Conformationally restricted TRH analogues: Constraining the pyroglutamate region

Article abstract of DOI:10.1016/S0968-0896(01)00287-5

A modified synthetic route has been developed so that the steric size of constraints added to the pyroglutamate region of TRH (pGluHisProNH₂) can be varied. Both an analogue with a smaller ethylene bridge and a larger, more flexible propane bri

Full text of DOI:10.1016/S0968-0896(01)00287-5

Bioorganic & Medicinal Chemistry 10 (2002) 291–302
Conformationally Restricted TRH Analogues:
Constraining the Pyroglutamate Region
Jill C. Simpson,a Chris Ho,b E.F. Berkley Shands,c Marvin C. Gershengorn,d,e
Garland R. Marshallb and Kevin D. Moellera,*
aDepartment of Chemistry, Department of Molecular Biology and Pharmacology, Washington University, St. Louis, MO 63130, USA
bCenter for Molecular Design, Washington University, St. Louis, MO 63130, USA
cDepartment of Computer Science, Washington University, St. Louis, MO 63130, USA
dDivision of Molecular Medicine, Department of Medicine, Cornell University Medical College, New York, NY 10021, USA
eThe New York Hospital, New York, NY 10021, USA
Received 15 May 2001; accepted 31 July 2001
Abstract—A modified synthetic route has been developed so that the steric size of constraints added to the pyroglutamate region of
TRH (pGluHisProNH2) can be varied. Both an analogue with a smaller ethylene bridge and a larger, more flexible propane bridge
in this region have been synthesized. These analogues were synthesized in order to probe why the initial incorporation of an ethane
bridge into this region of the molecule had led to an analogue with a binding constant and potency three times lower than that of an
directly analogous unconstrained analogue. The data for both analogues indicated that the fall off in activity caused by the ethane
bridge in the initial analogue was not caused by the size of the bridge. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
metics.5 This work led to the synthesis of a TRH
analogue with a constraint added to the pyroglutamate
region (bridge A),6 several analogues with constraints
added to the His-Pro region (bridge B),7 and a pair of
analogues with constraints added to both regions.8
While the initial synthetic routes developed allowed us
to start answering questions about the nature of TRH
binding to its endocrine receptor (TRH-R1), they were
limited in scope. This was especially true of the strategy
employed to constrain the pyroglutamate region of the
molecule. One one hand, the chemistry utilized to build
6 (Scheme 2) allowed for the synthesis in just six steps
from pyroglutamic acid. On the other, it was hampered
by both the use of an intermolecular reductive amina-
tion reaction that required a large excess of the NH2–
Lactam based peptidomimetics can be valuable tools for
probing the activity of peptide conformations.1 These
analogues, which exhibit increased hydrolytic stability
and preserve both backbone and sidechain function-
ality, can be designed by simply replacing spatially close
hydrogens within a conformation of interest with a car-
bon bridge. For example, the family of constrained
peptidomimetics (2) illustrated in Scheme
1 was
designed to mimic a proposed endocrine receptor bound
conformation (1) of thyrotropin-releasing hormone
(thyroliberin, TRH).2À4 However, while lactam based
peptidomimetics offer many advantages, their utility is
often limited by the difficulty associated with their
syntheses. Work to evaluate the effectiveness of TRH
analogues like 2 and probe the biological relevance of
the conformation represented by 1 stalled when the
constrained analogues could not be made.3b
In order to address this issue, we began to develop con-
venient synthetic routes to lactam based peptidomi-
*Corresponding author. Tel.: +1-314-935-4270; fax: +1-314-935-
4481; e-mail: moeller@wuchem.wustl.edu
Scheme 1.
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00287-5

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J. C. Simpson et al. / Bioorg. Med. Chem. 10 (2002) 291–302
PhePro–NH2 dipeptide, inconsistent yields and the
competitive formation of diketopiperazide products
during the last two steps of the synthesis. Because of
these problems the synthesis was difficult to scale up,
impractical for the synthesis of rings with larger, more
flexible constraints, and incompatible with the synthesis
of analogues containing a smaller ethylene bridge con-
straint.
amination and the diketopiperzide formation that hin-
dered the original route used for 6 (Scheme 2) would be
avoided.
This approach to analogues 6–8 proved very useful. The
synthesis of the TRH analogue constrained with an
ethylene bridge (7) is outlined in Scheme 4. As in the
earlier synthesis of 6, the allyl-substituted pyr-
oglutamate building block 5 was made by first functio-
nalizing menthyl pyroglutamic acid with the use of an
anodic amide oxidation reaction (Scheme 2),6a,10 and
then treating the resulting methoxylated product with
allylsilane and TiCl4 to afford a 2:1 mixture of stereo-
isomeris allylpyroglutamate derivatives. A separation by
fractional crystallization led to 5 in a 54% isolated
yield. Once in hand, the building block was saponified
with KOH in methanol to form the carboxylic acid and
the acid coupled to phenylalanine benzyl ester using
This lack of synthetic flexibility was particularly both-
ersome because analogue 6 showed both an affinity and
a level of potency for TRH-R1 that was approximately
three times lower than the corresponding unconstrained
Phe2-TRH.6 While 6 was still a full agonist for the
receptor, we were curious as to whether the reduction in
its activity was due to the bridge in 6 constraining the
analogue into a conformation that was not quite correct
or whether the reduction in its activity was due to the
steric size of the bridge. Adding to this interest was the
initial observation that the fully constrained TRH ana-
logue 2a (n=1, m=1, Ha=R-configuration) might be a
partial agonist for TRH-R1.8,9 One suggestion for
explaining this observation was that analogue 2a might
be too rigid to allow for the conformational changes in
the ligand–receptor complex needed for initiating sec-
ond messenger release. If this were the case, then
increasing the flexibility of the bridges might restore
potency to the analogue. But would a larger constraint
in the spirocyclic portion of 2a be tolerated by the
receptor? It was clear that a more versatile route to
building the spirocyclic ring skeleton was needed if
questions of this nature were to be answered.
Synthesis
With this in mind, a revised strategy for synthesizing the
spirocyclic lactam ring skeletons was proposed (Scheme
3). The key to this plan was the conversion of the 5-
allylpyroglutamate derivative (5) into an advanced
intermediate (9) that could be used to construct all of
the desired analogues. This would be accomplished by
using the olefin of the allyl group in 9 to set up either an
intramolecular reductive amination reaction or an
intramolecular Mitsunobu reaction. Once the spir-
ocyclic ring was in place, the benzyl ester would be
deprotected and the resulting acid coupled to prolina-
mide. In this way, both the intermolecular reductive
Scheme 3.
Scheme 2.
Scheme 4.

J. C. Simpson et al. / Bioorg. Med. Chem. 10 (2002) 291–302
293
HOBt/EDCI conditions in order to form 9. Oxidation
and cleavage of the olefin with OsO4 and NaIO4 led
directly to the cyclized a-hydroxyalkylamide which was
then protected as the t-butyldimethylsilyl ether (10). The
benzyl ester was deprotected using hydrogenolysis con-
ditions, the resulting acid coupled to prolinamide, and
the synthesis completed by eliminating the t-butyldi-
methylsilyl ether group with the use of reduced pressure
and silica gel as a catalyst.11
for avoiding competitive intramolecular cyclization
reactions involving this nitrogen later in the synthesis.
The PMB group was selected after several other groups
(Bzl, Cbz, and MOM) proved difficult to remove fol-
lowing formation of the spirocyclic ring skeleton. With
the pyroglutamate nitrogen protected, the menthyl ester
was hydrolyzed using lithium hydroxide, and the
resulting carboxylic acid coupled to the phenylalanine
benzyl ester. A hydroboration/oxidation sequence using
disiamylborane and hydrogen peroxide was employed
to convert the olefin into primary alcohol 11. The
desired spirocyclic ring was then generated with the use
of a Mitsunobu reaction. At this point, the PMB group
was removed with the use of ceric ammonium nitrate to
form 12. The revoval of the protecting group at this
stage of the synthesis was critical. Removal of the PMB
group during the final step of the synthesis failed when
the final CAN deprotection step led to an over-oxida-
tion of the PMB benzylic carbon prior to the desired
collapse of the hemiaminal intermediate. The result was
the formation of a PMB ester that could not be cleaved
from the product tripeptide. This problem did not
interfere with the deprotection reaction leading to 12,
and the synthesis was completed by deprotecting the
benzyl ester using a catalytic hydrogenolysis and cou-
pling the resulting acid to prolinamide with the use of
HOBt/EDCI conditions. As with the earlier analogues,
the purity of the analogue used for testing was estab-
lished by C, H, and N analysis.
Several items about this synthesis deserve comment.
First, the use of the benzyl protecting group for the
phenylalanine carboxylic acid was essential. Both acid
and base cleavage of alternative ester groups led to
competitive hydrolysis of the tertiary amide. Second,
protection of the N-a-hydroxyamide group was impor-
tant. Elimination prior to the final coupling steps was
problematic because of the incompatibility of the
enamide moiety with the reaction conditions required
for deprotection and coupling steps. Alternatively,
attempts to carry the alcohol through the final steps of
the synthesis were complicated by lactone formation.
Third, a number of elimination reactions were attemp-
ted using both the silyl ether and the corresponding
deprotected alcohol. Among the reagents tried were
TFA with heat, POCl3, SOCl2, Burgess reagent, and
methyltriphenylphosphonium iodide. None of these
alternatives was competitive with the silica gel based
strategy. Fourth, enamide analogue 7 was a very stable
compound. It could be stirred in methanol for 24 h,
chromatographed through silica gel, and recrystallized
without any decomposition. The purity of the analogue
used for biological testing was established by C, H, and
N analysis. Finally, the constrained analogue 7 could be
readily hydrogenated to form the previously synthesized
TRH analogue 6 having an ethane bridge in the pyr-
oglutamate region. In this way, both of the five-mem-
bered ring lactam pyroglutamate derivatives could be
made using the same synthetic route.
Biological Testing and Discussion
Analogues 7 and 8 were examined for both their affinitiy
for TRH-R1 and their ability to serve as agonists for the
receptor. Affinities, reported as Ki (mM) in Table 1, were
determined by measuring the concentration of the ana-
logue required to completely displace [NtMe-His]-TRH
from the receptor. [NtMe-His]-TRH is an unrestricted
TRH analogue in which the imidazole side chain of His
is methylated. [NtMe-His]-TRH is known to bind to
TRH-R1 with an affinity about 10 times as great as that
of the natural hormone. The agonist behavior of the
analogues was tested in HEK 293EM cells stably
expressing mTRH-R1 by incubating the cells with var-
ious doses of the analogues being examined.12 The
extent of agonist behaviour was then determined by
measuring the amount of second messenger (IP3)
released. The data are reported as EC50 (mM) values
(Table 1).
The synthesis of the six-membered ring analogue fol-
lowed a similar path (Scheme 5). In this synthesis, the
pyroglutamate nitrogen was first protected with a para-
methoxybenzyl ether. This protection step was essential
From the data in Table 1, two things become readily
apparent. First, the analogue having the ethylene bridge
(7) did not bind TRH-R1 with as high an affinity as did
the analogue with the ethane bridge (6). The same con-
clusion was reached about the potency of the analogues.
Second, the use of the larger propane bridge (8) in place
of the ethane bridge (6) did not significantly alter either
the affinity or the potency of the analogue for TRH-R1.
Initially, it was not clear why changing the ethane
bridge in 6 to the ethylene bridge found in 7 would
decrease the potency of the analogue. A direct compar-
Scheme 5.

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J. C. Simpson et al. / Bioorg. Med. Chem. 10 (2002) 291–302
Table 1. Biological data
Analogue
Ki (mM)
EC50 (mM)
TRH (p-GluHisPro-NH2)
Phe2TRH (p-GluPhePro-NH2)
Analogue 6b
Analogue 7
Analogue 8
0.0038 (0.0025–0.0055)a
0.45 (0.25–0.75)
1.6 (0.95–2.7)
0.0013 (0.00099–0.0018)
0.11 (0.080–0.150)
0.10 (0.057–0.19)
0.78 (0.55–1.1)
11 (7.1–15)
1.5 (0.95–2.4)
0.16 (0.10–0.26)
aThe numbers in parentheses represent the 95% confidence intervals for the data given.
bAnalogue 6 was re-examined in this study so that the activity of 6 could be directly compared to that of 7 and 8.
ison of analogues 6 and 7 suggested that the two analo-
gues were very similar. For example, Figure 1 compares
the minimized structures of the two analogues.13 In this
drawing, the molecules were aligned by overlaying the
key pharmacophoric groups (the pyroglutamate car-
bonyl, the phenyl ring of the second residue, and the
primary amide of the prolinamide) in the two molecules.
When this was done, the only difference between the
analogues appeared to be a slight twist of the pyr-
oglutamate group relative to the rest of the molecule.
The conclusion could be drawn for the overlap between
analogues 8 and 6 (Fig. 2). Again, the only difference
between these analogues was a slight twist of the pyr-
oglutamate group, although the twist in 8 was in the
opposite direction of the one observed for 7.
A more significant difference between the analogues
could be seen when their overlap with the receptor
model developed by Marshall and coworkers was taken
into account.2À4 This model was derived by determining
a common conformation for all of the active analogues
of TRH.14 Once the common conformation was found,
the analogues were aligned and a set of average dis-
tances between the pharmacophoric groups within the
molecules determined. The compatibility of analogues
6–8 with this model was then evaluated by using the
average distances as constraints for the distances
between the pharmacophoric groups in 6–8. The energy
for each analogue was then minimized and compared to
the minimized energy of the analogue without the dis-
tance constraints. In this way, a measure for what it
‘costs’ each analogue to overlap with the receptor was
obtained. Imposing the distance constraints associated
with the model for TRH-R1 onto analogue 6 led to an
increase in energy of 5.66 kcal/mol. For the analogue
having the ethylene bridge in the pyroglutamate region
(7), this increase in energy jumped to 10.85 kcal/mol.
For the analogue having the six-membered ring con-
straint (8), the increase in energy was only 1.87 kcal/
mol. This data suggested that while both 7 and 8 could
overlap with the previously active analogue 6 to roughly
the same extent, the two analogues were significantly
different with respect to their ability to overlap with the
receptor. It was more difficult for analogue 7 to obtain
the conformation necessary for binding the receptor; a
suggestion that was consistent with the lower level of
activity obtained for this analogue relative to that
obtained for analogue 8.
Figure 1. Compound 6=black; compound 7=grey.
The energy calculations were also consistent with the
relative affinities and potencies of 6 and 7; the largest
increase in energy corresponding to the less active ana-
logue (Table 2). Clearly, it was not beneficial to decrease
the steric size of the bridge in 6 at the expense of further
constraining the analogue. However, the analysis of 8
relative to the original 6 was not so straightforward.
The energy calculations using the model for TRH-R1
failed to predict the nearly identical activity of these two
analogues. In this case, the lower increase in energy
calculated for 8 (1.87 kcal/mol relative to 5.66 kcal/mol)
suggested that 8 should have had a higher affinity and
potency for TRH-R1 than 6. The most reasonable
explanation for this observation was that the increase in
the steric size of the propane bridge in 8 relative to the
ethane bridge undermined the ability of 8 to bind to
TRH-R1. When 8 was overlapped with the model for
TRH/TRH-R1 binding developed using all of the pre-
Figure 2. Compound 6=black; Compound 8=grey.

J. C. Simpson et al. / Bioorg. Med. Chem. 10 (2002) 291–302
295
Table 2. Energy calculations
unrestricted counterpart was most likely not due to the
size of the ethane bridge.
Analogue
Minimized Energy
(no distance
constraints)
Minimized Energy
(with distance
constraints)
Difference
(kcal/mol)
(kcal/mol)
(kcal/mol)
Experimental15
6
7
8
28.56
29.50
28.90
34.22
40.35
30.77
+5.66
+10.85
+1.87
(2R,S)-2-Methoxy-5-oxoproline-(1S,2R,5S)-5-methyl-2-
(1-methylethyl)cyclohexyl ester (4). An oven-dried 250
mL three-neck flask with stir bar was cooled under N2.
To the flask was added 11.5 g (43 mmol) of (+)-men-
thylpyroglutamate and 8.31 g (21.5 mmol, 0.5 equiv) of
nBuNPF6. The solids were dissolved in 86 mL of anhy-
drous MeOH, two platinum wire (0.8 mm diameter by
1.5 cm length) were inserted, and the entire reaction
setup was degassed by sonication for 10 min. The solu-
tion was then oxidized at a constant current of 26.8 mA
until 18,630 C (4.5 F/mol) had passed. The completed
reaction solution was transferred to a 500 mL flask
(single-neck), the MeOH was removed in vacuo, and
100 mL of Et2O was added. The electrolyte was then
filtered, and the filtrate was dried over anhydrous
MgSO4, filtered, and concentrated completely in vacuo.
The crude product was then purified by gravity-flow
chromatography through 350 g of slurry packed silica
gel using a 1:1 Et2O/hexane to 100% Et2O gradient
solvent system. Fractions containing 4 (TLC solvent
Et2O, Rf 0.74, yellow/blue spot with p-anisaldehyde
stain) were combined, dried over anhydrous MgSO4,
filtered, and concentrated in vacuo to yield 12.2 g (41.0
mmol, 95%) of pure product as a clear, viscous liquid.
1H NMR (300 MHz, CDCl3) two isomers, d 8.63 (br s,
0.6H), (8.53 (br s, 0.4H), 4.76 (tt, 1H, J=8.6, 2.4 Hz),
3.26 (s, 3H), 2.63–2.56 (m, 1H), 2.40–2.30 (m, 3H),
2.04–2.00 (m, 1H), 1.92–1.87 (m, 1H), 1.70 (dm, 2H,
Jd=11 Hz), 1.53–1.45 (m, 2H), 1.15–0.80 (unresolved
m, 3H), 0.92–0.89 (2 unresolved d, 6H), 0.77–0.74 (2
unresolved d, 3H); 13C NMR (75 MHz, CDCl3) two
isomers, d 178.3, 178.2, 168.3, 168.2, 91.8, 91.7, 76.1,
51.2, 51.0, 46.4, 40.1, 33.8, 31.3, 31.1, 29.0, 28.0, 25.8,
22.8, 21.6, 20.4, 20.4, 15.7, 15.6. IR (thin film) 3205 (bs),
2945 (s), 2877 (s), 1716 (s), 1460 (m), 1076 (s), 737 (m)
cmÀ1. LRFAB MS (3-NBA/gly/TFA matrix) m/e (rela-
tive intensity) identity of ion if known; 298 (28) MH+;
266 (52) MH+–HOCH3; 160 (35) 2-OMe-oxoproli-
neH+, 128 (100) MH+–OCH3–C10H19; 114 (90) M–
C11H9O2. HRFAB MS (3-NBA/gly/TFA matrix): m/e
calcd for C16H28NO4 (MH+) 298.2018; found 298.2017
(dev À0.4 ppm).
viously active analogues, it was found that the propane
bridge in 8 occupied a position in space that was unique
to 8. While the active analogue approach does not
involve a model for the receptor and can not directly
identify interactions between the receptor and elements
of a particular ligand, the observation that the bridge in
8 occupied space not utilized by the other analogues was
consistent with the suggestion that the bridge might
interfere with the receptor in ways not available to the
smaller ethane bridge in 6.
Like the biological results obtained using 5, the results
obtained for analogues 6 and 8 supported the idea that
the loss of affinity and potency for 6 relative to its
acyclic counterpart was due to its being constrained in a
conformation that was not quite correct and not sterics.
If the ethane bridge in 6 had decreased the activity of
the analogue through steric interactions, then the use of
a larger ring should have increased these interactions
and led to an even less active analogue; a result that was
not observed.
Conclusions
We have found that a revised strategy for adding con-
straints to the pyroglutamate region of TRH provides a
significantly improved method for varying the nature of
the constraint. The development of this approach
allowed for the synthesis of analogues having an ethyl-
ene, an ethane, and a propane bridge in this region.
From the biological data obtained, it was determined
that altering the original bridge used to constrain the
pyroglutamate region of TRH in 6 did not lead to an
improvement in the affinity or potency of the analogue
for TRH-R1. The use of a smaller, more highly con-
strained ethylene bridge (7) proved to be detrimental to
the affinity and potency of the analogue while the use of
a larger, more flexible propane bridge (8) made no sigi-
nificant change in the biological activity of the analo-
gue. The decrease in activity for the molecule containing
the ethylene bridge was consistent with the model for
TRH-R1 generated using the active analogue approach.
This model showed that it cost more energy for the
analogue with the ethylene bridge to overlap with the
receptor than it did for the corresponding analogue with
the ethane bridge. This model also suggested that the
use of the propane bridge should improve the potency
of the analogue. While this turned out not to be the
case, the fact that the use of the larger bridge led
to a level of affinity and potency for the analogue
equal to that of the smaller analogue 6 suggested
that the observation that 6 was less potent than its
(2r)-2-Prop-2-enyl-5-oxoproline (1S,2R,5S)-5-methyl-2-
(1-methylethyl)cyclohexyl ester (5). To a dry 250 mL
flask under an argon atmosphere containing compound
4 and a stir bar was added 40 mL of CH2Cl2. The
resulting solution was cooled to À78 ꢀC in an acetone/
dry ice bath. When cool and while vigorously stirring
under argon, 21.1 mL of a 1 M solution of TiCl4 in
CH2Cl2 (21.1 mmol, 1.1 equiv) was added dropwise via
syringe. The dark-brown solution was stirred for 5 min,
and then 14 mL (9.8 g, 86 mmol, 4.5 equiv) of allyl-TMS
was added via syringe. The reaction solution was allowed
to stir while warming slowly to room temperature over

296
J. C. Simpson et al. / Bioorg. Med. Chem. 10 (2002) 291–302
16 h, after which the solution was transferred to a 1 L
Erlenmeyer flask and diluted with 200 mL of CH2Cl2.
While stirring vigorously, 200 mL of saturated aqueous
NaHCO3 solution was slowly added to neutralize any
excess TiCl4. The layers were separated, and the aq layer
was extracted twice more with 200 mL portions of
CH2Cl2. All organic layers were combined, con-
centrated in vacuo to approximately 75 mL, dried over
MgSO4, filtered, and concentrated completely in vacuo.
The crude product was then purified by flash chroma-
tography through 175 g of slurry packed silica gel using
Et2O as eluent. Fractions containing product (TLC
solvent Et2O, Rf 0.30, blue spot with p-anisaldehyde
stain) were combined and concentrated in vacuo to a
yellow oil which was dissolved in 25 mL of hexane in a
250 mL Erlenmeyer flask The hexane solution was
cooled to 0 ꢀC at which time a seed crystal of 5 was
added, and the flask was placed in the freezer overnight
to complete the selective crystallization of the desired R
isomer. Careful filtration of the crystals with cold hex-
ane yielded 3.30 g (10.7 mmol, 56%) of pure diaster-
eomer 5. 1H NMR (300 MHz, CDCl3) d 6.03 (br s, 1H),
5.74–5.60 (m, 1H), 5.18–5.16 (m, 1H), 5.21 (s, 1H), 4.72
(dt, 1H, Jt=11.0 Hz, Jd=4.4 Hz), 2.69 (dd, 1H,
J=13.7, 6.3 Hz), 2.45–2.34 (m, 4H), 2.20–2.04 (m, 1H),
1.96 (dm, 1H, Jd=12.0 Hz), 1.92 (septet of d, 1H,
Jsep=6.8 Hz, Jd=2.7 Hz), 1.74–1.66 (m, 2H), 1.46–1.39
(m, 2H), 1.14–0.80 (m, 3H), 0.92–0.89 (2 unresolved d,
6H), 0.75 (d, 3H, J=7.1 Hz); 13C NMR (75 MHz,
CDCl3) d 176.6, 172.6, 131.0, 120.6, 76.0, 65.2, 46.8,
43.4, 40.6, 34.1, 31.4, 30.5, 29.7, 26.2, 23.05, 21.9, 20.8,
15.9. IR (thin film) 3343 (bm), 2952 (s), 2931 (s), 2870
(m), 1730 (s), 1699 (s), 1455 (m), 1370 (m), 1182 (m),
1065 (m), 930 (m), 915 (m), 731 (s) cmÀ1. LRFAB MS
(3-NBA/gly/TFA matrix) m/e (relative intensity) iden-
tity of ion if known; 308 (86) MH+; 170 (100) 2-allyl-
oxoprolineH+; 128 (12) 2-oxoprolineH+; 124 (64) M-
C11H18O. HRFAB MS (3-NBA/gly/TFA matrix): m/e
calcd for C18H29NO3 (MH+) 308.2226; found 308.2225
(dev +0.1 ppm).
filtered, and concentrated in vacuo to yield 1.71 g (10.1
mmol, 97%) of the acid as a white solid. 1H NMR
(300 MHz, CDCl3) d 11.11 (br s, 1H), 7,85 (br s, 1H),
5.74 (ddt, 1H, J=15.1, 11.8, 6.6 Hz), 5.205 (d, 1H,
J=11.8 Hz), 5.199 (d, 1H, J=15.1 Hz), 2.69 (A of
ABX, 1H, JAB=13.8 Hz, JAX=6.6 Hz), 2.53–2.41
(unresolved m, 4H), 2.19–2.06 (m, 1H); 13C NMR
(75 MHz, CDCl3) d 179.5, 176.1, 130.9, 120.6, 66.1,
42.8, 30.1, 29.5. IR (thin film) 3010 (bm), 3271 (bm),
3078 (m), 2557 (m), 1715 (s), 1644 (s), 1416 (m), 1234
(s), 995 (m), 924 (m), 728 (m) cmÀ1. LRFAB MS (3-
NBA/gly/TFA matrix) m/e (relative intensity) identity
of ion if known; 170 (94) MH+; 128 (16) MH+–pro-
pene; 124 (100) MH+–CO2. HRFAB MS (3-NBA/gly/
TFA matrix): m/e calcd for C8H12NO3 (MH+)
170.0817; found 170.0813 (dev À2.4 ppm).
(2R)-2-Prop-2-enyl-5-oxoprolyl-L-phenylalanine benzyl
ester (9). Into a dry 25 mL flask with stir bar was dis-
solved 0.505 g (2.98 mmol) of the allylated pyroglutamic
acid in 6 mL of CH2Cl2. The mixture was cooled to
0 ꢀC, and then 0.44 g (3.3 mmol, 1.1 equiv) of HOBt and
0.42 mL (0.38 g, 3.3 mmol, 1.1 equiv) of NEM was
added while stirring vigorously to completely dissolve
under N2. When all was dissolved, 0.63 g (3.3 mmol, 1.1
equiv) of EDC was added. After 5 min, 0.84 g (3.3
mmol, 1.1 equiv) of H2N-l-Phe-Obnꢁ HCl was added.
The mixture was allowed to warm to 25 ꢀC and stirred
for 2 h. The reaction was then diluted with 10 mL of
EtOAc and the CH2Cl2 was distilled in vacuo. The
remainder was diluted further by the addition of 40 mL
of EtOAc and then washed with 30 mL of 5% aq
NaHCO3 solution, 30 mL of 5% aq citric acid solution,
and 30 mL of saturated aq NaCl solution, extracting
each aq wash twice with additional EtOAc before
proceeding. The EtOAc extracts were combined, dried
over MgSO4, filtered, and concentrated in vacuo to a
sticky brown oil. The crude material was then grav-
ity-flow chromatographed through 125 g of slurry
packed silica gel using a 5:95 MeOH/EtOAc solvent
mixture. Fractions containing product (TLC solvent
same as column, Rf 0.54, UV-active spot or faint
yellow/white spot with ninhydrin stain) were combined
and concentrated in vacuo to afford 1.2 g (2.98 mmol,
(2R)-2-Prop-2-enyl-5-oxoproline. To a 100 mL flask with
stir bar was added 4.74 g of (15.4 mmol) menthyl ester 5
and 15 mL of anhydrous MeOH. The mixture was then
stirred to dissolve under N2 and then was cooled to
0 ꢀC. Meanwhile, 0.95 g (16.9 mmol, 1.1 equiv) of finely
ground KOH was dissolved in 20 mL of MeOH. This
KOH/MeOH solution was then added to the cold, stir-
ring ester/MeOH solution by quantitative transfer via
cannula. An additional 5 mL of MeOH was used for
rinsing. The reaction flask was allowed to slowly warm
to 25 ꢀC over several hours and then stirred 18 h more,
checking periodically by TLC (Et2O eluent) for com-
pletion as evidenced by disappearance of 5. Approxi-
mately 90 mL of H2O was added and the MeOh was
then removed in vacuo. The cloudy aq solution was
extracted with 50 mL of Et2O three times to remove the
liberated menthol and then acidified to a pH of 2 by the
addition of 150 mL of 2 N aq HCl. The solution was
concentrated in vacuo completely. To the resulting pro-
duct/salt mixture was added 100 mL of CH2Cl2, and
then the KCl salt was removed by filtration. The filtrate,
containing product, was dried over anhyd MgSO4,
1
100%) of pure 9 as a white solid. H NMR (300 MHz,
CDCl3) d 7.37–7.02 (m, 10H), 6.48 (br s, 1H), 5.68–5.54
(m, 1H), 5.21–5.08 (unresolved m, 4H), 4.96–4.88 (m,
1H), 3.24 (dd, A of ABX, 1H, JAB=13.9 Hz,
JAX=5.5 Hz), 2.99 (dd, B of ABX, 1H, JAB=13.9
Hz, JBX=8.5 Hz), 2.80 (dd,
A of ABX, 1H,
JAB=13.8 Hz, JAX=5.6 Hz), 2.48–1.91 (unresolved
m, 6H); 13C NMR (75 MHz, CDCl3) d 178.2, 173.3,
171.8, 136.9, 135.1, 131.8, 129.1, 128.6, 128.6, 128.5,
127.2, 120.4, 67.4, 65.4, 53.1, 42.7, 37.7, 31.8, 29.6.
IR (thin film) 3308 (bm), 3062 (m), 3027 (m), 2931
(m), 1743 (s), 1702 (s), 1671 (s), 1500 (m), 1455 (m),
1271 (m), 1100 (w), 997 (w), 922 (w), 737 (s), 699 (s)
cmÀ1. LRFAB MS (3-NBA/gly/TFA matrix) m/e (rela-
tive intensity) identity of ion if known; 407 (100) MH+;
365 (2) MH+–propene; 256 (6.5); 214 (8.5). HRFAB
MS (3-NBA/gly/TFA matrix): m/e calcd for
C24H27N2O4 (MH+) 407.1971; found 407.1977 (dev
+0.1 ppm).

J. C. Simpson et al. / Bioorg. Med. Chem. 10 (2002) 291–302
297
Nꢀ -[(5R,8RS)-8-Hydroxy-2,6-dioxo-1-azaspiro[4.4]-
octyl]-L-phenylalanine benzyl ester. Compound 9 (3.3 g,
8.12 mmol) was placed in a 500 mL flask with stir bar
and dissolved in 80 mL of distilled THF and 40 mL of
distilled/deionized H2O while stirring under N2 at 25 ꢀC.
Next, 1.75 mL of a 0.05 M solution of OSO4 in H2O (1
mol%) was added via syringe. The solution was allowed
to stir for 5 min over which time the reaction solution
slowly turned brown. Then, 4.34 g (20.3 mmol, 2.5
equiv) of solid NaIO4 was added. Almost immediately a
white precipitate began to form, and the reaction was
left to stir under N2 for 5 h more to ensure reaction
completion. The THF was removed in vacuo and the
remaining aq reaction solution was poured into a 250
mL separatory funnel and extracted eight times with
100 mL portions of CH2Cl2, checking by TLC for
complete transfer of product to organic phase
(15:85 MeOH/EtOAc as eluent, Rf 0.46, UV-active spot
or pink spot with ninhydrin stain). The CH2Cl2 extracts
were combined, dried over MgSO4, filtered, and con-
centrated in vacuo to afford a yellow viscous oil. This
crude product was then flash chromatographed through
100 g of slurry-packed silica gel using a 5:95 MeOH/
EtOAc solvent mixture. Fractions containing product
were combined, concentrated in vacuo, redissolved in 80
mL of CH2Cl2, dried over MgSO4, filtered, and con-
centrated in vacuo completely to give 3.29 g (8.12 mmol,
100%) of pure cyclized N-a-hydroxyamide as a white
solution was diluted to approximately 100 mL with
CH2Cl2 and washed with 50 mL of 5% aq citric acid
solution and 50 mL of 5% aq NaHCO3 solution, re-
extracting each aq wash once with additional CH2Cl2
before proceeding. All organic extracts were combined,
dried over MgSO4, filtered, and concentrated in vacuo.
The crude material was then flash chromatographed
through 250 g of slurry packed silica gel using a
5:94:1 MeOH/EtOAc/Et3N solvent mixture. Fractions
containing product (two diastereomers) were combined,
concentrated in vacuo, redissolved in 50 mL of CH2Cl2,
dried over MgSO4, filtered, and concentrated in vacuo
to give 4.70 g (8.99 mmol, 99%) of the silated product
10 as a viscous oil. 1H NMR (300 MHz, CDCl3) d 7.37–
7.14 (m, 10H), 5.75 (br s, 1H), 6.16 (d, A of AB, 1H,
J=12.0 Hz), 5.11 (d, B of AB, 1H, J=12.0 Hz), 4.39
(dd, X of ABX, 1H, J=1.8, 5.0 Hz), 4.23 (dd, X of
ABX, 1H, J=5.8, 10.3 Hz), 3.40–3.22 (m, 2H), 2.70–
2.60 (m, 1H), 2.45–2.28 (m, 2H), 2.03–1.78 (unresolved
m, 3H), 0.76 (s, 9H), À0.05 (s, 6H); 13C NMR (75 MHz,
CDCl3) d 176.8, 173.3, 169.0, 137.5, 129.1, 129.0, 128.9,
128.8, 128.7, 128.5, 128.4, 126.9, 81.6, 67.5, 61.7, 57.1,
43.5, 35.5, 29.5, 25.6, 17.5, À4.7, À5.2. IR (thin film)
3418 (m), 3375 (bm), 2952 (m), 2890 (w), 2856 (m), 1740
(s), 1709 (s), 1456 (m), 1387 (m), 1261 (s), 1223 (m),
1110 (m), 877 (m), 836 (s), 781 (m), 737 (s), 699 (s)
cmÀ1. LRFAB MS (3-NBA/gly/TFA matrix): m/e
(relative intensity) identity of ion if known; 523 (2)
MH+; 391 (92) MH+–HOSiR3; 91 (100) C7H+7 .
HRFAB MS (3-NBA/gly/TFA matrix): m/e calcd for
1
solid. H NMR (300 MHz, CDCl3) approx 1:1 mixture
of isomers, d 7.83 (br s, 0.5H), 7.38–7.11 (m, 10H), 6.23
(br s, 0.5H), 5.20 (dd, 1H, J=10.0, 6.8 Hz), 5.16–5.10
(m, 2H), 4.58 (dd, J=9.5, 6.8 Hz), 4.38 (dd, 0.5H,
J=6.1, 2.4 Hz), 4.6 (very br unresolved s, 1H), 3.48–
3.32 (m, 1.5H), 3.10 (A of ABX, 0.5H, JAB=14.4,
JAX=10.0), 2.93–2.02 (unresolved m, 5H), 1.92 (dd,
0.5H, J=13.3, 5.7 Hz), 1.83–1.76 (m, 0.5 H); 13C NMR
(75 MHz, CDCl3) two isomers, d 178.6, 178.4, 175.7,
173.7, 172.7, 169.7, 137.2, 135.3, 135.2, 134.3, 128.9,
128.7, 128.7, 128.6, 128.5, 128.4, 128.3, 128.1, 127.3,
126.8, 81.2, 77.7, 68.0, 67.3, 62.8, 62.2, 56.8, 55.4, 42.5,
42.0, 35.2, 34.8, 33.1, 31.5, 30.1, 29.8. IR (thin film)
3314 (bm), 3055 (m), 2986 (w), 1710 (s), 1440 (m), 1400
(m), 1265 (s), 880 (w), 720 (s), 690 (s) cmÀ1. LRFAB MS
(3-NBA/gly/TFA matrix) m/e (relative intensity) iden-
tity of ion if known; 409 (12) MH+; 391 (100) MH+–
H2O; 363 (10) MH+–H2O–CO; 322 (68); 273 (9) MH+–
CO–HOC7H7; 250 (20); 120 (68). HRFAB MS (3-NBA/
gly/TFA matrix): m/e calcd for C23H25N2O5 (MH+)
409.1763; found 409.1760 (dev À0.8 ppm).
Nꢀ-[(5R,8RS)-8-tert-Butyldimethylsilyloxy-2,6-dioxo-1-
azaspiro[4.4]-octyl]-L-phenylalanine benzyl ester (10). In
a dry 50 mL flask with stir bar under N2 was placed 3.73
g (9.13 mmol) of the N-a-hydroxy amide synthesized
above, 20 mL of CH2Cl2, and 2.13 mL (1.96 g, 18.3
mmol, 2 equiv) of 2,6-lutidine. The mixture was stirred
to dissolve and then cooled to 0 ꢀC. When cool, 4.5 mL
(5.65 g, 21.4 mmol, 2.3 equiv) of TBS-OTf was added
slowly via syringe. The reaction was then allowed to
warm to 25 ꢀC and stirred for 6 h, monitoring by TLC
for completion (5:95 MeOH/EtOAc, product Rf 0.63
and 0.51, two diastereomers, UV-active or pink spot
with ninhydrin stain). When complete, the reaction
C23H23N2O4
391.1668 (dev +2.6 ppm).
(MH+–HOSiR3)
391.1658;
found
Nꢀ-[(5R,8RS)-8-tert-Butyldimethylsilyloxy-2,6-dioxo-1-
azaspiro[4.4]-octyl]-L-phenylalanyl-L-prolinamide. Benzyl
ester compound 11 (1.26 g, 2.41 mmol) was placed in a
dry 100 mL flask with stir bar. Approximately 1 mL of
5% Pd/C and 5 mL of CH2Cl2 were added, and the
flask was filled with N2 and stoppered. A 2.5 L H2 bal-
loon fitted with a 0.5 mm syringe needle was placed on
the reaction, and the N2 was forced out of the flask. The
reaction mixture was then left to stir at 25 ꢀC under H2
for 18 h, after which the balloon was removed and the
flask flushed with N2. The reaction mixture was diluted
with 10 mL of CH2Cl2 and 10 mL of EtOAc and then
filtered through 250 g of well-packed Celite to remove
the Pd/C. The filtrate was dried over MgSO4, filtered,
and concentrated completely in vacuo to give 1.01 g
(97% by NMR) of crude carboxylic acid as a sticky
white solid which was not further purified.16 A 0.48 g
portion (1.11 mmol, 48% of the total) of the crude car-
boxylic acid made above was placed in a dry 25 mL
flask with 0.18 g (1.33 mmol, 1.2 equiv) of HOBt and
2.5 mL of CH2Cl2. The mixture was stirred under N2
and cooled to 0 ꢀC, at which time 0.16 mL (0.112 g, 1.11
mmol, 1 equiv) of Et3N and 0.23 g (1.22 mmol, 1.1
equiv) of EDC were added. After 5 min, 0.15 g (1.33
mmol, 1.2 equiv) of HN-l-Pro-NH2 was added, and the
reaction solution was allowed to slowly warm to 25 ꢀC
over 2.5 h. Then, the reaction solution was diluted with
10 mL of EtOAc and the CH2Cl2 was distilled in vacuo.
The remaining EtOAc mixture was washed with 30 mL
of 5% aq citric acid solution, 30 mL of 5% aq NaHCO3

298
J. C. Simpson et al. / Bioorg. Med. Chem. 10 (2002) 291–302
solution, and 30 mL of saturated aq NaCl solution,
extracting each aq wash twice with 60 mL of additional
EtOAc. The combined EtOAc extracts were dried over
MgSO4, filtered, and concentrated completely in vacuo.
The crude product was then purified by gravity-flow
chromatography through 35 g of slurry packed silica gel
using a 10:90:2 MeOH/EtOAc/Et3N solvent mixture.
Fractions containing product (TLC solvent same as
column, Rf 0.47 and 0.38 of two diastereomers, UV-
active spot or faint pink/brown spot with ninhydrin
stain) were combined, concentrated in vacuo, redis-
solved in 25 mL of CH2Cl2, dried over MgSO4, filtered,
and concentrated completely in vacuo to afford 0.406 g
(0.77 mmol, 67% over two steps) of the pure proline
pletely in vacuo to a brown viscous oil. The crude pro-
duct was purified by gravity-flow chromatography
through 20 g of slurry packed silica gel using a
20:80 MeOH/EtOAc solvent mixture. Fractions con-
taining product (TLC solvent same as column, Rf 0.43,
UV-active spot or pink spot with p-anisaldehyde stain)
were combined, concentrated in vacuo, redissolved in 20
mL of CH2Cl2, dried over MgSO4, filtered, and con-
centrated again in vacuo to afford 0.11 g (58%) of pure
7. The purified product could then be recrsytallized as
the monohydrate from hexane for use in biological
1
testing. H NMR (300 MHz, CDCl3) d 7.34–7.18 (m,
5H), 7.03 (d, 1H, J=4 Hz), 6.96 (d, minor isomer, J=5
Hz), 6.28 (br s, 1H), 5.98 (br s, minor isomer), 5.32 (d,
1H, J=5 Hz), 5.42 (d, minor isomer, J=5 Hz), 5.25 (br
s, 1H), 5.18 (br s, minor isomer), 5.17 (t, 1H, J=8.1
Hz), 4.88 (br s, 1H), 4.51 (m, 1H), 3.64 (dd, A of ABX,
1H, JAB=16 Hz, JAX=8.9 Hz), 3.46–3.38 (m, 1H), 3.25
(dd, A of ABX, 1H, JAB=13.7 Hz, JAX=7.5 Hz), 3.03
(dd, B of ABX, 1H, JAB=13.7 Hz, JBX=8.7 Hz), 2.72–
2.61 (m, 1H), 2.57–1.75 (unresolved m, 7H), 1.55 (br s
due to H2O, 1H); 13C NMR (75 MHz, CDCl3) d 178.0,
173.9, 169.2, 135.2, 132.0, 129.3, 128.9, 128.7, 127.6,
110.8, 65.0, 59.7, 52.9, 47.6, 37.1, 29.6, 29.3, 27.7, 12.2;
minor isomer peaks visible at d 130.2, 60.0, 54.5, 42.0,
35.3. IR (diffusion reflectance) 3310 (bm), 3197 (s),
2962 (m), 2879 (m), 1705 (s), 1656 (s), 1547 (w), 1445
(s), 1385 (s), 1343 (s), 1294 (s), 1268 (s), 1196 (m), 1154
(m), 1120 (m), 1086 (m), 1037 (w), 973 (w), 920 (mw),
871 (mw), 840 (mw), 792 (m), 750 (m), 697 (ms), 648
(m), 501 (m), 471 (m) cmÀ1. LRFAB MS (3-NBA/gly/
TFA matrix): m/e (relative intensity) identity of ion if
known; 397 (100) MH+; 371 (4) MH+–C2H2; 352 (3)
MH+–CONH3; 283 (8) MH+–prolinamide. HRFAB
MS (3-NBA/gly/TFA matrix): m/e calcd for
C21H25N4O4 (MH+) 397.1876; found 397.1876 (dev 0.0
ppm). Elemental analysis calcd for C21H24N4O4&z_-
rad;H2O: C, 60.86; H, 6.08; N, 13.52. Found: C, 60.93,
H, 6.10, N, 13.05.
1
amide derivative as a white solid. H NMR (300 MHz,
CDCl3) multiple indistinguishable isomers d 7.80 (br s,
0.1H), 7.34–7.22 (m, 5H), 6.90 (d, 0.1H, J=7.8 Hz),
6.72 (br s, 0.2H), 6.51 (br s, 0.2H), 6.25–6.10 (2 unre-
solved br s, 0.9H), 5.96 (br s, 0.1H), 5.92 (br s, 0.1H),
5.75 (d, 0.8H, J=4.4 Hz), 5.60 (br s, 0.8H), 5.22–5.20
(m 0.2H), 5.12 (m, 0.1H), 5.05 (dd, X of ABX, 0.8H,
J=5.4 Hz, 16.1 Hz), 4.62 (m, 0.2H), 4.52 (d, 0.2H,
J=4.4 Hz), 4.44 (t, 0.9H, J=5.6 Hz), 4.05 (d, 0.3H,
J=7.8 Hz), 3.80–3.70 (m, 0.3H), 3.61–3.43 (m, 1.6H),
3.40–3.36 (unresolved m 0.5H), 3.30 (dd, A of ABX,
1H, J=11.0, 12.9 Hz), 3.21–3.00 (m, 1H), 2.97 (0.7H,
dd, J=4.9, 6.6 Hz), 2.78–1.65 (unresolved m, 13H),
1.62–1.48 (m, 0.2H), 0.97–0.84 (unresolved m, 9H),
0.28–0.09 (unresolved m, 6H); 13C NMR (75 MHz,
CDCl3) d 177.5, 177.0, 174.4, 173.3, 173.0, 169.8, 151.9,
136.3, 136.0, 129.5, 129.3, 128.9, 127.4, 80.6, 78.2, 61.7,
60.4, 59.8, 54.0, 47.5, 43.6, 43.4, 37.3, 33.8, 29.6, 29.1,
27.8, 25.7, 24.5, 20.0, 17.6, À4.2, À4.7. IR (thin film)
3466 (m), 3418 (m), 3329 (bm), 3205 (bm), 3055 (m),
2959 (m), 2931 (m), 2884 (mw), 2863 (m), 1705 (s), 1647
(s), 1647 (s), 1422 (m), 1363 (w), 1343 (w), 1267 (s), 1188
(w), 1113 (m), 1079 (w), 1035 (w), 898 (w), 870 (w), 836
(m), 781 (mw), 733 (s), 703 (s) cmÀ1. LRFAB MS (3-
NBA/gly/TFA matrix): m/e (relative intensity) identity
of ion if known; 471 (14) MH+–tBuH; 415 (13) MH+–
SiR+3 H+; 398 (25) MH+–SiR3–NH2; 397 (100) MH+–
Nꢀ-[(5R)-2,6-Dioxo-1-azaspiro[4.4]-octyl]-L-phenylala-
nyl-L-prolinamide (6). To a 25 mL flask with stir bar
was added 90 mg (0.23 mmol) of 7 and excess 5% Pd/C
(approximately 1 mL). The flask was filled with N2 and
2 mL each of MeOH and EtOAc were added, then a 2.5
L H2 balloon fitted with a 0.2 mm syringe needle was
placed on the reaction flask. The N2 was forced out of
the flask, and the reaction mixture was then left to stir
under H2 at 25 ꢀC for 2 h after which the balloon was
removed and the flask flushed with N2. The reaction
mixture was filtered through 125 g of well-packed Celite
to remove the Pd/C using MeOH to rinse well, then the
filtrate was dried over MgSO4, filtered, and con-
centrated completely in vacuo. The crude product was
purified by gravity-flow chromatography through 3 g of
slurry-packed silica gel using a 20:80 MeOH/EtOAc
solvent mixture. Fractions containing product (TLC
solvent same as column, Rf 0.14, UV-active spot or pink
spot with p-anisaldehyde stain) were combined, con-
centrated in vacuo, redissolved in CH2Cl2, dried over
MgSO4, filtered, and concentrated in vacuo to give 82
mg (91%) of a white solid whose 1H NMR was identical
to the previously synthesized 6.6b
HOSiR3; 387 (9) M415–C2H4 (or CO); 370 (13) M398
–
CO; 369 (47) M397–C2H4 (or CO); 352 (12) M369–NH3
or M397 H+–CONH2; 324 (3) M352–C4H7N; 255 (27)
M238–CO; 240 (17); 227 (26); 130 (20); 115 (8); Sir3+; 91
(30) C7H+7 . HRFAB MS (3-NBA/gly/TFA matrix): m/e
calcd for C21H25N4O4 (MH+–HOSiR3) 397.1876;
found 397.1864 (dev À2.9 ppm).
Nꢀ-[(5R)-2,6-Dioxo-1-azaspiro[4.4]-oct-8-enyl]-L-phenyl-
alanyl-L-prolinamide (7). The N-a-siloxyamide synthe-
sized above (0.250 g, 0.47 mmol) was placed in a 25 mL
flask with about 2 mL of CH2Cl2 and 3 mL of silica gel
(200 mesh). The CH2Cl2 was then heat distilled to leave
the starting material adsorbed directly onto the silica
gel. The flask was fitted with a reflux condenser and
attached to a variable vacuum pump. The reaction
mixture was then heated slowly over 1 h to 200 ꢀC under
a 30–40 mm Hg argon atmosphere, then left at 200 ꢀC
for 15 min. The reaction mixture was allowed to cool to
25 ꢀC at 1 atm (Ar) and then filtered to remove the silica
gel, rinsing well with EtOAc and MeOH. The filtrate
was dried over MgSO4, filtered, and concentrated com-

J. C. Simpson et al. / Bioorg. Med. Chem. 10 (2002) 291–302
299
(2R)-1-(4-Methoxybenzyl)-2-prop-2-enyl-5-oxoproline
(1S,2R,5S)-5-methyl-2-(1-methylethyl)cyclohexyl ester.
Preparation of pMeOBnBr. To a 10 mL flask contain-
ing 1.75 mL (1.93 g, 14 mmol) of pMeOBnOH was
added 4 mL of concentrated HBr while vigorously stir-
ring. After 15 min, 80 mL of Et2O was added, and the
entire mixture was poured into a 125 mL separatory
funnel. The product pMeOBnBr was extracted into the
Et2O by shaking, and then the layers were separated.
The Et2O phase was washed with 50 mL of saturated aq
NaHCO3 solution and 50 mL of saturated aq NaCl
solution, then dried over CaCl2, filtered, and con-
centrated in vacuo to a pale yellow oil which was kept
under an argon atmosphere until used in the following
protection step. A flame-dried 250 mL flask with stir bar
was cooled under argon, then 0.41 g of a 60% NaH
dispersion of NaH in mineral oil (0.25 g NaH, 10.2
mmol, 1.1 equiv) was weighed into the flask under an
argon atmosphere. The dispersion was washed several
times with dry cyclohexane, decanting the oil/solvent
mixture each time. To the flask was then added 35 mL
of anhyd DMF and the mixture was cooled to 0 ꢀC.
Next, 2.85 g (9.3 mmol) of compound 5 was added as a
DMF solution (15 mL) by transferring via cannula. The
reaction mixture was stirred for 15 min, and then the
pMeOBnBr prepared above was added by transferring
via cannula as a solution in 20 mL of anhydrous DMF.
The mixture was stirred at 0 ꢀC for 1.5 h and then
allowed to warm to 25 ꢀC and stirred for 14 h. The
reaction was neutralized by the addition of 10 mL of
glacial HOAc, then poured into a 250 mL separatory
funnel containing 80 mL of ice water. The water was
extracted three times with 100 mL portions of Et2O, and
the combined organic extracts were dried over MgSO4,
filtered, and concentrated in vacuo to a brown oil. The
crude product was then partially purified by gravity-
flow chromatography through 200 g of slurry-packed
silica gel using a 3:1 to 1:1 hexane/Et2O gradient solvent
mixture. Fractions containing product (TLC solvent 3:1
hexane/Et2O, Rf 0.3, UV-active spot or blue spot with p-
anisaldehyde stain) were combined, dried over MgSO4,
filtered, and concentrated in vacuo to afford an 80:20
mixture (mol ratio) of the protected product (2.81 g, 6.5
mmol, 71%) and PMB-Br (0.33g, 1.6 mmol). In addi-
tion, 0.58 g (1.9 mmol, 20%) of the unprotected starting
material 5 was isolated from the reaction product mix-
20.8, 15.8, 15.7. IR (thin film) 3438 (w), 3075 (w), 2959
(s), 2918 (s), 2875 (s), 1730 (s), 1695 (s), 1606 (ms), 1586
(w), 1514 (s), 1432 (m), 1391 (s), 1446 (m), 1245 (s), 1172
(s), 1035 (s), 966 (m), 898 (m), 812 (m), 737 (s) cmÀ1
.
LRFAB MS (3-NBA matrix): m/e (relative intensity)
identity of ion if known; 428 (30) MH+; 386 (1) MH+–
propene; 308 (<1) MH+–PMB++H+; 244 (23) M–
CO2menthyl; 216 (1) M244–CO (or C2H4); 182 (12); 124
(6) M308–CO2menthyl–H+; 122 (18); 121 (100) PMB+;
91 (5) C7H+7 . HRFAB MS (3-NBA matrix): m/e calcd
for C26H38NO4 (MH+) 428.2801; found 428.2780 (dev
À4.8 ppm).
(2R)-1-(4-Methoxybenzyl)-2-prop-2-enyl-5-oxoproline.
In a 25 mL flask with stir bar was dissolved 1.47 g (3.44
mmol) of the PMB protected, allylated pyroglutamate
menthyl ester synthesized above in 4.5 mL of MeOH,
2.5 mL of THF, and 2.5 mL of H2O. The reaction
solution was cooled to 0 ꢀC while stirring under N2, and
0.36 g (8.6 mmol, 2.5 equiv) of LiOHꢁ H2O was added.
The reaction solution was allowed to warm to 25 ꢀC and
then was stirred for 48 h, at which time it was diluted
with 10 mL of H2O and 40 mL of Et2O. The menthol
and unreacted starting material were extracted into the
Et2O phase by shaking, the layers were separated, and
then the aq phase was acidified to a pH of 2 by the
addition of 0.5 mL of 2 N aq HCl. The product was
then extracted with Et2O (two 50 mL portions). The
combined Et2O extracts were dried over MgSO4, fil-
tered, and concentrated in vacuo to afford 0.89 g (3.1
mmol, 90%) of the crude acid. The product acid was
not further purified. 1H NMR (300 MHz, CDCl3) d
10.60 (br s, 1H), 7.27–7.22 (m, 2H), 6.82–6.78 (m, 2H),
5.46–5.35 (m, 1H), 5.11–5.05 (m, 2H), 4.60 (d, 1H,
J=15.2 Hz), 4.33 (d, 1H, J=15.2 Hz), 3.76 (s, 3H),
2.69–2.55 (m, 2H), 2.50–2.40 (m, 2H), 2.28–2.19 (m,
1H), 2.09–1.98 (m, 1H); 13C NMR (75 MHz, CDCl3) d
177.4, 176.1, 158.9, 131.2, 129.9, 129.1, 120.4, 113.7,
68.9, 55.2, 44.5, 39.0, 29.4, 27.8. IR (thin film) 2800 (v
bm), 3053 (ms), 2962 (ms), 2841 (ms), 2773 (bm), 2545
(bm), 2311 (w), 1724 (s), 1690 (s), 1641 (s), 1513 (s),
1449 (m), 1415 (ms), 1362 (mw), 1335 (mw), 1268 (s),
1241 (s), 1173 (s), 1116 (mw), 1033 (m), 928 (m), 890
(mw), 814 (mw), 739 (s), 709 (s) cmÀ1. LRFAB MS (3-
NBA/gly/TFA matrix) m/e (relative intensity) identity
of ion if known; 290 (39) MH+; 244 (7) MH+–H2O–
CO; 182 (6) M–H3COPh; 121 (100) H3COPhCH+2 ; 107
(12) H3COPh+. HRFAB MS (3-NBA/gly/TFA matrix):
m/e calcd for C16H20NO4 (MH+) 290.1392; found
290.1392 (dev À0.1 ppm).
1
ture. H NMR of the mixture (300 MHz, CDCl3) two
rotamers visible plus PMB-Br, d 7.27–7.21 (m, 2H),
6.83–6.79 (m, 2H), 5.42–5.29 (m, 1H), 5.08–4.98 (m,
2H), 4.80 (d, A of AB, 0.7H, J=15.1 Hz), 4.67 (td, 1H,
Jd=4.4 Hz, Jt=11 Hz), ꢂ4.67 (buried d, A of AB
0.3H), 4.13 (d, B of AB, 0.3H, J=15.1 Hz), 4.02 (d, B of
AB, 0.7H, J=15.1 Hz), 3.77 (s, 3H), 2.66–2.34 (m, 4H),
2.25–2.15 (m, 1H), 2.07–1.95 (m, 1H), 1.88–1.70 (m,
2H), 1.69–1.65 (m, 2H), 1.49–1.26 (m, 3H), 1.12–0.75
(unresolved m, 2H), 0.91–0.82 (3 unresolved d, 6H),
0.79–0.72 (3 unresolved d, 3H); PMB-Br peaks visible at
d 7.30–7.27 (m, 2H), 6.89–6.84 (m, 2H), 4.61 (s, 2H),
2.79 (s, 3H); 13C NMR (75 MHz, CDCl3) two rotamers
visible, d 176.2, 172.2, 158.7, 158.7, 131.3, 129.9, 129.7,
129.4, 128.4, 120.2, 120.0, 113.7, 113.6, 75.9, 75.7, 69.3,
68.9, 64.7, 55.1, 55.0, 46.7, 44.3, 40.3, 40.0, 39.7, 39.3,
33.9, 31.2, 29.5, 28.0, 27.8, 26.1, 26.0, 22.8, 21.8, 21.7,
(2R)-1-(4-Methoxybenzyl)-2-prop-2-enyl-5-oxoprolyl-L-
phenylalanine benzyl ester. The carboxylic acid made in
the previous experiment (1.41 g, 4.9 mmol) and 0.79 g
(5.8 mmol, 1.2 equiv) of HOBt were placed in a dry 50
mL flask with stir bar and 10 mL of CH2Cl2. While
stirring the mixture under N2, 1.4 mL (1.04 g, 10.3
mmol, 2.1 equiv) of Et3N was added and all was dis-
solved. The reaction solution was then cooled to 0 ꢀC,
and then 1.57 g (5.4 mmol, 1.1 equiv) of H2N-l-Phe-
Obnꢁ HCl and 1.03 g (5.4 mmol, 1.1 equiv) of EDC were
added. The reaction mixture was allowed to warm to
25 ꢀC and was stirred vigorously under N2 for 10 h. The

300
J. C. Simpson et al. / Bioorg. Med. Chem. 10 (2002) 291–302
CH2Cl2 was then removed in vacuo and the crude resi-
due was dissolved in 20 mL of EtOAc and 20 mL of
Et2O. The organic solution was washed with 25 mL of
5% aq citric acid solution followed by 25 mL of 5% aq
NaHCO3 solution. Both aq extracts were re-extracted
twice with 40 mL portions of Et2O before proceeding
with the next wash. The combined organic extracts were
dried over MgSO4, filtered, and concentrated in vacuo
to a thick brown oil. The crude product was then pur-
ified by gravity-flow chromatography through 125 g of
slurry packed silica gel using a 1:1 hexane/Et2O to
100% Et2O gradient solvent mixture. Fractions con-
taining product (TLC solvent Et2O, Rf 0.16, UV-active
spot or pale-yellow spot with p-anisaldehyde stain) were
combined, dried over MgSO4, filtered, and concentrated
in vacuo to afford 2.50 g (4.75 mmol, 96%) of the pure
slowly warmed to 25 ꢀC while stirring, and then the
layers were separated. The aq phase was extracted twice
with 50 mL portions of Et2O. The Et2O extracts were
combined, dried over MgSO4, filtered, and concentrated
in vacuo. The crude product was then purified by flash
chromatography through 125 g of slurry-packed silica
gel using a 1:1 Et2O/EtOAc solvent mixture. Fractions
containing product (TLC solvent same as column, Rf
0.20, UV-active spot or blue spot with p-anisaldehyde
stain) were combined, dried over MgSO4, filtered, and
concentrated in vacuo to afford 2.26 g (4.15 mmol,
1
89%) of pure 11 as a white solid. H NMR (300 MHz,
CDCl3) d 7.39–7.14 (m, 10H), 7.03–6.98 (m, 2H), 6.79–
6.74 (m, 2H), 6.19 and 6.17 (2 br s, 1H), 5.19 (d, A of
AB, 1H, J=12.1 Hz), 5.08 (d, B of AB, 1H, J=12.1
Hz), 4.76–4.68 (m, 1H), 4.53 (d, A of AB, 1H, J=15.1
Hz), 3.93 (d, B of AB, 1H, J=15.1 Hz), 3.74 (s, 3H),
3.32–3.27 (m, 2H), 3.14 (dd, A of ABX, 1H, JAB=14.0
Hz, JAX=5.9 Hz), 2.84 (dd, B of ABX, 1H, JAB=14.0
Hz, JAX=8.1 Hz), 2.36–2.22 (m, 2H), 2.04–1.87 (unre-
solved m, 3H), 1.67–1.57 (m, 1H), 1.43–1.35 (m, 1H),
1.28–1.23 (m, 1H), 1.10–1.01 (m, 1H); 13C NMR
(75 MHz, CDCl3) d 176.2, 173.0, 170.8, 158.8, 135.6,
134.8, 130.0, 129.6, 128.9, 128.6, 128.55, 128.5, 127.2,
113.6, 70.4, 67.4, 62.1, 55.3, 53.2, 44.3, 37.3, 30.5, 29.4,
29.0, 26.3. IR (diffusion reflectance) 3462 (s), 3280 (s),
3200 (bm), 3076 (m), 2992 (m), 2939 (m), 1732 (s), 1668
(s), 1555 (s), 1453 (m), 1407 (m), 1249 (m), 1116 (m),
1071 (m), 1037 (m), 965 (m), 845 (m), 814 (m), 746 (s),
705 (s), 584 (m), 524 (m) cmÀ1. LRFAB MS (3-NBA/
gly/TFA matrix) m/e (relative intensity) identity of ion if
known; 545 (100) MH+; 527 (5) MH+–H2O; 437 (14)
MH+–C7H7OH or MH+–CH3OPh+; 407 (18).
HRFAB MS (3-NBA/gly/TFA matrix): m/e calcd for
C32H37N2O6 (MH+) 545.2651; found 545.2640 (dev
À1.0 ppm).
Nꢀ-[(5R)-2,6-Dioxo-1-(4-methoxybenzyl)-1-azaspiro[5.4]
- nonyl] - L - phenylalanine benzyl ester. In a dry 10 mL
flask with stir bar were placed 0.37 g (0.8 mmol) of 11
and 0.36 g (1.4 mmol, 2 equiv) of Ph3P. The mixture
was dissolved in 3.4 mL of THF, and then 0.21 mL
(0.24 g, 1.4 mmol, 2 equiv) of DEAD was added slowly
via syringe while vigorously stirring under an atmo-
sphere of argon. The reaction solution was then allowed
to stir at 25 ꢀC for 12 h, after which the solution was
diluted with 5 mL of EtOAc and washed with 10 mL of
saturated aq NaHCO3 solution. The organic phase was
then dried over MgSO4, filtered, and concentrated in
vacuo. The crude product was partially purified by flash
chromatography through 40 g of slurry packed silica gel
using a 1:1 Et2O/hexane to 100% Et2O to 1:1 Et2O/
EtOAc gradient solvent mixture. Fractions containing
product (TLC solvent Et2O, Rf 0.17, UV-active spot or
blue spot with p-anisaldehyde stain) along with Ph3PO
byproduct were combined, dried over MgSO4, filtered,
and concentrated in vacuo to afford approximately 0.35
g (0.66 mmol, 98%) of the cyclized product in an 85:15
(by NMR) mixture with Ph3PO. The product was not
further purified. 1H NMR of mixture (300 MHz,
CDCl3) d 7.71–6.71 (m, 14H+Ph3PO contaminant),
5.31–5.13 (unresolved m, 3H), 4.93 (d, 0.5H, J=15.6
Hz), 4.76 (d, 0.5H, J=15.9 Hz), 3.76 and 3.74 (2 s, 3H),
1
coupled product as a white solid. H NMR (300 MHz,
CDCl3) two rotamers visible, d 7.39–7.15 (m, 11H),
7.03–6.96 (m 2H), 6.80–6.74 (m, 2H), 6.22 and 6.20 (2
unresolved br s, 1H), 5.33–4.94 (unresolved m, 4H),
4.72–4.65 (m, 1H), 4.49 (d, 0.7H, J=15.0 Hz), 4.48
(unresolved m, 0.3H), 4.07 (d, 0.7H, J=15.0 Hz), 3.82–
3.70 (unresolved m, 0.3H), 3.74 (s, 3H), 3.15 (dd, A of
ABX, 0.7H, JAB=14.1 Hz, JAX=5.6 Hz), 3.05–2.94 (m,
0.3H), 2.91–2.79 (m, 1H), 2.59 (dd, A of ABX, 1H,
JAB=14.9 Hz, JAX=8.5 Hz), 2.44 (dd, B of ABX, 1H,
JAB=14.9 Hz, JBX=5.4 Hz), 2.35–2.10 (unresolved m,
2.7H), 2.03–1.93 (unresolved m, 1.3H), 1.84–1.73 (m,
1H); 13C NMR (75 MHz, CDCl3) two rotamers visible,
d 176.8, 173.6, 170.8, 159.8, 136.0, 135.6, 131.9, 130.2,
130.1, 130.0, 129.9, 128.8, 128.7, 128.6, 128.5, 128.4,
126.7, 120.4, 114.0, 169.9, 169.8, 167.7, 167.6, 54.4, 53.8,
53.7, 44.4, 39.0, 38.9, 37.2, 29.8, 29.3, 29.0. IR (thin
film) 3322 (m), 3205 (w), 3055 (m), 3027 (m), 2925 (m),
1740 (s), 1671 (bs), 1613 (m), 1582 (m), 1514 (ms), 1456
(ms), 1404 (m), 1353 (mw), 1264 (m), 1247 (m), 1113
(mw), 1083 (w), 1035 (m), 1004 (mw), 925 (w), 819 (w),
733 (s), 703 (s) cmÀ1. LRFAB MS (3-NBA/gly/TFA
matrix) m/e (relative intensity) identity of ion if known;
527 (17) MH+; 244 (52) MH+–H2N-Phe-OBn–CO; 211
(11);
124
(13)
MH+–H2N-Phe-OBn–CO–
H3COPhCH+2 +H+; 121 (100) H3COPhCH+2 ; 107 (7)
H3COPh+; 91 (90) C7H+7 . HRFAB MS (3-NBA/gly/
TFA matrix): m/e calcd for C32H35N2O5 (MH+)
527.2546; found 527.2553 (dev +1.3 ppm).
(2R)-1-(4-Methoxybenzyl)-2-(3-hydroxypropyl)-5-oxo-
prolyl-L-phenylalanine benzyl ester (11). A 50 mL flask
with stir bar was flame dried and cooled under argon,
and then 4.5 mL of THF and 0.93 mL of a 10 M solu-
ꢁ
tion of BH3 Me2S in Me2S (9.3 mmol, 2 equiv) were
added to the flask. The solution was cool to 0 ꢀC under
argon and then 2.0 mL (1.3 g, 18.6 mmol, 4 equiv) of 2-
methyl-2-butene was added slowly via syringe. The
solution was stirred while slowly warming to 10 ꢀC over
45 min and then re-cooled to 0 ꢀC at which time 2.45 g
(4.65 mmol) of the olefinic product from the previous
step was added by quantitative transfer via cannula as a
solution in 19 mL of THF. The reaction solution was
then allowed to slowly warm to 25 ꢀC over 1.5 h and
then re-cooled to 0 ꢀC and diluted with 25 mL of Et2O.
Next, 23 mL of a 2:1 mixture of 3 N aq NaOH and 30%
aq H2O2 was added very slowly. The solution was

J. C. Simpson et al. / Bioorg. Med. Chem. 10 (2002) 291–302
301
3.74–3.42 (unresolved m, 1.5H), 3.16–3.07 (m, 2H), 2.97
(dm, 1H, Jd=4.4 Hz), 2.85 (d, 0.5H, J=15.9 Hz), 2.68–
2.55 (m, 0.5H), 2.68–2.55 (m, 0.5H), 2.43–2.18 (m, 2H),
1.80–1.33 (unresolved m, 5.5H). LRFAB MS of mixture
(3-NBA/gly/TFA matrix) m/e (relative intensity) iden-
tity of ion if known; 527 (14) MH+; 419 (8) MH+–
C7H7OH; 391 (12) MH+–C7H7OH–CO; 279 (70)
Ph3POH+; 121 (100) H3COPhCH+2 ; 91 (60) C7H+7 .
HRFAB MS (3-NBA/gly/TFA matrix): m/e calcd for
C32H35N2O5 (MH+) 527.2546; found 527.2789 (dev
À1.6 ppm).
balloon was removed and the flask flushed with argon
once more. The reaction mixture was then filtered
through 200 g of well-packed Celite to remove the cat-
alyst, and the filtrate was concentrated in vacuo to
afford 0.36 g (1.1 mmol, 98%) of the deprotected acid as
a white solid that was >98% pure by NMR. The pro-
duct was not purified further. 1H NMR (300 MHz,
CDCl3) d 7.99 (br s, 1H), 7.67 (br s, 1H), 7.33–7.17 (m,
5H), 3.93 (dd, 1H, J=6.1, 9.8 Hz), 3.45–3.30 (unre-
solved m, 3H), 2.72–2.60 (m, 2H), 2.44–2.32 (m, 2H),
2.04–1.96 (m, 1H), 1.88–1.46 (unresolved m, 4H). 13C
NMR (75 MHz, CDCl3) d 180.4, 172.5, 172.0, 138.1,
129.0, 128.6, 128.4, 126.6, 64.2, 62.2, 50.9, 34.2, 34.1,
32.9, 30.0, 19.5. IR (diffusion reflectance) 3250 (bs),
3000 (very bs), 2939 (s), 2864 (s), 2576 (bm), 1705 (s),
1649 (s), 1490 (m), 1449 (ms), 1354 (ms), 1328 (ms),
1283 (m), 1207 (s), 1139 (m), 1086 (mw), 1033 (w), 939
(w), 894 (w), 822 (w), 750 (m), 701 (m), 671 (mw), 644
(mw), 569 (w), 508 (w) cmÀ1. LRFAB MS (3-NBA/Li+
matrix) m/e (relative intensity) identity of ion if known;
323 (20) MLi+; 249 (5) MLi+–H2O–2CO; 202 (9); 160
(100); 136 (30); 91 (25) C7H+7 . LREI MS: m/e (relative
intensity) identity of ion if known; 316 (12) M+; 298
(34) M+–CHBn–CO2; 124 (93); 110 (100); 91 (74)
C7H+7 . HRFAB MS (3-NBA/Li+ matrix): m/e calcd for
C17H20N2O4Li (MLi+) 323.1583; found 323.1582 (dev-
0.3 ppm).
Nꢀ-[(5R)-2,6-Dioxo-1-azaspiro[5.4]-nonyl]-L-phenylala-
nine benzyl ester (12). The spirocyclic building block
made in the previous experiment (ca. 0.40 g, 0.77 mmol)
contaminated with approx 0.07 g (0.26 mmol) of Ph3PO
was dissolved in 3 mL of CH3CN and 3 mL of H2O in a
25 mL flask with stir bar. When all was dissolved, 1.05 g
(1.9 mmol, 2.5 equiv) of CAN was added, and the reac-
tion solution was left to stir at 25 ꢀC for 2 h. The solu-
tion was diluted with 5 mL of H2O, poured into a 125
mL separatory funnel, and extracted twice with 30 mL
portions of EtOAc. The EtOAc extract was then washed
with 15 mL of 5% aq NaHCO3 solution, and then was
dried over MgSO4, filtered, and concentrated in vacuo.
The crude product was purified by flash chromato-
graphy through 20 g of slurry-packed silica gel using
EtOAC as eluent. Fractions containing product (TLC
solvent same as column, Rf 0.14, UV-active spot or blue
spot with p-anisaldehyde stain) were combined, con-
centrated in vacuo, dried over MgSO4, filtered, and
concentrated again in vacuo to afford 0.20 g (0.49
mmol, 53% over two steps) of pure 12 as a white solid.
1H NMR (300 MHz, CDCl3) d 7.40–7.15 (m, 10H), 5.35
(d, A of AB, 1H, J=12.1 Hz), 5.10 (d, B of AB, 1H,
J=12.1 Hz), 4.86 (dd, X of ABX, 1H, J=5.8, 10.7 Hz),
3.39 (dd, A of ABX, 1H, JAB=14.3 Hz, JAX=5.8 Hz),
3.28–3.14 (unresolved m, 2H), 2.93–2.85 (m, 1H), 2.65–
2.56 (m, 1H), 2.39–2.22 (m, 2H), 1.82–1.58 (unresolved
m, 5H), 1.68 (s due to H2O); 13C NMR (75 MHz,
CDCl3) d 178.1, 171.7, 169.8, 136.8, 135.3, 128.8, 128.6,
128.4, 128.3, 128.1, 126.7, 66.9, 61.3, 59.9, 47.3, 34.7,
34.1, 33.4, 30.0, 19.5. IR (thin film) 3233 (bm), 3062 (m),
3072 (m), 2945 (s), 2877 (m), 1740 (s), 1702 (s), 1647 (s),
1493 (ms), 1452 (s), 1346 (m), 1326 (s), 1267 (m), 1209
(ms), 1083 (w), 1028 (mw), 980 (w) cmÀ1. LRFAB MS
(3-NBA/gly/TFA matrix) m/e (relative intensity) iden-
tity of ion if known; 407 (63) MH+; 299 (2) MH+–
C7H7OH; 271 (2) MH+–C7H7OH–CO; 243 (8) MH+–
C7H7OH–CO–CO (or C2H4); 154 (32); 136 (26); 124
(16); 107 (11); 91 (100) C7H+7 HRFAB MS (3-NBA/gly/
TFA matrix): m/e calcd for C24H27N2O4 (MH+)
407.1971; found 407.1963 (dev À1.9 ppm).
Nꢀ-[(5R)-2,6-Dioxo-1-azaspiro[5.4]-nonyl]-L-phenylala-
nyl-L-prolinamide (8). The carboxylic acid made in the
previous step (0.23 g, 0.71 mmol) and 0.12 g (0.85
mmol, 1.2 equiv) of HOBt were placed in a dry 50 mL
flask with stir bar and 1.5 mL of CH2Cl2, and the mix-
ture was cooled to 0 ꢀC. While stirring the mixture
under N2, 0.14 mL (0.12 g, 1.1 mmol, 1.5 equiv) of
NEM and 0.09 g (0.8 mmol, 1.1 equiv) of HN-l-Pro-
NH2 were added. When all was dissolved, 0.16 g (0.85
mmol, 1.2 equiv) of EDC was added. The reaction
mixture was allowed to slowly warm to 25 ꢀC and stir-
red 14 h under N2. The reaction solution was then dilu-
ted with 5 mL of EtOAc washed with 3 mL of 5% aq
citric acid solution followed by 3 mL of 5% aq
NaHCO3 solution. Both aq extracts were re-extracted
twice with 5 mL portions of EtOAc before proceeding
with the next wash. The combined EtOAc extracts were
dried over MgSO4, filtered, and concentrated in vacuo.
The crude product was then purified by flash chroma-
tography through 15 g of slurry packed silica gel using a
20:80 MeOH/EtOAc solvent mixture. Fractions con-
taining product (TLC solvent same as column, Rf 0.40,
faint UV-active spot or pale brown spot with p-ani-
saldehyde stain) were combined, concentrated in vacuo,
re-dissolved in 10 mL of CH2Cl2, dried over MgSO4,
filtered, and concentrated in vacuo to afford 0.21 g (0.51
mmol, 70%) of pure product 8 as a white solid that
could be recrystallized as the monohydrate from hexane
Nꢀ-[(5R)-2,6-Dioxo-1-azaspiro[5.4]nonyl]-L-phenylala-
nine. Compound 12 (0.48 g, 1.2 mmol) and approx 1 g
of Pd on activated carbon (Pd 5% by weight) were
placed in a dry 25 mL flask with stir bar. The flask was
flushed with argon, and then the contents were dissolved
in 2.5 mL of CH2Cl2. A 2.0 L H2 balloon was then
placed on the reaction flask and the argon was forced
out, filling the flask with H2. The reaction was then left
to stir at 25 ꢀC and 1 atm for 20 h, after which the H2
1
for biological testing. H NMR (300 MHz, CDCl3) two
rotamers, d 7.36–7.22 (m, 5H), 7.09 (br s, 0.25H), 6.71
(br s, 0.75H), 6.68 (br s, 0.25H), 6.55 (br s, 0.75H), 6.12
(br s, 0.25H), 5.47 (br s, 0.75H), 5.20–5.15 (m, 0.75H),
4.60–4.50 (m, 0.75H), 4.50–4.45 (m, 0.25H), 4.20–4.16
(m, 0.25H), 3.82–3.79 (m, 0.25H), 3.67–3.56 (m, 1H),
3.43–3.22 (unresolved m, 3.5H), 3.09 (dd, A of ABX,

302
J. C. Simpson et al. / Bioorg. Med. Chem. 10 (2002) 291–302
0.75H, JAB=13.7 Hz, JAX=8.5 Hz), 2.87–2.82 (m,
0.25H), 2.67–2.28 (unresolved m, 3H), 2.15–2.07 (unre-
solved m, 1.5H), 1.98–1.56 (unresolved m, 7.5H), 1.30–
1.20 (m, 0.25H); 13C NMR (75 MHz, CDCl3) two rota-
mers, d 178.7, 178.5, 174.0, 173.8, 172.4, 169.5, 169.2,
137.6, 129.4, 129.2, 128.6, 128.5, 127.2, 62.1, 61.7, 61.6,
59.8, 47.4, 45.5, 34.9, 34.8, 33.5, 32.2, 30.0, 28.0, 25.0,
21.9, 19.6, 19.2. IR (diffusion reflectance) 3196 (m),
3025 (w), 2960 (m), 2871 (m), 1687 (s), 1640 (s), 1625 (s),
1439 (m), 1350 (m), 1289 (m), 1187 (m), 915 (w), 704
(m), 500 (m) cmÀ1. LRFAB MS (3-NBA/Li+ matrix)
m/e (relative intensity) identity of ion if known; 419 (32)
MLi+; 313 (52); 160 (100) MLi+–C14H17N2O2–N; 127
(47); 119 (38). HRFAB MS (matrix): m/e calcd for
C22H28N4O4Li (MLi+) 419.2270; found 419.2253 (dev 5
ppm). Elemental analysis calcd for C22H28N4O4&z_-
rad;H2O: C, 61.38; H, 7.02; N, 13.01. Found: C, 61.05,
H, 7.08, N, 13.33.
4. (a) For alternate approaches to understanding TRH/TRH-
R1 binding, see Hinkle, P. M.; Woroch, E. L.; Tashjian, G. H.
J. Biol. Chem. 1974, 249, 3085. (b) Goren, H. J.; Bauce, L. G.;
Vale, W. Mol. Pharmacol. 1977, 13, 606. (c) Kamiya, K.;
Takamoto, M.; Wada, Y.; Fujino, M.; Nishikawa, M. J.
Chem. Soc., Chem. Commun. 1980, 438. (d) Sharif, N. A.; To,
Z.; Whiting, R. L. Biochem. Biophys. Res. Commun. 1989, 161,
1306. (e) Vonhof, S.; Feuerstein, G. Z.; Cohen, L. A.; Labroo,
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Acknowledgements
We thank the National Institutes of Health (RO1
GM5324001 and R01 DK43036) for their generous
financial support. In addition, we thank the Washington
University High Resolution NMR Facility, partially
supported by NIH grants RR02004, RR05018, and
RR07155, and the Washington University Mass Spec-
trometry Resource Center, partially supported by NIH
RR00954, for their assistance.
8. Chu, W.; Perlman, J. H.; Gershengorn, M. C.; Moeller,
K. D. Bioorg. Med. Chem. Lett. 1998, 8, 3093.
9. More recent studies using HEK 293EM cells stably expres-
sing mTRH-R1 have shown that the fully constrained analo-
gue (n=m=1) is a full agonist of the receptor. Gershengorn,
M. C.; Chu, W.; Moeller, K. D. Unpublished results.
10. (a) For reviews of anodic amide oxidations, see: Shono,
T.; Matsumura, Y.; Tsubata, K. In Organic Synthesis; Saucy,
G., Ed.; Organic Synthesis, 1984, Vol. 63, p 206, and refer-
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For applications to functionalizing amino acids, see: Moeller,
K. D. Top. Curr. Chem. 1997, 185, 49 and in particular refer-
ence 13 therein.
11. Shono, T.; Matsumura, Y.; Tsubata, K.; Sugihara, Y.;
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104, 6697.
12. (a) For published procedures, see: Perlman, J. H.; Thaw,
C. N.; Laakkonen, L.; Bowers, C. Y.; Osman, R.; Ger-
shengorn, M. C. J. Biol. Chem. 1994, 269, 1610. (b) Straub,
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References and Notes
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(b) For more recent references see: Polyak, F.; Lubell, W. D.
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references therein.
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Releasing Hormone: Biomedical Significance; In Metcalf
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Clarke, F. H., Ed.; Academic: New York, 1978; Vol. 13, p 227,
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15. For a description of general experimental details, see:
Wong, P. L.; Moeller, K. D. J. Am. Chem. Soc. 1993, 115,
11434.
16. If the acid is not used soon after synthesis, over time it will
lose the silyloxy protecting group and give rise to intramole-
cular lactone formation.