Synthesis and pharmacological evaluation of side chain modified glutamic acid analogues of the neuroprotective agent glycyl-L-prolyl-L-glutamic acid (GPE)

Article abstract of DOI:10.1016/j.bmc.2004.10.006

The synthesis of eight GPE* analogues, wherein the γ-carboxylic moiety of the glutamic residue has been modified, is described by coupling readily accessible N-benzyloxycarbonyl-glycyl-l-proline 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.2004.10.006

Bioorganic & Medicinal Chemistry 13 (2005) 519–532
Synthesis and pharmacological evaluation of side chain modified
glutamic acid analogues of the neuroprotective agent
glycyl-L-prolyl-L-glutamic acid (GPE)
Margaret A. Brimble,* Nicholas S. Trotter, Paul W. R. Harris 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
Abstract—The synthesis of eight GPE* analogues, wherein the c-carboxylic moiety of the glutamic residue has been modified, is
described by coupling readily accessible N-benzyloxycarbonyl-glycyl-L-proline with various analogues of glutamic acid. Pharmaco-
logical 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
has therefore been proposed that GPE 1, or analogues
thereof, may provide a novel class of pharmaceutical
agents for the treatment of CNS injuries, neurodegener-
ative diseases including Alzheimerꢀs, Parkinsonꢀs and
Huntingtonꢀs disease, multiple sclerosis and general
aging-induced cognitive dysfunction.29–32 Analogues of
GPE are of sufficiently low molecular weight and are
lipophilic enough to cross the blood–brain barrier. Their
metabolic stability and oral bioavailability can be im-
proved by synthetic modification of the parent tripep-
tide structure.
As described in our previous paper1, insulin-like growth
factor-1 (IGF-1) is a potent neurotrophic factor2,3 dis-
tributed within the mammalian central nervous system4
(CNS), which 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 1)5–11 in damaged
regions of the brain.12–17 IGF-1 is thought to function
as a prohormone for GPE 1, which acts on a yet un-
known receptor.18–20 Although there is no direct evi-
dence that GPE 1 is naturally present or produced in
the brain,21 it was observed to act as a neuronal rescue
factor following transient HI brain injury for cerebral,
cortical, striatal and hippocampal neurons,22 with
peripheral activity, producing wider neuroprotective
effects than local administration.20
We therefore herein report the preparation and pharma-
cological evaluation of several analogues of general
structure Gly-Pro-Glu-OH (GPE*), in which the c-posi-
tion of Glu (E*) has been modified. We also utilised our
in vitro brain cell culture in the screening of these ana-
logues.1 This work is part of a structure–activity rela-
tionship study to determine the significance of the Glu
residue of this tripeptide on neuroprotective activity,
aiding our selection of suitable candidates for continued
drug development.
In addition to in vitro studies by Sara and colleagues,5,18
GPE 1 was suggested to possess a neuromodulatory role
in the CNS, possibly through interaction with glutamate
receptors,5,6,11,18,19,22–25 which provide a potential target
for the rational design of neuroprotective agents.26–28 It
2. Results and discussion
So as to ascertain the importance of the c-carboxylic
acid functionality on the glutamate residue of GPE,
eight analogues with modifications at this site were syn-
thesised and evaluated for their neuroprotective effects.
Keywords: Neuroprotective agents; Peptides; Proline; GPE;
Glutamate.
*
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.2004.10.006

520
M. A. Brimble et al. / Bioorg. Med. Chem. 13 (2005) 519–532
H
X H3N
CO2H
N
CO2H
OH
N
N
+
γ
O
O
γ
R1
R1
O
O
R
R
NH2
NHCO2Bn
GPE* γ-glutamate modified analogues
2
Modified glutamates
Scheme 1. General retrosynthesis for GPE* c-glutamate modified analogues.
The synthetic strategy employed involved preparation of
known N-benzyloxycarbonyl-glycyl-L-proline33,34 2, and
effecting peptide bond construction with several modi-
fied benzyl-protected glutamic acid residues (Scheme
1). After assembly of the new pseudo-peptide bond, a
simple deprotection procedure furnished the desired
GPE* analogues.
chloride salt35,36 4, which was subsequently coupled with
commercial N-benzyloxycarbonylglycine 5, using 1,3-
dicyclohexylcarbodiimide (DCC) to realise protected
dipeptide37 6. Finally, saponification of the methyl ester
afforded acid 2.
Based on the structure of GPE 1, the first analogue 7 in-
volved replacement of the c-carboxylic acid group by a
hydrogen, starting from the modified glutamic acid 8
(Scheme 3). Elimination of the carboxylic acid group
in analogue 7 removes the amino acid character from
the glutamate residue of GPE 1, potentially suppressing
The synthesis of N-CBz peptide33,34 2 was achieved in
82% yield over two steps from readily available L-pro-
line methyl ester 4 (Scheme 2). Thus, treatment of 3 with
thionyl chloride in methanol gave quantitatively hydro-
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).
H2N
CO2H
H2N
CO2Bn
OH
(i)
N
.pTsOH
+
O
O
8
9
NHCO2Bn
H
N
H
2
CO2Bn
N
CO2H
N
N
(ii)
(iii)
O
O
O
O
NHCO2Bn
NH2
7 (83:17 trans:cis)
10
Scheme 3. Reagents and conditions: For 7: (i) pTsOH, BnOH, benzene, reflux, 17h (95%); (ii) DIPEA, EtOCOCl, CH2Cl2, 0ꢁC to rt, 17h (78%); (iii)
H2, 10% Pd/C, 2:1 MeOH–H2O, 18h (84%). For 8: (i) pTsOH, BnOH, benzene, reflux, 17h (93%); (ii) Et3N, EtOCOCl, CH2Cl2, 0ꢁC to rt, 17h
(94%); (iii) H2, 10% Pd/C, 2:1 MeOH–H2O, 18h (73%).

M. A. Brimble et al. / Bioorg. Med. Chem. 13 (2005) 519–532
521
O
H2N
CO2Bn
OH
O
N
(i)
N
N
+
O
O
O
O
O
CO2H
NHCO2Bn
NHCO2Bn
2
15
16
H
N
H
N
CO2Bn
CO2H
(iii)
(ii)
CO2Bn
N
N
O
O
O
O
R
NHCO2Bn
NHCO2Bn
18 R = CONH2
19 R = CONHMe
20 R = CONMe2
21 R = CH2OH
17
H
N
CO2H
N
(iv)
O
O
R
NH2
11 R = CONH2, R1 = H (85:15 trans:cis)
12 R = CONHMe, R1 = H (86:14 trans:cis)
13 R = CONMe2, R1 = H (87:13 trans:cis)
14 R = CH2OH, R1 = H (76:24 trans:cis)
Scheme 4. Reagents and conditions: (i) DME, N-hydroxysuccinimide, DCC, N2, 0ꢁC, 23h (85%); (ii) Et3N, H2O, THF, rt, 16h (95%); For 11: (iii)
Boc2O, NH4HCO3, pyridine, 1,4-dioxane, N2, rt, 21h (89%); (iv) H2, 10% Pd/C, 2:1 MeOH–H2O, rt, 18h (82%). For 12: (iii) Et3N, MeNHÆHCI,
CH3CN, TBTU, N2, rt, 16.5h (79%); (iv) H2, 10% Pd/C, 2:1 MeOH–H2O, rt, 18h (67%). For 13: (iii) Et3N, Me2NÆHCl, CH2Cl2, EtOCOCl, N2, 0ꢁC
to rt, 17h (89%); (iv) H2, 10% Pd/C, 2:1 MeOH–H2O, rt, 18h (95%). For 14: (iii) (a) Et3N, THF, EtOCOCl, N2, ꢀ5ꢁC, 30min; (b) NaBH4, MeOH,
ꢀ5ꢁC to rt, 30min (63%); (iv) H2, 10% Pd/C, 82:16:2 THF–H2O–Et3N, rt, 18h (100%).
the action of proteases, thereby imparting improved
metabolic stability. Additionally, the overall polarity
of the molecule is reduced, thus increasing lipophilicity
and membrane permeability of the analogue.
In the case of the primary amide 11, the precursor 17
was treated with di-tert-butyl dicarbonate and ammono-
lysis of the intermediate anhydride with ammonium
hydrogen carbonate under basic conditions afforded
fully protected tripeptide 18. Subsequent deprotection
yielded analogue 11 as an 85:15 trans:cis mixture of
rotamers in 82% yield over the two steps. Analogues
12 and 13 were afforded by coupling of acid 17 with
either methylamine hydrochloride using the coupling
The commercial amino acid (2S)-aminobutanoic acid 8,
was initially protected as a benzyl ester 9 by heating
with benzyl alcohol and p-toluenesulfonic acid in ben-
zene. Coupling of the aforementioned acid 2 with pro-
tected amino ester 9 using peptide coupling produced
protected tripeptide 10. Upon deprotection, GPE* ana-
logue 7 was produced as an 83:17 trans:cis mixture of
rotamers.
reagent,
2-(N-benzotriazole-1-yl)-1,1,3,3-tetramethy-
luronium tetrafluoroborate (TBTU) or with dimethyl-
amine hydrochloride using the mixed anhydride
method, respectively, followed by hydrogenolysis of
the resultant amides 19 and 20. In the case of the alcohol
14, reduction of the mixed anhydride formed from acid
17 with sodium borohydride followed by hydrogenolysis
yielded analogue 14. Analogues 11–14 all existed pre-
dominantly as the trans proline–glycine amide bond
conformers.
Another strategy for reducing the overall charge in the
GPE molecule whilst maintaining the steric bulk of the
glutamate c-position involves replacement of the side
chain carboxylic acid with an amide (analogues 11–13)
or an alcohol (analogue 14) (Scheme 4).
The syntheses of all four analogues 11–14 started from
L-glutamic acid a-benzyl ester 15. Reaction of amine
15 with succinimide 16 yielded dipeptide 17 in 95% yield.
Subsequent modifications of the c-carboxylic acid group
on dipeptide 17 and deprotection provided analogues
11–14.
Two further GPE* analogues 22 and 23 are also re-
ported herein in which the carboxylic acid side chain
of the glutamate residue is completely removed and
the two carboxylic acid groups of the glutamate are con-
verted to more lipophilic methyl esters (Schemes 5 and
6).

522
M. A. Brimble et al. / Bioorg. Med. Chem. 13 (2005) 519–532
H
N
CO2Bn
OH
N
H2N
CO2Bn
N
(i)
+
O
O
O
.pTsOH
O
NHCO2Bn
NHCO2Bn
24
25
2
H
N
CO2H
N
(ii)
O
O
NH2
22 (76:24 trans:cis)
Scheme 5. Reagents and conditions: (i) Et3N, EtOCOCl, CH2Cl2, 0ꢁC to rt, 17h (94%); (ii) H2, 10% Pd/C, MeOH, 18h (85%).
H
N
CO2Me
CO2Me
H2N
CO2Me
CO2Me
OH
N
(i)
N
+
O
O
O
O
NHCO2Bn
.HCl
NHCO2Bn
26
27
2
H
N
CO2Me
N
(ii)
O
O
CO2Me
NH2
23 (87:13 trans:cis)
Scheme 6. Reagents and conditions: (i) Et3N, EtOCOCl, CH2Cl2, 0ꢁC to rt, 17h (97%); (ii) H2, 10% Pd/C, 2:1 MeOH–H2O, 18h (97%).
Analogue 22 was afforded by coupling of acid 2 with
protected glycine 24 to give amide 25 followed by depro-
tection to afford GPG 22 as a 76:24 trans:cis mixture of
rotamers (Scheme 5). Diester 23, was obtained in 97%
yield as an 87:13 trans:cis mixture of rotamers via con-
densation of dimethyl L-glutamate hydrochloride 26
with acid 2 and subsequent hydrogenolysis of the prod-
uct 27 (Scheme 6).
logue 28 in 77% yield as an 82:18 trans:cis mixture of
conformers.
The eight GPE* analogues synthesised were subjected to
in vitro evaluation for prevention of neuronal cell death.
Of the eight analogues, GPG 22, lacking any c side
chain, showed neuroprotective activity at 1mM with a
recovery value of 20% (Fig. 1). To compare, GPE 1
recovery values range from 25% to 40%. Lower neuro-
protective values were obtained with 1mM of the unsub-
stituted amide 11, the alcohol 14 and the diesterified 23
where the recovery values ranged from 10% to 15%.
None of the other analogues exhibited neuroprotective
activity.
One further analogue 28 was prepared in which the two
carboxylic acid functionalities of the glutamic acid resi-
due were left intact but however an additional propyl
group was introduced to the c-position in order to in-
crease the lipophilic properties of the molecule (Scheme
7). Commercially available L-glutamic acid dibenzyl
ester p-toluenesulfonate 29 was transformed into Boc-
protected amino acid 30 by reaction with di-tert-butyl
dicarbonate. Subsequent alkylation38 with lithium hexa-
methyldisilazide and allyl bromide afforded the c-alkyl-
ated product 31 as a single diastereomer in 73% yield,
which then underwent Boc-deprotection to 32 and cou-
pling with acid 2 to yield fully protected tripeptide 33 in
87% yield. The sequence was completed by hydrogenoly-
sis of amide 33 under standard conditions affording ana-
3. Conclusion
To summarise, we described herein the synthesis of eight
analogues of GPE and their biological evaluation against
striatal cell survival post-apoptosis-induced injury. Com-
pound 22 exhibited the best recovery value, 20%, while
11, 14 and 23 had lower recovery values but all four
exhibited neuroprotective activity. The structure and

M. A. Brimble et al. / Bioorg. Med. Chem. 13 (2005) 519–532
523
H2N
CO2Bn
BocHN
CO2Bn
BocHN
CO2Bn
CO2Bn
(ii)
(i)
CO2Bn
CO2Bn
.pTsOH
29
30
31
H2N
.CF3CO2H
CO2Bn
CO2Bn
OH
N
+
(iii)
O
O
N
NHCO2Bn
32
2
H
N
H
CO2H
CO2H
N
CO2Bn
CO2Bn
N
(iv)
O
O
O
O
NH2
NHCO2Bn
33
28 (82:18 trans:cis)
Scheme 7. Reagents and conditions: (i) Et3N, Boc2O, CH2Cl2, 0ꢁC to rt, 19h (97%); (ii) (a) lithium hexamethyldisilazide, THF, ꢀ78ꢁC, N2, 40min;
(b) allyl bromide, ꢀ78ꢁC, 1.5h then ꢀ78ꢁC to rt, 1.5h (73%); (iii) (a) TFA, CH2Cl2, 0ꢁC, 2h; (b) CH2Cl2, Et3N, EtOCOCl, 0ꢁC to rt, N2, 17h (87%);
(iv) H2, 10% Pd/C, 2:1 MeOH–H2O, 18h (77%).
matography (TLC) was carried out on 0.20mm pre-
coated silica gel plates (ALUGRAMꢂ SIL G/UV254).
Products were visualised by UV fluorescence and heat-
ing of plates dipped in anisaldehyde in ethanolic 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 determined on an Electrothermalꢂ
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 Per-
kin–Elmer Spectrum One FT-IR spectrometer and the
samples were prepared as thin films between sodium
chloride 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
Figure 1. Cellular survival bioassay. MTT values were taken 24h after
injury when apoptotic nuclei become apparent within the cell culture.
GPG (1mM) 22 confers a significant increased cellular protection of
10.4%.
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, dd = doublet
of doublets, m = multiplet, and where br = broad), cou-
pling constant (J/Hz) and assignment. For 13C NMR
data, chemical shifts (ppm) are referenced internally to
CDCl3 (d 77.0), CD3OD (d 49.1), (CD3)2SO (d 39.4)
or externally to 3-(trimethylsilyl)-1-propanesulfonic acid
sodium salt (DSS) and are reported consecutively as
position (dC), degree of hybridisation and assignment.
Assignments were aided by DEPT135, COSY, NOESY,
spectra of the new analogues were determined by full
analysis of the NMR and mass spectral data.
4. Experimental
4.1. General method
All reagents were used as supplied. Solvents were puri-
fied by standard methods.39 Analytical thin layer chro-

524
M. A. Brimble et al. / Bioorg. Med. Chem. 13 (2005) 519–532
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
for the minor cis conformer is asterisked (*). Assignment
of the respective isomers is made according to Kessler40
and NOESY experiments. Mass spectra were recorded
on a VG-70SE mass spectrometer (EI, CI and FAB).
High-resolution mass spectra were recorded at a nomi-
nal resolution of 5000. Analytical HPLC studies were
performed on a Waters 600 system with a 2487 dual k
absorbance detector using an Altech Econosphere
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. L-Proline
methyl ester hydrochloride35,36 (4) and N-benzyloxycar-
bonyl-glycyl-L-proline33,34 (2) were prepared according
to experimental procedures given in the previous paper.1
All chemicals utilised in the bioassay were purchased
from Invitrogen.
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.
Representative procedure A for the benzylation of carb-
oxylic acids is as follows:
4.4. (2S)-Benzyl 2-aminobutanoate p-toluenesulfonate
(10)
A mixture of L-(+)-2-aminobutanoic acid 8 (0.48g,
4.56mmol), p-toluenesulfonic acid monohydrate
(98.5%) (1.29g, 6.68mmol) and benzyl alcohol (4.99g,
46.1mmol) in benzene (30cm3) was heated under reflux,
using a Dean Stark distilling receiver, for 22h, after
which the reaction mixture was reduced in volume by re-
moval of benzene (15cm3) by distillation. The solution
was cooled to room temperature, anhydrous diethyl
ether (20cm3) was added and the mixture left for 1h
at 0ꢁC to afford p-toluenesulfonate 9 (1.55g, 93%) as a
crystalline white solid: mp 125–127ꢁC; [a]D ꢀ5.7 (c
0.26 in CH2Cl2); mmax (film)/cmꢀ1 3579, 2963 br, 1748,
1602, 1523, 1497, 1450, 1400, 1367, 1302, 1259, 1212,
1188, 1125, 1038, 1013, 948, 811, 731 and 677; dH
(300MHz; CDCl3; Me4Si) 0.83 (3H, t, J 7.5, 4-H3),
1.83 (2H, quintet, J 7.1, 3-H2), 2.30 (3H, s, Ar–CH3),
4.2. 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).
3.87–4.02 (1H, m, 2-H), 5.00 (1H, d,
J 12.3,
OCHAHBPh), 5.12 (1H, d, J 12.2, OCHAHBPh), 7.08
(2H, d, J 8.0, 2 · 30-H), 7.22–7.34 (5H, m, Ph), 7.74
(2H, d, J 8.1, 2 · 20-H) and 8.25 (3H, br s, N+H3); dC
(75MHz; CDCl3) 9.0 (CH3, 4-C), 21.2 (CH3, Ar–
CH3), 23.6 (CH2, 3-C), 54.2 (CH, 2-C), 67.6 (CH2,
OCH2Ph), 126.1 (CH, Ar), 128.2 (CH, Ar), 128.3 (CH,
Ar), 128.4 (CH, Ar), 128.7 (CH, Ar), 134.9 (quat., Ph),
140.2 (quat., Ar), 141.5 (quat., Ar) and 169.2 (quat.,
CO);
m/z
(FAB+)
559.2483
[M2ÆpTsOHÆH+:
(C11H15NO2)2. C7H8O3SÆH requires 559.2478].
Representative procedure B for the preparation of pro-
tected tripeptides and analogues is as follows:
4.5. (2S)-Benzyl N-benzyloxycarbonyl-glycyl-L-prolyl-2-
aminobutanoate (10)
To a solution of acid 2 (0.65g, 2.12mmol) in methylene
chloride (26cm3), cooled to 0ꢁC under nitrogen, was
added dropwise triethylamine (0.33cm3, 2.37mmol)
and ethyl chloroformate (0.22cm3, 2.30mmol) and the
resultant mixture stirred at 0ꢁC for 45min to form the
mixed anhydride. Triethylamine (0.42cm3, 3.01mmol)
was added to a solution of benzyl 2-aminobutanoate
p-toluenesulfonate 9 (1.02g, 2.79mmol) in methylene
chloride (13cm3) under nitrogen at 0ꢁC and the result-
ing solution was added dropwise to the above mixed
anhydride. The resultant mixture was warmed to room
temperature and stirred for 16h then the methylene
chloride solution was extracted successively with 10%
aqueous hydrochloric acid (50cm3) and saturated aque-
ous sodium hydrogen carbonate (50cm3), dried
(MgSO4), filtered and evaporated to dryness in vacuo.
4.3. Cellular injury, drug administration and cellular
survival analysis
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 to-
gether 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 sulphate
(SDS) solution. After 16–24h incubation time, extinc-
tion values are read at 595nm.

M. A. Brimble et al. / Bioorg. Med. Chem. 13 (2005) 519–532
525
Purification of the ensuing residue by flash column chro-
matography (21g SiO2; 30–100% ethyl acetate–hexane;
gradient elution) yielded fully protected tripeptide ana-
logue 10 (0.96g, 94%) as a pale yellow oil. Tripeptide 10
was shown to be a 90:10 trans:cis mixture of conformers
and Prob-HAHB), 1.70 (1H, apparent octet, J 7.3, 3-
HAHB), 2.14–2.48 (1H, m, Prob-HAHB), 3.50–3.73
(2.17H, m, Prod-H2 and Glya-HAHB*), 3.91–4.16
*
(2.83H, m, Glya-HAHB Glya-H2 and 2-H) and 4.49
(1H, dd, J 8.2 and 4.1, Proa-H); dC (75MHz; D2O)
11.4 (CH3, 4-C), 11.6* (CH3, 4-C), 23.9* (CH2, Proc-
C), 26.1 (CH2, Proc-C), 26.5* (CH2, 3-C), 26.9 (CH2,
3-C), 31.3 (CH2, Prob-C), 33.6* (CH2, Prob-C), 42.1*
(CH2, Glya-C), 42.3 (CH2, Glya-C), 48.8 (CH2, Prod-
C), 49.5* (CH2, Prod-C), 58.6 (CH, 2-C), 58.9* (CH,
2-C), 62.0* (CH, Proa-C), 62.4 (CH, Proa-C), 167.5
(quat., Gly-CO), 168.0* (quat., Gly-CO), 174.6* (quat.,
Pro-CON), 175.0 (quat., Pro-CON), 180.5* (quat., 1-
CO) and 180.6 (quat., 1-CO); m/z (FAB+) 258.1446
(MH+. C11H20N3O4 requires 258.1454).
1
by H NMR analysis (the ratio was estimated from the
integration of the doublets at d 6.52 and 7.10, assigned
to the Ea-NH protons of the minor and major conform-
ers, respectively): Rf 0.50 (EtOAc); [a]D ꢀ74.2 (c 0.15 in
CH2Cl2); mmax (film)/cmꢀ1 3312 br, 3064, 2971, 2878,
1725, 1649, 1533, 1453, 1386, 1344, 1256, 1211, 1193,
1146, 1055, 986, 917, 740 and 697; dH (300MHz; CDCl3;
Me4Si) 0.86 (3H, t, J 7.5, 4-H3), 1.73 (1H, apparent oc-
tet, J 7.2, 3-HAHB), 1.63–2.40 (5H, m, 3-HAHB, Proc-H2
and Prob-H2), 3.40 (1H, dd, J 16.4 and 9.0, Prod-
HAHB), 3.47–3.70 (1H, m, Prod-HAHB), 3.80* (0.10H,
dd, J 16.8 and 4.0, Glya-HAHB), 3.95 (0.90H, dd, J
17.1 and 4.2, Glya-HAHB), 4.05 (1H, dd, J 17.3 and
4.6, Glya-HAHB), 4.32* (0.10H, t, J 5.3, Proa-H),
4.44–4.63 (1.90H, m, 2-H and Proa-H), 5.05–5.25 (4H,
m, 2 · OCH2Ph), 5.59* (0.10H, br s, Gly-NH), 5.70
(0.90H, br s, Gly-NH), 6.52* (0.10H, d, J 7.9, 2-NH),
7.10 (0.90H, d, J 7.5, 2-NH) and 7.23–7.50 (10H, m,
2 · Ph); dC (75MHz; CDCl3) 9.4 (CH3, 4-C), 9.8*
(CH3, 4-C), 22.1* (CH2, Proc-C), 24.5 (CH2, Proc-C),
24.8* (CH2, 3-C), 25.0 (CH2, 3-C), 27.7 (CH2, Prob-
C), 31.8* (CH2, Prob-C), 43.0* (CH2, Glya-C), 43.2
(CH2, Glya-C), 46.1 (CH2, Prod-C), 46.9* (CH2, Prod-
C), 53.4 (CH, 2-C), 59.8 (CH, Proa-C), 66.6 (CH2,
OCH2Ph), 66.6 (CH2, OCH2Ph), 66.9* (CH2, OCH2Ph),
127.7* (CH, Ph), 127.7 (CH, Ph), 127.8 (CH, Ph), 127.9
(CH, Ph), 128.0* (CH, Ph), 128.1 (CH, Ph), 128.2 (CH,
Ph), 128.3 (CH, Ph), 135.0* (quat., Ph), 135.3 (quat.,
Ph), 136.2 (quat., Ph), 156.1 (quat., NCO2), 156.2*
(quat., NCO2), 167.8 (quat., Gly-CO), 167.9* (quat.,
Gly-CO), 170.7 (quat., Pro-CON), 171.2* (quat., Pro-
CON) and 171.8 (quat., 1-CO); m/z (EI+) 481.2216
(M+. C26H31N3O6 requires 481.2213).
4.7. N-Benzyloxycarbonyl-glycyl-L-proline N-hydroxy-
succinimide ester1 (16)
To a mixture of acid 2 (4.40g, 14.36mmol) in anhydrous
1,2-dimethoxyethane (130mL) under nitrogen at 0ꢁC,
were successively added N-hydroxysuccinimide (1.94g,
16.35mmol) and a solution of 1,3-dicyclohexylcarbodii-
mide (3.49g, 16.91mmol) in 1,2-dimethoxyethane
(19mL) cooled to 0ꢁC. The reaction mixture was
warmed to room temperature, stirred for 23h then fil-
tered through a CeliteTM pad, to partially remove solid
1,3-dicyclohexylurea, and the filtrate concentrated to
dryness under reduced pressure. The resultant residue
was dissolved in methylene chloride, washed with 10%
aqueous hydrochloric acid (100mL) and saturated
aqueous sodium hydrogen carbonate (100mL), dried
(MgSO4), filtered and concentrated to dryness. Purifica-
tion of the residue by flash column chromatography
(SiO2; 30–90% ethyl acetate–hexane; gradient elution)
gave a white foam, which was dissolved in methylene
chloride and triturated with diethyl ether to yield succin-
imide 16 (4.94g, 85%) as a white solid. Compound 16
was shown to be an 85:15 trans:cis mixture of conform-
ers: Rf 0.40 (EtOAc); mp 102–104ꢁC; [a]D ꢀ80.7 (c 0.29
in CH2Cl2); mmax (film)/cmꢀ1 3583 wk, 3406 br, 3333 br,
2952, 2883, 1815, 1784, 1738, 1660, 1522, 1439, 1368,
1245, 1206, 1077, 992, 911, 813, 734, 699 and 645; dH
(400MHz; CDCl3; Me4Si) 1.98–2.25 (2H, m, Proc-H2),
2.28–2.39 (1.70H, m, Prob-H2), 2.39–2.49* (0.30H, m,
Prob-H2), 2.82 [4H, s, C(O)CH2CH2C(O)], 3.38–3.56
(0.85H, m, Prod-HAHB), 3.56–3.75 (1.15H, m, Prod-
HAHB and Prod-HAHB*), 3.89* (0.15H, dd, J 16.8 and
3.4, Glya-HAHB), 3.98 (0.85H, dd, J 17.2 and 3.8,
Glya-HAHB), 4.08 (0.85H, dd, J 17.3 and 5.0, Glya-
HAHB), 4.14* (0.15H, dd, J 17.0 and 6.0, Glya-HAHB),
4.76* (0.15H, br t, J 5.1, Proa-H), 4.83 (0.85H, t, J 6.2,
Proa-H), 5.11 (2H, s, OCH2Ph), 5.64* (0.15H, br s, Gly-
NH), 5.75 (0.85H, br s, Gly-NH) and 7.27–7.38 (5H, m,
Ph); dC (100MHz; CDCl3) 22.0* (CH2, Proc-C), 24.6
(CH2, Proc-C), 25.4 [CH2, C(O)CH2CH2C(O)], 29.1
(CH2, Prob-C), 31.8* (CH2, Prob-C), 43.1 (CH2, Glya-
C), 45.7 (CH2, Prod-C), 46.7* (CH2, Prod-C), 56.6*
(CH, Proa-C), 56.7 (CH, Proa-C), 66.6 (CH2, OCH2Ph),
66.7* (CH2, OCH2Ph), 127.8 (CH, Ph), 127.9 (CH, Ph),
128.3, (CH, Ph), 136.4 (quat., Ph), 156.2 (quat., NCO2),
167.3 (quat., CO), 167.5 (quat., CO), 167.6* (quat., CO)
Representative procedure C for the hydrogenation of
protected tripeptides and analogues is as follows:
4.6. (2S)-Glycyl-L-prolyl-2-aminobutanoic acid (7)
A mixture of protected tripeptide 10 (0.46g, 0.96mmol)
and 10wt% palladium on activated carbon (0.22g,
0.21mmol) in 2:1 methanol–water (30cm3) was stirred
under an atmosphere of hydrogen at room temperature,
protected from light, for 22h. The reaction mixture was
filtered through a CeliteTM pad, and the pad washed with
50:50 methanol–water (150cm3). The filtrate was con-
centrated to dryness under reduced pressure at ambient
temperature and the residue triturated with methanol to
afford 7 (0.18g, 73%) as a white amorphous solid. Com-
pound 7 was shown to be an 83:17 trans:cis mixture of
conformers by 13C NMR analysis (the ratio was esti-
mated from the relative intensities of the resonances at
d 23.9 and 26.1, assigned to the Proc-C atoms of the
minor and major conformers, respectively): mp 188–
198ꢁC (decomp.); [a]D ꢀ112.2 (c 0.18 in H2O); mmax (Nu-
jol mull)/cmꢀ1 3528, 3312, 3188, 2630, 1648, 1598, 1570,
1537, 1418, 1234, 1208 and 930; dH (300MHz; D2O) 0.91
(3H, t, J 7.4, 4-H3), 1.60–2.14 (4H, m, 3-HAHB Proc-H2

526
M. A. Brimble et al. / Bioorg. Med. Chem. 13 (2005) 519–532
and 168.8 [quat., C(O)CH2CH2C(O)]; m/z (FAB+)
ammonium hydrogen carbonate (0.21g, 2.66mmol).
The mixture was stirred for 21h, the solvent removed
in vacuo, and the resultant residue dissolved in methyl-
ene chloride, washed with 10% aqueous hydrochloric
acid (50mL) and saturated aqueous sodium hydrogen
carbonate (50mL), dried (MgSO4), filtered and evapo-
rated to dryness. Purification of the residue by flash
column chromatography (SiO2; 80–20% ethyl acetate–
hexane to methanol–methylene chloride; gradient elu-
tion) gave primary amide 18 (0.87g, 89%) as a white
foam. Compound 18 was shown to be an 83:17 trans:cis
mixture of conformers: Rf 0.50 (10% MeOH–CH2Cl2);
[a]D ꢀ53.2 (c 0.24 in CH2Cl2); mmax (film)/cmꢀ1 3583
wk, 3309 br, 3065, 2952, 1720, 1655, 1536, 1453, 1342,
1255, 1212, 1172, 1129, 1052, 986, 913, 736 and 697;
dH (400MHz; CDCl3; Me4Si) 1.67–2.46 (8H, m, Proc-
H2, Prob-H2, Glnb-H2 and Glnc-H2), 3.27–3.75 (2H,
m, Prod-H2), 3.86* (0.17H, dd, J 12.2 and 4.2, Glya-
HAHB), 3.95 (1H, dd, J 17.2 and 4.9, Glya-HAHB and
Glya-HAHꢁB), 4.04 (0.83H, dd, J 17.1 and 5.1, Glya-
HAHB), 4.24* (0.17H, dd, J 8.5 and 2.5, Proa-H), 4.47
(0.83H, br d, J 5.4, Proa-H), 4.57 (1H, td, J 8.3, 8.3
and 3.2, Glna-H), 5.04–5.20 (4H, m, 2 · OCH2Ph),
5.53 (0.83H, br s, CONHAHB), 5.69–5.90 (1.17H, br
404.1451 (MH+. C19H22N3O7 requires 404.1458).
4.8. a-Benzyl N-benzyloxycarbonyl-glycyl-L-prolyl-L-
glutamate (17)
Triethylamine (1.65mL, 11.84mmol) and water (75mL)
were successively added to a mixture of L-glutamic acid
a-benzyl ester 15 (2.21g, 9.31mmol) in tetrahydrofuran
(150mL) at room temperature. A mixture of succinimide
16 (3.00g, 7.44mmol) in tetrahydrofuran (95mL) was
slowly added to the resultant solution and the reaction
mixture stirred for 16h. The solvent was then removed
in vacuo, the residue partitioned between methylene
chloride (100mL) and 10% aqueous hydrochloric acid
(100mL), and the aqueous layer extracted twice with
methylene chloride. The combined organic fractions
were washed with 10% aqueous hydrochloric acid
(200mL) and brine (200mL), dried (MgSO4), filtered
and evaporated to dryness. Purification of the residue
by flash column chromatography (SiO2; 50–20% ethyl
acetate–hexane to methanol–methylene chloride; gradi-
ent elution) afforded acid 17 (3.72g, 95%) as an hygro-
scopic white foam. Compound 17 was shown to be a
77:23 trans:cis mixture of conformers: Rf 0.50 (10%
MeOH–CH2Cl2); [a]D ꢀ55.6 (c 0.48 in CH2Cl2); mmax
(film)/cmꢀ1 3583 wk, 3312 br, 2952, 1721, 1646, 1534,
1453, 1342, 1257, 1208, 1172, 1125, 1057, 983, 912, 739
and 697; dH (300MHz; CDCl3; Me4Si) 1.76–2.45 (8H,
m, Proc-H2, Prob-H2, Glub-H2 and Gluc-H2), 3.38
(0.77H, br dd, J 16.1 and 8.4, Prod-HAHB), 3.46–3.76
(1.23H, m, Prod-HAHB and Prod-HAHB*), 3.76*
(0.23H, dd, J 17.2 and 3.2, Glya-HAHB), 3.96 (1H, dd,
J 17.3 and 4.9, Glya-HAHB and Glya-HAHB*), 4.06
(0.77H, dd, J 17.1 and 4.9, Glya-HAHB), 4.24–4.31*
(0.23H, m, Proa-H), 4.49–4.64 (1.77H, m, Proa-H and
Glua-H), 5.03–5.21 (4H, m, 2 · OCH2Ph), 6.01 (1H,
br s, Gly-NH) and 7.27–7.39 (11H, m, 2 · Ph and
Glua-NH); dC (100MHz; CDCl3) 22.2* (CH2, Proc-
C), 24.6 (CH2, Proc-C), 25.5* (CH2, Glub-C), 26.5
(CH2, Glub-C), 28.2 (CH2, Prob-C), 30.2 (CH2, Gluc-
C), 30.6* (CH2, Gluc-C), 31.9* (CH2, Prob-C), 43.1*
(CH2, Glya-C), 43.2 (CH2, Glya-C), 46.4 (CH2, Prod-
C), 47.2* (CH2, Prod-C), 52.0 (CH, Glua-C), 52.5*
(CH, Glua-C), 60.1 (CH, Proa-C), 66.9 (CH2,
OCH2Ph), 67.1 (CH2, OCH2Ph), 67.3* (CH2, OCH2Ph),
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), 128.5 (CH, Ph), 135.1* (quat., Ph), 135.2 (quat.,
Ph), 136.1* (quat., Ph), 136.2 (quat., Ph), 156.8 (quat.,
NCO2), 156.9* (quat., NCO2), 168.7 (quat., Gly-CO),
171.2* (quat., CO), 171.3 (quat., CO), 171.3 (quat.,
CO), 171.5* (quat., CO), 176.0 (quat., Gluc-CO) and
176.3* (quat., Gluc-CO); m/z (FAB+) 526.2197 (MH+.
C27H32N3O8 requires 526.2189).
*
m, CONHAHB and Gly-NH), 6.28 (1H, br s, CON-
HAHB) and 7.25–7.38 (11H, m, 2 · Ph and Glna-NH);
dC (100MHz; CDCl3) 22.2* (CH2, Proc-C), 24.6 (CH2,
Proc-C), 25.9* (CH2, Glnb-C), 27.1 (CH2, Glnb-C),
28.5 (CH2, Prob-C), 31.0 (CH2, Glnc-C), 31.4* (CH2,
Glnc-C), 31.9* (CH2, Prob-C), 43.1* (CH2, Glya-C),
43.2 (CH2, Glya-C), 46.3 (CH2, Prod-C), 47.1* (CH2,
Prod-C), 51.8 (CH, Glna-C), 52.4* (CH, Glna-C),
59.7* (CH, Proa-C), 60.1 (CH, Proa-C), 66.7 (CH2,
OCH2Ph), 67.0 (CH2, OCH2Ph), 67.1* (CH2, OCH2Ph),
127.7 (CH, Ph), 127.9 (CH, Ph), 128.0 (CH, Ph), 128.1
(CH, Ph), 128.1* (CH, Ph), 128.3 (CH, Ph), 128.3*
(CH, Ph), 128.4 (CH, Ph), 128.4 (CH, Ph), 135.1* (quat.,
Ph), 135.2 (quat., Ph), 136.3 (quat., Ph), 156.5* (quat.,
NCO2), 156.6 (quat., NCO2), 167.6* (quat., Gly-CO),
168.2 (quat., Gly-CO), 171.4* (quat., CO), 171.5 (quat.,
CO), 171.6 (quat., CO), 171.7* (quat., CO) and 175.2
(quat., Glnc-CO); m/z (FAB+) 525.2355 (MH+.
C27H33N4O7 requires 525.2349).
4.10. Glycyl-L-prolyl-L-glutamine (11)
Following procedure C, hydrogenation of amide 18
yielded tripeptide 11 (0.23g, 82%) as a white solid. Com-
pound 11 was shown to be an 85:15 trans:cis mixture of
conformers: mp 167–169ꢁC; [a]D ꢀ80.6 (c 0.24 in H2O);
dH (400MHz; D2O) 1.80–2.23 (5H, m, Glnb-H2, Prob-
HAHB and Proc-H2), 2.23–2.45 (3H, m, Prob-HAHB
and Glnc-H2), 3.48–3.82 (2H, m, Prod-H2), 3.75*
(0.15H, d, J 16.2, Glya-HAHB), 3.94* (0.15H, d, J
16.0, Glya-HAHB), 3.97 (0.85H, d, J 18.1, Glya-HAHB),
4.01 (0.85H, d, J 17.4, Glya-HAHB), 4.17 (1H, dd, J 8.4
and 4.9, Glna-H) and 4.48 (1H, dd, J 7.9 and 3.7, Proa-
H); dC (100MHz; D2O) 23.9* (CH2, Proc-C), 26.1 (CH2,
Proc-C), 28.9* (CH2, Glnb-C), 29.6 (CH2, Glnb-C), 31.2
(CH2, Prob-C), 33.4 (CH2, Glnc-C), 33.6* (CH2, Prob-
C), 33.6* (CH2, Glnc-C), 42.1* (CH2, Glya-C), 42.3
(CH2, Glya-C), 48.8 (CH2, Prod-C), 49.4* (CH2, Prod-
C), 56.4 (CH, Glna-C), 56.9* (CH, Glna-C), 62.1*
4.9. N-Benzyloxycarbonyl-glycyl-L-prolyl-L-glutamine
a-benzyl ester (18)
To a solution of acid 17 (0.98g, 1.86mmol) and di-tert-
butyl dicarbonate (0.56g, 2.49mmol) in 1,4-dioxane
(28mL), under nitrogen at room temperature, were
added pyridine (0.21mL, 2.60mmol) dropwise and

M. A. Brimble et al. / Bioorg. Med. Chem. 13 (2005) 519–532
527
(CH, Proa-C), 62.5 (CH, Proa-C), 167.5 (quat., Gly-
CO), 168.0* (quat., Gly-CO), 174.8* (quat., Pro-CON),
175.2 (quat., Pro-CON), 179.4* (quat., Glna-CO),
179.5 (quat., Glna-CO), 180.2* (quat., Glnc-CO)
and 180.4 (quat., Glnc-CO); m/z (FAB+) 301.1519
(MH+. C12H21N4O5 requires 301.1512).
CONHCH3);
C28H35N4O7 requires 539.2506).
m/z
(FAB+)
539.2508
(MH+.
4.12. Glycyl-L-prolyl-L-N-methylglutamine (12)
Following procedure C, hydrogenation of N-methyl-
amide 19 yielded analogue 12 (0.20g, 67%) as a white
solid. Compound 12 was shown to be an 86:14 trans:
cis mixture of conformers: mp 193–196ꢁC (dec.); [a]D
ꢀ77.3 (c 0.19 in H2O); dH (300MHz; D2O) 1.82–2.46
(8H, m, Glnb-H2, Prob-H2, Proc-H2 and Glnc-H2),
2.72 (3H, s, CONHCH3), 3.51–3.73 (2H, m, Prod-H2),
3.77* (0.14H, d, J 16.3, Glya-HAHB), 3.96* (0.14H, d,
J 16.4, Glya-HAHB), 3.98 (0.86H, d, J 17.3, Glya-
HAHB), 4.04 (0.86H, d, J 17.2, Glya-HAHB), 4.11–
4.21* (0.14H, m, Glna-H), 4.15 (0.86H, dd, J 8.5 and
4.9, Glna-H) and 4.49 (1H, dd, J 8.2 and 4.0, Proa-
H); dC (75MHz; D2O) 23.9* (CH2, Proc-C), 26.1
(CH2, Proc-C), 27.7 (CH3, CONHCH3), 29.2* (CH2,
Glnb-C), 29.9 (CH2, Glnb-C), 31.3 (CH2, Prob-C),
33.6* (CH2, Prob-C), 34.1 (CH2, Glnc-C), 34.2* (CH2,
Glnc-C), 42.1* (CH2, Glya-C), 42.3 (CH2, Glya-C),
48.8 (CH2, Prod-C), 49.4* (CH2, Prodd-C), 56.5 (CH,
Glna-C), 56.9* (CH, Glna-C), 62.1* (CH, Proa-C),
62.5 (CH, Proa-C), 167.5 (quat., Gly-CO), 168.0*
(quat., Gly-CO), 174.7* (quat., Pro-CON), 175.2 (quat.,
Pro-CON), 177.7* (quat., CONHCH3), 177.8
(quat., CONHCH3), 179.4* (quat., CO2H) and 179.6
(quat., CO2H); m/z (FAB+) 315.1672 (MH+. C13H23-
N4O5 requires 315.1669).
4.11. N-Benzyloxycarbonyl-glycyl-L-prolyl-L-N-methyl-
glutamine a-benzyl ester (19)
Triethylamine (1.62mL, 11.62mmol) was added drop-
wise to a mixture of acid 17 (1.02g, 1.94mmol) and
methylamine hydrochloride (0.30g, 4.44mmol) in aceto-
nitrile (45mL), under nitrogen at room temperature,
and the reaction mixture stirred for 5min. 2-(N-Benzo-
triazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluorobo-
rate (TBTU) (0.64g, 1.95mmol) was added in one
portion, the mixture stirred for 16.5h then the solvent
removed under reduced pressure. The resultant residue
was partitioned between methylene chloride (50mL)
and 10% aqueous hydrochloric acid (50mL) and the
aqueous fraction extracted with methylene chloride
(2 · 50mL). The combined organic fractions were
washed with 10% aqueous hydrochloric acid (100mL)
and saturated aqueous sodium hydrogen carbonate
(100mL), dried (MgSO4), filtered and evaporated to
dryness. Trituration of the residue with methylene chlo-
ride and diethyl ether afforded N-methylamide 19
(0.83g, 79%) as a white solid. Compound 19 was shown
to be an 85:15 trans:cis mixture of conformers: Rf 0.45
(10% MeOH–CH2Cl2); mp 137–139ꢁC; [a]D ꢀ54.4 (c
0.25 in CH2Cl2); mmax (film)/cmꢀ1 3583 wk, 3312 br,
3065, 2951, 1725, 1649, 1543, 1453, 1411, 1377, 1335,
1258, 1213, 1171, 1054, 986, 914, 734 and 698; dH
(300MHz; CDCl3; Me4Si) 1.79–2.36 (8H, m, Proc-H2,
Glnb-H2, Prob-H2 and Glnc-H2), 2.66–2.73* (0.45H,
m, CONHCH3), 2.69 (2.55H, d, J 4.7, CONHCH3),
3.27–3.80 (2H, m, Prod-H2), 3.84* (0.30H, d, J 4.1,
Glya-H2), 3.96 (0.85H, dd, J 17.3 and 4.9, Glya-HAHB),
4.05 (0.85H, dd, J 17.3 and 4.9, Glya-HAHB), 4.21*
(0.15H, dd, J 8.5 and 2.6, Proa-H), 4.35–4.53 (0.85H,
m, Proa-H), 4.54 (1H, td, J 8.3, 8.3 and 3.6, Glna-H),
5.06–5.20 (4H, m, 2 · OCH2Ph), 5.70 (0.85H, br t, J
4.4, Gly-NH), 5.82* (0.15H, br t, J 3.8, Gly-NH),
6.23–6.48 (1H, br m, CONHCH3), 7.16–7.52 (10.85H,
m, 2 · Ph and Gln-NH) and 7.45* (0.15H, d, J 7.6,
Gln-NH); dC (75MHz; CDCl3) 22.4* (CH2, Proc-C),
24.8 (CH2, Proc-C), 26.2 (CH3, CONHCH3), 26.3*
(CH3, CONHCH3), 27.8 (CH2, Glnb-C), 28.4 (CH2,
Prob-C), 31.8 (CH2, Glnc-C), 31.9* (CH2, Glnc-C or
Prob-C), 32.0* (CH2, Prob-C or Glnc-C), 43.1* (CH2,
Glya-C), 43.3 (CH2, Glya-C), 46.3 (CH2, Prod-C),
47.2* (CH2, Prod-C), 51.8 (CH, Glna-C), 52.4* (CH,
Glna-C), 59.8* (CH, Proa-C), 60.3 (CH, Proa-C),
66.8* (CH2, OCH2Ph), 66.9 (CH2, OCH2Ph), 67.1
(CH2, OCH2Ph), 67.2* (CH2, OCH2Ph), 127.8* (CH,
Ph), 127.9 (CH, Ph), 128.1 (CH, Ph), 128.2 (CH, Ph),
128.3* (CH, Ph), 128.4 (CH, Ph), 128.4* (CH, Ph),
128.5 (CH, Ph), 128.5 (CH, Ph), 135.2 (quat., Ph),
136.2 (quat., Ph), 156.3* (quat., NCO2), 156.4 (quat.,
NCO2), 167.2* (quat., Gly-CO), 167.9 (quat., Gly-
CO), 171.3 (quat., CO), 171.4* (quat., CO), 171.5 (quat.,
CO), 172.7 (quat., CONHCH3) and 172.8* (quat.,
4.13. N-Benzyloxycarbonyl-glycyl-L-prolyl-L-N,N-
dimethylglutamine a-benzyl ester (20)
Following procedure B, coupling of acid 17 with
dimethylamine hydrochloride using triethylamine as
the base yielded N,N-dimethylamide 20 (0.48g, 89%)
as a colourless oil, which slowly crystallised to a white
solid. Compound 20 was shown to be a 79:21 trans:cis
mixture of conformers: Rf 0.50 (10% MeOH–CH2Cl2);
mp 110–111ꢁC; [a]D ꢀ56.9 (c 0.26 in CH2Cl2); mmax
(film)/cmꢀ1 3583 wk, 3297 br, 3063, 3033, 2946, 1724,
1645, 1530, 1453, 1340, 1255, 1214, 1185, 1055, 986,
915, 815, 737 and 697; dH (300MHz; CDCl3; Me4Si)
1.80–2.28 (6H, m, Proc-H2, Prob-H2 and Glnb-H2),
2.33 (2H, t, J 6.5, Glnc-H2), 2.82* [0.63H, s, CON(-
CH3)A(CH3)B], 2.84 [2.37H, s, CON(CH3)A(CH3)B],
2.86 [2.37H, s, CON(CH3)A(CH3)B], 2.91* [0.63H, s,
CON(CH3)A(CH3)B], 3.30–3.47 (0.79H, m, Prod-
HAHB), 3.50–3.67 (1H, m, Prod-HAHB and Prod-
HAHB*), 3.75–3.86* (0.21H, m, Prod-HAHB), 3.91*
(0.42H, dd, J 9.5 and 4.5, Glya-H2), 4.03 (1.58H, d, J
4.2, Glya-H2), 4.26* (0.21H, dd, J 7.3 and 3.5, Proa-
H), 4.39–4.56 (1.79H, m, Glna-H and Proa-H), 5.06–
5.22 (4H, m, 2 · OCH2Ph), 5.61* (0.21H, br t, J 4.5,
Gly-NH), 5.75 (0.79H, br t, J 3.8, Gly-NH), 7.27–7.39
(10H, m, 2 · Ph), 7.72 (0.79H, d, J 7.0, Glna-NH) and
8.52* (0.21H, d, J 5.6, Glna-NH); dC (75MHz; CDCl3)
22.2* (CH2, Proc-C), 24.5 (CH2, Proc-C), 24.9* (CH2,
Glnb-C), 26.3 (CH2, Glnb-C), 28.5 (CH2, Prob-C),
29.2 (CH2, Glnc-C), 29.5* (CH2, Glnc-C), 31.8* (CH2,
Prob-C), 35.4 [CH3, CON(CH3)A(CH3)B], 35.6* [CH3,
CON(CH3)A(CH3)B], 36.9 [CH3, CON(CH3)A(CH3)B],

528
M. A. Brimble et al. / Bioorg. Med. Chem. 13 (2005) 519–532
43.0* (CH2, Glya-C), 43.3 (CH2, Glya-C), 46.1 (CH2,
Prod-C), 46.9* (CH2, Prod-C), 52.3 (CH, Glna-C),
52.9* (CH, Glna-C), 60.1 (CH, Proa-C), 66.5* (CH2,
OCH2Ph), 66.6 (CH2, OCH2Ph), 66.8 (CH2, OCH2Ph),
66.9* (CH2, OCH2Ph), 127.8* (CH, Ph), 127.9* (CH,
Ph), 127.9 (CH, Ph), 127.9 (CH, Ph), 128.1 (CH, Ph),
128.2* (CH, Ph), 128.3 (CH, Ph), 128.3 (CH, Ph),
128.4 (CH, Ph), 135.4 (quat., Ph), 136.3 (quat., Ph),
136.4* (quat., Ph), 156.1 (quat., NCO2), 167.4 (quat.,
Gly-CO), 167.7* (quat., Gly-CO), 171.1* (quat., CO),
171.2 (quat., CO), 171.4 (quat., CO), 171.5* (quat.,
CO), 172.1 [quat., CON(CH3)2] and 172.4* [quat.,
CON(CH3)2]; m/z (EI+) 552.2577 (M+. C29H36N4O7
requires 552.2584).
ganic fraction washed with 10% aqueous hydrochloric
acid (50mL), saturated aqueous sodium hydrogen car-
bonate (50mL) and brine (50mL), dried (MgSO4), fil-
tered and evaporated to dryness. The resultant residue
was triturated with methylene chloride and diethyl ether
to yield acid-sensitive alcohol 21 (0.44g, 63%) as a white
solid. Compound 21 was shown to be an 85:15 trans:cis
mixture of conformers: Rf 0.70 (10% MeOH–CH2Cl2);
mp 116–117ꢁC; [a]D ꢀ65.5 (c 0.20 in CH2Cl2); mmax
(film)/cmꢀ1 3583 wk, 3314 br, 3064, 3033, 2952, 2877,
1725, 1649, 1535, 1453, 1339, 1256, 1213, 1172, 1057,
987, 913, 736 and 698; dH (300MHz; CDCl3; Me4Si)
1.39–1.65 (2H, m, 4-H2), 1.69–2.32 (6H, m, 3-H2,
Proc-H2 and Prob-H2), 2.69 (0.85H, br s, OH), 3.17*
(0.15H, br s, OH), 3.39 (0.85H, br dd, J 16.0 and 8.4,
Prod-HAHB), 3.46–3.71 (3.15H, m, Prod-HAHB, Prod-
4.14. Glycyl-L-prolyl-L-N,N-dimethylglutamine (13)
*
HAHB and 5-H2), 3.90* (0.30H, d, J 4.7, Glya-H2),
Following procedure C, hydrogenation of N,N-dimeth-
ylamide 20 yielded analogue 13 (0.26g, 95%) as an extre-
mely hygroscopic white solid, on trituration with
anhydrous diethyl ether. Compound 13 was shown to
be an 87:13 trans:cis mixture of conformers: mp 175ꢁC
(dec.); [a]D ꢀ65.6 (c 0.19 in H2O); dH (300MHz; D2O)
1.72–2.55 (8H, m, Glnb-H2, Proc-H2, Prob-H2 and
Glnc-H2), 2.86 [3H, s, CON(CH3)A(CH3)B], 2.99*
[0.39H, s, CON(CH3)A(CH3)B], 3.00 [2.61H, s, CON(-
CH3)A(CH3)B], 3.43–3.67 (2H, m, Prod-H2), 3.71*
(0.13H, d, J 16.5, Glya-HAHB), 3.82–3.99 (1.87H, m,
3.97 (1.70H, d, J 4.9, Glya-H2), 4.28* (0.15H, t, J 5.6,
Proa-H), 4.46–4.63 (1.85H, m, Proa-H and 2-H), 5.04–
5.23 (4H, m, 2 · OCH2Ph), 5.71* (0.15H, br t, J 5.0,
Gly-NH), 5.75 (0.85H, br t, J 4.7, Gly-NH), 7.22–7.45
(10.85H, m, 2-NH and 2 · Ph) and 7.76* (0.15H, d, J
7.6, 2-NH); dC (75MHz; CDCl3) 22.3* (CH2, Proc-C),
24.8 (CH2, Proc-C), 28.0 (CH2, Prob-C), 28.2 (CH2, 4-
C), 28.5 (CH2, 3-C), 32.0* (CH2, Prob-C), 43.3 (CH2,
Glya-C), 46.3 (CH2, Prod-C), 47.1* (CH2, Prod-C),
52.2 (CH, 2-C), 60.1 (CH, Proa-C), 61.8 (CH2, 5-C),
66.9 (CH2, OCH2Ph), 67.0 (CH2, OCH2Ph), 67.1*
(CH2, OCH2Ph), 128.0 (CH, Ph), 128.1 (CH, Ph),
128.2 (CH, Ph), 128.2* (CH, Ph), 128.3 (CH, Ph),
128.4* (CH, Ph), 128.5 (CH, Ph), 128.5 (CH, Ph),
135.4 (quat., Ph), 136.3 (quat., Ph), 156.6 (quat.,
NCO2), 156.7* (quat., NCO2), 168.0* (quat., Gly-CO),
168.2 (quat., Gly-CO), 171.0 (quat., CO), 171.4* (quat.,
CO) and 171.9 (quat., CO); m/z (FAB+) 512.2389
(MH+. C27H34N3O7 requires 512.2397).
*
Glya-HAHB and Glya-H2), 4.10 (1H, dd, J 8.2 and
5.1, Glna-H) and 4.42 (1H, dd, J 8.2 and 4.0, Proa-
H); dC (75MHz; D2O) 23.9* (CH2, Proc-C), 26.1
(CH2, Proc-C), 28.5* (CH2, Glnb-C), 29.3 (CH2,
Glnb-C), 31.3 (CH2, Prob-C), 31.3 (CH2, Glnc-C),
31.5* (CH2, Glnc-C), 33.6* (CH2, Prob-C), 37.3 [CH3,
CON(CH3)A(CH3)B], 39.3* [CH3, CON(CH3)A(CH3)B],
39.4 [CH3, CON(CH3)A(CH3)B], 42.2* (CH2, Glya-C),
42.3 (CH2, Glya-C), 48.8 (CH2, Prod-C), 49.4* (CH2,
Prod-C), 56.7 (CH, Glna-C), 57.2* (CH, Glna-C),
62.1* (CH, Proa-C), 62.5 (CH, Proa-C), 167.6 (quat.,
Gly-CO), 168.2* (quat., Gly-CO), 174.8* (quat., Pro-
CON), 175.2 (quat., Pro-CON), 176.7* [quat.,
CON(CH3)2], 176.8 [quat., CON(CH3)2], 179.6* (quat.,
CO2H) and 179.8 (quat., CO2H); m/z (FAB+)
329.1836 (MH+. C14H25N4O5 requires 329.1825).
4.16. (2S)-Glycyl-L-prolyl-2-amino-5-hydroxypentanoic
acid (14)
Following procedure C, hydrogenation of alcohol 21 in
82:16:2 tetrahydrofuran–water–triethylamine (24.5mL;
v/v) yielded analogue 14 (0.27g, 100%) as an hygroscopic
pale yellow solidꢁ on trituration with anhydrous diethyl
ether. Compound 14 was shown to be a 76:24 trans:cis
mixture of conformers: [a]D ꢀ80.2 (c 0.29 in H2O); dH
(300MHz; D2O) 1.48–2.48 (8H, m, 4-H2, 3-H2, Proc-
H2 and Prob-H2), 3.48–3.73 (4.24H, m, Prod-H2, 5-H2
4.15. (2S)-Benzyl N-benzyloxycarbonyl-glycyl-L-prolyl-2-
amino-5-hydroxypentanoate (21)
*
To a solution of acid 17 (0.72g, 1.37mmol) in anhy-
drous tetrahydrofuran (50mL), cooled to ca. ꢀ5ꢁC un-
der nitrogen, were successively added dropwise
triethylamine (0.32mL, 2.30mmol) and ethyl chlorofor-
mate (0.21mL, 2.20mmol) and the reaction mixture stir-
red at ca. ꢀ5ꢁC for 30min to form the mixed anhydride.
Powdered sodium borohydride (0.24g, 6.35mmol) was
added in one portion, followed by methanol (15mL)
dropwise, keeping the temperature at ca. ꢀ5ꢁC. The
effervescent reaction mixture was stirred for 15min,
warmed to room temperature and stirred for a further
15min, then quenched with saturated aqueous ammo-
nium chloride (5mL). The organic solvents were re-
moved in vacuo at ambient temperature, the aqueous
residue extracted with ethyl acetate (50mL) and the or-
and Glya-HAHB*), 3.79–4.00 (1.76H, m, Glya-HAHB
and Glya-H2), 4.11–4.26* (0.24H, m, 2-H), 4.17
(0.76H, dd, J 7.7 and 5.2, 2-H) and 4.40–4.57 (1H, m,
Proa-H); dC (75MHz; D2O) 24.5* (CH2, Proc-C), 26.7
(CH2, Proc-C), 30.3 (CH2, 4-C), 30.6* (CH2), 30.7
(CH2, 3-C), 31.9 (CH2, Prob-C), 34.2* (CH2, Prob-C),
43.3 (CH2, Glya-C), 49.3 (CH2, Prod-C), 50.0* (CH2,
Prod-C), 57.5 (CH, 2-C), 57.7* (CH, 2-C), 62.6* (CH,
Proa-C), 63.0 (CH, Proa-C), 63.6* (CH2, 5-C), 63.7
(CH2, 5-C), 169.5 (quat., Gly-CO), 170.3* (quat., Gly-
CO), 175.4* (quat., Pro-CON), 175.7 (quat., Pro-
ꢁ Due to the extremely hygroscopic nature of 14, its melting point could
not be determined under standard atmospheric conditions.

M. A. Brimble et al. / Bioorg. Med. Chem. 13 (2005) 519–532
529
CON), 180.9* (quat., 1-CO) and 181.1 (quat., 1-CO);
m/z (FAB+) 288.1551 (MH+. C12H22N3O5 requires
288.1560).
H2), 3.57–3.77 (2H, m, Prod-H2), 3.78–4.16 (4H, m,
2 · Glya-H2) and 4.53–4.63 (1H, m, Proa-H); dC
(75MHz; D2O) 23.8* (CH2, Proc-C), 26.0 (CH2, Proc-
C), 31.4 (CH2, Prob-C), 33.6* (CH2, Prob-C), 42.3*
(CH2, Glya-C), 42.4 (CH2, Glya-C), 45.1 (CH2, Glya-
CCO2H), 45.2* (CH2, Glya-CCO2H), 48.7 (CH2, Prod-
C), 49.3* (CH2, Prod-C), 62.0* (CH, Proa-C), 62.4
(CH, Proa-C), 168.2 (quat., Gly-CON), 168.6* (quat.,
Gly-CON), 175.2* (quat., Pro-CON), 175.4 (quat.,
Pro-CON) and 178.1 (quat., Gly-CO2H); m/z (FAB+)
230.1125 (MH+. C9H16N3O4 requires 230.1141).
4.17. Glycine benzyl ester p-toluenesulfonate41 (24)
Following procedure A, benzylation of glycine yielded
p-toluenesulfonate41 24 (11.22g, 99%) as a crystalline
white solid: mp 130–132ꢁC (lit.,41 132–134ꢁC); dH
(400MHz; CDCl3; Me4Si) 2.23 (3H, s, Ar–CH3), 3.69
(2H, br d, J 5.6, Glya-H2), 4.98 (2H, s, OCH2Ph), 6.98
(2H, d, J 8.0, 2 · 3-H), 7.15–7.28 (5H, m, Ph),
7.67 (2H, d, J 8.1, 2 · 2-H) and 8.08 (3H, br s, N+H3);
dC (100MHz; CDCl3) 21.2 (CH3, Ar–CH3), 40.5 (CH2,
Glya-C), 67.6 (CH2, OCH2Ph), 125.9 (CH, Ar), 128.2
(CH, Ar), 128.3 (CH, Ar), 128.4 (CH, Ar), 128.9 (CH,
Ar), 134.7 (quat., Ph), 140.3 (quat., Ar), 141.2 (quat.,
Ar) and 167.4 (quat., Gly-CO); m/z (FAB+) 166
(BnO2CCH2NH3+, 100%) and 91 (C7H7+, 53).
4.20. Dimethyl N-benzyloxycarbonyl-glycyl-L-prolyl-L-
glutamate (27)
Following procedure B, coupling of acid 2 with dimethyl
L-glutamate hydrochloride 26 using triethylamine as the
base yielded amide 27 (1.91g, 97%) as a colourless oil.
Compound 27 was shown to be an 89:11 trans:cis mix-
ture of conformers: Rf 0.30 (EtOAc); [a]D ꢀ62.3 (c
0.23 in CH2Cl2); mmax (film)/cmꢀ1 3305 br, 2953 br,
1732, 1650, 1532, 1440, 1256, 1213, 1173, 1054, 984
and 698; dH (300MHz; CDCl3; Me4Si) 1.84–2.48 (8H,
m, Prob-H2, Proc-H2, Glub-H2 and Gluc-H2), 3.42
(1H, br dd, J 16.2 and 8.6, Prod-HAHB), 3.51–3.82
(1H, m, Prod-HAHB), 3.64 (2.67H, s, OCH3), 3.68*
(0.33H, s, OCH3), 3.70* (0.33H, s, OCH3), 3.74
(2.67H, s, OCH3), 3.98 (1H, dd, J 17.2 and 4.1, Glya-
HAHB), 4.06 (1H, dd, J 17.3 and 4.7, Glya-HAHB),
4.29–4.37* (0.11H, m, Proa-H), 4.47–4.62 (1.89H, m,
Glua-H and Proa-H), 5.10* (0.22H, s, OCH2Ph), 5.12
(1.78H, s, OCH2Ph), 5.58* (0.11H, br s, Gly-NH),
5.72 (0.89H, br s, Gly-NH), 7.09–7.17* (0.11H, m,
Glu-NH), 7.22 (0.89H, d, J 7.5, Glu-NH) and 7.28–
7.41 (5H, m, Ph); dC (75MHz; CDCl3) 22.2* (CH2,
Proc-C), 24.6 (CH2, Proc-C), 26.0* (CH2, Glub-C),
26.8 (CH2, Glub-C), 27.8 (CH2, Prob-C), 29.7 (CH2,
Gluc-C), 30.1* (CH2, Gluc-C), 31.8* (CH2, Prob-C),
43.1* (CH2, Glya-C), 43.3 (CH2, Glya-C), 46.1 (CH2,
Prod-C), 47.0* (CH2, Prod-C), 51.6 (CH3, OCH3),
51.8* (CH3, OCH3), 52.2 (CH, Glua-C), 52.3* (CH,
Glua-C), 59.9 (CH, Proa-C), 60.1* (CH, Proa-C), 66.7
(CH2, OCH2Ph), 127.8 (CH, Ph), 127.9 (CH, Ph),
128.3 (CH, Ph), 136.3 (quat., Ph), 156.2 (quat.,
NCO2), 167.9 (quat., Gly-CO), 168.2* (quat., Gly-
CO), 170.9 (quat., Pro-CON), 171.5* (quat., Pro-
CON), 171.7* (quat., Glu-CO), 171.9 (quat., Glu-CO),
173.1 (quat., Glu-CO) and 173.3* (quat., Glu-CO); m/z
(EI+) 463.1958 (M+. C22H29N3O8 requires 463.1955).
4.18. N-Benzyloxycarbonyl-glycyl-L-prolyl-glycine benzyl
ester (25)
Following procedure B, coupling of acid 2 with p-tolu-
enesulfonate 24 using triethylamine as the base yielded
amide 25 (1.03g, 94%) as a slightly opaque oil. Com-
pound 25 was shown to be an 86:14 trans:cis mixture
of conformers: Rf 0.30 (EtOAc); [a]D ꢀ68.7 (c 0.58 in
CH2Cl2); mmax (film)/cmꢀ1 3317 br, 2952, 1743, 1722,
1650, 1534, 1453, 1256, 1188, 1051, 983, 908, 739 and
698; dH (300MHz; CDCl3; Me4Si) 1.77–2.29 (3H, m,
Prob-HAHB and Proc-H2), 2.32–2.45 (1H, m, Prob-
HAHB), 3.30–3.70 (2H, m, Prod-H2), 3.78–4.13 (4H,
m, 2 · Glya-H2), 4.28–4.38* (0.14H, m, Proa-H), 4.61
(0.86H, dd, J 8.1 and 1.7, Proa-H), 5.05–5.20 (4H, m,
2 · OCH2Ph), 5.53* (0.14H, br s, NH), 5.67 (0.86H, br
s, NH), 6.78* (0.14H, br s, NH) and 7.13–7.40
(10.86H, m, 2 · Ph and NH); dC (75MHz; CDCl3)
21.9* (CH2, Proc-C), 24.4 (CH2, Proc-C), 27.9 (CH2,
Prob-C), 31.8* (CH2, Prob-C), 41.1 (CH2, Glya-
CCO2–), 43.0* (CH2, Glya-C), 43.1 (CH2, Glya-C),
46.0 (CH2, Prod-C), 46.9* (CH2, Prod-C), 59.8 (CH,
Proa-C), 66.6 (CH2, OCH2Ph), 66.7 (CH2, OCH2Ph),
66.8* (CH2, OCH2Ph), 127.6* (CH, Ph), 127.7 (CH,
Ph), 127.8 (CH, Ph), 127.9* (CH, Ph), 128.0 (CH, Ph),
128.1* (CH, Ph), 128.2 (CH, Ph), 128.3 (CH, Ph),
128.6* (CH, Ph), 128.8* (CH, Ph), 135.0* (quat., Ph),
135.1 (quat., Ph), 136.2 (quat., Ph), 156.3 (quat.,
NCO2), 156.5* (quat., NCO2), 168.2 (quat., Gly-
CON), 169.4 (quat., Gly-CO2), 171.3 (quat., Pro-
CON) and 171.8* (quat., Pro-CON); m/z (EI+)
453.1893 (M+. C24H27N3O6 requires 453.1900).
4.21. Dimethyl glycyl-L-prolyl-L-glutamate (23)
Following procedure C, hydrogenation of amide 27
yielded analogue 23 (1.31g, 97%) as an hygroscopic
white foam following purification by flash column chro-
matography (SiO2; 5–40% methanol–methylene chlo-
ride; gradient elution). Compound 23 was shown to be
an 87:13 trans:cis mixture of conformers: Rf 0.30 (20%
MeOH–CH2Cl2); [a]D ꢀ90.7 (c 0.20 in H2O); mmax
(film)/cmꢀ1 3583, 3199 br, 2954 br, 1738, 1660, 1535,
1454, 1436, 1353, 1259, 1207, 1173, 1125, 987 and 917;
dH (400MHz; D2O) 1.77–2.12 (4H, m, Prob-HAHB
Proc-H2 and Glub-HAHB), 2.14–2.45 (2H, m,
4.19. Glycyl-L-prolyl-glycine42 (22)
Following procedure C, hydrogenation of amide 25 in
methanol yielded tripeptide42 22 (0.15g, 85%) as a white
solid on trituration with diethyl ether. Compound 22
was shown to be a 76:24 trans:cis mixture of conformers:
mp 175–185ꢁC (decomp.); [a]D ꢀ89.8 (c 0.17 in H2O);
mmax (Nujol mull)/cmꢀ1 3543, 3311 br, 2659, 1649,
1595, 1570, 1533, 1317, 1230, 1201 and 930; dH
(300MHz; D2O) 1.90–2.54 (4H, m, Prob-H2 and Proc-

530
M. A. Brimble et al. / Bioorg. Med. Chem. 13 (2005) 519–532
Glub-HAHB and Prob-HAHB), 2.45–2.61 (2H, m, Gluc-
H2), 3.50–3.64 (2.13H, m, Prod-H2 and Glya-HAHB*),
3.70 (3H, s, OCH3), 3.74 (3H, s, OCH3), 3.87–4.03
(1.87H, m, Glya-H2 and Glya-HAHB*), 4.42–4.52
(1.87H, m, Glua-H and Proa-H) and 4.54* (0.13H, dd,
J 8.6 and 2.6, Proa-H); (100MHz; D2O) 24.7* (CH2,
Proc-C), 27.0 (CH2, Proc-C), 27.9* (CH2, Glub-C),
28.3 (CH2, Glub-C), 32.2 (CH2, Prob-C), 32.6 (CH2,
Gluc-C), 32.8* (CH2, Gluc-C), 34.6* (CH2, Prob-C),
43.1* (CH2, Glya-C), 43.3 (CH2, Glya-C), 49.6 (CH2,
Prod-C), 50.3* (CH2, Prod-C), 54.8 (CH, Glua-C),
54.9* (CH, Glua-C), 55.1 (CH3, OCH3), 55.7 (CH3,
OCH3), 55.8* (CH3, OCH3), 62.6* (CH, Proa-C), 63.0
(CH, Proa-C), 168.7 (quat., Gly-CO), 169.3* (quat.,
Gly-CO), 176.0* (quat., CO), 176.2 (quat., CO),
176.5* (quat., CO), 177.0 (quat., CO), 178.2* (quat.,
CO) and 178.3 (quat., CO); m/z (EI+) 329.1584 (M+.
C14H23N3O6 requires 329.1587).
+19.4 (c 0.23 in CH2Cl2); dH (300MHz; CDCl3; Me4Si)
1.43 [9H, s, C(CH3)3], 2.06 (2H, m, allyl-CH2), 2.30–2.44
(2H, m, Glub-H2), 2.64 (1H, quintet, J 6.7, Gluc-H),
4.41 (1H, br d, J 7.6, Glua-H), 5.01–5.71 (7H, m,
2 · OCH2Ph, @CH2, Glua-NH), 5.60–5.74 (1H, m,
CH@CH2) and 7.31–7.35 (10H, m, 2 · Ph); dC
(75MHz; CDCl3) 28.2 [CH3, C(CH3)3], 33.4 (CH2, al-
lyl-CH2), 36.2 (CH2, Glub-C), 41.7, (CH, Gluc-C),
52.2 (CH, Glua-C), 66.4 (CH2, OCH2Ph), 67.0 (CH2,
OCH2Ph), 80.0 [quat., C(CH3)3], 117.6 (CH2, @CH2),
128.2 (CH, Ph), 128.2 (CH, Ph), 128.3 (CH, Ph), 128.4
(CH, Ph), 128.5 (CH, Ph), 134.1 (CH, CH@CH2),
135.2 (quat., Ph), 135.7 (quat., Ph), 155.3 (quat.,
NCO2), 172.1 (quat., Glua-CO) and 174.6 (quat.,
Gluc-CO); m/z (FAB+) 468.2382 (MH+. C27H34NO6 re-
quires 468.2386).
4.24. (cS)-Dibenzyl N-benzyloxycarbonylglycyl-L-prolyl-
L-c-allylglutamate (33)
4.22. Dibenzyl N-tert-butyloxycarbonyl-L-glutamate43
(30)
Trifluoroacetic acid (3mL) was added to a solution of
alkene 31 (0.48g, 1.07mmol) in methylene chloride
(15mL) at 0ꢁC and the reaction mixture stirred for 2h
then the volatiles were removed in vacuo to yield tenta-
tively trifluoroacetate 32 as a cream solid. To a solution
of acid 2 (0.30g, 0.94mmol) in methylene chloride
(20mL), cooled to 0ꢁC under nitrogen, were successively
added dropwise triethylamine (0.15mL, 1.07mmol) and
ethyl chloroformate (0.10mL, 1.07mmol) and the solu-
tion stirred at 0ꢁC for 45min to form the mixed anhy-
dride. Triethylamine (0.15mL, 1.07mmol) was added
to the crude trifluoroacetate 32 in methylene chloride
(15mL), under nitrogen at 0ꢁC, and the resultant solu-
tion added dropwise to the above mixed anhydride.
The reaction mixture was warmed to room temperature,
stirred for 17h then washed with 10% aqueous hydro-
chloric acid and saturated aqueous sodium hydrogen
carbonate, dried (MgSO4), filtered and evaporated to
dryness in vacuo. Purification of the resultant residue
by flash column chromatography (SiO2; 50–80% ethyl
acetate–hexane; gradient elution) yielded amide 33
(0.54g, 87%) as a colourless oil, which crystallised at
0ꢁC to a white solid. Compound 33 was shown to be
an 88:12 trans:cis mixture of conformers: mp 42–45ꢁC;
[a]D ꢀ29.7 (c 0.28 in CH2Cl2); dH (300MHz; CDCl3;
Me4Si) 1.70–1.91 (2H, m, Prob-HAHB and Proc-HAHB),
1.96–2.14 (4H, m, Prob-HAHB, Proc-HAHB and Glu-
H2), 2.30 (2H, t, J 6.6, allyl-CH2), 2.64 (1H, quintet, J
6.7, Gluc-H), 3.24–3.60 (2H, m, Prod-H2), 3.76–3.98
(2H, m, Glya-H2), 4.33* (0.12H, br s, Proa-H), 4.49
(0.88H, d, J 5.7, Proa-H), 4.60 (1H, q, J 7.0, Glua-H),
4.94–5.68 (8H, m, 3 · OCH2Ph and @CH2), 5.55–5.68
(1H, m, CH@CH2), 5.94 (1H, br s, Gly-NH), 7.29
(15H, s, 3 · Ph) and 7.38 (1H, d, J 7.8, Glu-NH); dC
(75MHz; CDCl3) 22.3* (CH2, Proc-C), 24.8 (CH2,
Proc-C), 28.0 (CH2, Prob-C), 31.9* (CH2, Prob-C),
32.8 (CH2, Glub-C), 35.9 (CH2, allyl-CH2), 41.4 (CH,
Gluc-C), 41.9* (CH, Gluc-C), 43.4 (CH2, Glya-C),
46.2 (CH2, Prod-C), 47.1* (CH2, Prod-C), 50.8 (CH,
Glua-C), 51.2* (CH, Glua-C), 60.0 (CH, Proa-C),
60.2* (CH, Proa-C), 66.4 (CH2, OCH2Ph), 66.5*
(CH2, OCH2Ph), 66.8 (CH2, OCH2Ph), 67.1 (CH2,
OCH2Ph), 67.3* (CH2, OCH2Ph), 67.4 (CH2, OCH2Ph),
Triethylamine (2.25mL, 2.20mmol) and di-tert-butyl
dicarbonate (2.10g, 9.61mmol) were successively added
to a solution of L-glutamic acid dibenzyl ester p-toluene-
sulfonate 29 (4.00g, 8.01mmol) in methylene chloride
(80mL) at 0ꢁC. The reaction mixture was warmed to
room temperature, stirred for 19h then washed with
2M aqueous hydrochloric acid, saturated aqueous so-
dium hydrogen carbonate, dried (MgSO4), filtered and
the solvent removed in vacuo. The resultant residue
was purified by crystallisation from ether–hexane to give
carbamate43 30 (3.32g, 97%) as a white solid: mp 73–
76ꢁC; dH (400MHz; CDCl3; Me4Si) 1.46 [9H, s,
C(CH3)3], 1.96–2.07 (1H, m), 2.22–2.27 (1H, m), 2.38–
2.52 (2H, m), 4.40 (1H, br d, J 3.6, Glua-H), 5.12–5.19
(5H, m, 2 · OCH2Ph and Glua-NH) and 7.29–7.39
(10H, m, 2 · Ph); dC (100MHz; CDCl3) 27.6 (CH2,
Glub-C), 28.1 [CH3, C(CH3)3], 30.1 (CH2, Gluc-C),
52.8 (CH, Glua-C), 66.4 (CH2, OCH2Ph), 67.1 (CH2,
OCH2Ph), 79.7 [quat., C(CH3)3], 128.1 (CH, Ph),
128.2 (CH, Ph), 128.3 (CH, Ph), 128.5 (CH, Ph), 128.5
(CH, Ph), 135.2 (quat., Ph), 135.7 (quat., Ph), 155.3
(quat., NCO2), 172.0 (quat., CO) and 172.4 (quat., CO).
4.23. (cS)-Dibenzyl N-tert-butyloxycarbonyl-L-c-allylgl-
utamate (31)
A solution of lithium hexamethyldisilazide (1molLꢀ1
,
10.30mL, 10.30mmol) in tetrahydrofuran was added
dropwise to a solution of carbamate 30 (2.00g,
4.68mmol) in anhydrous tetrahydrofuran (20mL) at
ꢀ78ꢁC under an atmosphere of nitrogen. The resultant
yellow solution was stirred for 40min, allyl bromide
(1.31mL, 14.0mmol) was added dropwise and stirring
continued for a further 1.5h at ꢀ78ꢁC. Saturated aque-
ous ammonium chloride (20mL) was then added and
the reaction mixture warmed to room temperature over
1.5h. The white precipitate was filtered, washed with
ethyl acetate and the filtrate concentrated to dryness in
vacuo. The ensuing residue was purified by flash column
chromatography (SiO2; 6:1 to 5:1 hexane–ethyl acetate)
to afford alkene 31 (1.59g, 73%) as a colourless oil: [a]D

M. A. Brimble et al. / Bioorg. Med. Chem. 13 (2005) 519–532
531
6. Sara, V. R.; Carlsson-Skwirut, C.; Drakenberg, K.;
Giacobini, M. B.; Hakansson, L.; Mirmiran, M.; Nord-
berg, A.; Olson, L.; Reinecke, M.; Stahlbom, P. A.;
Sandberg Nordqvist, A. C. Ann. N.Y. Acad. Sci. 1993,
692, 183.
7. Yamamoto, H.; Murphy, L. J. Endocrinology 1994, 135,
2432.
8. Yamamoto, H.; Murphy, L. J. J. Endocrinol. 1995, 146,
141.
9. Sara, V. R.; Carlsson-Skwirut, C.; Andersson, C.; Hall, E.;
Sjogren, B.; Holmgren, A.; Jornvall, H. Proc. Natl. Acad.
Sci. U.S.A. 1986, 83, 4904.
10. Carlsson-Skwirut, C.; Jornvall, H.; Holmgren, A.; Anders-
son, C.; Bergman, T.; Lindquist, G.; Sjogren, B.; Sara, V.
R. FEBS Lett. 1986, 201, 46.
67.4 (CH2, OCH2Ph), 117.6 (CH2, @CH2), 117.9* (CH2,
@CH2), 128.0 (CH, Ph), 128.1 (CH, Ph), 128.2 (CH,
Ph), 128.2 (CH, Ph), 128.4 (CH, Ph), 128.5 (CH, Ph),
128.5 (CH, Ph), 128.6 (CH, Ph), 134.2* (CH,
CH@CH2), 134.4 (CH, CH@CH2), 135.2* (quat., Ph),
135.4 (quat., Ph), 135.7* (quat., Ph), 135.9 (quat., Ph),
136.6 (quat., Ph), 156.4 (quat., NCO2), 156.6*
(quat., NCO2), 168.1 (quat., Gly-CO), 168.5* (quat.,
Gly-CO), 171.3 (quat., Pro-CON), 171.5* (quat., Pro-
CON), 171.6 (quat., Glua-CO), 171.7* (quat., Glua-
CO), 174.4* (quat., Gluc-CO) and 174.5 (quat.,
Gluc-CO); m/z (FAB+) 656.2964 (MH+. C37H42N3O8
requires 656.2972).
11. Bourguignon, J. P.; Gerard, A. Brain Res. 1999, 847,
247.
4.25. (cS)-Glycyl-L-prolyl-L-c-propylglutamic acid (28)
12. Gluckman, P. D.; Klempt, N. D.; Guan, J.; Mallard, C.
E.; Sirimanne, E. S.; Dragunow, M.; Klempt, M.; Singh,
K.; Williams, C. E.; Nijolics, K. Biochem. Biophys. Res.
Commun. 1992, 182, 593.
13. Lee, W.-H.; Bondy, C. Ann. N.Y. Acad. Sci. 28 1993, 679,
418.
14. Galli, C.; Meucci, O.; Scorziello, A.; Werge, T. M.;
Calissano, P.; Schettini, G. J. Neurosci. 1995, 15, 1172.
15. Mason, J. L.; Ye, P.; Suzuki, K.; DꢀErcole, A. J.;
Matsushima, G. K. J. Neurosci. 2000, 20, 5703.
16. Pons, S.; Rejas, M. T.; Torres-Aleman, I. Dev. Brain Res.
1991, 62, 169.
17. Walter, H. J.; Berry, M.; Hill, D. J.; Logan, A. Endocr-
inology 1997, 138, 3024.
18. Nilsson-Hakansson, L.; Civalero, I.; Zhang, X.; Carlsson-
Skwirut, C.; Sara, V. R.; Nordberg, A. Neuroreport 1993,
4, 1111.
Following procedure C, hydrogenation of amide 33
yielded analogue 28 (0.084g, 77%) as an off white solid
when freeze-dried from toluene–methanol. Compound
28 was shown to be an 82:18 trans:cis mixture of con-
formers: mp 160–165ꢁC; [a]D ꢀ76.6 (c 0.06 in H2O);
dH (400MHz; D2O) 0.91 (3H, t,
J 7.0, Gluc-
CH2CH2CH3), 1.29–1.38 (2H, m, Gluc-CH2CH2CH3),
1.58 (2H, d, J 6.4, Gluc-CH2CH2CH3), 1.91–2.51 (7H,
m, Glub-H2, Gluc-H, Prob-H2 and Proc-H2), 3.55–
3.68 (2H, m, Prod-H2), 3.78* (0.18H, d, J 16.0, Glya-
HAHB), 3.97–4.08 (1.82H, m, Glya-H2 and Glya-
HAHB*), 4.31 (1H, br s, Glua-H) and 4.53 (1H, d, J
5.5, Proa-H); dC (100MHz; D2O) 13.1 (CH3, Gluc-
CH2CH2CH3), 19.6 (CH2, Gluc-CH2CH2CH3), 21.9*
(CH2, Proc-C), 24.0 (CH2, Proc-C), 29.2 (CH2, Prob-
C), 31.6* (CH2, Prob-C), 33.5 (2 · CH2, Glub-C and
Gluc-CH2CH2CH3), 33.9* (CH2), 40.1* (CH2, Glya-
C), 40.3 (CH2, Glya-C), 42.5 (CH, Gluc-C), 43.6*
(CH, Gluc-C), 46.8 (CH2, Prod-C), 47.4* (CH2, Prod-
C), 52.8 (CH, Glua-C), 53.5* (CH, Glua-C), 60.0*
(CH, Proa-C), 60.4 (CH, Proa-C), 165.5 (quat., Gly-
CO), 166.0* (quat., Gly-CO), 173.0* (quat.,
Pro-CON), 173.4 (quat., Pro-CON), 176.9 (quat.,
Glua-CO) and 181.0 (quat., Gluc-CO); m/z (FAB+)
344.1833 (MH+. C15H26N3O6 requires 344.1822).
19. Ikeda, T.; Waldbillig, R. J.; Puro, D. J. Brain Res. 1995,
686, 87.
20. Sizonenko, S. V.; Sirimanne, E. S.; Williams, C. E.;
Gluckman, P. D. Brain Res. 2001, 922, 42.
21. Saura, J.; Curatolo, L.; Williams, C. E.; Gatti, S.; Benatti,
L.; Peters, C.; Guan, J.; Dragunow, M.; Post, C.; Faull, R.
L. M.; Gluckman, P. D.; Skinner, S. J. M. Neuroreport
1999, 10, 161.
22. Guan, J.; Waldvogel, H. J.; Faull, R. L. M.; Gluckman, P.
D.; Williams, C. E. Neuroscience 1999, 89, 649.
23. Guan, J.; Krishnamurthi, R.; Waldvogel, H. J.; Faull, R.
L. M.; Clark, R.; Gluckman, P. Brain Res. 2000, 859,
286.
24. Bourguignon, J. P.; Alvarez Gonzalez, M. L.; Gerard, A.;
Franchimont, P. Endocrinology 1994, 134, 1589.
25. Bourguignon, J. P. U.S. Patent 5,804,550, 1998.
26. Knopfel, T.; Kuhn, R.; Allgeier, H. J. Med. Chem. 1995,
38, 1417.
Acknowledgements
The authors thank Neuren Pharmaceuticals Ltd for
financial support.
27. Riedel, G. Trends Neurosci. 1996, 19, 219.
28. Nicoletti, F.; Bruno, V.; Copani, A.; Casabona, G.;
Knopfel, T. Trends Neurosci. 1996, 19, 267.
29. Abood, N. A.; Brimble, M. A. PCT Int. Appl. 0294856,
2002.
30. Gluckman, P.; Alexi, T. PCT Int. Appl. 0216408, 2002.
31. Gluckman, P. D.; Williams, C. E.; Guan, J.; Krishnamur-
thi, R. V. M. U.S. Pat. Appl. Publ. 20020035066, 2002.
32. Gluckman, P. D.; Sirimanne, E. S.; Krissansen, G. W.;
Kanwar, J. R. U.S. Pat. Appl. Publ. 2003027760, 2003.
33. Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M. J.
Am. Chem. Soc. 1964, 86, 1839.
34. Savrda, J. J. Org. Chem. 1977, 42, 3199.
35. El-Abadelah, M. M.; Hussein, A. Q.; Thaher, B. A.
Heterocycles 1991, 32, 1879.
36. Williamson, D. A.; Bowler, B. E. J. Am. Chem. Soc. 1998,
120, 10902.
References and notes
1. Trotter, N. S.; Brimble, M. A.; Harris, P. W. R.; Callis,
D. J.; Sieg, F. Bioorg. Med. Chem., preceding paper, see
doi:10.1016/j.bmc.2004.10.005.
2. Sara, V. R.; Carlsson-Skwirut, C. Prog. Brain Res. 1988,
73, 87.
3. De Pablo, F.; De La Rosa, E. J. Trends Neurosci. 1995, 18,
143.
4. Baskin, D. G.; Wilcox, B. J.; Figlewicz, D. P.; Dorsa, D.
M. Trends Neurosci. 1988, 11, 107.
5. Sara, V. R.; Carlsson-Sdwirut, C.; Bergman, T.; Jornvall,
H.; Roberts, P. J.; Crawford, M.; Hakansson, L. N.;
Civalero, I.; Nordberg, A. Biochem. Biophys. Res. Com-
mun. 1989, 165, 766.

532
M. A. Brimble et al. / Bioorg. Med. Chem. 13 (2005) 519–532
37. Lenman, M. M.; Lewis, A.; Gani, D. J. Chem. Soc., Perkin
Trans. 1 1997, 2297.
38. Hanessian, S.; Margarita, R. Tetrahedron Lett. 1998, 39,
5887–5890.
39. Perrin, D. D.; Armarego, W. L. F.; Perrin, D. R.
Purification of Laboratory Chemicals; Pergamon: Oxford,
1980.
40. Kessler, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 512.
41. Servas, L.; Winitz, M.; Greenstein, J. P. J. Org. Chem.
1957, 22, 1515.
42. Commercially available from Indofine Chemical Com-
pany, and Bachem: Registry Number 2441-63-6.
43. Chen, S. T.; Chen, S. Y.; Wang, K. T. J. Org. Chem. 1992,
57, 6960.