Dipeptides as effective prodrugs of the unnatural amino acid (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740), a selective group II metabotropic glutamate receptor agonist

J. Med. Chem. 2005, 48, 5305-5320

5305

Dipeptides as Effective Prodrugs of the Unnatural Amino Acid

(+)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (LY354740), a Selective

Group II Metabotropic Glutamate Receptor Agonist

Ana Bele´n Bueno,*^,†Iva´n Collado,^†Alfonso de Dios,^†Carmen Dom´ınguez,^†Jose´ Alfredo Mart´ın,^†

Luisa M. Mart´ın,^†Mar´ıa Angeles Mart´ınez-Grau,^†Carlos Montero,^†Concepcio´n Pedregal,^†John Catlow,^‡

D. Scott Coffey,^‡Michael P. Clay,^‡Anne H. Dantzig,^‡Terry Lindstrom,^‡James A. Monn,^‡Haiyan Jiang,^‡

Darryle D. Schoepp,^‡Robert E. Stratford,^‡Linda B. Tabas,^‡Joseph P. Tizzano,^‡,§Rebecca A. Wright,^‡and

Marc F. Herin^#

Lilly, S.A., Avda. de la Industria 30, 28108 Alcobendas, Madrid, Spain, Lilly Research Laboratories,

Indianapolis, Indiana 46285, and Lilly Development Centre, 11 Rue Granbonpre´, 1348 Mont-Saint-Guibert, Belgium

Received March 15, 2005

(+)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (1), also known as LY354740, is a highly

potent and selective agonist for group II metabotropic glutamate receptors (mGlu receptors 2

and 3) tested in clinical trials. It has been shown to block anxiety in the fear-potentiated startle

model. Its relatively low bioavailability in different animal species drove the need for an effective

prodrug form that would produce a therapeutic response at lower doses for the treatment of

anxiety disorders. We have investigated the increase of intestinal absorption of this compound

by targeting the human peptide transporter hPepT1 for active transport of di- and tripeptides

derived from 1. We have found that oral administration of an N dipeptide derivative of 1 (12a)

in rats shows up to an 8-fold increase in drug absorption and a 300-fold increase in potency in

the fear-potentiated startle model in rats when compared with the parent drug 1.

Introduction

istration of 1 was shown to prevent lactate-induced

panic-like responses in panic-prone rats.⁵

The treatment of neurological or psychiatric disorders,

such as anxiety, has been linked to selective activation

of metabotropic excitatory amino acid receptors. Glu-

tamate receptors are classified into ionotropic glutamate

receptors (iGlu receptors),¹which are ligand-gated ion

channels, and metabotropic glutamate receptors (mGlu

receptors),²which are G-protein-coupled receptors in-

volved in the production of second messengers. There

are currently eight different subtypes of mGlu receptors

that have been cloned from human and rat species,

which are designated mGlu1-mGlu8. These have been

subdivided into three subgroups (I, II, and III) based

on higher structural homology and shared pharmacology

within each mGlu receptor group. Group II mGlu

receptors include mGlu2 and mGlu3 receptors, which

are negatively coupled to cyclic adenosine monophos-

phate (cAMP) formation.

(+)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid

(1), also known as LY354740,³is a potent group II mGlu

receptor agonist tested in clinical trials. It has been

shown to suppress enhanced glutamatergic excitations

in brain synapses (e.g., hippocampus, prefrontal cortex,

amygdala, locus coeruleus) involved in anxiety/stress

disorders. Oral or parenteral administration of 1 also

demonstrated anxiolytic activity in the fear-potentiated

startle model in rats.⁴More recently, systemic admin-

In vivo studies in rats and dogs⁶show that 1 is a safe

drug that is not metabolized by either species but

excreted unchanged in the urine. Brain penetration

following systemic administration⁷has been reported.

Pharmacokinetic studies demonstrate that the oral

bioavailability is relatively low in rats (10%) and moder-

ate in dogs (40%) apparently because of low transfer

across the intestinal epithelial membrane. This fact

made it advisable to search for a prodrug that would

increase the absorption of this amino acid in the

intestinal tract.

Most orally administered drugs rely on passive dif-

fusion to cross cell membranes. However, developments

in biotechnology and peptide synthesis as well as new

screening strategies in the past years have led to the

exploitation of the unique pharmacological activities of

peptides and peptidomimetics as potential drugs. These

developments have spurred great interest in the human

intestinal peptide transporter, hPepT1, as being ap-

pealing for oral drug delivery.⁸Although intestinal

peptide transport is not fully understood, it is known

that, unlike the structurally restrictive amino acid

mediated transport systems,⁹the peptide transport

carrier hPepT1 has broad substrate specificity and

recognizes not only di- or tripeptides^10,11but also pep-

tidomimetics such as â-lactam antibiotics,¹²cepha-

losporins,¹³angiotensin-converting enzyme (ACE) in-

hibitors,¹⁴renin inhibitors¹⁵and even compounds without

an obvious peptide bond or equivalent, such as δ-ami-

nolevulinic acid¹⁶and ω-amino fatty acids.¹⁷The peptide

carrier has also been targeted for increasing the avail-

ability of drugs with low permeability by designing

* To whom correspondence should be addressed. Phone: +34-91-

6633402. Fax: +34-91-6633411. E-mail: bueno_ana_belen@lilly.com.

^†Lilly, S.A.

^‡Lilly Research Laboratories.

^§Current address: DOV Pharmaceutical, Inc., Continental Plaza,

433 Hackensack Avenue, Hackensack, New Jersey 07601.

^#Lilly Development Centre.

Published on Web 07/15/2005

5306 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16

Bueno et al.

of the amino acids, using different reaction conditions

depending on the nature of the protecting groups (see

Experimental Section), provided the free peptides or the

HCl salts. The C-2 dipeptides synthesized using either

route, along with the amino acids utilized, and the yields

in each step are collected in Table 1.

Synthesis of C-6 Peptides through Intermediate

4. The N-Alloc of 1 in the presence of paraformaldehyde

under acid catalysis²³provided intermediate 4, where

the R-amino acid functionality has been protected as an

oxazolidinone. Coupling of the free acid on C-6 under

standard conditions with the L-phenylalanine allyl ester

provided 9. Reaction of this compound with Pd(0) in the

presence of dimethylbarbituric acid (DMBA) removed

the two allyl groups and the oxazolidinone in a single

step. The zwitterion thus formed was transformed into

the hydrochloride (10‚HCl) with HCl in EtOAc. Com-

pound 10 was the only C-6 peptide synthesized for this

study (Scheme 3).

Synthesis of N Peptides through Intermediate

5. The reaction of 1 with thionyl chloride in methanol²⁴

led to the dimethyl ester 5 in quantitative yield.

Coupling of 5 with N-Boc protected amino acids or

dipeptides provided fully protected di- and tripeptides

11a-o that, after basic treatment to remove the methyl

esters followed by acid deprotection of the N-Boc, yielded

the final peptides 12a-o as hydrochlorides (Scheme 4

and Table 1).

Synthesis of N,C-2 Peptides through Intermedi-

ate 3. Compound 3 was coupled with L-Leu-OMe fol-

lowing standard conditions to provide the protected

dipeptide 6k. Treatment of the dipeptide with HCl to

remove the Boc protecting group followed by the cou-

pling of the free amine with Boc-L-Ala afforded the fully

protected tripeptide 13. Hydrolysis of the esters with

LiOH and removal of the Boc group with HCl gave rise

to the hydrochloride 14‚HCl (Scheme 5).

Figure 1.

peptide or peptidomimetic prodrugs that are transported

through the intestine by the hPepT1 and then hydro-

lyzed prior to or after the delivery of the drug to the

systemic circulation. This strategy has been used to

increase the availability of L-acyclovir and azidothymi-

dine (AZT) by making 5′-amino acid ester prodrugs¹⁸

and the availability of L-methyldopa by making L-

methyldopa-L-Phe¹⁹and D-phenylglycine-L-methyldopa.²⁰

Interestingly, the studies made on L-methyldopa re-

vealed that both approaches, N dipeptides and C dipep-

tides of this amino acid, succeeded in increasing the

uptake of the drug. In addition to the increased bio-

availability compared to the parent drug, this approach

is also attractive because of the low potential for side

effects following release of the drug, because these

promoieties would be natural amino acids.

The goal of this work was to increase the oral

bioavailability of 1 by making oligopeptides that would

be substrates for the intestinal peptide transporter

hPepT1. Since there are three points for attachment of

the amino acids to 1, we have synthesized C-2, C-6, and

N peptides with a variety of amino acids and dipeptides

as potential prodrugs of 1 (see Figure 1).

Chemistry

The starting material for the synthesis of all the

prodrugs was the parent drug 1, which was synthesized

as described in the literature.³Regio- and chemoselec-

tive protections of the parent provided four key inter-

mediates (2-5) in which only one functionality remains

unprotected (Scheme 1). Coupling of these intermediates

with the conveniently protected amino acids or dipep-

tides followed by full deprotection provided all the

prodrugs.

Synthesis of C-2 Peptides through Intermedi-

ates 2 and 3. We developed two different routes to

access to C-2 peptides. Both were based on the protec-

tion of the free amine and the acid on C-6 prior to the

coupling with amino acids. First, the free amine of 1

was protected as an allyl carbamate by standard

methods. The resulting diacid was regioselectively²¹

esterified with 1 equiv of allyl alcohol in the presence

of EDCI, providing pure compound 2 after column

chromatography (Scheme 2).

All the peptides synthesized (collected in Table 1 and

Schemes 3 and 5) are white solids as the free zwitterion

or the HCl salt.

In Vivo Exposure

Rat Screen. Studies measuring the plasma concen-

trations of the parent in rats were performed by col-

lecting blood samples 0.5, 1, and 3 h after oral admin-

istration of a single dose of the peptide. This assay

provided a rank order of the relative efficacy of the

different peptides in delivering 1 to the plasma. The

results are summarized in Table 2. These data suggest

a high specificity in the transport and/or hydrolysis of

the different families of peptides in the rat model. While

a high plasma concentration of 1 was obtained when N

dipeptides were orally administered to the animals, the

compound was almost undetected when the C-6 dipep-

tide 10 or the N,C-2 tripeptide 14 was administered. It

is unknown whether the low plasma exposure of 1 after

oral administration of the N,C dipeptide or the C-6

dipeptide is due to poor absorption or poor bioconversion

of the prodrugs because we did not pursue further

studies with these two peptides. With respect to C-2

dipeptides, only two C-2 dipeptides (8b and 8f) provided

plasma concentrations of the parent drug in a range

similar to those obtained with the N dipeptides.

Alternatively, 1 was reacted with HCl in MeOH²²to

provide the C-6 methylated derivative as the only

regioisomer. Protection of the free amine with Boc₂O led

to 3 in 75% yield (Scheme 2).

The free acid on intermediates 2 and 3 was coupled

with acid-protected amino acids to provide fully pro-

tected C-2 dipeptides 6 and 7. When commercially

available, the amino acids were selected as allyl esters

for reacting with 2 or as methyl esters for reacting with

3 so that both esters in 6 and 7 (R₂and R₃; see Scheme

2) could be hydrolyzed in a single step. Full deprotection

Rat Bioavailabilities. The absolute oral bioavail-

ability in rats was determined for those dipeptides that

Dipeptides as Effective Prodrugs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5307

Scheme 1

Scheme 2^a

of 8b was 3-fold lower than that after intravenous

administration of 1, explaining the higher plasma

concentrations of 8b observed in Figure 2.

In comparison, the N-linked prodrug 12a was rapidly

absorbed and completely biotransformed when admin-

istered to Fischer rats. Indeed the maximum plasma

concentration of 1 appeared 1 h after oral administra-

tion and only a very low amount of the prodrug was

detectable 30 min after administration (Figure 2). A

comparison of the area under the curve (AUC) for 1 with

the AUC determined after intravenous administration

of 1 indicated that the oral bioavailability of 1 was 84%

following oral administration of the N-linked prodrug.

^a(a) (i) AllocCl, NaHCO₃; (ii) EDCI, HOBT, Et₃N, DMAP,

AllylOH, 30%; (b) (i) HCl(g), MeOH; (ii) Boc₂O, K₂CO₃, 75%; (c)

(coupling) EDCI, DMAP, HOBT, Et₃N, H₂N-(amino acid)-OR₃.

In Vitro Bioconversion

Two selected peptides (8b and 12a) were incubated

with rat liver homogenate and human jejunum in order

to assess the stability of the prodrugs to enzymatic

hydrolysis. The results are presented in Figure 3. Only

the N dipeptide prodrug 12a was unstable when ex-

posed to enzymes located in the brush-border and citosol

of the enterocytes (jejunum homogenate) or to the liver

enzymes. These data are in agreement with the expo-

sure studies in which it was shown that only N dipeptide

prodrugs were completely bioconverted in vivo to the

parent drug in rats. Compound 8b is transported to the

blood stream, but it is not fully hydrolyzed by peptidases

to the parent drug.

had shown high plasma concentration of parent in the

three-time-point rat screen. The results are collected in

Table 2. The concentration of prodrug circulating was

also measured.

The only three C-2 dipeptides evaluated in this study,

8b, 8c, and 8f, did not show a complete in vivo

bioconversion to the parent 1, with high concentrations

of the prodrug detected at all time points. However, for

two of them (8b and 8f, Table 2), the systemic avail-

ability of 1 was high after oral administration. This

indicates that in some cases at least, C-2 peptides are

well-absorbed, presumably via the PepT1 transporter.

The evaluated N dipeptides (12a, 12c, 12e, 12f, and

12j), on the other hand, were completely bioconverted

in rats to the parent 1 with high bioavailability (71-

105%).

The curves of two representative dipeptides, 8b as an

example of a C-linked dipeptide and 12a as an example

of an N-linked dipeptide, are shown for comparison

(Figure 2). When 8b was orally administered to Fischer

rats at a dose of 8 mg/kg (27 µM), it was only partially

converted into 1. High concentrations of the prodrug

remained in the rat plasma. These data were compared

to equimolar intravenous administrations of 8b or 1.

This allowed us to determine that the oral bioavailabil-

ity of the prodrug was 42% while the systemic avail-

ability of 1 was 74%. The intravenous data also dem-

onstrated that 29% of the iv dose of 8b was recovered

as 1 in rat plasma. This indicates that 1 formed from

8b after oral administration was partly formed during

the first pass and partly from 8b available systemically.

The intravenous data suggest that the bioconversion is

the rate-limiting step of the elimination of 8b because

8b was eliminated at the same rate as 1 formed from

8b. These data also indicate that the plasma clearance

In Vitro Transport Results

The in vivo results presented in the previous section

demonstrated the enhanced exposure after oral admin-

istration to rats of dipeptides of 1 when compared to

the parent drug itself. We wondered if an active

transport mechanism such as the intestinal peptide

transporter, PepT1, might be responsible for the greater

oral absorption that was observed. With this aim we

examined the uptake of a known substrate of the

intestinal peptide transporter, radiolabeled glycylsar-

cosine (Gly-Sar), into Caco-2 cells and into Chinese

hamster ovary cells transfected with human PepT1

(CHO/hPepT1) in the presence of selected peptides.

Transport into Caco-2 Cell Line. Initially, uptake

of 100 µM Gly-Sar into Caco-2 cells was measured in

the absence and presence of a selected peptide at a final

concentration of 1 mM. This assay provided a rank order

of the relative affinity of the transporter for the various

peptides. As shown in Figure 4, 1 and the C-2 peptides

showed weaker inhibition of Gly-Sar uptake than the

corresponding N peptides (p < 0.05).²⁵

5308 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16

Table 1. Synthesis of C-2 Peptides and N Peptides

Bueno et al.

series

protected amino acid

intermediate

protected peptide (% yield)

peptide (% yield)

L-Ala-OEt

2

3

6a (92%)

6b (98%)

6c (79%)

6d (82%)

6e (76%)

6f (90%)

6g (69%)

6h (72%)

7i (95%)

7j (85%)

8a (38%)

8b (50%)

8c (48%)

8d (56%)

8e (70%)

8f (50%)

8g (89%)

8h (31%)

8i (87%)

L-Leu-OAllyl

L-Phe-OAllyl

L-Val-OAllyl

Gly-OEt

L-Met-OMe

L-Pro-OMe

L-Ile-OAllyl

L-Tyr-OMe

L-Ser(OTBDMS)-OMe

8j‚HCl (20%)

Boc-L-Ala

5

11a (50%)

11b (93%)

11c (95%)

11d (88%)

11e (87%)

11f (87%)

11 g (88%)

11h (89%)

11i (78%)

11j (79%)

11k (73%)

11l (82%)

11m (94%)

11n (90%)

11o (95%)

12a‚HCl (80%)

12b (80%)

Boc-^D-Ala

Boc-^L-Leu

12c‚HCl (48%)

12d‚HCl (71%)

12e‚HCl (44%)

12f‚HCl (64%)

12g (44%)

Boc-^D-Leu

Boc-^L-Phe

Boc-^L-Val

Boc-Gly

Boc-^L-Met

12h (78%)

Boc-^L-Pro

12i‚HCl (90%)

12j‚HCl (64%)

12k (35%)

Boc-^L-Ile

Boc-^L-Tyr

Boc-^L-Ser(OTBDMS)-OH

R-Boc-ꢀ-Boc-^L-Lys

Boc-^L-Ala-L-Ala

Boc-^L-Leu-L-Ala

12l (11%)

12m‚2HCl (82%)

12n (45%)

12o (30%)

Scheme 3^a

^a(a) (i) AllocCl, NaHCO₃; (ii) CH₂O, p-TsOH, 65% two steps; (b) EDCI, DMAP, HOBT, Et₃N, H₂N-Phe-Oallyl, 47%; (c) (i) DMBA,

(Ph₃P)₄Pd cat.; (ii) saturated HCl in EtOAc, 91% two steps.

Scheme 4^a

^a(a) MeOH, SOCl₂; (b) EDCI, DMAP, HOBT, Et₃N, BocHN-(amino acid)_n-OH; (c) (i) LiOH; (ii) saturated HCl in EtOAc.

Scheme 5^a

Gly-Sar into Caco-2 cells or CHO/hPepT1 cells. The

results are summarized in Table 3.

Some interesting observations can be made from these

results: (a) Gly-Sar uptake was inhibited by the pro-

drugs (di- and tripeptides) but not by the amino acid

analogue 1. (b) Compounds that show a high concentra-

tion of parent in the three-point rat screen show high

affinity for the transporter, such as 12a (0.12 mM), 12c

(1.43 mM), and 12e (0.52 mM). Compounds with lower

affinities (12b and 12o) show lower concentrations of

parent in the same assay (entries 12 and 25 of Table

2), although further studies would be necessary to

determine whether these compounds are poorly trans-

ported or hydrolyzed or both. (c) The uptake assay by

CHO/hPepT1 is more predictive of the absorption of C-2

dipeptides than the Caco-2 assay because compounds

8b and 8c are absorbed, as demonstrated by the high

concentrations of prodrug detected in plasma (see

entries 2 and 3, Table 2). These in vivo data correlate

with a good affinity for the transporter shown in the

^a(a) EDCI, HOBT, Et₃N, DMAP, L-Leu-OMe, 40%; (b) (i) HCl(g),

MeOH; (ii) EDCI, HOBT, Et₃N, DMAP, BocHN-L-Ala, 48%; (c) (i)

LiOH; (ii) saturated HCl in EtOAc, 81%.

To further determine the affinity of the peptide

transporter, dose-response curves of selected com-

pounds were examined for their effect on the uptake of

Dipeptides as Effective Prodrugs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5309

Table 2. Plasma Concentrations (ng/mL) and AUC Values (ng‚h/mL) of Prodrug and 1 in Rats after an Oral Dose of 5 mg equiv 1/kg

series

entry

aa^a

Ala

Leu

Phe

Val

Gly

Met

Pro

Ile

1 at 0.5 h (ng/mL)^b1 at 1 h (ng/mL)^b1 at 3 h (ng/mL)^boral F,^c% AUC of prodrug

1

2

8a

8b

8c

8d

8e

8f

0

450

20

10

19

602

0

798

45

17

0

919

10

0

74

21264

11367

3

0.3

4

5

6

808

0

889

0

66

16648

7

8g

8h

8i

8

18

30

36

34

44

0

20

124

53

9

Tyr

Ser

10

8j

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

12a Ala

3732

81

3381

191

4323

894

2763

2124

1992

2107

455

1457

2163

1365

147

1770

525

1152

877

1274

1136

ND^d

670

2032

ND^d

246

734

660

85

94

0

12b D-Ala

12c Leu

12d D-Leu

12e Phe

4488

1478

1710

2387

1707

3720

319

2009

1915

1497

74

71

94

194

12f

Val

ND^d

12g Gly

12h Met

12i

12j

Pro

Ile

105

ND^d

12k Tyr

12l

12m Lys

Ser

12n Ala-Ala

12o Leu-Ala

3620

2315

1554

1337

26

10

14

Phe

0

27

Ala-Leu

44

26

55

^aThe configuration of the amino acid is L unless otherwise indicated. ^bPlasma concentration of 1 after oral dosing of corresponding

prodrug. ^cBioavailability of parent from prodrug. ^dND: not determined.

Figure 2. Comparison of plasma concentrations vs time profiles for 8b and 12a in rats following an oral dose of 5 mg equiv 1/kg.

CHO/hPepT1 assay. (d) N Dipeptides gave better inhibi-

tion of uptake by the human intestinal PepT1 trans-

porter than N tripeptides (compare IC₅₀for dipeptides

12a (0.12 mM) and 12c (1.43 mM) with IC₅₀for

tripeptides 12n (2.88 mM) and 12o (5.26 mM) and their

correlation with entries 11, 13, 24, and 25 in Table 2).

(e) Although only two examples of unnatural D-peptides

are available (compare 12a with 12b and 12c with 12d,

Table 3), dipeptides with the L-configuration on the

amino acid seem to have better affinity for the carrier

than the corresponding dipeptides with the unnatural

D-configuration.

These in vitro data and their correlation with in vivo

results suggest that these dipeptides are actively trans-

ported into the intestinal enterocyte by the peptide

carrier hPepT1 prior to exiting the enterocyte into the

blood stream.

In Vitro Binding to mGlu Receptors 2/3

To test the affinity for mGlu receptors 2/3, compound

12a (tested as the HCl salt) was examined for displace-

ment of mGlu receptors 2/3 ligand binding. For these

studies, 12a‚HCl displacement of high-affinity mGlu

receptors 2/3 antagonist ligand [³H]LY341495 binding

5310 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16

Bueno et al.

Table 4. Affinity of 1 versus Prodrug 12a‚HCl for

Recombinant Human and Native Rat Brain mGluR2 Receptors

displacement of [³H]LY341495

binding (N ) 3)

mean K_i( SE (nM)

for 1

K_i(nM)

for 12a‚HCl

receptor preparation

human mGlu2

human mGlu3

rat forebrain

84.7 ( 11.1

125.6 ( 4.8

80.0 ( 7.3

>10 000

Figure 3. In vitro bioconversion of 8b and 12a incubated for

1 h at 10 µM in liver or jejunum homogenates.

branes with nanomolar potencies. In contrast, 12a‚HCl

did not appreciably displace [³H]LY341495 binding at

up to 10 000 nM, which is 80- to 120-fold higher than

the K_ivalues for the parent molecule in these tissues.

[³H]LY341495 binding to rat forebrain membranes

represents mGlu receptors 2/3 binding to native tissue.

Rat brain tissue also represents the species used for in

vivo efficacy data with 12a‚HCl (see below). As shown

in Figure 5 (right panel) and Table 4, 1 displaced [³H]-

LY341495 binding to rat forebrain membranes with a

potency similar to that observed in human recombinant

receptors. In contrast, 12a‚HCl did not appreciably

displace [³H]LY341495 binding to rat forebrain mem-

branes at up to 10 000 nM.

In summary, ligand binding data demonstrate that

12a‚HCl, unlike 1, had no intrinsic affinity at human

recombinant or native rat mGlu receptors 2/3.

Figure 4. Comparison of the influence of C-2 and N dipeptide

prodrugs on 100 µM ¹⁴C Gly-Sar uptake in Caco-2 cells. 1 and

Gly-Pro controls are also shown. Results are presented as

percentage inhibition (mean ( SC of three to six filters) of ¹⁴

C

In Vivo Actions in Rat Fear-Potentiated Startle

Anxiety Model

Gly-Sar uptake relative to control filters that were exposed

only to ¹⁴C Gly-Sar.

To study the oral potencies of 12a (tested as the HCl

salt) in comparison to 1 in an mGlu receptors 2/3 linked

therapeutic animal model, studies in the rat fear-

potentiated startle assay were performed. This model

was specifically chosen because it is highly sensitive to

mGlu receptors 2/3 agonists such as 1²⁷and served as

the basis for the development of 1 for anxiety disorders

in humans. To verify that the actions of compound 12a‚

HCl in this model were mGlu receptors 2/3 mediated,

as we have shown previously for the parent,^27bthe

ability of LY341495 (an mGlu receptors 2/3 antagonist)²⁸

to block compound-mediated suppression of fear-poten-

tiated startle was also determined. As a positive control

in each experiment, diazepam (0.6 mg/kg ip) was used.

All experiments were performed in fed rats.

Table 3. Affinity of the Transport Carrier for Selected Di- and

Tripeptides and 1

IC₅₀(mM)

series

Caco-2

hPepT1

1

>10

>19

C-2 dipeptides

8b

8c

>10

0.62

1.54

2.68

N peptides

12a

12b

12c

12d

12e

12n

12o

0.30

>10

5.01

6.31

0.42

7.29

>10

0.12

6.66

1.43

1.7

0.52

2.88

5.26

N,C-2 tripeptides

14

3.05

1.34

As shown in Figure 6, the parent compound 1

significantly suppresses rat fear-potentiated startle with

a minimally effective dose (MED or lowest dose where

p < 0.05 compared to control) of 3.0 mg/kg (Table 5).

This is compared to 0.3 mg/kg 1 reported by Helton et

al.^27aHowever, in this more recent experiment (Figure

6, left panel), 0.3 mg/kg 1 suppressed fear-potentiated

to cell membranes from human mGlu2, human mGlu3,

and native rat brain tissues was determined.²⁶1 was

used as a positive control/comparator in these studies.

As shown in Figure 5 (left and middle panels) and

Table 4, 1 potently displaced [³H]LY341495 binding to

human mGlu2 and human mGlu3 expressing cell mem-

Figure 5. Unlike 1, 12a‚HCl does not displace [³H]LY341495 binding to human recombinant or rat brain mGluR2 receptors.

Dipeptides as Effective Prodrugs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5311

Figure 6. Potency, onset, and duration of 1 in rat fear-potentiated startle. 1 was dosed orally in fed rats. The positive control in

each experiment, diazepam (0.6 mg/kg) was given ip 30 min prior to testing.

Table 5. Comparison 1 and 12a‚HCl in the Rat Fear-Potentiated Startle Assay

parameter measured

1

12a‚HCl

0.01 mg/kg po

e1 h @ 0.01 and 0.1 mg/kg po

4-8 h @ 0.01 mg/kg po

g24 h @ 0.1 mg/kg po

MED (1 h pretreatment)

onset of effect

duration of effect

0.3^a-3.0 mg/kg po

e1 h @ 3.0 mg/kg po

8-24 h @ 3.0 mg/kg po

^aData from ref 27a.

Figure 7. Oral potency of 12a‚HCl in rat fear-potentiated startle. 12a‚HCl was dosed orally (po) in fed rats. Two separate

experiments with overlapping doses of 12a‚HCl were performed to determine the MED for oral activity. The positive control in

each experiment, diazepam (0.6 mg/kg), was given ip 30 min prior to testing.

Figure 8. Time course for 12a‚HCl suppression of rat fear-potentiated startle. 12a‚HCl (0.01 mg/kg po (left panel), 0.1 mg/kg po

(right panel)) was administered to fed rats at the designated pretreatment times. The positive control in the experiment, diazepam

(0.6 mg/kg), was given ip 30 min prior to testing.

startle by ∼50%, but this was not statistically signifi-

cant (p ) 0.08). At 3.0 mg/kg 1 had an onset of 1 h and

was active between 8 and 24 h following administration

(Figure 6, right panel).

As shown in Figure 7, in two separate experiments

with overlapping doses, 12a‚HCl significantly blocked

fear-potentiated startle with an MED of 0.01 mg/kg po.

As shown in Figure 8, at an MED dose of 0.01 mg/kg

po, 12a‚HCl had an onset of 1 h and was still active at

up to 4 h in this test. At a dose of 0.1 mg/kg po 12a‚

HCl, a significant effect was observed within 1 h, and

12a‚HCl was still active at up to 24 h postadministra-

tion. Thus, in this test, 12a‚HCl was observed to be

∼300-fold more potent than the parent compound 1.

Furthermore, a dose of 0.1 mg/kg po 12a‚HCl produced

a longer-lasting effect when compared to a 30-fold

higher dose (3.0 mg/kg) of the parent. Figure 9 shows

that the suppression of fear-potentiated startle by 12a‚

HCl was reversed by LY341495, an mGlu receptors 2/3

antagonist.

Summary of Efficacy Data

In vitro studies showed that the prodrug of 1, 12a‚

HCl, had no appreciable affinity for mGlu receptors 2/3.

5312 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16

Bueno et al.

bioconversion model) suggest that a dipeptide prodrug-

like 12a could also be a suitable prodrug for 1 in

humans.

Experimental Section

Biology Methods. In Vivo Results. Male Fisher 344 rats

weighing 190-250 g were obtained from Harlan (Indianapolis,

IN) and housed in polycarbonate cages in a light-controlled

environment (12 h of light, 12 h of dark) with free access to

food and water. The animals were acclimated to the housing

environment for 3 days before drug administration. Water was

provided ad libitum throughout the study, and the animals

were fasted overnight prior to dosing, with the food being

returned approximately 4 h postdose. The rats received either

an oral dose of the prodrug that was the molar equivalent to

a 5 mg/kg dose of compound 1 dissolved in pH 2.5 20 mM

KHPO₄or an intravenous dose of compound 1 at 2.5 mg/kg

dissolved in water. Blood was collected from the orbital sinus

for the first two time points, and the last time point blood was

collected from carbon dioxide anesthetized animals by cardiac

puncture. The blood samples were combined with 50 mL of a

100 mM phenylmethylsulfonyl fluoride solution to inhibit

hydrolysis of the prodrug. The sample was then centrifuged,

and the resulting plasma was stored at -20 °C or below until

analysis. There were three plasma samples per time point, and

each rat was collected from three time points.

In Vitro Bioconversion. 1 prodrugs were incubated for 1

h with liver and jejunum homogenates. The liver homogenate

was prepared by probe-sonicating a fresh liver sample diluted

3-fold with pH 7.4 100 mM phosphate buffer with 5 mM MgCl₂

while being kept on ice. The jejunum homogenate was pre-

pared by scraping an approximate 2 in. section (human) with

a razor blade, diluting the preparation in 2 mL of 5 mM Tris

and 50 mM manitol, probe-sonicating, and diluting with 2 mL

of 100 mM phosphate pH 7.4 with 5 mM MgCl₂while being

kept on ice. The incubation was then performed in a 96-well

plate by gently shaking 0.8 mL of homogenate with 40 mL of

a 200 mM prodrug solution at 37 °C for 1 h. The reaction was

stopped with 50 mL of a 100 mM phenylmethylsulfonyl

fluoride solution, and the samples were stored at -20 °C or

below until analysis.

Figure 9. Reversal 12a‚HCl suppression of fear-potentiated

startle by the mGluR2 antagonist LY341495. 12a‚HCl (0.1 mg/

kg) was dosed orally (po) in fed rats 1 h prior to testing.

LY341495 was given sc at 1 mg/kg 1 h prior to testing. The

positive control, diazepam (0.6 mg/kg), was given ip 30 min

prior to testing.

This indicates that the in vivo pharmacology of this

compound in rats and humans likely reflects the con-

version of the prodrug to the parent molecule 1, which

then acts at mGlu receptors 2/3 to produce a therapeutic

effect.

When given orally to fed rats, 12a‚HCl was active in

the rat fear-potentiated startle test at ∼30-300 times

lower doses when compared to 1. Importantly, antago-

nist studies with LY341495 also demonstrated that the

actions of 12a‚HCl in the anxiety test in vivo were group

II mGlu receptors mediated. These animal data suggest

that anxiolytic doses of 12a‚HCl in humans would be

considerably lower than that of the parent compound.

If this in vivo animal model data directly predict human

anxiety responses, 12a‚HCl would produce anxiolytic

effects in humans at 30- to 300-fold lower doses than

the parent compound.

Conclusions

A series of di- and tripeptides of 1 have been inves-

tigated in this study as prodrugs of the parent amino

acid 1 in order to increase the relatively low oral

bioavailability of this compound in rats (10%). In vitro

transport studies have shown the broad specificity of

the human PepT1 transporter that recognizes a variety

of peptides (N dipeptides, N tripeptides, and C-2 dipep-

tides of 1) suggesting that these peptides are actively

transported by the peptide carrier. In vitro bioconver-

sion studies of two model dipeptides, 8b and 12a, show

complete hydrolysis of 12a in the presence of rat liver

or human jejunum, while 8b is more stable to enzymatic

hydrolysis. In vivo studies performed with rats correlate

with the in vitro results. Thus, while N dipeptides are

rapidly absorbed and bioconverted in vivo to the parent

drug, C-2 dipeptides fail to be transported or to com-

pletely biotransform.²⁹As a result, N dipeptides are

more suitable prodrugs of 1, improving significantly the

intestinal uptake of the parent drug. N-Ala dipeptide

(12a) shows a 84% oral bioavailability in rats, with no

prodrug circulating 30 min after oral administration.

The released promoiety is the safe L-Ala. In vivo efficacy

experiments performed with 12a have shown this

compound to be 300-fold more active in the fear-

potentiated startle model of anxiety in rats than the

parent compound 1.

Samples were extracted utilizing solid-phase extraction and

analyzed by LC/MS/MS. One hundred microliters of a sample

was combined with 20 mL of internal standard (LY459477)

and 500 mL of water. The sample was then slowly extracted

at approximately 1 drop/3 s on a SAX 100 mg cartridge (IST)

that had been preconditioned with 0.5 mL of methanol and

0.5 mL of water. The cartridge was then washed with 0.5 mL

of water and 1 mL of methanol. To elute of the prodrug, the

cartridge was washed with 0.5 mL of ethyl acetate (1%

trifluoroacetic acid and 1% water), dried for 10 min under full

vacuum, and eluted with 0.25 mL of 0.2% acetic acid. To

extract compound 1, the cartridge was eluted with 0.8 mL of

3% trifluroracetic acid, 5% water, and 93% methanol.

Uptake by the Intestinal Human PepT1 Transporter.

Caco-2 Cells. For cells grown on filters, inhibition of 100 mM

[¹⁴C]Gly-Sar was measured in the absence or presence of 1 mM

of prodrugs, dipeptide control, or 1. The flux buffer was 25

mM MES, pH 6.0, which also contained 125 mM NaCl, 5.2

mM KCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, and 10 mM glucose.

Prior to an experiment, cells were washed once in pH 7.4 buffer

(25 mM HEPES used as the buffering agent) and starved for

30-60 min. This buffer was removed, and 0.3 mL of the pH 6

buffer containing Gly-Sar, 100 mM mannitol, and 1 mM of a

competing substrate was added to the cells and incubated for

15 min on an orbital shaker at 37 °C. Cells and filters were

washed twice in cold pH 7.4 buffer, 0.3 mL on top and 1 mL

on the bottom. Individual filters were then removed from the

plastic filter cup housing using a scalpel and placed in

scintillation vials with 10 mL of ReadySolv HP (Beckman

Coulter, Fullerton, CA). Samples were then counted for 5 min.

With mannitol as a marker, extracellular fluid remaining on

the cells was <0.1% of the initial amount applied. Alternatively

for cells grown in 96-well plates, inhibition of 40 mM [3H]Gly-

In vitro results obtained with human cells (human

PepT1 for absorption model and human jejunum for

Dipeptides as Effective Prodrugs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5313

Sar was measured over a concentration range of 0.125-2.0

mM for the prodrugs or 0.5-10 mM for 1. On the day of an

experiment, the medium was removed and replaced with 0.1

mL of the 25 mM HEPES pH 7.4 buffer used in the 24-well

plate filter experiments. Sixty minutes later, this buffer was

replaced with 40 mL of the 25 mM MES pH 6 buffer also

described above. After 15 min at 37 °C in a humidified orbital

shaker, cells were washed three times with 0.2 mL of ice-cold

pH 7.4 buffer. Preliminary studies demonstrated linear uptake

of 40 mM Gly-Sar between 5 and 15 min. Cells were then lysed

by adding 50 mL of 1% Triton-X 100, and the plates were

allowed to sit overnight at ambient temperature. Subse-

quently, an aliquot of 10 mL was removed for analysis of

protein content using the Pierce BCA protein assay reagent.

A volume of 200 mL of Packard Microscint 40 was added to

the remaining 40 mL, sealed using Packard Top Seal and

mixed, and tritium was counted for 5 min on a Perkin-Elmer

Top Count NXT microplate scintillation and luminescence

counter (Perkin-Elmer Life Sciences, Boston, MA). Uptake rate

was calculated on the basis of total protein content. Residual

extracellular fluid remaining on the cells was estimated using

[³H]inulin as the marker. IC₅₀values were calculated using a

computer-generated curve-fitting program, BRAVO/SAS, and

represent the concentration that gave 50% inhibition of the

maximum inhibition observed in the experiment. For a given

experiment, each inhibitor concentration was measured in

duplicate and the average percent inhibition at each concen-

tration was used in the curve-fitting. For most inhibitors, at

least two experiments (on different days) were performed.

CHO/PepT1 Cells. Inhibition of 40 mM [³H]Gly-Sar was

measured over a concentration range of 0.01-3.15 mM for the

prodrugs or 0.59-19 mM for 1. The flux buffer was sodium-

free modified EBSS buffer at pH 6.0 as previously published.²⁵

Cells were washed once in EBSS pH 7.4 and starved for 30-

45 min. Buffer was removed, and an aliquot of 40 mL of flux

buffer was added to cells and incubated for 5-20 min on an

orbital shaker at 37 °C. Cells were washed three times with

ice-cold EBSS pH 7.4 using a Perkin-Elmer Filtermate 196

with the air and vacuum off. Excess fluid was removed, and

cells were lysed as described above except with 40 mL of

Triton-X 100. After the plates were allowed to sit overnight,

30 mL was transferred to an opaque bottom Packard culture

plate with the addition of 200 mL of Microscint 40 per well.

The methods for analysis of protein content and extracellular

volume, as well as IC₅₀determinations, were the same as

described for the Caco-2 96-well experiments. For each inhibi-

tor, at least two independent determinations of IC₅₀(from

separate experiment days) were made.

In Vitro Receptor Binding. Membranes from cells ex-

pressing recombinant human mGlu receptors were prepared

by scraping attached cells from T-150 flasks, centrifuging at

approximately 1000g for 5 min, and freezing resultant pellets

at -20 °C until the day of assay. Rat brain tissue was obtained

by decapitation from adult male Sprague-Dawley rats (150-

250 g, Harlan/Sprague-Dawley, Indianapolis, IN). Forebrain

tissue (cortex, hippocampus, and striatum) was dissected and

homogenized in 30 mM Tris-HCl + 2.5 mM CaCl₂buffer (pH

7.6 at 5 °C). The homogenized tissue was washed three times

by centrifugation, then incubated for 30 min at 37 °C followed

by three more washes, then resuspended in 10 volumes of

buffer and frozen at -20 °C.

Frozen tissue preparations were thawed on the day of assay,

suspended in ice-cold assay buffer (10 mM potassium phos-

phate pH 7.6 + 100 mM potassium bromide), homogenized,

and washed 3× by centrifugation at 50000g for 10 min. To

start the binding reaction, washed tissue (0.015-0.020 mg of

protein) was added to deep-well polypropylene microtiter

plates containing [³H]LY341495 (1 nM) and appropriate

concentrations of test compounds in assay buffer. The final

assay volume was 0.5 mL. Nonspecific binding was defined

with 1 mM ^L-glutamate. Assay plates were incubated on ice

for 30-45 min, and the reaction was terminated by rapid

filtration. Filters were placed in minivials with scintillation

cocktail and counted on a Beckman LS6000 counter. Protein

concentration was determined using the Pierce Coomassie

microassay. Affinity constants (K_i) were calculated using the

GraphPad Prism program.

Fear-Potentiated Startle in Rats. Apparatus and Pro-

cedures. SR-LAB (San Diego Instruments, San Diego, CA)

chambers were used for conditioning sessions and for produc-

ing and recording startle responses. A respondent conditioning

procedure was used to produce potentiation of startle re-

sponses. The fear-potentiated startle paradigm was conducted

over 3 consecutive days. All 3 days began with a 5 min

adaptation period before the trial started. On day 1 (baseline

startle) after the adaptation period, the animals received 30

trials of 120 dB auditory noise. Startle responding was

measured through transducers located under the startle

platforms. Recorded values represent a maximum change in

voltage (V_max). The mean startle amplitude (V_max) was used to

assign animals to groups (n ) 8) with similar mean values

before conditioning began. Day 2 consisted of conditioning the

animals. Each animal received 0.5 mA of shock for 500 ms

preceded by a 5 s presentation of light (15 W) that remained

on for the duration of the shock. Ten presentations of the light

and shock were administered. Animals tested in the precon-

ditioning paradigm were administered compound prior to

conditioning. On day 3, 24 h after conditioning, startle testing

sessions were conducted. Ten trials of acoustic startle (120 dB),

non-light-paired, were presented at the beginning of the

session to minimize the influence of the initial rapid phase of

habituation to the stimulus. This was followed by 20 random

trials of the noise alone and 20 random trials of noise preceded

by light. Animals tested in the postconditioning paradigm were

administered compounds prior to testing on day 3.

Statistical Analysis. With exclusion of the first 10 trials,

the startle response amplitudes for each trial type were

averaged for each animal. Data were presented as the differ-

ence between light + noise and noise alone. Differences in

startle response amplitudes were analyzed by JMP statistical

software using a one-way ANOVA (analysis of variance),

followed by a Dunnett’s post hoc analysis to determine

differences between control and treatment groups. Interaction

experiments between agonists and antagonists were analyzed

using the Student’s T test. Group differences were considered

to be significant at p < 0.05.

Chemistry Methods. (1S,2S,5R,6S)-2-Allyloxycarbonyl-

aminobicyclo[3.1.0]hexane-2,6-dicarboxylic Acid 6-Allyl

Ester 2. (1S,2S,5R,6S)-2-Aminobicyclo[3.1.0]hexane-2,6-di-

carboxylic acid monohydrate 1 (15.0 g, 73.9 mmol) was slowly

dissolved in 250 mL of saturated NaHCO₃(250 mL) in a round-

bottom flask provided with a stopper. After complete solution,

dioxane (100 mL) and allyl chloroformate (15.7 mL, 147.8

mmol) were added at room temperature, and the mixture was

stirred overnight. The reaction mixture was diluted with water

(100 mL) and washed with ethyl acetate (3×). The organic

layer was extracted once with saturated NaHCO₃. The com-

bined aqueous layers were acidified to pH 1 with 4 N HCl and

extracted with ethyl acetate (2×). The organic layer was dried

over magnesium sulfate, filtered, and concentrated to provide

an oil (13.4 g, 67% yield) that was used without further

1

purification. H NMR (CD₃OD) δ: 6.01-5.82 (m, 2 H), 5.35-

5.13 (m, 4 H), 4.51 (d, J ) 5.1 Hz, 4 H), 2.48-1.78 (m, 5 H),

1.69-1.62 (m, 1 H), 1.45-1.29 (m, 1 H). ¹³C NMR (CD₃OD) δ:

176.7, 176.6, 158.3, 134.2, 117.4, 67.3, 66.3, 35.8, 33.1, 29.9,

27.0, 22.0.

To a suspension of the compound obtained in the previous

step (the Alloc derivative of 1) (13.4 g, 49.8 mmol) in dichlo-

romethane (400 mL), N-ethyl-N′-dimethylaminopropylcarbo-

diimide (9.55 g, 49.8 mmol) and (dimethylamino)pyridine (0.61

g, 5.0 mmol) were added at room temperature under nitrogen.

Allyl alcohol (3.4 mL, 49.8 mmol) was added, and the mixture

was stirred overnight at room temperature. The reaction

mixture was diluted with dichloromethane and washed with

water (2×). The organic layer was dried over magnesium

sulfate, filtered, and concentrated to provide 2 (6.8 g, 44%

yield) as an oil. ¹H NMR (CD₃OD) δ: 6.01-5.82 (m, 1 H), 5.35-

5314 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16

Bueno et al.

5.13 (m, 2 H), 4.51 (d, J ) 5.1 Hz, 2 H), 2.48-1.78 (m, 5 H),

1.69-1.62 (m, 1 H), 1.45-1.29 (m, 1 H).

solution) under nitrogen. The resulting solution was stirred

at room temperature overnight. The solvent was removed

under vacuum, the residue was diluted with ethyl acetate (50

mL) and washed with saturated KHSO₄(2×) and with

NaHCO₃and brine. The organic layer was dried over MgSO₄,

filtered, and concentrated under vacuum. The residue was

purified by column chromatography on silica gel (eluent

hexane/ethyl acetate).

General Procedure B: Deprotection of Allyl Deriva-

tives. The corresponding allylated compound (1.0 equiv) was

dissolved in dry dichloromethane (0.1 M solution) under

nitrogen. 1,3-Dimethylbarbituric acid (0.5 equiv for each allyl

group to be removed) and tetrakis(triphenylphosphine)-

palladium(0) (0.02 equiv) were added, and the resulting

solution was heated at 35 °C for 2 h. After the mixture was

cooled to room temperature, if a solid was formed, it was

filtered, washed with dichloromethane, ethyl acetate, and

diethyl ether, and dried under vacuum to provide the product.

In case the solid did not appear, the solvent was removed

under vacuum and a large amount of diethyl ether was added.

After the mixture was stirred for 30 min, the solid was filtered,

washed with ether and ethyl acetate, and dried to provide the

product.

General Procedure C: Deprotection of N-Boc, 6-Meth-

yl Derivatives. The corresponding 2-N-Boc-6-methyl ester

dipeptide (1.0 equiv) was dissolved in THF and an equal

volume of 2.5 N aqueous LiOH (10-20 equiv) was added. The

reaction mixture was stirred at room temperature for 1-3 h.

After dilution with water, the mixture was washed with ethyl

acetate. The aqueous layer was acidified to pH 2 with 1 N HCl

and extracted with ethyl acetate. The combined organic

extracts were washed with brine, dried over sodium sulfate,

filtered, and concentrated. The foamy solid was dissolved in a

solution of ethyl acetate saturated with hydrogen chloride (g)

(5-10 mL/mmol), and the resulting mixture was stirred

overnight. The solid was filtered, rinsed with diethyl ether,

and dried under high vacuum to provide the product.

General Procedure D: Deprotection of 2,6-Dimethyl

Esters. The corresponding 2′-N-Boc-2,6-dimethyl ester dipep-

tide derivative (1.0 equiv) was dissolved in THF, and an equal

volume of 2.5 N aqueous LiOH (10-20 equiv) was added. The

reaction mixture was stirred at room temperature for 1-3 h.

The solution was diluted with water and washed with EtOAc

(2×). The aqueous layer was acidified to pH 1-2 with 1 N

hydrochloric acid and extracted with ethyl acetate (5×). The

combined organic layer was washed with brine, dried over

sodium sulfate, filtered, and evaporated under vacuum to

dryness. The crude N-Boc-dicarboxylic acid was dissolved in

a saturated solution of hydrogen chloride gas in ethyl acetate

(5-10 mL/mmol), and the mixture was stirred for 16 h. The

resulting white precipitate was filtered, rinsed with ethyl

ether, and dried under high vacuum to afford the hydrochloride

salt as a fine white powder.

(1S,2S,5R,6S)-2-tert-Butoxycarbonylaminobicyclo[3.1.0]-

hexane-2,6-dicarboxylic Acid 6-Methyl Ester (3).

A

250 mL flask provided with a stopper was charged with

(1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid

monohydrate 1 (18.08 g, 89.0 mmol), and 90 mL of a 2.5 M

HCl(g) in methanol was added. The suspension was vigorously

stirred, and methanol (50 mL) was added after 10 min. The

solution was stirred for 5 h. Diethyl ether was added, and the

solid was filtered and rinsed with diethyl ether. After the

sample was dried under high vacuum, the 6-methyl ester

derivative of 1 was obtained as a fine powder (5.5 g, 87%

1

yield): mp 245 °C (dec). [R]²⁵+2° (c 1.25, MeOH). H NMR

D

(D₂O) δ: 3.65 (s, 3H), 2.30-2.00 (m, 6H), 1.56 (m, 1H). ¹³C

NMR (D₂O) δ: 174.4, 172.9, 65.9, 52.6, 32.6, 30.0, 29.2, 26.2,

21.2.

The 6-methyl ester derivative (8.6 g, 43.2 mmol), di-tert-

butyl dicarbonate (18.8 g, 86.3 mmol), and potassium carbon-

ate (11.9 g, 86.3 mmol) were dissolved in dioxane (200 mL)

and water (100 mL). The solution was stirred at room

temperature for 2 days. Hydrochloric acid (1 N) was added

dropwise to give pH 1, and it was extracted with ethyl acetate.

The organic layer was washed with brine, dried over MgSO₄,

filtered, and concentrated to provide 3 as a white solid (9.7 g,

75% yield): mp 164-165 °C. [R]²⁵-30° (c 1.6, MeOH). ¹H

D

NMR (CDCl₃) δ: 3.65 (s, 3 H), 2.45-1.87 (m, 5 H), 1.71 (t, J )

2.9 Hz, 1 H), 1.41 (s, 9 H), 1.30-1.09 (m, 1 H). ¹³C NMR

(CDCl₃) δ: 177.8, 172.9, 156.2, 81.6, 66.4, 51.7, 34.7, 32.5, 28.4,

28.1 (3C), 26.4, 21.0.

(1S,2S,5R,6S)-3-Allyloxycarbonyl-5-oxooxazolidine-4-

spiro-2′-bicyclo[3,1,0]-hexane-6′-carboxylic Acid (4). The

Alloc derivative of 1 (see synthesis of 2) (19.2 g, 64.3 mmol),

paraformaldehyde (7.7 g, 257.3 mmol), and p-toluenesulfonic

acid (0.61 g, 3.2 mmol) were refluxed in 200 mL of benzene

with azeotropic removal of water for 2 h. The mixture was

cooled to room temperature, diluted with 200 mL of ethyl

acetate, washed with brine, dried over MgSO₄, filtered, and

concentrated to afford a slightly hygroscopic solid (17.6 g, 97%).

¹H NMR (CDCl₃) δ: 6.10-5.90 (m, 1 H), 5.41-5.21 (m, 4 H),

4.65 (dt, J ) 5.6, 1.3 Hz, 2 H), 2.51-2.49 (m, 1 H), 2.33-2.18

(m, 2 H), 2.04-1.92 (m, 3 H), 1.75-1.70 (m, 1 H). ¹³C NMR

(CD₃OD) δ: 176.1, 175.4, 153.0, 133.5, 118.9, 78.4, 67.8, 67.4,

33.1, 27.2 (×2), 26.0, 23.9.

(1S,2S,5R,6S)-2-Aminobicyclo[3.1.0]hexane-2,6-dicar-

boxylic Acid Dimethyl Ester Hydrochloride (5). Thionyl

chloride (807 mL, 11.1 mol) was added to methanol (9.5 L) over

a period of 1 h while maintaining the temperature between 2

and 20 °C. The solution was maintained for 30 min, then

(1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid

monohydrate 1 (1.61 kg, 7.92 mol) was added. The resulting

solution was heated to 47 °C and maintained between 47 and

50 °C for 17 h. An amount of approximately 7.3 L of methanol

was then removed by vacuum distillation (47-50 °C, 240-

275 mmHg). The remaining methanol was removed by azeo-

tropic distillation with tert-butyl methyl ether (MTBE) at

atmospheric pressure (added MTBE (10 L), removed 8.5 L;

added MTBE (10 L), removed 8.5 L; added MTBE (8 L),

removed 5.1 L). During the course of the distillations a white

solid began to precipitate from the solution. After completion

of the distillations, MTBE (2 L) was added to the resulting

slurry, which was cooled to 22 °C. The solid was filtered, rinsed

with MTBE (2 L), and dried under vacuum to afford 1.94 kg

If further purification was needed, the crude amino diacid

was chromatographed over a C8 reverse-phase support, eluting

with acetonitrile in water with 0.05% of trifluoroacetic acid to

provide, after drying, the amino diacid as a zwitterion.

(1S,2S,5R,6S)-2-Amino-2-[(1′S)-carboxyethyl]carbamoyl-

bicyclo[3.1.0]hexane-6-carboxylic Acid (8a). (1S,2S,5R,6S)-

2-Allyloxycarbonylamino-2-[(1′S)-ethoxycarbonylethyl]-

carbamoylbicyclo[3.1.0]hexane-6-carboxylic Acid Allyl

Ester (6a). The reaction of ^L-alanine ethyl ester and 2 as

described in general procedure A provided compound 6a as a

white solid that was used for the next step without further

purification (92% yield).

(98%) of 5 as a white solid: mp 193-194 °C. [R]²⁵+22° (c

D

1.0, MeOH). ¹H NMR (D₂O) δ: 3.86 (s, 3H), 3.67 (s, 3H), 2.31-

2.04 (m, 6H), 1.57 (m, 1H). ¹³C NMR (methanol-d₄) δ: 171.9,

170.2, 65.6, 52.8, 51.2, 32.4, 29.9, 28.5, 26.2, 20.7. Anal. (C₁₀H₁₆-

NO₄Cl) C, H, N, Cl.

The allyl groups of compound 6a were removed following

general procedure B. The residue was suspended in 2 mL of

THF, and 4 mL of 2.5 N LiOH was added. The reaction mixture

was stirred for 2 h, acidified to pH 3 with 6 N HCl, and

evaporated to dryness. Purification by ion exchange chroma-

General Procedure A: Coupling 2-5 with Protected

Amino Acids. In a round-bottom flask intermediates 2-5 (1.0

equiv), N-ethyl-N′-dimethylaminopropylcarbodiimide (1.4 equiv),

(dimethylamino)pyridine (0.1 equiv), N-hydroxybenzotriazole

(1.2 equiv), the protected amino acid (1.2 equiv), and triethy-

lamine (2 equiv) were dissolved in dichloromethane (0.1 M

tography provided 8a as a white solid (38% yield): mp >300

1

°C (changed color at 290 °C). [R]²⁵-55° (c 0.5, 1 N HCl). H

D

NMR (D₂O) δ: 4.12 (q, J ) 7.3 Hz, 1 H), 2.14-2.13 (m, 2 H),

2.00-1.90 (m, 3 H), 1.79 (t, J ) 3.0 Hz, 1 H), 1.59-1.41 (m, 1

Dipeptides as Effective Prodrugs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5315

H), 1.25 (d, J ) 7.3 Hz, 3 H). ¹³C NMR (D₂O) δ: 174.8, 177.2,

Compound 6d was deprotected as described in the general

procedure B. The solid was stirred in MeOH for 15 min and

filtered to provide 8d as a white solid (56% yield): mp >300

170.1, 66.1, 50.8, 30.9, 29.7, 28.4, 24.9, 22.8, 16.8.

(1S,2S,5R,6S)-2-Amino-2-[(1′S)-carboxy-3′-methylbutyl]-

carbamoylbicyclo[3.1.0]hexane-6-carboxylic Acid (8b).

(1S,2S,5R,6S)-2-Allyloxycarbonylamino-2-[(1S)-allyloxy-

carbonyl-3-methylbutyl]carbamoylbicyclo[3.1.0]hexane-

6-carboxylic Acid Allyl Ester (6b). ^L-Leucine allyl ester

hydrochloride (1.3 equiv), (dimethylamino)pyridine (0.1 equiv),

a slurry of 5-([1,4′]bipiperidinyl-1′-sulfonyl)benzotriazol-1-ol in

20 mL of dimethylformamide previously heated at 60 °C (1.2

equiv), a solution of N-ethyl-N′-dimethylaminopropylcarbodi-

imide (1.4 equiv) and 2 (1.0 equiv) in 20 mL of dichloromethane

and triethylamine (1.2 equiv) were mixed in a screw-capped

tube and stirred in an orbital agitator overnight. Dichlo-

romethane was removed under vacuum, and the DMF solution

was diluted with 150 mL of ethyl acetate and washed 1 N HCl

and brine. The organic layer was dried over MgSO₄, filtered,

and concentrated to provide 6b (98% yield) as an oil that was

°C. [R]²⁵-48° (c 1, 1 N HCl). ¹H NMR (DMSO-d₆) δ: 4.05

D

(m, 1 H), 2.18-1.85 (m, 7 H), 1.28-1.17 (m, 1 H), 0.86 (d, J )

6.4 Hz, 3 H), 0.84 (d, J ) 6.5 Hz, 3 H). ¹³C NMR (DMSO-d₆) δ:

174.2, 173.4, 173.2, 65.7, 58.1, 35.4, 33.3, 30.9, 28.7, 26.8, 21.4,

19.7, 18.3.

(1S,2S,5R,6S)-2-Amino-2-[(1′S)-carboxymethyl]car-

bamoylbicyclo[3.1.0]hexane-6-carboxylic Acid (8e).

(1S,1′S,2S,5R,6S)-2-Allyloxycarbonylamino-2-(ethoxy-

carbonylmethylcarbamoyl)bicyclo[3.1.0]hexane-6-carbox-

ylic Acid Allyl Ester (6e). The reaction of glycine ethyl ester

and 3 as described in general procedure A provided compound

6e as a white solid that was used for the next step without

further purification (76% yield). ¹H NMR (CDCl₃) δ: 7.02 (m,

1 H), 5.99-5.94 (m, 2 H), 5.64 (bs, 1 H), 5.34-5.16 (m, 4 H),

4.54 (dt, J ) 5.6, 1.1 Hz, 4 H), 4.19 (q, J ) 7.3 Hz, 2 H), 4.07-

3.99 (m, 2 H), 2.52 (b, J ) 3.5 Hz, 1 H), 2.42 (dd, J ) 13.2, 8.3

Hz, 1 H), 2.21-2.07 (m, 2 H), 1.94 (dd, J ) 12.9, 7.8 Hz, 1 H),

1.74 (t, J ) 3.0 Hz, 1 H), 1.27 (t, J ) 7.0 Hz, 3 H), 1.30-1.15

(m, 1H).

1

used for the next step without further purification. H NMR

(CDCl₃) δ: 7.13 (bs, 1 H), 5.95-5.79 (m, 2 H), 5.60 (s, 1 H),

5.34-5.07 (m, 7 H), 4.63-4.50 (m, 6 H), 2.49-2.34 (m, 3 H),

2.10-1.85 (m, 4 H), 1.71 (t, J ) 2.7 Hz, 1 H), 1.41 (d, J ) 7.3

Hz, 3 H), 1.28-1.12 (m, 1 H).

Compound 6e was deprotected as described in the general

procedure B. The residue was suspended in 2 mL of THF, and

an amount of 4 mL of 2.5 N aqueous lithium hydroxide was

added. The reaction mixture was stirred for 2 h, acidified to

pH 3 with 6 N HCl, and concentrated to dryness. Purification

by ion exchange chromatography provided 8e as a white solid

Compound 6b (1.0 equiv) was dissolved in dry dichlo-

romethane (0.1 M solution) under nitrogen. 1,3-Dimethylbar-

bituric acid (6.0 equiv) and tetrakis(triphenylphosphine)-

palladium(0) (0.03 equiv) were added, and the solution was

heated at 35 °C for 2 h. After the mixture was cooled to room

temperature, the solvent was removed under vacuum and the

resulting residue was dissolved in a solution of ethyl acetate

saturated with hydrogen chloride gas and stirred for 2 h. The

reaction mixture was filtered, and the filtrate was washed with

ethyl acetate and ether and dried to provide 8b as a white

solid (50% yield): mp 130.5-131.7 °C. ¹H NMR (DMSO-d₆/

TFA-d) δ: 4.45-4.38 (m, 1 H), 2.27-2.21 (m, 2 H), 2.11-1.90

(m, 4 H), 1.77-1.54 (m, 4 H), 0.95 (dd, J ) 6.18, 5.10 Hz, 6

H). ¹³C NMR (DMSO-d₆/TFA-d) δ: 173.7, 173.2, 170.1, 65.9,

51.2, 31.7, 31.1, 29.5, 25.7, 24.9, 23.4, 21.6, 21.2. MS (electro-

spray), m/z: 299.16 (M⁺+ H).

(70% yield): mp 240 °C (with decomposition). [R]²⁵-40° (c

D

1

0.5, 1 N HCl). H NMR (D₂O + pyridine-d₅) δ: 3.75 (s, 2 H),

2.10-2.00 (m, 5 H), 1.76 (t, J ) 2.7 Hz, 1 H), 1.68-1.55 (m, 1

H). ¹³C NMR (D₂O + pyridine-d₅) δ: 179.0, 175.9, 171.2, 66.5,

43.6, 31.1, 29.5, 27.9, 25.0, 24.3.

(1S,2S,5R,6S)-2-Amino-2-[(1′S)-carboxy-3′-methylthio-

propyl]carbamoylbicyclo[3.1.0]hexane-6-carboxylic Acid

(8f). (1S,2S,5R,6S)-2-Allyloxycarbonylamino-2-[(1′S)-meth-

oxycarbonyl-3′-methylthiopropyl]carbamoylbicyclo[3.1.0]-

hexane-6-carboxylic Acid Allyl Ester (6f). The reaction of

L-methionine methyl ester hydrochloride and 2 as described

in general procedure A provided compound 6f as a white foamy

solid that was used for the next step without further purifica-

tion (90% yield). ¹H NMR (CDCl₃) δ: 7.21 (brs, 1 H), 5.89 (m,

2 H), 5.40-5.20 (m, 4 H), 4.67 (m, 1 H), 4.52 (d, 4 H, J ) 7.0

Hz), 3.74 (s, 3 H), 2.55-2.37 (m, 4 H), 2.08 (s, 3 H), 2.30-1.89

(m, 5 H), 1.71 (t, 1 H, J ) 2.4 Hz), 1.21 (m, 1 H). ¹³C NMR

(CDCl₃) δ: 172.5, 172.2, 171.9, 155.7, 132.4, 131.9, 118.3, 117.9,

67.0, 65.9, 65.3, 52.4, 51.5, 34.3, 32.7, 31.2, 28.9, 26.4, 21.2,

15.3.

(1S,1′S,2S,5R,6S)-2-Amino-2-[(1′S)-carboxy-2-phenyl-

ethyl]carbamoylbicyclo[3.1.0]hexane-6-carboxylic Acid

(8c). (1S,2S,5R,6S)-2-Allyloxycarbonylamino-2-[(1′S)-al-

lyloxycarbonyl-2′-phenylethyl]carbamoylbicyclo[3.1.0]-

hexane-6-carboxylic Acid Allyl Ester (6c). The reaction of

L-phenylalanine allyl ester and 2 as described in general

procedure A provided compound 6c as a white solid that was

used for the next step without further purification (79%

1

yield): mp 220 °C (with decomposition). H NMR (CDCl₃) δ:

7.27-7.08 (m, 5 H), 7.01-6.97 (bd, 1 H), 5.96-5.75 (m, 3 H),

5.64 (bs, 1 H), 5.32-5.17 (m, 6 H), 4.83 (c, J ) 6.4 Hz, 1 H),

4.59-4.49 (m, 6 H), 3.17-3.09 (m, 2 H), 2.42 (bs, 1 H), 2.30

(dd, J ) 13.2, 7.5 Hz, 1 H), 2.03-1.84 (m, 3 H), 1.66 (t, J )

3.0 Hz, 1H), 1.24-1.08 (m, 1H).

Compound 6c was deprotected as described in the general

procedure B. The solid was stirred in MeOH for 15 min and

filtered to provide 8c as a white solid (48% yield): mp >300

°C. ¹H NMR (D₂O) δ: 7.07-7.04 (m, 5 H), 4.35-4.32 (m, 1 H),

3.15-3.08 (m, 1 H), 2.85-2.74 (m, 1 H), 1.88 (m, 1 H), 1.74-

1.74 (m, 1 H), 1.52-1.10 (m, 5 H). ¹³C NMR (DMSO-d₆) δ:

174.0, 173.3, 171.6, 138.5, 129.7 (2C), 128.3 (2C), 126.5, 65.6,

54.7, 37.0, 33.8, 32.8, 28.6, 26.1, 21.7.

Compound 6f was deprotected as described in the general

procedure B. The residue obtained was dissolved in 10 mL of

2.5 N lithium hydroxide and stirred overnight. The solution

was acidified to pH 2 with 1 N aqueous HCl and extracted

with ethyl acetate (4×). The combined organic extracts were

dried over MgSO₄and concentrated to dryness. The resulting

material was purified by anion exchange chromatography to

provide 8f as a white solid (50% yield): mp 72-74 °C. [R]²⁵

D

1

-62° (c 0.95, 1 N HCl). H NMR (D₂O) δ: 4.26 (dd, 1 H, J )

8.0, 4.6 Hz), 2.54-2.31 (m, 2 H), 2.00 (s, 3 H), 2.10-1.85 (m,

7 H), 1.77 (t, 1 H, J ) 2.7 Hz), 1.58 (m, 1 H). ¹³C NMR (D₂O)

δ: 178.4, 177.3, 170.6, 66.4, 54.6, 31.2, 30.2, 29.7, 29.5, 28.0,

25.3, 23.9, 14.2. MS (electrospray), m/z: 317 (M⁺+ 1).

(1S,2S,5R,6S)-2-Amino-2-[(1′S)-carboxy-2′-methylpropyl]-

carbamoylbicyclo[3.1.0]hexane-6-carboxylic Acid (8d).

(1S,2S,5R,6S)-2-Allyloxycarbonylamino-2-[(1′S)-allyloxy-

carbonyl-2′-methylpropyl]carbamoylbicyclo[3.1.0]hexane-

6-carboxylic Acid Allyl Ester (6d). The reaction of ^L-valine

allyl ester and 2 as described in general procedure A provided

compound 6d as a white solid that was used for the next step

(1′S,2′S,5′R,6′S)-2′-Amino-6′-carboxybicyclo[3.1.0]-

hexane-2′-carbonylpyrrolidine-2S-carboxylic Acid (8g).

(1S′,2S′,5R′,6S′)-1-(6′-Allyloxycarbonyl-2′-allyloxycarbo-

nylamino-2′-bicyclo[3.1.0]hexane-2′-carbonylpyrrolidine-

2S carboxylic Acid Methyl Ester (6g). The reaction of

L-proline methyl ester hydrochloride and 2 as described in

general procedure A provided compound 6g as a white foamy

solid that was used for the next step without further purifica-

tion (69% yield). ¹H NMR (CDCl₃) δ: 5.87 (m, 2 H), 5.25 (m, 4

H), 4.54 (d, 4 H, J ) 5.9 Hz), 3.70 (s, 3 H), 3.69 (m, 1 H), 2.54

1

without further purification (82% yield). H NMR (CDCl₃) δ:

5.88 (m, 3H), 5.49-5.17 (m, 7H), 4.64-4.51 (m, 7H), 2.55 (bs,

1H), 2.43 (dd, J ) 13.2, 8.3 Hz, 1H), 2.30-1.87 (m, 4H), 1.71

(t, J ) 2.7 Hz, 1H), 1.29-1.13 (m, 1H), 0.96 (d, J ) 6.7 Hz,

3H), 0.90 (d, J ) 7.0 Hz, 3H).

(m, 2 H), 2.13-1.85 (m, 7 H), 1.64 (m, 1 H), 1.19 (m, 1 H). ¹³

NMR (CDCl₃) δ: 172.8, 172.2, 170.0, 155.8, 154.7, 132.5, 132.0,

C

5316 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16

Bueno et al.

118.0, 117.7, 66.8, 65.6, 65.1, 60.4, 51.9, 47.5, 34.6, 32.5, 31.5,

28.9, 27.8, 25.4, 20.8.

The reaction mixture was stirred at room temperature for 1

h, the solvent was evaporated, and the residue was purified

by chromatography (eluent EtOAc). The solid was subjected

to the general procedure of deprotection C. The final hydro-

chloride was purified by HPLC to provide 8j as a very

hygroscopic white solid (11% yield). [R]²⁵_D-14° [c 1, 1 N HCl].

¹H NMR (CDCl₃) δ: 4.01-3.71 (m, 3 H), 2.39 (dd, J ) 6.4, 3.0

Hz, 1 H), 2.19-1.86 (m, 4 H), 1.59 (t, J ) 2.7 Hz, 1 H), 1.41-

1.25 (m, 1 H). ¹³C NMR (D₂O) δ: 177.0, 175.9, 167.8, 66.4, 60.2,

54.4, 34.2, 31.4, 29.1, 25.5, 20.7. MS (electrospray) m/z: 273

(M⁺+ 1).

Compound 6g was deprotected as described in the general

procedure B, providing 8g as a white solid (89% yield): mp

274 °C. [R]²⁵_D-103° (c 0.92, 1 N HCl). ¹H NMR (D₂O) δ: 4.25

(m, 1 H), 2.08 (m, 2 H), 1.82-2.30 (m, 10 H), 1.70 (m, 1 H),

1.44 (m, 1 H). ¹³C NMR (D₂O) δ: 172.1, 169.5, 125.7, 67.1, 58.6,

45.8, 32.7, 32.3, 27.9, 27.1, 25.7, 22.3, 21.8. MS (electrospray),

m/z: 303, 265.

(1S,2S,5R,6S)-2-Amino-2-[(1′S)-carboxy-(2′R)-methyl-

butyl]carbamoylbicyclo[3.1.0]hexane-6-carboxylic Acid

(8h). (1S,2S,5R,6S)-2-Allyloxycarbonylamino-2-[(1′S)-al-

lyloxycarbonyl-(2′R)-methylbutyl]carbamoylbicyclo[3.1.0]-

hexane-6-carboxylic Acid Allyl Ester (6h). The reaction

of ^L-isoleucine allyl ester and 2 as described in general

procedure A provided compound 6h as a white solid that was

used for the next step without further purification (72% yield).

¹H NMR (CDCl₃) δ: 5.90-5.87 (m, 3 H), 5.53 (bs, 1 H), 5.37-

5.16 (m, 6 H), 4.63-4.52 (m, 7 H), 2.53 (bs, 1 H), 2.40 (dd, J )

13.4, 8.3 Hz, 1 H), 2.12-1.87 (m, 4 H), 1.70 (t, J ) 3.0 Hz, 1

H), 1.48-1.05 (m, 3 H), 0.90 (t, J ) 7.3 Hz, 3 H), 0.90 (d, J )

7.0 Hz, 3 H).

Compound 6h was deprotected as described in the general

procedure B. The residue was purified by cation exchange

chromatography, eluting with 2 N ammonia in methanol. The

yellow solid obtained was stirred with methanol for 15 min,

filtered, and dried to provide 8h as a white solid (31% yield):

mp >300 °C (changed color at 291 °C). [R]²⁵_D-40° (c 0.5, 1 N

HCl). ¹H NMR (DMSO-d₆) δ: 4.03 (bs, 1 H), 1.82 (m, 7 H),

1.54-1.38 (m, 1 H), 1.29-1.02 (m, 2 H), 0.87-0.80 (m, 6 H).

¹³C NMR (DMSO-d₆) δ: 174.4, 173.5, 173.2, 65.7, 57.6, 37.7,

35.7, 33.7, 28.5, 27.0, 25.2, 21.6, 16.1, 12.0.

(1S,2S,5R,6S)-2-Amino-2-[(1′S)-carboxy-2′-(4′′-hydroxy-

phenyl)ethyl]carbamoylbicyclo[3.1.0]hexane-6-carboxylic

Acid (8i). (1S,2S,5R,6S)-2-tert-Butoxycarbonylamino-2-

[2-(4-hydroxyphenyl)-1′S-methoxycarbonylethyl]carbam-

oylbicyclo[3.1.0]hexane-6-carboxylic Acid Methyl Ester

(7i). The reaction of l-tyrosine methyl ester hydrochloride and

3 as described in general procedure A provided compound 7i

as a white solid that was used for the next step without further

purification (95% yield). ¹H NMR (CDCl₃) δ: 6.98 (d, 2 H, J )

8.2 Hz), 6.73 (d, 2 H, J ) 8.3 Hz), 5.11 (brs, 1 H), 4.81 (dd, 1

H, J ) 7.5, 6.1 Hz), 3.71 (s, 3 H), 3.67 (s, 3 H), 3.15 (dd, 1 H,

J ) 13.7, 6.2 Hz), 2.99 (dd, 1 H, J ) 13.9, 6.2 Hz), 2.45 (dd, 1

H, J ) 12.6, 7.5 Hz), 2.25-1.82 (m, 4 H), 1.64 (t, 1 H, J ) 2.9

Hz), 1.40 (s, 9 H), 1.12 (m, 1 H). ¹³C NMR (CDCl₃) δ: 173.0,

172.0, 155.5, 155.5, 155.2, 130.2 (2 C), 127.0, 115.6 (2 C), 80.7,

67.0, 53.4, 52.2, 51.8, 37.1, 35.2, 32.8, 28.7, 28.0 (3 C), 26.8,

21.3.

Because of the hygroscopicity of the compound, it was

dissolved in 10 mL of a solution of saturated HCl(g) in EtOAc

and stirred for 2 h. The white precipitate was filtered and

washed with EtOAc. The 8j‚HCl salt was more stable to

moisture than the corresponding zwitterion: mp 197-200 °C.

IR (KBr): 3436, 3213, 1711, 1684 cm^-1

.

(1S,2S,5R,6S)-2-Amino-6-[(1′S)-carboxy-2′-phenylethyl]-

carbamoylbicyclo[3.1.0]hexane-2-carboxylic Acid Hy-

drochloride (10‚HCl). (1S,2S,5R,6S)-3-Allyloxycarbonyl-

5-oxo-4-spiro-6′-[(1′′S)-carboxy-2′′-phenylethyl]carbamoyl-

2′(bicyclo[3,1,0]hexane)oxazolidine (9). The reaction of

L-phenylalanine allyl ester and 4 as described in general

procedure A provided compound 9 as a white foamy solid

(47%). ¹H NMR (CD₃OD) δ: 7.33-7.24 (m, 3 H), 7.15-7.10

(m, 2 H), 6.17 (d, J ) 7.8 Hz, 1 H), 6.00-5.75 (m, 2 H), 5.34-

5.18 (m, 6 H), 4.87 (dt, J ) 8.1, 5.9 Hz, 1 H), 4.61-4.57 (m, 4

H), 3.12 (d, J ) 6.2 Hz, 2 H), 2.43-2.15 (m, 3 H), 2.07-1.86

(m, 3 H), 1.77-1.70 (m, 1 H).

Compound 9 was deprotected as described in the general

procedure B. The residue was dissolved in a solution of ethyl

acetate saturated with hydrogen chloride gas and stirred for

2 h. The reaction mixture was filtered, and the filtrate was

washed with ethyl acetate and ether providing 10‚HCl as a

1

white solid (91%). H NMR (CD₃OD) δ: 7.33-7.23 (m, 5 H),

4.71 (dd, J ) 8.41, 5.40 Hz, 1 H), 3.22 (dd, J ) 13.81, 5.20 Hz,

1 H), 3.00 (dd, J ) 14.01, 8.41 Hz, 1 H), 2.21-1.86 (m, 6 H),

1.58-1.43 (m, 1 H). ¹³C NMR (CD₃OD) δ: 173.8, 171.5, 171.3,

128.9 128.0, 126.4, 65.4, 53.6, 37.1, 31.4, 30.1, 27.4, 26.3, 22.2.

(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)propionyl]aminobi-

cyclo[3.1.0]hexane-2,6-dicarboxylicAcid(12a).(1S,2S,5R,6S)-

2-[(2′S)-(2′-tert-Butoxycarbonylamino)propionyl]-

aminobicyclo[3.1.0]hexane-2,6-dicarboxylic Acid Dimeth-

yl Ester (11a). The reaction of 5 with N-Boc-^L-alanine

following general procedure A provided 11a as a foamy white

solid (50% yield): mp 51-52 °C. [R]²⁵-28° (c 0.52, CHCl₃).

D

¹H NMR (CDCl₃) δ: 7.28 (bs, 1H), 5.04 (brd, 1H, J ) 7.6 Hz),

4.16 (m, 1H), 3.74 (s, 3H), 3.66 (s, 3H), 2.49 (dd, 1H, J ) 13.9,

8.3 Hz), 2.42 (dd, 1H, J ) 6.3, 2.8 Hz), 2.18-1.89 (m, 3H), 1.70

(t, 1H, J ) 2.9 Hz), 1.45 (s, 9H), 1.33 (d, 3H, J ) 7.0 Hz), 1.19

(m, 1H). ¹³C NMR (CDCl₃) δ: 172.8, 172.6, 172.6, 155.7, 80.2,

66.3, 52.6, 51.8, 49.5, 34.4, 32.0, 28.2, 28.1, 26.6, 21.1, 17.6.

Compound 7i was deprotected as described in the general

procedure C to provide 8i as a white solid (87% yield): mp

1

174-176 °C. [R]²⁵-47° (c 1.1, 1 N HCl). H NMR (D₂O) δ:

D

7.13 (d, 2 H, J ) 8.0 Hz), 6.80 (d, 2 H, J ) 6.8 Hz), 4.79 (m, 1

H), 3.32 (dd, 1 H, J ) 14.2, 5.4 Hz), 2.96 (dd, 1 H, J ) 14.2,

10.7), 2.25-2.10 (m, 2 H), 1.92-1.79 (m, 2 H), 1.59-1.41 (m,

3 H). ¹³C NMR (CD₃OD) δ: 173.1, 173.0, 169.5, 156.0, 129.7,

114.9, 65.9, 53.7, 35.2, 31.1, 30.4, 28.9, 24.9, 21.4. MS (elec-

trospray), m/z: 349 (M⁺+ 1), 332.

(1S,2S,5R,6S)-2-Amino-2-[(1′S)-carboxy-(2′R)-hydroxy-

propyl]carbamoylbicyclo[3.1.0]hexane-6-carboxylic Acid

Hydrochloride (8j). (1S,2S,5R,6S)-Methyl 2-[(2′S)2-tert-

Butoxycarbonylamino-3-(tert-butyldimethylsilanyloxy)-

propionylamino]bicyclo[3.1.0]hexane-2,6-dicarboxy-

late (7j). The reaction of (2S)-2-amino-3-(tert-butyldimethyl-

silanyloxy)propionic acid methyl ester³⁰and 3 as described in

general procedure A provided compound 7j as a white foamy

solid (82% yield). ¹H NMR (CDCl₃) δ: 7.44 (bs, 1 H), 5.38 (bs,

1 H), 4.16-4.05 (m, 1 H), 3.94 (dd, J ) 9.7, 4.3 Hz, 1 H), 3.72

(s, 3 H), 3.65 (s, 3 H), 3.60-3.51 (m, 1 H), 2.64 (dd, J ) 14.0,

8.6 Hz, 1 H), 2.30-2.11 (m, 2 H), 2.03-1.97 (m, 2 H), 1.72 (m,

1 H), 1.43 (s, 9 H), 1.26-1.09 (m, 1 H), 0.88 (s, 9 H), 0.11 (s, 3

H), 0.10 (s, 3 H).

Compound 11a was deprotected following general procedure

D to provide 12a‚HCl as a white solid (80% yield): mp >250

°C, dec. [R]²⁵_D-7.8° (c 1.0, MeOH). ¹H NMR (CD₃OD) δ: 3.96

(q, 1H, J ) 7.0 Hz), 2.47 (dd, 1H, J ) 6.3, 2.7 Hz), 2.37 (dd,

1H, J ) 13.6, 8.2 Hz), 2.18-1.92 (m, 3H), 1.66 (t, 1H, J ) 3.0

Hz), 1.53 (d, 3H, J ) 7.0 Hz), 1.46-1.34 (m, 1H). ¹³C NMR

(CD₃OD) δ: 175.2, 174.7, 170.2, 66.4, 49.0, 36.6, 32.0, 28.5,

26.3, 21.2, 16.6.

For complete characterization it was transformed into the

crystalline sulfonate salt (12a‚HSO₃Me). (1S,2S,5R,6S)-2-

[(2′S)-(2′-Amino)propionyl]aminobicyclo[3.1.0]hexane-2,6-dicar-

boxylic acid hydrochloride (12a‚HCl) (1.0 g, 3.42 mmol) was

dissolved in water (1 mL), and 1.0 N NaOH (3.42 mL, 3.42

mmol) was added. The solution was maintained in the

refrigerator for 24 h. The solution remained clear. Acetone (2

mL) was added, and the solution was stored in the refrigerator

for 16 h. A white solid precipitated out of solution, and the

mixture could not be stirred. Acetone (4 mL) was added, and

the mixture was stirred at room temperature, then filtered

and dried to afford 630 mg of the title compound as a white

crystalline solid corresponding to the zwitterion (72% yield),

which contained 2-4% NaCl. ¹H NMR (CD₃OD) δ: 3.93 (q, J

Compound 7j was dissolved in dry THF (10 mL) under

nitrogen, and 1 M tetrabutylammonium fluoride was added.

Dipeptides as Effective Prodrugs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5317

2.60 (1H, bs), 2.30-1.70 (8H, m), 1.43 (9H, s), 0.99-0.90 (6H,

) 7.1 Hz, 1 H), 2.48 (dd, J ) 6.6, 2.9 Hz, 1 H), 2.32 (dd,, J )

13.5, 8.4 Hz, 1 H), 2.20-2.08 (m, 1 H), 2.01-1.90 (m, 2 H),

1.61 (t, J ) 2.9 Hz, 1 H), 1.51 (d, J ) 7.0 Hz, 3 H), 1.48-1.33

(m, 1 H). ¹³C NMR (CD₃OD) δ: 176.9 (2 C), 171.1, 68.0, 50.1,

35.9, 33.2, 29.7, 27.3, 22.5, 17.6.

t

m). MS m/z: 327 [M + H - CO₂Bu].

Compound 11d was deprotected following general procedure

1

D to provide 12d‚HCl as a white solid (71% yield). H NMR

(CD₃OD) δ: 3.62 (1H, t, 7.2 Hz), 3.02 (1H, t), 2.18 (1H, dd, 2.3

Hz, 6.4 Hz), 2.04-1.91 (1H, m), 1.90-0.80 (7H, m), 0.68 (6H,

2 × t, 6.7 Hz, 6.8 Hz). MS m/z: 299 [M + H].

A solution of 12a (1.07 g, 3.00 mmol), methanesulfonic acid

(0.584 mL, 9.00 mmol), and dioxane (10 mL) was stirred for

48 h. The mixture was filtered and dried to afford 12a‚HSO₃-

Me as a crude, white, amorphous solid (1.05 g). A sample of

this solid (1.0 g) was dissolved in MeOH (10 mL). The solution

was concentrated to 3.3 g total weight, and seed crystals were

added. Ethyl acetate (10 mL) was then added to the mixture

over a period of 15 min. The mixture was stirred for 30 min,

filtered, and dried under vacuum to afford 830 mg of the title

(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-3′-phenylpropionyl]amino-

bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid Hydrochlo-

ride (12e‚HCl). (1S,2S,5R,6S)-2-[(2′S)-(2′-tert-Butoxycar-

bonylamino)-3′-phenylpropionyl]aminobicyclo[3.1.0]-

hexane-2,6-dicarboxylic Acid Dimethyl Ester (11e). The

reaction of 5 with N-Boc-^L-phenylalanine following general

procedure A provided 11e as a foamy white solid (87% yield):

1

mp 60-61 °C. [R]²⁵-2° (c 1.0, CHCl₃). H NMR (CDCl₃) δ:

1

compound as a white, crystalline solid (78% yield). H NMR

D

7.33-7.21 (m, 5H), 6.64 (bs, 1H), 5.13 (brd, 1H, J ) 6.9 Hz),

4.32 (q, 1H, J ) 7.0 Hz), 3.72 (s, 3H), 3.66 (s, 3H), 3.03 (d, 2H,

J ) 7.1 Hz), 2.48-2.38 (m, 2H), 2.08-1.84 (m, 3H), 1.60 (t,

1H, J ) 2.9 Hz), 1.41 (s, 9H), 1.14-1.02 (m, 1H). ¹³C NMR

(CDCl₃) δ: 172.5, 172.4, 171.3, 155.4, 136.8, 129.4, 128.5, 126.7,

80.1, 66.1, 55.3, 52.5, 51.7, 38.1, 34.1, 31.7, 28.2, 28.0, 26.3,

20.9.

(CD₃OD) δ: 3.96 (q, J ) 7.1 Hz, 1 H), 2.71 (s, 3 H), 2.45 (dd,

J ) 6.4, 2.7 Hz, 1 H), 2.38 (dd, J ) 13.9, 8.4 Hz, 1 H), 2.20-

2.08 (m, 1 H), 2.01-1.93 (m, 2 H), 1.67 (t, J ) 2.9 Hz, 1 H),

1.52 (d, J ) 7.0 Hz, 3 H), 1.46-1.35 (m, 1 H). ¹³C NMR (CD₃-

OD) δ: 176.3, 175.7, 171.2, 67.4, 50.0, 39.5, 35.7, 33.1, 29.5,

27.4, 22.2, 17.6. Anal. (C₁₂H₂₀N₂O₈S) C, H, N.

(1S,2S,5R,6S)-2-[(2′R)-(2′-Amino)propionyl]aminobi-

cyclo[3.1.0]hexane-2,6-dicarboxylic Acid Hydrochloride

(12b‚HCl).(1S,2S,5R,6S)-2-[(2′R)-tert-Butoxycarbonylamino)-

propionyl]aminobicyclo[3.1.0]hexane-2,6-dicarboxylic

Acid Dimethyl Ester (11b). The reaction of 5 with N-Boc-

D-alanine following general procedure A provided 11b as a

Compound 11e was deprotected following general procedure

D to provide 12e‚HCl as a white solid (44% yield): mp 122-

123 °C. [R]²⁵-2° (c 1.0, MeOH). ¹H NMR (methanol-d₄) δ:

D

7.41-7.26 (m, 5H), 4.13 (dd, 1H, J ) 7.6, 6.1 Hz), 3.28 (dd,

1H, J ) 14.2, 6.2 Hz), 3.07 (dd, 1H, J ) 14.2, 7.6 Hz), 2.55

(dd, 1H, J ) 6.4, 2.8 Hz), 2.31-1.89 (m, 4H), 1.64 (t, 1H, J )

2.8 Hz), 1.35 (m, 1H). ¹³C NMR (methanol-d₄) δ: 178.8, 178.3,

172.2, 137.9, 133.2, 132.6, 131.3, 70.0, 57.9, 41.0, 37.7, 35.4,

32.3, 29.4, 24.7.

1

foamy white solid (93% yield). H NMR (CDCl₃) δ: 7.03 (s, 1

H), 5.00 (s, 1 H), 4.12 (br m, 1 H), 3.72 (s, 3 H), 3.64 (s, 3 H),

2.52 (dd, J ) 14.0, 8.5 Hz, 1 H), 2.39 (dd, J ) 6.5, 3.0 Hz, 1

H), 2.18-2.10 (m, 1 H), 2.01 (app. quintet, J ) 3.5 Hz, 1 H),

1.94 (dd, J ) 13.5, 8.0 Hz, 1 H), 1.67 (t, J ) 3.0 Hz, 1 H), 1.44

(s, 9 H), 1.31 (d, J ) 7.0 Hz, 3 H), 1.24-1.17 (m, 1 H). ¹³C

NMR (CDCl₃) δ: 172.8, 172.7, 172.6, 155.7, 80.2, 66.1, 52.7,

51.8, 49.6, 34.4, 32.0, 28.2, 28.1, 26.6, 21.0, 17.4.

(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-3′-methylbutyryl]amino-

bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid Hydrochlo-

ride (12f‚HCl). (1S,2S,5R,6S)-2-[(2′S)-(2′-tert-Butoxycar-

bonylamino)-3′-methylbutyryl]aminobicyclo[3.1.0]hexane-

2,6-dicarboxylic Acid Dimethyl Ester (11f). The reaction

of 5 with N-Boc-^L-valine following general procedure A pro-

Compound 11b was deprotected following general procedure

1

D to provide 12b‚HCl as a white solid (80% yield). H NMR

vided 11f as a foamy white solid (87% yield): mp 63-65 °C.

(CD₃OD) δ: 3.92 (q, J ) 7.1 Hz, 1 H), 2.56 (dd, J ) 6.4, 2.7

Hz, 1 H), 2.25 (J ) 14.0, 8.5 Hz, 1 H), 2.13-2.06 (m, 1 H),

2.01 (m, 1 H), 1.95 (dd, J ) 12.8, 8.0 Hz, 1 H), 1.62 (t, J ) 3.0

Hz, 1 H), 1.51 (d, J ) 6.9 Hz, 3 H), 1.43-1.37 (m, 1 H). ¹³C

NMR (CD₃OD) δ: 176.4, 175.7, 171.2, 67.3, 50.0, 35.5, 32.8,

30.0, 26.7, 21.9, 17.7.

1

[R]²⁵-1° (c 1.16, CHCl₃). H NMR (CDCl₃) δ: 6.75 (bs, 1H),

D

5.06 (bd, 1H, J ) 8.8 Hz), 3.90 (dd, 1H, J ) 8.9, 8.8 Hz), 3.73

(s, 3H), 3.66 (s, 3H), 2.54 (dd, 1H, J ) 13.8, 8.3 Hz), 2.41 (dd,

1H, J ) 5.9, 2.4 Hz), 2.23-1.88 (m, 4H), 1.71 (t, 1H, J ) 2.9

Hz), 1.44 (s, 9H), 1.30-1.19 (m, 1H), 0.97 (d, 3H, J ) 6.8 Hz),

0.93 (d, 3H, J ) 6.8 Hz). ¹³C NMR (CDCl₃) δ: 172.5, 172.4,

171.8, 155.8, 79.8, 66.2, 59.3, 52.4, 51.7, 34.3, 31.9, 30.9, 28.2,

27.9, 26.5, 21.1, 18.9, 17.6.

(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino-4′-methyl)pentanoyl]-

aminobicyclo[3.1.0]hexane-2,6-dicarboxylic Acid Hydro-

chloride (12c‚HCl). (1S,2S,5R,6S)-2-[(2′S)-(2′-tert-Butoxy-

carbonylamino-4′-methyl)pentanoyl]aminobicyclo[3.1.0]-

hexane-2,6-dicarboxylic Acid Dimethyl Ester (11c). The

reaction of 5 with N-Boc-^L-leucine following general procedure

Compound 11f was deprotected following general procedure

E to provide 12f‚HCl as a white solid (64% yield): mp 217-

219 °C. [R]²⁵_D+ 6° (c 0.86, MeOH). ¹H NMR (D₂O) δ: 3.78 (d,

1H, J ) 5.8 Hz), 2.56 (dd, 1H, J ) 6.9, 2.9 Hz), 2.19-1.95 (m,

5H), 1.66 (t, 1H, J ) 3.0 Hz), 1.50 (m, 1H), 0.99 (d, 3H, J )

6.9 Hz), 0.98 (d, 3H, J ) 6.9 Hz). ¹³C NMR (D₂O + methanol-

d₄) δ: 177.0, 176.0, 169.3, 66.3, 58.1, 34.0, 31.3, 30.0, 29.5,

25.1, 20.9, 17.3, 16.9.

A provided 11c as a foamy white solid (95% yield): mp 62-63

1

°C. [R]²⁵-27° (c 1.0, CHCl₃). H NMR (CDCl₃) δ: 7.00 (bs,

D

1H), 4.97 (bd, 1H, J ) 8.4), 4.05 (m, 1H), 3.69 (s, 3H), 3.62 (s,

3H), 2.40 (m, 2H), 2.08-1.84 (m, 3H), 1.67-1.58 (m, 3H), 1.41

(m, 1H), 1.41 (s, 9H), 1.22 (m, 1H), 0.90 (d, 3H, J ) 6.2 Hz),

0.89 (d, 3H, J ) 6.1 Hz). ¹³C NMR (CDCl₃) δ: 172.8, 172.6,

172.5, 155.8, 80.0, 66.2, 52.5, 51.7, 40.7, 34.3, 31.9, 28.2, 28.1,

26.4, 24.6, 22.7, 22.0, 21.0.

(1S,2S,5R,6S)-2-(2-Aminoacetylamino)bicyclo[3.1.0]-

hexane-2,6-dicarboxylic Acid (12 g). (1S,2S,5R,6S)-2-[(2-

tert-Butoxycarbonylaminoacetylamino)bicyclo[3.1.0]-

hexane-2,6-dicarboxylic Acid Dimethyl Ester (11g). The

reaction of 5 with N-Boc-glycine following general procedure

Compound 11c was deprotected following general procedure

1

D to provide 12c‚HCl as a white solid (48% yield): mp 135-

A provided 11g as a foamy white solid (88% yield). H NMR

1

137 °C. [R]²⁵+0.3° (c 1.0, MeOH). H NMR (methanol-d₄) δ:

(CDCl₃) δ: 7.01 (bs, 1 H), 5.28 (bs, 1 H), 3.75 (d, J ) 5.6 Hz,

2 H), 3.72 (s, 3 H), 3.63 (s, 3 H), 2.54-2.38 (m, 2 H), 2.15-

1.87 (m, 3 H), 1.68 (t, J ) 2.9 Hz, 1 H), 1.43 (s, 9 H), 1.31-

1.11 (m, 1 H).

D

3.84 (bt, 1H, J ) 6.6 Hz), 2.52 (dd, 1H, J ) 6.2, 2.8 Hz), 2.31

(dd, 1H, J ) 13.5, 8.6 Hz), 2.21-1.89 (m, 3H), 1.74-1.59 (m,

4H), 1.44 (m, 1H), 1.02 (d, 3H, J ) 6.4 Hz), 0.99 (d, 3H, J )

6.2 Hz). ¹³C NMR (methanol-d₄) δ: 175.3, 174.9, 169.7, 66.6,

51.8, 40.6, 34.4, 32.0, 28.7, 26.1, 24.4, 21.8, 21.5, 21.3.

Compound 11g was deprotected following general procedure

D to provide 12g‚HCl as a white solid (64% yield). Further

purification of the chlorohydrate with a C8 reverse-phase

support, eluting with MeOH/H₂O, provided, after drying, the

(1S,2S,5R,6S)-2-[(2′R)-Amino-4′-methyl)pentanoyl]amino-

bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid Hydrochlo-

ride (12d‚HCl). (1S,2S,5R,6S)-2-[2′R-tert-Butoxycarbonyl-

amino-4′-methyl)pentanoyl]aminobicyclo[3.1.0]hexane-2,6-

dicarboxylic Acid Dimethyl Ester (11d). The reaction of 5

with N-Boc-^D-leucine following general procedure A provided

zwitterion 12g as a white solid (44% yield): mp 149-156 °C.

1

[R]²⁵-6° (c 1, MeOH). H NMR (MeOH-d₄) δ: 3.68 (s, 2 H),

D

2.37 (dd, J ) 6.2, 3.0 Hz, 1 H), 2.19-2.08 (m, 1 H), 2.01-1.85

(m, 3 H), 1.59 (t, J ) 2.7 Hz, 1 H), 1.37-1.21 (m, 1 H).

(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-(4′-methylthio)butyryl]-

aminobicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (12h).

1

11d as a foamy white solid (88% yield). H NMR (CD₃OD) δ:

7.92 (1H, bs), 4.57-4.48 (1H, m), 3.75 (3H, s), 3.70 (3H, s),

5318 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16

Bueno et al.

(1S,2S,5R,6S)-2-[(2′S)-(2′-tert-Butoxycarbonylamino)-(4′-

methylthio)butyryl]aminobicyclo[3.1.0]hexane-2,6-di-

carboxylic Acid Dimethyl Ester (11h). The reaction of 5

with N-Boc-^L-methionine following general procedure A pro-

vided 11h as a foamy white solid (89% yield). ¹H NMR (CDCl₃)

δ: 7.04 (brs, 1 H), 5.15 (d, 1 H, J ) 8.3 Hz), 4.13 (m, 1 H),

3.61 (s, 3 H), 3.53 (s, 3 H), 2.48-2.25 (m, 4 H), 1.97 (s, 3 H),

2.15-1.71 (m, 5 H), 1.57 (t, 1 H, J ) 3.0 Hz), 1.31 (s, 9 H),

1.13 (m, 1 H). ¹³C NMR (CDCl₃) δ: 172.4, 172.3, 171.5, 155.4,

79.9, 66.1, 52.7, 52.4, 51.6, 34.2, 31.8, 31.4, 29.7, 28.1 (3 C),

27.9, 26.4, 20.9, 15.0.

Compound 11k was deprotected following general procedure

D to provide 12k‚HCl as a white solid. Further purification of

the chlorohydrate with a C8 reverse-phase support, eluting

with acetonitrile in water with 0.05% of trifluoroacetic acid,

provided, after drying, 12k as a zwitterion (35% yield): mp

1

169 °C. [R]²⁵-2° (c 0.95, MeOH). H NMR (methanol-d₄) δ:

D

7.13 (d, 2 H, J ) 8.6 Hz), 6.79 (d, 2 H, J ) 8.6 Hz), 4.02 (dd,

1 H, J ) 7.5, 5.9 Hz), 3.18 (dd,1 H, J ) 14.2, 5.9 Hz), 2.94 (dd,

1 H, J ) 14.4, 7.5 Hz), 2.54 (dd, 1 H, J ) 6.3, 2.8 Hz), 2.34-

1.89 (m, 4 H), 1.66 (t, 1 H, J ) 3.0 Hz), 1.35 (m, 1 H). ¹³C

NMR (methanol-d₄) δ: 174.9, 174.3, 168.5, 156.7, 130.3, 124.5,

115.4, 66.0, 54.2, 36.3, 33.9, 31.5, 28.3, 25.5, 20.8. MS (elec-

trospray), m/z: 349 (M⁺+ 1), 331.

Compound 11h was deprotected following general procedure

D to provide 12h‚HCl as a white solid (64% yield). Further

purification of the chlorohydrate with a C8 reverse-phase

support, eluting with acetonitrile in water with 0.05% of

(1S,2S,5R,6S)-2-[(2′S)-2′-Amino-3′-hydroxypropionyl-

amino)bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (12l).

(1S,2S,5R,6S)-2-[(2′S)-2-tert-Butoxycarbonylamino-3-(tert-

butyldimethylsilanyloxy)propionylamino]bicyclo[3.1.0]-

hexane-2,6-dicarboxylic Acid Dimethyl Ester (11l). The

reaction of 5 with (2S)-2-tert-butoxycarbonylamino-3-(tert-

butyldimethylsilanyloxy)propionic acid methyl ester³¹following

general procedure A provided 11l as a foamy white solid (82%

yield). ¹H NMR (CDCl₃) δ: 7.44 (bs, 1 H), 5.38 (bs, 1 H), 4.16-

4.05 (m, 1 H), 3.94 (dd, J ) 9.7, 4.3 Hz, 1 H), 3.72 (s, 3 H),

3.65 (s, 3 H), 3.60-3.51 (m, 1 H), 2.64 (dd, J ) 14.0, 8.6 Hz, 1

H), 2.30-2.11 (m, 2 H), 2.03-1.97 (m, 2 H), 1.72 (m, 1 H), 1.43

(s, 9 H), 1.26-1.09 (m, 1 H), 0.88 (s, 9 H), 0.11 (s, 3 H), 0.10

(s, 3 H).

Compound 11l was dissolved in dry THF (10 mL) under

nitrogen, and 1 M tetrabutylammonium fluoride in THF was

added. The reaction mixture was stirred at room temperature

for 1 h, the solvent was evaporated, and the residue was

purified by silica gel chromatography, eluting with ethyl

acetate. The desilylated material was subjected to general

procedure D. The resulting chlorohydrate was purified by

HPLC to provide 12l as a very hygroscopic white solid (11%

yield): mp (HCl salt) 197-200 °C. [R]²⁵_D-14° [c 1, 1 N HCl].

¹H NMR (CDCl₃) δ: 4.01-3.71 (m, 3 H), 2.39 (dd, J ) 6.4, 3.0

Hz, 1 H), 2.19-1.86 (m, 4 H), 1.59 (t, J ) 2.7 Hz, 1 H), 1.41-

1.25 (m, 1 H). ¹³C NMR (D₂O) δ: 177.0, 175.9, 167.8, 66.4, 60.2,

54.4, 34.2, 31.4, 29.1, 25.5, 20.7.

trifluoroacetic acid, provided, after drying, 12h as a zwitterion

1

(78% yield): mp 164 °C. [R]²⁵+13° (c 1.1, MeOH). H NMR

D

(methanol-d₄) δ: 3.97 (t, 1 H, J ) 6.4 Hz), 2.64-2.56 (m, 2 H),

2.48-2.38 (m, 2 H), 2.11 (s, 3 H), 2.19-1.91 (m, 5 H), 1.66 (t,

1 H, J ) 3.0 Hz), 1.38 (m, 1 H). ¹³C NMR (methanol-d₄) δ:

174.7, 174.1, 168.3, 66.1, 52.0, 34.1, 31.5, 30.8, 28.1, 28.0, 25.9,

20.8, 13.6. MS (electrospray), m/z: 317 (M⁺+ 1), 229.

(1S,2S,5R,6S)-2-[(2′S)-(Pyrrolidine-2-carbonyl)]amino-

bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid Hydrochlo-

ride (12i‚HCl). (1S,2S,5R,6S)-2-[(2′S)-(1′-tert-Butoxycar-

bonylpyrrolidine-2′-carbonyl)amino]bicyclo[3.1.0]hexane-

2,6-dicarboxylic Acid Dimethyl Ester (11i). The reaction

of 5 with N-Boc-^L-proline following general procedure A

provided 11i as a foamy white solid (78% yield). ¹H NMR

(CDCl₃) δ: 4.2 (bs, 1H), 3.71 (s, 3H), 3.63 (s, 3H), 3.30 (bs,

2H), 2.47-2.45 (m, 2H), 2.03-1.77 (m, 8H), 1.65 (t, 1H, J ) 3

Hz), 1.47 (s, 9H), 1.20-1.00 (m, 1H).

Compound 11i was deprotected following general procedure

D to provide 12i‚HCl as a white solid (90% yield): mp 237-

240 °C. [R]²⁵_D-49° (c 0.5, 1 N HCl). ¹H NMR (methanol-d₄) δ:

4.31-4.24 (m, 1H), 3.37-3.28 (m, 2H), 2.45-2.41 (m, 3H),

2.14-1.98 (m, 6H), 1.65 (t, 1H, J ) 2 Hz), 1.44-1.32 (m, 1H).

¹³C NMR (methanol-d₄) δ:174.7, 174.1, 168.4, 66.2, 59.5, 46.1,

34.3, 31.6, 29.6, 28.0, 26.0, 23.4, 20.8.

(1S,2S,5R,6S)-2-[(2′S,3′S)-(2′-Amino-3′-methylpentan-

oylamino)bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid Hy-

drochloride (12j‚HCl). (1S,2S,5R,6S)-2-[(2′S,3′S)-(2′-(2-

tert-Butoxycarbonylamino-3-methylpentanoylamino)-

bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid Dimethyl Ester

(11j). The reaction of 5 with N-Boc-^L-isoleucine following

general procedure A provided 11j as a foamy white solid (79%

yield). ¹H NMR (CDCl₃) δ: 6.75 (bs, 1 H), 5.06 (bd, J ) 8.3

Hz, 1 H), 3.90 (dd, J ) 8.9, 6.7 Hz, 1 H), 3.70 (s, 3H), 3.63 (s,

3 H), 2.54-2.39 (m, 2 H), 2.21-1.72 (m, 4 H), 1.67 (t, J ) 3.2

Hz, 1 H), 1.60-1.40 (m, 1H), 1.41 (s, 9H), 1.22-1.0 (m, 2H),

0.91 (d, J ) 6.7 Hz,3 H), 0.87 (t, J ) 7.3 Hz, 3 H).

(1S,2S,5R,6S)-2-[(2′S)-(2′,6′-Diamino)hexanoyl]amino-

bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid Dihydrochlo-

ride(12m‚2HCl).(1S,2S,5R,6S)-2-[(2′S)-(2′,6′-Bis-tert-butoxy-

carbonylamino)hexanoyl]aminobicyclo[3.1.0]hexane-2,6-

dicarboxylic Acid Dimethyl Ester (11m). The reaction of

5 with NR-Boc-Nꢀ-Boc-^L-lysine following general procedure A

provided 11m as a foamy white solid (94% yield): mp 65-67

°C. [R]²⁵_D-11° (c 1.05, CHCl₃). ¹H NMR (CDCl₃) δ: 6.97 (brs,

1H), 5.12 (brd, 1H, J ) 7.0 Hz), 4.76 (brs, 1H), 4.06 (brq, 1H,

J ) 7.4 Hz), 3.75 (s, 3H), 3.66 (s, 3H), 3.11 (AB system, 2H),

2.50 (dd, 1H, J ) 13.8, 8.3 Hz), 2.42 (dd, 1H, J ) 6.5, 2.7 Hz),

2.23-1.76 (m, 5H), 1.69 (t, 1H, J ) 2.9 Hz), 1.63-1.11 (m, 5H),

Compound 11j was deprotected following general procedure

1.43 (s, 9H), 1.42 (s, 9H).

¹³C NMR (CDCl₃) δ: 172.6, 172.5,

D to provide 12j‚HCl as a white solid (64% yield): mp >300

172.3, 156.1, 155.8, 80.1, 66.3, 53.7, 52.7, 51.9, 39.8, 34.4, 32.0,

31.7, 29.5, 28.4, 28.3, 28.1, 26.6, 22.3, 21.1.

1

°C. [R]²⁵-5° (c 1, 1 N HCl). H NMR (MeOH-d₄) δ: 8.93 (s,

D

1 H), 3.73 (d, J ) 5.4 Hz, 1 H), 2.54 (dd, J ) 6.4, 3.0 Hz, 1 H),

2.35-1.91 (m, 5 H), 1.67-1.15 (m, 4 H), 1.06 (d, J ) 7.0 Hz, 3

H), 0.98 (t, J ) 7.3 Hz, 3 H). ¹³C NMR (MeOH-d₄) δ: 176.2,

175.7, 169.6, 67.4, 58.7, 38.1, 35.3, 32.9, 29.8, 26.9, 25.2, 22.2,

15.1, 11.7.

Compound 11m was deprotected following general proce-

dure D to provide 12m‚2HCl as a white solid (82% yield): mp

75-77 °C. [R]²⁵_D-1° (c 1.0, MeOH). ¹H NMR (D₂O) δ: 3.90 (t,

1H, J ) 6.3 Hz), 2.84 (bt, 2H, J ) 7.6 Hz), 4.42 (dd, 1H, J )

6.3, 2.8 Hz), 2.09-1.73 (m, 6H), 1.59-1.26 (m, 6H). ¹³C NMR

(D₂O + methanol-d₄) δ: 177.1, 176.3, 169.4, 66.1, 52.4, 38.8,

33.8, 31.2, 30.2, 29.3, 26.3, 25.1, 20.8, 20.6.

(1S,2S,5R,6S)-2-[2′(S)-(2′′(S)-Aminopropionylamino)-

propionylamino]bicyclo[3.1.0]hexane-2,6-dicarboxylic

Acid (12n). 2(S)-(2′(S)-tert-Butoxycarbonylaminopropio-

nylamino)propionic Acid (N-Boc-^L-alanyl-L-alanine). In

a flask containing ^L-alanyl-L-alanine (5 mmol) were added di-

tert-butyl dicarbonate (10 mmol) dissolved in 15 mL of dioxane

and 15 mL of saturated aqueous sodium bicarbonate. The

reaction mixture was stirred at room temperature overnight.

It was diluted with water (100 mL) and washed with EtOAc

(9 × 50 mL). The aqueous layer was acidified to pH 1 with 6

N hydrochloric acid and extracted with EtOAc. The organic

layer was washed with brine, dried over MgSO₄, filtered, and

(1S,2S,5R,6S)-2-[(2′S)-(2′-Amino)-(3′-p-hydroxyphenyl)-

propionyl]aminobicyclo[3.1.0]hexane-2,6-dicarboxylic

Acid (12k). (1S,2S,5R,6S)-2-[(2′S)-(2′-tert-Butoxycarbo-

nylamino)-(3′-p-hydroxyphenyl)propionyl]aminobicyclo-

[3.1.0]hexane-2,6-dicarboxylic Acid Dimethyl Ester (11k).

The reaction of 5 with N-Boc-^L-tyrosine following general

procedure A provided 11k as a foamy white solid (73% yield).

¹H NMR (methanol-d₄) δ: 7.03 (d, 2 H, J ) 8.0 Hz), 6.72 (d, 2

H, J ) 8.0 Hz), 5.21 (bs, 1 H), 4.28 (m, 1 H), 3.75 (s, 3 H), 3.63

(s, 3 H), 2.91 (m, 2 H), 2.39 (m, 2 H), 2.15-1.80 (m, 3 H), 1.59

(t, 1 H, J ) 4.0 Hz), 1.39 (s, 9 H). ¹³C NMR (methanol-d₄)

δ: 173.6, 171.7, 155.3, 130.4 (2 C), 127.8, 121.2, 115.4 (2 C),

80.3, 66.2, 52.6, 51.8, 37.4, 34.1, 31.7, 28.1 (3 C), 26.3, 22.5,

21.0.

Dipeptides as Effective Prodrugs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5319

1

evaporated to provide the dipeptide as an oil (88% yield). H

NMR (CDCl₃) δ: 9.90(bs, 1H), 7.04 (bs, 1 H), 5.46 (bs, 1 H),

4.54 (m, 1 H), 4.20 (m, 1 H), 1.50-1.30 (m, 15 H).

7.7 Hz, 5.0 Hz), 1.58-1.52 (4H, m), 1.42 (9H, s), 1.08-1.02

(1H, m), 0.82 (6H, 2 × t, 6.5 Hz). LCMS m/z: 327 [M + H -

CO2^tBu]⁺.

(1S,2S,5R,6S)-2-[(2S)-tert-Butoxycarbonylamino)pro-

pionyl]amino-2-[(1S)-3-methyl-1-methoxycarbonylbutyl]-

carbamoylbicyclo[3.1.0]hexane-6-carboxylic Acid Meth-

yl Ester (13). Compound 6k was dissolved in 1.0 N HCl in

EtOAc and stirred at room temperature for 3 h. The solvent

was removed under vacuum, and the residue was reacted with

N-Boc-^L-alanine following general procedure A, providing 13

(1S,2S,5R,6S)-2-[2′(S)-(2′′(S)-tert-butoxycarbonylamino-

propionylamino)propionylamino]bicyclo[3.1.0]hexane-2,6-

dicarboxylic Acid Dimethyl Ester (11n). The reaction of

5 with N-Boc-^L-alanyl-L-alanine following general procedure

1

A provided 11n as a foamy white solid (90% yield). H NMR

(CDCl₃) δ: 7.26 (bs, 1 H), 6.66 (bd, J ) 7.5 Hz, 1 H), 4.98 (bd,

J ) 7.0 Hz, 1 H), 4.46 (quint, J ) 7.2 Hz, 1 H), 4.13 (m, J )

7.2 Hz, 1 H), 3.74 (s, 3H), 3.65 (s, 3H), 2.48-2.36 (m, 2H),

2.14-1.87 (m, 3H), 1.69 (t, J ) 2.4 Hz, 1H), 1.43 (s, 9 H), 1.36

(d, J ) 7.0 Hz, 3 H), 1.35 (d, J ) 7.0 Hz, 3 H).

1

as a foamy white solid (48% yield). H NMR (CDCl₃) δ: 7.30

(1H, d, 6.0 Hz), 6.79 (1H, bs), 4.95 (1H, d, 6.3 Hz), 4.54-4.46

(2H, m), 3.63 (3H, s), 3.60 (3H, s), 2.45-2.39 (2H, m), 2.04-

2.00 (2H, m), 1.89-1.83 (2H, m), 1.59-1.53 (4H, m), 1.42 (9H,

s), 1.23 (6H, 2 × t, 6.7 Hz), 1.18-1.14 (1H, m), 0.80 (3H, d, 7.0

Compound 11n was deprotected following general procedure

D to provide 12n‚HCl as a white solid. Purification by C8

reverse-phase chromatography, eluting with MeOH/H₂O, pro-

vided the zwitterion 12n as a white solid (45% yield): mp 193

Hz). LCMS m/z: 498 [M + H]⁺and 398 [M + H - CO₂Bu]⁺.

t

Compound 13 was deprotected following general procedure

D to provide 14‚HCl as a white solid (81% yield). ¹H NMR

(D₂O) δ: 4.34-4.29 (1H, m), 3.95 (1H, q, 6.8 Hz), 2.41 (1H,

dd, 6.6 Hz, 2.7 Hz), 2.09-2.01 (2H, m), 1.9-1.3 (7H, m), 1.40

(3H, d, 6.8 Hz), 0.76 (6H, 2 × t, 5.9 Hz). ¹³C NMR (D₂O) δ:

173.7, 173.4, 173.1, 172.6, 52.5, 52.2, 51.4, 41.6, 33.1, 29.1, 28.7,

27.0, 25.4, 23.2, 22.3, 21.7. LCMS m/z: 369 M⁺.

°C. [R]²⁵-25° (c 1.05, MeOH). IR (KBr): 3380, 3285, 1664

D

1

cm^-1. H NMR (MeOH-d₄) δ: 8.67 (bs, 1 H), 4.43 (q, J ) 7.3

Hz, 1 H), 3.93 (q, J ) 7.0 Hz, 1 H), 2.45-2.34 (m, 2 H), 2.16-

1.88 (m, 3 H), 1.66 (t, J ) 3.2 Hz, 1 H), 1.50 (d, J ) 7.3 Hz, 3

H), 1.35 (d, J ) 7.3 Hz, 3 H), 1.33 (m, 1 H). ¹³C NMR (MeOH-

d₄) δ: 176.8, 176.3, 175.0, 171.0, 67.7, 50.6, 50.4, 36.1, 33.3,

29.8, 27.8, 22.7, 18.6, 18.0.

Acknowledgment. We thank Dr. Dennis Zimmer-

man his helpful collaboration. We are grateful to Dr.

Maria Luz de la Puente, Dr. Alfonso Espada, and Dr.

Aranzazu Mar´ın for the purification and HPLC analysis

of compounds.

(1S,2S,5R,6S)-2-[(2′S)-((2′′S)-Amino-4-methylpentano-

ylamino)propionylamino]bicyclo[3.1.0]hexane-2,6-dicarbox-

ylicAcid(12o).2(S)-(2′(S)-tert-Butoxycarbonylamino-4-meth-

ylpentanoylamino)propionic Acid (N-Boc-^L-leucinyl-L-

alanine). In a flask containing ^L-leucinyl-L-alanine (5 mmol)

were added di-tert-butyl dicarbonate (10 mmol) dissolved in

15 mL of dioxane and 15 mL of saturated aqueous sodium

bicarbonate. The reaction mixture was stirred at room tem-

perature overnight. It was diluted with water (100 mL) and

washed with EtOAc (2 × 50 mL). The aqueous layer was

acidified to pH 1 with 6 N hydrochloric acid and extracted with

EtOAc. The organic layer was washed with brine, dried over

MgSO₄, filtered, and evaporated to provide acid (N-Boc-L-

leucinyl-^L-alanine as a foamy white solid (78% yield). ¹H

NMR (CDCl₃) δ: 6.90 (bs, 1 H), 5.15 (bs, 1 H), 4.63-4.46 (m,

1 H), 4.17-4.06 (m, 2 H), 1.75-1.43 (m, 15 H), 0.95-0.91 (m,

6 H).

(1S,2S,5R,6S)-2-[(2′S)-(2′′S)-tert-Butoxycarbonylamino-

4-methylpentanoylamino)propionylamino]bicyclo[3.1.0]-

hexane-2,6-dicarboxylic Acid Dimethyl Ester (11o). The

reaction of 5 with N-Boc-^L-leucinyl-L-alanine following general

procedure A provided 11o as a foamy white solid (95% yield).

¹H NMR (CDCl₃) δ: 7.09 (s, 1 H), 6.62 (d, J ) 7.3 Hz, 1 H),

4.90 (d, J ) 7.3 Hz, 1 H), 4.45 (m, 1 H), 4.08 (m, 1 H), 3.72 (s,

3 H), 3.65 (s, 3 H), 2.47-2.37 (m, 2 H), 2.07-1.53 (m, 7 H),

1.43 (s, 9 H), 1.34 (d, J ) 7.0 Hz, 3 H), 1.40-1.20 (m, 1H),

0.95-0.92 (m, 6 H).

Supporting Information Available: Additional informa-

tion on the uptake of the intestinal human PepT1 transporter

and the fear-potentiated startle model and analytical instru-

mentation and methods for HPLC purity determination. This

material is available free of charge via the Internet at http://

pubs.acs.org.

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Compound 11o was deprotected following general procedure

D to provide 12o‚HCl as a white solid. Purification by C8

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JM050235R

Article Doi

Dipeptides as effective prodrugs of the unnatural amino acid (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740), a selective group II metabotropic glutamate receptor agonist

DOI: 10.1021/jm050235r

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