iso-Lactam and reduced amide analogues of the peptidomimetic dopamine receptor modulator 3(R)-[(2(S)-Pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide

Article abstract of DOI:10.1016/S0968-0896(03)00396-1

An analogue of the highly potent γ-lactam Pro-Leu-Gly-NH₂ peptidomimetic, 3(R)-[(2(S)-pyrrolidinylcarbonyl) amino]-2-oxo-1-pyrrolidineacetamide (2), 4(R)-[[2(S)-pyrrolidinylcarbonyl]amino]-2-oxo-1-pyrrolidineacetamide (3), in which the lactam carbonyl moiety has been placed in a different position with respect to the 3-amino group was synthesized. Also, a series of analogues of 2, compounds 4-6, were synthesized in which each of the amide bonds of 2 were systematically replaced with a reduced amide bond surrogate. The analogues were tested for their ability to enhance the binding of [ ³H]N-propylnorapomorphine to dopamine receptors in a functional in vitro assay utilizing bovine striatal membranes. Peptidomimetic 3 was shown to be more potent than 2, while 4 and 5 were significantly less effective than 2. Peptidomimetic 6 had a pharmacological profile similar to that of 2.

Full text of DOI:10.1016/S0968-0896(03)00396-1

Bioorganic & Medicinal Chemistry 11 (2003) 4103–4112
iso-Lactam and Reduced Amide Analogues of the Peptidomimetic
Dopamine Receptor Modulator 3(R)-[(2(S)-
Pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide
Kristine Dolbeare,a Giuseppe F. Pontoriero,b Suresh K. Gupta,b Ram K. Mishrab and
Rodney L. Johnsona,*
aDepartment of Medicinal Chemistry, University of Minnesota, 308Harvard St. SE, Minneapolis, MN 55455, USA
bDepartment of Psychiatry and Behavioural Neurosciences, McMaster University, 1200 Main St. W.,
Hamilton, Ontario Canada L8N 3Z5
Received 28 January 2003; accepted 28 April 2003
Abstract—An analogue of the highly potent g-lactam Pro-Leu-Gly-NH2 peptidomimetic, 3(R)-[(2(S)-pyrrolidinylcarbonyl) amino]-
2-oxo-1-pyrrolidineacetamide (2), 4(R)-[[2(S)-pyrrolidinylcarbonyl]amino]-2-oxo-1-pyrrolidineacetamide (3), in which the lactam
carbonyl moiety has been placed in a different position with respect to the 3-amino group was synthesized. Also, a series of analo-
gues of 2, compounds 4–6, were synthesized in which each of the amide bonds of 2 were systematically replaced with a reduced
amide bond surrogate. The analogues were tested for their ability to enhance the binding of [3H]N-propylnorapomorphine to
dopamine receptors in a functional in vitro assay utilizing bovine striatal membranes. Peptidomimetic 3 was shown to be more
potent than 2, while 4 and 5 were significantly less effective than 2. Peptidomimetic 6 had a pharmacological profile similar to that
of 2.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
cyclase activity,18 enhance N-propylnorapomorphine
(NPA)-stimulated low Km GTPase activity in rat striatal
membranes,18 potentiate the contralateral rotational
behavior induced by apomorphine in the rat nigro-
striatal 6-hydroxydopamine lesion model of Parkinson’s
disease,19,20 protect C57 BL/6 mice against MPTP-
induced dopaminergic degeneration,21 and attenuate
haloperidol-induced c-fos and Fos expression.22
l-Prolyl-l-leucyl-glycinamide (1, PLG) has the unique
ability to modulate D2 dopamine receptors within the
central nervous system.1À7 This neuropharmacological
activity has prompted the synthesis of numerous peptide
and peptidomimetic analogues of PLG.8À16 One of the
most potent PLG peptidomimetics to be made is the g-
lactam 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-
1-pyrrolidineacetamide (2).11 The pharmacological pro-
file of 2 resembles that of PLG except that it is 100–
1000Â more potent. For example, both PLG and 2
induce an increase in agonist binding to the D2 dopa-
mine receptor by increasing the affinity of the receptor
for agonists and by increasing the proportion of D2
receptors existing in the high-affinity state.6,17 Like
PLG, 2 is able to inhibit dopamine-stimulated adenylyl
Analogues of 2 in which the carbonyl moieties of the
peptidomimetic have been modified were designed to
determine the role that these moieties might play in the
activity and potency of 2. One such analogue was the
iso-g-lactam 3 in which the lactam carbonyl group
found in 2 has been shifted from the 2-position to the 5-
position of the lactam ring. It was felt that this modifi-
cation would maintain the ability of the g-lactam to
function as a b-turn mimic, but would orient the lac-
tam carbonyl in a different area of space. Analogues
4–6 represent the systematic replacement of each of
the amide bonds in 2 with a reduced amide bond
surrogate.
*Corresponding author. Tel.: +1-612-624-7997; fax: +1-612-624-
0139; e-mail: johns022@umn.edu
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00396-1

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K. Dolbeare et al. / Bioorg. Med. Chem. 11 (2003) 4103–4112
Results
Synthesis
Scheme 1.
Synthesis of the iso-g-lactam 3 began with the dipeptide
ethyl N-(tert-butoxycarbonyl)-b-ethyl-d-aspartyl-glyci-
nate (7) as outlined in Scheme 1. Compound 7 was
made by the same method as that reported for the
synthesis of its enantiomer.23 Treatment of 7 with Law-
esson’s reagent formed thiopeptide 8 in a 58% yield.
Reductive desulfurization of 8 by the reaction of nickel
borohydride, formed in situ from sodium borohydride
and nickel chloride was accomplished by the procedure
of Ede et al.24 This procedure was immediately followed
by a subsequent thermal closure in refluxing toluene to
give iso-g-lactam 9. The tert-butoxycarbonyl group was
removed from 9 and the product was coupled to Boc-l-
proline to give 10. This ester was converted to amide 11.
Trifluoroacetic acid deprotection of 11 gave the desired
proline yielded only 22% of 15. The difference in cou-
pling efficiencies are suspected to be due to the differ-
ential solubility properties of the ditrifluoracetate salt
versus the dihydrochloride salt in CH2Cl2. Aminolysis
of methyl ester 15 with ammonia gave a 79% yield of
amide 16. Deprotection of 16 with trifluoroacetic acid
followed by reverse phase chromatography gave pepti-
domimetic 4 as its ditrifluoroacetate salt.
Synthesis of reduced amide peptidomimetic 5 was car-
ried out as outlined in Scheme 3. 3-Amino-g-lactam 17,
which was obtained through the deprotection of 12,11
and Cbz-l-prolinal (18), which was obtained through
diisobutylaluminum hydride reduction of Cbz-l-Pro-
OMe,27 were reacted together in the presence of sodium
cyanoborohydride to give 19. This material was con-
verted to primary amide 20, which was then subjected to
hydrogenolysis to give pyrrolidinyl g-lactam 5.
iso-g-lactam PLG peptidomimetic
fluoroacetate salt.
3
as its tri-
The synthesis of the amino pyrrolidinyl peptidomimetic
4 began with the known g-lactam 1211 (Scheme 2). This
material was converted to thiolactam 13 with Law-
esson’s reagent25 in refluxing benzene. Desulfurization
of 13 to give pyrrolidine 14 was accomplished overnight
with Raney nickel in diethyl ether.26 The tert-butoxy-
carbonyl group of 14 was removed with trifluoroacetic
acid and the material that was obtained was coupled to
Boc-l-proline using 1-[3-(dimethylamino)propyl]-3-
The synthesis of 6 (Scheme 4) began with the Boc-d-
methionine amide 21, which was obtained through the
coupling of Boc-d-Met with mono benzyloxycarbonyl
protected diaminoethane.28 Formation of the g-lactam
22 was accomplished by the methodology of Freidinger
et al.29,30 as modified by Yu et al.11 Treatment of 22
with 4 N HCl in dioxane followed by EDC coupling of
the product to Cbz-l-proline yielded two distinct pro-
ducts. Detailed 1D and 2D NMR analysis, combined
with MS and elemental analysis identified these com-
pounds to be the expected prolyl-lactam 23 and the bis-
prolyl-lactam 24 in yields of 32and 26%, respectively. It
.
ethylcarbodiimide hydrochloride (EDC HCl) to give 15
in an excellent yield (86%). Interestingly, when 4 N HCl
in dioxane was used instead of trifluoroacetic acid to
remove the tert-butoxycarbonyl group from 14 the sub-
sequent coupling reaction of the product with Boc-l-

K. Dolbeare et al. / Bioorg. Med. Chem. 11 (2003) 4103–4112
4105
appears that during the removal of the tert-butoxy-
Pharmacology
carbonyl group from 22 some of the benzyloxycarbonyl
protecting group also is being removed despite the fact
that this protecting group is normally stable under
these conditions. Hydrogenolysis of 23 in methanol
with catalytic 10% palladium on carbon gave the
desired peptidomimetic 6.
iso-g-Lactam peptidomimetic 3 and the reduced amide
bond peptidomimetics 4–6 were evaluated in a func-
tional in vitro assay utilizing bovine striatal mem-
branes.6,31 These analogues were tested for their ability
to enhance the binding of [3H]NPA to dopamine receptors
at three concentrations: 1, 10, and 100 nM. The results,
along with the results previously obtained for 2,32 are
summarized in Figure 1. Peptidomimetics 3–6 all retained
some ability to enhance the binding of [3H]NPA to dopa-
mine receptors isolated from bovine striatal membranes
when compared to control preparations.
Peptidomimetics 2 and 3 at a concentration of 100 nM
showed similar ability to enhance [3H]NPA binding to
dopamine receptors. At a concentration of 10 nM, 2
was more active than 3, but at 1 nM 3 became sig-
nificantly more active than 2. Because of its enhanced
activity at the 1 nM concentration, 3 also was evaluated
at a concentration of 0.1 nM (data not shown). The
results were that in going from 1 to 0.1 nM the percen-
tage enhancement of [3H]NPA binding dropped from 37
to 7%. Thus, the maximum effect of 3 occurred at 1 nM.
Like 2, compounds 4 and 5 showed very low activity at
a concentration of 0.1 nM. At concentrations of 10 nM
and 100 nM, the activities of 4 and 5 were significantly
lower than that observed for 2. Peptidomimetic 6
showed an activity profile similar to that of 2.
Scheme 2.
Scheme 3.
Scheme 4.

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K. Dolbeare et al. / Bioorg. Med. Chem. 11 (2003) 4103–4112
peptidomimetic to its recognition site. However, the fact
that 25 previously had been shown to potentiate the
effects of l-dopa in rats35 would seem to argue against
this possibility. Alternatively, the reduced activity seen
with 4 may be the result of changes in conformation
that are allowed by the more flexible pyrrolidinyl ring as
compared to the lactam ring.
The decrease in the activity seen with 5 is consistent
with the loss of activity seen with 26, the corresponding
reduced amide bond analogue of PLG. An NMR study
on 26 showed that the compound did not exist in a b-
turn because of the lack of the prolyl carbonyl needed to
form the 10-membered hydrogen bonded system.35 The
reduced amide bond that has been incorporated in 5
also eliminates the carbonyl group that may help stabi-
lize, through a 10-membered hydrogen bond, the
postulated b-turn bioactive conformation of 2.11,33
Figure 1. Stimulation of [3H]NPA binding to bovine striatal mem-
branes by the PLG peptidomimetics 2–6. Data represent the percent
increase in specific [3H]NPA binding over the control value (the bind-
ing of [3H]NPA in the absence of any PLG peptidomimetic) when the
indicated concentration of peptidomimetic was added directly to the
assay buffer. Data for 2 are from ref 32. Results are the meansÆSEM
of 3–4 separate experiments carried out in triplicate. aSignificantly
different (p <0.01) from control value. bSignificantly different
(p<0.001) from control value. cSignificantly different (p<0.0001) from
the corresponding concentration of 2. dSignificantly different
(p<0.001) from the corresponding concentration of 2. eSignificantly
In previous structure–activity studies on PLG, the car-
boxamide moiety was shown to be necessary for the
dopamine receptor modulating activity of the molecule
as measured in the Dopa potentiation test and the oxo-
tremorine antagonism test.36 It was postulated that the
carboxamide group was participating in important
hydrogen bonding interactions. The retention of activity
seen with 6 suggests that the reduced amide bond pres-
ent in this analogue can substitute for the carboxamide
moiety present in 2. This may be because the amino
group of 6, whether it is in its protonated or unproto-
nated form possesses the ability to participate in hydro-
gen bonding interactions either intramolecularly or
intermolecularly as does the amide NH2 group of 2.
f
different (p<0.01) from the corresponding concentration of 2. Signi-
ficantly different (p<0.05) from the corresponding concentration of 2.
Discussion
It was anticipated that the iso-g-lactam constraint of
peptidomimetic 3 would possess the potential to serve as
a b-turn mimic in the same manner as the g-lactam
constraint in 2 does,33 since previous studies with
human growth hormone fragments that incorporated
iso-g-lactam and g-lactam residues that are enantio-
meric to those of this study showed the ability of both
residues to assume a b-turn.23,34 Thus, the difference
between the iso-g-lactam and g-lactam constraints
would be the position in space in which the carbonyl
would be projected. The significant activity seen with 3
suggests that the lactam carbonyl in 2 is not an absolute
requirement for the activity seen with 2. Although at a
concentartion of 10 nM 2 produced a greater enhance-
ment of [3H]NPA binding to dopamine receptors than
3, the effect of 3 at 1 nM was significantly greater than
that of 2 at 1 nM. The drop in the activity of 3 when
going from 1 to 0.1 nM indicates that the maximal
enhancement of [3H]NPA binding for this peptidomi-
metic occurs at 1 nM, a concentration that is ten times
lower than the concentration at which the maximum
effect for 2 is seen.
Conclusion
In summary, this study has shown that replacing the g-
lactam moiety of 2 with the iso-g-lactam constraint
leads to a potent modulator of dopamine receptors,
peptidomimetic 3. The study also has shown that the
amide bond involving the prolyl residue and the lactam
amide bond are important structural features of 2, since
replacing these with reduced amide bond surrogates
result in peptidomimetics with significantly lower activ-
ity. Only the C-terminal carboxamide group of 2 can be
replaced with a reduced amide bond without a
significant decrease in activity.
Experimental
General aspects
The reduced amide bond analogues 4 and 5 are analo-
gous to the reduced amide bond PLG analogues that
Thin-layer chromatography was performed on Analtech
250 mm silica gel GHLF Uniplates and were visualized
either by UV, I2, ninhydrin spray (amines), 2,6-
dichlorophenol indophenol spray (acids), or 2,4-dini-
trophenylhydrazine (aldehydes). Chromatographic pur-
ification on silica gel (Merck, grade 60, 240–400 mesh, 60
A) was done by flash or gravity methods. Optical rota-
tions were measured on a Rudolph Research Autopol III
polarimeter at the 589 nm Na D-Line. Melting points
have
been
synthesized
previously:
Pro-
Leuc[NHCH2]Gly-NH2 (25) and Proc[NHCH2]Leu-
Gly-NH2 (26), respectively.35 The significant loss of
activity seen with 4 could be due to the fact that in going
from 2 to 4 the amide nitrogen atom in the lactam ring
becomes an amine that would be protonated to a sig-
nificant extent at physiological pH. Protonation could
have an adverse impact on the binding of the PLG

K. Dolbeare et al. / Bioorg. Med. Chem. 11 (2003) 4103–4112
4107
were obtained on a Thomas-Hoover melting point
apparatus and are uncorrected. Elemental analyses were
performed by M-H-W Laboratories, Phoenix, AZ,
USA.
13.8, 28.1, 37.4, 43.4, 44.2, 54.2, 61.2, 79.4, 155.2, 168.3,
173.0. FAB MS (glycerol matrix) m/z 287 [M+H]+.
Anal. calcd for C13H22N2O5: C, 54.53; H, 7.74; N, 9.78.
Found: C, 54.54; H, 7.59; N, 9.81.
N-(tert-Butoxycarbonyl)-ꢀ-ethyl-D-aspartyl- [CSNH]-
glycine ethyl ester (8). N-(tert-Butoxycarbonyl)-b-ethyl-
d-aspartyl-glycine ethyl ester23 (7, 17.2g, 0.05 mol) was
dissolved in 300 mL dry benzene and 10.0 g of Law-
esson’s reagent (0.025 mol) was added. The reaction was
heated at reflux for 4 h, then it was cooled and con-
centrated in vacuo to a yellow oil. The oil was twice
chromatographed on a 4Â40 cm silica gel column elut-
ing with 20% Et2O in CH2Cl2 to yield 10.5 g (58%) of 8
as a yellow oil. [a]d À12.9 (c 1.15, CHCl3) (lit23 [a]D for
the enantiomer of 8 was 7.5 in CDCl3). 1H NMR
(CDCl3) d 1.22–1.31 (m, 6H), 1.44 (s, 9H), 2.87 (dd, 1H,
J=7.2and 17.1 Hz), 3.19 (dd, 1H, J=3.6 and 17.1 Hz),
4.14 (q, 2H, J=6.9 Hz), 4.24 (q, 2H, J=7.2Hz), 4.34–
4.36 (m, 2H), 4.80–4.86 (m, 1H), 5.79–5.82 (br d, 1H),
8.83–8.85 (br m, 1H); 13C NMR (CDCl3) d 14.2, 14.3,
28.4, 39.6, 47.4, 56.6, 61.3, 62.0, 80.8, 155.4, 168.4,
171.7, 202.7; FAB MS (glycerol matrix) m/z 363
[M+H]+. Anal. calcd for C15H26N2O6S: C, 49.71; H,
7.23; N, 7.73; S, 8.85. Found: C, 49.51; H, 6.97; N, 8.00;
S, 8.63.
Ethyl
4(R)-[[1-(tert-butoxycarbonyl)-2(S)-pyrrolidinyl-
carbonyl]amino]-2-oxo-1-pyrrolidineacetate (10). iso-
Lactam 9 (0.14 g, 0.49 mmol) was treated with excess
HCl (4 N in dioxane, 10 mL) overnight at room tem-
perature. The excess HCl and dioxane were removed in
vacuo. The residue was dissolved in CH2Cl2 and this
solution was evaporated to dryness. This process was
repeated twice and yielded a white foam. The hydro-
chloride salt was dried under vacuum overnight and the
salt was dissolved in 40 mL dry CH2Cl2 and 5 mL dry
.
DMF. HOBt H2O (0.08 g, 0.58 mmol) and Boc-l-Pro-
OH (0.126 g, 0.58 mmol) were added and the stirred
ꢀ
.
solution chilled to À78 C. EDC HCl (0.112g, 0.58
mmol) was added to the mixture followed by Et3N (0.16
mL, 1.17 mmol) and the reaction was allowed to warm
to room temperature overnight. The reaction was fur-
ther stirred under nitrogen at room temperature for 7
days. The solvent was removed in vacuo and the residue
dissolved in CH2Cl2. The reaction was washed with
10% citric acid solution, 1 M NaHCO3 solution, and
saturated NaCl solution successively. The organic layer
was dried (MgSO4), filtered and concentrated in vacuo
to a yellow oil that was chromatographed on a 2Â2 0 cm
silica gel column eluted with 5% EtOH/CH2Cl2 to yield
0.10 g (55%) of 10 as a clear oil. [a]d À50.5 (c 2.08,
Ethyl 4(R)-[N-(tert-butoxycarbonyl)amino]-2-oxo-1-pyr-
rolidineacetate (9). Thiopeptide 8 (10.4 g, 0.029 mol)
was dissolved in 250 mL dry THF and 250 mL 100%
ꢀ
1
.
EtOH. The solution was cooled to 0 C. NiCl2 6H2O
(54.6 g, 0.23 mol) was added to the solution which then
turned emerald green. NaBH4 (26.0 g, 0.67 mol) was
added in portions over 15 min causing the solution to
turn black, bubble and expel gas. The reaction was
allowed to warm to room temperature where upon it
was stirred for 30 min. The reaction was filtered through
a pad of silica gel in a 4Â10 cm column. The black
residue on the silica gel was eluted with 500 mL EtOH,
500 mL THF, 500 mL Et2O, and 500 mL EtOAc, suc-
cessively. The organic layers were combined and con-
centrated in vacuo to give a black residue that was
dissolved in CH2Cl2. The solution was washed with 200
mL of saturated NaHCO3 solution. An emulsion was
formed that was broken up by the addition of 150 mL
of saturated EDTA solution. The organic layer was
washed twice with saturated EDTA solution and once
with H2O, then dried (MgSO4), filtered and con-
centrated in vacuo to a yellow oil. This oil in 100 mL
toluene was heated at reflux for 4 days after which time
the solvent was removed in vacuo, and the resulting oil
chromatographed on a 4Â40 cm silica gel column elut-
ing with 25% EtOAc in hexanes followed by 60%
EtOAc in hexanes. The total yield of 9 was 3.39 g (41%)
as a white solid. Mp: 95–96 ꢀC (lit24 mp for the enantio-
mer of 9 was 71.5–72 ꢀC); [a]D 17.3 (c 1.24, CHCl3) (lit24
CHCl3). H NMR (DMSO, rotamers present) d 1.18 (t,
3H, J=7.2Hz), 1.31 and 1.37 (s, 9H), 1.70–1.79 (m,
3H), 2.04–2.16 (m, 1H), 2.22 (dd, 1H, J=8.4 and 16.8
Hz), 2.59 (dd, 1H, J=8.4 and 16.8 Hz), 3.15–3.31 (m,
3H), 3.67 (dd, 1H, J=7.5 and 9.6 Hz), 3.93–4.02(m,
3H, Pro), 4.09 (q, 2H, J=7.2Hz), 4.31–4.35 (br m, 1H),
8.27 and 8.31 (d, 1H, J=6.9 Hz); 13C NMR (DMSO) d
14.5, 23.6, 28.5, 31.5, 36.8, 42.9, 43.8, 47.0, 53.9, 60.1,
61.2, 78.9, 153.7, 169.1, 172.9, 173.1; FAB MS
(m-nitrobenzylalcohol matrix) m/z 384 [M+H]+. Anal.
calcd for C18H29N3O6: C, 56.38; H, 7.62; N, 10.96.
Found: C, 56.17; H, 7.63; N, 10.72.
4(R)-[[1-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]carbony-
l]amino]-2-oxo-1-pyrrolidineacetamide (11). A solution
of 10 (1.13 g, 2.95 mmol) in 50 mL of a concentrated
solution of methanolic ammonia was stirred overnight.
Solvent was removed in vacuo to yield a white foam
that was chromatographed on a 4Â20 cm silica gel col-
umn with 5% MeOH in CH2Cl2 as the eluting solvent.
The product was isolated as a white solid (0.83 g, 79%).
Crystallization was achieved from EtOAc/hexanes. Mp:
122–123 ꢀC; [a]d À20.0 (c 0.86, MeOH). 1H NMR
(DMSO, rotamers present) d 1.32and 1.38 (s, 9H),
1.71–1.77 (m, 3H), 2.07–2.11 (m, 1H), 2.16 (dd, 1H,
J=4.0 and 16.5 Hz), 2.60 (dd, 1H, J=9.0 and 16.5 Hz),
3.12–3.17 (m, 1H), 3.23–3.28 (m, 1H), 3.34–3.38 (m,
1H), 3.65 (dd, 1H, J=7.5 and 9.5 Hz), 3.74 (s, 2H), 3.96
(dd, 1H, J=3.5 and 8.5 Hz), 4.33–4.35 (m, 1H), 7.13 (s,
1H), 7.37 (s, 1H), 8.25 and 8.30 (d, 1H, J=7.5 Hz); 13C
NMR (DMSO) d 23.2, 28.1, 31.0, 36.7, 42.1, 44.7, 46.5,
53.9, 59.7, 78.4, 153.2, 169.5, 172.3, 172.5; FAB MS
(glycerol matrix) m/z 355 [M+H]+. Anal. calcd for
1
[a]D for the enantiomer of 9 was À14.3 in CHCl3). H
NMR (CDCl3) d 1.18 (t, 3H, J=7.5 Hz), 1.33 (s, 9H),
2.27 (dd, 1H, J=5.1 and 17.1 Hz), 2.64 (dd, 1H, J=8.4
and17.1 Hz), 3.32(dd, 1H, J=3.6 and 9.6 Hz), 3.67 (dd,
1H, J=6.0 and 9.6 Hz), 3.85 (d, 1H, J=18.3 Hz), 4.02–
4.10 (br d, 1H), 4.09 (q, 2H, J=7.5 Hz), 4.19–4.26 (br
m, 1H), 5.57 (d, 1H, J=7.2Hz); 13C NMR (CDCl3) d

4108
K. Dolbeare et al. / Bioorg. Med. Chem. 11 (2003) 4103–4112
C16H26N4O5: C, 54.22; H, 7.39; N, 15.81. Found: C,
54.10; H, 7.29; N, 15.76.
column and eluting with 10% hexanes in EtOAc gave
1.36 g (80%) of product. mp: 59–60 ꢀC; [a]D 10.9 (c 0.51,
MeOH). 1H NMR (CDCl3) d 1.44 (s, 9H), 1.66–1.67 (m,
1H), 2.26–2.30 (m, 1H), 2.47–2.48 (m, 1H), 2.69–2.71
(m, 1H), 2.76–2.79 (m, 1H), 2.96–2.97 (m, 1H), 3.35 (s,
2H), 3.73 (s, 3H), 4.20 (br m, 1H), 4.95 (br s, 1H); 13C
NMR (CDCl3) d 28.3, 32.7, 49.9, 51.6, 52.2, 55.8, 60.3,
79.2, 155.3, 170.8; FAB MS (glycerol matrix) m/z 259
[M+H]+. Anal. calcd for C12H22N2O4: C, 55.80; H,
8.58; N, 10.84. Found: C, 56.00; H, 8.33; N, 10.59.
4(R)-[[2(S)-Pyrrolidinylcarbonyl]amino]-2-oxo-1-pyrroli-
.
dineacetamide trifluoroacetate (3 CF3CO2H). Boc-pro-
tected isolactam 11 (66 mg, 0.19 mmol) was dissolved in
20 mL dry CH2Cl2 and 286 mL TFA (3.7 mmol) was
added. The solution was stirred at room temperature
overnight and then the solvent was removed in vacuo.
For a total of three times, the residue was dissolved in
CH2Cl2 and the solvent then evaporated. The clear oil
which was obtained was chromatographed on a 1Â20
cm silica gel column eluting with 10% MeOH/CHCl3 to
give a white solid. The solid was dissolved in H2O and
the solution lyophilized to give 48 mg (70%) of 3 as a
hygroscopic white solid. The product was shown to be
pure by analytical HPLC analysis on a LiChrosorb1
RP-18 4.6Â250 mm analytical column using 40% H2O/
acetonitrile with 0.1% TFA as the eluting solvent:
tR=2.2 min. HPLC analysis on the RP-18 column elut-
ing with 20% H2O/MeOH and 0.1% TFA gave a
tR=3.6 min. [a]D 3.3 (c 0.57, MeOH). 1H NMR (D2O) d
1.84–1.89 (m, 3H), 2.17–2.26 (buried m, 1H), 2.25 (dd,
1H, J=4.2and 17.7 Hz), 2.74 (dd, 1H, J=8.4 and 17.7
Hz), 3.15–3.25 (m, 3H), 3.69 (dd, 1H, J=7.2and 10.8
Hz), 3.81 (d, 1H, J=17.1), 3.88 (d, 1H, J=17.1 Hz),
4.12(t, 1H, J=7.2Hz), 4.33–4.38 (m, 1H); 13C NMR
(D2O) d 23.6, 29.5, 36.6, 43.3, 44.8, 46.3, 54.3, 59.6,
169.1, 172.3, 175.9; FAB HRMS (glycerol matrix) m/z
255.1461 (C11H18N4O3+H+ requires 255.1458).
Methyl 3(R)-[[1-(tert-butoxycarbonyl)-2(S)-pyrrolidinyl-
carbonyl]amino]-1-pyrrolidineacetate (15). Pyrrolidine
14 (1.26 g, 4.9 mmol) was dissolved in 100 mL dry
CH2Cl2 and 5 mL TFA (65.3 mmol) was added to the
solution. The solution was stirred under N2 at room
temperature for 5 h and then the solvent was removed in
vacuo. The residue was dissolved in CH2Cl2 and the
solution was evaporated. This was done for a total of
4Â to ultimately yield to a white solid. This solid was
dried under vacuum overnight and then dissolved in 60
.
mL dry CH2Cl2 and 30 mL dry DMF. HOBt HO (0.79
g, 5.8 mmol) and Boc-l-Pro-OH (1.26 g, 5.8 mmol) were
ꢀ
(1.12g, 5.8 mmol) was added followed by 1.6 mL Et N
.
added and the solution was cooled to À78 C. EDC HCl
3
(11.7 mmol). The reaction and workup was the same as
that used in the synthesis of 10. The oil obtained was
chromatographed on a 4Â40 cm silica gel column elut-
ing with 5% MeOH/EtOAc to yield 1.49 g (86%) of 15
as a golden oil. A small sample was further purified on a
2Â20 cm silica gel column eluting with 20% hexanes in
EtOAc and 0.1% NH4OH to yield analytically pure
product. [a]d À36.2( c 1.62, MeOH). 1H NMR (DMSO,
rotamers present) d 1.42(s, 9H), 1.63–1.70 (m, 1H),
1.83–1.93 (m, 2H), 2.06 and 2.14 (br s, 2H), 2.19–2.26
(m, 1H), 2.52–2.57 (br m, 1H), 2.62–2.68 (br m, 1H),
2.82–2.90 (br m, 2H), 3.35 (s, 2H), 3.40–3.45 (br m, 2H),
3.70 (s, 3H), 4.15 and 4.21 (br s, 1H), 4.40–4.50 (br m,
1H), 6.78 and 7.27 (s, 1H); 13C NMR (DMSO, 60 ꢀC) d
23.0, 28.0, 30.8, 31.2, 46.4, 48.0, 51.0, 51.6, 55.2, 58.9,
59.4, 78.2, 153.3, 170.6, 172.1; FAB MS (m-nitrobenzyl-
alcohol matrix) m/z 356 [M+H]+. Anal. calcd for
C17H29N3O5: C, 57.45; H, 8.22; N, 11.82. Found: C,
57.19; H, 8.03; N, 12.05.
Methyl 3(R)-[N-(tert-butoxycarbonyl)amino]-2-thioxo-1-
pyrrolidineacetate (13). Lactam 1211 (1.0 g, 3.67 mmol)
was dissolved in 100 mL dry benzene and Lawesson’s
reagent (0.73 g, 1.8 mmol) was added. The reaction was
heated at reflux for 4 h after which it was cooled to
room temperature. Concentration of the reaction in
vacuo yielded a yellow oil. The oil was chromato-
graphed on 4Â30 cm silica gel column eluting with
CH2Cl2 followed by 20% Et2O in CH2Cl2 to yield 0.6 g
(58%) product. Mp: 79–81 ꢀC; [a]D 58.9 (c 1.41,
MeOH). 1H NMR (CDCl3) d 1.46 (s, 9H), 1.90–1.98 (m,
1H), 2.83–2.84 (br m, 1H), 3.68 (t, 1H, J=9.5 Hz), 3.77
(s, 3H), 3.78–3.82(m, 1H), 4.30–4.44 (br m, 1H), 4.50 (d,
1H, J=17.5 Hz), 4.63 (d, 1H, J=17.5 Hz), 5.49 (br s,
1H); 13C NMR (CDCl3) d 28.4, 30.0, 49.4, 52.2, 52.6,
61.7, 79.9, 155.6, 167.2, 201.9; FAB MS (m-nitrobenzyl-
alcohol matrix) m/z 288 [M]+ and 287 [M-H]À.
3(R)-[[1-(tert-Butoxycarbonyl)-2(S)-pyrrolidinylcarbon-
yl]amino]-1-pyrrolidineacetamide (16). A solution of 15
(1.07 g, 3.0 mmol) in 40 mL concentrated methanolic
ammonia was stirred for 2days. The solvent was
removed in vacuo and the oily residue that was obtained
was dissolved in CH2Cl2 and then the solvent was
removed. This procedure was carried out 3Â. The foam
that was obtained was chromatographed on a 2Â30 cm
silica gel column eluting with 10% MeOH in EtOAc to
yield 0.86 g (84%) of product. An analytical sample was
obtained through purification on a 1Â20 cm silica gel
column eluting with 10% MeOH in EtOAc and 0.1%
NH4OH. [a]d À21.6 (c 3.46, MeOH). 1H NMR (CDCl3,
50ꢀC) d 1.44 (s, 9H), 1.60–1.67 (m, 1H), 1.85–1.95 (m,
3H), 2.23–2.27 (m, 2H), 2.48–2.52 (m, 1H), 2.58–2.61
(m, 1H), 2.70–2.80 (m, 1H), 2.88–2.95 (m, 1H), 3.14 (s,
2H), 3.34–3.40 (m, 2H), 4.16–4.23 (m, 1H), 4.35–4.45
(m, 1H), 5.77 (s, 1H), 6.91 (s, 1H); 13C NMR (CDCl3) d
Methyl 3(R)-[N-(tert-butoxycarbonyl)amino]-1-pyrrolidi-
neacetate (14). Thiolactam 13 (1.9 g, 6.5 mmol) was
dissolved in 50 mL anhydrous Et2O. Raney nickel (50%
in H2O, 10 g) was washed with 100% EtOH, and twice
with Et2O, carefully decanting the solution away from
the catalyst each time. Et2O (50 mL) was added to the
Raney nickel and this slurry was added to the solution
of 13. The solution was vigorously stirred overnight
upon which time the solvent was decanted away and the
Raney nickel was washed with Et2O. The organic layers
were combined and filtered through a pad of wet Celite
which was then washed with Et2O. Concentration of the
filtrate in vacuo yielded a clear oil that solidified to a
white solid. Silica gel chromatography using a 4Â40 cm

K. Dolbeare et al. / Bioorg. Med. Chem. 11 (2003) 4103–4112
4109
24.5, 28.4, 31.2, 32.5, 47.2, 48.8, 53.0, 58.4, 59.8, 60.7,
80.5, 156.7, 171.4, 173.3; FAB MS (m-nitrobenzylalco-
hol matrix) m/z 341 [M+H]+. Anal. calcd for
C16H28N4O4 0.5H2O: C, 55.00; H, 8.36; N, 16.03.
Found: C, 55.22; H, 8.11; N, 16.09.
lactam 17. Molecular sieves (4 A, 10 g) were added and
the reaction was stirred at room temperature, under
nitrogen, for 4 h. NaCNBH3 (0.76 g, 12.1 mmol) was
added in one portion and the reaction was stirred under
N2 for 6 days. The reaction was acidified with 10%
citric acid solution to pH 4 and then made basic (pH 10)
by the addition of saturated NaHCO3 solution. The
aqueous solution was extracted 4Â with EtOAc. The
organic layer was washed with H2O and saturated NaCl
solution, dried (MgSO4), filtered and concentrated in
vacuo to give a yellow oil. This oil was chromato-
graphed on a 2Â40 cm silica gel column eluting with
10% MeOH in hexanes and 0.1% NH4OH to yield 0.86
g (55%) of 19 as a clear oil. [a]d À33.7 (c 2.11, MeOH).
1H NMR (CDCl3, rotamers present) d 1.65–1.69 (m,
1H), 1.81–1.97 (m, 3H), 2.09–2.12 (m, 1H), 2.34–2.37
(m, 1H), 2.50 and 2.59 (t, 1H, J=10.0 Hz), 2.87 and
3.02(d, 1H, J=8.5 Hz), 3.20–3.45 (m, 4H), 3.54–3.57 (t,
1H, J=7.5 Hz), 3.72(s, 3H), 3.87–3.97 (m, 2H), 4.19
(dd, 1H, J=8.0 and 17.0 Hz), 5.07–5.19 (m, 2H), 7.27–
7.36 (m, 5H); 13C NMR (CDCl3, rotamers present) d
22.9 (23.8), 26.8, 28.9 (29.5), 44.2, 44.9, 46.7 (46.9), 49.6
(50.0), 52.2, 57.0 (57.6), 57.9, 66.6 (66.9), 127.8, 127.9,
128.0, 128.3, 128.5, 137.0, 155.1 (155.2), 169.0, 174.9;
FAB MS (m-nitrobenzylalcohol matrix) m/z 390
[M+H]+. Anal. calcd for C20H27N3O5: C, 61.68; H,
6.99; N, 10.79. Found: C, 61.48; H, 6.76; N, 10.57.
.
3(R) - [[2(S) - Pyrrolidinylcarbonyl]amino] - 1 - pyrrolidine-
.
acetamide ditrifluoroacetate (4 2CF3CO2H). Compound
16 (212 mg, 0.62 mmol) was deprotected in the same
manner as that described for the synthesis of 3. Puri-
fication on a 1Â5 cm C-8 reverse-phase column eluting
with 10% MeOH/acetonitrile and a 1Â10 cm silica gel
column eluting with 10% MeOH/CHCl3 gave an off-
white solid. The solid was dissolved in H2O and lyophi-
lized to give 68.4 mg (23%) of analytically pure 4 as a
hygroscopic off-white solid. The product was shown to
be pure by analytical HPLC analysis on a Waters
Spherisorb1 4.6Â150 mm analytical column. Product
was eluted with 25% MeOH/CHCl3 and 0.1% NH4OH
and had a tR of 7.2min. Further HPLC analysis on a
LiChrosorb1 RP-18 4.6Â250 mm analytical column
with 40% H2O/acetonitrile and 0.1% TFA gave a tR of
2.8 min. HPLC analysis on the RP-18 column eluting
with 20% H2O/MeOH and 0.1% TFA gave a tR of 3.5
min. [a]d À26.0 (c 0.50, MeOH). 1H NMR (D2O) d
1.66–1.72(m, 1H), 1.86–1.92(m, 3H), .218–.230 (m,
2H), 2.74–2.81 (m, 2H), 2.97–3.02 (m, 1H), 3.05 (dd,
1H, J=7.0 and 10.5 Hz), 3.18–3.29 (m, 2H), 3.34 (d,
1H, J=15.5 Hz), 3.45 (d, 1H, J=15.5 Hz), 4.14–4.17
(m, 1H), 4.22–4.27 (m, 1H); 13C NMR (D2O) d 23.7,
29.6, 30.1, 46.4, 49.0, 52.8, 56.5, 58.7, 59.6, 169.2, 172.7;
CI HRMS m/z 241.1664 (C11H20N4O2+H+ requires
241.1665).
3(R)-[[1-(Benzyloxycarbonyl)-2(S)-pyrrolidinylmethyl]-
amino]-2-oxo-1-pyrrolidineacetamide (20). A solution of
19 (0.7 g, 1.8 mmol) in 40 mL concentrated methanolic
ammonia was stirred overnight. The solvent was
removed in vacuo to yield an oil that was chromato-
graphed on a 2Â40 cm silica gel column eluting with
10% MeOH and 0.1% NH4OH in EtOAc to yield 0.59
g (86%) of product as a white solid. Mp 49–50 ꢀC; [a]d
À28.7 (c 0.79, MeOH). 1H NMR (CDCl3, rotamers
present) d 1.61–1.65 (m, 1H), 1.78–1.95 (m, 3H), 2.02-
2.09 (m, 1H), 2.30–2.40 (m, 1H), 2.54–2.58 and 2.47–
2.51 (m, 1H), 2.81–2.83 and 2.93–2.96 (m, 1H), 3.24–
3.27 (m, 1H), 3.34–3.43 (m, 3H), 3.50 (t, 1H, J=8.5 Hz),
3.81–3.98 (m, 3H), 5.03–5.26 (m, 2H), 6.28 (s, 1H), 6.61
(s, 1H), 6.75 (s, 1H), 7.25–7.32 (m, 5H); 13C NMR
(DMSO, rotamers present) d 22.3 (23.2), 26.4, 28.0
(28.7), 44.4, 45.0, 46.2 (46.6), 49.0 (49.1), 56.7 (57.5),
57.8, 65.6 (65.9), 127.4, 127.7, 128.4, 137.2, 154.1, 169.5,
174.1; FAB MS (m-nitrobenzylalcohol matrix) m/z 375
[M+H]+. Anal. calcd for C19H26N4O4: C, 60.95; H,
7.00; N, 14.96. Found: C, 60.71; H, 7.02; N, 14.91.
Methyl 3(R)-[[1-(benzyloxycarbonyl)-2(S)-pyrrolidinyl-
methyl]amino]-2-oxo-1-pyrrolidineacetate (19). Cbz-Pro-
OMe27 (2.6 g, 9.9 mmol) was dissolved in 100 mL dry
toluene and this solution was cooled to À78 ꢀC.
DIBAL-H (13.4 mL of 1.5 M toluene solution, 20.1
mmol) was added dropwise to this solution via an
addition funnel. The chilled solution was stirred under
N2 for 3 h and then MeOH (30 mL) was added drop-
wise to quench the reaction. This was followed by 80
mL 1 N HCl. The reaction was allowed to warm to
room temperature whereupon the layers were separated.
The aqueous layer was extracted 4Â with EtOAc and
the organic layers were combined and washed with
saturated NaCl solution. The organic layer was dried
(MgSO4), filtered and concentrated in vacuo (under
40 ꢀC) to give 2(S)-N-(benzyloxycarbonyl)-pyrrolidine-
2-carboxaldehyde (18, 2.2 g, 95%) that was used
directly without purification in the next step.
3(R)-[[2(S)-Pyrrolidinylmethyl]amino]-2-oxo-1-pyrroli-
dineacetamide (5). Cbz-protected lactam 20 (80 mg,
0.21 mmol) was dissolved in 30 mL dry MeOH. Pd/C
(100 mg) was added to a Parr hydrogenation flask and
the mixture was shaken on a hydrogenator under 40 psi
H2 overnight. The solution was filtered and con-
centrated in vacuo. MeOH was added to the residue and
the mixture was filtered through an ultrafiltration disk
attached to a syringe. The solvent was removed in vacuo
to yield a yellow oil which was chromatographed on a
1Â20 cm silica gel column eluting with 33% MeOH/
CHCl3 and 0.1% NH4OH. Further purification on a
1Â5 cm column of C-8 reverse-phase silica gel eluting
Lactam 1211 (1.1 g, 4.0 mmol) was dissolved in 10 mL
4 N HCl/dioxane and the solution was stirred overnight.
The solvent was removed in vacuo and the residue that
was obtained was dissolved in CH2Cl2 and the solution
stripped of solvent. This addition and evaporation of
CH2Cl2 was done an additional 3Â. The white solid of
the deprotected lactam 17 that was obtained was dried
under high vacuum overnight. Aldehyde 18 (2.2 g, 9.4
mmol) was dissolved in 50 mL dry MeOH and this
solution was added to the flask containing deprotected

4110
K. Dolbeare et al. / Bioorg. Med. Chem. 11 (2003) 4103–4112
with 10% MeOH/acetonitrile and a 1Â10 cm silica gel
column eluting with 10% MeOH/CHCl3 gave an off-
white solid. The solid was dissolved in H2O and lyophi-
lized to give 7.5 mg (15%) of analytically pure 5 as a
hygroscopic off-white solid. HPLC analysis on a Waters
Spherisorb1 4.6Â150 mm analytical column gave a tR
of 1.9 min when eluted with 25% MeOH and 0.1%
NH4OH in CHCl3. Further HPLC analysis on a
LiChrosorb1 RP-18 4.6Â250 mm analytical column
using 40% H2O and 0.1% TFA in acetonitrile gave a tR
of 3.3 min and HPLC analysis on the RP-18 column
eluting with 20% H2O and 0.1% TFA in MeOH gave a
tR of 3.7 min. [a]D 22.4 (c 0.42, MeOH). 1H NMR
(D2O) d 1.28–1.35 (m, 1H), 1.62–1.79 (m, 3H), 1.83–
1.90 (m, 1H), 2.26–2.32 (m, 1H), 2.59 (dd, 1H, J=6.0
and 12.0 Hz), 2.65 (dd, 1H, J=6.2 and 12.0 Hz), 2.79–
2.88 (m, 2H), 3.13–3.19 (m, 1H), 3.32 (dd, 2H, J=5.0
and 8.7 Hz), 3.52(t, 1H, J=8.5 Hz), 3.88 (d, 1H,
J=17.2Hz), 3.92(d, 1H, J=17.2Hz); 13C NMR (D2O)
d 24.0, 25.0, 28.8, 45.3, 45.4, 45.7, 49.6, 57.9, 58.2, 172.4,
176.8; FAB HRMS (m-nitrobenzylalcohol matrix) m/z
241.1666 (C11H20N4O2+H+ requires 241.1665).
excess MeI was removed in vacuo. The resulting oil was
dissolved in CH2Cl2 and the solvent removed in vacuo.
This procedure was carried out 3Â to give a yellow
foam. The residue was dried under high vacuum over-
night after which it was dissolved in 150 mL dry CH2Cl2
and 30 mL dry DMF. After the solution was cooled to
0 ꢀC, NaH (60% in oil, 0.49 g, 12.3 mmol) was added in
portions over 10 min. The reaction was stirred under N2
at 0 ꢀC for 3 h and then at room temperature for 2days.
Citric acid (1.40 g, 12.3 mmol) was added to quench the
reaction and removal of the solvent in vacuo gave a
yellow slurry. The slurry was partitioned between 10%
citric acid solution and EtOAc. The aqueous layer was
extracted 4Â with EtOAc and the organic layers were
combined and washed with 1 M NaHCO3 solution fol-
lowed by saturated NaCl solution. The organic layer
was dried (MgSO4), filtered and concentrated in vacuo
to a yellow oil that was chromatographed on a 4Â40 cm
silica gel column eluting with 10% MeOH in EtOAc to
yield 2.35 g (53%) of product as a white powder. Mp
147–149 ꢀC; [a]D 14.8 (c 0.94, MeOH). 1H NMR
(CDCl3) d 1.44 (s, 9H), 1.82–1.93 (m, 1H), 2.48–2.55 (m,
1H), 3.33–3.60 (m, 6H), 4.07–4.12(m, 1H), 5.08–5.13
(m, 2H), 5.25–5.28 (br m, 1H), 5.54–5.56 (br m, 1H),
7.29–7.35 (m, 5H); 13C NMR (CDCl3) d 27.8, 28.3, 38.6
43.3, 44.5, 52.5, 66.7, 80.0, 128.1, 128.5, 136.6, 155.8,
156.8, 173.2; FAB MS (m-nitrobenzylalcohol matrix) m/z
378 [M+H]+. Anal. calcd for C19H27N3O5: C, 60.46; H,
7.21; N, 11.13. Found: C, 60.50; H, 6.95; N, 10.87.
N1-[(tert-Butoxycarbonyl)-D-methionyl]- N2-(benzyloxy-
carbonyl)-ethane-1,2-diamine (21). Boc-Met-OH (8.0 g,
32mmol), was dissolved in 300 mL dry CH Cl2 and 50
2
mL dry DMF. N-(Benzyloxycarbonyl)-ethane-1,2-dia-
28
.
.
mine HCl (11.1 g, 48 mmol) and HOBt H2O (6.50 g,
48 mmol) were added and the solution was cooled to
ꢀ
.
À78 C. EDC HCl (5.25 g, 27 mmol) and Et3N (13.3
mL, 96 mmol) were then added and the reaction was
allowed to warm to room temperature under N2 over-
night. The reaction was then stirred for 3 days. The
solvent was removed in vacuo and the residue dissolved
in CH2Cl2. This solution was washed with saturated
NaHCO3 solution, 10% citric acid solution, H2O, and
saturated NaCl solution successively. The organic layer
was dried (MgSO4), filtered and concentrated in vacuo
to a white solid that was chromatographed on a 4Â30
cm silica gel column eluting with 10% MeOH in EtOAc.
The material obtained from this chromatographic pro-
cedure was crystallized from EtOAc/hexanes to give
7.52g of 21. An additional 2.4 g of 21 was obtained by
chromatography of the mother liquor on a 4Â30 cm
silica gel column eluting with a gradient of 500 mL 20%
EtOAc in hexanes, 500 mL 50% EtOAc in hexanes, and
1 L 10% MeOH in EtOAc. The total yield of 21 was 9.9
N-[((1-Benzyloxycarbonyl)amino)ethyl]-3(R)-[[1-(benzy-
loxycarbonyl)-2(S)-pyrrolidinylcarbonyl]amino]-pyrroli-
din-2-one (23). Lactam 22 (1.57 g, 4.16 mmol) was
treated with 20 mL 4 N HCl/dioxane for 5 h. The sol-
vent was removed in vacuo from the cloudy white mix-
ture and the residue was dissolved in CH2Cl2 which was
then removed in vacuo. The salt was dried under high
vacuum overnight and then dissolved in 100 mL dry
.
CH2Cl2 and 60 mL dry DMF. HOBt H2O (0.67 g, 5
mmol) and Cbz-l-Pro-OH (1.24 g, 5 mmol) were added
ꢀ
.
and the solution was cooled to À78 C. EDC HCl (0.96
g, 5 mmol) was added followed by 1.4 mL Et3N (10
mmol) and the reaction was allowed to warm to room
temperature overnight. The reaction was further stirred
under nitrogen at room temperature for 3 days. The
solvent was removed in vacuo and the residue dissolved
in CH2Cl2. The organic layer was washed with 1 M
NaHCO3 solution, 10% citric acid solution and satu-
rated NaCl solution and then dried (MgSO4), filtered
and concentrated in vacuo to a yellow oil. This oil was
chromatographed on a 3Â40 cm silica gel column elut-
ing with 10% MeOH/EtOAc to give 0.66 g (26%) of a
side product that was determined to be N-[[1-(benzyl-
oxycarbonyl) - 2(S) - pyrrolidinylcarbonyl]amino]ethyl]-
3(R)-[[1-(benzyloxycarbonyl)-2(S)-pyrrolidinylcarbony-
l]amino]-pyrrolidin-2-one (24) [mp 75–77 ꢀC; [a]d À72.9
(c 1.23, MeOH); FAB MS (m-nitrobenzylalcohol
matrix) m/z 606 [M+H]+] and 0.68 g (32%) of the
desired product 23 as a white foam. Mp 54–55 ꢀC; [a]d
g (73%). Mp 132–133 ꢀC; [a]D 12.5 (c 0.99, MeOH). H
1
NMR (CDCl3) d 1.45 (s, 9H), 1.86–1.95 (m, 1H), 2.04–
2.15 (m, 1H), 2.10 (s, 3H), 2.52–2.57 (m, 2H), 3.35–3.42
(m, 4H), 4.22–4.24 (m, 1H), 5.11 (s, 2H), 5.30 (d, 1H,
J=7.2Hz), 5.42–5.45 (br m, 1H), 6.81–6.84 (br m, 1H),
7.32–7.38 (m, 5H); 13C NMR (CDCl3) d 15.4, 28.4, 30.2,
31.6, 40.3, 40.8, 53.8, 66.9, 80.3, 128.2, 128.5, 136.4,
155.7, 157.0, 172.4; FAB MS (m-nitrobenzylalcohol
matrix) m/z 426 [M+H]+. Anal. calcd for
C20H31N3O5S: C, 56.45; H, 7.34; N, 9.87; S, 7.53.
Found: C, 56.62; H, 7.06; N, 10.11; S, 7.91.
1
N-[((1-Benzyloxycarbonyl)amino)ethyl]-3(R)-[N-(tert-bu-
toxycarbonyl)amino]-pyrrolidin-2-one (22). Compound
21 (5.0 g, 11.8 mmol) was stirred at room temperature in
excess neat MeI (30 mL) overnight after which the
À54.1 (c 0.94, MeOH). H NMR (DMSO-d6, rotamers
present) d 1.66–1.80 (m, 4H), 2.06–2.18 (m, 2H), 3.07–
3.11 (br m, 3H), 3.14–3.38 (m, 5H), 4.17–4.31 (m, 2H),
4.95–5.10 (m, 4H), 7.25–7.36 (m, 11H), 8.18 and 8.25 (d,

K. Dolbeare et al. / Bioorg. Med. Chem. 11 (2003) 4103–4112
4111
1H, J=8.1 Hz); 13C NMR (DMSO-d6, rotamers pre-
sent) d 23.4 (24.2), 26.2, 30.7 (31.7), 38.3 (43.0), 43.9,
467.0 (47.6), 50.6, 59.9 (60.4), 65.8 (66.2), 127.5, 127.8,
127.9, 128.2, 128.2, 128.7, 128.8, 137.5, 137.6, 154.3
(156.6), 172.1, 172.4 (172.7); FAB MS (m-nitrobenzyl-
alcohol matrix) m/z 509 [M+H]+. Anal. calcd for
filters. The filters were washed with 3Â5 mL of Tris–
EDTA buffer (pH 7.4, 50 mM Tris–HCl, 1 mM EDTA)
and the radioactivity was determined on a Beckman
scintillation counter (Model 1780). The percent increase
in NPA binding produced by compounds 3–6 was com-
pared against the control value (the binding of [3H]NPA
in the absence of any PLG peptidomimetic) and against
the activity of peptidomimetic 2. All data were analyzed
using the student t test in the GraphPad Prism (Version
2.01) software package.
.
C27H32N4O6 MeOH: C, 62.21; H, 6.71; N, 10.36.
Found: C, 62.54; H, 6.51; N, 10.72.
3(R)-[[2(S)-Pyrrolidinylcarbonyl]amino]-1-(aminoethyl)-
pyrrolidin-2-one (6). Diprotected Cbz lactam 23 (190.4
mg, 0.37 mmol) was dissolved in 50 mL dry MeOH. Pd/
C (100 mg) was added to a Parr hydrogenation flask
and the flask was shaken on the hydrogenator under 60
psi H2 overnight. The solution was filtered and con-
centrated in vacuo. The residue was dissolved in MeOH
and then filtered through an ultrafiltration disk attached
to a syringe. The solvent was removed in vacuo to yield
a yellow oil which was chromatographed on a 1Â2 0 cm
silica gel column eluting with 25% MeOH and 0.1%
NH4OH in CHCl3. Further purification on a 1Â5 cm
column of C-8 reverse-phase silica gel eluting with 10%
MeOH/acetonitrile and a 0.5Â3 cm silica gel column
eluting with 20% MeOH/CHCl3 gave an off-white solid.
The material was dissolved in H2O and lyophilized to
give 32mg (36%) of analytically pure product as a
hygroscopic off-white solid. HPLC analysis using a
Water Spherisorb1 4.6Â150 mm analytical column
gave a tR of 4.7 min when the product was eluted with
25% MeOH and 0.1% NH4OH in CHCl3. HPLC ana-
lysis using a LiChrosorb1 RP-18 4.6Â250 mm analy-
tical column using 40% H2O and 0.1% TFA in
acetonitrile gave a tR of 2.9 min. HPLC analysis on the
RP-18 column eluting with 20% H2O/MeOH with 0.1%
TFA gave a tR of 4.0 min. [a]d À5.6 (c 0.72, MeOH). 1H
NMR (DMSO-d6) d 1.56–1.71 (m, 3H), 1.74–1.81 (m,
1H), 1.84–1.97 (m, 1H), 2.19–2.29 (m, 1H), 2.69 (t, 2H,
J=6.3 Hz), 2.72–2.86 (m, 2H), 3.21 (t, 2H, J=6.3 Hz),
3.25–3.30 (m, 2H), 3.42 (br s, 2H), 3.50 (dd, 1H, J=5.4,
8.7 Hz), 4.31 (dd, 1H, J=9.0 and 18.0 Hz), 8.15 (d,
1H, J=8.0 Hz); 13C NMR (DMSO-d6) d 25.7, 26.2,
30.4, 38.4, 43.5, 44.3, 46.7, 49.9, 60.2, 172.1, 174.6; CI
HRMS m/z 241.1666 (C11H20N4O2+H+ requires
241.1665).
Acknowledgements
This work was supported in part by a NIH grant
(NS20036) to R.L.J.
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