Design, synthesis, and proposed active site binding analysis of monocyclic 2-azetidinone inhibitors of prostate specific antigen

J . Med. Chem. 2001, 44, 1491-1508

1491

Design , Syn th esis, a n d P r op osed Active Site Bin d in g An a lysis of Mon ocyclic

2-Azetid in on e In h ibitor s of P r osta te Sp ecific An tigen

Robert M. Adlington,^†J ack E. Baldwin,^†Gerald W. Becker,^§Beining Chen,^†Leifeng Cheng,^†Stephen L. Cooper,^†

Robert B. Hermann,^§Trevor J . Howe,^‡William McCoull,^†Ann M. McNulty,^§Blake L. Neubauer,^§and

Gareth J . Pritchard^†,|,

*

The Dyson Perrins Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QY, U.K., Eli Lilly and Company,

Lilly Research Centre Ltd., Erl Wood Manor, Sunninghill Road, Windlesham, Surrey GU20 6PH, U.K., and Lilly Research

Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285

Received March 30, 2000

A homology derived molecular model of prostate specific antigen (PSA) was created and refined.

The active site region was investigated for specific interacting functionality and a binding model

postulated for the novel 2-azetidinone acyl enzyme inhibitor 1 (IC₅₀) 8.98 ( 0.90 µM) which

was used as a lead compound in this study. A single low energy conformation structure II

(Figure 2) was adopted as most likely to represent binding after minimization and dynamics

calculations. Systematic analysis of the binding importance of all three side chains appended

to the 2-azetidinone was conducted by the synthesis of several analogues. A proposed salt bridge

to Lys-145 with 4 (IC₅₀) 5.84 ( 0.92 µM) gave improved inhibition, but generally the binding

of the N-1 side chain in a specific secondary aromatic binding site did not tolerate much

structural alteration. A hydrophobic interaction of the C-4 side chain afforded inhibitor 6 (IC₅₀

) 1.43 ( 0.19 µM), and polar functionality could also be added in a proposed interaction with

Gln-166 in 5 (IC₅₀) 1.34 ( 0.05 µM). Reversal of the C-4 ester connectivity furnished inhibitors

7 (IC₅₀) 1.59 ( 0.15 µM), 11 (IC₅₀) 3.08 ( 0.41 µM), and 13 (IC₅₀) 2.19 ( 0.36 µM) which

were perceived to bind to PSA by a rotation of 180° relative to the C-4 ester of normal

connectivity. Incorporation of hydroxyl functionality into the C-3 side chain provided 16 (IC₅₀

) 348 ( 50 nM) with the greatest increase in PSA inhibition by a single modification. Multiple

copy simultaneous search (MCSS) analysis of the PSA active site further supported our model

and suggested that 18 would bind strongly. Asymmetric synthesis yielded 18 (IC₅₀) 226 ( 10

nM) as the most potent inhibitor of PSA reported to date. It is concluded that our design

approach has been successful in developing PSA inhibitors and could also be applied to the

inhibition of other enzymes, especially in the absence of crystallographic information.

In tr od u ction

previously communicated a series of PSA inhibitors.¹¹

In this report, we detail our active site analysis of a

homology derived model of PSA, with subsequent design

and synthesis of a range of novel monocyclic 2-azetidi-

none inhibitors of PSA.

Our approach to the inhibition of PSA was based upon

the lead compound 1 (IC₅₀) 8.98 ( 0.90 µM.). This slow

binding, time dependent inhibitor was shown by elec-

trospray ionization mass spectrometry and peptide

mapping experiments to form a stable 1:1 acyl enzyme

complex at the active site with Ser-189. Computer

Prostate specific antigen (PSA) is a serine protease

produced in the epithelial cells of the prostate gland.¹

It exhibits proteolytic action upon semenogelin and

fibronectin, resulting in the liquifaction of seminal

fluid.²In addition, PSA has also been shown to be

responsible for the degradation of insulin-like growth

factor binding protein-3 (IGFBP-3) in seminal fluid.³

This stimulates mitogenesis by insulin-like growth

factors I and II (IGF-I, II) in prostate epithelial cells

which are normally blocked by IGFBP-3.⁴Such action

may be responsible for enhanced growth of the prostatic

cancer cell line DU-145,⁵and PSA may play a causative

rather than merely descriptive role in prostate cancer.^6,7a

(Recent work has shown that this may not be the case.^7b)

This, and the implication of PSA in female breast

tumors^8,9and bone metastasis,¹⁰makes the design of

PSA inhibitors important synthetic targets. We have

* To whom correspondence should be addressed. Tel: 44 1509 222

586. Fax: 44 1509 223 925. E-mail: G.J .Pritchard@lboro.ac.uk.

^†Dyson Perrins Laboratory, U.K.

simulated docking and subsequent molecular modeling

calculations allowed us to create a description of binding

of monocyclic 2-azetidinones to the active site of PSA

and design modified structures predicted to bind more

strongly. Such target molecules were then synthesized,

^§Lilly Research Center, U.S.A.

^‡Lilly Research Centre, Ltd., U.K. Present address: Novartis

Horsham Research Centre, Wimblehurst Road, Horsham, W. Sussex

RH12 4AB, U.K.

^|Present address: Department of Chemistry, Loughborough Uni-

versity, Loughborough, Leicestershire LE11 3TU, U.K.

Published on Web 04/03/2001

1492 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10

Adlington et al.

F igu r e 1.

F igu r e 2.

and their inhibitory value against PSA was assessed.

By treating this as an iterative process, biological data

could be used to refine our model and further design

compounds predicted to be more potent inhibitors.

44) which formed a region we termed the “arginine

bank.” The role of each location around the active site

on docking of inhibitor structures will be discussed.

The structure of each enantimomer of compound 1

was minimized to an energy gradient tolerance of 0.01

using CHARMm 21.3¹⁷on a Silicon Graphics R4600

Indy workstation. The low energy conformations were

manually docked into the active site of PSA using two

criteria. First, the 2-azetidinone carbonyl was directed

toward the oxyanion hole of PSA with the carbonyl

carbon proximal to the hydroxyl group of Ser-189.

Second, it was expected that one of the three 2-azeti-

dinone side chains would occupy the S₁specificity pocket

and, subject to the first constraint, only the C-3 benzyl

side chain could be fitted into this binding subsite. Such

filling of the S₁specificity pocket with the C-3 side chain

of a 2-azetidinone has also been proposed in the inhibi-

tion of human leukocyte elastase^22,23and thrombin.²⁴

The flexible nature of 1 allowed the N-1 side chain to

be fitted either in the valley or cleft regions or toward

the arginine bank. The remaining C-4 side chain

naturally orientated toward the groove regions near the

active site, and which groove was occupied depended

upon the N-1 side chain orientation.

The six fitted structures of 1 (three for each enanti-

omer) were minimized to an energy gradient tolerance

of 0.01 using CHARMm 21.3¹⁷within a constrained

model of PSA. Dynamics calculations were then per-

formed again, only allowing the inhibitor structure to

move. Typically the system was heated to 300 K over

30 ps followed by a 10 ps equilibration period at this

temperature. From the resulting structure, 10-30 ps

simulations were performed, and low energy structures

thus identified were further subjected to minimizations.

The final conformer structures, shown in Figures 2 and

3 for the (3S,4S), (3R,4R) enantiomers, respectively,

were analyzed for the position of each side chain and,

in the case of the C-4 side chain, whether a polar

interaction with the enzyme could be detected (Table

1). The distance from the carbonyl carbon to the closest

NH group of the oxyanion hole (D_oh, Table 1) was

measured with only conformer structure II exhibiting

a formal hydrogen bonding distance.²⁵Each inhibitor

conformer structure was removed from the enzyme and

allowed to freely release for 50 steps of minimization to

gauge how far they were in energy from the free

Molecu la r Mod elin g

A homology derived molecular model of PSA was

created by appropriate substitution for PSA from por-

cine kallikrein using AMBER^12,13(version 3.0A). Two

main loop insertions were necessary, and conformations

were twisted to point hydrophilic groups toward the

outside of the enzyme. Minimization and dynamics

calculations were simulated within a monolayer of water

with the substrate molecules SGAWYYVPLG or casein

trypsin cleavage peptide inserted in the active site to

maintain rigidity. Where residues were conserved, the

hydrogen bonding structure was also conserved, and no

major steric clashes were observed during this refine-

ment procedure. Other homology derived models for

PSA have been created by similar procedures.^14-16

A

Ramachandran plot was generated within QUANTA17

(version 4.0) on a Silicon Graphics R4400 Indigo work-

station and showed little deviation from the allowed æ

and ψ angles of the protein backbone.¹⁸Inspection of

our PSA model revealed that the active site comprising

the catalytic residues His-41, Asp-96, and Ser-189 was

contained within a crevice along the enzyme surface.

On each side of this crevice was a domain of six-stranded

antiparallel â-barrels as typical of serine proteases. The

main binding subsite, the S₁specificity pocket, was

clearly identified proximal to the active site with Ser-

183 located at the very base of this pocket, thus

explaining the restricted chymotrypsin-like activity of

PSA.^19,20A solvent accessible surface, using a probe

radius of 1.4 Å and centered on the oxygen of Ser-189,

was calculated according to the method of Connolly.²¹

The simplified topology of the active site area is repre-

sented in Figure 1. The cleft and valley regions form

the active site crevice with the former being quite open

but the latter displaying a more narrow opening in the

enzyme surface, especially at one point where Ala-24

and Thr-143 residues protrude. Two less distinguished

and shallow grooves in the enzyme surface were also

detected close to the active site. Finally, in a flexible

loop region which would be able to fold over the active

site were a series of arginine residues (Arg-21, 23, and

2-Azetidinone Inhibitors of Prostate Specific Antigen

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1493

F igu r e 4.

Ta ble 2

F igu r e 3.

Ta ble 1

minimized form. This energy difference (∆_bind) reflects

the unfavorable conformational strain energy, which is

countered by a favorable van der Waals energy differ-

ence between free and bound states.²⁶Conformer struc-

ture IV was immediately discarded as an unrealistic

binding possibility since the inhibitor was orientated

above rather than within the active site. This again left

conformer structure II as potentially the best description

of inhibitor binding with the lowest conformational

strain on binding.

hydration sphere of benzene from Monte Carlo simula-

tion experiments.²⁸

In an attempt to both verify and utilize our active site

analysis, compounds 2-4 were synthesized (Table 2).

Without the second aromatic B ring on the nitrogen side

chain as in compounds 2 and 3, poorer binding to PSA

was predicted, as reflected in significantly decreased

IC₅₀values. Incorporation of a second carboxyl group

in 4 was envisaged to allow salt bridging with Lys-145

in the valley region without dislodging either the A or

B ring binding interactions, and an improvement in PSA

inhibition was achieved.

Discu ssion

Our initial modeling experiments indicated that

conformer structure II, indicating the (3S,4S) enanti-

omer, should be used as the basis for modification of

lead inhibitor 1. In addition to strong interaction of the

2-azetidinone carbonyl in the oxyanion hole and the C-3

benzyl side chain in the S₁specificity pocket, the

extended N-1 side chain proved a snug fit in the valley

region (Figure 2). With four aromatic rings in inhibitor

1 we expected that aromatic-aromatic interaction

would be important in binding.²⁷Notably, conformer

structure II shows edge to face aromatic interaction of

the A and B rings with Phe-141 and Phe-57, respec-

tively, (Figure 4). Further, the B ring appeared firmly

lodged between protons from the methyl group of both

Ala-24 and Thr-143 in the manner found for the first

In conformer structure II, the C-4 side chain was

forced by the geometric constraints of the 2-azetidinone

ring to lie in the lower groove. Although a formal

hydrogen bond was not detected with Gln-166, confor-

mational changes on binding and acyl enzyme formation

could permit a strong interaction with the carboxyl

group. Investigation of inhibitor-PSA interaction at the

C-4 side chain was conducted with compounds 5 and 6

(Table 3). First, by changing the carboxyl to the more

flexible aminomethylene group in 5, a better distance

and directionality of perceived hydrogen bonding to Gln-

166 could be achieved, and indeed a significant lowering

of the IC₅₀value was obtained. This vindicated the

decision to base structure modification on binding

conformation II. Conformer structure III could be

1494 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10

Adlington et al.

Ta ble 3

Ta ble 4

argued to explain the potency of inhibitor 4 via an

interaction of the new carboxyl group at the “arginine

bank” despite a poorer fit in the oxyanion hole and

greater conformational strain on binding. However, in

conformer structure III the geometric constraints of the

2-azetidinone ring forces the C-4 substituent into the

upper groove area where no possible polar interactions

could be detected, hence the potency of 5 could only be

explained by binding in the manner of conformer

structure II. Alteration of the substitution pattern of

the aryl ring resulted in much poorer inhibitors. Large

conformational changes would be necessary to allow

hydrogen bonding to Gln-166 in this instance, and

occupation of the lower groove region is sterically

prohibited for either ortho or meta substituents, result-

ing in a significant decrease of binding potential of the

C-4 side chain.

Since there appeared to be strict distance require-

ments for interaction with Gln-166 at the end of the

lower groove, compound 6 was prepared which could

utilize hydrophobic interactions only within the groove,

without attempting to create a polar interaction. Com-

pound 6 showed significant improvement over the lead

inhibitor 1. It is probable that if a precise interaction

with Gln-166 is not possible, then polar functionalities

will experience an attraction out of the enzyme toward

external solvation, explaining the potency of compound

6.

Further information concerning structural and chemi-

cal requirements were elucidated by biological testing

of various synthetic intermediates. As expected, potent

inhibitors containing amino or carboxyl functionality

were inactive if these groups were chemically protected.

Also, the 2-azetidinones which were unsubstituted at

nitrogen did not display any PSA activity, presumably

due not only to a lack of a side chain binding interaction

but also to a low reactivity of the 2-azetidinone carbonyl

toward acylation in the absence of an electron with-

drawing group at N-1.

The C-4 ester group allowed the possibility of polar

binding interactions to PSA. No such interaction was

obvious from inspection of our model, and the carbonyl-

oxymethylene group was reversed to probe binding

interactions. The simple azetidinone 7 was found to be

a potent inhibitor of PSA. We attempted to combine this

“reverse ester” functionality with the extended N-1 side

chain and C-4 carbonyl functionality from our lead

inhibitor 1 to give compounds 8 and 9, respectively.

Unfortunately neither compound exhibited realistic PSA

inhibition (Table 4).

It was probable that “reverse ester” 7 had a different

mode of binding to that of our previous inhibitors which

we termed “normal esters.” By 180° rotation around the

2-azetidinone carbonyl of 7 in the binding conformation

II, the N-1 side chain could be positioned within the S₁

specificity pocket. This binding hypothesis was sup-

ported by comparison with the crystal structure of tosyl-

R-chymotrypsin where the tosyl group occupies the S₁

specificity pocket.²⁹Since reverse esters of cis relative

stereochemistry were prepared, such a manipulation

would necessitate the opposite enantiomer (i.e., (3R,4R)

as shown in Figure 5 for 10) to achieve a conformation

able to bind at the active site. Such a manipulation

suggests that the (3R,4R) enantiomer would have a

more energetically favorable binding conformation for

this reverse ester compared to the predicted (3S,4S)

enantiomer for the normal ester compounds. The com-

pounds were prepared as cis racemates to prevent the

exclusion of a possible binding conformer. Thus, tosyl

functionality attached at N-1 of a reverse ester would

also be expected to inhibit PSA.

In accord with this model, 10 showed good PSA

inhibition. Molecular models showed that the angle

2-Azetidinone Inhibitors of Prostate Specific Antigen

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1495

F igu r e 6.

F igu r e 5.

propane, methionine, nitro, and threonine were all used.

For each ligand, 1000 copies were used initially with a

convergence criterion of 0.216, and the procedure was

iterated three times to allow complete searching of the

active site. In most cases the MCSS functionality maps

were inconclusive for the identification of local minima

due to large numbers of clusters. However, in the case

of toluene, only two low energy binding sites were

identified as depicted in Figure 6 on the Connolly

surface. In view of the PSA inhibitors synthesized thus

far, the importance of the binding of such aromatic

functionality is evident. The lowest energy toluene

cluster (red and yellow) shows a strong aromatic binding

potential within the S₁specificity pocket as utilized by

the C-3 benzyl side chain in the normal ester series of

PSA inhibitors and the N-1 aryl sulfonyl side chain in

the reverse ester series in our model. The other toluene

binding site (green) identified by the MCSS technique

corresponds to the proposed B ring binding site of

conformer structure II. An overlay of the toluene MCSS

results and conformer structure II shows a good fit,

further verifying this conformation as representative of

binding in the normal ester series of inhibitors (Figure

7).

It was clear that the C-3 benzyl aromatic ring could

fit further into the S₁specificity pocket to give stronger

binding. Several residues (Ser-183, Ser-217, and Thr-

184) at the bottom of this pocket create a hydrogen

bonding network. One of many sites identified by the

MCSS method for the methanol ligand was in this area,

suggesting that a p-hydroxyl substitution of the aro-

matic ring would improve binding. The conformer

structure II of the (3S,4S) enantiomer of the lead

inhibitor 1 was altered to include such a p-hydroxyl

group as in 15 and was minimized within the active site

of PSA. The resulting model of 15 is depicted within all

possible binding regions within the active site of PSA

in Figure 8. After energy minimization calculations the

C-3 aromatic ring was indeed pulled further into the

S₁specificity pocket at the expense of interaction in the

lower groove with Gln-166. Consequently, although 15

was a potent PSA inhibitor, compound 16 with no polar

substituent at C-4 was predicted to improve PSA

binding and consequently inhibition. Biological data

(Table 5) confirmed this hypothesis. Indeed the sole

introduction of the p-hydroxyl group converted a com-

pletely inactive compound into the good inhibitor 17,

subtended between the N-1 substituent and the average

plane of the 2-azetidinone ring was not as steep as for

the C-3 substituent. Therefore, when the N-1 substitu-

ent of 10 was oriented in the direction of the S₁

specificity pocket, the angle of approach over the lip of

the pocket caused the p-methyl substituent to violate

the solvent accessible boundary. Conformational changes

that occur on acylation could alleviate this problem, but

we considered the unsubstituted analogue 11, which

would create less steric clashes. Slight, but not greatly

improved, potency was observed. However, introduction

of a polar functionality such as a nitro group in 12 did

decrease PSA inhibition significantly. This would be

expected to result from the bulkier nitro versus hydro-

gen substituent. Also the para substituent cannot reach

the polar region at the base of the specificity pocket

because of the angle of entry dictated by the pyramidal

nitrogen atom. The electronic nature of para substitu-

ents in 10-12 did not indicate any trend in inhibition

values, presumably since all contained a suitably reac-

tive 2-azetidinone carbonyl functionality to allow acyl-

ation of the enzyme. However, with a more flexible

hydrophobic ethyl group, a further improvement in PSA

inhibition by 13 was found.

It was now apparent that we did indeed have two

distinct series of monocyclic 2-azetidinone inhibitors of

PSA which bound to the active site in a different

manner. In further confirmation, normal ester 14 was

prepared with tosyl functionality at N-1 and was found

to be largely inactive against PSA. This clearly showed

that the characteristic binding groups from these nor-

mal and reverse esters could not be transposed in a

structure-activity relationship analysis, and it strongly

indicated different binding modes for these types of

esters.

Mu ltip le Cop y Sim u lta n eou s Sea r ch

A de novo design approach to the inhibition of PSA

was undertaken using a multiple copy simultaneous

search (MCSS) method.³⁰A sphere of radius 15 Å

centered on the amide proton of Gly-187 was selected

such that the active site and possible binding subsites

were included. The functional groups methanol, acetate,

methylammonium, toluene, N-methylacetamide, ether,

1496 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10

Adlington et al.

Ta ble 5

F igu r e 7.

Sch em e 1^a

a

(a) NaOH, H₂O, Ac₂O, 0 °C, 80%; (b) R ) H, PCl₅, 100 °C,

80%; R ) OAc, (COCl)₂, DCM, cat. DMF, 90%; (c) NEt₃, (p-MeO-

C₆H₄-NdCH)₂(22), 35-40 °C; (d) 5% aq. HCl, R ) H, 61%; R )

OAc, 85%.

F igu r e 8.

even in the absence of an extended secondary aromatic

binding group.

The conformer structure II, used in the design of more

potent PSA inhibitors, represents only the (3S,4S)

enantiomer. The calculated conformers of the (3R,4R)

enantiomer are not able to utilize the secondary toluene

binding subsite identified by MCSS calculations. Thus

we were able to predict that the (3S,4S) enantiomer of

the cis racemic normal ester compounds that we had

prepared would be more active than their (3R,4R)

counterparts. Assuming little inhibitory activity of the

(3R,4R) enantiomers, an increase of up to 50% inhi-

bition over the racemic mixture could be achieved by

the preparation of a single (3S,4S) enantiomer. In

practice, the homochiral (3S,4S)-2-azetidinone 18 ex-

hibited significant lowering (35%) of the IC₅₀value for

inhibition, emphasizing the predictive accuracy of our

model.

reaction using a modification of the method used by

Alcaide (Scheme 1).³¹Protection of 3-(4-hydroxyphenyl)-

propionic acid 20, as phenolic acetate 21, using acetic

anhydride, then allowed acid chloride formation using

oxalyl chloride with a catalytic quantity of N,N-di-

methylformamide.³²Hydrocinnamic acid 19 was con-

verted to its acid chloride using phosphorus penta-

chloride. 1,4-Bis(4′-Methoxyphenyl)-1,4-diazabuta-1,3-

diene 22³³was prepared and underwent Stau¨dinger

reaction, with acid chlorides derived from 19 and 21,

respectively, and subsequent imine hydrolysis to give

23 and 24 in good yields. A moderate temperature range

of 35-40 °C was found necessary to minimize competing

Ch em istr y

The monocyclic 2-azetidinones 23 and 24, with cis

relative stereochemistry, were prepared via a Stau¨dinger

2-Azetidinone Inhibitors of Prostate Specific Antigen

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1497

Sch em e 2^a

borohydride in methanol selectively reduced the alde-

hyde group to give alcohol 30 in good yield (Scheme 2).³⁵

Consequently, diverse modification of the C-4 side

chain could be easily incorporated into our synthetic

plan.

For both normal and reverse ester compounds, 4-car-

boxybenzaldehyde 31 was used as a starting material.

The carboxyl functionality was protected as the tert-

butyl ester using N,N-dimethylformamide di-tert-butyl

acetal.³⁶For the preparation of gram quantities of 32,

N,N-dimethylformamide dineopentyl acetal and tert-

butyl alcohol were used with only a small reduction in

yield (56%). Then either sodium borohydride in metha-

nol afforded alcohol 33, or J ones oxidation³⁴gave acid

34, both in high yields (Scheme 3). Notably, no ester

hydrolysis was detected under the latter acidic reaction

conditions despite the ease of removal of the tert-butyl

group with trifluoroacetic acid. To minimize reaction

steps, the amine functionality of 4-[N-(tert-butyloxycar-

bonyl)aminomethyl]benzoic acid 35 was protected with

a tert-butyloxycarbonyl group which could also be

removed under trifluoroacetic acid conditions. Acid 36

underwent borane reduction to give alcohol 37 in good

yield (Scheme 3).

a

(a) CrO₃-H₂SO₄, acetone-H₂O, 0 °C; (b) NaBH₄, MeOH.

Sch em e 3^a

The N-1 side chains were prepared from isophthalic

acid which contained the meta substitution pattern

required to realize secondary subsite binding (Scheme

4). Modification of the literature procedure allowed

preparation of acid 41.³⁷Monoesterification of 38 could

be achieved in moderate yield to give 39 with recovery

of unreacted 38 (and diester) which could be recycled.

Arndt-Eistert homologation was conducted using 2,2,2-

trichloroethanol to trap the intermediate ketene and

form 40 which enabled purification on silica gel. Each

acid functionality could be selectively revealed due to

the orthogonal nature of the protecting groups. Zinc

reduction in acetic acid³⁸followed by acid chloride

formation gave the required acid chloride 42. Alterna-

tively, hydrogenation quantiatively yielded 43 which

was re-esterified with alcohol 33 allowing formation of

acid chloride 44. Acid 43 could also be reprotected as

the tert-butyl ester 45 using O-tert-butyl trichloro-

acetimidate.³⁹Treatment of 43 with N,N-dimethyl-

formamide di-tert-butyl acetal resulted in enamine

a

(a) (^tBuO)₂CHNMe₂, PhH, reflux; (b) NaBH₄, MeOH, 0 °C; (c)

CrO₃-H₂SO₄, acetone-H₂O, 0 °C; (d) Boc₂O, NaOH, H₂O-dioxan,

0 °C; (e) BH₃-THF, THF, 25 °C.

ketene dimerization to the 1,3-cyclobutadiones 25 and

26 and bias formation of 2-azetidinones 23 and 24 over

amides 27 and 28. No trans-2-azetidinones were de-

tected from these transformations.

The required C-3 benzyl functionalities were now in

place, and functionalization at C-4 was conducted next.

Aldehyde 23 could be oxidized to acid 29 using a slight

excess of J ones reagent.³⁴In the presence of excess

J ones reagent, cis to trans epimerization, presumably

at the C-4 position, resulted. Alternatively, sodium

Sch em e 4^a

a

(a) NEt₃, MeOH; (b) BnBr, DMF, 100 °C; (c) (COCl)₂, cat. DMF, DCM; (d) CH₂N₂, Et₂O, 0 °C-RT; (e) Ag₂O, Cl₃CCH₂OH, 60 °C; (f)

Zn, AcOH-H₂O, THF; (g) H₂, Pd-C, EtOAc; (h) DCCI, (33), cat. DMAP, THF; (i) Cl₃CC(O^tBu)dNH, excess BF₃‚OEt₂, DCM-cyclohexane.

1498 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10

Adlington et al.

Sch em e 5^a

a

(a) DCCI, (33), cat. DMAP, THF; (b) CeIV(NH₄)₂(NO₃)₆, MeCN-H₂O, 0 °C; (c) NaN(SiMe₃)₂, THF, -78 °C then RCOCl, -78 °C; (d)

CF₃CO₂H, 0 °C.

Sch em e 6^a

catalytic quantity of (dimethylamino)pyridine. Nitrogen

dearylation with ceric ammonium nitrate gave 2-azeti-

dinone 54, which was both acylated to give 7 and 8 and

sulfonylated to give 10-13. The aryl sulfonlyation

reaction initially gave poor yields due to competing

oligomerization reactions for the formation of dimers 55

and trimers 56.⁴¹It was found that the aryl sulfonyl-

ation reactions proceeded in good yields in the presence

of excess aryl sulfonyl chloride at low concentrations of

2-azetidinone 54.⁴¹The formation of 13 proceeded in

poor yield due to the poor reactivity of ethanesulfonyl

chloride toward S_N2 displacement reactions. The 2-

azetidinones 9 and 14 were prepared by analogous

reaction sequences.

a

(a) PhCOCl, cat. DMAP, NEt₃, DCM; (b) CeIV(NH₄)₂(NO₃)₆,

MeCN-H₂O, 0 °C; (c) NaN(SiMe₃)₂, THF, -78 °C then RCOCl,

-78 °C; (d) NaN(SiMe₃)₂, THF, -78 °C then R′SO₂Cl, -78 °C.

formation at the benzylic position in addition to the

desired ester formation. Subsequent zinc reduction and

acid chloride formation gave 46 in high yield.

Manipulation of the 2-azetidinone C-4 side chain

proceeded smoothly (Scheme 5). Dicyclohexylcarbodi-

imide mediated esterification of 29 with 33 gave the N-1

protected 2-azetidinone 47. Oxidative nitrogen dearyl-

ation with ceric ammonium nitrate⁴⁰gave 2-azetidinone

48 in good yield, and a variety of acyl groups were

attached by deprotonation with sodium bis(trimethyl-

silylamide) and reaction with acid chlorides. Variable

yields of 49 to 52 were obtained for such acylations,

presumably due to the steric bulk of the acid chlorides

used and the presence of acidic impurities from their

formation. Storage under high vacuum overnight im-

mediately following preparation of the acid chloride,

prior to reaction, generally afforded higher yields al-

though these reactions were not optimized. Deprotection

with trifluoroacetic acid gave target compounds 1-4 in

good yields. Reaction times were generally less than 20

min, otherwise ring opening of the reactive 2-azetidi-

none occurred. Other normal esters with different

functionalities at C-4 (e.g., 5 and 6) were prepared by

analogous reaction steps.

In the case of C-3 p-hydroxybenzyl 2-azetidinones,

additional synthetic steps were required (Scheme 7).

The acetyl protecting group was found suitable to

withstand J ones oxidation to give acid 57, esterification,

and also nitrogen dearylation to give 2-azetidinone 59.

However, after N-1 acylation afforded the activated

2-azetidinone 60, it was not found possible to remove

the acetyl protecting group without ring opening of the

reactive 2-azetidinone. Thus the acetyl group was

exchanged for an organosilicon protecting group after

investigation of several deacylation procedures. Alkaline

hydrolysis (LiOH, K₂CO₃) of the acetyl group in 58a

resulted in competing hydrolysis of the C-4 benzyl ester.

Sodium borohydride⁴²and potassium trimethyl-

silanoate⁴³also resulted in unselective hydrolysis, al-

though 61a was always the main product. However,

using a mild activated zinc procedure, excellent yields

of 61a ,b were obtained after 4 days.⁴⁴Silyl protection

to give 62a ,b then allowed selective oxidative removal

of the N-1 aromatic ring. The tert-butyldiphenylsilyl

protecting group was used in preference to tert-butyl-

dimethylsilyl, as the latter did not withstand the acidic

dearylation conditions. Acylation with benzoyl chloride

The synthesis of reverse ester inhibitors proceeded

by similar reaction methods (Scheme 6). Esterification

of 30 at C-4 gave 53 using benzoyl chloride and a

2-Azetidinone Inhibitors of Prostate Specific Antigen

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1499

Sch em e 7^a

a

(a) CrO₃-H₂SO₄, acetone-H₂O, 0 °C; (b) DCCI, BnOH, cat. DMAP, THF; (c) CeIV(NH₄)₂(NO₃)₆, MeCN-H₂O, 0 °C; (d) NaN(SiMe₃)₂,

THF, -78 °C then RCOCl, -78 °C; (e) activated Zn, MeOH-THF, 35 °C; (f) TBDPSCl, imidazole, DMF, 40 °C; (g) NH₄F‚HF, NMP-DMF;

(h) CF₃CO₂H, 0 °C.

gave imide 63 which was used as a model for attempted

desilylation reactions. Decomposition resulted when

trifluoroacetic acid or 48% hydrogen fluoride was used.

However, the mild desilylating agent ammonium hy-

drogen difluoride was found to afford 17 in 50% yield.⁴⁵

The desired extended N-1 side chain was attached, and

ammonium hydrogen difluoride was used again suc-

cessfully without 2-azetidinone ring opening to give

phenols 16 and 64, and also 15, after an additional

trifluoroacetic acid deprotection step.

did not lead to separable diastereomers by chromatog-

raphy or crystallization. Failure of 66 to undergo the

reaction was attributed to the need of a more reactive

imine component to allow the reaction to proceed at

temperatures lower than that which caused extensive

dimerization of the acid chloride used (<40 °C).

Initial attempts to convert our cis racemic 2-azet-

idinone synthesis into an asymmetric process using

the chiral diimines 65⁴⁶and 66⁴⁷in an asymmetric

Stau¨dinger reaction were unsuccessful. A high yield

(82%) of a 1:1 diastereomeric mixture 67 could be

obtained using 65 at a moderate temperature (35-40

°C), but the diastereomers were found to be inseparable

on silica gel. Lower temperatures, which are generally

required for diastereoselectivity, resulted in poor chemi-

cal yield. Subsequent elaboration to a hydroxymethyl

and phenylcarbonyloxymethyl substituent at C-4 also

Consequently, a new synthetic route was adopted for

the preparation of 18 (Scheme 8). The homochiral

2-azetidinone 68 with the required (4S) stereochemistry

was prepared from L-aspartic acid by the literature

method.⁴⁸The C-3 arylidene 2-azetidinones 69a ,b were

prepared from a Peterson reaction with 4-tert-butyl-

diphenylsilyloxybenzaldehyde and the (3S) stereochem-

istry created in a subsequent hydrogenation step.⁴⁹The

Sch em e 8^a

a

(a) i. LDA (2.1 equiv), THF, -9 ° C, ii. TMSCl (1.1 equiv), THF, -9 °C, iii. LDA (1.1 equiv), THF, -9 °C to 0 °C; (b) p-TBDPSO-

C₆H₄-CHO, THF, 0 °C; (c) EDCI, BnOH, cat. DMAP, DCM; (d) H₂, Pd-C, THF; (e) 48% aq. HF, MeCN; (f) NaN(SiMe₃)₂, THF, -78 °C

then m-BnO₂C-C₆H₄-CH₂COCl (42), -78 °C to RT, 45%; (g) NH₄F‚HF, NMP-DMF.

1500 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10

Adlington et al.

a slope (reaction rate) for each inhibitor concentration. The

reaction rates (mO.D. min^-1) for each inhibitor concentration

were transferred to J MP for nonlinear analysis using a four-

parameter logistic model. Estimated IC₅₀values were defined

as the serial dilution required to produce a half-maximal effect.

All data for active compounds that have been reported were

acquired using the spectrophotometric assay, but for prelimi-

nary screening of many compounds an HPLC assay was also

used. In an HPLC assay, the peptide substrate (SGAWYYV-

PLG) was dissolved in PBS to a concentration of 0.1 mg/mL.

PSA stock was diluted to a concentration of 0.1 mg/mL with

PBS containing 50% glycerol. A total of 1 to 5 mg of each

compound was dissolved in DMSO and made up to 300 and

30 µM stock solutions. Serial dilutions of inhibitors with DMSO

were prepared (30, 10, 3, 1 µM), and PSA was added. After

incubation at 37 °C for 2 h, the reaction was terminated by

addition of 100 µL of trifluoroacetic acid. A 100 µL sample was

injected onto a reversed-phase HPLC column (Vydac C-18 solid

phase with particle size of 5 mm.) A gradient elution using

the mobile phase A ) 0.1% TFA in water, B ) 0.1% TFA in

acetonitrile was conducted as follows:

(4S) benzyloxycarbonyl group was thought to sterically

prevent addition of hydrogen from one face of the

2-azetidinone, and subsequent rebenzylation afforded

70 as the sole isolable product. Both E and Z isomers

of 69 could be isolated and converted to 70. The overall

yield for conversion of 68 to 70 (6% over seven steps)

was disappointing, and material losses were ascribed

to partial desilylation at N-1 during purification proce-

dures. Consequently, N-1 desilylation of 70 proved facile

and selective in respect of the tert-butyldiphenylsilyl

ether at C-3, using 48% hydrogen fluoride, giving

2-azetidinone 71 in excellent yield. Comparison of the

¹H (500 MHz) NMR spectra of 71, the cis racemic

material, and a doped sample of both, all in the presence

of 3 mol % of Eu(hfc)₃,⁵⁰clearly showed the enantio-

purity of 71. Finally, acylation and desilylation proce-

dures as described for the racemic synthesis afforded

18 in good yield.

Con clu sion

time (min)

%B

In summary, a homology derived model was created

for PSA which led to the design and selection of a

number of novel monocyclic 2-azetidinone PSA inhibitor

targets, predicted to show strong binding properties

with the active site of PSA. A further, distinct, reverse

ester series of monocylic 2-azetidinones were also pre-

pared on the basis of this model and by the use of simple

structure-activity analysis (SAR). These reverse esters

were found to be inhibitors of PSA, and a different mode

of binding was proposed. Furthermore, MCSS calcula-

tions were conducted to enhance interpretation of the

homology model for normal ester (3S,4S)-2-azetidinone

inhibitors, and the C-3 p-hydroxybenzyl substituent was

predicted to exhibit greatest binding at the active site

of PSA. Subsequent synthesis of the pure enantiomer

18 realized increased PSA inhibition (IC₅₀) 226 ( 10

nM), further vindicating the active site binding model.

Thus, by use of molecular modeling, we were able to

augment SAR analysis and prepare the most potent

inhibitors of PSA known to date. Further structural

modification of the 2-azetidinone side chains could yield

other potent PSA inhibitors. In addition, such a design

process may prove successful in predicting inhibitors of

other enzymes, particularly when crystallographic struc-

tures are not available.

0

5

35

36

40

15

30

15

Enzyme cleavage was monitored by detection at 280 nm.

Estimated IC₅₀values were defined as the serial dilution

required to produce a half-maximal effect.

Ch em istr y. All solvents were distilled before use. Anhy-

drous dichloromethane (DCM), toluene, benzene, acetonitrile,

DMSO, 1-methyl-2-pyrrolidinone (NMP), and tert-butyl alcohol

were obtained by refluxing over calcium hydride followed by

distillation under an inert atmosphere of argon. Anhydrous

methanol was obtained by distillation from magnesium meth-

oxide. Anhydrous diethyl ether and anhydrous tetrahydrofuran

(THF) were obtained by distillation from sodium/benzophenone

ketyl under nitrogen immediately prior to use. LP and LP 40-

60 refer to the fractions of light petroleum ether which boil

within the ranges 30-40 °C and 40-60 °C, respectively.

Solvents were evaporated under reduced pressure on a Bu¨chi

RE111 rotavapor; high boiling solvents were evaporated on a

Bu¨chi RE111 rotavapor fitted with a dry ice condenser at less

than 2 mmHg. Kugelro¨hr distillations were performed at the

recorded oven temperature and pressure.

All other reagents were used as obtained from commercial

sources or purified by methods described in D. P. Perrin and

W. L. F. Armarego’s Purification of Laboratory Chemicals, 3rd

ed.; Pergamon Press: New York, 1988. All chemicals were

handled in accordance with safety instructions as detailed in

Good Laboratory Practice; Dyson Perrins Laboratory: Oxford,

1993-1996.

Exp er im en ta l Section

Biology. IC₅₀values were run on an individual as opposed

to batch basis. As such, each IC₅₀value has its own quoted

standard error limits; thence these values are not strictly

quantitative. A spectrophotometric assay was used to deter-

mine the ability of compounds to inhibit PSA cleavage of

p-nitroaniline (p-NA) from MeO-Suc-Arg-Pro-Tyr-p-NA‚HCl,

a chromogenic substrate for chymotrypsin (Pharmacia Hepar

Chromogenix S-2586). All experiments were carried out in flat

bottom 96-well plates coated with 10 µL of PBS/BSA (6.3 µg/

mL BSA) and analyzed by a microtiter plate reader (Bio-Tec

Instruments). A total of 1 to 5 mg of each compound was

dissolved in DMSO and made up to 300 µM stock solutions.

Serial dilutions of inhibitors with DMSO were evaluated (300

to 0.01 µM). A total of 10 µL of control or inhibitor was

preincubated with 10 µL of PSA (40 nM) for 2 h at 37 °C. The

reaction was initiated with 70 µL of 1 mM substrate. Twenty

plate scans during a 10 min period at 450 and 405 nm were

used to monitor the progress of enzyme cleavage. Software for

the Bio-Tek plate reader (CERES UV900HDi) plotted reaction

time on the X-axis versus mO.D. on the Y-axis and calculated

All reactions were performed under an inert atmosphere of

argon unless otherwise stated. Low temperatures were ob-

tained either by the use of dry ice/acetone or dry ice/2-propanol

cooling baths, or by the use of a J ulabo FT901 Immersion

Cooler.

Thin-layer chromatography was performed on glass plates

precoated with Merck silica gel 60F₂₅₄0.2 mm, which were

visualized by quenching of ultraviolet fluorescence (λ_max254

nm), by staining with iodine vapor, or by staining with 5%

w/v phosphomolybdic acid in ethanol, 10% w/v ammonium

molybdate in 2 N sulfuric acid, or 10% aqueous bromocreosol

green, all followed by heat. R_fvalues are quoted to the nearest

0.05. Flash chromatography was performed on J anssen silica

(0.035-0.070 nm) or Sorbsil C60 silica gel (30-60 mm).

Melting points (mp) were determined using a Bu¨chi 510 or

a Cambridge Instruments Galen III Ko¨fler Block apparatus.

Optical rotations ([R]^t_D) were determined at temperature t

using a Perkin-Elmer 241 polarimeter in 1 dm path length

cells at 589 nm. Values are given in 10^-1deg cm²g with

2-Azetidinone Inhibitors of Prostate Specific Antigen

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1501

concentrations given in g/100 mL. Ultraviolet spectra were

recorded on a Perkin-Elmer Lambda 2 UV-vis spectropho-

tometer with absorbances given as λ_max. Infrared spectra were

recorded on a Perkin-Elmer 781 spectrometer or a Perkin-

Elmer 1750 Fourier Transform spectrophotometer. Only se-

lected absorbances are listed.

with 5% aqueous hydrochloric acid (2 × 50 mL), water (100

mL), and brine (100 mL), dried (MgSO₄), filtered, and concen-

trated under reduced pressure to yield a black oil. The oil was

passed through a silica pad, eluting with DCM-ether (10:1) to

yield a yellow solid. Recrystallization from DCM-LP afforded

23 as a white crystalline solid (2.16 g, 61%): (R_f0.45, DCM);

mp 98-100 °C; IR (KBr, cm^-1) 3031, 2935, 1748, 1724; MS

(CI, NH₃) m/z 296 [MH⁺, 100], 149 [67], 134 [27], 124 [23].

Anal. (C₁₈H₁₇NO₃) C, H, N.

Proton nuclear magnetic resonance (¹H NMR) spectra were

recorded on a Varian Gemini 200 operating at 200 MHz, a

Bru¨ker AC200 operating at 200 MHz, or a Bru¨ker AM500 or

AMX500 operating at 500 MHz. The spectra were referenced

to residual protonated solvent residues, for example, CHCl₃

δ_H) 7.27 ppm, as an internal standard. Proton decoupled

carbon-13 nuclear magnetic resonance (¹³C NMR) spectra were

recorded on either a Varian Gemini 200 operating at 50.3 MHz

or a Bru¨ker AMX500 operating at 125.7 MHz. The spectra

were referenced to solvent carbon residues. Distortionless

enhancement by polarization transfer (DEPT) editing was

generally used for spectra obtained at 50.3 MHz. All chemical

shifts (δ_H, δ_C) are quoted in parts per million (ppm) downfield

from tetramethylsilane, in the solvent indicated in parenthe-

ses. Values are quoted to 0.01 ppm for δ_Hand δ_C. Signal

multiplicities are described as s - singlet, d - doublet, t -

triplet, and q - quartet, with through n bond coupling

cis-3-(4′-Acetoxyben zyl)-4-for m yl-1-(4′-m eth oxyph en yl)-

2-a zetid in on e (24). To a stirred solution of acid (21) (17.0 g,

81.6 mmol) in DCM (250 mL) were added DMF (3 drops) then

oxalyl chloride (45.1 g, 355 mmol). The resulting yellow

solution was stirred for 1.5 h and concentrated under reduced

pressure to give an orange oil. Distillation under reduced

pressure afforded the 3-(4′-acetoxyphenyl)propionyl chloride

as a colorless oil (16.7 g, 90%): bp 141 °C (0.06 mmHg); IR

(NaCl, cm^-1) 3039, 2936, 1796, 1763). To a stirred suspension

of diimine (22) (3.22 g, 12.0 mmol) and triethylamine (2.43 g,

24.0 mmol) in dry toluene (110 mL) at 35-40 °C was added

3-(4′-acetoxyphenyl)propionyl chloride (5.44 g, 24.0 mmol) in

toluene (5 mL), dropwise over 2.5 h. The color changed from

brown to orange as the reaction mixture was stirred for a

further 1.5 h at 35-40 °C. Then 5% aqueous hydrochloric acid

(150 mL) was added and the resulting heterogeneous mixture

stirred for 2.5 h at room temperature. The solution was diluted

with toluene (50 mL) and the aqueous layer separated then

extracted with toluene (2 × 50 mL). The organic layers were

combined and washed with water (100 mL) and brine (100

mL), dried (MgSO₄), filtered, and concentrated under reduced

pressure to yield a black gum. Ether was added, and after

scratching the glass flask, a pale brown solid precipitated and

was filtered from the solution. Further purification was

possible using flash chromatography (SiO₂, DCM-ether, 25:1)

to afford 24 as a pale yellow crystalline solid (3.61 g, 85%):

n

constants J being reported to the nearest 0.5 hertz (Hz). The

symbol br indicates that the peak was broad.

Low resolution mass spectra (MS) were recorded on either

a V. G. ZAB1F, V. G. Masslab 20-250, V. G. TRIO1 (GCMS),

V. G. Platform, or a V. G. BIO-Q instrument, with the modes

of ionization being indicated as direct chemical ionization

(DCI), probe chemical ionization (CI), atmospheric pressure

chemical ionization (APCI), fast atom bombardment (FAB), or

electrospray (ES). Intensities were recorded as percentages of

the largest signal and only major peaks were recorded, as the

mass-to-charge (m/z) ratio. APCI measurements were obtained

using a mixed solvent system [MeOH:MeCN:H₂O, 40:40:20].

3-(4′-Acetoxyp h en yl)p r op ion ic Acid (21). To a stirred

suspension of phloretic acid [3-(4′-hydroxyphenyl)propionic

acid] 20 (24.9 g, 15.0 mol) in water (120 mL) was added

powdered sodium hydroxide (15.0 g, 37.5 mol), and a clear pale

brown solution resulted after stirring for 30 min. Ice (150 mL)

was added to cool the solution, and after this had melted, acetic

anhydride (23.0 g, 22.5 mol) was added in one portion. After

the mixture was stirred for 17 h, a brown precipitate had

formed. The mixture was extracted with ether (2 × 120 mL)

to remove the precipitate, and the aqueous layer was gradually

acidified by careful addition of 5% aqueous hydrochloric acid.

The solution was extracted at each integral pH with ether (2

× 120 mL). It was noted that on reaching pH 4 a white

precipitation occurred. The ether solutions obtained at pH 4-5

were combined, dried (MgSO₄), filtered, and concentrated

under reduced pressure to yield a pale brown solid. Recrys-

tallization from ether-LP 40-60 afforded 21 as white needles

(24.9 g, 80%): mp 97-98 °C; IR (KBr, cm^-1) 2480-3280, 1756,

1704; MS (CI, NH₃) m/z 226 [MNH₄⁺, 100], 208 [M⁺, 8], 166

[30], 107 [73]. Anal. (C₁₁H₁₂O₄) C, H.

(R_f0.45, DCM-ether; 10:1); mp 114.5-116 °C; IR (KBr, cm^-1

)

3007, 2955, 2934, 1746; MS (CI, NH₃) m/z 371 [MNH₄⁺, 100],

354 [MH⁺, 32], 222 [28]. Anal. (C₂₀H₁₉NO₅) C, H, N.

Gen er a l P r oced u r e for th e P r ep a r a tion of Acid s 29

a n d 57: cis-3-Ben zyl-4-ca r boxy-1-(4′-m eth oxyp h en yl)-2-

a zetid in on e (29). To a stirred solution of aldehyde 23 (2.50

g, 8.5 mmol) in acetone (65 mL), at 0 °C, was added freshly

prepared J ones reagent dropwise until an orange color per-

sisted. [J ones reagent was prepared by dissolving chromium

trioxide (6.7 g) in 12.5 mL of distilled water and adding 5.8

mL of concentrated sulfuric acid.] The reaction mixture was

concentrated under reduced pressure, and the residue was

then dissolved in ethyl acetate (50 mL) and washed with water

(3 × 30 mL). The resulting orange solution was dried (MgSO₄),

filtered, and concentrated under reduced pressure to yield an

orange solid. Trituration with ether afforded 29 as a yellow

crystalline solid (1.82 g, 69%); mp 207-208 °C; IR (KBr, cm^-1

)

3240-2420, 1729, 1687; MS (CI, NH₃) m/z 329 [MNH₄⁺, 5],

312 [MH⁺, 100], 296 [3], 239 [18], 134 [18]. Anal. (C₁₈H₁₇NO₄)

C, H, N.

cis-3-(4′-Acetoxyben zyl)-4-ca r boxy-1-(4′-m eth oxyp h en -

yl)-2-a zetid in on e (57). The title compound was prepared in

89% isolated yield from aldehyde 24 (9.37 mmol scale) follow-

ing a procedure similar to that described above for 29: mp

193-194 °C; IR (KBr, cm^-1) 3640-2760, 3008, 2933, 1767,

cis-3-Ben zyl-4-for m yl-1-(4′-m eth oxyp h en yl)-2-a zetid i-

n on e (23). Hydrocinnamic acid 19 (60.0 g, 400 mmol) was

carefully added to phosphorus pentachloride (83.3 g, 400 mmol)

and heated to 100 °C. The reaction was extremely vigorous,

and the resulting solution was stirred under reflux for 1 h.

Phosphorus oxychloride was distilled off under atmospheric

pressure (107 °C), and then distillation under reduced pressure

afforded hydrocinnamoyl chloride as a colorless oil (53.6 g,

80%): [bp 146 °C (25 mmHg), lit.⁵¹107 °C (11 mmHg)]; IR

(NaCl, cm^-1) 3030, 2931, 1795. To a stirred suspension of 1,4-

bis(4′-methoxyphenyl)-1,4-diazabuta-1,3-diene⁴⁶(22) (3.22 g,

12.0 mmol) and triethylamine (2.43 g, 24.0 mmol) in dry

toluene (110 mL) was added hydrocinnamoyl chloride (4.05 g,

24.0 mmol) dissolved in toluene (10 mL) via slow addition over

10 min, maintaining the temperature within the range 35-

40 °C. The color changed from brown to orange as the reaction

mixture was stirred for 1 h. Aqueous hydrochloric acid (5%,

100 mL) was added and the resulting heterogeneous mixture

stirred vigorously for a further 3 h. The solution was washed

1747, 1727; MS (ES) m/z 368 [(M - H)^-, 100]. Anal. (C₂₀H₁₉

-

NO₆) C, H, N.

cis-3-Ben zyl-4-h yd r oxym eth yl-1-(4′-m eth oxyp h en yl)-2-

a zetid in on e (30). A stirred suspension of aldehyde 23 (886

mg, 3.00 mmol) in methanol (12 mL) was cooled to 0 °C.

Sodium borohydride (113 mg, 2.99 mmol) was added portion-

wise over 10 min, allowing dissolution to occur after each

addition. A white precipitate soon formed, and the reaction

was allowed to stir at 0 °C for a further 1 h. Water (7 mL)

was added and the reaction mixture allowed to warm to room

temperature. Methanol was removed under reduced pressure

and the remaining solution extracted with ethyl acetate (3 ×

15 mL). The solution was washed with water (20 mL), dried

(MgSO₄), filtered, and concentrated under reduced pressure

1502 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10

Adlington et al.

to yield a white solid. Flash chromatography (SiO₂, LP-ethyl

acetate, 3:1; 1:1, gradient elution) afforded 30 as a white

crystalline solid (717 mg, 80%): (R_f0.15, LP-ethyl acetate; 4:1);

mp 115 °C; IR (KBr, cm^-1) 3474, 3081, 1708; MS (CI, NH₃)

m/z 315 [MNH₄⁺, 10], 298 [MH⁺, 100]. Anal. (C₁₈H₁₉NO₃) C,

H, N.

under reduced pressure to yield a white solid. Flash chroma-

tography (SiO₂, LP-ether; 2:1; 1:2, gradient elution) afforded

37 as a white crystalline solid (955 mg, 67%): (R_f0.45, LP-

ether; 1:5); mp 100-101 °C; IR (KBr, cm^-1) 3348, 2981, 2924,

1685; MS (APCI, NH₃) m/z 255 [MNH₄⁺, 14], 238 [MH⁺, 4],

199 [100], 138 [67], 106 [22]. Anal. (C₁₃H₁₉NO₃) C, H, N.

4-(ter t-Bu tyloxycar bon yl)ben zaldeh yde (32). To a stirred

solution of 4-carboxybenzaldehyde 31 (1.20 g, 7.99 mmol) in

refluxing benzene (15 mL) was added N,N-dimethylformamide

di-tert-butylacetal (6.50 g, 32.0 mmol) over 1 h. The mixture

was refluxed for a further 30 min, then allowed to cool, and

washed with water (15 mL), 5% aqueous sodium hydrogen

carbonate (2 × 10 mL), and brine (10 mL). The organic layer

was separated, dried (MgSO₄), filtered, and concentrated under

reduced pressure to yield a white solid. Flash chromatography

(SiO₂, LP-ether, 5:1) afforded 32 as a white crystalline solid

Isop h th a lic Acid -m on oben zylester (39).³⁷To a stirred

solution of isophthalic acid (38) (1.66 g, 10 mmol) in a methanol

(20 mL)-water (2 mL) mixture was added triethylamine (1.02

g, 10.1 mmol) dissolved in methanol (10 mL). After the mixture

was stirred overnight, the solvent was removed under reduced

pressure and the residue dissolved in DMF (25 mL). Benzyl

bromide (1.88 g, 11.0 mmol) was added dropwise and the

mixture stirred for 2 h at 100 °C. After cooling, the mixture

was poured into 5% aqueous sodium hydrogen carbonate (50

mL) and extracted with ethyl acetate (3 × 30 mL). The aqueous

layer was acidified with 1 N hydrochloric acid to pH 6 and

further extracted with ethyl acetate (3 × 30 mL) to remove

diester impurities. Further acidification of the aqueous layer

to the range pH 5-pH 3 allowed ethyl acetate extraction of

the monoester product. Then acidification to pH 1 permitted

recovery of unreacted 38. The organic layers containing

monoester were combined, washed with brine (50 mL), dried

(MgSO₄), filtered, and concentrated under reduced pressure

to yield a white solid. Recrystallization from ethanol-water

afforded 39 as a white crystalline solid (1.25 g, 49%): (R_f0.25,

LP-ether; 1:1); mp 107 °C; IR (KBr, cm^-1) 3300-2400, 1719,

1705, 1690; MS (CI, NH₃) m/z 274 [MNH₄⁺, 24], 257 [MH⁺, 4],

149 [60], 108 [94], 91 [100]. Anal. (C₁₅H₁₂O₄) C, H.

2,2,2-Tr ich lor oe t h yl-(3-b e n zyloxyca r b on yl)p h e n yl-

a ceta te (40).³⁷To a stirred solution of monoester 39 (2.60 g,

10.1 mmol) in DCM (30 mL) was added 2 drops of DMF. Oxalyl

chloride (6.35 g, 50.0 mmol) was added, and after being stirred

for 1 h at room temperature the reaction mixture was

concentrated under reduced pressure to afford 3-(benzyloxy-

carbonyl)benzoyl chloride as a colorless thick oil which was

used without further purification [(R_f0.5, LP-ether; 1:1); IR

(NaCl, cm^-1) 3069, 3035, 2957, 1762, 1724.]

An alcohol free, ethereal solution of diazomethane (ca. 3.4

g, 0.32 M prepared from 24.5 g of Diazald) was cooled with

stirring in an ice-salt bath to a temperature between -5 and

0 °C. The acid chloride dissolved in benzene (30 mL) was added

dropwise over 15 min, and stirring continued at a temperature

below 0 °C for a further 30 min. The mixture was then allowed

to warm to room temperature and stirred overnight. Excess

diazomethane was removed under reduced pressure (bubbling

gases were passed through an acetic acid reservoir) and then

the reaction mixture concentrated under reduced pressure to

dryness, affording benzyl-3-(diazomethylcarbonyl)benzoate in

quantitative yield as a bright yellow solid which was used

without further purification.

(1.05 g, 64%): (R_f0.7, LP-ether; 1:1); mp 52 °C; IR (KBr, cm^-1

)

1707, 1700; MS (GCMS, CI, NH₃) m/z 224 [MNH₄⁺, 12], 207

[MH⁺, 100], 133 [15], 74 [7], 57 [3]. Anal. (C₁₂H₁₄O₃) C, H.

4-(ter t-Bu tyloxyca r bon yl)ben zyl Alcoh ol (33). A stirred

solution of aldehyde 32 (4.12 g, 20.0 mmol) in methanol (30

mL) was cooled in an ice bath at 0 °C. Sodium borohydride

(832 mg, 22.0 mmol) was then added in ca. 50 mg portions

over 10 min, allowing dissolution to occur after each addition,

and the reaction was allowed to stir at 0 °C for a further 1 h.

Solvent was removed under reduced pressure, and the residue

was dissolved in ethyl acetate (300 mL). The solution was

washed with water (2 × 250 mL) and 5% aqueous sodium

hydrogen carbonate (200 mL), dried (MgSO₄), filtered, and

concentrated under reduced pressure to yield a gray solid.

Flash chromatography (SiO₂, LP-ether, 3:2) afforded 33 as a

white crystalline solid (3.55 g, 85%): (R_f0.4, LP-ether; 1:1);

mp 51-52 °C; IR (KBr, cm^-1) 3600-3200, 2975, 1713; MS (CI,

NH₃) m/z 226 [MNH₄⁺, 25], 209 [MH⁺, 82], 170 [70], 153 [42],

135 [100]. Anal. (C₁₂H₁₆O₃) C, H.

Ter ep h th a lic Acid -m on o(ter t-bu tyl)ester (34). To a

stirred solution of aldehyde 32 (412 mg, 2.0 mmol) in acetone

(15 mL), in an ice bath, was added freshly prepared J ones

reagent dropwise until an orange color persisted.³⁴The reac-

tion mixture was concentrated under reduced pressure, and

the residue was dissolved in ethyl acetate (30 mL) and washed

with water (3 × 20 mL) until the organic layer was colorless.

The resulting solution was dried (MgSO₄), filtered, and

concentrated under reduced pressure to yield a white solid.

Recrystallization from LP-ether afforded 34 as white needles

(374 mg, 84%): (R_f0.4, LP-ether; 1:1); mp 238-240 °C; IR

(KBr, cm^-1) 3600-2290, 2977, 1714, 1690; MS (CI, NH₃) m/z

240 [MNH₄⁺, 100], 223 [ MH⁺, 12], 74 [28]. Anal. (C₁₂H₁₄O₄)

C, H.

4-[N-(ter t-Bu tyloxycar bon yl)am in om eth yl]ben zoic Acid

(36). To a stirred solution of 4-(aminomethyl)benzoic acid (5.00

g, 33.1 mmol) in 1,4-dioxan (60 mL), water (30 mL), and 1 M

NaOH (34 mL, 34 mmol), at 0 °C, was added di-tert-butyl-

pyrocarbonate(7.94 g, 36 mmol), and the mixture was stirred

at 0 °C for 40 min. The solution was concentrated under

reduced pressure to 40 mL, then ethyl acetate (80 mL) was

added, and the mixture was acidified to pH 4 with 1 M KHSO₄.

The organic layer was dried (Na₂SO₄), filtered, and concen-

trated under reduced pressure to yield a white solid. Recrys-

talization from ethyl acetate gave 36 as white crystalline

needles (7.46 g, 90%); mp 167-168 °C; IR (KBr, cm^-1) 3356,

2982, 2931, 1686; MS (APCI, NH₃) m/z 252 [MH⁺, 100%], 222

[30], 172 [25]. Anal. (C₁₃H₁₇NO₄) C, H, N.

4-(ter t-Bu tyloxyca r bon yla m in om eth yl)ben zyl Alcoh ol

(37). A stirred solution of acid 36 (1.51 g, 6.0 mmol) in THF

(60 mL) was prepared in a flask immersed in a water bath at

ca. 25 °C. Borane-THF complex (15 mL of a 1 M solution, 15

mmol) was carefully added dropwise over 10 min, and evolu-

tion of hydrogen was observed. After being stirred overnight

(20 h), the reaction mixture was quenched with water (30 mL)

and extracted with ethyl acetate (2 × 60 mL). The organic

layers were combined and washed sequentially with water (30

mL), 5% aqueous sodium hydrogencarbonate (2 × 30 mL), and

water (30 mL), then dried (MgSO₄), filtered, and concentrated

The solid was dissolved in 2,2,2-trichloroethanol (35 mL, 365

mmol) and heated to 50-60 °C. Silver oxide (ca. 2 g) [freshly

prepared from silver nitrate and sodium hydroxide precipitate,

thoroughly washed sequentially with water, acetone, and ether

then dried under vacuum over P₂O₅] was added in 250 mg

portions to the reaction mixture until addition no longer

resulted in evolution of nitrogen. Heating was continued for a

further 30 min, and then the mixture was allowed to cool to

room temperature after a total of 210 min. Chloroform (30 mL)

was added to dilute the mixture which was filtered through

Celite with chloroform (100 mL) to remove silver residues and

concentrated under reduced pressure to yield an orange oil.

Flash chromatography (SiO₂, LP-ether, 10:1; 2:1, gradient

elution) afforded 40 as a colorless oil (2.75 g, 68%): (R_f0.75,

LP-ether; 1:1); IR (NaCl, cm^-1) 3035, 2955, 1755, 1718; MS

+

(CI, NH₃) m/z MNH₄

: {424 [0.5%], 422 [3], 420 [9], 418

[³⁵Cl₃MNH₄⁺,10]}, 293 [22], 295 [20], 297 [7], 108 [87], 91 [100].

Anal. (C₁₈H₁₅Cl₃O₄) C, H.

3-(Ben zyloxyca r bon yl)p h en yla cetic Acid (41).³⁷To a

stirred solution of trichloroethylester 40 (2.44 g, 6.08 mmol)

in THF (180 mL), in an ice bath at 0 °C, was added 90%

aqueous acetic acid (30 mL). Zinc dust (24.0 g, 367 mmol) was

added portionwise every 10 min over 1 h. After 3 h the reaction

2-Azetidinone Inhibitors of Prostate Specific Antigen

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1503

mixture was filtered through Celite and partitioned with DCM

(200 mL)-water (200 mL). The aqueous layer was separated

and further extracted with DCM (2 × 100 mL). The organic

extacts were combined, dried (MgSO₄), filtered, and concen-

trated under reduced pressure to yield a gray solid. Recrys-

tallization from ether afforded 41 as a white crystalline solid

2,2,2-Tr ich lor oeth yl-[(3-ter t-bu tyloxyca r bon yl)p h en yl]-

a ceta te (45). To a stirred solution of acid 43 (78 mg, 0.25

mmol) in DCM (1 mL) was added O-(tert-butyl)-trichloro-

acetimidate (110 mg, 0.503 mmol) in cyclohexane (2 mL). Upon

addition of boron trifluoride etherate (5 mL), the reaction

mixture became cloudy. After the mixture was stirred for 22

h, solid sodium hydrogencarbonate (5 mg) was added and the

resulting solution filtered through a short flash silica pad.

Concentration under reduced pressure afforded 45 as a color-

(1.38 g, 84%): (R_f0.45, LP-ether; 1:1); mp 95 °C; IR (KBr, cm^-1

)

+

3240-2200, 2955, 1720, 1700; MS (CI, NH₃) m/z 288 [MNH₄

,

23], 271 [MH⁺, 5], 163 [22], 108 [82], 91 [100]. Anal. (C₁₆H₁₄O₄)

C, H.

less oil (64 mg, 70%): (R_f0.45, LP-ether; 5:1); IR (NaCl, cm^-1

)

2978, 1758, 1713; MS (CI, NH₃) m/z: {388 [31], 386 [100], 384

[³⁵Cl₃MNH₄⁺, 100]}, 332 [20], 330 [74], 328 [71], 152 [28].

HRMS (CI, C₁₅H₂₁³⁵Cl₃NO₄⁺): found, 384.0536; requires,

384.0536.

3-(Ben zyloxyca r bon yl)p h en yla cetyl Ch lor id e (42). To

a stirred solution of acid 41 (108 mg, 0.40 mmol) in DCM (50

mL), was added DMF (2 drops) then oxalyl chloride (254 mg,

2.00 mmol). The resulting orange solution was stirred for 3 h

and concentrated under reduced pressure to yield 3-(benzyl-

oxycarbonyl) phenylacetyl chloride 42 as a gray solid (quant)

3-(ter t-Bu tyloxyca r bon yl)p h en yla cetyl Ch lor id e (46).

To a stirred solution of trichloroethylester 45 (218 mg, 0.60

mmol) in THF (16 mL), in an ice bath at 0 °C, was added 90%

aqueous acetic acid (3 mL). Zinc dust (2.36 g, 36.1 mmol) was

added in ca. 400 mg portions every 10 min over 1 h. A further

portion of zinc dust (1.18 g, 18.0 mmol) and 90% acetic acid

(1.5 mL) was added after 90 min. After 3 h the reaction

mixture was filtered through Celite and partitioned with DCM

(50 mL)-water (50 mL). The aqueous layer was separated and

further extracted with DCM (2 × 50 mL). The organic extacts

were combined, dried (MgSO₄), filtered, and concentrated

under reduced pressure to yield a gray solid. Recrystallization

from ether afforded 3-(tert-butyloxycarbonyl)phenylacetic acid

as a white crystalline solid (132 mg, 94%): (R_f0.25, LP-ether;

5:1); mp 76-78 °C; IR (KBr, cm^-1) 3420-2460, 2977, 2927,

1716, 1705; MS (CI, NH₃) m/z 254 [45%, MNH₄⁺], 198 [100,

which was used without further purification [IR (NaCl, cm^-1

3034, 1798, 1718].

)

2,2,2-Tr ich lor oeth yl-(3-car boxyph en yl)acetate (43). The

trichloroethyl ester 40 (100 mg, 0.25 mmol) was dissolved in

ethyl acetate (5 mL). To this stirred solution was added

palladium on activated carbon (10 mg, 10% w/w), and the

reaction vessel sequentially evacuated and flushed with

hydrogen 3 times. After 3 h of stirring under an atmosphere

of hydrogen (balloon pressure), the reaction mixture was

filtered through Celite and concentrated under reduced pres-

sure to afford 43 as a white crystalline solid (78 mg, quant):

(R_f0.35, LP-ether; 1:1); mp 113-114 °C; IR (KBr, cm^-1) 2959,

2232, 2072, 1753, 1694; MS (DCI, NH₃) m/z MNH₄⁺{334 [4%],

332 [33], 330 [92], 328 [³⁵Cl₃MNH₄^+,100]}, 294 [15], 226 [25],

152 [33]. Anal. (C₁₁H₉Cl₃O₄) C, H.

+

MNH₄- C₄H₈]. Anal. (C₁₃H₁₆O₄) C, H.

To a stirred solution of the above acid (78 mg, 0.330 mmol)

in DCM (50 mL) were added DMF (2 drops) then oxalyl

chloride (190 mg, 1.50 mmol). The resulting orange solution

was stirred for 3 h and concentrated under reduced pressure

to yield 3-(tert-butyloxycarbonyl)phenylacetyl chloride 46 as

a gray solid (quant) which was used without further purifica-

tion [IR (NaCl, cm^-1) 2980, 1800, 1712].

3-[4′-(t er t -B u t y lo x y c a r b o n y l)b e n zy lo x y c a r b o n y l]-

p h en yla cetyl Ch lor id e (44). To a stirred solution of acid 43

(75 mg, 0.24 mmol) in dry THF (4 mL) cooled in an ice bath

was added 4-(tert-butyloxycarbonyl)benzyl alcohol 33 (50 mg,

0.24 mmol) and DMAP (ca. 1 mg). Dicyclohexylcarbodiimide

(53 mg, 0.26 mmol) was then added in ca. 10 mg portions over

10 min and the solution stirred overnight. A white precipitate

of dicyclohexylurea was removed by filtration and the filtrate

concentrated under reduced pressure. The residue was redis-

solved in DCM (10 mL), washed with water (3 × 8 mL), dried

(MgSO₄), filtered, and concentrated under reduced pressure

to yield a white solid. Flash chromatography (SiO₂, LP-ether;

4:1; 2:1, gradient elution) afforded 2,2,2-trichloroethyl-{3-[4′-

(tert-butyloxycarbonyl)benzyloxycarbonyl]phenyl}acetate as a

white crystalline solid (81 mg, 67%): (R_f0.75, LP-ether; 1:1);

mp 69 °C; IR (KBr, cm^-1) 3050, 2933, 1757, 1718; MS (CI, NH₃)

Gen er a l P r oced u r e for th e P r ep a r a tion of C-4 Es-

t er s: cis-3-Ben zyl-4-[4′-(ter t-b u t yloxyca r b on yl)b en zyl-

oxyca r bon yl]-1-(4′-m eth oxyp h en yl)-2-a zetid in on e (47).

To a stirred solution of acid 29 (1.56 g, 5.02 mmol) in dry THF

(75 mL), cooled in an ice bath, was added 4-(tert-butyloxycar-

bonyl)benzyl alcohol 33 (1.05 g, 5.05 mmol) and DMAP (ca.

10 mg). Dicyclohexylcarbodiimide (1.12 g, 5.48 mmol) was

added portionwise over 10 min and the solution stirred

overnight. A white precipitate of dicyclohexylurea was removed

by filtration and the filtrate concentrated under reduced

pressure. The residue was redissolved in DCM (50 mL),

washed with water (3 × 30 mL), dried (MgSO₄), filtered, and

concentrated under reduced pressure to yield an orange solid.

Flash chromatography (SiO₂, LP-ether, 3:2) afforded 47 as a

white crystalline solid (2.07 g, 82%): (R_f0.6, LP-ether; 1:1);

mp 98-99 °C; IR (KBr, cm^-1) 3027, 2970, 2933, 1751, 1735,

1711; MS (DCI, NH₃) m/z 519 [MNH₄⁺, 12%], 501 [M⁺, 21],

446 [100], 238 [28]. Anal. (C₃₀H₃₁NO₆) C, H, N.

m/z MNH₄{524 [4%], 522 [24], 520 [73], 518 [³⁵Cl₃MNH₄

,

+

65]}, 468 [5], 466 [35], 464 [100], 462 [92], 428 [27], 152 [34].

Anal. (C₂₃H₂₃Cl₃O₆) C, H.

To a stirred solution of the above trichloroethylester (513

mg, 1.02 mmol) in THF (38 mL), in an ice bath at 0 °C, was

added 90% aqueous acetic acid (5 mL). Zinc dust (2.70 g, 41.3

mmol) was added in ca. 500 mg portions every 10 min over 1

h. A further amount of 90% aqueous acetic acid (2 mL) and

zinc dust (500 mg, 7.65 mmol) was added after 150 min. After

3 h, the reaction mixture was filtered through Celite and

partitioned with DCM (50 mL)-water (50 mL). The aqueous

layer was further extracted with DCM (2 × 50 mL). The

organic extacts were combined, dried (MgSO₄), filtered, and

concentrated under reduced pressure to yield a gray solid.

Recrystallization from ether afforded 3-[4′-(tert-butyloxycar-

bonyl)benzyloxycarbonyl]phenylacetic acid as a white crystal-

line solid (295 mg, 78%): (R_f0.65, LP-ether; 1:1); mp 122 °C;

IR (KBr, cm^-1) 3600-2400, 3015, 2979, 1716, 1707, 1695; MS

(DCI, NH₃) m/z 388 [MNH₄⁺, 65], 332 [100], 163 [11]. Anal.

(C₂₁H₂₂O₆) C, H.

cis-3-(4′-Ace t oxyb e n zyl)-4-b e n zyloxyca r b on yl-1-(4′-

m eth oxyp h en yl)-2-a zetid in on e (58a ). The title compound

was prepared in 82% isolated yield (7.72 mmol scale) from acid

57 and benzyl alcohol following a procedure similar to that

described above for 47: (R_f0.55, LP-ether; 1:5); mp 136-137

°C; IR (KBr, cm^-1) 3025, 2955, 1759, 1736; MS (CI, NH₃) m/z

477 [MNH₄⁺, 6], 460 [MH⁺, 15], 107 [66], 91 [100]. Anal.

(C₂₇H₂₅NO₆) C, H, N.

cis-3-(4′-Acetoxyben zyl)-4-[4′-(ter t-bu tyloxyca r bon yl)-

b e n zyloxyca r b on yl]-1-(4′-m e t h oxyp h e n yl)-2-a ze t id i-

n on e (58b). The title compound was prepared in 75% isolated

yield (10.5 mmol scale) from acid 57 and alcohol 33 following

a procedure similar to that described above for 47: (R_f0.7,

DCM-ether; 10:1); mp 46-48 °C; IR (KBr, cm^-1) 2980, 1761,

1711; MS (DCI, NH₃) m/z 577 [MNH₄⁺, 16], 559 [M⁺, 25], 521

[28], 504 [29], 326 [46], 296 [35], 135 [78], 107 [100], 91 [49],

56 [30]. Anal. (C₃₂H₃₃NO₈) C, H, N.

To a stirred solution of the above acid (148 mg, 0.40 mmol)

in DCM (50 mL) was added DMF (2 drops) then oxalyl chloride

(254 mg, 2.00 mmol). The resulting orange solution was stirred

for 3 h and concentrated under reduced pressure to yield 44

as a gray solid (quant) which was used without further

purification [IR (NaCl, cm^-1) 2979, 1800, 1713].

1504 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10

Adlington et al.

Gen er a l P r oced u r e for N-1 Dea r yla tion of 2-Azetid i-

n on es: cis-3-Ben zyl-4-[4′-(ter t-bu tyloxyca r bon yl)ben zyl-

oxyca r bon yl]-2-a zetid in on e (48). The protected 2-azetidi-

none 47 (1.51 g, 3.01 mmol) was dissolved in acetonitrile (70

mL) and cooled in an ice-salt bath to between -5 and 0 °C.

At this temperature a solution of ceric ammonium nitrate (4.93

g, 8.99 mmol) in water (40 mL) was added over 3 min. The

reaction mixture was stirred for a further 10 min then diluted

with water (30 mL) and extracted with ethyl acetate (3 × 30

mL). The organic solution was then washed sequentially with

5% aqueous sodium hydrogen carbonate (20 mL), 10% aqueous

sodium sulfite (3 × 30 mL), and water (40 mL), dried (MgSO₄),

filtered, and concentrated under reduced pressure to yield an

orange solid. Flash chromatography (SiO₂, DCM-ether, 6:1)

afforded 48 as a white foam (993 mg, 83%): (R_f0.3, LP-ether;

1:5); mp 112 °C; IR (CHCl₃, cm^-1) 3020, 1776, 1748, 1709; MS

(DCI, NH₃) m/z 413 [MNH₄⁺, 100], 357 [59], 340 [18], 131 [17].

Anal. (C₂₃H₂₅NO₅) C, H, N.

cis-3-Be n zyl-4-p h e n ylca r b on yloxym e t h yl-2-a ze t id i-

n on e (54). The title compound was prepared in 73% isolated

yield (0.60 mmol scale) from 2-azetidinone 53 following a

procedure similar to that described above for 48: (R_f0.3, LP-

ethyl acetate; 2:1); mp 117-119 °C; IR (KBr, cm^-1) 3207 (NH),

3087, 2925, 1751, 1713; MS (CI, NH₃) m/z 313 [MNH₄⁺, 47],

296 [MH⁺, 100], 130 [26], 105 [45], 91 [3]. Anal. (C₁₈H₁₇NO₃)

C, H, N.

Gen er a l P r oced u r e for th e P r ep a r tion of N-1 Acyla ted

2-Azetid in on es: cis-3-Ben zyl-4-ben zyloxyca r bon yl-1-[3′-

(b en zyloxyca r b on yl)p h en yla cet a t e]-2-a zet id in on e (6).

cis-3-Benzyl-4-benzyloxycarbonyl-1-(4′-methoxyphenyl)-2-azeti-

dinone was prepared in 69% isolated yield (3.00 mmol scale)

from acid 29 and benzyl alcohol following a procedure similar

to that described above for 47: (R_f0.3, LP-ether; 3:2); mp 113-

114 °C; IR (KBr, cm^-1) 2970, 1737; MS (DCI, NH₃) m/z 402

[MH⁺, 100], 238 [48], 91 [48]. Anal. (C₂₅H₂₃NO₄) C, H, N.

cis-3-Benzyl-4-benzyloxycarbonyl-2-azetidinone was pre-

pared in 88% isolated yield (1.49 mmol scale) from cis-3-benzyl-

4-benzyloxycarbonyl-1-(4′-methoxyphenyl)-2-azetidinone fol-

lowing a procedure similar to that described above for 48: (R_f

0.55, LP-ether; 3:2); mp 111 °C; IR (KBr, cm^-1) 3209 (NH),

3102, 2974, 1752; MS (CI, NH₃) m/z 313 [MNH₄⁺, 11], 296

[MH⁺, 72], 268 [55], 91 [100]. Anal. (C₁₈H₁₇NO₃) C, H, N.

To a stirred solution of cis-3-benzyl-4-benzyloxycarbonyl-2-

azetidinone (118 mg, 0.40 mmol) in THF (12 mL), at -78 °C

was added dropwise sodium bis(trimethylsilyl)amide (0.40 mL

of a 1.0 M solution in THF, 0.40 mmol). After 20 min, the acid

chloride 42(0.40 mmol), dissolved in THF (2 mL), was added

dropwise with stirring continued at -78 °C. Water (1 mL) was

added to the reaction at -78 °C after 2 h followed by ethyl

acetate (30 mL), and the solution was allowed to warm to room

temperature with stirring. The mixture was washed with

water (2 × 10 mL), dried (MgSO₄), filtered, and concentrated

under reduced pressure to yield a gray solid. Flash chroma-

tography (SiO₂, LP-ether, 3:1; 2:1, gradient elution) afforded

the unreacted 2-azetidinone (50 mg, 42%) and 6 as a pale green

gummy solid (87 mg, 40%): (R_f0.2, LP-ether; 1:1); mp 60-61

°C; IR (NaCl, cm^-1) 3065, 3033, 2954, 1796, 1746, 1714; MS

(DCI, NH₃) m/z 565 [MNH₄⁺, 100], 548 [MH⁺, 21], 350 [5], 198

[9], 108 [35], 91 [68]. HRMS (CI, C₃₄H₃₃N₂O₆⁺): found,

565.2340; requires, 565.2339. Anal. (C₃₄H₂₉NO₆) C, H, N.

cis-1-Ben zoyl-3-b en zyl-4-[4′-(ter t-b u t yloxyca r b on yl)-

ben zyloxyca r bon yl]-2-a zetid in on e (49). The title com-

pound was prepared in 83% isolated yield (0.30 mmol scale)

from 2-azetidinone 48 and benzoyl chloride following a pro-

cedure similar to that described above for 6: (R_f0.3 LP-ether;

1:1); IR (NaCl, cm^-1) 3064, 2977, 1801, 1748, 1711, 1676; MS

(APCI) m/z 522 [M + Na⁺, 4], 475[11], 444[12], 443[15],

264[38], 222[12], 220[10], 122[100]. Anal. (C₃₀H₂₉NO₆) C, H,

N.

procedure similar to that described above for 6: (R_f0.2, LP-

ether; 2:1); mp 52-53 °C; IR (NaCl, cm^-1) 3065, 2978, 2933,

1798, 1749, 1713; MS (DCI, NH₃) m/z 631 [ MNH₄⁺, 30], 575

[7], 519 [39], 197 [94], 154 [93], 91 [100], 78 [33], 58 [99]. HRMS

(C₃₆H₄₃N₂O₈⁺): found, 631.3020; requires, 631.3019.

cis-3-Ben zyl-4-[(4′-ter t-b u t yloxyca r b on yl)b en zyloxy-

ca r b on yl]-1-{3′-[b en zyloxyca r b on yl]p h en yla cet a t e)-2-

a zetid in on e (51). The title compound was prepared in 38%

isolated yield (0.40 mmol scale) from 2-azetidinone 48 and acid

chloride 42 following a procedure similar to that described

above for 6: (R_f0.3 LP-ether; 4:6); IR (NaCl, cm^-1) 2977, 2933,

1797, 1748, 1715; MS (APCI) m/z 670[M + Na⁺, 8], 592[12],

574[52], 438[8], 124[24], 122[100]. Anal. (C₃₉H₃₇NO₈) C, H, N.

cis-3-Ben zyl-4-[(4′-ter t-b u t yloxyca r b on yl)b en zyloxy-

ca r bon yl]-1-{3′-[(4′′-ter t-bu tyloxyca r bon yl)ben zyloxyca r -

bon yl]p h en yla ceta te}-2-a zetid in on e (52). The title com-

pound [and unreacted 48 (18%)] was prepared in 36% isolated

yield (0.40 mmol scale) from 2-azetidinone 48 and acid chloride

44 following a procedure similar to that described above for

6: (R_f0.35, LP-ether; 1:1); mp 105-106 °C; IR (KBr, cm^-1

)

2980, 1786, 1744, 1730, 1714; MS (FAB) m/z 636 [MH⁺- (C₄H₈

× 2), 3], 618 [8], 484 [6], 269 [7], 135 [100], 118 [13], 91 [22],

57 [99]. Anal. (C₄₄H₄₅NO₁₀) C, H, N.

cis-1-Ben zoyl-3-ben zyl-4-p h en ylca r bon yloxym eth yl-2-

a zetid in on e (7). The title compound was prepared in 94%

isolated yield (0.68 mmol scale) from 2-azetidinone 54 and

benzoyl chloride following a procedure similar to that described

above for 6: R_f0.65, LP-EtAc; 1:2); mp 124-126 °C; IR (KBr,

cm^-1) 3058, 3028, 2954, 1790, 1720, 1671; MS (APCI) m/z

400[M + H⁺, 34], 296[6], 278[13], 174[7], 157[6], 122[13],

105[100]. Anal. (C₂₅H₂₁NO₄) C, H, N.

cis-3-Ben zyl-1-[3′-(ben zyloxyca r bon yl)p h en yla ceta te]-

4-p h en ylca r bon yloxym eth yl-2-a zetid in on e (8). The title

compound was prepared in 38% isolated yield (0.70 mmol

scale) from 2-azetidinone 54 and acid chloride 42 following a

procedure similar to that described above for 6: (R_f0.25, LP-

ethyl acetate; 4:1); IR (NaCl, cm^-1) 3032, 1789, 1719; MS (CI,

NH₃) m/z 565 [MNH₄⁺, 74], 548 [MH⁺, 54], 296 [33], 108 [37],

105 [100], 91 [40], 78 [12]. HRMS (CI, C₃₄H₃₀NO₆⁺): found,

548.2070; requires, 548.2073.

cis-1-Ben zoyl-4-b en zyloxyca r b on yl-3-(4′-ter t-b u t yld i-

p h en ylsilyloxyb en zyl)-2-a zet id in on e (63). cis-4-Benzyl-

oxycarbonyl-3-(4′-tert-butyldiphenylsilyloxybenzyl)-2-azetidi-

none was prepared in 63% isolated yield (0.016 mmol scale)

from 2-azetidinone 62a following a procedure similar to that

described above for 48: (R_f0.25, DCM-ether; 10:1); mp 47-

48 °C; IR (NaCl, cm^-1) 3304, 3078, 2932, 1766, 1259, 1114;

MS (DCI, NH₃) m/z 567 [MNH₄⁺, 10], 550 [MH⁺, 4], 91 [100].

HRMS (CI, C₃₄H₃₉N₂O₄Si⁺): found, 567.2680; requires, 567.2679.

Anal. (C₃₄H₃₅NO₄Si) C, H, N.

The title compound 63 was prepared in 54% isolated yield

(0.346 mmol scale) from cis-4-benzyloxycarbonyl-3-(4′-tert-

butyldiphenylsilyloxybenzyl)-2-azetidinone and benzoyl chlo-

ride following a procedure similar to that described above for

6: (R_f0.75, DCM); IR (NaCl, cm^-1) 3071, 2932, 1799, 1747,

1258, 1114; MS (DCI, NH₃) m/z 671 [6%, MNH₄⁺], 654 [MH⁺,

18], 312 [29], 286 [30], 122 [41], 105 [72], 91 [100], 78 [33].

HRMS (CI, C₄₁H₄₀NO₅Si⁺): found, 654.2680; requires, 654.2676.

Gen er a l P r oced u r e for Dep r otection s Usin g Tr i-

flu or oa cetic Acid : cis-3-Ben zyl-4-[(4′-ca r boxy)ben zyloxy-

ca r bon yl]-1-{3′-[(4"-ca r boxy)ben zyloxyca r bon yl]p h en yl-

a ceta te}-2-a zetid in on e (4). To diester 52 (22 mg, 0.029

mmol) stirred at 0 °C was added trifluoroacetic acid (0.5 mL).

After 25 min, 52 had been consumed and the solution was

concentrated under reduced pressure. Sequential trituration

with ether and concentration under reduced pressure afforded

4 as a white crystalline solid (16 mg, 87%): (R_f0.3, LP-ether;

1:5); mp 206 °C; IR (KBr, cm^-1) 3560-2420, 2927, 1799, 1746,

1718, 1694; MS (ES) m/z 635 [M^-, 36], 634 [(M - H)^-, 100].

cis-3-Ben zyl-4-[(4′-ca r b oxy)b en zyloxyca r b on yl]-1-[3′-

(ben zyloxycar bon yl)ph en ylacetate]-2-azetidin on e (1). The

title compound was prepared in 88% isolated yield (0.067 mmol

scale) from diester 51 following a procedure similar to that

described above for 4: (R_f0.35, LP-ethyl acetate; 2:3) mp 136

cis-3-Ben zyl-4-[(4′-ter t-b u t yloxyca r b on yl)b en zyloxy-

ca r bon yl]-1-{3′-[ter t-bu tyloxyca r bon yl]p h en yla ceta te)-2-

a zet id in on e (50). The title compound [and unreacted 48

(14%)] was prepared in 42% isolated yield (0.30 mmol scale)

from 2-azetidinone 48 and acid chloride 46 following a

2-Azetidinone Inhibitors of Prostate Specific Antigen

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1505

°C (dec); IR (NaCl cm^-1) 3600-3000, 3065, 3032, 1797, 1747,

1714; MS (APCI) m/z 590[M - H⁺, 100], 410[6], 338[8], 187[22],

186[16]. Anal. (C₃₅H₂₉NO₈) C, H, N.

idinone following a procedure similar to that described above

for 48: (R_f0.35, LP-ethyl acetate; 1:1); mp 202-203 °C; IR

(KBr, cm^-1) 3193, 2981, 1737, 1722, 1705; MS (CI, NH₃) m/z

413 [MNH₄⁺, 54], 396 [MH⁺, 39], 357 [52], 340 [66], 264 [26],

174 [100], 130 [88]. HRMS (CI, C₂₃H₂₅NO₅⁺): found, 396.1811;

requires, 396.1811.

cis-1-Benzoyl-3-benzyl-4-[4′-(tert-butyloxycarbonyl)phenyl-

carbonyloxymethyl]-2-azetidinone was prepared in 66% iso-

lated yield (0.250 mmol scale) from cis-3-benzyl-4-[4′-(tert-

butyloxycarbonyl)phenylcarbonyloxymethyl]-2-azetidinone and

benzoyl chloride following a procedure similar to that described

above for 6: (R_f0.4, LP-ethyl acetate; 4:1); mp 115-116 °C;

IR (KBr, cm^-1) 2978, 2925, 1802, 1723, 1704; MS (CI, NH₃)

m/z 517 [MNH₄⁺, 13], 500 [MH⁺, 74], 122 [100], 105 [91], 91

[20], 58 [18]. Anal. (C₃₀H₂₉NO₆) C, H, N.

cis-1-Ben zoyl-3-b en zyl-4-[(4′-ca r b oxy)b en zyloxyca r -

bon yl]-2-a zetid in on e (2). The title compound was prepared

in 84% isolated yield (0.16 mmol scale) from diester 49

following a procedure similar to that described above for 4:

(R_f0.35, LP-ethyl acetate; 2:3); mp 99 °C (dec); IR (NaCl, cm^-1

)

3600-2700, 3064, 3300, 1798, 1748, 1686; MS (APCI) m/z

442[M - H⁺], 262[13], 187[28].

cis-3-Ben zyl-4-[(4′-ca r b oxy)b en zyloxyca r b on yl]-1-[3′-

ca r boxyp h en yla ceta te]-2-a zetid in on e (3). The title com-

pound was prepared in 63% isolated yield (0.043 mmol scale)

from diester 50 following a procedure similar to that described

above for 4: (R_f0.6, LP-ether; 1:5); mp 214 °C (dec); IR (KBr,

cm^-1) 3600-2480, 2920, 1782, 1739, 1718, 1694; MS (ES) m/z

501 [M^-, 32], 500 [(M - H)^-, 100].

cis-3-Ben zyl-1-[3′-(ben zyloxyca r bon yl)p h en yla ceta te]-

4-[4′-(a m m on iu m m et h yl)b en zyloxyca r b on yl]-2-a zet id i-

n on e, Tr iflu or oa ceta te (5). cis-3-Benzyl-4-{4′-[N-(tert-butyl-

oxycarbonyl)aminomethyl]benzyloxycarbonyl}-1-(4′-methoxy-

phenyl)-2-azetidinone was prepared in 89% isolated yield (0.73

mmol scale) from acid 29 and alcohol 37 following a procedure

similar to that described above for 47: (R_f0.4, LP-DCM; 1:20);

mp 93-94 °C; IR (KBr, cm^-1) 3382, 3061, 1741, 1696; MS

(FAB, Na) m/z 553 [MNa⁺, 6], 530 [M⁺, 38], 429 [13], 164 [100],

91 [29], 57 [99]. Anal. (C₃₁H₃₄N₂O₆) C, H, N.

cis-3-Benzyl-4-{4′-[N-(tert-butyloxycarbonyl)aminomethyl]-

benzyloxycarbonyl}-2-azetidinone was prepared in 71% iso-

lated yield (1.80 mmol scale) from cis-3-benzyl-4-{4′-[N-(tert-

butyloxycarbonyl)aminomethyl]benzyloxycarbonyl}-1-(4′-

methoxyphenyl)-2-azetidinone following a procedure similar

to that described above for 48: (R_f0.25, LP-ether; 1:5); mp

109-111 °C; IR (KBr, cm^-1) 3373, 3355, 2981, 1771, 1724,

1687; MS (DCI, NH₃) m/z 442 [MNH₄⁺, 3], 369 [59], 341 [56],

325 [100], 120 [55]. Anal. (C₂₄H₂₈N₂O₅) C, H, N.

cis-3-Benzyl-1-[3′-(benzyloxycarbonyl)phenylacetate]-4-{4′-

[N-(tert-butyloxycarbonyl)aminomethyl]benzyloxycarbonyl}-2-

azetidinone was prepared in 61% isolated yield (0.45 mmol

scale) from cis-3-benzyl-4-{4′-[N-(tert-butyloxycarbonyl)amino-

methyl]benzyloxycarbonyl}-2-azetidinone and acid chloride 42

following a procedure similar to that described above for 6:

The title compound 9 was prepared in 71% isolated yield

(0.086 mmol scale) from cis-1-benzoyl-3-benzyl-4-[4′-(tert-bu-

tyloxycarbonyl)phenylcarbonyloxymethyl]-2-azetidinone fol-

lowing a procedure similar to that described above for 4: (R_f

0.55, LP-ethyl acetate; 1:4); mp 178 °C; IR (KBr, cm^-1) 3630-

+

2720, 3066, 2925, 1813, 1717; MS (CI, NH₃) m/z 461 [MNH₄

,

5], 444 [MH⁺, 23], 400 [12], 280 [55], 190 [100], 176 [28], 122

[89], 105 [88], 91 [28]. HRMS (CI, C₂₆H₂₂NO₆⁺): found,

444.1450; requires, 444.1447. Anal. (C₂₆H₂₁NO₆) C, H, N.

cis-1-[3′-(Ben zyloxyca r bon yl)p h en yla ceta te]-4-(4′-ca r -

boxyben zyloxyca r bon yl)-3-(4′-h yd r oxyben zyl)-2-a zetid i-

n on e (15). The title compound was prepared in 98% isolated

yield (0.0235 mmol scale) from ester 64 following a procedure

similar to that described above for 4: (R_f0.2, DCM-ether; 10:

1); mp 78-80 °C; IR (KBr, cm^-1) 3362, 3440-2480, 3061, 2927,

1799, 1743, 1717; MS (ES) m/z 606 [(M - H)^-, 100], 561 [10].

cis-3-Ben zyl-4-p h en ylca r b on yloxym et h yl-1-(4′-m et h -

oxyp h en yl)-2-a zetid in on e (53). To a stirred solution of

alcohol 30 (4.55 g, 15.3 mmol) in dry DCM (150 mL), at room

temperature, were added DMAP (ca. 5 mg) and triethylamine

(1.55 g, 15.3 mmol). Benzoyl chloride (2.15 g, 15.3 mmol) was

added dropwise over 5 min and the solution stirred for 20 h.

Further amounts of triethylamine (310 mg, 3.06 mmol) and

benzoyl chloride (430 mg, 3.06 mmol) were added to the

reaction mixture, and stirring was continued for 24 h. The

reaction mixture was washed with water (3 × 60 mL), dried

(MgSO₄), filtered, and concentrated under reduced pressure

to yield a white solid. Flash chromatography (SiO₂, LP-ether,

2:1; 1:2, gradient elution) afforded 53 as a white crystalline

(R_f0.3, DCM-ethyl acetate; 20:1); mp 94-95 °C; IR (KBr, cm^-1

)

3346, 1781, 1733, 1725, 1713, 1686; MS (FAB) m/z 677 [MH⁺,

20], 577 [76], 164 [100], 91 [96]. Anal. (C₄₀H₄₀N₂O₈) C, H, N.

The title compound 5 was prepared in 99% isolated yield

(0.075 mmol scale) from cis-3-benzyl-1-[3′-(benzyloxycarbonyl)-

phenylacetate]-4-{4′-[N-(tert-butyloxycarbonyl)aminomethyl]-

benzyloxycarbonyl}-2-azetidinone following a procedure simi-

solid (5.18 g, 84%): (R_f0.5, LP-ether; 1:2); mp 113 °C; IR (KBr,

cm^-1) 3027, 2937, 1741, 1707; MS (CI, NH₃) m/z 419 [MNH₄

5], 402 [MH⁺, 100], 270 [11], 149[12], 105 [37]. Anal. (C₂₅H₂₃

NO₄) C, H, N.

,

+

-

Gen er a l P r oced u r e for N-Ar ylsu lfon yla tion of 2-Azet-

id in on es: cis-3-Ben zyl-4-p h en ylca r b on yloxym et h yl-1-

(4′′-tolu en esu lfon yl)-2-a zetid in on e (10) a n d cis-3-Ben zyl-

1-{2′-b en zyl-1′-oxo-4′-p h en ylca r b on yloxy-3′-[N-(4′′-t olu -

en esu lfon yl)a m in e]bu tyl}-4-p h en ylca r bon yloxym eth yl-

2-a zetid in on e (55), (Ar ) p-C₆H₄-Me). To a stirred solution

of 2-azetidinone 54 (93.6 mg, 0.32 mmol) in THF (20 mL), at

-72 °C, was added dropwise sodium bis(trimethylsilyl)amide

(0.32 mL of a 1.0 M solution in THF, 0.32 mmol). After 25

min, p-toluenesulfonyl chloride (604 mg, 3.2 mmol), dissolved

in THF (1 mL), was added all in one portion and stirring

continued for 21 h at -72 °C. Water (2 mL) was added to the

reaction and the mixture allowed to warm to room temperature

with stirring. The mixture was diluted with water (15 mL)

and extracted with ethyl acetate (2 × 20 mL), and the organic

extracts were combined, washed with water (3 × 15 mL), dried

(MgSO₄), filtered, and concentrated under reduced pressure

to yield a green oil. Flash chromatography (SiO₂, DCM; DCM-

ether, neat; 10:1, gradient elution) afforded the title com-

pounds: 10 as a white foam (73.2 mg, 52%) and 55 (Ar )

p-C₆H₄-Me) as a white foam (23.5 mg, 20%). 10: (R_f0.4, LP-

ethyl acetate; 2:1); mp 99-100 °C; IR (KBr, cm^-1) 3063, 2921,

1793, 1728, 1360, 1171; MS (CI, NH₃) m/z 467 [MNH₄⁺, 20],

450 [MH⁺, 2], 345 [18], 328 [36], 189 [100], 174 [96], 108 [32],

105 [82], 91 [37], 78 [23]. HRMS (CI, C₂₅H₂₇N₂O₅S⁺): found,

467.1641; requires, 467.1641. Anal. (C₂₅H₂₃NO₅S) C, H, N. 55:

lar to that described above for 4: mp 58-59 °C; IR (KBr, cm^-1

)

3620-3245, 2860, 1798, 1745, 1713; MS (ES) m/z 577 [MH⁺,

100].

cis-1-Ben zoyl-3-b en zyl-4-(4′-ca r b oxyp h en ylca r b on yl-

oxym eth yl)-2-a zetid in on e (9). To a stirred solution of acid

34 (111 mg, 0.50 mmol) and alcohol 30 (164 mg, 0.55 mmol)

in dry DCM (10 mL), in an ice bath at 0 °C, were added DMAP

(ca. 2 mg) and EDCI (105 mg, 0.55 mmol). After being stirred

for 22 h, the solution was allowed to warm to room tempera-

ture. Further EDCI (20 mg, 0.10 mmol) was added, and the

reaction mixture was stirred for a further 2 h. The reaction

mixture was washed with water (2 × 10 mL), dried (MgSO₄),

filtered, and concentrated under reduced pressure to yield a

white foam. Flash chromatography (SiO₂, LP-ether, 2:1; 1:2,

gradient elution) afforded cis-3-benzyl-4-[4′-(tert-butyloxy-

carbonyl)phenylcarbonyloxymethyl]-1-(4′-methoxyphenyl)-2-

azetidinone as a white crystalline solid (143 mg, 57%): (R_f0.3,

LP-ether; 1:1); mp 122-123 °C; IR (KBr, cm^-1) 3063, 2981,

2934, 1740, 1729, 1714; MS (CI, NH₃) m/z 519 [MNH₄⁺, 9],

502 [MH⁺, 92], 446 [37], 298 [41], 280 [75], 150 [98], 149 [100],

124 [86]. Anal. (C₃₀H₃₁NO₆) C, H, N.

cis-3-Benzyl-4-[4′-(tert-butyloxycarbonyl)phenylcarbonyl-

oxymethyl]-2-azetidinone was prepared in 67% isolated yield

(1.80 mmol scale) from cis-3-benzyl-4-[4′-(tert-butyloxycar-

bonyl)phenylcarbonyloxymethyl]-1-(4′-methoxyphenyl)-2-azet-

1506 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10

Adlington et al.

(Ar ) p-C₆H₄-Me) (R_f0.25, LP-ethyl acetate; 2:1); mp 84-86

°C; IR (KBr, cm^-1) 3282, 3033, 2924, 1793, 1719, 1704, 1339,

1161; MS (FAB, NH₃) m/z 745 [MH⁺, 94], 623 [12], 105 [100].

HRMS (FAB, C₄₃H₄₁N₂O₈S⁺): found, 745.253; requires, 745.258.

DCM-ether; 10:1); mp 158-159 °C; IR (KBr, cm^-1) 3293, 2977,

1739, 1713; MS (DCI, NH₃) m/z 462 [MH⁺- C₄H₈, 6], 342 [8],

328 [8], 284 [20], 124 [83], 107 [100], 91 [72]. Anal. (C₃₀H₃₁

NO₇) C, H, N.

-

Gen er a l P r oced u r e for th e P r ep a r a tion of Silyl Eth er s

62a a n d 62b: cis-4-Ben zyloxyca r bon yl-3-(4′-ter t-bu tyl-

d ip h en ylsilyloxyben zyl)-1-(4′-m eth oxyp h en yl)-2-a zetid i-

n on e (62a ). To a stirred slurry of phenol 61a (1.74 g, 4.17

mmol) in DMF (4 mL), heated at 40 °C, were added imidazole

(708 mg, 10.4 mmol) and tert-butyldiphenylsilyl chloride (1.37

g, 4.98 mmol). After being stirred at 40 °C for 17 h, the reaction

mixture was diluted with water (40 mL) and extracted with

ethyl acetate (3 × 50 mL). The organic layers were combined,

washed with water (40 mL), dried (MgSO₄), filtered, and

concentrated under reduced pressure to yield a colorless oil.

Flash chromatography (SiO₂, DCM) afforded 62a as a white

gummy foam (2.57 g, 94%): (R_f0.8, ether-LP; 1:1); mp 33-35

°C; IR (NaCl, cm^-1) 2932, 1757, 1753, 1251, 1115; MS (DCI,

NH₃) m/z 656 [MH⁺, 10], 91 [100], 78 [12]. Anal. (C₄₁H₄₁NO₅-

Si ) C, H, N.

cis-3-(4′-ter t-Bu t yld ip h en ylsilyloxyb en zyl)-4-[4′-(ter t-

b u t yloxyca r b on yl)b en zyloxyca r b on yl]-1-(4′-m et h oxy-

p h en yl)-2-a zetid in on e (62b). The title compound was pre-

pared in 78% isolated yield from 2-azetidinone 61b (0.435

mmol scale) following a procedure similar to that described

above for 62a : (R_f0.7, DCM-ether; 10:1); mp 63-65 °C; IR

(KBr, cm^-1) 2932, 2858, 1761, 1713, 1251, 1114; MS (FAB, Na)

m/z 778 [MNa⁺, 84], 755 [M⁺, 15], 722 [14], 564 [28], 537 [25],

521 [35], 509 [47], 497 [67], 481 [64], 469 [83], 455 [68], 441

[71], 413 [100]. Anal. (C₄₆H₄₉NO₇Si) C, H, N.

cis-1-Ben zen esu lfon yl-3-ben zyl-4-p h en ylca r bon yloxy-

m eth yl-2-a zetid in on e (11) a n d cis-3-Ben zyl-1-{2′-ben zyl-

1′-oxo-3′-[N-(ben zen esu lfon yl)am in e]-4′-[ph en ylcar bon yl-

oxy]b u t yl}-4-p h en ylca r b on yloxym et h yl-2-a zet id in on e

(55), (Ar ) P h ). The title compounds, 11 and 55 (Ar ) Ph),

were prepared in 61% and 8% isolated yields, respectively (0.30

mmol scale), from 2-azetidinone 54 following a procedure

similar to that described above for 10 and 55 (Ar ) p-C₆H₄-

Me). 11: (R_f0.35, LP-ethyl acetate; 2:1); mp 44-46 °C; IR

(NaCl, cm^-1) 3034, 1793, 1722, 1315, 1171; MS (CI, NH₃) m/z

453 [MNH₄⁺, 77], 436 [MH⁺, 5], 331 [15], 314 [18], 130 [29],

105 [100]. HRMS (CI, C₂₄H₂₅N₂O₅S⁺): found, 453.1484; re-

quires, 453.1484. Anal. (C₂₄H₂₁NO₅S) C, H, N. 55: (Ar ) Ph)

(R_f0.25, LP-ethyl acetate; 2:1); IR (NaCl, cm^-1) 3282, 3031,

2929, 1790, 1722, 1704, 1339, 1163; MS (CI, NH₃) m/z 748

[MNH₄⁺, 0.3], 731 [MH⁺, 0.6], 626 [3], 609 [15], 452 [37], 428

[32], 314 [47], 296 [38], 175 [41], 105 [100]. HRMS (CI,

C

₄₂H₃₉N₂O₈S⁺): found, 731.2430; requires, 731.2427.

cis-3-Ben zyl-1-(4′-n itr oben zen esu lfon yl)-4-p h en ylca r -

bon yloxym eth yl-2-a zetid in on e (12). The title compound

was prepared in 67% isolated yield from 2-azetidinone 54 (0.25

mmol scale) following a procedure similar to that described

above for 10: (R_f0.55, LP-ethyl acetate; 2:1); mp 60-62 °C;

IR (KBr, cm^-1) 3105, 2955, 1796, 1724, 1350, 1175; MS (DCI,

NH₃) m/z 498 [MNH₄⁺, 25], 359 [22], 105 [100]. Anal.

(C₂₄H₂₀N₂O₇S) C, H, N.

Gen er a l P r oced u r e for th e P r ep a r a tion of P h en ols

16-18 a n d 64: cis-1-Ben zoyl-4-ben zyloxyca r bon yl-3-(4′-

h yd r oxyben zyl)-2-a zetid in on e (17). To a stirred solution

of silyl ether 63 (100 mg, 0.153 mmol) in DMF (4 mL) and

N-methylpyrolidin-2-one (1.5 mL) was added ammonium

hydrogen difluoride (87 mg, 1.53 mmol). The resulting suspen-

sion was stirred at room temperature for 16 h. The reaction

mixture was diluted with water (10 mL) and extracted with

ethyl acetate (3 × 15 mL). The organic layers were combined,

washed with water (2 × 20 mL) and brine (2 × 20 mL), dried

(MgSO₄), filtered, and concentrated under reduced pressure

to yield a colorless oil. Flash chromatography (SiO₂, DCM-

ether, 25:1) afforded 17 as a white crystalline solid (31.7 mg,

50%): (R_f0.7, DCM-ether, 10:1); mp 162-163 °C; IR (KBr,

cm^-1) 3415, 1802, 1737; MS (DCI, NH₃) m/z 433 [MNH₄⁺, 2],

416 [MH⁺, 12], 147 [40], 122 [41], 105 [100], 91 [77], 78 [22].

HRMS (CI, C₂₅H₂₂NO₅⁺): found, 416.1498; requires, 416.1498.

Anal. (C₂₅H₂₁NO₅) C, H, N.

cis-1-[3′-(Ben zyloxyca r bon yl)p h en yla ceta te]-4-ben zyl-

oxyca r bon yl-3-(4′-h yd r oxyben zyl)-2-a zetid in on e (16). cis-

4-Benzyloxycarbonyl-1-[3′-(benzyloxycarbonyl)phenylacetate]-

3-(4′-tert-butyldiphenylsilyloxybenzyl)-2-azetidinone was pre-

pared in 56% isolated yield (0.80 mmol scale) from cis-4-

benzyloxycarbonyl-3-(4′-tert-butyldiphenylsilyloxybenzyl)-2-

azetidinone (36% recovery) and acid chloride 42 following a

procedure similar to that described above for 6: (R_f0.3, DCM);

mp 47-48 °C; IR (KBr, cm^-1) 3033, 2931, 2857, 1796, 1747,

1714, 1269, 1112; MS (DCI, NH₃) m/z 819 [MNH₄⁺, 8], 802

[MH⁺, 2], 91 [100]. Anal. (C₅₀H₄₇NO₇Si) C, H, N.

cis-3-Ben zyl-1-et h a n esu lfon yl-4-p h en ylca r b on yloxy-

m eth yl-2-a zetid in on e (13). The title compound was prepared

in 29% isolated yield from 2-azetidinone 54 (0.42 mmol scale)

following a procedure similar to that described above for 10:

(R_f0.25, DCM; DCM-ether, neat; 10:1); IR (NaCl, cm^-1) 2936,

1794, 1723, 1355, 1156; MS (CI, NH₃) m/z 405 [MNH₄⁺, 55],

388 [MH⁺, 11], 283 [37], 266 [53], 105 [100]. HRMS (CI,

C

₂₀H₂₅N₂O₅S⁺): found, 405.1484; requires, 405.1484.

cis-3-Ben zyl-4-ben zyloxycar bon yl-1-(4′-tolu en esu lfon yl)-

2-a zetid in on e (14). The title compound was prepared in 61%

isolated yield (0.30 mmol scale) from cis-3-benzyl-4-benzyloxy-

carbonyl-2-azetidinone following a procedure similar to that

described above for 10: (R_f0.5, DCM); mp 129-130 °C; IR

(KBr, cm^-1) 2927, 1808, 1744, 1351, 1163; MS (CI, NH₃) m/z

467 [MNH₄⁺, 100], 131 [35], 108 [44], 91 [82]. Anal. (C₂₅H₂₃

NO₅S) C, H, N.

-

Gen er a l P r oced u r e for th e P r ep a r a tion of P h en ols 61a

a n d 61b: cis-4-Ben zyloxyca r bon yl-3-(4′-h yd r oxyben zyl)-

1-(4′-m eth oxyp h en yl)-2-a zetid in on e (61a ). To a stirred

solution of acetate 58a (525 mg, 1.14 mmol) in a methanol (35

mL)-THF (20 mL) solution, at 35 °C, was added activated zinc.

[Activated zinc was prepared by stirring zinc dust in 10%

aqueous hydrochloric acid (50 mL) for 4 min. The zinc was

filtered, washed sequentially with acetone (2 × 40 mL), ether

(3 × 40 mL), and then methanol (2 × 40 mL), and added as a

moist slurry in methanol.⁴⁴] The reaction mixture was stirred

for 4 days at 35 °C and then filtered through Celite to remove

excess zinc. The solvent was removed under reduced pressure

and the resulting gray oil dissolved in an ethyl acetate (20

mL)-water (20 mL) mixture. The solution was acidified to pH

3, extracted with ethyl acetate (3 × 40 mL), dried (MgSO₄),

filtered, and concentrated under reduced pressure to yield a

gray solid. Flash chromatography (SiO₂, DCM-ether, 25:1)

afforded 61a as a white crystalline solid (419 mg, 88%): (R_f

The title compound 16 was prepared in 68% isolated yield

from cis-4-benzyloxycarbonyl-1-[3′-(benzyloxycarbonyl)phenyl-

acetate]-3-(4′-tert-butyldiphenylsilyloxybenzyl)-2-azetidinone

(0.14 mmol scale) following a procedure similar to that

described above for 17: (R_f0.2, DCM-ether, 25:1); mp 43-45

°C; IR (KBr, cm^-1) 3434, 2923, 1795, 1746, 1717, 1703; MS

(FAB) m/z 564 [MH⁺, 46], 546 [3], 287[19], 147 [11], 107 [9],

91 [100]. Anal. (C₃₄H₂₉NO₇) C, H, N.

0.45, DCM-ether; 10:1); mp 147.5-148.5 °C; IR (KBr, cm^-1

)

3343, 2962, 1735; MS (CI, NH₃) m/z 435 [MNH₄⁺, 3], 418 [MH⁺,

100], 342 [20], 284 [19], 124 [50], 108 [41], 91 [59], 78 [11].

Anal. (C₂₅H₂₃NO₅) C, H, N.

(3S,4S)-4-Ben zyloxyca r bon yl-1-[3′-(ben zyloxyca r bon -

yl)ph en ylacetate]-3-[4′-h ydr oxyben zyl]-2-azetidin on e (18).

(3S,4S)-4-Benzyloxycarbonyl-1-[3′-(benzyloxycarbonyl)phenyl-

acetate]-3-[4′-(tert-butyldiphenylsilyloxy)benzyl]-2-azetidinone

was prepared in 45% isolated yield (0.11 mmol scale) from

2-azetidinone 71 and acid chloride 42 following a procedure

cis-4-[4′-(ter t-Bu t yloxyca r b on yl)b en zyloxyca r b on yl]-

3-(4′-h yd r oxyb e n zyl)-1-(4′-m e t h oxyp h e n yl)-2-a ze t id i-

n on e (61b). The title compound [and also 4-(tert-butyloxycar-

bonyl)benzyl alcohol 33 (17%)] was prepared in 71% isolated

yield (0.643 mmol scale) from 2-azetidinone 58b following a

procedure similar to that described above for 61a : (R_f0.45,

similar to that described above for 6: (R_f0.3, DCM); [R]^22.5

D

2-Azetidinone Inhibitors of Prostate Specific Antigen

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1507

-2.5 (c 0.88, CHCl₃); IR (NaCl, cm^-1) 3071, 2932, 2858, 1797,

1747, 1715, 1269, 1113; MS (APCI, NH₃) m/z 824 [MNa⁺, 8],

819 [MNH₄⁺, 22], 802 [MH⁺, 45], 664 [12], 345[14], 122 [100].

The title compound 18 was prepared in 67% isolated yield (0.35

mmol scale) from (3S,4S)-4-benzyloxycarbonyl-1-[3′-(benzyl-

oxycarbonyl)phenylacetate]-3-[4′-(tert-butyldiphenylsilyloxy)-

benzyl]-2-azetidinone following a procedure similar to that

described above for 17: (R_f0.2, DCM-ether, 25:1); [R]_D^22.5

-14.1 (c 0.18, CHCl₃); Spectral data as for 16.

cis-1-[3′-(Ben zyloxyca r bon yl)p h en yla ceta te]-4-[4′-(ter t-

b u t yloxyca r b on yl)b e n zyloxyca r b on yl]-3-(4′-h yd r oxy-

ben zyl)-2-a zetid in on e (64). cis-3-(4′-tert-Butyldiphenylsilyl-

oxybenzyl)-4-[4′-(tert-butyloxycarbonyl)benzyloxycarbonyl]-2-

azetidinone was prepared in 68% isolated yield (0.34 mmol

scale) from 2-azetidinone 62b following a procedure similar

to that described above for 48: (R_f0.25, DCM-ether; 10:1); mp

62-64 °C; IR (KBr, cm^-1) 3397, 3072, 2932, 2858, 1773, 1714,

1257, 1114; MS (DCI, NH₃) m/z 667 [MNH₄⁺, 100], 611 [28],

594 [6], 477 [52], 460 [87], 345 [51], 193 [52], 119 [66], 78 [48].

Anal. (C₃₉H₄₃NO₆Si) C, H, N.

cis-1-[3′-(Benzyloxycarbonyl)phenylacetate]-3-(4′-tert-butyl-

diphenylsilyloxybenzyl)-4-(4′-tert-butyloxycarbonylbenzyloxy-

carbonyl)-2-azetidinone was prepared in 52% isolated yield

(0.21 mmol scale) from cis-3-(4′-tert-butyldiphenylsilyloxyben-

zyl)-4-[4′-(tert-butyloxycarbonyl)benzyloxycarbonyl]-2-azetidi-

none and acid chloride 42 following a procedure similar to that

described above for 6: (R_f0.3, DCM; DCM-ether, neat; 50:1);

mp 74-75 °C; IR (KBr, cm^-1) 2930, 1797, 1751, 1714, 1269,

1113; MS (APCI, NH₃) m/z 919 [MNH₄⁺, 23], 846 [100], 828

[78], 690 [56], 662 [83], 624 [63], 570 [65]. Anal. (C₅₅H₅₅NO₉-

Si) C, H, N.

The title compound 64 was prepared in 82% isolated yield

(0.09 mmol scale) from cis-1-[3′-(benzyloxycarbonyl)phenyl-

acetate]-3-(4′-tert-butyldiphenylsilyloxybenzyl)-4-(4′-tert-butyl-

oxycarbonylbenzyloxycarbonyl)-2-azetidinone following a pro-

cedure similar to that described above for 17: (R_f0.3, DCM-

ether, 10:1); mp 59-61 °C; IR (KBr, cm^-1) 3436, 3034, 2977,

2931, 1800, 1750, 1714; MS (FAB) m/z 664 [MH⁺, 34], 608 [50],

590 [43], 500 [8], 197 [18], 135, [13], 107 [19], 91 [100]. Anal.

(C₃₉H₃₇NO₉) C, H, N.

(4S)-4-Ben zyloxyca r bon yl-1-(ter t-bu tyld im eth ylsilyl)-

3-[Z-4′-(ter t-bu tyld ip h en ylsilyloxy)p h en ylm eth ylen e]-2-

a zetid in on e (69a ) a n d (4S)-4-Ben zyloxyca r bon yl-1-(ter t-

bu tyld im eth ylsilyl)-3-[E-4′-(ter t-bu tyld ip h en ylsilyloxy)-

p h en ylm eth ylen e]-2-a zetid in on e (69b). To a stirred solu-

tion of di-isopropylamine (1.67 g, 2.31 mL, 16.5 mmol) in dry

THF (16.3 mL) cooled to 0 °C, was added n-butyllithium (11.4

mL of a 1.45 M solution in hexanes, 16.5 mmol). After being

stirred for 5 min at 0 °C, the reaction mixture containing lith-

ium di-iso-propylamide was cooled to -78 °C. A solution of

(4S)-1-(tert-butyldimethylsilyl)-4-carboxy-2-azetidinone 68⁴⁸

(1.15 g, 5.01 mmol) dissolved in dry THF (50 mL) was stirred

at -9 °C, freshly prepared lithium di-isopropylamide solution

(20 mL, 11.0 mmol) was added, and stirring of the resulting

brown solution continued at -9 °C for 15 min. Trimethylsilyl

chloride (0.599 g, 5.52 mmol) was added, resulting in a yellow

solution, which was stirred for 15 min at -9 °C. A further

amount of freshly prepared lithium di-isopropylamide solution

(10 mL, 5.5 mmol) was added, and stirring of the resulting

brown solution continued for 5 min, allowing the solution to

warm to 0 °C. A solution of 4′-tert-butyldiphenylsilyloxy

benzaldehyde (1.99 g, 5.52 mmol) in THF (10 mL) was added

dropwise over 5 min which resulted in a temporary deep red

coloration, and stirring was continued at 0 °C for a further 1

h. The reaction mixture was removed from the cooling bath,

diluted with water (50 mL), then acidified with 5% aqueous

hydrochloric acid to pH 3, and extracted with ethyl acetate (2

× 50 mL). The organic extracts were combined and washed

with brine (100 mL), dried (MgSO₄), filtered, and concentrated

under reduced pressure to yield a brown foam. This foam was

dissolved in dry DCM (50 mL) and stirred at room tempera-

ture. Benzyl alcohol (0.596 g, 5.51 mmol) and DMAP (ca.2 mg)

were added, and then EDCI (1.06 g, 5.53 mmol) was added

portionwise over 10 min. After being stirred for a further 3 h,

the reaction mixture was washed with brine (3 × 30 mL), dried

(MgSO₄), filtered, and concentrated under reduced pressure

to yield a green gum. Flash chromatography (SiO₂, DCM)

afforded 69a as a colorless gum (246 mg, 7%), and 69b as a

white foam (436 mg, 13%). 69a : (R_f0.75, DCM); [R]²¹-33.3

D

(c 0.91, CHCl₃); UV λ_max(CHCl₃) 316 nm (ꢀ 13500); IR (NaCl,

cm^-1) 3071, 2956, 2931, 2858, 1740, 1675, 1252; MS (APCI,

NH₃) m/z 694 [MH⁺+ MeOH, 5], 662 [MH⁺, 100], 634 [12],

345 [15]. HRMS (CI, C₄₀H₄₈NO₄Si₂⁺): found, 662.3120; re-

quires, 662.3122. Anal. (C₄₀H₄₇NO₄Si₂) C, H. 69b: (R_f0.2,

DCM); mp 43-44 °C; [R]²_D¹+81.1 (c 0.92, CHCl₃); UV λ_max

(CHCl₃) 310 nm (ꢀ 18130); IR (NaCl, cm^-1) 3071, 2956, 2931,

2858, 1749, 1683, 1257; MS (APCI, NH₃) m/z 694 [MH⁺+MeOH,

6], 662 [MH⁺, 100], 634 [38], 401 [8], 345 [9]. HRMS (CI,

C₄₀H₄₈NO₄Si₂⁺): found, 662.3120; requires, 662.3122. Anal.

(C₄₀H₄₇NO₄Si₂) C, H.

(3S,4S)-4-Ben zyloxyca r b on yl-1-(ter t-b u t yld im et h yl-

silyl)-3-[4′-(tert-butyldiphenylsilyloxy)benzyl]-2-azetidinone

(70). The E-alkene 69b (0.298 g, 0.45 mmol) was dissolved in

THF (5 mL). To this stirred solution was added palladium on

activated carbon (67 mg, 10% w/w), and the reaction vessel

was evacuated and flushed with hydrogen. After being stirred

for 30 h under an atmosphere of hydrogen, the reaction

solution was filtered through Celite and concentrated under

reduced pressure to afford a brown foam. This foam was

dissolved in dry DCM (10 mL) and stirred at room tempera-

ture. Benzyl alcohol (54.1 mg, 0.50 mmol) and DMAP (ca. 2

mg) were added, then EDCI (95.9 mg, 0.50 mmol) was added

portionwise over 10 min. After being stirred for a further 14

h, the reaction mixture was washed with brine (20 mL), dried

(MgSO₄), filtered, and concentrated under reduced pressure

to yield a yellow oil. Flash chromatography (SiO₂, DCM)

afforded 70 as a colorless gum (98 mg, 33%): (R_f0.55, DCM);

[R]²_D^0.5+4.5 (c 0.96, CHCl₃); IR (NaCl, cm^-1) 3034, 2956, 2931,

2858, 1753, 1256; MS (APCI, NH₃) m/z 686 [MNa⁺, 18], 664

[MH⁺, 99], 636 [100], 550 [12], 435 [74], 345 [46], 122 [46].

HRMS (CI, C₄₀H₅₀NO₄Si₂⁺): found, 664.3280; requires, 664.3278.

A similar procedure was conducted using the Z-alkene 69a ,

affording 70 in 30% yield with spectroscopic data as above.

(3S,4S)-4-Ben zyloxyca r bon yl-3-[4′-(ter t-bu tyld ip h en yl-

silyloxy)ben zyl]-2-a zetid in on e (71). To a stirred solution

of 2-azetidinone 70 (84.2 mg, 0.127 mmol) in acetonitrile (3.5

mL), at room temperature, was added 48% aqueous hydrogen

fluoride (3 drops). After being stirred for 20 min, the reaction

mixture was diluted with water (3.5 mL), neutralized with 5%

aqueous sodium hydrogen carbonate, and extracted with ethyl

acetate (2 × 20 mL). The organic extracts were combined,

washed with brine (20 mL), dried (MgSO₄), filtered, and

concentrated under reduced pressure to yield 71 as a colorless

gum (64.8 mg, 93%): (R_f0.25, DCM-ether; 10:1); [R]^20.5+9.8

D

(c 0.62, CHCl₃); IR (NaCl, cm^-1) 3402, 3072, 2931, 1747, 1259,

1114; MS (APCI, NH₃) m/z 567 [MNH₄⁺, 100], 550 [MH⁺, 5],

345 [13], 122 [30]. HRMS (CI, C₃₄H₃₉N₂O₄Si⁺): found, 567.2679;

requires, 567.2679.

Ack n ow led gm en t. We thank the EPSRC (formerly

SERC) for a studentship to W.M., Eli Lilly and Company

for CASE support, the EPSRC mass spectrometry

service (Swansea) for high resolution mass spectra, and

J . G. Vaughan for the preparation of 68.

Su p p or tin g In for m a tion Ava ila ble: NMR data. This

material is available free of charge via the Internet at

http://pubs.acs.org.

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J M000145G

Article Doi

Design, synthesis, and proposed active site binding analysis of monocyclic 2-azetidinone inhibitors of prostate specific antigen

DOI: 10.1021/jm000145g

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Article abstract of DOI:10.1021/jm000145g

Full text of DOI:10.1021/jm000145g

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