; Davies, J. E.; Holmes, A. B. J. Chem. Soc.,
Perkin 1 2000, 2943–2956; (b) Derrer, S.; Davies, J. E.;
Holmes, A. B. J. Chem. Soc., Perkin 1 2000, 2957–2967;
(c) Park, K.-H.; Kurth, M. J. Tetrahedron 2002, 58, 8629–
8659, general review, see references cited therein; (d)
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Acknowledgements
We thank Victor Day (Crystalytics, PO Box 82286
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