Synthesis of macrocyclic analogues of the neuroprotective agent glycyl-l-prolyl-l-glutamic acid (GPE)

Article abstract of DOI:10.1039/b605293b

The syntheses of seven macrocyclic analogues of the neuroprotective tripeptide glycyl-l-prolyl-l-glutamic acid (GPE) 1 are described. Macrocycles 6 and 7 mimic the cis conformer of GPE whereas macrocycles 2-5, 8, and 9 mimic the trans conformer of GPE. The macrocyclic peptides of well-defined geometry were prepared via Grubbs ring closing metathesis of an appropriate diene precursor. In turn each of the diene precursors were prepared from the readily available allyl-substituted amino acid building blocks 12, 13, 14, 27, 36 and 51. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b605293b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of macrocyclic analogues of the neuroprotective agent
glycyl-L-prolyl-L-glutamic acid (GPE)†
Paul W. R. Harris and Margaret A. Brimble*
Received 12th April 2006, Accepted 10th May 2006
First published as an Advance Article on the web 1st June 2006
DOI: 10.1039/b605293b
The syntheses of seven macrocyclic analogues of the neuroprotective tripeptide glycyl-L-prolyl-L-
glutamic acid (GPE) 1 are described. Macrocycles 6 and 7 mimic the cis conformer of GPE whereas
macrocycles 2–5, 8, and 9 mimic the trans conformer of GPE. The macrocyclic peptides of well-defined
geometry were prepared via Grubbs ring closing metathesis of an appropriate diene precursor. In turn
each of the diene precursors were prepared from the readily available allyl-substituted amino acid
building blocks 12, 13, 14, 27, 36 and 51.
Glu. These studies demonstrated that the neuroprotective activity
Introduction
of most of the GPE analogues was of lower potency than that
of the endogenous tripeptide. Moreover, it was concluded that
the prevention of neuronal death by these GPE analogues after
NMDA injury is not directly linked to their affinity for glutamate
receptors.
The use of cyclic peptides provides an elegant solution for the
synthesis of peptides of restricted geometry that can be used to
probe the bioactive conformation of a given peptide.16
The unique properties that proline17 imparts on the overall
structure of proteins indicate that proline-containing sequences
act as molecular hinges, swivels, and switches and play a pivotal
role in biological signaling processes.18 The fact that the Xaa–
Pro amide bond exists as a mixture of cis and trans isomers,19
whereas most peptide bonds adopt the trans form, also sug-
gests that catalysis of cis/trans-prolyl isomerization by rotamase
enzymes20 is critical for the protein folding process. Given that
the conformation of the proline residue of the GPE molecule
play a pivotal role in the observed biological activity we decided
to synthesize a small library of GPE analogues in which the
conformation of the proline ring was restricted by its incorporation
into a macrocyclic structure. The synthesis of these macrocyclic
analogues allows the relationship between cis–trans conformers
and peptide bioactivity21,22 to be probed.
Protease-mediated metabolism of IGF-1 (insulin-like growth fac-
tor type-1) is reported to lead to the formation of the endogenous
N-terminal tripeptide Gly-Pro-Glu (GPE 1) along with the 67
amino acid des(1–3)IGF-1 fragment.1–3 Although GPE does not
bind to IGF-1 receptors and its mode of action is unclear, in vitro
studies have demonstrated its ability to stimulate acetylcholine and
dopamine release,4,5 and to protect different types of neurons from
diverse induced injuries (e.g. hypoxia-ischemia and glutamate).6–8
More importantly, GPE shows neuroprotective properties in
different animal models of neurodegenerative diseases such as
Huntington’s, Parkinson’s and Alzheimer’s diseases.5,6,9 Prelim-
inary observations suggested that GPE possibly exhibited its
neuromodulatory role in the CNS through interaction with one
or more glutamate receptors and it has been demonstrated1
that it binds to the N-methyl-D-aspartate (NMDA) receptor but
not to the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) or kainate receptors.
Results and discussion
The structural simplicity of GPE renders it a suitable lead
molecule for the development of novel strategies for the de-
velopment of effective neuroprotective drugs based on non-
peptide analogues. GPE peptidomimetics could also be used as
pharmacological probes to investigate the mechanism of action
of this tripeptide. With these aims in mind, we10–13 and others14,15
have embarked on a synthetic program focused on the systematic
modification of the individual amino acid residues, Gly, Pro and
The ring closing metathesis reaction (RCM) is a powerful tool23
for the synthesis of macrocyclic systems and has proven a
popular method for the synthesis of cyclic peptides from an
acyclic precursor.24–32 We therefore decided to prepare a series of
macrocycles 2–7 (Fig. 1) in which a short alkyl chain attached
to the nitrogen atom or the alpha carbon of the glycine residue
is linked to the C-5 and C-2 positions of the proline residue.
Additionally, two macrocycles 8 and 9 containing a short alkyl
chain linking the glycine unit (through the nitrogen atom or the
alpha carbon) to the glutamate side chain were also prepared.33
Initially, our attention focused on the synthesis of macrocycles
2, 3, 4 and 5 that mimic the trans conformation of GPE 1.
Macrocycles 2 and 3 were prepared via ring closing metathesis
of dienes 10 and 11 that are, in turn, derived from the union
Department of Chemistry, University of Auckland, 23 Symonds St.,
Auckland, New Zealand. E-mail: m.brimble@auckland.ac.nz; Fax: +64 9
3737422
† Electronic supplementary information (ESI) available: general experi-
1
mental details together with full experimental procedures, H NMR, 13C
NMR and mass spectral data for compounds 10, 11, 13, 16, 23, 28, 29, 37,
50, 52, 54, 55 and 56. See DOI: 10.1039/b605293b
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stereochemistry between C-2 and C-5 on the proline ring but with
trans stereochemistry about the glycine C(O)–NPro bond.
The synthesis of the analogous macrocycle 3 that also exhibits
trans stereochemistry about the glycine C(O)–NPro bond but with
trans stereochemistry between C-2 and C-5 on the proline ring,
followed a similar strategy to macrocycle 2 (Scheme 2). In this
case, allylation39 of the mixture of aminols formed upon reduction
of the lactam carbonyl group of tert-butyl ester 2240 afforded a 57 :
43 mixture of cis-5-allylproline 23a : trans-5-allylproline 23b that,
upon deprotection of the Boc group, afforded separable amines 13.
The trans amine 13b was subjected to DCC coupling with Boc-
(S)-allylglycine 14 to afford diene 11 that underwent sluggish ring
closing metathesis, despite using a high catalyst loading, affording
the cyclic olefin 24 in low yield. Hydrolysis of the tert-butyl ester
afforded acid 25 that was immediately coupled with di-tert-butyl
(S)-glutamate (HCl salt) 20 using BoP–Cl to afford olefin 36
in 60% yield over two steps. Finally hydrogenation and global
deprotection afforded the desired macrocycle 3 albeit in low yield.
Our attention next focused on the synthesis of macrocycles 4
and 5 in which an alkyl chain linked the glycine nitrogen to the
C-5 on the proline ring. These two macrocycles also mimic the
trans conformation of GPE 1 and were both accessible via ring
closing metathesis of dienes 28 and 29 followed by appendage of a
glutamate fragment. Dienes 28 and 29 in turn were available from
the union of 5-allylproline 13 with N-allylglycine 27 (Schemes 3,
4 and 5).
Benzyloxycarbamate protected N-allylglycine 27 was prepared
via slight modification of the literature preparation41 and coupled
with a mixture of diastereomeric 5-allylprolines 13 using EDCI
to afford the separable dienes 28 and 29 in moderate yield
(60%) (Scheme 3). The cis/trans ratio of the newly created
amide (Pro) bond was 1 : 1 in both of these flexible acyclic
dipeptides. Subsequent ring closing metathesis of cis-diene 28
afforded cyclic olefin 30 (Scheme 4). Hydrogenation of the olefin
effected concomitant removal of the benzyloxycarbamate group
hence the cyclic amine was reprotected as a Boc carbamate 31 after
hydrolysis of the tert-butyl ester. Coupling acid 31 with di-tert-
butyl (S)-glutamate (HCl salt) 20 using BoP–Cl afforded amide
32 that afforded the desired macrocycle 4 upon removal of the
tert-butyl esters and the tert-butyl carbamate.
In a similar fashion, ring closing metathesis of trans-diene 29
afforded cyclic olefin 33 that was readily converted to macrocycle
5 via the intermediacy of acid 34 and amide 35 (Scheme 5). The
ring closure was not affected by the nature of the stereochemistry
of the allyl group at C-5 on the proline ring, with ring closure
of dienes 28 and 29 giving similar yields of the respective cyclic
olefins 30 and 33 with comparable reaction times and catalyst
loadings. Very little deallylation/isomerization was observed in
the ring closing metathesis reaction and both cyclic olefins 30
and 33 only adopted the trans conformation about the amide
bond thus affording macrocycles 4 and 5 exhibiting only the trans
conformation about the glycine C(O)–NPro bond.
Fig. 1
of cis-5-allylproline 12a or trans-5-allylproline 13 with Boc-(S)-
allylglycine 14 respectively (Schemes 1 and 2). The preparation
of the trans mimic of GPE with cis stereochemistry at C-2
and C-5 of the proline ring, namely macrocycle 2, commenced
with the union of cis-5-allylproline 12a with Boc-(S)-allylglycine
14. Pyroglutamic acid 15 was converted to a Boc protected
ethyl ester derivative 16.34 Selective reduction of the lactam
carbonyl group with lithium triethylborohydride yielded a mixture
of aminols that underwent BF3·Et2O mediated allylation with
allyltributylstannane to afford an inseparable 66 : 33 mixture
of cis-5-allylproline 17a : trans-5-allylproline 17b.35 Liberation
of the free amines 12 upon removal of the Boc group followed
by direct coupling with Boc-(S)-allylglycine 1436,37 using DCC
afforded an inseparable 77 : 23 mixture of cis-diene 10a : trans-
diene 10b. Subjection of this mixture of dienes to ring closing
metathesis using Grubbs’ first generation catalyst with heating
in dichloromethane for 24 h followed by stirring overnight in
dimethyl sulfoxide38 resulted in exclusive formation of olefin 18
with cis stereochemistry between C-2 and C-5 of the proline ring
due to the much slower rate of trans-diene 10b to undergo the
ring closing metathesis reaction. Hydrolysis of the ethyl ester
afforded acid 19, which was immediately reacted with di-tert-
butyl (S)-glutamate 20 using BoP–Cl to afford olefin 21 in 70%
yield over two steps. Finally hydrogenation of the olefin over PtO2
in THF followed by immediate deprotection of the Boc group
and tert-butyl esters afforded the desired macrocycle 2 with cis
Macrocycles 6 and 7, in which a side chain linked C-2 of
the proline unit to the glycine nitrogen or the a-carbon, were
identified as possible mimics for the cis (Pro) conformation of
GPE 1. (S)-2-Allylproline methyl ester 36 was prepared using
Seebach’s procedure,42,43 however subsequent coupling with Boc-
(S)-allylglycine 14 using DCC with HOBt as additive only afforded
diene 37 in low yield (Scheme 6). Ring closing metathesis of
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Scheme 1 Reagents, conditions and yields: (i) EtOH, SOCl2, 0 ◦C to room temp, overnight; (ii) DMAP, Boc2O, CH3CN, room temp, 18 h, 85% over
2 steps; (iii) LiEt3BH, THF, −78 ◦C, 1 h, then 30% H2O2, 0 ◦C, 0.5 h; (iv) BF3·OEt2, allyltributylstannane, CH2Cl2, −78 ◦C, 2 h, 54% over 2 steps;
(v) CF3CO2H, CH2Cl2, room temp, 4 h; (vi) DCC, Et3N, CH2Cl2, 0 ◦C to room temp, overnight, 55% over 2 steps; (vii) Cl2(PCy3)2Ru CHPh, CH2Cl2,
=
45 ◦C, 24 h then DMSO, room temp, overnight, 74%; (viii) 1 M aq. NaOH, H2O, dioxane, room temp, 23 h; (ix) BoP–Cl, Et3N, CH2Cl2, 0 ◦C to room
temp, 21 h, 70% over 2 steps; (x) PtO2, H2, THF, room temp, overnight, then CF3CO2H, CH2Cl2, room temp, 5 h, 60% over 2 steps.
diene 37 required the use of Grubbs’ second generation catalyst44
affording bicyclic cyclooctene 3845 as a single cis (Pro) conformer.
Hydrolysis of the methyl ester and direct coupling with di-tert-
butyl (S)-glutamate (HCl salt) 20 using BoP–Cl afforded olefin
39. Finally, hydrogenation over PtO2 followed by removal of the
Boc and tert-butyl ester groups using trifluoroacetic acid afforded
the desired macrocycle 6.
Our attention next focused on the synthesis of macrocycle 7
that contains a side chain linking C-2 of the proline unit to the
glycine nitrogen. Attempts to effect ring closing metathesis of the
dipeptides 43–47, formed from the union of (S)-2-allylproline 36
with N-allylglycine derivatives 14, 27 or 40–42, using Grubbs’ first
and second generation catalysts were unsuccessful (Scheme 7).
Whilst the presence of the electron-withdrawing Boc, CO2Bn, Ac
or Fmoc groups may have accelerated competing deallylation (or
isomerization in the case of 42), use of N,Nꢀ-diallylglycine 40
in an effort to overcome this problem only afforded recovered
starting material possibly due to quenching of the catalyst by the
more basic nitrogen atom.46 Conversion of diallylamine 40 into
its hydrochloride salt followed by ring closing metathesis was also
unsuccessful.47 The synthesis of macrocycle 7 thus required an
alternative strategy.
The ring-closing metathesis of dienes contained within the GPE
1 scaffold was next examined by focusing on the preparation
of macrocycles 8 and 9 in which an alkyl chain links the
glutamate residue to the glycine a-carbon or nitrogen respec-
tively. C-allylglycyl dipeptide 50 was prepared via the union of
proline methyl ester 48 with Cbz-(S)-allylglycine 4948 followed by
hydrolysis (Scheme 8). Subsequent union with c-allylglutamate
51 (prepared from dibenzyl (S)-glutamate following a similar
procedure to that reported49 using dimethyl glutamate) afforded
diene 52 that underwent ring closing metathesis to afford a mixture
of isomeric cyclic olefins 53. Hydrogenation of the olefin 53
effected global deprotection of the benzyloxycarbamate and the
benzyl esters affording the desired macrocycle 8 in 58% yield as a
single trans conformer over two steps.
In a similar fashion, N-allylglycyl dipeptide 55 was prepared
via the union of proline methyl ester 48 with Cbz-N-allylglycine
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Scheme 2 Reagents, conditions and yields: (i) LiEt3BH, THF, −78 ◦C, 1 h, then 30% H2O2, 0 ◦C, 0.5 h; (ii) allyltributylstannane, Me3SiOTf, −78 ◦C, 2 h,
◦
◦
=
70% over 2 steps; (iii) 4 M HCl, dioxane, 0 C, 1 h then room temp, 40 min, 72%; (iv) DCC, CH2Cl2, 0 C to room temp, 18 h, 81%; (v) Cl2(PCy3)2Ru CHPh,
CH2Cl2, 45 ◦C, 4 days, then DMSO, room temp, overnight (39%); (vi) CF3CO2H, CH2Cl2, room temp, 5 h then NaHCO3, H2O, dioxane, Boc2O, room
temp, 21 h; (vii) 20, BoP–Cl, Et3N, CH2Cl2, 0 ◦C to room temp, 19 h, 60% over 3 steps; (viii) PtO2, EtOAc, H2, CF3CO2H, room temp, 6 h then PtO2,
THF–MeOH, H2, room temp, overnight, then CH2Cl2, CF3CO2H, room temp, 4 h (12%).
catalyst followed by hydrogenation of the resultant cyclic olefin
57 afforded macrocycle 9 in 58% yield after purification by
HPLC together with the heterolysis product 58. Cyclotetradecene
9 existed as a 65 : 35 mixture of trans : cis (Pro) conformers.
The increased proportion of the cis conformer may reflect the
increased flexibility of the Pro amide bond when it is embedded
in a larger 14 membered ring. The observation of only the trans
amide conformer may be a consequence of the smaller ring size
and/or stabilisation of the structure by the formation of a c-turn.
A peptide c-turn is a structural motif present in many biologically
active cyclic peptides that occurs to reverse the orientation of
the peptide chain. c-Turns contain three residues held together
in a seven membered cyclic conformation by an intramolecular
hydrogen bond.50
Scheme 3 Reagents, conditions and yields: (i) 4 M HCl, dioxane, 0 ◦C, 1 h
then room temp, 40 min; (ii) 27, EDCI, CH2Cl2, Et3N, 0 ◦C to room temp,
16 h, 60% over 2 steps.
In summary, the synthesis and olefin metathesis of several
allylated tripeptides incorporating proline has been accomplished,
affording macrocyclic structures related to the neuroprotective
tripeptide GPE 1. The proline-containing tripeptides cyclized to
afford cyclic products that adopted a single conformation about
the Gly–Pro amide bond (with the exception of 9). N-Allyl dienes
27 followed by hydrolysis of the methyl ester 54 (Scheme 9).
Subsequent union with c-allylglutamate 51 afforded N-allylglycyl
diene 56. Exposure of diene 56 to second generation Grubbs’
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=
Scheme 4 Reagents, conditions and yields: (i) Cl2(PCy3)2Ru CHPh, CH2Cl2, reflux, 48 h, then DMSO, room temp, 24 h, 46%; (ii) PtO2, THF, H2, room
temp, 16 h then 30% HBr–HOAc, room temp, 2 h then NaHCO3, H2O, dixoane, Boc2O, room temp, 4 days, 78% over 3 steps; (iii) BoP–Cl, Et3N, CH2Cl2,
0 ◦C to room temp, 24 h, 64%; (iv) CF3CO2H, CH2Cl2, room temp, 4 h, 80%.
=
Scheme 5 Reagents, conditions and yields: (i) Cl2(PCy3)2Ru CHPh, CH2Cl2, reflux, 48 h, then DMSO, room temp, 24 h, 42%; (ii) PtO2, THF, H2, room
temp, 16 h then 30% HBr–HOAc, room temp, 2 h then NaHCO3, H2O, dixoane, Boc2O, room temp, 4 days, 58% over 3 steps; (iii) BoP–Cl, Et3N, CH2Cl2,
0 ◦C to room temp, 17 h; (iv) CF3CO2H, CH2Cl2, room temp, 4 h, 55% over 2 steps.
28 and 29 afforded cyclononenes 30 and 33 with similar yields
under identical reaction conditions. The cis [C-2/C-5 (Pro)] isomer
of C-allyl diene 10a underwent smooth cyclisation to cyclooctene
18 in good yield in contrast to the trans [C-2/C-5 (Pro)] isomer
11 that required high catalyst loading to achieve a moderate
yield of cyclooctene 24. In the two cases involving cyclization
of dienes in which one allyl group was located at the more
hindered C-2 position on proline, C-allyl diene 37 readily formed
the metathesis product 38 whereas N-allyl dienes 43–47 failed to
undergo cyclisation to the analogous diazabicyclic product.
resultant purple solution heated at reflux for 24 h. The or-
ange/brown solution was cooled to room temperature, dimethyl
sulfoxide (0.160 cm3, 2.26 mmol) was added and the solution
stirred overnight. The solvent was removed in vacuo and the
residue purified by chromatography (SiO2, 2 : 1, 1 : 1, hexanes–
ethyl acetate) to give alkene 18 (0.044 g, 74%) as a colourless oil.
Alkene 18 existed exclusively as the trans C(O)–NPro conformer:
[a]D −93.2 (c 0.29 in CH2Cl2); dH (400 MHz; CDCl3; Me4Si) 1.25
(3H, t, J 7.1, OCH2CH3), 1.42 [9H, s, C(CH3)3], 1.89–1.98 (2H,
m, 10-HAHB and 9-HAHB), 2.01–2.09 (1H, m, 9-HAHB), 2.11–2.16
(1H, m, 10-HAHB), 2.24–2.33 (1H, m, 4-HAHB), 2.42 (1H, dq, J
15.2 and 3.4, 7-HAHB), 2.70–2.80 (2H, m, 4-HAHB and 7-HAHB),
4.15 (3H, q, J 7.1, OCH2CH3 and 8-H obscured), 4.47 (1H, dd,
J 8.4 and 2.8, 11-H), 4.84 (3H, br q, J 7.8, 3-H), 5.59 (1H, d, J
7.3, N–H), 5.67–5.73 (1H, m, 6-H) and 5.77–5.83 (1H, m, 5-H); dC
(100 MHz; CDCl3) 14.0 (CH3, OCH2CH3), 27.1 (CH2, 10-C), 28.2
[CH3, C(CH3)3], 32.77 (CH2, 7-C or 9-C), 32.84 (CH2, 9-C or 7-C),
35.2 (CH2, 4-C), 51.7 (CH, 3-C), 58.6 (CH, 8-C), 60.2 (CH, 11-C),
60.8 (CH2, OCH2CH3), 79.4 [quat., C(CH3)3], 125.7 (CH, 6-C),
129.1 (CH, 5-C), 155.1 (quat., NCO2), 170.9 (quat., 11-CO) and
Experimental
(8R,3S,11S)-1-Aza-3-(tert-butyloxycarbonylamino)-11-
ethoxycarbonyl-2-oxobicyclo[6.3.0]undec-5-ene 18
To a degassed solution of dienes 10 (0.064 g, 0.170 mmol)
in dry dichloromethane (43 cm3) was added bis(tricyclohexyl-
phosphine)benzylidineruthenium dichloride (Grubbs’ catalyst)
(0.014 g, 0.0170 mmol) under a nitrogen atmosphere and the
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Scheme 6 Reagents, conditions and yields: (i) DCC, HOBt, Et3N, CH2Cl2, 0 ◦C to room temp, overnight (ca. 23%); (ii) Grubbs’ catalyst II (10 mol%),
benzene, 45 ◦C, 48 h then DMSO overnight, 58%; (iii) 1 M aq. NaOH, dioxane, H2O, room temp, then 39, BoP–Cl, Et3N, CH2Cl2, 0 ◦C to room temp,
20 h, 69% over 2 steps; (iv) PtO2, H2, THF, room temp, 16 h, then CF3CO2H, CH2Cl2, room temp, 5 h, 81%.
◦
=
Scheme 7 Reagents, conditions and yields: (i) DCC, HOBt, Et3N, CH2Cl2, 0 C to room temp, overnight (ii) 10–30% Cl2(PCy3)2Ru CHPh, CH2Cl2,
reflux, 24–96 h, or 30% Grubbs catalyst II, CH2Cl2, reflux, 48 h.
171.7 (quat., 2-C); m/z (EI+) 352.2001 (M+. C18H28N2O5 requires
352.1998).
(0.033 cm3, 0.325 mmol) and bis(2-oxo-3-oxazolidinyl)phosphinic
chloride (BoP–Cl) (0.041 g, 0.163 mmol) were added and the
mixture stirred for 21 h. The reaction mixture was washed with
saturated aqueous sodium hydrogen carbonate, 2 M aqueous hy-
drochloric acid, dried (Na2SO4), filtered and the solvent removed
to yield an oil (0.078 g) which was purified by chromatography
(SiO2, 1 : 1, hexane–ethyl acetate) to afford amide 21 (0.050 g, 70%
over 2 steps) as a colourless oil. Amide 21 existed exclusively as
the trans C(O)–NPro conformer: [a]D −79.4 (c 0.47 in CH2Cl2);
dH (400 MHz; CDCl3; Me4Si) 1.39–1.50 [27H, m, 3 × C(CH3)3],
1.78–1.91 (3H, m, 10ꢀ-HAHB, 3-HAHB and 9ꢀ-HAHB), 2.0–2.33 (7H,
m, 10ꢀ-HAHB, 3-HAHB, 4-H2, 9ꢀ-HAHB, 7ꢀ-HAHB and 4ꢀ-HAHB),
2.80–2.95 (2H, br m, 7ꢀ-HAHB and 4ꢀ-HAHB), 4.15 (1H, br q, J
7.1, 8ꢀ-H), 4.42 (1H, ddd, J 7.8, 7.8 and 5.2, 2-H), 4.66 (1H,
d, J 7.5, 11ꢀ-H), 4.97 (1H, br q, J 7.4, 3ꢀ-H), 5.57 (1H, d, J
7.8, N–H), 5.61–5.67 (1H, m, 6ꢀ-H), 5.81 (1H, br t, J 7.8, 5ꢀ-H)
(2S,3ꢀS,8ꢀR,11ꢀS)-Di-tert-butyl 2-{[1ꢀ-aza-3ꢀ-(tert-
butyloxycarbonylamino)-2ꢀ-oxobicyclo[6.3.0]undec-5ꢀ-ene-11-
carbonyl]amino}-1,5-pentanedioate 21
1 M Aqueous sodium hydroxide (0.63 cm3, 0.63 mmol) was
added to a solution of ester 18 in dioxane (1.3 cm3) and the
opaque solution stirred for 23 h at room temperature. The reaction
mixture was diluted with water, extracted with dichloromethane
and the aqueous layer acidified with solid citric acid and extracted
with dichloromethane. The combined organic layers were dried
(Na2SO4), filtered and the solvent removed to yield acid 19
(0.040 g). Hydrochloride salt 20 (0.048 g, 0.1625 mmol) was added
to a solution of 19 and the solution cooled to 0 ◦C. Triethylamine
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Scheme 8 Reagents, conditions and yields: (i) DCC, HOBt, Et3N, CH2Cl2, 0 ◦C to room temp, overnight, then 1 M aq. NaOH, dioxane, room temp,
24 h, 64% over 2 steps; (ii) 50, EtOCOCl, Et3N, CH2Cl2, 0 ◦C, 40 min, then 51, 0 ◦C to room temp, overnight, 60%; (iii) Cl2(PCy3)2Ru CHPh, CH2Cl2,
=
reflux, 48 h then DMSO, 24 h; (iv) 10% Pd/C, H2, THF–H2O (4 : 1), room temp, 21 h or 10% PtO2, H2, THF, room temp, 16 h, then 10% Pd/C, H2,
MeOH–H2O (4 : 1), room temp, 5 h, 58% over 2 steps.
Scheme 9 Reagents, conditions and yields: (i) EDCI, Et3N, CH2Cl2, 0 ◦C to room temp, 19 h, 66%; (ii) 1 M aq. NaOH, dioxane, room temp, 20 h, 55%;
(ii) 55, EtOCOCl, Et3N, CH2Cl2, 0 ◦C then 51, overnight, 94%; (iii) Grubbs’ catalyst II, benzene, 40 ◦C, 65 h; (iv) 10% Pd/C, H2, MeOH–H2O (4 : 1),
room temp, 17 h, 58% over 2 steps.
and 7.47 (1H, d, J 8.0, N–H); dC (100 MHz; CDCl3) 26.1 (CH2,
10ꢀ-C), 27.9 [CH3, C(CH3)3], 27.95 [CH3, C(CH3)3], 28.2 [CH3,
C(CH3)3], 28.1, (CH2, 3-C), 31.3 (CH2, 4-C), 32.0 (CH2, 7ꢀ-C),
33.2 (CH2, 9ꢀ-C), 35.7 (CH2, 4ꢀ-C), 51.0 (CH, 3ꢀ-C), 51.9 (CH,
2-C), 58.6 (CH, 8ꢀ-C), 60.8 (CH, 11ꢀ-C), 79.6 [quat., C(CH3)3],
80.4 [quat., C(CH3)3], 81.9 [quat., C(CH3)3], 124.9 (CH, 6ꢀ-C),
130.4 (CH, 5ꢀ-C), 155.0 (quat., NCO2), 169.9 (quat., 1-C), 170.5
(quat., 11ꢀ-CO), 171.8 (quat., 2ꢀ-C or 5-C) and 171.9 (quat., 2ꢀ-
C or 5-C); m/z (FAB+) 566.3454 [MH+. C29H48N3O8 requires
566.3441].
2702 | Org. Biomol. Chem., 2006, 4, 2696–2709
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and 171.2 (quat., 11-CO); m/z (EI+) 380.2304 (M+. C20H32N2O5
requires 380.2311).
(2S,3ꢀS,8ꢀR,11ꢀS)-2-{[(3ꢀ-Amino-1ꢀ-aza-2ꢀ-oxobicyclo-
[6.3.0]undecyl)-11ꢀ-carbonyl]amino}-1,5-pentanedioic acid
trifluoroacetate salt 2
(2S,8ꢀS,11ꢀS)-Di-tert-butyl 2-{[(1-aza-2-oxo-3-tert-butoxy-
carbonylaminobicyclo[6.3.0]undec-5-ene)-11-carbonyl]amino}-
1,5-pentanedioate 26
PtO2 (0.00184 g, 0.0081 mmol) was added to a stirred solution
of amide 21 (0.046 g, 0.081 mmol) in tetrahydrofuran (4 cm3)
under a nitrogen atmosphere. The mixture was hydrogenated
(1 atm. of hydrogen) overnight, filtered through CeliteTM, and
the solvent removed in vacuo. The residue was dissolved in
dichloromethane (5 cm3), trifluoroacetic acid (3 cm3) added and
the solution stirred for 5 h at room temperature. Removal of the
volatiles in vacuo, purification by RP HPLC [10% acetonitrile :
90% water (containing 0.05% trifluoroacetic acid)] and trituration
from ether–toluene gave 2 (0.0228 g, 60%, 2 steps) as a colourless
oil. Macrocycle 2 existed exclusively as the trans C(O)–NPro
conformer: [a]D −27.9 (c 0.33 in MeOH); dH (400 MHz; D2O)
1.37–1.85 (8H, m, 5ꢀ-H2, 6ꢀ-H2, 4ꢀ-H2 and 9ꢀ-H2), 1.98–2.28 (5H,
m, 3-H2, 7ꢀ-H2 and 10ꢀ-HAHB), 2.36 (1H, dt, J 12.4 and 7.2, 10ꢀ-
HAHB), 2.57 (2H, t, J 7.6, 4-H2), 4.23 (1H, br t, J 8.2, 8ꢀ-H) and
4.41 (3H, m, 2-H, 11ꢀ-H, 3ꢀ-H); dC (100 MHz; D2O) 21.6 (CH2,
5ꢀ-C or 6ꢀ-C), 24.2 (CH2, 5ꢀ-C or 6ꢀ-C), 25.5 (CH2, 3-C), 27.1
(CH2, 10ꢀ-C), 29.7 (CH2, 4-C), 31.5 (CH2, 9ꢀ-C), 32.8 (CH2, 7ꢀ-
C), 35.0 (CH2, 4ꢀ-C), 51.2 (CH, 3ꢀ-C), 51.9 (CH, 2-C), 59.9 (CH,
8ꢀ-C), 61.4 (CH, 11ꢀ-C), 116.2 (quat., q, J 291, CF3), 162.7 (quat.,
q, J 35.2, CF3CO2H), 168.5 (quat., 2ꢀ-C), 173.7 (quat., 11ꢀ-CO),
174.7 (quat., 1-C) and 176.8 (quat., 5-C); m/z (FAB+) 356.1816
[MH(free base)+. C16H26N3O6 requires 356.1822].
Alkene 24 (0.021 g, 0.055 mmol) was stirred at room temperature
in dichloromethane–trifluoroacetic acid (3 : 1, v/v, 4 cm3) for
5 h. Removal of the volatiles in vacuo yielded an oil that
was dissolved in saturated aqueous sodium hydrogen carbonate
(1.5 cm3). Dioxane (1 cm3) was added followed by di-tert-butyl
dicarbonate (0.015 g, 0.066 mmol) and the milky suspension
was stirred at room temperature for 21 h. The reaction was
diluted with water until a solution was obtained and extracted
with dichloromethane. The aqueous layer was acidified with 2 M
aqueous hydrochloric acid, extracted with dichloromethane and
the combined organic extracts were dried (Na2SO4). Removal of
the solvent in vacuo gave acid 25 (0.012 g) that was dissolved
in dichloromethane (3 cm3). Glutamic acid di-tert-butyl ester
hydrochloride◦20 (0.013 g, 0.044 mmol) was added and the solution
cooled to 0 C. Triethylamine (0.013 cm3, 0.092 mmol) and
bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoP–Cl) (0.012 g,
0.048 mmol) were added and the mixture stirred for 19 h. The reac-
tion mixture was washed with saturated aqueous sodium hydrogen
carbonate, 2 M aqueous hydrochloric acid, dried (Na2SO4) and the
solvent removed to yield an oil (0.033 g), which was purified by
chromatography (silica gel, hexane–ethyl acetate, 2 : 1, 1 : 1, 1 : 2)
to afford amide 26 (0.0125 g, 60% over 3 steps) as a colourless oil:
[a]D +12.1 (c 0.247 in CH2Cl2); dH (400 MHz; CDCl3; Me4Si) 1.45–
1.48 [27H, s, 3 × C(CH3)3], 1.65–1.75 (1H, m, 9ꢀ-HAHB), 1.87–1.96
(1H, m, 3-HAHB), 2.02–2.41 (7H, m, 10ꢀ-H2, 4-H2, 3-HAHB, 9ꢀ-
HAHB and 7ꢀ-HAHB), 2.51 (1H, br d, J 17.4, 7ꢀ-HAHB), 2.57–2.64
(1H, m, 4ꢀ-HAHB), 2.70–2.80 (1H, m, 4ꢀ-HAHB), 4.39–4.48 (2H, m,
11ꢀ-H and 2-H), 4.52–4.62 (2H, m, 8ꢀ-H and 3ꢀ-H), 5.63–5.69 (1H,
m, 6ꢀ-H), 5.78–5.84 (1H, m, 5ꢀ-H), 5.91 (1H, br s, N–H) and 6.73
(1H, d, J 7.0, N–H); dC (100 MHz; CDCl3) 25.4 (CH2, 10ꢀ-C), 27.2
(CH2, 3-C), 27.9 [CH3, C(CH3)3], 28.0 [CH3, C(CH3)3], 28.3 [CH3,
C(CH3)3], 31.3 (CH2, 4-C), 32.5 (CH2, 5ꢀ-C), 33.3 (CH2, 9ꢀ-C),
33.7 (CH2, 7ꢀ-C), 52.3 (CH, 2-C), 56.3 (CH, 3ꢀ-C), 57.8 (CH, 8ꢀ-C),
61.9 (CH, 11ꢀ-C), 79.7 [quat., C(CH3)3], 80.6 [quat., C(CH3)3], 82.0
[quat., C(CH3)3], 127.9 (CH, 5ꢀ-C), 128.6 (CH, 6ꢀ-C), 155.4 (quat.,
NCO2), 170.4 (quat., 2ꢀ-C), 170.8 (quat., CO), 171.7 (quat., CO)
and 172.6 (quat., CO); m/z (FAB+) 566.3423 [MH(free base)+.
C29H48N3O8 requires 566.3441].
(8S,11S)-1-Aza-2-oxo-3-tert-butoxycarbonylamino-11-tert-
butoxycarbonylbicyclo[6.3.0]undec-5-ene 24
To a degassed solution of diene 11 (0.053 g, 0.129 mmol)
in dry dichloromethane (32 cm3) was added bis(tricyclohexyl-
phosphine)benzylidieneruthenium dichloride (Grubbs’ catalyst)
(0.011 g, 0.0129 mmol) under a flow of nitrogen and the resultant
purple solution heated at reflux under a nitrogen atmosphere for
24 h. Further bis(tricyclohexylphosphine)benzylidieneruthenium
dichloride (Grubbs’ catalyst) (0.011 g, 0.0129 mmol) was added
and refluxing continued for a further 48 h after which time another
portion of Grubbs catalyst (0.011 g, 0.0129 mmol) was added and
the solution refluxed for an additional 24 h. The orange/brown
solution was cooled to room temperature, dimethyl sulfoxide
(0.151 cm3, 1.935 mmol) was added and the solution stirred
overnight. The solvent was removed in vacuo and the residue
purified by chromatography (silica gel, hexanes–ethyl acetate, 4 :
3, 3 : 1, 2 : 1) to give alkene 24 (0.0237 g, 39%) as a colourless
solid. Alkene 24 was shown to be exclusively trans C(O)–NPro
conformer: mp 175–195 ◦C; [a]D −24.9 (c 0.237 in MeOH); dH
(400 MHz; CDCl3; Me4Si) 1.45 [9H, s, C(CH3)3], 1.47 [9H, s,
C(CH3)3], 1.68–1.74 (1H, m, 9-HAHB), 1.82 (1H, td, J 11.0 and 5.9,
10-HAHB), 2.17–2.26 (2H, m, 9-HAHB and 10-HAHB), 2.27–2.40
(1H, br m, 7-HAHB), 2.49 (1H, br d, J 17.6, 7-HAHB), 2.55–2.63
(1H, m, 4-HAHB), 2.66–2.80 (1H, m, 4-HAHB), 4.40 (1H, dd, J 8.6
and 4.2, 11-H), 4.45–4.62 (2H, m, 3-H and 8-H), 5.62–5.68 (2H, m,
H-6 and N–H) and 5.80–5.90 (1H, m, 5-H); dC (100 MHz; CDCl3)
25.5 (CH2, 10-C), 27.9 [CH3, C(CH3)3], 28.3 [CH3, C(CH3)3], 32.6
(CH2, 4-C), 33.4 (CH2, 7-C and 9-C), 55.8 (CH, 3-C), 57.2 (CH, 8-
C), 61.5 (CH, 11-C), 79.5 [quat., C(CH3)3], 81.2 [quat., C(CH3)3],
128.6 (CH, 5-C and 6-C), 155.1 (quat., NCO2), 169.6 (quat., 2-C)
(2S,3ꢀS,8ꢀS,11ꢀS)-2-{[(3ꢀ-Amino-1ꢀ-aza-2ꢀ-oxobicyclo-
[6.3.0]undecyl)-11ꢀ-carbonyl]amino}-1,5-pentanedioic acid
trifluoroacetate salt 3
PtO2 (0.001 g, 0.0042 mmol) was added to a stirred solution of
amide 26 (0.012 g, 0.021 mmol) in ethyl acetate–trifluoroacetic
acid (5 : 3, v/v, 8 cm3) under a nitrogen atmosphere. The mixture
was hydrogenated (1 atm. of hydrogen) overnight, filtered through
CeliteTM, and the solvent removed in vacuo. NMR and HPLC
analysis showed the reaction to be incomplete (both double
bond and protecting groups remaining). Thus PtO2 (0.0003 g,
0.0126 mmol) was added to a stirred solution of the residue in
tetrahydrofuran–methanol (1 : 1, v/v, 2 cm3) under a nitrogen
atmosphere. The mixture was hydrogenated (1 atm. of hydrogen)
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overnight, filtered through CeliteTM, and the solvent removed in
vacuo. The residue was dissolved in dichloromethane (3 cm3),
trifluoroacetic acid (1 cm3) added and the solution stirred for 4 h at
room temperature. Removal of the volatiles in vacuo, purification
by RP HPLC [90% water (containing 0.05% trifluoroacetic acid) :
10% acetonitrile, 13 ml min−1] and drying on a freeze drier gave 3
(1.4 mg 12%) as a thin film. Due to the small amount of sample
obtained an optical rotation was not recorded: dH (400 MHz;
D2O) 1.74–2.42 (14H, m, 5ꢀ-H2, 6ꢀ-H2, 4ꢀ-H2, 9ꢀ-H2, 3-H2, 7ꢀ-
H2 and 10ꢀ-H2), 2.52 (2H, t, J 8.8, 4-H2), 4.3–4.4 (1H, m, 2H),
4.43–4.5 (1H, m) and 4.62–4.69 (1H, m); dC (100 MHz; D2O)
22.3 (CH2), 25.4 (CH2), 28.4 (CH2), 29.0 (CH2), 30.2 (CH2), 32.4
(CH2), 33.7 (CH2), 53.9 (CH), 55.1 (CH), 60.2 (CH), 62.8 (CH),
168.1 (quat., CO) and 173.7 (quat., CO); 2 quaternary carbons
and trifluoroacetate signals not detected; m/z (FAB+) 356.1816
[MH(free base)+. C16H26N3O6 requires 356.1822].
under a flow of nitrogen. The mixture was hydrogenated (1 atm of
H2) for 16 h, filtered through CeliteTM, and the solvent removed in
vacuo. The residue was dissolved in a solution of hydrobromic acid
in acetic acid (30% w/w, 3 cm3) and stirred at room temperature
for 2 h. Removal of the volatiles in vacuo at 30 ◦C followed by
addition and evaporation of methanol–water (3 : 1) several times
yielded the hydrobromide salt which was dissolved in a solution
of saturated sodium hydrogen carbonate (3 cm3). Dioxane (2 cm3)
was then added and di-tert-butyl dicarbonate added to the opaque
solution (0.076 g, 0.350 mmol). The resultant suspension was
stirred for 24 h, di-tert-butyl dicarbonate (0.076 g, 0.3504 mmol),
added and stirring continued for a further 72 h. Water (10 cm3)
was added, the solution washed with dichloromethane, and the
aqueous layer acidified with 2 M aqueous hydrochloric acid and
extracted with dichloromethane. The combined organic layers
were dried (Na2SO4), filtered and the solvent removed to yield an
oil (0.08 g) that was purified by chromatography (SiO2, 2 : 1 : 0, 2 :
1 : 0.3, hexanes–ethyl acetate–acetic acid) to give acid 31 (0.074 g,
ca. 78%) as a colourless oil. Acid 31 existed exclusively as the trans
C(O)–NPro conformer. In addition, restricted rotation about the
N–CO carbamate bond was also observed resulting in a 1 : 1
mixture of conformers: dH (400 MHz; CDCl3; Me4Si) 1.25–1.48
[11H, m, C(CH3)3, 8-HAHB and 6-HAHB or 7-HAHB], 1.54–1.82
(5H, m, 8-HAHB, 10-HAHB and 6-HAHB or 7-HAHB), 1.90–2.03
(1H, m, 10-HAHB), 2.10–2.33 (2H, m, 11-H2), 2.78 (1H, td, J 12.7
and 2.6, 5-HAHB), 3.78–3.95 (2H, m, 5-HAHB and 3-H), 4.20–4.30
(1H, m, 9-H), 4.52 (0.5H, d, J 17.7, 3-HAHB), 4.62 (1H, t, J 8.6,
12-H) and 4.76 (0.5H, d, J 17.2, 3-HAHB); dC (100 MHz; CDCl3)
22.2 (CH2, 6-C or 7-C), 27.6 (CH2, 6-C or 7-C), 24.3 (CH2, 11-C),
24.8 (CH2, 11-C), 26.8 (CH2, 6-C or 7-C), 27.9 (CH2, 6-C or 7-
C), 28.3 [CH3, C(CH3)3], 32.6 (CH2, 10-C), 33.1 (CH2, 10-C), 34.8
(CH2, 8-C), 35.3 (CH2, 8-C), 51.1 (CH2, 5-C), 51.7 (CH2, 5-C), 54.7
(CH2, 3-C), 55.9 (CH2, 3-C), 56.6 (CH, 9-C), 56.7 (CH, 9-C), 60.8
(CH, 12-C), 61.1 (CH, 12-C), 80.7 [quat., C(CH3)3], 154.8 (quat.,
NCO2), 155.6 (quat., NCO2), 170.0 (quat., 2-C), 170.5 (quat., 2-
C), 174.1 (quat., 12-CO) and 174.7 (quat., 12-CO); m/z (FAB+)
327.1925 (MH+. C16H27N2O5 requires 327.1920).
(6Z,9R,12S)-1,4-Diaza-4-benzyloxycarbonyl-12-tert-
butoxycarbonyl-2-oxobicyclo[7.3.0]dodec-6-ene 30
To a degassed solution of diene 28 (0.11 g, 0.242 mmol)
in dry dichloromethane (60 cm3) was added bis(tricyclohexyl-
phosphine)benzylidineruthenium dichloride (Grubbs’ catalyst)
(0.020 g, 0.024 mmol) under an atmosphere of nitrogen and
the resultant purple solution heated at reflux for 24 h. Fur-
ther bis(tricyclohexylphosphine)benzylidineruthenium dichloride
(Grubbs’ catalyst) (0.020 g, 0.024 mmol) was then added, and
refluxing continued for a further 24 h. The solution was cooled to
room temperature, dimethyl sulfoxide (0.16 cm3, 2.26 mmol) was
added and the orange/brown solution stirred for 22 h. The solvent
was removed in vacuo and the residue purified by chromatography
(SiO2, hexanes–ethyl acetate, 3 : 1, 2 : 1, 1 : 1) to give a colourless
oil which was further purified by chromatography (C18 RP silica,
100 : 0, 9 : 1, 9 : 3, 1 : 1, 1 : 9, water–acetonitrile,) to give alkene 30
(0.046 g, 46%) as a colourless oil. Alkene 30 existed exclusively as
the trans C(O)–NPro conformer. In addition, restricted rotation
about the N–CO carbamate bond was also observed resulting in
a 1 : 1 mixture of conformers: [a]D −243.9 (c 0.27 in CH2Cl2);
dH (400 MHz; CDCl3; Me4Si) 1.49 [9H, s, C(CH3)3], 1.84–2.34
(5H, m, 8-HAHB, 10-H2 and 11-H2), 2.98–3.08 (1H, m, 8-HAHB),
3.69–3.88 (2H, m, 5-HAHB and 3-HAHB), 4.0 (1H, p, J 7.6, 9-H),
4.33–4.61 (3H, m, 5-HAHB, 3-HAHB and 12-H), 5.10–5.30 (2H,
m, OCH2Ph), 5.51–5.65 (1H, m, 6-H), 5.98–6.09 (1H, m, 7-H)
and 7.29–7.38 (5H, m, Ph); dC (100 MHz; CDCl3) 27.2 (CH2,
11-C), 28.0 [CH3, C(CH3)3], 33.9 (CH2, 8-C), 35.1 (CH2, 10-C),
42.2 (CH2, 5-C), 42.3 (CH2, 5-C), 45.7 (CH2, 3-C), 45.9 (CH2, 3-
C), 59.5 (CH, 9-C), 59.6 (CH, 9-C), 61.2 (CH, 12-C), 61.3 (CH,
12-C), 67.4 (CH2, OCH2Ph), 67.5 (CH2, OCH2Ph), 81.4 [quat.,
C(CH3)3], 81.5 [quat., C(CH3)3], 125.8 (CH, 6-C), 126.0 (CH, 6-C),
127.7 (CH, Ph), 127.8 (CH, Ph), 127.81 (CH, Ph), 127.9 (CH, Ph),
128.3 (CH, Ph), 132.0 (CH, 7-C), 132.4 (CH, 7-C), 136.5 (quat.,
Ph), 155.8 (quat., NCO2), 156.2 (quat., NCO2), 167.2 (quat., 2-C),
167.4 (quat., 2-C), 171.2 (quat., 12-CO) and 171.4 (quat., 12-CO);
m/z (EI+) 414.2154 (M+. C23H30N2O5 requires 414.2154).
(2S,9ꢀS,12ꢀS)-Di-tert-butyl 2-{[(1ꢀ,4ꢀ-diaza-
4ꢀ-tert-butyloxycarbonyl-2ꢀ-oxobicyclo[7.3.0]dodecyl)-12ꢀ-
carbonyl]amino}-1,5-pentanoate 32
Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoP–Cl) (0.08 g,
0.31 mmol) was added to a solution of acid 31 (0.07 g,
0.20 mmol), L-glutamic acid di-tert-butyl ester hydrochloride 20
(0.085 g, 0.289 mmol) and triethylamine (0.084 cm3, 0.60 mmol)
◦
in dichloromethane (7 cm3) at 0 C. The mixture was stirred for
24 h, washed with saturated aqueous sodium hydrogen carbonate,
2 M aqueous hydrochloric acid, dried (Na2SO4), filtered and the
solvent removed under reduced pressure. The resultant oil (0.141
g) was purified by chromatography (SiO2, 1 : 1, 1 : 2, hexane–ethyl
acetate) to afford amide 32 (0.077 g, ca. 64%) as a colourless oil.
Amide 32 existed exclusively as the trans C(O)–NPro conformer.
In addition, restricted rotation about the N–CO carbamate bond
was also observed resulting in a 46 : 54 mixture of conformers:
dH (300 MHz; CDCl3; Me4Si) 1.14–1.30 [2H, m, 8ꢀ-HAHB and Hꢀ-
6(1H) or Hꢀ-7(1H)], 1.34–1.41 [27H, m, 3 × C(CH3)3], 1.54–2.28
([11H, m, 8ꢀ-HAHB, 10ꢀ-H2, 3-H2, 4-H2, 6ꢀ-H(3H) or 7ꢀ-H(3H)]
and 11ꢀ-HAHB), 2.31–2.49 (1H, m, 11ꢀ-HAHB), 2.60–2.70 (1H, m,
(9R,12S)-1,4-Diaza-4-tert-butyloxycarbonyl-2-
oxobicyclo[7.3.0]dodec-2-carboxylic acid 31
To a stirred solution of alkene 30 (0.12 g, 0.29 mmol) in tetrahy-
drofuran (4 cm3) was added platinum oxide (0.0066 g, 0.029 mmol)
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5ꢀ-HAHB), 3.65–3.87 (2H, m, 5ꢀ-HAHB and 3ꢀ-HAHB), 4.18 (1H,
br s, 9ꢀ-H), 4.39 (1H, q, J 7.6, 2-H), 4.48 (0.5H, d, J 17.5, 3ꢀ-
HAHB), 4.65 (1H, t, J 7.8, 12ꢀ-H), 4.74 (0.5H, d, J 17.0, 3ꢀ-HAHB),
7.45 (0.46H, d, J 7.4, N–H), and 7.64 (0.54H, d, J 7.7, N–H); dC
(75 MHz; CDCl3) 22.7 (CH2, 11ꢀ-C), 23.3 (CH2, 6ꢀ-C or 7ꢀ-C), 23.4
(CH2, 6ꢀ-C or 7ꢀ-C), 27.0 (CH2, 3-C, 6ꢀ-C or 7ꢀ-C), 27.7 (CH2, 3-C,
6ꢀ-C or 7ꢀ-C), 27.8 (CH2, 3-C, 6ꢀ-C or 7ꢀ-C), 28.2 (CH2, 3-C, 6ꢀ-C
or 7ꢀ-C), 27.9 [CH3, C(CH3)3], 28.0 [CH3, C(CH3)3], 28.4 [CH3,
C(CH3)3], 31.4 (CH2, 4-C), 32.5 (CH2, 10ꢀ-C), 32.8 (CH2, 10ꢀ-C),
34.4 (CH2, 8ꢀ-C, 35.1 (CH2, 8ꢀ-C), 50.7 (CH2, 5ꢀ-C), 51.5 (CH2,
5ꢀ-C), 52.25 (CH, 2-C), 52.33 (CH, 2-C), 54.6 (CH2, 3ꢀ-C), 55.0
(CH2, 3ꢀ-C), 56.6 (CH, 9ꢀ-C), 56.8 (CH, 9ꢀ-C), 60.2 (CH, 12ꢀ-C),
60.8 (CH, 12ꢀ-C), 80.4 [quat., C(CH3)3], 80.6 [quat., C(CH3)3], 81.9
[quat., C(CH3)3], 154.7 (quat., NCO2), 155.5 (quat., NCO2), 169.4
(quat., 2ꢀ-C), 169.8 (quat., 2ꢀ-C), 170.6 (quat., 12ꢀ-CO, 1-C, 5-C),
170.8 (quat., 12ꢀ-CO, 1-C, 5-C) and 171.8 (quat., 12ꢀ-CO, 1-C, 5-C);
m/z (FAB+) 568.3592 (MH+. C29H50N3O8 requires 568.3598).
as a colourless oil. Alkene 33 existed exclusively as the trans C(O)–
NPro conformer. In addition, restricted rotation about the N–CO
carbamate bond was also observed resulting in a 1 : 1 mixture of
conformers: [a]D −118.2 (c 0.34 in CH2Cl2); dH (400 MHz; CDCl3;
Me4Si) 1.46 [9H, s, C(CH3)3], 1.74 (1H, dd, J 11.8 and 5.7, 10-
HAHB), 1.93 (1H, dd, J 12.5 and 6.5, 11-HAHB), 2.11–2.45 (4H,
m, 8-H2, 10-HAHB and 11-HAHB), 3.53 (1H, dd, J 15.7 and 8.0,
5-HAHB), 4.13–4.37 (4H, m, 3-H2, 9-H and 12-H), 4.50 (1H, d, J
15.4, 5-HAHB), 5.12–5.29 (2H, m, OCH2Ph), 5.54–5.82 (2H, m,
6-H and 7-H) and 7.31–7.43 (5H, m, Ph); dC (100 MHz; CDCl3)
26.7 (CH2, 11-C), 27.8 [CH3, C(CH3)3], 34.0 (CH2), 34.2 (CH2),
34.3 (CH2), 34.4 (CH2), 43.9 (CH2, 5-C), 44.5 (CH2, 5-C), 48.5
(CH2, 3-C), 49.5 (CH2, 3-C), 57.2 (CH, 9-C), 57.6 (CH, 9-C), 60.1
(CH, 12-C), 67.6 (CH2, OCH2Ph), 81.2 [quat., C(CH3)3], 126.8
(CH, 6-C), 126.9 (CH, 6-C), 128.0 (CH, Ph), 128.4 (CH, Ph),
128.7 (CH, Ph), 129.9 (CH, 7-C), 136.3 (quat., Ph), 136.4 (quat.,
Ph), 155.7 (quat., NCO2), 167.8 (quat., 2-C), 167.9 (quat., 2-C)
and 170.7 (quat., 12-CO); m/z (EI+) 414.2157 (M+. C23H30N2O5
requires 414.2155).
(2S,9ꢀR,12ꢀS)-2-{[(1ꢀ,4ꢀ-Diaza-2ꢀ-oxobicyclo[7.3.0]dodecyl)-12ꢀ-
carbonyl]amino}-1,5-pentanedioic acid trifluoroacetate 4
(9S,12S)-1,4-Diaza-4-tert-butyloxycarbonyl-2-oxo-
Amide 32 (0.072 g, 0.126 mmol) was dissolved in a mixture of
dichloromethane and trifluoroacetic acid (5 : 2, v/v) and stirred at
room temperature for 4 h. Evaporation of the volatiles, and sub-
sequent purification by RP-HPLC [water (0.05% trifluoroacetic
acid)–acetonitrile, 90 : 10, 13 ml min−1], gave 6 (0.059 g, 80%) as a
colourless wax after drying on a freeze drier. Macrocycle 4 existed
exclusively as the trans C(O)–NPro conformer: [a]D −36.9 (c 0.195
in MeOH); dH (400 MHz; CDCl3; Me4Si) 1.71–2.29 (11H, m, 6ꢀ-
H2, 7ꢀ-H2, 8ꢀ-H2, 10ꢀ-H2, 3-H2 and 11ꢀ-HAHB), 2.33–2.39 (1H, m,
11ꢀ-HAHB), 3.27 (1H, dt, J 10.2 and 4.1, 5ꢀ-HAHB), 3.39 (1H, td,
J 12.0 and 2.2, 5ꢀ-HAHB), 3.76 (1H, d, J 13.6, 3ꢀ-HAHB), 4.0–4.25
(2H, m, 3ꢀ-HAHB and 9ꢀ-H), 4.45 (1H, dd, J 9.3 and 5.1, 2-H) and
4.51 (1H, dd, J 9.2 and 8.3, 12ꢀ-H); dC (100 MHz; CDCl3) 24.06
(CH2, 6ꢀ-C or 7ꢀ-C), 24.1 (CH2, 6ꢀ-C or 7ꢀ-C), 25.7 (CH2, 3-C), 27.7
(CH2, 11ꢀ-C), 29.6 (CH2, 4-C), 32.5 (CH2, 10ꢀ-C), 33.4 (CH2, 8ꢀ-
C), 43.3 (CH2, 5ꢀ-C), 46.8 (CH2, 3ꢀ-C), 51.8 (CH, 2-C), 61.9 (CH,
12ꢀ-C), 62.8 (CH, 9ꢀ-C), 116.2 (quat., q, J 290, CF3), 162.6 (quat.,
q, J 35.2, CF3CO2H), 164.7 (quat., 2ꢀ-C), 173.4 (quat., 12ꢀ-CO),
174.6 (quat., 1-C), and 176.8 (quat., 5-C); m/z (FAB+) 356.1823
[MH(free base)+. C16H26N3O6 requires 356.1822].
bicyclo[7.3.0]dodecyl-12-carboxylic acid 34
To a stirred solution of alkene 33 (0.11 g, 0.263 mmol) in tetrahy-
drofuran (4 cm3) was added platinum oxide (0.006 g, 0.026 mmol)
under a flow of nitrogen. The mixture was hydrogenated (1 atm.
of hydrogen) for 16 h, filtered over CeliteTM, and the solvent
removed in vacuo. The residue was dissolved in a solution of
30% hydrobromic acid in acetic acid (3 cm3) and stirred at room
temperature for 2 h. Removal of the volatiles in vacuo at 40 ◦C
followed by repeated evaporation from methanol–water (3 : 1)
yielded the hydrobromide salt which was dissolved in a solution
of saturated sodium hydrogen carbonate (3 cm3). Dioxane (2 cm3)
and di-tert-butyl dicarbonate (0.07 g, 0.32 mmol) were added and
the resultant suspension was stirred for 24 h, then further di-tert-
butyl dicarbonate (0.07 g, 0.32 mmol) was added and stirring
continued for a further 48 h. Water (10 cm3) was added, the
solution washed with dichloromethane and the aqueous layer
was acidified with 2 M aqueous hydrochloric acid and extracted
with dichloromethane. The combined organic layers were dried
(Na2SO4), filtered and the solvent removed to yield an oil (0.072 g)
that was purified by chromatography (SiO2, 1 : 1 : 0.3, hexanes–
ethyl acetate–acetic acid) to give acid 34 (0.05 g, 58%) as a
colourless oil. Acid 34 existed exclusively as the trans C(O)–
NPro conformer. In addition, restricted rotation about the N–
CO carbamate bond was also observed resulting in a 72 : 28
mixture of conformers: [a]D +3.9 (c 0.23 in CH2Cl2); dH (400 MHz;
CDCl3; Me4Si) 1.30–1.48 [11H, m, C(CH3)3, 8-HAHB and 11-
HAHB], 1.63–1.79 (5H, m, 8-HAHB, 6-H2 and 7-H2), 2.01–2.07
(1H, m, 11-HAHB), 2.12–2.32 (2H, m, 10-H2), 2.62–2.85 (1H, m,
5-HAHB), 3.73–3.83 (1.28 H, 3-HAHB and 5-HAHB*), 4.0 (0.72H,
br d, J 14.2, 5-HAHB), 4.20–4.32 (1H, m, 9-H), 4.41* (0.28H,
d, J 8.0, 12-H), 4.50 (0.72H, d, J 8.8, 12-H), 4.57 (0.72H, d, J
17.8, 3-HAHB), 4.69* (0.28H, d, J 16.5, 3-HAHB) and 8.40 (1H,
br s, OH); dC (100 MHz; CDCl3) 22.1 (CH2, 7-C), 22.6* (CH2,
7-C), 25.1 (CH2, 11-C), 25.7* (CH2, 11-C), 26.6 (CH2, 6-C), 28.1
[CH3, C(CH3)3], 28.3* [CH3, C(CH3)3], 32.5 (CH2, 10-C), 32.7*
(CH2, 10-C), 35.9* (CH2, 8-C), 36.3 (CH2, 8-C), 50.3* (CH2,
5-C), 51.8 (CH2, 5-C), 54.9* (CH2, 3-C), 55.4 (CH, 9-C), 56.0*
(6Z,9S,12S) N-Benzyloxycarbonyl-1,4-diaza-12-tert-
butoxycarbonyl-2-oxobicyclo[7.3.0]dodec-6-ene 33
To a degassed solution of diene 29 (0.10 g, 0.23 mmol) in
dry dichloromethane (56 cm3) was added bis(tricyclohexyl-
phosphine)benzylidineruthenium dichloride (Grubbsꢀ catalyst)
(0.018 g, 0.023 mmol) under an atmosphere of nitrogen and
the resultant purple solution heated at reflux for 24 h. Fur-
ther bis(tricyclohexylphosphine)benzylidineruthenium dichloride
(0.018 g, 0.023 mmol) was added and heating continued for 24 h,
then the reaction mixture was cooled to room temperature and
dimethyl sulfoxide (0.160 cm3, 2.3 mmol) was added and the
orange/brown solution stirred for 24 h. The solvent was removed
in vacuo and the residue purified by chromatography (SiO2, 3 : 1,
2 : 1, 1 : 1, hexanes–ethyl acetate) to give a colourless oil which was
further purified by chromatography (C18 RP silica, 100 : 0, 9 : 1, 9 :
3, 1 : 1, 1 : 9, water–acetonitrile,) to give alkene 33 (0.039 g, 42%)
This journal is
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Org. Biomol. Chem., 2006, 4, 2696–2709 | 2705
©

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(CH, 9-C), 56.3 (CH2, 3-C), 60.5* (CH, 12-C), 60.8 (CH, 12-C),
80.6* [quat., C(CH3)3], 81.0 [quat., C(CH3)3], 155.2 (quat., NCO2),
155.5* (quat., NCO2), 169.5* (quat., 2-C), 169.8 (quat., 2-C), 174.9
(quat., 12-CO) and 175.1* (quat., 12-CO); m/z (EI+) 326.1837
(M+. C16H26N2O5 requires 326.1842).
was added and the purple solution heated at 80 ◦C for 35 min.
The dark brown solution was cooled to room temperature and a
solution of diene 37 (0.041 g, 0.112 mmol) in dry benzene (40 cm3)
added and the mixture heated at 45 ◦C for 48 h. The brown
solution was cooled to room temperature, dimethyl sulfoxide
(0.043 g, 0.56 mmol) was added and the mixture stirred overnight.
The solvent was removed in vacuo and the residue purified by
chromatography (SiO2, 2 : 1, 1 : 1, hexane–ethyl acetate) to give
alkene 38 (0.022 g, 58%) as a pale yellow oil. Alkene 38 existed
exclusively as the trans C(O)–NPro conformer: [a]D −86.3 (c 0.183
in CH2Cl2) [lit.,45 −87.4 (c 0.35 in CHCl3]; dH (400 MHz; CDCl3;
Me4Si) 1.44 [9H, s, C(CH3)3], 1.66–1.86 (2H, m, 10-H2), 1.96–2.11
(2H, m, 9-HAHB and 4-HAHB), 2.46 (1H, dd, J 12.3 and 6.1, 9-
HAHB), 2.66 (1H, dd, J 14.8 and 8.2, 7-HAHB), 2.80–2.90 (1H, m,
4-HAHB), 3.04 (1H, dd, J 15.1 and 8.6, 7-HAHB), 3.40 (1H, ddd, J
11.6, 11.1 and 7.4, 11-HAHB), 3.76–3.83 (1H, m, 11-HAHB), 3.78
(3H, s, OCH3), 4.97–5.04 (1H, m, 3-H), 5.52–5.63 (2H, m, 6-H
and N–H) and 5.74–5.79 (1H, m, 5-H); dC (100 MHz; CDCl3) 20.6
(CH2, 10-C), 28.2 [CH3, C(CH3)3], 35.0 (CH2, 7-C), 35.4 (CH2, 4-
C), 38.4 (CH2, 9-C), 48.4 (CH2, 11-C), 50.8 (CH, 3-C), 53.0 (CH3,
OCH3), 69.7 (quat., 8-C), 79.4 [quat., C(CH3)3], 122.8 (CH, 5-C),
132.2 (CH, 6-C), 154.8 (quat., NCO2), 171.5 (quat., 2-C) and 173.7
(quat., 8-CO).
(2S,9ꢀS,12ꢀS)-2-{[(1ꢀ,4ꢀ-Diaza-2ꢀ-oxobicyclo[7.3.0]dodecyl)-12ꢀ-
carbonyl]amino}-1,5-pentanedioic acid trifluoroacetate salt 5
Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoP–Cl) (0.057 g,
0.225 mmol) was added to a solution of acid 2 (0.05 g,
0.15 mmol), L-glutamic acid di-tert-butyl ester hydrochloride 20
(0.062 g, 0.21 mmol) and triethylamine (0.061 cm3, 0.44 mmol)
◦
in dichloromethane (5 cm3) at 0 C. The mixture was stirred for
17 h, washed with saturated aqueous sodium hydrogen carbonate,
2 M aqueous hydrochloric acid, dried (Na2SO4), filtered and the
solvent removed to yield an oil (0.108 g) which was purified by
chromatography (SiO2, 1 : 1, 1 : 2, 1 : 3, hexane–ethyl acetate) to
afford an inseparable mixture (0.074 g) of the desired amide 35 con-
taminated with a glutamate–bis(2-oxo-3-oxazolidinyl)phosphinic
chloride adduct. The mixture was dissolved in dichloromethane
(5 cm3), trifluoroacetic acid (2 cm3) was added and the solution
stirred for 4 h. Further trifluoroacetic acid (1 cm3) was then
added and stirring continued for 2 h. The volatiles were removed
in vacuo, the residue suspended in water and filtered through
a plug of cotton wool. The filtrate was subsequently purified
by RP-HPLC [water (0.05% trifluoroacetic acid) : acetonitrile,
90 : 10, 13 ml min−1] to afford trifluoroacetate 5 (0.039 g, 55%
from 34) as a colourless wax after trituration from diethyl ether–
toluene. Macrocycle 5 existed exclusively as the trans C(O)–NPro
conformer: [a]D −22.3 (c 0.35 in MeOH); dH (400 MHz; CDCl3;
Me4Si) 1.68–2.04 (9H, m, 7ꢀ-H2, 6ꢀ-H2, 10ꢀ-H2, 3ꢀ-HAHB, 8ꢀ-HAHB
and 11ꢀ-HAHB), 2.19–2.26 (2H, 8ꢀ-HAHB and 3ꢀ-HAHB), 2.42–2.48
(1H, m, 11ꢀ-HAHB), 2.50–2.58 (2H, m, 4-H2), 3.04 (1H, td, J 11.2
and 2.5, 5ꢀ-HAHB), 3.33 (1H, dt, J 14.1 and 4.9, 5ꢀ-HAHB), 3.82
(1H, d, J 14.1, 3ꢀ-HAHB), 4.32 (1H, d, J 14.0, 3ꢀ-HAHB), 4.38–
4.45 (2H, m, 9ꢀ-H and 2-H) and 4.59 (1H, dd J 9.6 and 1.8, 12ꢀ-
H); dC (100 MHz; CDCl3) 25.6 (CH2, 6ꢀ-C or 7ꢀ-C), 26.0 (CH2,
6ꢀ-C or 7ꢀ-C), 28.1 (CH2, 3-C), 29.9 (CH2, 11ꢀ-C), 32.3 (CH2, 4-
C), 34.0 (CH2, 10ꢀ-C or 8ꢀ-C), 35.2 (CH2, 10ꢀ-C or 8ꢀ-C), 46.9
(CH2, 5ꢀ-C), 48.7 (CH2, 3ꢀ-C), 54.3 (CH, 2-C), 62.9 (CH, 12ꢀ-
C), 64.6 (CH, 9ꢀ-C), 118.6 (quat., q, J 291, CF3), 165.1 (quat.,
q, J 36.2, CF3CO2H), 167.9 (quat., 2ꢀ-C), 175.9 (quat., 12ꢀ-CO),
177.1 (quat., 1-C), and 179.4 (quat., 5-C); m/z (FAB+) 356.1824
[MH(free base)+. C16H26N3O6 requires 356.1822].
(2S,3ꢀS,8ꢀS)-Di-tert-butyl 2-{[(1ꢀ-aza-3ꢀ-(tert-butyloxy-
carbonylamino)-2ꢀ-oxobicyclo[6.3.0]undec-5ꢀ-ene)-8ꢀ-
carbonyl]amino}-1,5-pentanedioate 39
To a solution of alkene 38 (0.022 g, 0.065) in dioxane (0.7 cm3)
was added 1 M aqueous sodium hydroxide (0.32 cm3, 0.32 mmol)
and the opaque solution stirred at room temperature for 17 h.
Water was added and the mixture washed with dichloromethane.
The aqueous layer was acidified with citric acid and the prod-
uct extracted with dichloromethane. The organic layers were
pooled, dried (MgSO4) and the solvent removed to afford an
oil (0.020 g). To a solution of this oil in dichloromethane
(4 cm3) was added L-glutamic acid di-tert-butyl ester hydrochloride
20 (0.025 g, 0.085 mmol) was added and the solution cooled
to 0 ◦C. Triethylamine (0.017 g, 0.17 mmol) and bis(2-oxo-
3-oxazolidinyl)phosphinic chloride (0.021 g, 0.085 mmol) were
added and the solution stirred for 20 h. The reaction mixture was
washed with 2 M aqueous hydrochloric acid, saturated aqueous
sodium hydrogen carbonate, dried (Na2SO4), filtered and the
solvent removed to yield an oil (0.041 g) which was purified by
chromatography (SiO2, 1 : 1, 2 : 3, hexane–ethyl acetate) to give
amide 39 (0.025 g, 69% in 2 steps) as a colourless oil. Amide 39
existed exclusively as the cis C(O)–NPro conformer: [a]D −49.6 (c
0.25 in CH2Cl2); dH (400 MHz; CDCl3; Me4Si) 1.40–1.44 [27H, m,
3 × C(CH3)3], 1.55–1.66 (1H, m), 1.78 (1H, p, J 6.4), 1.89 (1H,
td, J 13.2 and 6.1), 2.05–2.39 (5H, m), 2.65–2.75 (2H, m, 4ꢀ-H2),
2.80 (1H, dd, J 15.0 and 7.9, 7ꢀ-HAHB), 2.92 (1H, dd, J 14.9 and
8.9, 7ꢀ-HAHB), 3.55 (1H, ddd, J 11.8, 11.8 and 7.0, 11ꢀ-HAHB),
3.65–3.70 (1H, m, 11ꢀ-HAHB), 4.40–4.46 (1H, m, 3ꢀ-H), 4.88–4.97
(2H, m, 6ꢀ-H and 2-H), 5.67–5.79 (2H, m, 5ꢀ-H and N–H) and 8.36
(1H, d, J 8.4, N–H); dC (100 MHz; CDCl3) 20.4 (CH2, 10ꢀ-C), 25.4
(CH2, 3-C), 27.9 [CH3, C(CH3)3], 28.0 [CH3, C(CH3)3], 28.2 [CH3,
C(CH3)3], 31.5 (CH2, 4-C), 32.2 (CH2, 4ꢀ-C), 34.6 (CH2, 7ꢀ-C), 37.4
(CH2, 9ꢀ-C), 49.3 (CH2, 11ꢀ-C), 52.1 (CH, 3ꢀ-C), 52.8 (2-C), 71.8
(quat., 8ꢀ-C), 80.4 [quat., C(CH3)3], 81.1 [quat., C(CH3)3], 125.0
(3S,8S)-1-Aza-3-(tert-butyloxycarbonylamino)-8-methoxy-
carbonyl-2-oxobicyclo[6.3.0]undec-5-ene45 38
A solution of freshly sublimed potassium tert-butoxide (0.0018 g,
0.0157 mmol) in dry tetrahydrofuran (1 cm3) was added
to a stirred suspension of 1,3-bis(2,4,6-trimethylphenyl)-4,5-
dihydroimidazolium tetrafluoroborate (0.07 g, 0.0177 mmol) in
dry tetrahydrofuran (2 cm3) under an atmosphere of nitro-
gen. Dry tetrahydrofuran (1 cm3) was used to rinse the re-
maining potassium tert-butoxide from the reaction flask. The
resultant suspension was stirred for 2 min then a solution
of bis(tricyclohexylphosphine)benzylidineruthenium dichloride
(Grubbsꢀ catalyst) (0.010 g, 0.011 mmol) in dry benzene (10 cm3)
2706 | Org. Biomol. Chem., 2006, 4, 2696–2709
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©

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(CH, 5ꢀ-C), 130.2 (CH, 6ꢀ-C), 156.3 (quat., NCO2), 170.5 (quat.,
2ꢀ-C), 171.9 (quat., 8ꢀ-CO), 171.2 (quat., 1-C) and 173.5 (quat.,
5-C); m/z (EI+) 565.3365 (M+. C29H47N3O8 requires 565.3363).
temperature. The reaction mixture was filtered through a pad of
CeliteTM, washed with methanol–water (4 : 1) and concentrated
in vacuo to yield a film. The second sample was dissolved in
tetrahydrofuran (3 cm3) and platinum(IV) oxide‡ (0.00078 g,
0.0034 mmol) added under an atmosphere of nitrogen. To the
reaction flask was fitted a balloon of hydrogen and stirred for 16 h
at room temperature. The reaction mixture was filtered through
a pad of CeliteTM, washed with methanol and concentrated in
vacuo to yield a film that was dissolved in methanol–water (4 :
1, 5 cm3) and 10 wt% palladium on activated carbon (0.008 g
0.00749 mmol) was added under an atmosphere of nitrogen. To
the reaction flask was fitted a balloon of hydrogen and stirred
for 5 h at room temperature. The reaction mixture was filtered
through a pad of CeliteTM, washed with methanol–water (4 : 1)
and concentrated in vacuo to yield a film. Both samples were
combined, purified by RP HPLC [10% acetonitrile–90% water
(containing 0.05% trifluoroacetic acid) then 80 : 20, 70 : 30] and
dried on a freeze drier to give macrocycle 8 (0.016 g, 58% from
53) as a colourless wax. Macrocycle 8 existed exclusively as the
trans conformer: [a]D −39.4 (c 0.0864 in water); dH (400 MHz;
D2O), 1.20–1.62 (6H, m, 5-H2, 14-H2, 7-H2), 1.81–1.90 (1H, m,
4-HAHB), 2.04–2.36 (7H, m, 4-HAHB, 9-H2, 6-H2, 15-H2), 2.43
(1H, br t, J 6.2, 8-H), 3.68–3.79 (2H, m, 16-H2), 4.48–4.49 (1H,
m, H-3) and 4.51–4.55 (1H, m, 10-H) [13-H obscured by HOD];
dC (100 MHz; D2O) 18.2 (CH2, 5-C), 23.9 (CH2, 14-C), 24.2 (CH2,
15-C), 26.0 (CH2, 6-C), 26.8 (CH2, 4-C), 28.0 (CH2, 7-C), 30.3
(CH2, 9-C), 38.2 (CH, 8-C), 47.1 (CH2, 16-C), 50.6 (CH, 10-C),
50.9 (CH, 3-C), 59.5 (CH, 13-C), 116.25 (q, J 291.7, CF3), 162.8
(q, J 35.2, CF3CO2H), 169.2 (quat., 2-C), 174.0 (quat., 10-CO),
and 179.8 (quat., 8-CO); m/z (FAB+) 356.1811 [MH(free base)+.
C16H26N3O6 requires 356.1821].
(2S,3ꢀS,8ꢀS)-2-{[(1ꢀ-Aza-3ꢀ-amino-2ꢀ-oxobicyclo[6.3.0]undecyl)-8ꢀ-
carbonyl]amino}-1,5-pentanedioic acid trifluoroacetate salt 6
PtO2 (0.001 g, 0.004 mmol) was added to a stirred solution
of amide 7 (0.025 g, 0.044 mmol) in tetrahydrofuran (4 cm3)
under a nitrogen atmosphere. The mixture was hydrogenated
(1 atm. of hydrogen) for 17 h, filtered through CeliteTM, and
the solvent removed in vacuo. The residue was dissolved in
dichloromethane (3 cm3), trifluoroacetic acid (1 cm3) added and
the solution stirred for 4 h at room temperature. Removal of the
volatiles in vacuo and analysis by HPLC and NMR showed the
reaction to be incomplete. The residue was therefore redissolved in
dichloromethane–trifluoroacetic acid (3 : 1, 4 cm3) and the solution
stirred at room temperature for 2.5 h. The volatiles were removed
in vacuo, and the residue purified by RP HPLC [20% acetonitrile–
80% water (containing 0.05% trifluoroacetic acid)] to give an
oil which was triturated from ether–toluene to give macrocycle
6 (0.0167 g, 81% over 2 steps) as a hygroscopic white solid.
Macrocycle 6 existed exclusively as the cis C(O)–NPro conformer:
◦
mp 75–85 C; [a]D −4.4 (c 0.16 in MeOH); dH (400 MHz; D2O)
1.30–1.39 (1H, m, 5ꢀ-HAHB), 1.57–1.65 (1H, m, 6ꢀ-HAHB), 1.79–
1.91 (4H, m, 5ꢀ-HAHB, 6ꢀ-HAHB, 4ꢀ-HAHB and 10ꢀ-HAHB), 1.98–
2.13 (4H, m, 4ꢀ-HAHB, 10ꢀ-HAHB, 7ꢀ-HAHB and 3-HAHB), 2.23–
2.37 (4H, m, 9ꢀ-H2, 3-HAHB and 7ꢀ-HAHB), 2.53 (2H, t, J 7.5, 4-H2),
3.68 (1H, dt, J 14.2 and 7.4, 11ꢀ-HAHB), 3.79 (1H, dt, J 14.0 and
7.5, 11ꢀ-HAHB), 4.20 (1H, dd, J 11.2 and 6.3, 3-H) and 4.53 (1H,
dd, J 10.2 and 4.9, 2-H); dC (100 MHz; D2O) 20.4 (CH2, 10ꢀ-C),
21.2 (CH2, 6ꢀC), 22.1 (CH2, 5ꢀ-C), 24.8 (CH2, 3-C), 30.3 (CH2, 4-
C), 31.1 (CH2, 4ꢀ-C), 36.4 (CH2, 7ꢀ-C), 40.9 (CH2, 9ꢀ-C), 49.1 (CH2,
11ꢀ-C), 51.2 (CH, 3ꢀ-C), 52.4 (CH, 2-C), 70.9 (quat., 8ꢀ-C), 116.1
(quat., q, J 291, CF3), 162.7 (quat., q, J 36.0, CF3CO2H), 169.4
(quat., 2ꢀ-C), 174.6 (quat., 8ꢀ-CO), 175.3 (quat., 1-C) and 177.0
(quat., 5-C); m/z (FAB+) 356.1830 [MH(free base)+. C16H16N3O6
requires 356.1822].
(9R,11S,14S)-1,4,12-Triaza-9,11-carboxy-2,13-
dioxobicyclo[14.3.0]heptadecane trifluoroacetate 9
Freshly sublimed potassium tert-butoxide (0.009 g, 0.0792 mmol)
was added to a stirred suspension of 1,3-bis(2,4,6-trimethyl-
phenyl)-4,5-dihydroimidazolium tetrafluoroborate (0.031 g,
0.0792 mmol) in tetrahydrofuran (6 cm3) under an atmosphere
of nitrogen. The resultant suspension was stirred for 2 min then
a solution of bis(tricyclohexylphosphine)benzylideneruthenium
dichloride (0.054 g, 0.066 mol) in dry benzene (6 cm3) was added
and the purple solution was heated at 80 ◦C for 35 min. The
dark brown solution was cooled to room temperature and a
solution of protected tripeptide 56 (0.155 g, 0.22 mmol) in dry
benzene (85 cm3) added and the mixture heated at 45 ◦C for
65 h. The solvent was removed in vacuo and the residue purified
by chromatography (silica gel, hexane–ethyl acetate, 1 : 1, 1 : 2,
1 : 3, 1 : 4) to give the metathesis product 57 and unidentified
product(s) (0.110 g) as a brown oil: [m/z (FAB+) 668.2982 (MH+.
C38H42N3O8 requires 668.2972]. The mixture was subsequently
dissolved in methanol–water (4 : 1, 50 cm3) and 10 wt% palladium
on activated carbon (0.035 g, 0.033 mmol) was added under an
atmosphere of nitrogen. To the reaction flask was fitted a balloon
of hydrogen and the mixture stirred for 17 h at room temperature.
(3S,8R,10S,13S)-3-Amino-1,11-diaza-8,10-carboxy-2,12-
oxobicyclo[13.3.0]hexadecane trifluoroacetate 8
To a solution of protected tripeptide 52 (0.041 g, 0.0589 mmol)
in dry dichloromethane (7.5 cm3) was added bis(tricyclo-
hexylphosphine)benzylidineruthenium dichloride (0.015 g,
0.0126 mmol) under an atmosphere of nitrogen. The purple
solution was heated at reflux for 48 h, cooled to room temperature
and dimethyl sulfoxide (0.045 cm3, 0.63 mmol) added. The
orange/brown solution was stirred for 24 h, filtered through a
short plug of silica gel (eluting with hexanes–ethyl acetate, 1 : 3),
and purified by chromatography (C18 RP silica, water–acetonitrile,
20–70%) to give the metathesis product 53 (48 mg) and other
unidentified product(s) as a yellow oil, [m/z (FAB+) 668.2977
(MH+. C38H42N3O8 requires 668.2972]. The metathesis product
53 was divided into 2 samples (∼25 mg each); the first sample
was dissolved in tetrahydrofuran–water (4 : 1, 5 cm3) and 10 wt%
palladium on activated carbon (0.008 g, 0.00749 mmol) was
added under an atmosphere of nitrogen. To the reaction flask
was fitted a balloon of hydrogen and stirred for 21 h at room
‡ PtO2 cleanly reduces the double bond; the subsequent debenzylation
with 10% Pd/C is much cleaner than carrying out a one pot hydrogena-
tion/debenzylation using 10% Pd/C.
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The reaction mixture was filtered through a pad of CeliteTM
,
References
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vacuo to yield a film (0.064 g) that was purified by RP HPLC [20%
acetonitrile–80% water (containing 0.05% trifluoroacetic acid)].
Repeated evaporation from a toluene–ether mixture and drying
on a freeze drier gave macrocycle 9 (0.045 g, 58% from 56) as a
colourless solid. Macrocycle 9 was shown to be a 65 : 35 trans–
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1
cis mixture of conformers by H NMR analysis (the ratio was
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78 : 22 trans–cis mixture of conformers by 13C NMR analysis
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conformers respectively): dH (400 MHz; D2O), 0.90 (3H, t, J 6.5,
Gluc-CH2CH2CH2CH3), 1.33 (4H, br s, Gluc-CH2CH2CH2CH3),
1.64 (2H, br q, J 7.0, Gluc-CH2CH2CH2CH3), 2.03–2.17 (5H,
m, Glub-H2, Proc-H2, Prob-HAHB), 2.31–2.35 (0.78H, m, Prob-
HAHB), 2.57 (1H, p, J 6.9, Gluc-H), 3.59–3.75 (2.18H, m, Prod-
H2, Glya-HAHB*), 3.98–4.10 (1.82H, Glya-H2, Glya-HAHB*),
4.47 (1H, t, J 7.5, Glua-H) and 4.51–4.56 (1H, m, Proa-H); dC
(100 MHz; D2O) 13.0 (CH3, Gluc-CH2CH2CH2CH3), 21.7 (CH2,
Gluc-CH2CH2CH2CH3), 21.8* (CH2, Gluc-CH2CH2CH2CH3),
24.0 (CH2, Proc-C), 28.1 (CH2, Gluc-CH2CH2CH2CH3), 28.2*
(CH2, Gluc-CH2CH2CH2CH3), 29.3 (CH2, Prob-C), 30.9 (CH2,
Gluc-CH2CH2CH2CH3), 31.3*, 31.6*, 32.1* (CH2), 32.3 (CH2,
Glub-C), 40.1* (CH2, Glya-C), 40.3 (CH2, Glya-C), 42.0 (CH,
Gluc-C), 43.0* (CH, Gluc-C), 46.8 (CH2, Prod-C), 47.4* (CH2,
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Acknowledgements
The authors thank Neuren Pharmaceuticals Ltd. for financial
support.
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