Synthesis and neuroprotective activity of analogues of glycyl-L-prolyl-L- glutamic acid (GPE) modified at the α-carboxylic acid

Article abstract of DOI:10.1016/j.bmc.2004.10.005

The synthesis of nine GPE* analogues, wherein the α-carboxylic acid group of glutamic acid has been modified, is described by coupling readily accessible N-benzyloxycarbonyl-glycyl-l-proline 2 with various analogues of glutamic acid. Pharmacological evaluation of the novel compounds was undertaken to further understand the role of the glutamate residue on the observed neuroprotective properties of the endogenous tripeptide GPE.

Full text of DOI:10.1016/j.bmc.2004.10.005

Bioorganic & Medicinal Chemistry 13 (2005) 501–517
Synthesis and neuroprotective activity of analogues of
glycyl-L-prolyl-L-glutamic acid (GPE) modified
at the a-carboxylic acid
Nicholas S. Trotter, Margaret A. Brimble,* Paul W. R. Harris,
David J. Callis and Frank Sieg
Neuren Pharmaceuticals Medicinal Chemistry Group, Department of Chemistry, University of Auckland, 23 Symonds Street,
Auckland 1000, New Zealand
Received 11 August 2004; revised 1 October 2004; accepted 4 October 2004
Available online 28 October 2004
Abstract—The synthesis of nine GPE* analogues, wherein the a-carboxylic acid group of glutamic acid has been modified, is
described by coupling readily accessible N-benzyloxycarbonyl-glycyl-L-proline 2 with various analogues of glutamic acid. Pharma-
cological evaluation of the novel compounds was undertaken to further understand the role of the glutamate residue on the observed
neuroprotective properties of the endogenous tripeptide GPE.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
to the significantly lower affinity of des-N(1–3)-IGF-1
for IGF binding proteins,10,12,26,27 hypothesised to
transport IGF-1 to the injured region for neuronal res-
cue.12,28 Together with the neuroprotective actions of
the aforementioned des-N(1–3)-IGF-1, binding proteins
and the IGF-1 receptor,24,27,29,30 IGF-1 is believed to
function as a prohormone for GPE 1, which acts on a
yet unknown receptor.31–33 GPE 1 is not known to modu-
late or cross-react with the brainꢀs IGF-1 receptor.15
Insulin-like growth factor-1 (IGF-1) is broadly distrib-
uted within the mammalian central nervous system1
(CNS) and is a potent neurotrophic factor.2,3 In experi-
mental animal models, IGF-1, produced endogenously
in damaged regions of the brain, has potent antiapop-
totic effects on neurons and glial cells and is postulated
to restrict progressive cell death.4–9 When administered
following hypoxic-ischemic (HI) brain injury, IGF-1 re-
duced neuronal loss and cortical infarction.10–14
GPE 1 was observed to act as a neuronal rescue factor
following transient HI brain injury for cerebral cortical,
striatal and hippocampal neurons,34 with peripheral
activity, relative to local administration, producing
wider neuroprotective effects.33 Although the precise
mechanism of action of GPE 1 remains obscure, it
exhibits preferential binding to glia upon local injection,
potentially representing a complementary conduit for
central and systemic IGF-1ꢀs antiapoptotic effects.33 In
vitro studies by Sara and co-workers15,31 indicated that
GPE 1 potentiated the K+-stimulated release of dopam-
ine and acetylcholine through the N-methyl-D-aspartate
(NMDA) receptor system and a nonelucidated mecha-
nism, respectively.
Much of the endogenous IGF-1 in the CNS is thought
to be proteolytically cleaved into des-N(1–3)-IGF-1, a
truncated IGF-1 comprised of 67 amino acids, and the
N-terminal tripeptide Gly-Pro-Glu-OH (GPE 1)
(Scheme 3).15–21 There is, however, no direct evidence
that GPE is naturally present or produced in the brain.22
Although des-N(1–3)-IGF-1 is more neurotrophically
potent than its precursor IGF-1,23–25 and both exhibit
similar neuroprotective actions in rats,12 the former is
a less potent protective agent in the treatment of post-
HI brain injury. This reduced efficacy can be attributed
In addition, some preliminary observations suggest that
GPE 1 has a neuromodulatory role in the CNS, poss-
ibly through interaction with glutamate recep-
tors.15,16,21,31,32,34–37 L-Glutamate, being the main
Keywords: Neuroprotective agents; Peptides; Proline; GPE.
*
Corresponding author. Tel.: +64 09 3737599; fax: +64 09 3737422;
e-mail: m.brimble@auckland.ac.nz
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.10.005

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N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
excitatory amino acid neurotransmitter, functions
through a heterogeneous family of two major receptor
types—the ionotropic (iGluRs)38,39 and metabotropic
(mGluRs)40–42 glutamate receptors.43–46 Malfunctioning
of the glutaminergic system may be involved in psychiat-
ric and neurodegenerative brain disorders, with gluta-
tool of these GPE analogue compounds. This work is
part of a structure–activity relationship study to deter-
mine the significance of the Glu residue of this tripeptide
on neuroprotective activity, greatly aiding the selection
of suitable candidates for continued drug development.
mate accumulation after brain injury having
a
prominent role in developing excitotoxic neuronal
death.47–51 Thus the glutamate receptors provide an
excellent target for rational design of neuroprotective
agents.52–54
2. Results and discussion
In order to ascertain the importance of the a-carboxylic
acid functionality on the glutamate residue of GPE, nine
novel analogues were synthesised and evaluated for their
neuroprotective effects. The synthetic strategy employed
involved preparation of known N-benzyloxycarbonyl-
glycyl-L-proline60,61 2, and effecting peptide bond
construction with several modified benzyl-protected
glutamic acid residues (Scheme 1).
Based on these scientific findings, it has been proposed
that GPE 1, or analogues thereof, may provide a novel
class of pharmaceutical agents for the treatment of
CNS injuries; neurodegenerative diseases including Alz-
heimerꢀs, Parkinsonꢀs and Huntingtonꢀs Diseases; multi-
ple sclerosis and general ageing-induced cognitive
dysfunction.55–58 Analogues of GPE should be low
molecular weight and must be lipophilic to cross the
blood–brain barrier. The ideal synthetic analogue of
GPE 1 would result in an enhanced pharmacokinetic
profile, and, depending on the nature of the modifica-
tion, lead to improved metabolic stability and increased
oral bioavailability. Ultimately, this would lead to less
intrusive routes of administration.
After assembly of the new pseudo-peptide bond, a sim-
ple deprotection procedure furnishes the desired GPE*
analogues.
The synthesis of N-CBz peptide60,61 2 was achieved in
82% yield over three steps from readily available L-pro-
line 3 (Scheme 2). Thus, treatment of 3 with thionyl
chloride in methanol gave quantitatively hydrochloride
salt62,63 4, which was subsequently coupled with com-
mercial N-benzyloxycarbonylglycine 5, using 1,3-di-
cyclohexylcarbodiimide (DCC) to realise protected
dipeptide64 6. Finally, saponification of the methyl ester
afforded acid 2.
We herein report the preparation and pharmacological
evaluation of a variety of analogues of general structure
Gly-Pro-Glu-OH (GPE*), in which the a-position of
Glu (E*) has been modified. We also report the develop-
ment of a mixed neuronal, astrocytic brain cell culture
(modified method from Chen et al.59) deriving from
the striatum, to gain an efficient first, in vitro screening
An initial model study was carried out to assess the reli-
ability of the key proline–glutamate bond forming step
H
X H3N
R
R1
N
R
R1
OH
N
N
+
O
O
O
O
CO2Bn
CO2H
NH2
NHCO2Bn
GPE* analogues
Modified glutamates
2
Scheme 1. General retrosynthesis for GPE* analogues.
OCH3
OH
(i)
N
H
.HCl
BnO2CNH
CO2H
N
H
+
O
O
3
4
5
OCH3
OH
N
N
O
(ii)
(iii)
O
O
O
NHCO2Bn
NHCO2Bn
2
DCU
+
6
Scheme 2. Reagents and conditions: (i) SOCl2, MeOH, ꢀ5ꢁC to rt, N2, 19h (98%); (ii) Et3N, 1,3-dicyclohexylcarbodiimide (DCC), CH2Cl2, 0ꢁC to
rt, N2, 18h; (iii) 1M NaOH (aq), 1,4-dioxane, rt, 21h (82% in two steps from 4).

N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
503
H
H2N
CO2Bn
.pTsOH
N
CO2Bn
CO2Bn
OH
(i)
N
N
+
O
O
O
O
CO2Bn
NHCO2Bn
NHCO2Bn
2
7
8
γ
β
H
N
α
δ
CO2H
N
O
(ii)
α
N
O
O
γ
α
O
N
CO2H
NH2
H
1 (80:20 trans:cis)
γ-turn
Scheme 3. Reagents and conditions: (i) Et3N, EtOCOCl, CH2Cl2, N2, 0ꢁC to rt, 17h (100%); (ii) H2, 10% Pd/C, 80:20 MeOH–H2O, rt, 18h (94%).
by first preparing GPE 1 itself using a mixed anhydride
protocol.65–67 Reaction of acid 2 with ethyl chlorofor-
mate under basic conditions generated the mixed anhy-
dride in situ, which was then treated with dibenzyl
glutamate 7 to provide perbenzylated tripeptide 8 as a
single optical isomer in 100% yield. The final step in
the sequence involved hydrogenolysis of amide 8 over
activated palladium on charcoal to afford glycyl-L-prol-
yl-L-glutamic acid15 (GPE) 1 after trituration from
diethyl ether (Scheme 3). The deprotection step was per-
formed in a mixture of water and methanol in order to
solubilise the product as it formed.
teases, thereby imparting improved metabolic stability.
Additionally, the overall polarity of the molecule is
reduced, thus increasing lipophilicity and membrane
permeability of the analogue.
The commercial amino acid 12 and variants 1370 and
14,71 as synthesised according to literature procedures,
were initially protected as benzyl esters 15, 16 and 17,
respectively, by heating with benzyl alcohol and p-tolu-
enesulfonic acid in benzene. Coupling of the aforemen-
tioned acid 2 with amino ester 15 using the mixed
anhydride method described above gave an amide that
underwent hydrogenolysis to afford analogue 9 as an
86:14 trans:cis mixture of rotamers in 80% yield over
the two steps. Similarly, subjection of the pseudo-glut-
amate residues 16 and 17 to a peptide coupling reaction
with acid 2, followed by deprotection, afforded GPE*
analogues 10 and 11, respectively.
In a deuterium oxide solution, GPE 1 existed as an 80:20
trans:cis mixture of the two proline–glycine peptide
bond-rotamers. The major conformer was assigned as
having the trans-configuration, or c-turn, by compari-
son of the differences in the 13C NMR chemical shifts
observed for the b and c proline carbons of the major
and minor isomers. With small acyclic or cyclic peptides
the c-turn functions to facilitate stabilising hydrogen
bonding between the first and third amino acids in the
sequence.68 In a review by Kessler69 on cyclic peptide
conformations, it is stated that generally there is an
approximate 10ppm difference between the C-b and C-c
proline resonances for the cis conformation but only
5ppm for the alternative trans form. In our case, the
two major signals were separated by 5.1ppm thereby
establishing the trans conformation. This assignment
was supported by a strong nuclear Overhauser effect
(NOE) observed between the glycine-a and proline-d
protons.
Another strategy for reducing the overall charge in the
GPE molecule whilst maintaining the steric bulk of the
glutamate a-position involves replacement of the acid
with an amide moiety. To this end, preparation of ana-
logues 18, 19 and 20, with increasing degrees of amide
substitution, were investigated (Scheme 5).
The syntheses of all three analogues 18, 19 and 20
started from Boc-protected benzyl glutamic acid 21.72
Reaction of acid 21 with di-tert-butyl dicarbonate and
ammonolysis of the intermediate anhydride with ammo-
nium hydrogen carbonate under basic conditions
yielded primary amide 2273 in 98% yield. Subsequent tri-
fluoroacetic acid-mediated carbamate cleavage quantita-
tively afforded trifluoroacetate salt 25. Treatment of this
salt 25 with acid 2 was carried out using bis(2-oxo-3-
oxazolidinyl)phosphinic chloride (BoPCl) to provide
amide 28 in good yield. The successful employment of
BoPCl as the coupling agent to effect peptide bond for-
mation, without concomitant isomerisation of either of
the two stereogenic centres, provided an alternative to
the previously used mixed anhydride protocol. Hydro-
genation of the fully protected pseudo-tripeptide 28
under standard conditions gave analogue 1856 (65%),
Having successfully prepared GPE 1 our attention then
focused on the synthesis of various GPE* analogues
using a similar strategy. The first set of three analogues
9, 10 and 11 involved replacement of the a-carboxylic
acid group by a hydrogen, a methyl group and a geminal
dimethyl group, starting from the modified glutamic
acids 12, 1370 and 14,71 respectively (Scheme 4). Elimi-
nation of the carboxylic acid in this fashion removes
the amino acid character from the glutamate residue
of GPE 1, potentially suppressing the action of pro-

504
N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
H2N
R
R1
H2N
R
R1
OH
(i)
N
.pTsOH
+
O
O
CO2H
CO2Bn
NHCO2Bn
15 R = R1 = H
12 R = R1 = H
2
16 R = H, R1 = CH3
17 R = R1 = CH3
13 R = H, R1 = CH3, HCl salt
14 R = R1 = CH3
H
N
α
δ
R
R1
4
N
(ii), (iii)
O
α
O
1CO2H
NH2
1
9 R = R = H (86:14 trans:cis)
10 R = H, R1 = CH3 (76:24 trans:cis)
11 R = R1 = CH3 (80:20 trans:cis)
Scheme 4. Reagents and conditions: For 9: (i) pTsOHÆH2O, benzene, 80ꢁC, 68h (86%); (ii) Et3N, EtOCOCl, CH2Cl2, 0ꢁC to rt, N2, 17h (86%); (iii)
H2, 10% Pd/C, 80:20 MeOH–H2O, 16h (97%). For 10: (i) ion exchange resin then benzyl alcohol, pTsOHÆH2O, benzene, 80ꢁC, 19h (86%); (ii) Et3N,
EtOCOCl, CH2Cl2, 0ꢁC to rt, N2, 17h (80%); (iii) H2, 10% Pd/C, MeOH, 20h (93%). For 11: (i) benzyl alcohol, pTsOHÆH2O, benzene, 80ꢁC, 43h
(65%); (ii) Et3N, EtOCOCl, CH2Cl2, 0ꢁC to rt, N2, 18h (93%); (iii) H2, 10% Pd/C, 1.6:1.3:1.0 THF–MeOH–H2O, rt, 20h (89%).
BocHN
CO2H
BocHN
R
H2N
R
α
(i)
(ii)
.CF3CO2H
γ
CO2Bn
CO2Bn
CO2Bn
25 R = CONH2
26 R = CONHMe
27 R = CONMe2
21
22 R = CONH2
23 R = CONHMe
24 R = CONMe2
H
N
OH
R
(iv)
N
(iii)
N
+
O
O
O
O
CO2Bn
NHCO2Bn
NHCO2Bn
28 R = CONH2
29 R = CONHMe
30 R = CONMe2
2
H
N
R
N
O
O
CO2H
NH2
18 R = CONH2 (87:13 trans:cis)
19 R = CONHMe (83:17 trans:cis)
20 R = CONMe2 (84:16 trans:cis)
Scheme 5. Reagents and conditions: For 18: (i) [(CH3)3COC(O)]2O, NH4HCO3, pyridine, 1,4-dioxane, N2, rt, 17h (98%); (ii) CF3CO2H, CH2CH2,
0ꢁC, 1.5h (ca. 100%); (iii) BoPCl, i-Pr2NEt, CH2Cl2, N2, 0ꢁC to rt, 21.5h (69%); (iv) H2, 10% Pd/C, MeOH, rt, 20h (65%). For 19: (i) PyBoP,
MeNHÆHCl, Et3N, CH2Cl2, N2, rt, 3days (75%); (ii) CF3CO2H, CH2CH2, 0ꢁC, 1.5h (ca. 100%); (iii) BoPCl, i-Pr2NEt, CH2Cl2, N2, 0ꢁC to rt, 18.5h
(55%); (iv) H2, 10% Pd/C, MeOH, rt, 20h (97%). For 20: (i) EtOCOCl, Et3N, Me2NHÆHCl, CH2Cl2, N2, 0ꢁC to rt, 20.5h (100%); (ii) CF3CO2H,
CH2CH2, 0ꢁC, 1.5h (ca. 100%); (iii) BoPCl, i-Pr2NEt, CH2Cl2, N2, 0ꢁC to rt, 18.5h (25%); (iv) H2, 10% Pd/C, MeOH, rt, 20h (78%).
which existed predominantly as the trans proline–glycine
amide bond conformer.
The next target in this series was analogue 19. Using lit-
erature precedence, amino acid 21 was transformed into

N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
505
secondary amide74 23 by reaction with N-methylamine
using the coupling reagent (benzotriazole-1-yloxy)tri-
pyrrolidinophosphonium hexafluorophosphate (PyBoP)
in methylene chloride. Amide 23 was next treated with
TFA and the resultant salt 26 coupled to acid 2 with
BoPCl under basic conditions to afford amide 29 that
underwent deprotection to give analogue 19. The third
analogue 20 in this series was prepared from tertiary
amide 24 following a similar sequence. Inexplicably,
the BoPCl-promoted coupling reaction of 2 with 27 gave
compound 30 in low yield (25%). The precursor amide
24 was efficiently synthesised by formation of a mixed
anhydride of acid 21 with ethyl chloroformate and
triethylamine, and subsequent amidation with N,N-
dimethylamine.
the succinimide ester76 of acid 2 in the presence of diiso-
propylethylamine gave pure amide 34, albeit in 31%
yield after purification by flash column chromatogra-
phy. The sequence was completed by hydrogenolysis
of amide 34 under standard conditions affording ana-
logue 31 in 93% yield as a white solid.
The final two GPE* analogues 35 and 36 reported herein
differ from the seven described above in that the a-carb-
oxylic acid functionality of the glutamic acid residue was
left intact, however the nature of the a-carbon itself was
modified (Schemes 7 and 8). In the first case, the natu-
rally occurring L-glutamic acid was replaced by its opti-
cal antipode (analogue 35) and in the second, an
additional methyl group is introduced at the a-position
(analogue 36).
Acid 21 was next used for the production of the GPE*
analogue 31, in which the a-carboxylic acid was replaced
with an hydroxylmethyl group. N-Hydroxysuccinimide
(NHS) ꢁactive estersꢀ were used for the reduction of the
a-carboxylic acid functionality of glutamic acid and
for the subsequent coupling to the GP unit (Scheme
6). The requisite stable NHS esters,75,76 were easily pre-
pared following minor modifications of the original
literature procedure,60 from the reaction of corre-
sponding acids 21 and 2 with NHS and DCC in
EtOAc and DME, respectively. Rapid sodium borohy-
dride reduction of the intermediate NHS ester75 of 21
in methanolic THF gave the desired N-Boc amino alco-
hol77 32 (72%), with typical N-Boc deprotection using
TFA successfully furnishing the amine salt 33. In spite
of the acidic environment, no intramolecular condensa-
tion of the resultant alcohol with the c-benzyl ester to af-
ford a d-lactone was observed. Coupling of amine 33 to
Following the detailed synthesis of the putative neuro-
protective agent GPE 1, protected D-glutamic acid 37
was coupled with acid 2 to give amide 38 which was sub-
sequently deprotected in a 70:30 mixture of THF and
water to afford GP-(D)-E 35, a diastereoisomer of
GPE 1 (Scheme 7). It was rationalised that analogue
35, in which an unnatural D-amino acid residue had
been incorporated, would not be recognised by pro-
teases.78 Similarly, it was envisaged that analogue 36,
which bears an additional alkyl group at the a-position,
thereby increasing steric hindrance about the proline–
glutamic acid amide bond, would also be resistant to
proteolytic cleavage (Scheme 8).
Dibenzylation of racemic a-methylglutamic acid 39 af-
forded a-methylglutamate 40 in 55% yield. Condensa-
tion of glutamate 40 with acid 2 and subsequent
BocHN
CO2H
BocHN
H2N
OH
OH
α
(iii)
(i), (ii)
.CF3CO2H
γ
CO2Bn
CO2Bn
CO2Bn
33
21
32
H
OH
N
(iv),(v)
(vi)
OH
N
N
O
+
O
O
O
CO2Bn
NHCO2Bn
NHCO2Bn
2
34
H
N
OH
N
O
O
CO2H
NH2
31 (84:16 trans:cis)
Scheme 6. Reagents and conditions: (i) N-hydroxysuccinimide, DCC, EtOAc, N2, 0ꢁC to rt, 18h (ca. 100%); (ii) NaBH4, THF, MeOH, 0ꢁC,
1h (72% in two steps from 21); (iii) CF3CO2H, CH2Cl2, 0ꢁC, 1.5h (ca. 100%); (iv) N-hydroxysuccinimide, DCC, DME, N2, 0ꢁC to rt, 20h (69%);
(v) i-Pr2NEt, CH2Cl2, N2, 0ꢁC to rt, 2.5h (31%); (vi) H2, 10% Pd/C, MeOH, rt, 20h (93%).

506
N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
H
H2N
CO2Bn
.pTsOH
N
CO2Bn
CO2Bn
OH
(i)
N
N
+
O
O
O
O
CO2Bn
NHCO2Bn
NHCO2Bn
2
37
38
H
N
CO2H
(ii)
N
O
O
CO2H
NH2
35 (80:20 trans:cis)
Scheme 7. Reagents and conditions: (i) Et3N, EtOCOCl, CH2Cl2, N2, 0ꢁC to rt, 18.5h (64%); (ii) H2, 10% Pd/C, 70:30 THF–H2O, rt, 18h (68%).
H2N
CO2H
CH3
H2N
CO2Bn
CH3
OH
(i)
N
+
.pTsOH
O
O
CO2H
CO2Bn
NHCO2Bn
40
39
2
H
H
N
N
CO2Bn
CH3
CO2H
CH3
(ii)
N
(iii)
N
O
O
O
O
CO2Bn
CO2H
NHCO2Bn
NH2
36 (77:23 trans:cis)
41
Scheme 8. Reagents and conditions: (i) benzyl alcohol, pTsOHÆH2O, benzene, 80ꢁC, 54.5h (55%); (ii) Et3N, EtOCOCl, CH2Cl2, N2, 0ꢁC to rt, 22h
(97%); (iii) H2, 10% Pd/C, 80:20 MeOH–H2O, 21h (81%).
hydrogenolysis of the product 41 afforded analogue 36
in excellent yield as a 77:23 trans:cis mixture of rota-
mers. Unfortunately however, the respective esters 41
and acids 36 were found to be inseparable mixtures of
two diastereoisomers due to the lack of control of the
stereochemistry of the a-methyl group. Reverse-phase
HPLC analysis, showed that analogue 36 eluted as an
equimolar set of two diastereoisomers at similar reten-
tion times.
10 where the recovery value was around 20%. None of
the other compounds exhibited neuroprotective activity.
3. Conclusions
To summarise, we described herein the synthesis of nine
analogues of GPE and their biological evaluation
against striatal cell survival post apoptosis-induced in-
jury. Compound 20 exhibited similar efficacy as GPE
while 10 showed activity but was not as potent as 20.
The structure and spectra of the new analogues were
determined by full analysis of the NMR and mass spec-
tral data.
The nine GPE* analogues synthesised were subjected to
in vitro evaluation for prevention of neuronal cell death.
Of the nine analogues, only the N,N-dimethylamide, 20,
showed neuroprotective activity at 1–100lM with
recovery values ranging from 20% to 40%. Amide 20
exhibits similar efficacy as the best dose of GPE
(1mM). However, analogue 20 may not be more effec-
tive than GPE 1 because of the low significance values
(best recovery at 58% evaluated against the injured
vehicle—see Fig. 1). In repeated experiments the
recovery values vary from 20% to 40% whereas GPE 1
recovery values vary from 25% to 40%. Lower neuro-
protective values were obtained with 1mM of analogue
4. Experimental
All reagents were used as supplied. Solvents were
purified by standard methods.79 Analytical thin layer
chromatography (TLC) was carried out on 0.20mm
pre-coated silica gel plates (ALUGRAMꢂ SIL
G/UV254). Products were visualised by UV fluorescence

N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
507
of the respective isomers is made according to Kessler
et al.69 and NOESY experiments. Mass spectra were re-
corded on a VG-70SE mass spectrometer (EI, CI and
FAB). High-resolution mass spectra were recorded at
a nominal resolution of 5000. Analytical HPLC studies
were performed on a Waters 600 system with a 2487
dual k absorbance detector using an Altech Econo-
sphere C18Si column (150 · 4.6mm; 5lm), with a
5min H2O flush (0.05% TFA) then steady gradient over
25min to CH3CN as eluent at a flow rate of 1mL/min.
All chemicals utilised in the bioassay were purchased
from Invitrogen.
4.1. Striatal cell culture
Two pregnant Wistar rats (gestational day 18) under-
went Caesarean section according to approved proce-
dures from the animal ethics committee of the
University of Auckland. The Wistar dams were anaes-
thetised in a CO2-enriched atmosphere and sacrificed
by subsequent spinal cord dislocation. The embryos
were removed from their embryonic sacs and decapi-
tated. After incisions into the skull with fine scissors,
the embryonic brain was removed with a fine spatula.
Striatal tissue was separated from neocortical tissue un-
der the microscope and placed into serum-free DMEM/
F-12 medium supplemented with penicillin/streptomycin
(100u/mL). Tissue underwent 15 trituration steps using
a P1000 pipettor to obtain dissociated cells, which were
centrifuged for 5min at 250 G (4ꢁC) and the superna-
tant was discarded. Cells were re-suspended into 1mL
DMEM/F-12 medium and were kept on ice. Cell suspen-
sion (400,000cells/cm2) was applied to 0.1mg/mL poly-
L-lysine pre-coated 96-well plates (3h at 37ꢁC) and the
volume was increased up to 100lL with DMEM/F-
12 + 5% FBS. Cells were cultivated at 37ꢁC in 100%
humidity and saturated 5% CO2 atmosphere. After
24h the medium was changed to serum-free Neuroba-
sal/B27. Cells were fed every 3days and maintained until
8days in vitro (DIV).
Figure 1. Cellular survival bioassay. MTT-values were taken after 24h
when apoptotic nuclei become apparent within the cell culture. The
neuroprotective dose-response for GP-N,N-diMeE lies between 1 and
100lM.
and heating of plates dipped in anisaldehyde in etha-
nolic sulfuric acid or alkaline potassium permanganate
solution. Flash chromatography was performed using
Scharlau 60 (40–60lm mesh) silica gel. Melting points
in degrees Celsius (ꢁC) were measured on an Electro-
thermalꢂ melting point apparatus and are uncorrected.
Optical rotations were measured at the sodium D line
(589nm), at 20ꢁC, with a Perkin Elmer 341 polarimeter
using a 1dm path length cell and are given in units of
10ꢀ1 degcm2 gꢀ1. IR spectra were recorded on a Perkin
Elmer Spectrum One FT-IR spectrometer and the sam-
ples were prepared as thin films between sodium chlo-
ride plates. Nuclear Magnetic Resonance (NMR)
spectra were recorded on
a
Bruker AVANCE
DRX400 (1H, 400MHz; 13C, 100MHz), a Bruker
AVANCE 300 (1H, 300MHz; 13C, 75MHz) or a Bruker
AC200 (1H, 200MHz; 13C, 50MHz) spectrometer at
4.2. Cellular injury, drug administration and cellular
survival analysis
1
298K. For H NMR data, chemical shifts are described
in parts per million (ppm) relative to tetramethylsilane
(d 0.00), DOH (d 4.75), CHD2OD (d 3.30) or
CHD2S(O)CD3 (d 2.50) and are reported consecutively
as position (dH), relative integral, multiplicity (s = sin-
glet, d = doublet, t = triplet, q = quartet, p = pentet,
q = quintet, s = sextet, dd = doublet of doublets,
m = multiplet and where br = broad), coupling constant
(J/Hz) and assignment. For 13C NMR data, chemical
shifts (ppm) are referenced internally to CDCl3 (d
77.0), CD3OD (d 49.1) and (CD3)2S(O) (d 39.4) or exter-
nally to 3-(trimethylsilyl)-1-propanesulfonic acid so-
dium salt (DSS) and are reported consecutively as
position (dC), degree of hybridisation and assignment.
Assignments were aided by DEPT135, COSY, NOESY,
HSQC and HMBC experiments. When two sets of peaks
arise in the NMR spectra due to cis/trans isomerisation
about the glycine–proline amide bond, the chemical shift
Striatal cells were injured after 7 DIV. The injury para-
digm involved 30nM okadaic acid treatment with simul-
taneous drug administration (GPE analogues) for 24h.
Afterward, 1lL of 3lM okadaic acid stock solution
together with 1lL of GPE analogue dissolved in PBS
(vehicle received 2lL of PBS) were incubated for 24h
with the striatal cells. Subsequently, 20lL MTT (5mg/
mL in PBS) was added for 4h. The reaction was termi-
nated by addition of 100lL 4% sodium dodecyl sulfate
(SDS) solution. After 16–24h incubation time, extinc-
tion values are read at 595nm.
The difference between the vehicle and the okadaic acid
injury condition was calculated. This value is set as the
theoretical 100% recovery value. Measured values for
the tested compounds are expressed in percentage of
the 100% value. The unpaired Studentꢀs t-test is used
for statistical analysis.
for the minor cis conformer is asterisked ( ). Assignment
*

508
N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
4.3. L-Proline methyl ester hydrochloride62,63 (4)
ers: mp 154–157ꢁC (lit.,60 157–159ꢁC); dH (300MHz;
CDCl3; Me4Si) 1.75–2.35 (4H, m, Prob-H2 and Proc-
H2), 3.25–3.70 (2H, m, Prod-H2), 3.78–4.17 (2H, m,
Glya-H2), 4.39* (0.25H, t, J 5.7, Proa-H), 4.53 (0.75H,
t, J 5.5, Proa-H), 5.10 (2H, s, OCH2Ph), 5.80–6.02
(0.75H, m, Gly-NH), 6.02–6.15* (0.25H, m, Gly-NH),
7.20–7.45 (5H, m, Ph) and 8.06 (1H, br s, COOH); dC
(75MHz; CDCl3; Me4Si) 22.2* (CH2, Proc-C), 24.5
(CH2, Proc-C), 28.5 (CH2, Prob-C), 31.2* (CH2, Prob-
C), 43.1* (CH2, Glya-C), 43.2 (CH2, Glya-C), 46.2
(CH2, Prod-C), 46.8* (CH2, Prod-C), 58.5* (CH, Proa-
C), 59.2 (CH, Proa-C), 66.9 (CH2, OCH2Ph), 67.1*
(CH2, OCH2Ph), 127.9 (CH, Ph), 128.0 (CH, Ph),
128.4 (CH, Ph), 136.2* (quat., Ph), 136.3 (quat., Ph),
156.5 (quat., NCO2), 156.8* (quat., NCO2), 167.8*
(quat., Gly-CO), 168.4 (quat., Gly-CO), 173.8* (quat.,
CO2H) and 174.0 (quat., CO2H); m/z (EI+) 306.1213
(M+ÆC15H18N2O5 requires 306.1216).
Thionyl chloride (34mL, 0.47mol) was cautiously added
dropwise to a stirred solution of L-proline 3 (13.49g,
0.18mol) in anhydrous methanol (180mL) at ꢀ5ꢁC
under an atmosphere of nitrogen. The reaction mixture
was warmed to room temperature, stirred for 19h and
concentrated to dryness under reduced pressure. The
resultant oil was dissolved in toluene (200mL) and con-
centrated to dryness to remove residual thionyl chloride
and methanol to yield hydrochloride62,63 4 (18.95g, 98%)
as a colourless oil. Cooling of the crude oil to 0ꢁC for
12h afforded an hygroscopic white solid: dH (300MHz;
CDCl3; Me4Si) 1.98–2.28 (3H, br m, Prob-HAHB and
Proc-H2), 2.32–2.53 (1H, br m, Prob-HAHB), 3.43–
3.71 (2H, br m, Prod-H2), 3.85 (3H, s, OCH3), 4.41–
4.60 (1H, br m, Proa-H), 9.28 (1H, br s, NH) and
10.73 (1H, br s, HCl); dC (75MHz; CDCl3; Me4Si)
23.1 (CH2, Proc-C), 28.0 (CH2, Prob-C), 45.4
(CH2, Prod-C), 52.9 (CH3, OCH3), 58.7 (CH, Proa-C)
and 168.7 (quat., CO); m/z (EI+) 129.0788
(M+ꢀHClÆC6H11NO2 requires 129.0790).
Representative procedure A for the preparation of pro-
tected tripeptides and analogues from acid 2 is as
follows:
4.4. N-Benzyloxycarbonyl-glycyl-L-proline60,61 (2)
4.5. Dibenzyl N-benzyloxycarbonyl-glycyl-L-prolyl-L-
glutamate (8)
Anhydrous triethylamine (0.44mL, 3.16mmol) was
added dropwise to a stirred solution of hydrochloride
4 (0.51g, 3.08mmol) and acid 5 (0.63g, 3.01mmol) in
methylene chloride under an atmosphere of nitrogen.
The colourless solution was cooled to 0ꢁC, a solution
of 1,3-dicyclohexylcarbodiimide (0.67g, 3.25mmol) in
methylene chloride (9mL) at 0ꢁC added dropwise and
the reaction mixture warmed to room temperature and
stirred for 18h. The resultant white mixture was filtered
through a CeliteTM pad to partially remove 1,3-di-
cyclohexylurea, and the pad washed with methylene chlo-
ride (50mL). The filtrate was washed successively with
10% aqueous hydrochloric acid (50mL) and saturated
aqueous sodium hydrogen carbonate (50mL), dried
(MgSO4), filtered and concentrated to dryness in vacuo.
Further purification of the residue by flash column chro-
matography (SiO2; 30–80% ethyl acetate–hexane; gradient
elution) afforded N-benzyloxycarbonyl-glycyl-L-pro-
line methyl ester64 6 (0.88g), containing residual
1,3-dicyclohexylurea, as a white semi-solid: Rf 0.50
(EtOAc); m/z (EI+) 320.1371 (M+ÆC16H20N2O5 requires
320.1372).
To a solution of acid 2 (0.12g, 0.39mmol) in methylene
chloride (8mL), cooled to 0ꢁC under nitrogen, were
added dropwise triethylamine (0.07mL, 0.50mmol)
and ethyl chloroformate (0.05mL, 0.52mmol) and the
resultant mixture stirred at 0ꢁC for 40min to form the
mixed anhydride. Triethylamine (0.075mL, 0.54mmol)
was added to a suspension of p-toluenesulfonate 7
(0.26g, 0.52mmol) in methylene chloride (8mL) under
nitrogen at 0ꢁC and the resulting mixture was added
dropwise to the above mixed anhydride. The reaction
mixture was warmed to room temperature, stirred for
17h then successively washed with 10% aqueous hydro-
chloric acid (3 · 50mL) and saturated aqueous sodium
hydrogen carbonate (50mL), dried (MgSO4), filtered
and evaporated to dryness in vacuo. Purification of the
ensuing residue by flash column chromatography
(SiO2; 20–80% ethyl acetate–hexane; gradient elution)
yielded amide 8 (0.24g, 100%) as a colourless oil, which
was cooled to 0ꢁC to give a white solid. Compound 8
was shown to be an 86:14 trans:cis mixture of conform-
ers: Rf 0.55 (EtOAc); mp 116–118ꢁC; [a]D ꢀ55.9 (c 0.37,
CH2Cl2); mmax (film)/cmꢀ1 3316 br (NH), 1732 (ester
CO), 1650 (amide CO), 1532, 1453, 1257, 1168, 747
and 698; dH (300MHz; CDCl3; Me4Si) 1.75–2.50 (8H,
m, Prob-H2, Proc-H2, Glub-H2 and Gluc-H2), 3.28–
3.54 (1.72H, m, Prod-H2), 3.25–3.74* (0.28H, m, Prod-
H2), 3.90 (1H, dd, J 17.0 and 3.7, Glya-HAHB), 4.02
(1H, dd, J 17.2 and 4.7, Glya-HAHB), 4.23–4.30*
(0.14H, m, Proa-H), 4.44–4.63 (1.86H, m, Proa-H and
Glua-H), 5.06 (2H, s, OCH2Ph), 5.10 (2H, s, OCH2Ph),
5.14 (2H, s, OCH2Ph), 5.60* (0.14H, br s, Gly-NH), 5.71
(0.86H, br s, Gly-NH) and 7.15–7.45 (16H, m, 3 · Ph
and Glu-NH); dC (75MHz; CDCl3; Me4Si) 22.2*
(CH2, Proc-C), 24.6 (CH2, Proc-C), 25.9* (CH2, Glub-
C), 26.8 (CH2, Glub-C), 27.8 (CH2, Prob-C), 30.0
(CH2, Gluc-C), 30.3* (CH2, Gluc-C), 31.8* (CH2,
Prob-C), 43.3 (CH2, Glya-C), 46.1 (CH2, Prod-C),
To a solution of methyl ester 6 (0.88g, ca. 2.75mmol) in
1,4-dioxane (30mL) was added dropwise 1M aqueous
sodium hydroxide (14mL, 14mmol) and the mixture
was stirred for 21h at room temperature. Methylene
chloride (100mL) was then added and the organic layer
extracted with saturated aqueous sodium hydrogen car-
bonate (3 · 100mL). Careful acidification of the com-
bined aqueous layers with hydrochloric acid (32%),
extraction with methylene chloride (3 · 100mL), drying
of the combined organic layers (MgSO4), filtration and
concentration to dryness gave acid 60,61 2 (0.74g, 82%
in two steps from 4). Addition of carbon tetrachloride
and its removal in vacuo allowed for removal of residual
1,4-dioxane to give acid 2 as a white solid. Compound 2
was shown to be a 75:25 trans:cis80 mixture of conform-

N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
509
47.0* (CH2, Prod-C), 51.7 (CH, Glua-C), 52.0* (CH,
Glua-C), 60.0 (CH, Proa-C), 60.1* (CH, Proa-C), 66.3
(CH2, OCH2Ph), 66.6* (CH2, OCH2Ph), 66.7 (CH2,
OCH2Ph), 67.0 (CH2, OCH2Ph), 67.2* (CH2, OCH2Ph),
127.9 (CH, Ph), 128.0 (CH, Ph), 128.1 (CH, Ph), 128.2
(CH, Ph), 128.3 (CH, Ph), 128.4 (CH, Ph), 135.2 (quat.,
Ph), 135.7 (quat., Ph), 136.3 (quat., Ph), 156.2
(quat., NCO2), 167.9 (quat., Gly-CO), 168.2* (quat.,
Gly-CO), 170.9 (quat., Pro-CON), 171.0* (quat., Pro-
CON), 171.2 (quat., Glua-CO), 171.5* (quat.,
Glua-CO), 172.5 (quat., Glu-CO) and 172.8* (quat.,
Gluc-CO); m/z (EI+) 615.2588 (M+ÆC34H37N3O8
requires 615.2581).
(2.02g, 10.5mmol) and benzyl alcohol (1.30g,
12.00mmol) in benzene (20mL) was heated under reflux,
using a Dean and Stark distilling receiver, for 68h. The
reaction mixture was cooled to room temperature and
anhydrous diethyl ether (20mL) was added to afford
p-toluenesulfonate 15 (3.04g, 86%) as a crystalline white
solid: mp 103–106ꢁC; mmax (film)/cmꢀ1 3579, 3039 br,
2942 br, 1732, 1643, 1532, 1450, 1416, 1393, 1344,
1296, 1181, 1127, 1037, 1011, 996, 948, 819, 773, 732
and 687; dH (300MHz; CDCl3; Me4Si) 1.87 (2H, quin-
tet, J 7.3, 3-H2), 2.30 and 2.32 (5H, overlapping s and
t, J 7.4, CH3 and 2-H2), 2.80–2.97 (2H, m, 4-H2), 5.02
(2H, s, OCH2Ph), 7.09 (2H, d, J 8.0, 2 · 30-H), 7.23–
7.37 (5H, m, Ph) and 7.67–7.84 and 7.73 (5H, overlap-
ping br m and d, J 8.1, N+H3 and 2 · 20-H); dC
(75MHz; CDCl3) 21.3 (CH3, CH3), 22.4 (CH2, 3-C),
30.8 (CH2, 2-C), 39.2 (CH2, 4-C), 66.3 (CH2, OCH2Ph),
125.9 (CH, Ar), 128.1 (CH, Ar), 128.2 (CH, Ar), 128.5
(CH, Ar), 129.0 (CH, Ar), 135.8 (quat., Ph), 140.6
(quat., Ar), 141.2 (quat., Ar) and 172.2 (quat., CO);
m/z (FAB+) 559.2452 [M2ÆpTsOHÆH+: (C11H15NO2)2Æ
C7H8O3SÆH requires 559.2478].
Representative procedure B for the hydrogenation of
protected tripeptides and analogues is as follows:
4.6. Glycyl-L-prolyl-L-glutamic acid15 (1)
A mixture of protected tripeptide 8 (0.13g, 0.21mmol)
and 10wt% palladium on activated carbon (0.07g,
0.07mmol) in 80:20 methanol–water (24mL; v/v) was
stirred under an atmosphere of hydrogen at room tem-
perature, protected from light, for 18h. The reaction
mixture was filtered through a CeliteTM pad and the
pad washed with 50:50 methanol–water (100mL; v/v).
The filtrate was concentrated to dryness under reduced
pressure and the residue triturated with anhydrous
diethyl ether to afford tripeptide15 1 (0.06g, 94%) as a
white solid. Compound 1 was shown to be an 80:20
trans:cis mixture of conformers: mp 209–211ꢁC (lit.,81
216ꢁC); (Found: C, 47.57; H, 6.29; N, 13.68.
C12H19N3O6 requires C, 47.84; H, 6.36; N, 13.95); [a]D
ꢀ79.1 (c 0.07, H2O) [lit.,81 ꢀ84.8 (c 0.034, H2O)]; mmax
(KBr disc)/cmꢀ1 3467 br, 3317 br, 3170 br, 2932, 1960,
1646 (amide CO), 1595, 1538, 1310, 1263, 639 and
470; dH (400MHz; D2O) 1.80–2.07 (4H, m, Glub-HAHB,
Prob-HAHB and Proc-H2), 2.07–2.23 (1H, m, Glub-
HAHB), 2.23–2.32 (1H, m, Prob-HAHB), 2.40 (2H, t, J
7.6, Gluc-H2), 3.47–3.67 (2H, m, Prod-H2), 3.72*
(0.20H, d, J 16.2, Glya-HAHB), 3.94* (0.20H, d, J
16.4, Glya-HAHB), 3.96 (0.80H, d, J 16.6, Glya-HAHB),
4.00 (0.80H, d, J 16.6, Glya-HAHB), 4.21 (1H, dd, J 8.7
and 4.9, Glua-H) and 4.47 (1H, dd, J 8.3 and 4.0, Proa-
H); dC (100MHz; D2O) 24.8* (CH2, Proc-C), 27.0 (CH2,
Proc-C), 29.3* (CH2, Glub-C), 29.8 (CH2, Glub-C), 32.1
(CH2, Prob-C), 33.9 (CH2, Gluc-C), 34.5* (CH2, Gluc-
C), 43.0* (CH2, Glya-C), 43.2 (CH2, Glya-C), 49.6
(CH2, Prod-C), 50.3* (CH2, Prod-C), 57.0 (CH, Glua-
C), 57.4* (CH, Glua-C), 62.9* (CH, Proa-C), 63.3
(CH, Proa-C), 168.4 (quat., Gly-CO), 168.8* (quat.,
Gly-CO), 175.7* (quat., Pro-CON), 176.2 (quat., Pro-
CON), 180.1 (quat., Glua-CO) and 181.4 (quat., Gluc-
CO); m/z (FAB+) 302.1355 (MH+ÆC12H20N3O6 requires
302.1352).
4.8. Benzyl N-benzyloxycarbonyl-glycyl-L-prolyl-4-
aminobutanoate (42)
Following procedure A, coupling of acid 2 with p-tolu-
enesulfonate 15 yielded amide 42 (1.12g, 86%) as a
slightly opaque oil. Compound 42 was shown to be an
88:12 trans:cis mixture of conformers: Rf 0.15 (EtOAc);
[a]D ꢀ66.1 (c 0.39, CH2Cl2); mmax (film)/cmꢀ1 3314 br,
2950, 1727, 1649, 1535, 1452, 1255, 1167, 1054, 985,
740 and 698; dH (300MHz; CDCl3; Me4Si) 1.65–2.27
(3H, m, Prob-HAHB and Proc-H2), 1.83 (2H, quintet,
J 7.0, 3-H2), 2.28–2.47 (1H, m, Prob-HAHB), 2.39 (2H,
t, J 7.2, 2-H2), 3.14–3.70 (4H, m, 4-H2 and Prod-H2),
3.72–4.07 (2H, m, Glya-H2), 4.19–4.27* (0.12H, m,
Proa-H), 4.50 (0.88H, dd, J 8.0 and 1.5, Proa-H), 5.10
(2H, s, OCH2Ph), 5.11 (2H, s, OCH2Ph), 5.58*
(0.12H, br s, Gly-NH), 5.68 (0.88H, br s, Gly-NH),
6.70* (0.12H, br s, 4-NH), 6.95 (0.88H, br s, 4-NH)
and 7.24–7.43 (10H, m, 2 · Ph); dC (75MHz; CDCl3)
22.1* (CH2, Proc-C), 24.1* (CH2, 3-C), 24.2 (CH2, 3-
C), 24.5 (CH2, Proc-C), 28.0 (CH2, Prob-C), 31.4
(CH2, 2-C), 31.8* (CH2, Prob-C), 38.6 (CH2, 4-C),
38.9* (CH2, 4-C), 43.0* (CH2, Glya-C), 43.2 (CH2,
Glya-C), 46.1 (CH2, Prod-C), 46.8* (CH2, Prod-C),
60.1 (CH, Proa-C), 66.0 (CH2, OCH2Ph), 66.1* (CH2,
OCH2Ph), 66.6 (CH2, OCH2Ph), 127.7* (CH, Ph),
127.7 (CH, Ph), 127.9 (CH, Ph), 127.9 (CH, Ph), 128.2
(CH, Ph), 128.3 (CH, Ph), 135.6* (quat., Ph), 135.7
(quat., Ph), 136.2 (quat., Ph), 156.2 (quat., NCO2),
156.4* (quat., NCO2), 167.9* (quat., Gly-CO), 168.0
(quat., Gly-CO), 170.9 (quat., Pro-CON), 171.0* (quat.,
Pro-CON) and 173.0 (quat., 1-CO); m/z (EI+) 481.2201
(M+ÆC26H31N3O6 requires 481.2213).
Representative procedure C for the benzylation of carb-
oxylic acids is as follows:
4.9. Glycyl-L-prolyl-4-aminobutanoic acid (9)
4.7. Benzyl 4-aminobutanoate p-toluenesulfonate (15)
Following procedure B, hydrogenation of amide 42
yielded analogue 9 (0.26g, 97%) as an extremely hygro-
scopic, white amorphous solid. Compound 9 was shown
to be an 86:14 trans:cis mixture of conformers: [a]D
A mixture of 4-aminobutanoic acid 12 (1.00g,
9.70mmol), p-toluenesulfonic acid monohydrate

510
N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
ꢀ92.0 (c 0.19, H2O); mmax (film)/cmꢀ1 3262 br, 2952 br,
2644, 1659, 1558, 1457, 1429, 1401, 1355, 1302, 1245,
1191, 1092 and 919; dH (400MHz; D2O) 1.79 (2H, quin-
tet, J 7.0, 3-H2), 1.87–2.14 (3H, m, Prob-HAHB and
Proc-H2), 2.24 (2H, t, J 7.4, 2-H2), 2.14–2.48 (1H,
m, Prob-HAHB), 3.17–3.33 (2H, m, 4-H2), 3.53–3.72
(2H, m, Prod-H2), 3.92–4.12 (2H, m, Glya-H2), 4.44
(0.86H, dd, J 8.3 and 4.0, Proa-H) and 4.40–4.53*
(0.14H, m, Proa-H); dC (100MHz; D2O) 23.9* (CH2,
Proc-C), 26.0 (CH2, Proc-C), 26.9 (CH2, 3-C), 27.1*
(CH2, 3-C), 31.5 (CH2, Prob-C), 33.8* (CH2, Prob-C),
36.6 (CH2, 2-C), 41.2 (CH2, 4-C), 41.3* (CH2, 4-C),
42.2* (CH2, Glya-C), 42.4 (CH2, Glya-C), 48.7 (CH2,
Prod-C), 49.4* (CH2, Prod-C), 62.0* (CH, Proa-C),
62.7 (CH, Proa-C), 168.0 (quat., Gly-CO), 168.3*
(quat., Gly-CO), 175.0* (quat., Pro-CON), 175.6 (quat.,
Pro-CON), 184.2* (quat., 1-CO) and 184.5 (quat., 1-
CO); m/z (FAB+) 258.1470 (MH+ÆC11H20N3O4 requires
258.1454).
Glya-H2), 3.95 (3H, s, Glya-H2 and 4-H), 4.24*
(0.12H, br s, Proa-H), 4.46 (0.88H, d, J 7.5, Proa-H),
5.09 (4H, d, J 6.9, 2 · OCH2Ph), 5.80 (1H, br s, Gly-
NH), 6.64* (0.12H, d, J 8.3, 4-NH), 6.76 (0.88H, d, J
7.9, 4-NH) and 7.32–7.34 (10H, m, 2 · Ph); dC
(75MHz; CDCl3) 20.4* (CH3, 5-C), 20.6 (CH3, 5-C),
22.3* (CH2, Proc-C), 24.7 (CH2, Proc-C), 27.7 (CH2,
Prob-C), 29.6* (CH2, 3-C), 30.8 (CH2, 3-C), 31.3
(CH2, 2-C), 31.9* (CH2, 2-C), 43.3 (CH2, Glya-C),
44.9 (CH, 4-C), 45.4* (CH, 4-C), 46.2 (CH, Prod-C),
47.1* (CH, Prod-C), 60.4 (CH, Proa-C), 66.2 (CH2,
OCH2Ph), 66.4* (CH2, OCH2Ph), 66.8 (CH2, OCH2Ph),
127.9 (CH, Ph), 128.0 (CH, Ph), 128.1 (CH, Ph), 128.4
(CH, Ph), 128.44 (CH, Ph), 135.8 (quat., Ph), 136.6
(quat., Ph), 156.3 (quat., NCO2), 168.2 (quat., Gly-
CO), 170.2 (quat., Pro-CON) and 173.4 (quat., 1-CO);
m/z (FAB+) 495.2373 (MH+ÆC27H33N3O6 requires
495.2370).
4.12. (4R)-Glycyl-L-prolyl-4-aminopentanoic acid (10)
4.10. (4R)-Benzyl 4-aminopentanoate p-toluenesulfonate
(16)
Following procedure B, hydrogenation of amide 43 in
methanol yielded analogue 10 (0.13g, 93%) as a white
foam. Compound 10 was shown to be a 76:24 trans:cis
mixture of conformers: mp 65–75ꢁC; [a]D ꢀ57.1 (c
0.11, CH3OH); dH (300MHz; D2O) 1.19 (3H, d, J 6.5,
5-H3), 1.69–2.39 (8H, m, 3-H2, Prob-H2, Proc-H2 and
2-H2), 3.30–3.38 (1H, m, Prod-HAHB), 3.57–2.67
(1.24H, m, Prod-HAHB and Glya-HAHB*), 3.88–4.13
(3.24H, m, Glya-H2, 4-H and Glya-HAHB*), 4.44
(0.76H, dd, J 8.8 and 4.8, Proa-H) and 4.50* (0.24H,
dd, J 8.8 and 3.2, Proa-H); dC (75MHz; D2O) 19.5
(CH3, 5-C), 19.5* (CH3, 5-C), 21.9* (CH2, Proc-C),
24.1 (CH2, Proc-C), 29.5 (CH2, Prob-C), 31.2* (CH2,
3-C), 31.4 (CH2, 3-C), 32.0* (CH2, 2-C), 32.4 (CH2, 2-
C), 40.0* (CH2, Glya-C), 40.3 (CH2, Glya-C), 45.5
(CH, 4-C), 45.8* (CH, 4-C), 46.8 (CH2, Prod-C), 47.5*
(CH2, Prod-C), 59.8* (CH, Proa-C), 60.7 (CH, Proa-
C), 165.4 (quat., Gly-CO), 173.0 (quat., Pro-CON)
and 180.7 (quat., 1-CO); m/z (FAB+) 272.1617
(MH+ÆC12H22N3O4 requires 272.1610).
Hydrochloride70 13 (0.50g, 3.25mmol) was dissolved in
water and added to an Amberlite IR-120(H) ion ex-
change column (2cm · 15cm). The column was eluted
with water (200mL) then 2M ammonia solution. Nin-
hydrin-positive fractions (TLC, 1:1, methanol–methyl-
ene chloride, 3–4 drops ammonia solution) were
collected, combined and the solvent removed to afford
an oil (0.45g). A solution of this oil, benzyl alcohol
(5mL) and p-toluenesulfonic acid (0.62g, 3.25mmol)
in benzene (20mL), were heated under reflux, using a
Dean and Stark distilling receiver, for 19h. The solution
was cooled to room temperature and anhydrous diethyl
ether added to give p-toluenesulfonate 16 (1.06g, 86%) as
an off-white solid: mp 134–137ꢁC; [a]D +9.5 (c 0.20,
CH3OH); dH (300MHz; CDCl3; Me4Si) 1.20 (3H, d, J
6.6, 5-H3), 1.75–2.06, (2H, m, 3-H2), 2.32 (3H, s, Ar-
CH3), 2.41 (2H, t, J 7.6, 2-H2), 3.24–3.27 (1H, m,
4-H), 5.00–5.09 (2H, m, OCH2Ph), 7.09 (2H, d, J 8.0,
30-H); 7.27–7.38 (5H, m, Ph), 7.76 (2H, d, J 8.0, 20-H)
and 7.82 (2H, br s, NH2); dC (75MHz; CDCl3) 18.3
(CH3, 5-C), 21.2 (CH3, Ar-CH3) 29.4 (CH2), 29.8
(CH2), 47.5 (CH, 4-C), 66.3 (CH2, OCH2Ph), 125.9
(CH, Ph), 128.1 (CH, Ph), 128.15 (CH, Ph), 128.5
(CH, Ph), 128.9 (CH, Ph), 135.8 (quat., Ph), 140.6
(quat., Ph), 141.2 (quat., Ph) and 172.3, (quat., 1-CO);
m/z (FAB+) 587.2781 [M2ÆpTsOHÆH+: C12H17NO2)2Æ
C7H8O3SÆH requires 587.2791].
4.13. Benzyl 4-amino-4-methylpentanoate p-toluene-
sulfonate (17)
Following procedure C, benzylation of acid71 14 yielded
p-toluenesulfonate 17 (1.29g, 65%) as a crystalline white
solid: mp 113–115ꢁC; mmax (film)/cmꢀ1 3582, 2913 br,
2623 br, 2326 br, 2028 br, 1729, 1631, 1530, 1447,
1400, 1378, 1313, 1205, 1183, 1125, 1035, 1017, 811,
724 and 681; dH (300MHz; CDCl3; Me4Si) 1.24 (6H, s,
4-CCH3 and 5-H3), 1.95 (2H, br t, J 8.0, 3-H2), 2.30
(3H, s, Ar-CH3), 2.45 (2H, br t, J 8.0, 2-H2), 5.05 (2H,
s, OCH2Ph), 7.01 (2H, d, J 7.5, 2 · 30-H), 7.16–7.40
(5H, m, Ph), 7.70 (2H, d, J 7.8, 2 · 20-H) and 7.84
(3H, br s, N+H3); dC (100MHz; CDCl3) 21.3 (CH3,
Ar-CH3), 25.1 (CH3, 4-CCH3 and 5-C), 28.6 (CH2, 3-
C), 34.7 (CH2, 2-C), 54.0 (quat., 4-C), 66.4 (CH2,
OCH2Ph), 125.9 (CH, Ar), 128.1 (CH, Ar), 128.5 (CH,
Ar), 128.9 (CH, Ar), 135.8 (quat., Ph), 140.3 (quat.,
Ar), 141.1 (quat., Ar) and 172.6 (quat., 1-CO); m/z
(FAB+) 615.3123 [M2ÆpTsOHÆH+: (C13H19NO2)2Æ
C7H8O3SÆH requires 615.3104].
4.11. (4R)-Benzyl N-benzyloxycarbonyl-glycyl-L-prolyl-
4-aminopentanoate (43)
Following procedure A, coupling of acid 2 with p-tolu-
enesulfonate 16 yielded amide 43 (0.39g, 80%) as a col-
ourless oil. Compound 43 was shown to be an 88:12
trans:cis mixture of conformers: [a]D ꢀ58.2 (c 0.59,
CH2Cl2); dH (300MHz; CDCl3; Me4Si) 1.12 (3H, d, J
6.0, 5-H3), 1.72–2.29 (6H, m, 3-H2, Prob-H2 and Proc-
H2), 2.37 (2H, t, J 7.6, 2-H2), 3.30–3.38 (0.88H, m,
Prod-HAHB), 3.37–3.56 (0.88H, m, Prod-HAHB), 3.59–
3.64* (0.24H, m, Prod-H2), 3.72–3.79* (0.24H, m,

N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
511
4.14. Benzyl N-benzyloxycarbonyl-glycyl-L-prolyl-4-
amino-4-methylpentanoate (44)
3.62 (2H, m, Prod-H2), 3.83* (0.20H, d, J 16.6, Glya-
HAHB), 3.87 (0.80H, d, J 16.8, Glya-HAHB), 3.94
(0.80H, d, J 16.5, Glya-HAHB), 4.31 (0.80H, dd, J 8.3
and 4.5, Proa-H) and 4.36* (0.20H, dd, J 8.4 and 3.2,
Proa-H); dC (100MHz; D2O) 23.9* (CH2, Proc-C),
26.0 (CH2, Proc-C), 27.3 [CH3, 4-C(CH3)A(CH3)B],
27.5* [CH3, 4-C(CH3)A(CH3)B], 27.6* [CH3, 4-
C(CH3)A(CH3)B], 27.8 [CH3, 4-C(CH3)A(CH3)B], 31.6
(CH2, Prob-C), 34.1* (CH2, Prob-C), 34.2 (CH2, 3-C),
34.5* (CH2, 3-C), 37.7* (CH2, 2-C), 38.1 (CH2, 2-C),
42.3* (CH2, Glya-C), 42.5 (CH2, Glya-C), 48.7 (CH2,
Prod-C), 49.5* (CH2, Prod-C), 55.6 (quat., 4-C), 56.0*
(quat., 4-C), 62.1* (CH, Proa-C), 63.0 (CH, Proa-C),
168.1 (quat., Gly-CO), 168.8* (quat., Gly-CO), 174.1*
(quat., Pro-CON), 174.9 (quat., Pro-CON), 184.8*
(quat., 1-CO) and 185.1 (quat., 1-CO); m/z (FAB+)
286.1775 (MH+ÆC13H24N3O4 requires 286.1767).
Following procedure A, coupling of acid 2 with p-tolu-
enesulfonate 17 yielded amide 44 (0.91g, 93%) as a crys-
talline white solid. Compound 44 was shown to be a 92:8
trans:cis mixture of conformers: mp 140–142ꢁC; Rf 0.50
(EtOAc); [a]D ꢀ71.3 (c 0.49, CH2Cl2); mmax (film)/cmꢀ1
3305 br, 2971 br, 1724, 1650, 1545, 1452, 1284,
1253, 1162, 1043, 984, 735 and 697; dH (400MHz;
CDCl3; Me4Si) 1.27 [2.76H, s, 4-C(CH3)A(CH3)B], 1.30
[2.76H, s, 4-C(CH3)A(CH3)B], 1.36* [0.24H, s, 4-
C(CH3)A(CH3)B], 1.37* [0.24H, s, 4-C(CH3)A(CH3)B],
1.77–2.22 (5H, m, Prob-HAHB, Proc-H2 and 3-H2),
2.24–2.47 (3H, m, Prob-HAHB and 2-H2), 3.35 (1H,
dd, J 16.7 and 8.9, Prod-HAHB), 3.43–3.72 (1H, m,
Prod-HAHB), 3.79* (0.08H, dd, J 17.0 and 4.5, Glya-
HAHB), 3.87* (0.08H, dd, J 17.0 and 5.2, Glya-HAHB),
3.95 (0.92H, dd, J 17.1 and 4.2, Glya-HAHB), 4.02
(0.92H, dd, J 17.3 and 4.8, Glya-HAHB), 4.07–4.13*
(0.08H, m, Proa-H), 4.42 (0.92H, dd, J 8.0 and 1.7,
Proa-H), 5.09 (2H, s, OCH2Ph), 5.11 (2H, s, OCH2Ph),
5.62* (0.08H, br s, Gly-NH), 5.68 (0.92H, br s, Gly-
NH), 6.52* (0.08H, br s, 4-NH), 6.70 (0.92H, br s, 4-
NH) and 7.21–7.41 (10H, m, 2 · Ph); dC (100MHz;
CDCl3) 22.4* (CH2, Proc-C), 24.7 (CH2, Proc-C),
25.9* [CH3, 4-C(CH3)A(CH3)B], 26.3* [CH3, 4-
C(CH3)A(CH3)B], 26.6 [CH3, 4-C(CH3)A(CH3)B], 26.6
[CH3, 4-C(CH3)A(CH3)B], 27.6 (CH2, Prob-C), 29.1*
(CH2, 3-C), 29.4 (CH2, 3-C), 32.2* (CH2, Prob-C),
34.7 (CH2, 2-C), 35.2* (CH2, 2-C), 43.2* (CH2, Glya-
C), 43.4 (CH2, Glya-C), 46.2 (CH2, Prod-C), 47.1*
(CH2, Prod-C), 53.0 (quat., 4-C), 53.6* (quat., 4-C),
60.2* (CH, Proa-C), 60.8 (CH, Proa-C), 66.3 (CH2,
OCH2Ph), 66.7* (CH2, OCH2Ph), 66.8 (CH2, OCH2Ph),
127.2* (CH, Ph), 127.4* (CH, Ph), 128.0 (CH, Ph), 128.0
(CH, Ph), 128.1 (CH, Ph), 128.2* (CH, Ph), 128.4 (CH,
Ph), 128.5 (CH, Ph), 135.6* (quat., Ph), 135.8 (quat.,
Ph), 136.3 (quat., Ph), 156.2 (quat., NCO2), 167.8*
(quat., Gly-CO), 168.1 (quat., Gly-CO), 170.0 (quat.,
Pro-CON), 170.4* (quat., Pro-CON) 173.7 (quat., 1-
CO) and 174.2* (quat., 1-CO); m/z (FAB+) 510.2599
(MH+ÆC28H36N3O6 requires 510.2604).
4.16. Benzyl N-tert-butyloxycarbonyl-L-c-glutamate72
(21)
To a solution of benzyl L-c-glutamate 45 (2.00g,
8.40mmol) in 1:1 dioxane–water (30mL, v/v) at 0ꢁC
was successively added di-tert-butyl dicarbonate
(1.95g, 8.90mmol) in one portion, and triethylamine
(1.40mL, 10.00mmol) dropwise. The solution was stir-
red for 1h, warmed to room temperature and stirred
for a further 20h then washed with diethyl ether
(20mL). The aqueous layer was acidified with 5M
HCl solution (2mL) to pH1 then extracted with ethyl
acetate (2 · 50mL) and the combined organic fractions
washed with brine (20mL), dried (MgSO4), filtered
and concentrated to dryness in vacuo. The resultant
oil was purified by flash column chromatography (ethyl
acetate) to afford acid 21 (2.75g, 97%) as a light green
oil: dH (200MHz; CDCl3; Me4Si) 1.25 [9H, s,
C(CH3)3], 1.95–2.00 (1H, m, Glub-HAHB), 2.10–2.20
(1H, m, Glub-HAHB), 2.37–2.46 (2H, m, Gluc-H2),
4.00–4.05 (1H, m, Glua-H), 5.03 (2H, s, OCH2Ph),
5.10–5.20 (1H, m, Glua-NH), 7.25 (5H, s, Ph) and
9.63 (1H, br s, CO2H); dC (50MHz; CDCl3) 27.4
(CH2, Glub-C), 28.2 [CH3, C(CH3)3], 30.3 (CH2, Gluc-
C), 52.7 (CH, Glua-C), 66.5 (CH2, OCH2Ph), 80.3
[quat., C(CH3)3], 128.2 (CH, Ph), 128.3 (CH, Ph),
128.5 (CH, Ph), 135.7 (quat., Ph), 155.6 (quat.,
NCO2), 172.7 (quat., Gluc-CO) and 176.0 (quat.,
Glua-CO); m/z (EI+) 338 (MH+, 20 %).
4.15. Glycyl-L-prolyl-4-amino-4-methylpentanoic acid
(11)
Following procedure B, hydrogenation of amide 44 in
1.6:1.3:1.0 tetrahydrofuran–methanol–water yielded
analogue 11 (0.23g, 89%) as an hygroscopic, off-white
amorphous solid following purification by reverse-phase
flash column chromatography (10g C18SiO2; 0–20%
methanol–water; gradient elution). Compound 11 was
shown to be an 80:20 trans:cis mixture of conformers:
Rf 0.20 (MeOH) and 0.20 (C18SiO2, 30% MeOH/H2O);
mp 144–172ꢁC (decomp.); [a]D ꢀ82.9 (c 0.23, H2O),
[a]D ꢀ112.0 (c 0.19, MeOH); mmax (film)/cmꢀ1 3266 br,
3068 br, 2969, 2652, 1658, 1560, 1457, 1398, 1360,
1307, 1266, 1203, 1169, 1139, 1035, 921 and 845; dH
(400MHz; D2O) 1.28 and 1.31* (6H, overlapping s
and s, 2 · 4-CCH3), 1.77–2.08 (5H, m, Prob-HAHB,
Proc-H2 and 3-H2), 2.09–2.42 (3H, m, Prob-HAHB
and 2-H2), 3.42* (0.20H, d, J 16.3, Glya-HAHB), 3.46–
4.17. Benzyl N-tert-butyloxycarbonyl-L-glutamate
a-amide73 (22)
To a solution of acid 21 (0.87g, 2.57mmol) in 1,4-diox-
ane (25mL), at room temperature under nitrogen, was
successively added di-tert-butyl dicarbonate (0.73g,
3.33mmol), ammonium hydrogen carbonate (0.27g,
3.35mmol) and pyridine (0.27mL, 3.33mmol) dropwise.
The resultant solution was stirred for 17h, the solvent
removed in vacuo and the residue partitioned between
water (20mL) and ethyl acetate (20mL). The organic
layer was dried (MgSO4), filtered and concentrated to
dryness to give a solid which was washed with hexane
(2 · 10mL) to afford amide73 22 (0.85g, 98%) as a white

512
N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
solid: mp 123–125ꢁC (lit.,73 122–123ꢁC); [a]D +4.7 (c
0.06, MeOH) [lit.,73 +4.1 (c 1.00, EtOH)]; dH
(200MHz; CDCl3; Me4Si) 1.42 [9H, s, C(CH3)3], 1.87–
2.00 (1H, m, Glub-HAHB), 2.11–2.21 (1H, m, Glub-
HAHB), 2.45–2.57 (2H, m, Gluc-H2), 4.21–4.23 (1H,
m, Glua-H), 5.12 (2H, s, OCH2Ph), 5.40–5.42 (1H, m,
Glua-NH), 5.82 (1H, br s, CONHAHB), 6.42 (1H, br
s, CONHAHB) and 7.35 (5H, s, Ph); dC (50MHz;
CDCl3) 27.8 (CH2, Glub-C), 28.2 [CH3, C(CH3)3], 30.4
(CH2, Gluc-C), 53.2 (CH, Glua-C), 66.5 (CH2,
OCH2Ph), 80.1 [quat., C(CH3)3], 128.2 (CH, Ph),
128.3 (CH, Ph), 128.5 (CH, Ph), 135.6 (quat., Ph),
155.7 (quat., NCO2), 173.1 (quat., Gluc-CO) and
174.0 (quat., Glua-CO); m/z (FAB+) 337 (MH+, 37%).
(quat., Ph), 136.2 (quat., Ph), 156.9 (quat., NCO2),
169.8 (quat., Gly-CO), 172.1 (quat., Gluc-CO), 174.2
(quat., Glua-CO) and 174.4 (quat., Pro-CON);
m/z (FAB+) 525.2381 (MH+ÆC27H33N4O7 requires
525.2349).
4.20. Glycyl-L-prolyl-L-glutamic acid a-amide56 (18)
Following procedure B, hydrogenation of amide 28 in
methanol yielded analogue56 18 (0.19g, 65%) as a white
solid. Compound 18 was shown to be an 87:13 trans:cis
mixture of conformers: mp 112–114ꢁC; [a]D ꢀ68.5 (c
0.05, MeOH); dH (200MHz; D2O) 1.85–2.03 (6H, m,
Prob-H2, Proc-H2 and Glub-H2), 2.20–2.29 (2H, m,
Gluc-H2), 3.48–3.61 (2H, m, Prod-H2), 3.94–3.96 (2H,
m, Glya-H2), 4.14 (1H, m, Glua-H) and 4.39–4.43
(1H, m, Proa-H); dC (50MHz; D2O) 24.9 (CH2, Proc-
C), 27.6 (CH2, Glub-C), 30.0 (CH2, Prob-C), 33.9
(CH2, Gluc-C), 41.1 (CH2, Glya-C), 47.6 (CH2, Prod-
C), 54.5 (CH, Glua-C), 61.0* (CH, Proa-C), 61.3 (CH,
Proa-C), 166.8 (quat., Gly-CO), 174.7 (quat., Pro-
CON), 176.9 (quat., Glua-CO) and 182.2 (quat., Gluc-
CO); m/z (FAB+) 301.1507 (MH+ÆC12H21N4O5 requires
301.1512).
Representative procedure D for amine deprotection is as
follows:
4.18. Benzyl L-glutamate a-amide trifluoroacetate (25)
Trifluoroacetic acid (1mL) was added dropwise to a
solution of amide 22 (0.10g, 0.31mmol) in methylene
chloride (4mL), under nitrogen at 0ꢁC, and the reaction
mixture stirred for 1.5h then concentrated to dryness in
vacuo. The resultant residue was repeatedly dissolved in
toluene and the solvent removed (2 · 3mL) to give tri-
fluoroacetate 25 (0.10g) as an orange oil.
4.21. Benzyl N-tert-butyloxycarbonyl-L-glutamate
a-N-methylamide74 (23)
To a solution of acid 21 (0.80g, 2.40mmol) and (benzo-
triazole-1-yloxy)tripyrrolidinophosphonium hexafluoro-
phosphate (PyBoP) (1.25g, 2.40mmol) in methylene
chloride (40mL), at room temperature under nitrogen,
was successively added methylamine hydrochloride
(0.20g, 2.80mmol), and triethylamine (0.92mL,
2.40mmol) dropwise. The reaction mixture was stirred
for 3days, washed with saturated sodium hydrogen car-
bonate (25mL) and aqueous 2M citric acid (25mL),
dried (MgSO4), filtered and concentrated to dryness in
vacuo. The resultant oil was purified by flash column
chromatography (ethyl acetate) and trituration with
diethyl ether–10% hexane (10mL) to give amide74 23
(0.63g, 75%) as a white solid: mp 120–122ꢁC (lit.,74
120–123ꢁC); [a]D +4.9 (c 0.06, MeOH) [lit.,74 +6.0 (c
0.10, CDCl3)]; dH [200MHz, (CD3)2SO] 1.37 [9H, s,
C(CH3)3], 1.74–1.93 (2H, m, Glub-H2), 2.36 (2H, t, J
7.4, Gluc-H2), 2.58 (3H, d, J 4.7, NHCH3), 3.89–3.92
(1H, m, Glua-H), 5.09 (2H, s, OCH2Ph), 6.88 (1H, d,
J 8.1 Glua-NH), 7.36 (5H, s, Ph) and 7.71–7.76 (1H,
m, NHCH3); dC [50MHz, (CD3)2SO] 25.5 (CH3,
NCH3), 27.1 (CH2, Glub-C), 28.1 [CH3, C(CH3)3],
30.1 (CH2, Gluc-C), 53.4 (CH, Glua-C), 65.3 (CH2,
OCH2Ph), 78.0 [quat., C(CH3)3], 127.8 (CH, Ph),
127.9 (CH, Ph), 128.3 (CH, Ph), 136.1 (quat., Ph),
155.2 (quat., NCO2), 171.7 (quat., Glua-CO)
and 172.1 (quat., Gluc-CO); m/z (FAB+) 351 (MH+,
40 %).
Representative procedure E for the preparation of pro-
tected tripeptide analogues from acid 2 is as follows:
4.19. Benzyl N-benzyloxycarbonyl-glycyl-L-prolyl-L-
glutamate a-amide (28)
Diisopropylethylamine (0.16mL, 0.92mmol) was added
dropwise to a solution of acid 2 (0.10g, 0.31mmol),
crude trifluoroacetate 25 (0.07g, 0.31mmol) and bis(2-
oxo-3-oxazolidinyl)phosphinic chloride (BoPCl) (0.08g,
0.31mmol) in methylene chloride (5mL) under nitrogen
at 0ꢁC. The solution was stirred for 1.5h, warmed to
room temperature and stirred for a further 20h then
washed with saturated sodium hydrogen carbonate
(10mL), aqueous 2M citric acid (10mL), dried
(MgSO4), filtered and concentrated to dryness in vacuo.
The resultant residue was purified by flash column chro-
matography (10% methanol–ethyl acetate) to yield
amide 28 (0.11g, 69%) as a colourless oil, which solidi-
fied on standing to a white solid: mp 118–120ꢁC; [a]D
ꢀ58.1 (c 0.05, MeOH); dH (200MHz; CDCl3; Me4Si)
1.88–2.04 (6H, m, Prob-H2, Proc-H2 and Glub-H2),
2.42–2.44 (2H, m, Gluc-H2), 3.40–3.50 (1H, m, Prod-
HAHB), 3.50–3.60 (1H, m, Prod-HAHB), 3.89–3.92
(2H, m, Glya-H2), 4.30–4.40 (2H, m, Proa-H and
Glua-H), 5.00–5.09 (4H, m, 2 · OCH2Ph), 6.20–6.29
(2H, m, Pro-NH and CONHAHB), 6.89 (1H, br s, CON-
HAHB), 7.27–7.29 (10H, m, 2 · Ph) and 7.67 (1H, d, J
8.0, Glu-NH); dC (50MHz; CDCl3) 24.6 (CH2, Proc-
C), 25.7 (CH2, Glub-C), 29.1 (CH2, Prob-C), 30.5
(CH2, Gluc-C), 43.4 (CH2, Glya-C), 46.7 (CH2, Prod-
C), 52.9 (CH, Glua-C), 61.3 (CH, Proa-C), 66.6 (CH2,
OCH2Ph), 66.9 (CH2, OCH2Ph), 127.9 (CH, Ph),
128.2 (CH, Ph), 128.4 (CH, Ph), 128.5 (CH, Ph), 135.5
4.22. Benzyl L-glutamate a-N-methylamide trifluoro-
acetate (26)
Following procedure D, treatment of amide 23 with tri-
fluoroacetic acid yielded trifluoroacetate 26 (0.63g) as an
orange oil.

N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
513
4.23. Benzyl N-benzyloxycarbonyl-glycyl-L-prolyl-L-
glutamate a-N-methylamide (29)
gen carbonate (10mL), aqueous 2M citric acid
(10mL), dried (MgSO4), filtered and concentrated to
dryness in vacuo. The resultant residue was purified by
flash column chromatography (10% diethyl ether–meth-
ylene chloride) to yield amide 24 (1.08g, 100%) as a
green oil: dH (200MHz; CDCl3; Me4Si) 1.42 [9H, s,
C(CH3)3], 1.73–1.74 (1H, m, Glub-HAHB), 2.00–2.10
(1H, m, Glub-HAHB), 2.44–2.54 (2H, m, Gluc-H2),
2.90 (3H, s, NCH3), 3.10 (3H, s, NCH3), 4.70–4.71
(1H, m, Glua-H), 5.13 (2H, s, OCH2Ph), 5.48 (1H, d,
J 8.1, Glua-NH) and 7.36 (5H, s, Ph); dC (50MHz,
CDCl3) 28.1 (CH2, Glub-C), 28.2 [CH3, C(CH3)3], 29.5
(CH2, Gluc-C), 35.6 (CH3, NCH3), 36.9 (CH3, NCH3),
49.2 (CH, Glua-C), 66.3 (CH2, OCH2Ph), 79.5 [quat.,
C(CH3)3], 128.2 (CH, Ph), 128.5 (CH, Ph), 135.8 (quat.,
Ph), 155.6 (quat., NCO2), 171.6 (quat., Glua-CO) and
172.8 (quat., Gluc-CO); m/z (FAB+) 364.1999
(M+ÆC19H28N2O5 requires 364.1998).
Following procedure E, coupling of acid 2 with trifluoro-
acetate 26 yielded amide 29 (0.52g, 55%) as a colourless
oil: [a]D ꢀ43.8 (c 0.05, MeOH); dH (200MHz; CDCl3;
Me4Si) 1.93–2.25 (6H, m, Prob-H2, Proc-H2 and Glub-
H2), 2.46–2.54 (2H, m, Gluc-H2), 2.69–2.75 (3H, d, J
7.5, NCH3), 3.42–3.47 (1H, m, Prod-HAHB), 3.60–3.65
(1H, m, Prod-HAHB), 3.93–4.17 (2H, m, Glya-H2),
4.42–4.45 (2H, m, Proa-H and Glua-H), 4.98 (4H, s,
OCH2Ph), 5.10 (2H, s, OCH2Ph), 5.77 (1H, br s, Gly-
NH), 6.85 (1H, br s, NHCH3), 7.32 (5H, s, Ph), 7.45
(5H, s, Ph) and 7.63–7.67 (1H, m, Glu-NH); dC
(50MHz, CDCl3) 24.7 (CH2, Proc-C), 25.7 (CH3,
NCH3), 26.2 (CH2, Glub-C), 28.9 (CH2, Prob-C), 30.6
(CH2, Gluc-C), 43.5 (CH2, Glya-C), 46.7 (CH2, Prod-
C), 53.0 (CH, Glua-C), 61.2 (CH, Proa-C), 66.7 (CH2,
OCH2Ph), 67.1 (CH2, OCH2Ph), 127.9 (CH, Ph),
128.0 (CH, Ph), 128.4 (CH, Ph), 128.5 (CH, Ph), 135.4
(quat., Ph), 138.0 (quat., Ph), 156.5 (quat., NCO2),
169.1 (quat., Gly-CO), 171.2 (quat., Glua-CO), 171.3
(quat., Gluc-CO) and 174.7 (quat., Pro-CON); m/z
4.26. Benzyl-L-glutamate a-N,N-dimethylamide trifluoro-
acetate (27)
Following procedure D, treatment of amide 24 with tri-
fluoroacetic acid yielded trifluoroacetate 27 (0.98g) as an
orange oil.
(FAB+)
539.2504
(MH+ÆC28H35N4O7
requires
539.2506).
4.24. Glycyl-L-prolyl-L-glutamic acid a-N-methylamide
(19)
4.27. Benzyl N-benzyloxycarbonyl-glycyl-L-prolyl-L-
glutamate a-N,N-dimethylamide (30)
Following procedure B, hydrogenation of amide 29 in
methanol yielded analogue 19 (0.19g, 97%) as a white
solid. Compound 19 was shown to be an 83:17 trans:cis
mixture of conformers: mp 42–44ꢁC; [a]D ꢀ58.2 (c 0.08,
MeOH); dH (200MHz; D2O) 1.92–2.40 (6H, m, Prob-
H2, Proc-H2 and Glub-H2), 2.46 (2H, t, J 7.5,
Gluc-H2), 2.72 (3H, s, NHCH3), 3.55–3.65 (2H, m,
Prod-H2), 3.99 (2H, s, Glya-H2), 4.26–4.27 (1H, m,
Glua-H), 4.47–4.50 (1H, m, Proa-H); dC (50MHz;
D2O) 25.2 (CH2, Proc-C), 26.7 (CH3, NCH3), 27.0
(CH2, Glub-C), 30.4 (CH2, Prob-C), 31.1 (CH2, Gluc-
C), 41.0* (CH2, Glya-C), 41.3 (CH2, Glya-C), 47.8
(CH2, Prod-C), 54.3 (CH, Glua-C), 61.3 (CH, Proa-
C), 166.6 (quat., Gly-CO), 174.4 (quat., Glua-CO),
175.0 (quat., Gluc-C) and 178.2 (quat., Pro-CON); m/z
Following procedure E, coupling of acid 2 with trifluoro-
acetate 27 yielded amide 30 (0.36g, 25%) as a colourless
oil: Compound 30 was shown to be an 84:16 trans:cis
mixture of conformers: [a]D ꢀ67.0 (c 0.06, MeOH); dH
(200MHz; CDCl3; Me4Si) 1.73–2.13 (6H, m, Prob-H2,
Proc-H2 and Glub-H2), 2.14–2.53 (2H, m, Gluc-H2),
2.84* (0.48H, s, NCH3), 2.91 (2.52H, s, NCH3), 3.03*
(0.48 H, s, NCH3), 3.07 (2.52 H, s, NCH3), 3.30–3.40
(1H, m, Prod-HAHB), 3.40–3.50 (1H, m, Prod-HAHB),
3.92–4.13 (2H, m, Glya-H2), 4.46–4.50 (1H, m, Proa-
H), 4.95–5.00 (1H, m, Glua-H), 5.04 (2H, m, OCH2Ph),
5.09 (2H, m, OCH2Ph), 5.89 (1H, br s, Gly-NH) and
7.27–7.41 (10H, m, 2 · Ph); dC (50MHz, CDCl3) 24.7
(CH2, Proc-C), 27.6 (CH2, Glub-C), 28.6 (CH2, Prob-
C), 29.3 (CH2, Gluc-C), 35.7 (CH3, NCH3), 36.9
(CH3, NCH3), 43.4 (CH2, Glya-C), 46.3 (CH2, Prod-
C), 48.2 (CH, Glua-C), 60.4 (CH, Proa-C), 66.3 (CH2,
OCH2Ph), 66.8 (CH2, OCH2Ph), 127.9 (CH, Ph),
128.4 (CH, Ph), 135.8 (quat., Ph), 136.0 (quat., Ph)
156.2 (quat., NCO2), 167.7 (quat., Gly-CO), 171.0
(quat., Glua-CO), 171.1 (quat., Gluc-CO) and 172.9
(quat., Pro-CON); m/z (FAB+) 553.2661 (MH+ÆC29H37-
N4O7 requires 553.2662).
(FAB+)
315.1668
(MH+ÆC13H23N4O5
requires
315.1669).
4.25. Benzyl N-tert-butyloxycarbonyl-L-glutamate
a-N,N-dimethylamide (24)
To a solution of acid 21 (1.00g, 2.97mmol) in methylene
chloride (20mL), cooled to 0ꢁC under nitrogen, were
successively added dropwise triethylamine (0.50mL,
3.90mmol) and ethyl chloroformate (0.37mL,
3.90mmol), and the resultant mixture stirred at 0ꢁC
for 45min to form the mixed anhydride. Triethylamine
(0.90mL, 6.46mmol) was added to a suspension of
dimethylamine hydrochloride (0.53g, 6.50mmol) in
methylene chloride (20mL) under nitrogen at 0ꢁC and
the resulting solution added dropwise to the above
mixed anhydride. The reaction mixture was stirred for
30min, warmed to room temperature and stirred for a
further 20h then washed with saturated sodium hydro-
4.28. Glycyl-L-prolyl-L-glutamic acid a-N,N-dimethyl-
amide (20)
Following procedure B, hydrogenation of amide 30 in
methanol yielded analogue 20 (0.22g, 78%) as a white
solid. Compound 20 was shown to be an 83:17 trans:cis
mixture of conformers: mp 28–30ꢁC; [a]D ꢀ56.9 (c 0.05,
MeOH); dH (200MHz; CD3OD) 1.69–1.90 (6H, m,
Prob-H2, Proc-H2 and Glub-H2), 2.33 (2H, t, J 7.5,

514
N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
Gluc-H2), 2.82 (3H, s, NCH3), 3.05 (3H, s, NCH3),
3.34–3.50 (2H, m, Prod-H2), 3.78 (2H, m, Glya-H2),
4.34–4.37 (1H, m, Glua-H) and 4.70–4.80 (1H, m,
Proa-H); dC (50MHz, CD3OD) 25.7 (CH2, Proc-C),
28.1 (CH2, Glub-C), 30.6 (CH2, Prob-C), 30.7 (CH2,
Gluc-C), 31.2* (CH2, Gluc-C), 36.2 (CH3, NCH3),
37.6 (CH3, NCH3), 41.6 (CH2, Glya-C), 47.7 (CH2,
Prod-C), 48.5* (CH2, Prod-C), 50.1 (CH, Glua-C),
50.8* (CH, Glua-C), 61.2* (CH, Proa-C), 61.6 (CH,
Proa-C), 166.2 (quat., Gly-CO), 173.3 (quat., Gluc-
CO), 174.1 (quat., Glua-CO) and 176.8 (quat., Pro-
CON); m/z (FAB+) 329.1818 (MH+ÆC14H25N4O5
requires 329.1825).
4.32. N-Benzyloxycarbonyl-glycyl-L-proline N-hydroxy-
succinimide ester76 (47)
1,3-Dicyclohexylcarbodiimide (1.48g, 7.17mmol) and
N-hydroxysuccinimide (0.75g, 6.50mmol) were succes-
sively added to a solution of acid 2 (2.00g, 6.50mmol)
in dimethoxyethane (25mL) at 0ꢁC under nitrogen.
The reaction mixture was stirred for 3h, warmed to
room temperature and stirred for a further 17h. The
resultant solid was filtered, washed with diethyl ether
(150mL) and the filtrate concentrated to dryness in
vacuo to give succinimide ester76 47 (1.81g, 69%) as a
white solid.
4.29. Benzyl N-tert-butyloxycarbonyl-L-a-succinimido-
4.33. (4S)-Benzyl N-benzyloxycarbonyl-glycyl-L-prolyl-4-
amino-5-hydroxypentanoate (34)
glutamate75 (46)
To a solution of acid 21 (0.20g, 0.60mmol) in ethyl ace-
tate (8mL) at 0ꢁC under nitrogen, was successively
added N-hydroxysuccinimide (0.08g, 0.71mmol) and
1,3-dicyclohexylcarbodiimide (0.15g, 0.71mmol). The
reaction mixture was stirred for 1h, warmed to room
temperature and stirred for a further 17h then washed
with saturated aqueous sodium hydrogen carbonate
(10mL) and brine (10mL), dried (MgSO4), filtered and
concentrated to dryness in vacuo. The resultant colour-
less oil solidified to give succinimide ester75 46 (0.30g) as
a white solid.
Diisopropylethylamine (0.67mL, 3.84mmol) was added
dropwise to a solution of trifluoroacetate 33 (1.04g,
3.08mmol) and succinimide ester 47 (1.35g, 3.38mmol)
in methylene chloride (40mL) at 0ꢁC under nitrogen.
The reaction mixture was stirred for 30min, warmed
to room temperature and stirred for a further 2h, then
washed with saturated aqueous hydrogen carbonate
(25mL) and 2M citric acid (25mL), dried (MgSO4), fil-
tered and the solvent removed in vacuo. The resultant
residue was purified by flash column chromatography
(10% methanol–ethyl acetate) to afford amide 34
(0.49g, 31%) as a colourless oil: [a]D ꢀ43.0 (c 0.05,
MeOH); dH (200MHz; CDCl3; Me4Si) 1.83–2.16 (6H,
m, Prob-H2, Proc-H2 and 3-H2), 2.41 (2H, t, J 7.3, 2-
H2), 3.40–3.66 (5H, m, Prod-H2, Glya-H2 and OH),
3.90–3.95 (3H, 5-H2 and 4-H), 4.40–4.50 (1H, m,
Proa-H), 5.08 (2H, s, OCH2Ph), 5.09 (2H, s, OCH2Ph),
5.90 (1H, m, Gly-NH), 6.90 (1H, m, 4-NH) and 7.26–
7.32 (10H, m, 2 · Ph); dC (50MHz, CDCl3) 24.7 (CH2,
Proc-C), 25.7 (CH2, 3-C), 28.5 (CH2, Prob-C), 30.8
(CH2, 2-C), 43.4 (CH2, Glya-C), 46.5 (CH2, Prod-C),
51.4 (CH, 4-C), 60.7 (CH, Proa-C), 64.3 (CH2, 5-C),
66.4 (CH2, OCH2Ph), 67.0 (CH2, OCH2Ph), 128.0
(CH, Ph), 128.1 (CH, Ph), 128.5 (CH, Ph), 135.7 (quat.,
Ph), 136.2 (quat., Ph), 156.6 (quat., NCO2), 168.6 (quat.,
Gly-CO), 171.5 (quat., 1-CO) and 173.7 (quat., Pro-
CON); m/z (EI+) 512.2398 (MH+ÆC27H34N3O7 requires
512.2397).
4.30. (4S)-Benzyl N-tert-butyloxycarbonyl-4-amino-5-
hydroxypentanoate77 (32)
Sodium borohydride (0.03g, 0.66mmol) was added por-
tionwise over 15min to a solution of crude succinimide
ester 46 (0.30g, 0.60mmol) in tetrahydrofuran (5mL)
at 0ꢁC. Methanol (2.5mL) was added dropwise over
30min and the solution stirred for a further 15min then
quenched with saturated aqueous ammonium chloride
(10mL). The reaction mixture was extracted with ethyl
acetate (2 · 30mL) and the combined organic fractions
were washed with brine (10mL), dried (MgSO4), filtered
and concentrated to dryness in vacuo. Purification of
resultant residue by flash column chromatography (1:1
ethyl acetate–hexane) afforded alcohol 77 32 (0.14g,
72% in two steps from 21) as a white semi-solid: [a]D
ꢀ19.6 (c 0.06, MeOH); dH (200MHz; CDCl3; Me4Si)
1.43 [9H, s, C(CH3)3], 1.75–1.95 (2H, m, 3-H2), 2.46
(2H, t, J 7.3, 2-H2), 3.55–3.65 (3H, m, 4-H and 5-H2),
4.86–4.89 (1H, m, NH), 5.12 (2H, s, OCH2Ph) and
7.35 (5H, s, Ph); dC (50MHz, CDCl3) 26.3 (CH2, 3-C),
28.3 [CH3, C(CH3)3], 30.8 (CH2, 2-C), 52.2 (CH, 4-C),
65.0 (CH2, OCH2Ph), 66.4 (CH2, 5-C), 79.6 [quat.,
C(CH3)3], 128.2 (CH, Ph), 128.5 (CH, Ph), 135.7 (quat.,
Ph), 156.2 (quat., NCO2) and 173.5 (quat., 1-CO); m/z
(FAB+) 324.1817 (M+ÆC17H26NO5 requires 324.1811).
4.34. (4S)-Glycyl-L-prolyl-4-amino-5-hydroxypentanoic
acid (31)
Following procedure B, hydrogenation of amide 34 in
methanol yielded analogue 31 (0.27g, 93%) as a white
solid. Compound 31 was shown to be an 84:16 trans:cis
mixture of conformers: mp 63–65ꢁC; [a]D ꢀ56.1 (c 0.05,
MeOH); dH (200MHz; CD3OD) 1.94–2.03 (4H, m,
Prob-H2 and 3-H2), 2.15–2.25 (4H, m, Proc-H2 and 2-
H2), 3.51–3.56 (4H, m, Prod-H2 and 5-H2), 3.84–3.87
(1H, m, 4-H), 3.95 (2H, s, Glya-H2) and 4.40–4.50
(1H, m, Proa-H); dC (50MHz, CD3OD) 23.1* (CH2,
Proc-C), 25.1 (CH2, Proc-C), 25.3 (CH2, 3-C), 30.7
(CH2, Prob-C), 33.3* (CH2, 2-C), 34.8 (CH2, 2-C),
41.5 (CH2, Glya-C), 47.9 (CH2, Prod-C), 52.6 (CH,
4-C), 60.5* (CH, Proa-C), 61.8 (CH, Proa-C), 64.3
4.31. (4S)-Benzyl 4-amino-5-hydroxypentanoate trifluoro-
acetate (33)
Following procedure D, treatment of alcohol 32 with
trifluoroacetic acid yielded trifluoroacetate 33 (1.00g)
as an orange oil.

N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
515
(CH2, 5-C), 166.8 (quat., Gly-CO), 175.1 (quat., Pro-
CON) and 183.5 (quat., 1-CO); m/z (FAB+) 288.1551
(M+ÆC12H22N3O5 requires 288.1559).
8.7 and 3.1, Proa-H); dC (100MHz; D2O) 24.6* (CH2,
Proc-C), 26.6 (CH2, Proc-C), 29.3* (CH2, Glub-C),
29.6 (CH2, Glub-C), 32.2 (CH2, Prob-C), 34.2 (CH2,
Gluc-C), 34.4* (CH2, Gluc-C), 34.6* (CH2, Prob-C),
42.7* (CH2, Glya-C), 43.0 (CH2, Glya-C), 49.4 (CH2,
Prod-C), 50.0* (CH2, Prod-C), 57.1 br (CH, Glua-C),
57.5* br (CH, Glua-C), 62.3* (CH, Proa-C), 63.2 (CH,
Proa-C), 168.3 (quat., Gly-CO), 168.4* (quat., Gly-
CO), 175.6* (quat., Pro-CON), 175.8 (quat., Pro-
CON), 180.2* br (quat., Glua-CO), 180.2 br (quat.,
Glua-CO), 181.5* br (quat., Gluc-CO) and 181.9 br
(quat., Gluc-CO); m/z (FAB+) 302.1346 (MH+ÆC12H20-
N3O6 requires 302.1352).
4.35. Dibenzyl N-benzyloxycarbonyl-glycyl-L-prolyl-D-
glutamate (38)
Following procedure A, coupling of acid 2 with p-tolu-
enesulfonate 37 yielded amide 38 (1.62g, 64%) as a white
solid. Compound 38 was shown to be a 92:8 trans:cis
mixture of conformers: Rf 0.50 (EtOAc); [a]D ꢀ54.8 (c
0.29, CH2Cl2); mmax (film)/cmꢀ1 3583 wk, 3317 br,
3063, 3033, 2953, 2881, 1732, 1658, 1528, 1454, 1255,
1213, 1167, 1055, 985, 915, 739 and 698; dH (400MHz;
CDCl3; Me4Si) 1.78–2.11 (4H, m, Prob-HAHB, Proc-
H2 and Glu-HAHB), 2.13–2.49 (4H, m, Glub-HAHB,
Prob-HAHB and Gluc-H2), 3.33 (0.92H, br dd, J 16.6
and 9.3, Prod-HAHB), 3.36–3.46 (0.92H, m, Prod-
HAHB), 3.52–3.71* (0.16H, m, Pro-H2), 3.79* (0.08H,
dd, J 16.8 and 4.7, Glya-HAHB), 3.89 (1H, dd, J 17.1
and 4.0, Glya-HAHB and Glya-HAHB*), 4.00 (0.92H,
dd, J 17.2 and 4.9, Glya-HAHB), 4.27* (0.08H, br t, J
5.3, Proa-H), 4.52–4.64 (1.92H, m, Proa-H and Glua-
H), 5.02–5.21 (6H, m, 3 · OCH2Ph), 5.58–5.63*
(0.08H, br m, Gly-NH), 5.63–5.71 (0.92H, br m, Gly-
NH), 7.28–7.38 (15H, m, 3 · Ph) and 7.52 (1H, d, J
7.7, Glu-NH); dC (100MHz; CDCl3) 22.0* (CH2,
Proc-C), 24.4 (CH2, Proc-C), 25.8* (CH2, Glub-C),
26.4 (CH2, Glub-C), 27.9 (CH2, Prob-C), 30.1 (CH2,
Gluc-C), 30.3* (CH2, Gluc-C), 32.0* (CH2, Prob-C),
43.3 (CH2, Glya-C), 46.1 (CH2, Prod-C), 46.9* (CH2,
Prod-C), 51.8 (CH, Glua-C), 52.2* (CH, Glua-C), 60.0
(CH, Proa-C), 60.1* (CH, Proa-C), 66.4 (CH2,
OCH2Ph), 66.6* (CH2, OCH2Ph), 66.7 (CH2, OCH2Ph),
67.0 (CH2, OCH2Ph), 67.2* (CH2, OCH2Ph), 127.9
(CH, Ph), 127.9 (CH, Ph), 128.0 (CH, Ph), 128.1 (CH,
Ph), 128.1 (CH, Ph), 128.2* (CH, Ph), 128.3 (CH, Ph),
128.4 (CH, Ph), 128.4 (CH, Ph), 135.0* (quat., Ph),
135.1 (quat., Ph), 135.4* (quat., Ph), 135.5 (quat.,
Ph), 136.2 (quat., Ph), 156.2 (quat., NCO2), 156.3*
(quat., NCO2), 168.0* (quat., Gly-CO), 168.3 (quat.,
Gly-CO), 170.9 (quat., Pro-CON), 171.0* (quat., Pro-
CON), 171.1 (quat., Glua-CO), 171.5* (quat., Glua-
CO), 172.9 (quat., Gluc-CO) and 173.1* (quat.,
Gluc-CO); m/z (FAB+) 616.2653 (MH+ÆC34H38N3O8
requires 616.2659).
4.37. Dibenzyl (DL)-2-methylglutamate p-toluenesulfonate
(40)
Following procedure C, benzylation of acid 39 yielded
p-toluenesulfonate 40 (0.82g, 55%) as a crystalline white
solid: mp 112–116ꢁC; mmax (film)/cmꢀ1 3579, 3028 br,
2949 br, 2166 br, 1739, 1649, 1602, 1537, 1494, 1450,
1393, 1313, 1212, 1181, 1125, 1031, 1009, 966, 814,
749, 739 and 677; dH (300MHz; CDCl3; Me4Si) 1.56
(3H, s, Glua-CCH3), 2.13–2.38 (6H, m, Ar-CH3, Glub-
H2 and Gluc-HAHB), 2.46–2.68 (1H, m, Gluc-HAHB),
4.94 [1H, d, J 12.4, (OCHAHBPh)A], 4.99 [1H, d,
J
12.3, (OCHAHBPh)A], 5.05 [1H, d,
J
12.2,
(OCHAHBPh)B], 5.11 [1H, d, J 12.2, (OCHAHBPh)B],
6.98 (2H, d, J 7.9, 2 · 3-H), 7.16–7.40 (10H, m,
2 · Ph), 7.70 (2H, d, J 8.1, 2 · 2-H) and 8.53 (3H, br
s, N+H3); dC (75MHz; CDCl3) 21.3 (CH3, Ar-CH3),
22.0 (CH3, Glua-CCH3), 28.3 (CH2, Glub-C), 32.0
(CH2, Gluc-C), 59.7 (quat., Glua-C), 66.4 (CH2,
OCH2Ph), 68.1 (CH2, OCH2Ph), 126.1 (CH, Ar),
128.1 (CH, Ar), 128.1 (CH, Ar), 128.3 (CH, Ar), 128.4
(CH, Ar), 128.5 (CH, Ar), 128.5 (CH, Ar), 128.8 (CH,
Ar), 134.7 (quat., Ph), 135.7 (quat., Ph), 140.2 (quat.,
Ar), 141.3 (quat., Ar), 170.3 (quat., CO) and 171.9
(quat., CO); m/z (FAB+) 855.3555 [M2ÆpTsOHÆH+:
(C20H23NO4)2ÆC7H8O3SÆH requires 855.3527].
4.38. Dibenzyl N-benzyloxycarbonyl-glycyl-L-prolyl-(DL)-
2-methylglutamate (41)
Following procedure A, coupling of acid 2 with p-tolu-
enesulfonate 40 yielded amide 41 (0.40g, 97%) as a
slightly opaque oil. Compound 41 was shown to be an
87:13 trans:cis mixture of conformers: Rf 0.70 (EtOAc);
[a]D ꢀ59.0 (c 0.26, CH2Cl2); mmax (film)/cmꢀ1 3319 br,
3063, 3033, 2951, 1730, 1655, 1527, 1454, 1382, 1258,
1170, 1119, 1055, 985, 913, 736 and 698; dH (300MHz;
CDCl3; Me4Si) 1.53 (2.61H, s, Glua-CCH3), 1.57
(2.61H, s, Glua-CCH3), 1.61* (0.39H, s, Glu-CCH3),
1.62* (0.39H, s, Glua-CCH3), 1.69–2.47 (16H, m,
2 · Prob-H2, 2 · Proc-H2, 2 · Glub-H2 and 2 · Gluc-
H2), 3.15–3.40 (3.48H, m, 2 · Prod-H2), 3.45–3.75*
(0.52H, m, 2 · Prod-H2), 3.75–4.06 (4H, m, 2 · Glya-
H2), 4.16* (0.26H, br d, J 6.7, 2 · Proa-H), 4.47
(1.74H, br t, J 6.6, 2 · Proa-H), 4.98–5.21 (12H, m,
6 · OCH2 · Ph), 5.50* (0.26H, br s, 2 · Gly-NH), 5.65
(1.74H, br s, 2 · Gly-NH), 7.27–7.38 (30H, m, 6Ph),
7.41 (1H, br s, Glu-NH) and 7.52 (1H, br s, Glu-NH);
4.36. Glycyl-L-prolyl-D-glutamic acid (35)
Following procedure B, hydrogenation of amide 38 in
70:30 tetrahydrofuran–water yielded tripeptide 35
(0.22g, 68%) as an off-white solid. Compound 35 was
shown to be an 80:20 trans:cis mixture of conformers:
mp 198–200ꢁC; [a]D ꢀ70.6 (c 0.27, H2O); dH
(400MHz; D2O) 1.83–2.18 (5H, m, Glub-H2, Prob-
HAHB and Proc-H2), 2.21–2.45 (3H, m, Prob-HAHB
and Gluc-H2), 3.49–3.68 (2.20H, m, Prod-H2 and
Glya-HAHB*), 3.92* (0.20H, d, J 16.3, Glya-HAHB),
3.95 (0.80H, d, J 16.4, Glya-HAHB), 4.03 (0.80H, d, J
16.5, Glya-HAHB), 4.17* (0.20H, dd, J 9.3 and 4.7,
Glua-H), 4.20 (0.80H, dd, J 9.1 and 4.5, Glua-H),
4.42–4.53 (0.80H, m, Proa-H) and 4.52* (0.20H, dd, J

516
N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
dC (75MHz; CDCl3) 21.9* (CH3, Glua-CCH3), 22.0*
(CH2, Proc-C), 22.2* (CH2, Proc-C), 22.5 (CH3, Glua-
CCH3), 22.6* (CH3, Glua-CCH3), 22.7 (CH3, Glua-
CCH3), 24.4 (CH2, Proc-C), 24.5 (CH2, Proc-C), 27.5
(CH2, Prob-C), 27.7 (CH2, Prob-C), 28.7* (CH2,
Glub-C), 28.8 (CH2, Glub-C), 28.9 (CH2, Glub-C),
29.5* (CH2, Glub-C), 31.3 (CH2, Gluc-C), 31.8 (CH2,
Gluc-C), 43.2 (CH2, Glya-C), 45.9 (CH2, Prod-C),
46.0 (CH2, Prod-C), 46.9* (CH2, Prod-C), 46.9* (CH2,
Prod-C), 58.9 (quat., Glua-C), 58.9 (quat., Glua-C),
59.1* (quat., Glua-C), 59.1* (quat., Glua-C), 60.1
(CH, Proa-C), 60.1 (CH, Proa-C), 66.2 (CH2, OCH2Ph),
66.3 (CH2, OCH2Ph), 66.7 (CH2, OCH2Ph), 67.1 (CH2,
OCH2Ph), 67.3* (CH2, OCH2Ph), 127.8 (CH, Ph), 127.8
(CH, Ph), 127.9 (CH, Ph), 128.0 (CH, Ph), 128.0 (CH,
Ph), 128.0 (CH, Ph), 128.1 (CH, Ph), 128.1 (CH, Ph),
128.2 (CH, Ph), 128.3 (CH, Ph), 128.3 (CH, Ph), 128.4
(CH, Ph), 135.2* (quat., Ph), 135.4 (quat., Ph), 135.4
(quat., Ph), 135.5 (quat., Ph), 135.6 (quat., Ph), 136.3
(quat., Ph), 156.1 (quat., NCO2), 167.8 (quat., Gly-
CO), 168.0 (quat., Gly-CO), 170.0 (quat., Pro-CON),
170.6* (quat., Pro-CON), 170.6* (quat., Pro-CON),
172.6* (quat., Glu-CO), 172.8 (quat., Glu-CO), 172.9
(quat., Glu-CO), 173.0 (quat., Glu-CO), 173.1 (quat.,
Glu-CO), 173.5* (quat., Glu-CO) and 173.6* (quat.,
Glu-CO); m/z (FAB+) 630.2811 (MH+ÆC35H40N3O8
requires 630.2815).
Acknowledgements
The authors thank Neuren Pharmaceuticals Ltd for
financial support.
References and notes
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Following procedure B, hydrogenation of amide 41
yielded analogue 36 (0.11g, 81%) as a white solid. Com-
pound 36 was shown to be a 77:23 trans:cis mixture of
conformers of two equimolar diastereoisomers. Analyt-
ical reverse-phase HPLC studies indicated that 36 was a
1:1 mixture of SS and SR diastereoisomers with two
peaks of approximately equal integration eluting with
retention times of 13.52 and 13.95min at 207 and
205nm, respectively. Analogue 36: mp 164–180ꢁC
(decomp.); [a]D ꢀ71.3 (c 0.15, H2O); mmax (Nujol mull)/
cmꢀ1 3583, 3318 br, 2725, 1651, 1562, 1546, 1299,
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2 · Prob-H2,
2 · Proc-HAHB,
2 · Glub-H2
and
2 · Gluc-H2), 3.47–3.75 (4.46H, m, 2 · Prod-H2 and
2 · Glya-HAHB*), 3.87–4.13 (3.54H, m, 2 · Glya-HAHB
and 2 · Glya-HAHB) and 4.38–4.52 (2H, m, 2 · Proa-
H); dC (75MHz; D2O) 23.7* (CH3, Glua-CCH3), 23.9*
(CH2, Proc-C), 24.2 (CH3, Glua-CCH3), 24.3 (CH3,
Glua-CCH3), 26.0 (CH2, Proc-C), 26.0 (CH2, Proc-C),
31.2 (CH2, Prob-C), 33.7* (CH2, Prob-C), 33.8* (CH2,
Prob-C), 33.9 (CH2, Gluc-C), 34.2 (CH2, Gluc-C),
34.8* (CH2, Gluc-C), 42.1* (CH2, Glya-C), 42.3 (CH2,
Glya-C), 48.7 (CH2, Prod-C), 49.4* (CH2, Prod-C),
61.9* (CH, Proa-C), 62.0* (CH, Proa-C), 62.7(quat.,
Glua-C), 62.9 (CH, Proa-C), 63.0 (CH, Proa-C), 167.5
(quat., Gly-CO), 167.8* (quat., Gly-CO), 173.5* (quat.,
Pro-CON), 173.5* (quat., Pro-CON), 174.0 (quat., Pro-
CON), 181.5 br (quat., Glu-CO), 181.8 br (quat., Glu-
CO) and 182.9 br (quat., Glu-CO); m/z (FAB+)
316.1517 (MH+ÆC13H22N3O6 requires 316.1509).

N. S. Trotter et al. / Bioorg. Med. Chem. 13 (2005) 501–517
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