Functionalised 2,3-dimethyl-3-aminotetrahydrofuran-4-one and N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)acylamide based scaffolds: Synthesis and cysteinyl proteinase inhibition

Bioorganic & Medicinal Chemistry 12 (2004) 2903–2925

Functionalised 2,3-dimethyl-3-aminotetrahydrofuran-4-one

and N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)acylamide

based scaﬀolds: synthesis and cysteinyl proteinase inhibition

John Watts, Alex Benn, Nick Flinn, Tracy Monk, Manoj Ramjee, Peter Ray,^ꢀ

Yikang Wang and Martin Quibell^*

Amura Therapeutics Limited, Incenta House, Horizon Park, Barton Road, Comberton, Cambridge CB3 7AJ, UK

Received 21 January 2004; accepted 16 March 2004

Available online 30 April 2004

Abstract—A stereoselective synthesis of functionalised (2R,3R)-2,3-dimethyl-3-amidotetrahydrofuran-4-one, its (2S,3R)-epimer and

(3aR,6aR)-N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)acylamide cysteinyl proteinase inhibitors has been developed using Fmoc-

protected scaﬀolds 6–8 in a solid-phase combinatorial strategy. Within these scaﬀolds, the introduction of an alkyl substituent a to

the ketone aﬀords chiral stability to an otherwise conﬁgurationally labile molecule. Preparation of scaﬀolds 6–8 required stereo-

selective syntheses of suitably protected a-diazomethylketone intermediates 9–11, derived from appropriately protected a-meth-

ylthreonines (2R,3R)-12, (2R,3S)-13 and a protected analogue of (1R,2R)-1-amino-2-hydroxycyclopentanecarboxylic acid 14.

Application of standard methods for the preparation of amino acid a-diazomethylketones, through treatment of the mixed

anhydride or pre-formed acyl ﬂuorides of intermediates 12–14 with diazomethane, proved troublesome giving complex mixtures.

However, the desired a-diazomethylketones were isolated and following a lithium chloride/acetic acid promoted insertion reaction

provided scaﬀolds 6–8. Elaboration of 6–8 on the solid phase gave a,b-dimethyl monocyclic ketone based inhibitors 38a–f,

39a,b,d,e,f and bicyclic inhibitors 40a–e that exhibited low micromolar activity against a variety of cysteinyl proteinases.

1. Introduction

the CPB proteinases associated with Leishmaniasis.²

Selective inhibition of any of these CAC1 proteinases

oﬀers enormous therapeutic potential and consequently

there has been a concerted drive within the pharma-

ceutical industry towards the development of com-

pounds suitable for human administration.⁴These

eﬀorts have largely focused on low molecular weight

substrate based peptidomimetics and to date the most

advanced inhibitors are in early clinical assessment.⁵

The GSK candidate SB-462795 1, a potent inhibitor of

cathepsin K indicated for the treatment of osteoporosis,

emerged from the progression of early linear pepti-

domimetic ketones through to cyclic constrained ketone

inhibitors.⁶The initial cyclic inhibitors of GSK were

based upon potent, selective and reversible 3-amido-

tetrahydrofuran-4-ones 2a, 3-amidopyrrolidin-4-ones

2b, 4-amidotetrahydropyran-3-ones 2c and 4-amido

piperidin-3-ones 2d (Fig. 1).

Over the last two decades, cysteinyl proteinases have

been shown to exhibit a wide range of disease-related

biological functions and as such the inhibition of cys-

teinyl proteinase activity has evolved into an area of

intense current interest.¹In particular, proteinases of the

clan CA/family C1 (CAC1) have been implicated in a

multitude of disease processes.^2;3Examples include hu-

man proteinases such as cathepsin K (osteoporosis),

cathepsins S and F (autoimmune disorders), cathepsin B

(tumour invasion/metastases) and cathepsin L (metas-

tases/autoimmune disorders), as well as parasitic pro-

teinases such as falcipain (malaria parasite Plasmodium

falciparum), cruzipain (Trypanosoma cruzi infection) and

Keywords: Cysteinyl proteinase inhibitors; Cyclic ketones.

* Corresponding author. Tel.: +44-01223-264211; fax: +44-01223-26-

5662; e-mail: martin.quibell@incenta.co.uk

Upon further examination, it became apparent that

cyclic ketones 2, in particular the ﬁve-membered ring

analogues 2a and 2b, suﬀered from conﬁgurational

ꢀ

Present address: Organon Research Scotland, Newhouse, Lanark-

shire, Scotland ML1 5SH, UK.

doi:10.1016/j.bmc.2004.03.042

2904

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

O

N

O

S

X

O

n

O

H

N

Ph

R

N

H

Cbz

N

H

O

2a. X = O, n = 1

2b. X = NR', n = 1

2c. X = O,

n = 2

1. SB-462795

2d. X = NR', n = 2

2e. X = NR', n = 3

Figure 1. GSK cyclic ketone inhibitors of cathepsin K.

Figure 3. Predicted binding conformations for si (a) and re (b) ste-

reofacial thiolate addition to a model a-methyl monocyclic 3a, piper-

azine-1-carboxylic acid [3-methyl-1S-(3S-methyl-4-oxo-tetrahydro-

R²

O

R¹

6a

3a

R¹

O

2

3

O

2

3

furan-3-yl-carbamoyl)butyl]amide.^9;11The residues Gly²³, Trp²⁶, Cys⁶³

,

R

NH

R

N

H

R

N

H

O

Gly⁶⁴and Gly⁶⁵that make the P1 wall of cathepsin K structure 1mem

are shown as an electrostatic potential coloured surface. The hydrogen

atoms of the intermediate hydroxyl, a-methyl and a-NH are added and

residue Gly²³labelled.

O

4a. R¹, R²= H

(3R)

3a. R¹, R²= H

(3S)

5 (3aR, 6aR)

4b. R¹= Me, R²= H (2R, 3R)

4c. R¹= H, R²= Me (2S, 3R)

3b. R¹= Me, R²= H (2R, 3S)

3c. R¹= H, R²= Me (2S, 3S)

Figure 2. Stereochemical assignment of a,b-dimethyl monocyclic and

N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)acylamide bicyclic ket-

one inhibitors of CAC1 cysteinyl proteinases.

number of conclusions were apparent.¹¹Stereoisomer 3a

modelled through either the si (addition of thiolate to

face opposite the a-Me) or re (addition of thiolate to

same face as the a-Me) tetrahedral intermediate pro-

duced an unfavourable spatial clash between the S1 wall

of the proteinase comprised of Gly²³, Trp²⁶, Cys⁶³, Gly⁶⁴

and Gly⁶⁵and the a-methyl group. In particular, there

was a clear spatial overlap with the carbonyl of Gly²³

(Fig. 3).

instability due to facile epimerisation at the centre sit-

uated a to the ketone.^6b;7;8This physiochemical charac-

teristic generally precluded the pre-clinical optimisation

of inhibitors of formulae 2a–d and led to the development

of the conﬁgurationally stable azepanone series 2e.^6b

As an alternative to the ring expansion approach,

alkylation of the a-carbon would remove the ability of

cyclic ketones 2 to undergo a-enolisation and hence lead

to conﬁgurational stability. A preliminary examination

of a-methylation in the 3-amidopyrrolidin-4-one 2b

system has been reported, resulting in a substantial loss

in potency versus cathepsin K from K_i;appꢀ 0:18 to

50 nM.^6bHowever, it was unclear from this single

example whether the a-stereochemistry examined was of

the S conﬁguration as depicted in 3 or the R conﬁgu-

ration as depicted in 4 (Fig. 2). Our in-house molecular

modelling of 3-methyl-3-amidotetrahydrofuran-4-ones

3a and 4a has highlighted the importance of the a-ste-

reochemistry within these cyclic systems.⁹Herein we

report our design strategy, building block preparation,

solid-phase synthesis and the inhibition kinetics for a

series of conﬁgurationally stable a,b-dimethyl monocy-

clic 4 and N-(3-oxo-hexahydrocyclopenta[b]furan-3a-

yl)acylamide bicyclic ketones 5 as inhibitors of CAC1

proteinases.¹⁰

Molecular modelling of si thiolate addition to stereo-

isomer 3a (Fig. 3a) predicted that the oxygen of the

hemithioketal would be stabilised by a hydrogen bond

to the NH₂of the primary carboxamide side chain of

glutamine¹⁹and a second hydrogen bond to the back-

bone NH of cysteine²⁵. Additional hydrogen bonds were

predicted between the P1 NH of 3a and the backbone

C@O of asparagine¹⁵⁸; the P2 carbonyl oxygen of 3a and

the backbone NH of glycine⁶⁶and the P2 NH of 3a and

the backbone carbonyl of glycine⁶⁶. In contrast, re thio-

late addition to stereoisomer 3a (Fig. 3b) was predicted

to exhibit the same P2/P1 interactions as for si addition,

however the hydroxyl of the hemithioketal was now

stabilised by two hydrogen bonds from the p-NH of the

imidazole side chain of histidine¹⁵⁹

.

When considering stereoisomer 4a, binding conforma-

tions for both si (addition of thiolate to same face as the

a-Me) and re (addition of thiolate to face opposite the a-

Me) thiolate addition were predicted to retain the P2/P1

hydrogen-bonding network between the proteinase

backbone and the inhibitor (compare Figs. 3 and 4).

However, in contrast to stereoisomer 3a, both binding

modes predicted that the a-methyl of R-stereochemistry

projects away from the proteinase and into the solvent

exposed cavity (Fig. 4). A further contrast between ste-

reoisomers 3a and 4a was evident in the si thiolate ad-

duct (compare Figs. 3a and 4a), which predicted that the

hydroxyl of the hemithioketal would again be stabilised

by one hydrogen bond to the backbone NH of cysteine²⁵

but for 4a the second hydrogen bond would now be to

the backbone carbonyl of glycine²³.

2. Results and discussion

2.1. Design strategy

Our design strategy commenced with in silico con-

struction of each of the four possible hemithioketal

tetrahedral intermediates (si or re stereofacial thiolate

addition to both prime and nonprime binding orienta-

tions) for 3-methyl-3-amidotetrahydrofuran-4-one 3a,

then repeating the process for stereoisomer 4a.⁹When

considering the possible nonprime binding models, a

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

2905

Figure 4. Predicted binding conformations for si (a) and re (b) ste-

reofacial thiolate addition to a model a-methyl monocyclic 4a, piper-

azine-1-carboxylic acid [3-methyl-1S-(3R-methyl-4-oxo-tetrahydro-

Figure 5. Predicted binding conformations for si (a) and re (b) ste-

reofacial thiolate addition to a model bicycle 5, (3aR,6aR) piperazine-1

carboxylic acid [3-methyl-1S-(3-oxo-hexahydrocyclopenta[b]furan-3a-

ylcarbamoyl)butyl]amide.^9;10aThe residues Gly²³, Trp²⁶, Cys⁶³, Gly⁶⁴

and Gly⁶⁵that make the P1 wall of cathepsin K structure 1mem are

shown as an electrostatic potential coloured surface. The hydrogen

atoms of the intermediate hydroxyl, ring bridgehead 6a and -NH are

added.

furan-3-yl-carbamoyl)butyl]amide.^9;11The residues Gly²³, Trp²⁶, Cys⁶³

,

Gly⁶⁴and Gly⁶⁵that make the P1 wall of cathepsin K structure 1mem

are shown as an electrostatic potential coloured surface. The hydrogen

atoms of the intermediate hydroxyl, a-methyl and a-NH are added and

residue Gly²³labelled.

teinase. Prime-side binding models for compounds 3b,c

and 4b,c were complicated by predicted clashes between

either the C-2 or C-3 methyl with either Gly²³or Gly⁶⁴

of the proteinase for six of the eight possible confor-

mations. Only 3b si and 3c re gave solvent exposed

methyl substituents, however, as before, all prime-side

models were predicted to lose the major backbone

hydrogen-bonding network. Thus, from the 16 binding

conformations considered for 2,3-dimethyl-3-amido-

tetra-hydrofuran-4-ones 3b,c and 4b,c only the non-

prime-side model of 4b was predicted to exhibit a

reasonable energy conformer that retained a good

hydrogen-bonding network to the proteinase backbone

along with the absence of spatial clashes.¹⁴

When considering the possible prime-side binding

models, a number of conclusions were apparent.¹¹The

general spatial ﬁt of models generated from 3a with si or

re thiolate addition and 4a with re thiolate addition were

predicted to be reasonable with no obvious clash with

the proteinase. However, apart from the tetrahedral

hydroxyl, few hydrogen-bonding interactions were pre-

dicted between the compounds and proteinase.¹²Po-

tential binding modes of this nature, with the loss of

major backbone hydrogen-bonding networks would

probably lead to a dramatic loss of potency. Prime-side

binding models for stereoisomer 4a with si thiolate

addition were predicted to give an unfavourable clash

with Gly²³of the proteinase. Thus, from the eight

binding conformations considered for 3-methyl-3-

amidotetrahydrofuran-4-ones 3a and 4a, only the

nonprime-side models of 4a are predicted to exhibit a

reasonable energy conformer that retained a good

hydrogen-bonding network to the proteinase backbone

along with the absence of spatial clashes (Fig. 4).¹³

In the ﬁnal part of our design process, the nonprime-side

models of 4b were used to formulate the bicyclic ketone

5, containing the cis-fused N-(3-oxo-hexahydrocyclo-

penta[b]furan-3a-yl)acylamide. Models of si thiolate

addition to diastereoisomer 5 (Fig. 5a) predict stabili-

sation of the oxygen of the hemithioketal by a hydrogen

bond to the backbone NH of cysteine²⁵and a second

hydrogen bond between the hydroxyl and the backbone

carbonyl of glycine²³, along with the standard P2/P1

interactions (e.g., see earlier description for 3a). Again,

similar to the observations made for re addition to ste-

reoisomer 3a, re thiolate addition to diastereoisomer 5

(Fig. 5a) was predicted to exhibit the same P2/P1

interactions as for si addition, with the hydroxyl of the

hemithioketal being stabilised by two hydrogen bonds

from the p-NH of the imidazole side chain of histi-

The design process was further extended through the

introduction of an alkyl substituent to C-2 within gen-

eral formulae 3 and 4. Building upon the models de-

picted in Figures 3 and 4, a methyl substituent was

added to give 3b (C-2 R), 3c (C-2 S), 4b (C-2 R) and 4c

(C-2 S) and modelling assessment of all 16 possible

binding conformations conducted. As detailed previ-

ously, results indicated that nonprime-side binding of all

compounds of general formulae 3 gave an unfavourable

overlap between the a-methyl substituent and the pro-

teinase. In contrast, nonprime-side binding of com-

pounds of general formulae 4 gave a mixture of results.

Compound 4b was predicted to bind similarly to 4a (Fig.

4a and b), with the addition of a synclinal relationship

(ꢁ45ꢁ looking along the bond from C-2 ﬁ C-3) between

the C-2 and C-3 methyl substituents. However, if com-

pound 4c bound in a similar orientation to 4a (Fig. 4),

the addition of an antiperiplanar relationship (180ꢁ)

between the C-2 and C-3 methyl, would result in the C-2

methyl exhibiting an unfavourable clash with the pro-

dine¹⁵⁹

.

2.2. Chemistry

2.2.1. Synthesis of Fmoc-protected scaﬀolds 6–8. Syn-

thesis of compounds of general formulae 4 and 5 was

envisaged via solid-phase chemistries utilising the key

scaﬀold intermediates (2R,3R)-6, (2S,3R)-7 and

(3aR,6aR)-8 (Fig. 6).

2906

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

R²

H

N

(S)

Me

NC

Me

(R)

⁺H₃N

OH

R¹

Me

N

O

6a

3a

O

Ph

Ph_{iv, v}

2

3

-

CO₂

Fmoc

O

N

H

Me

O

20

O

Me

NH

O

Fmoc

16

O

6. R¹= Me, R²= H

7. R¹= H, R²= Me

8

H₂

N

Me

i, ii

iii

18

O

Figure 6. Protected mono- and bicyclic building blocks towards solid-

phase assembly of compounds of general formulae 4 and 5.

H

N

H

N

(R)

Me

NC

Me

(R)

⁺H₃N

OH

Me

Ph

-

CO₂

Retrosynthetic analysis indicated that the adaptation of

a literature method for the preparation of compounds of

general formula 2a, through a lithium chloride/acetic

acid promoted insertion reaction of protected amino

acid a-diazomethylketones,⁸may provide scaﬀolds 6–8

via a-diazomethylketones 9–11. It was thought that a-

diazomethylketones 9–11 would be available from pro-

tected amino acids 12–14 (Fig. 7), which in turn are

available from the free amino acids 15–17.

Me

O

19

O

Me

O

15

Scheme 1. Synthesis of (2R,3S)-16. Reagents and conditions: (i) TFA,

CH₂Cl₂; (ii) 2 equiv powdered NaCN, 2-propanol, 2 h; (iii) 1 equiv

TFA, 2-propanol then recrystallisation from Et₂O/hexane; (iv) 2 equiv

tert-BuOCl, Et₂O, then triethylamine; (v) cHCl; Dowex^â50W · 4

(elution with 1 M NH₃), then recrystallisation from H₂O/EtOH/Et₂O.

in anhydrous 2-propanol with powdered sodium cyanide

for 2 h, in our hands resulted in an improved formation

of the mixture of nitriles 19 and 20 (46%). Flash chro-

matography followed by recrystallisation from diethyl

ether/heptane gave enantiopure 20 (18%). Despite the

low overall yield of 20, the above modiﬁcation was

reproducible and provided suﬃcient quantities of

material (ꢂ3 g). Attempts to reproduce the reported

isomerisation of enantiopure 20 (as well as the mother

liquor obtained after recrystallisation, which contained

19 and 20) failed to provide any useful quantities of 19.¹⁷

Finally, removal of the phenylalanyl moiety from 20 was

accomplished by oxidation with freshly prepared tert-

butylhypochlorite and triethylamine, followed by

hydrolysis with concentrated hydrochloric acid to give

(2R,3S)-16 (33% from 20) after ion-exchange chroma-

tography and recrystallisation from water (trace)/etha-

nol/diethyl ether.

Routes towards amino acids 15–17 were sought and a

survey of the literature identiﬁed the work of Ohfune

et al. who have described the synthesis of a-alkylated

serine and threonine analogues¹⁵and the cyclic amino

acid (1R,2S)-17b.¹⁶The reported synthesis of (2R,3R)-15

and (2R,3S)-16 was appealing for our synthetic strategy

since it oﬀered a stereodivergent route to the desired

diastereoisomers (Scheme 1). However, following the

literature procedure, we were unable to achieve the

quoted yields, particularly for the intramolecular

Strecker reaction and equilibrium isomerisation (it may

be that the methodology is highly sensitive to scale-up).

Therefore, we adapted the literature procedures as fol-

lows. Treatment of diastereomeric N-Boc-

nine acetoin ester 18 (78% yield by the coupling of dl-

acetoin and N-Boc- -phenylalanine using 1-[3-(dimeth-

L-phenylala-

L

ylamino)propyl]-3-carbodiimideÆHCl (EDC) activation

in the presence of 4-(dimethylamino)pyridine (DMAP))

with triﬂuoroacetic acid (TFA) in dichloromethane gave

the corresponding TFA salt. Treatment of the TFA salt

Due to the diﬃculties encountered with the above

preparation of 19, the methodology reported by Seebach

R²

R¹

R²

R³

R¹

R²

O^-

R²

R¹

OR³

R⁴

OH

2

3

2

1

3

2

1

O^-

Fmoc

⁺H₃N

N

H

⁺H₃N

NH

Fmoc

O

9a. R¹= Me, R²= H, R³= H, R⁴= -CH=N₂

9b. R¹= Me, R²= H, R³= Bu^t, R⁴= -CH=N₂

10a. R¹= H, R²= Me, R³= H, R⁴= -CH=N₂

10b. R¹= H, R²= Me, R³= Bu^t, R⁴= -CH=N₂

12a. R¹= Me, R²= H, R³= H, R⁴= OH

12b. R¹= Me, R²= H, R³= Bu^t, R⁴= OH

12c. R¹= Me, R²= H, R³= H, R⁴= OAllyl

12d. R¹= Me, R²= H, R³= Bu^t, R⁴= OAllyl

13a. R¹= H, R²= Me, R³= H, R⁴= OH

13b. R¹= H, R²= Me, R³= Bu^t, R⁴= OH

13c. R¹= H, R²= Me, R³= H, R⁴= OAllyl

13d. R¹= H, R²= Me, R³= Bu^t, R⁴= OAllyl

13e. R¹= H, R²= Me, R³= Bu^t, R⁴= F

11a. R¹= H, R²= OH, R³= -CH=N₂

11b. R¹= H, R²= OBu^t, R³= -CH=N₂

14a. R¹= H, R²= OH, R³= OH

14b. R¹= H, R²= OBu^t, R³= OH

14c. R¹= OH, R²= H, R³= OAllyl

14d. R¹= H, R²= OH, R³= OAllyl

14e. R¹= H, R²= OBu^t, R³= OAllyl

14f. R¹= H, R²= OBu^t, R³= F

17a (1R, 2R) R¹= H, R²= OH

17b (1R, 2S) R¹= OH, R²= H

15 (2R, 3R) R¹= Me, R²= H

16 (2R, 3S) R¹= H, R²= Me

Figure 7. Amino acid building blocks towards mono- and bicyclic scaﬀolds 6–8.

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

2907

catalysed conditions described by Kunz and co-work-

ers,²¹providing 12c in excellent yield (94%). Protection

of the hydroxyl group of 12c as the tert-butyl ether to

give 12d was accomplished using isobutene in the pres-

ence of catalytic concd sulfuric acid (87% yield based on

recovered starting material). The allyl ester was then

smoothly cleaved using Pd-catalysed allyl transfer to

give acid 12b (98%).²²Activation of 12b as the mixed

anhydride using iso-butylchloroformate and N-meth-

ylmorpholine proceeded smoothly (similar to that ob-

served for 12a with HPLC-MS and analytical HPLC

monitoring showing >95% activation). However, in

contrast to activated 12a, the addition of freshly pre-

pared diazomethane to the mixed anhydride of 12b gave

no indication of the desired a-diazomethylketone 9b,

with a new major product identiﬁed as the N-carboxy-

anhydride 25 isolated (61%).²³This route was not

examined further since the desired scaﬀold 6 was already

in hand.

Me

OH

Me

O

N

Me

OH

i

O

N

iii

ii

CO₂Me

Ph

-

⁺H₃N

CO₂

CO₂Me

Me

^-Cl⁺H₃N

CO₂Me

Me

21

22

15

23

Scheme 2. Synthesis of (2R,3R)-15. Reagents and conditions: (i) H₂O,

EtOCNHPh, Et₂O; (ii) Li(i-Pr)₂N, THF, hexane, then CH₃I; (iii) 6 M

HCl, then Dowex^â50W · 4 (elution with 1 M NH₃) and recrystalli-

sation from H₂O/EtOH/Et₂O.

et al. (Scheme 2) was used in the synthesis of (2R,3R)-

15.¹⁸5R-Methyl-2-phenyl-4,5-dihydrooxazole-4S-car-

boxylic acid methyl ester 21 was prepared (67%) by a

slight modiﬁcation of the literature protocol, from the

commercially available ethyl benzimidate hydrochloride

salt (which was rendered salt free by washing a diethyl

ether solution with saturated aqueous sodium hydrogen

carbonate) and L-threonine methyl ester hydrochloride

22. Stereo-controlled methylation gave 23 (88%), which

underwent facile hydrolysis with 6 M hydrochloric acid

to give (2R,3R)-15 (99%) after ion-exchange chroma-

tography. Recrystallisation from water (trace)/ethanol/

diethyl ether gave 15 in multigram quantities.

H

OBu^t

Me

HN

O

H

Me

O

Me

O

N

O

N

H

CO₂Allyl

O

Me

24

Fm

O

26

25

Fmoc Na-protection of amino acids 15 and 16 pro-

ceeded smoothly under standard Schotten–Baumann

conditions to obtain amino acids 12a (92%) and 13a

(80%). In situactivation of 12a as the mixed anhydride

using iso-butylchloroformate and N-methylmorpholine

also proceeded smoothly (HPLC-MS and analytical

HPLC monitoring showed >95% activation). However,

the addition of freshly prepared diazomethane to the

mixed anhydride of 12a gave a complex mixture of

products from which the desired a-diazomethylketone

9a (11%), methyl ester (12a wherein R⁴¼ OMe, 18%)

and an uncharacterised mixture of products, which ap-

peared to polymerise on standing/attempted recrystalli-

sation were isolated.¹⁹Literature precedent appears to

suggest that the formation of methyl esters under these

conditions is common for hindered carboxylic acids,

even though the activation of 12a appeared near quan-

titative.²⁰Treatment of 9a with a solution of lithium

chloride in water and acetic acid gave the desired

scaﬀold (2R,3R)-(2,3-dimethyl-4-oxo-tetrahydrofuran-

For the synthesis of scaﬀold (2S,3R)-7 we took into

consideration the observations and conclusions detailed

above, in particular the low yield of a-diazomethylke-

tone 9a from 12a and the high yield of N-carboxyan-

hydride 25 from the activation of 12b. We chose to

investigate the preparation of a-diazomethylketone 10b

from protected acid 13b, through acyl ﬂuoride 13e.

Literature reported that activation of N-protected hin-

dered a,a-disubstituted amino acids as acyl ﬂuorides was

highly favourable due to their inherent stability after

puriﬁcation (even in the presence of tert-butyl-based

protecting groups), their slow conversion to 4H-oxazol-

5-one in the presence of tertiary bases and their ability to

react in high yield using relatively short reaction times.²⁴

Thus, Na-protected amino acid 13a was converted to

13b following the optimised protocols described for the

conversion of 12a to 12b. Treatment of acid 13b with

pyridine and cyanuric ﬂuoride gave the corresponding

acyl ﬂuoride 13e in near quantitative yield. Frustrat-

ingly, in contrast to the general literature precedence,²⁴

treatment of the 13e with diazomethane gave 4-(1S-tert-

butoxyethyl)-2-(9H-ﬂuoren-9-ylmethoxy)-4R-methyl-4H-

oxazol-5-one 26 as the major product (45% yield from

13e), which was partially separated from a mixture of

the desired a-diazomethylketone 10b and methyl ester

(13b wherein R⁴¼ OMe). Treatment of 10b with a

solution of lithium chloride in acetic acid and water gave

the desired scaﬀold (2S,3R)-7 (5.4% from acid 13b)

following chromatography. Furthermore, 4H-oxazol-5-

one 26 could be converted back into acyl ﬂuoride 13e

using a combination of HF/pyridine and cyanuric ﬂuo-

ride,²⁴which opens the possibility of recycling of 26 into

13e.

3-yl)carbamic acid 9H-ﬂuoren-9-ylmethyl ester

(50%, overall yield 5.5% from 12a) following chroma-

6

tography.

Since formation of a-diazomethylketone 9a was low

yielding, we investigated the preparation of a-diazo-

methylketone 9b containing tert-butyl ether protection

of the b-hydroxyl. It was envisaged that the precursor

acid 12b would be available from the intermediate allyl

ester 12d, in turn prepared by tert-butylation of allyl

ester 12c. Preparation of 12c was attempted from 12a

using para-toluenesulfonic acid, allyl alcohol and tolu-

ene under Dean–Stark reﬂux conditions. However, the

carbamate 4,5-dimethyl-2-oxo-oxazolidine-4-carboxylic

acid allyl ester 24 was obtained as the major product

(84%), together with a minor amount of the desired allyl

ester 12c (2%). The formation of carbamate was avoided

by the use of allyl bromide under mild phase transfer

Having synthesised scaﬀolds 6 and 7, we turned our

attention to synthesis of the ring constrained analogue 8.

2908

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

It was envisaged that 8 would be obtained from the

cyclisation of a-diazomethylketones 11a or 11b, that in

turn would be obtained from the corresponding b-hy-

droxy amino acid 17a (see Fig. 7). Synthesis of the epi-

meric b-hydroxy amino acid 17b has been reported by

Ohfune et al. and the method is outlined in Scheme 3.¹⁶

The methodology makes use of the previously discussed

intramolecular Strecker reaction. Since there were no

literature methods for the preparation of 17a, we chose

to synthesise 17b according to Scheme 3 with the aim of

inverting the hydroxyl group at a later stage. Thus,

alcohol 27, prepared by oxidation of cyclopentanone,

was coupled to BocPheOH (using EDC and DMAP) to

give 28. Hydrolysis of the dimethyl ketal was accom-

plished with para-toluenesulfonic acid monohydrate in

wet acetone to give the corresponding ketone 29 (40%

yield from cyclopentanone). Removal of the Boc pro-

tecting group and cyclisation under a modiﬁcation of

the reported literature protocol gave the desired

(4aS,7aS)-carbonitrile 30 (36%), together with ketimine

intermediates (see parentheses in Scheme 3). Carbonit-

rile 30 was then oxidised with freshly prepared tert-but-

ylhypochlorite and triethylamine to give crude 31 and

the resultant product mixture was hydrolysed with

concentrated hydrochloric acid and puriﬁed by ion-ex-

change chromatography to obtain the desired b-hydroxy

amino acid 17b (67%).

methoxycarbonylamino)-2S-hydroxycyclopentanecarb-

oxylic acid allyl ester 14c (approximately 9:1, 55% yield

in total). To facilitate puriﬁcation, the mixture of alco-

hols was heated at 100 ꢁC in toluene in the presence of

para-toluenesulfonic acid. Whilst the trans-(R,R)-epimer

14d remained intact, the cis-(R,S)-epimer 14c cyclised to

form the bicycle oxazolidine 32 that was readily sepa-

rated from the desired product by chromatography.

Additionally, following the Fmoc protection of 17b,

further evidence for the stereochemical assignment of

alcohols 14c and 14d was obtained upon treatment with

allyl alcohol and para-toluenesulfonic acid in reﬂuxing

toluene. Here the isolated products from the reaction

included 32 (49%) together with a trans epimer, which

based upon the precedence of Ohfune et al.¹⁶was as-

signed as the desired allyl-protected (R,R)-epimer 14d

(9%), presumably formed from a minor component

present in the starting material, namely the (R,R)-acid

14a. It is likely that this (R,R)-acid 14a would have

arisen from a small amount of the (R,R)-epimer 17a

being present following HCl hydrolysis of 31 (Scheme

3). The minor (R,R)-component was probably formed

from one of the alternative ketimine intermediates

shown in Scheme 3, with the stereochemistry of the C-2

carbon atom being incompletely controlled by the ste-

reochemistry of the phenylalanine.

t

H

OBu

O

H

Fmoc protection of 17b, followed by allylation under

the previously described phase transfer conditions gave

allyl ester 14c (68%). Attempts to invert the hydroxyl

group of 14c under Mitsunobu conditions using formic

acid as the nucleophile were unsuccessful.²⁵However,

inversion of the hydroxyl was achieved by Swern oxi-

dation of 14c, followed by sodium borohydride reduc-

O

N

O

H

O

N

NH

CO₂Allyl

Fmoc

Fm

34

32

33

The alcohol functionality of the (R,R)-epimer 14d was

protected as the tert-butyl ether using isobutene in the

presence of concentrated sulfuric acid to obtain 14e

(75%). Then the allyl ester of both 14d and 14e were

smoothly cleaved using Pd-catalysed allyl transfer to

give acids 14a (80%) and 14b (78%).²²In situactivation

of acid 14a as the mixed anhydride using iso-butyl-

chloroformate and N-methylmorpholine proceeded

smoothly (HPLC-MS and analytical HPLC monitoring

showed >95% activation). However, following treat-

ment with diazomethane, no evidence for the desired

diazomethylketone 11a was observed,¹⁹with the only

isolable product being tentatively identiﬁed as the 4,5-

bicyclic lactone 33 (41%). Similarly, in situactivation of

acid 14b as the mixed anhydride proceeded smoothly,

but following treatment with diazomethane a complex

inseparable mixture was now observed.¹⁹The desired

scaﬀold 8 was however prepared via the pre-formed acyl

ﬂuoride 14f, which was prepared from acid 14b by

treatment with pyridine and cyanuric ﬂuoride. In a

similar manner to that described earlier for acyl ﬂuoride

13e, treatment of the crude acyl ﬂuoride 14f with dia-

zomethane led to the recovery of mixtures of 14f, oxa-

zolone 34 and (1R,2R)-2-tert-butoxy-1-(9H-ﬂuoren-9-

ylmethoxycarbonylamino)cyclopentanecarboxylic acid

methyl ester (14b wherein R³¼ OMe) as the major

products (approximately 50%), together with the desired

a-diazomethylketone 11b (13% yield from 14f). The

oxazolone 34 containing mixture above could again be

tion to give

a

mixture of 1R-(9H-ﬂuoren-9-

ylmethoxycarbonylamino)-2R-hydroxycyclopentanecarb-

oxylic acid allyl ester 14d and 1R-(9H-ﬂuoren-9-yl-

O

MeO

MeO OMe

OH

OMe

O

ii

i

NHBoc

Ph

(S)

NHBoc

Ph

O

28

29

27

^-CN

H⁺

N

H⁺

N

H

N

Ph

O

Ph

iii, iv

2

O

H

N

H

NC

HO

Ph

N

O

Ph

H

v

vii

vi

-

O

CO₂

⁺H₃N

17b

H

30

31

Scheme 3. Synthesis of 17b. Reagents and conditions: (i) BocPheOH,

EDC, DMAP, dichloromethane; (ii) para-toluenesulfonic acid mono-

hydrate, (CH₃)₂CO; (iii) TFA, CH₂Cl₂, then concentration in vacuo

and azeotropic removal of excess TFA with toluene; (iv) MgSO₄,

NaOAc CH₃CN, then ﬁltration and concentration in vacuo; (v)

TMSCN, ZnCl₂; (vi) 2 equiv tert-BuOCl, Et₂O, followed by triethyl-

amine; (vii) cHCl, Dowex^â50W · 4 (elution with 1 M NH₃).

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

2909

re-cycled to the acyl ﬂuoride 14f by using a combination

of HF/pyridine and cyanuric ﬂuoride. Finally, 11b was

treated with a solution of lithium chloride in acetic acid

and water to give the desired ring constrained analogue

8 (83% from 11b).

compared to nonhindered ketones.^28bFinal compounds

38–40 were prepared by a series of sequential washing

and coupling reaction steps involving removal of Fmoc,

coupling of an activated Fmoc-NHCHR³-COOH, re-

moval of Fmoc, coupling of an activated R⁴-COOH and

acidolytic cleavage. The coupling of activated Fmoc-

amino acids to the amine functionality of the a,a-dialk-

ylated loaded scaﬀolds was expected to be a diﬃcult

reaction due to steric hinderance.³⁰However, the coupling

was found to be surprisingly facile, exhibiting complete

acylation under standard uronium activation conditions.

This facile acylation is probably due to the restricted

conformational freedom of the loaded scaﬀold providing

relatively easy access to the a-amine functionality.

2.2.2. Solid-phase synthesis. Two main strategies to-

wards the solid-phase assembly of ketones have recently

been developed.²⁶Firstly, the ketone functionality has

been transiently protected as the dimethylketal, then the

scaﬀold linked to the solid phase through an amine via

an acid labile backbone linker.^26bAlternatively, the ke-

tone containing scaﬀold has been derivatised as an acid

labile semicarbazone, through reaction with a hydra-

zide.^26c;dOur solid-phase strategy was based upon the

utilisation of the hydrazide linker described by Murphy

et al.,²⁷(Scheme 4).

2.2.3. Enzyme inhibition studies. Following the methods

detailed in Scheme 4, monocyclic inhibitors 38a–f,

39a,b,d,e,f and bicyclic inhibitors 40a–e were prepared

and then screened against cathepsins B, K, L and S as

well as the parasitic proteinases cruzain and CPB.^10bThe

preliminary steady-state inhibition constants (K_i^ss) are

shown in Table 1 (mean of n ¼ 3 determinations). The

substituents detailed in Table 1 were chosen to exemplify

binding groups that provide potent inhibitors when combined

with the unsubstituted monocyclic scaﬀold 2a of GSK.³¹

Semicarbazones 35a–37a were prepared by reﬂuxing the

respective ketones 6–8 with the hydrazide linker of

Murphy et al.,²⁷in aqueous ethanol/sodium acetate. For

scaﬀolds 6 and 7 optimal formation of the linker con-

structs 35a and 36a was obtained after 24 h. For bicyclic

scaﬀold 8 optimal formation of the linker construct 37a

was obtained after 8 h.^28aSemicarbazones 35a–37a

provided a carboxylic acid for attachment to an ami-

nomethyl functionalised polymer support (SP) and also

protection of the ketone from nucleophilic attack during

the synthetic steps prior to cleavage. The eﬃciency of

formation of loaded linker constructs 35b–37b was

monitored based by spectrophotometric determination

of the Fmoc-derived chromophore liberated upon

treatment with 20% piperidine in dimethylformamide

and shown to be essentially quantitative.²⁹The eﬃciency

of acidolytic cleavage was monitored by treatment of the

loaded construct with TFA/H₂O, followed by an

assessment of the residual (i.e., noncleaved) Fmoc-de-

rived chromophore. Constructs 35b–37b were found to

require signiﬁcantly extended cleavage times when

In general, the results show a marked loss of potency for

both the a-methyl monocyclic analogues (38 and 39) and

the cis-fused bicyclic analogues (40) when compared

with the non-a-substituted equivalents. However, clear

structure–activity trends are observed within an active

series against a particular proteinase. For example,

when comparing series a for cathepsin K, series b for

cathepsin S, series d for cathepsin L and series e for

cathepsins K and L, generally bicyclic analogues 40 were

more potent than the monocyclic analogues (2R,3R)-38,

which in turn were more potent than monocyclic ana-

logues (2S,3R)-39. These experimental ﬁnding are con-

sistent with the modelling conclusions formulated

during our design process. As detailed previously, it was

clear that stereoisomer (2R,3R)-4b was predicted to have

superior binding when compared to stereoisomer

(2S,3R)-4c and this has translated into generally im-

proved potency for series (2R,3R)-38 when compared to

series (2S,3R)-39. Also, during the design process the

possibility of cyclising the cis-methyl substituents of

(2R,3R)-4b providing bicyclic 5 was proposed (see Fig.

5). Experimentally, this has translated into improved

potency for series (3aR,6aR)-40 when compared to ser-

ies (2R,3R)-38. Given the reasonable binding confor-

mations⁹and comparable scoring functions¹¹for

inhibitors such as 38a and 40a against cathepsin K or

38b and 40b against cathepsin S when compared with

the equivalent non-a-methylated analogues, we were

intrigued to understand why such a marked loss of

potency has occurred. Thus, individual association (k_on)

and dissociation (k_off) rate constants were determined

for series b against cathepsin S and the results detailed in

Table 2.

R²

R¹

O

H

N

R⁴

N

H

O

R³

O

N

38 R¹= Me, R²= H

39 R¹= H, R²= Me

i, ii

Fmoc

iii, iv, iii, v, vi

6-8

O

NH

N

H

N

H

and

COR

35-37

a. R = OH

b. R = NH-SP

O

H

O

R⁴

N

O

R³

40

H

O

Scheme 4. Synthesis and use of supported linker constructs 35b–37b.

Reagents and conditions: (i) trans-4[(hydrazinocarbonyl)amino]meth-

ylcyclohexanecarboxylic acid. triﬂuoro-acetate, EtOH, H₂O, NaOAc,

reﬂux; (ii) 3 equiv 35a–37a, 3 equiv HBTU, 3 equiv HOBt, 6 equiv

NMM, H₂N-solid phase, DMF; (iii) 20% piperidine/DMF (v/v),

30 min; (iv) 10 equiv Fmoc-NHCHR³-COOH, 10 equiv HBTU,

10 equiv HOBt, 20 equiv NMM, DMF, rt, o/n; (v) 10 equiv R⁴-COOH,

10 equiv HBTU, 10 equiv HOBt, 20 equiv NMM, DMF, rt, o/n; (vi)

TFA/H₂O, (95:5, v/v), rt, 2 · 24 h (35b, 36b), 1 · 24 h (37b).

Clearly, once associated, inhibitors 38b, 40b and 41 ex-

hibit comparable hydrolysis rates as reﬂected by their

2910

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

Table 1. Preliminary inhibitory activities (K_i^ss, lM) for mono- and bicyclic inhibitors 38–40³¹

Compound no P3–P2 Structure

Cat. K

Cat. L

Cat. S

Cat. B

Cruz.

CPB

N

38a

39a

40a

8.7

NI

1.8

>65

26

NI^a

NI

>50

NI

21

O

H

N

(S)

21

>40

12.3

O

S

O

H

N

(S)

38b

39b

40b

NI

>80

>60

>50

9.9

NI

>85

5.6

O

H

38c

40c

NI

2.8

>65

>50

NI

>40

NI

>50

NI

>75

NI

>50

N ^(S)

S

O

H

(S)

N

38d

39d

40d

NI

>35

>30

2.8

NI

Br

O

>60

>55

>70

OH

O

H

(S)

H₂N

N

38e

39e

40e

22

11.3

>80

9.2

>40

NI

>70

NI

>60

NI

15.7

O

>70

>60

O

H

38f

39f

>50

NI

>30

>60

>30

NI

>30

NI

>30

NI

5.5

NI

N ^(S)

O

OH

^aWhere NI stands for no observed inhibition.

broadly similar k_offrates. However, the association rate

of inhibitors 38b and 40b was found to be signiﬁcantly

slower than that of the parent non-a-methylated ana-

logue 41 and this explains the loss in potency since the

steady-state inhibition constant K_i^ssꢀ k_off=k_on. The eﬀect

on association rate is most clearly seen in 39b where a

dramatically reduced k_onis observed, which is consistent

with the modelling conclusions formulated during our

design process.

However, introduction of the simple a-methyl group or

cyclisation of simple a,b-dimethyl substituents had a

profound eﬀect upon both the synthesis and cysteinyl

proteinase inhibition properties of these new scaﬀolds.

Synthetically, manipulation of a-methylthreonines

towards the (2R or S,3R)-2,3-dimethyl-3-amido-

tetrahydrofuran-4-one scaﬀolds proved challenging due

to their tendency to undergo multiple strain-relieving

side reactions that were not observed when reacting the

corresponding threonine analogues. Similarly, multiple

side reactions were observed when manipulating the

cyclic amino acid (1R,2R)-1-amino-2-hydroxycyclopen-

tanecarboxylic acid towards the bicyclic (3aR,6aR)-N-(3-

oxo-hexahydrocyclopenta[b]furan-3a-yl)-acylamide scaf-

fold. Additionally, utilisation of the new a-methyl

monocyclic and cis-5,5-bicyclic scaﬀolds on the solid

3. Conclusions

The introduction of an a-alkyl group into the 3-amido-

tetrahydrofuran-4-one scaﬀold removed the possibility

of enolisation, thus stabilising the a-stereochemistry.

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

2911

Table 2. Association (k_on) and dissociation rate constants (k_off) for series b inhibitors against cathepsin S

a

Compound

k_on(M^ꢁ1s^ꢁ1) (·10³)

k_off(s^ꢁ1) (·10^ꢁ3

)

K_i(M) (·10^ꢁ6

)

K_i^ss(M) (·10^ꢁ6

)

38b

39b

40b

41^b

2.6 Æ 0.2

0.009 Æ 0.001

1.1 Æ 0.6

8.8 Æ 1.8

4.9 Æ 0.9

16 Æ 2

3.3

9.9 Æ 2.5

86.2 Æ 24.2

5.6 Æ 0.4

544

14.5

0.37

100 Æ 30

37 Æ 8

0.23 Æ 0.08

^aInhibition constants calculated from the individual k_onand k_offrates.

^bThe parent non-a-methylated analogue (2R,3R)-furan-3-carboxylic acid [2-cyclohexyl-1S-(2-methyl-4-oxo-tetrahydrofuran-3-ylcarbamoyl)-

ethyl]amide.

phase highlighted that the introduction of a simple a-

methyl group had led to a dramatic eﬀect upon the steric

accessibility of the ketone moiety through signiﬁcantly

extended linker formation and cleavage times. The in-

creased chemical steric hinderance of the ketone moiety

was observed biologically through signiﬁcantly reduced

association rates for the a-alkylated inhibitor analogues

with the active site thiolate nucleophile. However, low

micromolar potency analogues were obtained, with

good selectivity, for a range of therapeutically attractive

CAC1 proteinases. Further exempliﬁcation of the a-

alkylation principle that ensures a-chiral integrity,

through spanning of the active site into both prime and

nonprime sides, could possibly access additional binding

parameters leading to an improvement in inhibitor

potency into the nanomolar range.

synthesis. Biochemical protocols together with enzyme

assays were carried out as previously described.^10b

Substrates utilising ﬂuorescence resonance energy

transfer methodology (i.e., FRET-based substrates)

were synthesised using standard solid-phase Fmoc

chemistry methods,³²and employed Abz (2-amin-

obenzoyl) as the ﬂuorescence donor and 3-nitrotyrosine

[Tyr(NO₂)] as the ﬂuorescence quencher.³³

4.1.1.

(2S)-2-tert-Butoxycarbonylamino-3-phenylprop-

ionic acid 1-methyl-2-oxopropyl ester (18). 1-[3-(Di-

methylamino)propyl]-3-ethylcarbodiimide hydrochlo-

ride (10.9 g, 57 mmol) and 4-(dimethylamino)pyridine

(700 mg, 5.7 mmol) were added to a solution of (2R)-2-

tert-butoxycarbonylamino-3-phenylpropionic acid (11 g,

41.8 mmol) in anhydrous dichloromethane (20 mL) at

0 ꢁC and under a nitrogen atmosphere. The mixture was

stirred for 5 min and then 3-hydroxybutan-2-one (6.6 g,

75 mmol, assuming monomeric form) was added at 0 ꢁC.

The mixture was allowed to warm to ambient temper-

ature then stirred for 3 days. The mixture was then

evaporated in vacuo to give a residue. Flash chroma-

tography of the residue over silica gel (500 g) using ethyl

acetate/heptane (3:7) as the eluent gave 18 (10.9 g, 78%),

TLC (single UV spot, R_f¼ 0:40, 50% ethyl acetate in

heptane) and HPLC-MS (single main UV peak with

R_t¼ 9:16 min, 358.2 [MþNa]^þ, 693.3 [2MþNa]^þ)

(lit.¹⁵).

4. Experimental

4.1. General procedures

Standard vacuum techniques were used in handling of

air sensitive materials. Solvents were purchased from

ROMIL Ltd, UK at SpS or Hi-Dry grade unless

otherwise stated. ¹H NMR and ¹³C NMR: Bruker

DPX400 (400 MHz ¹H frequency and 100 MHz ¹³C

frequency; QXI probe) in the solvents indicated.

Chemical shifts are expressed in parts per million (d) and

are referenced to residual signals of the solvent. Cou-

pling constants (J) are expressed in Hz. All analytical

HPLC were obtained on Phenomenex Jupiter C₄, 5 lm,

4.1.2. (2S,3S,5S)-5-Benzyl-2,3-dimethyl-6-oxomorpho-

line-3-carbonitrile (20). Triﬂuoroacetic acid (77 mL)

was added to a solution of (2R)-2-tert-butoxycarbon-

ylamino-3-phenylpropionic acid 1-methyl-2-oxopropyl

ester 18 (7.99 g, 23.8 mmol) in anhydrous dichlorome-

thane (140 mL) at 0 ꢁC under a nitrogen atmosphere.

The mixture was stirred for 1 h at 0 ꢁC then evaporated

in vacuo to give a residue. Toluene (40 mL) was then

added to the residue and evaporated in vacuo. This

procedure was repeated twice, to remove excess triﬂuo-

roacetic acid. The residue was then dissolved in anhy-

ꢀ

300 A, 250 · 4.6 mm, using mixtures of solvent A ¼ 0.1%

aq triﬂuoroacetic acid (TFA) and solvent B ¼ 90% ace-

tonitrile/10% solvent A on automated Agilent systems

with 215 and/or 254 nm UV detection. Unless otherwise

stated a gradient of 10–90% B in A over 25 min at

1.5 mL/min was performed for full analytical HPLC.

HPLC-MS analysis was performed on an Agilent 1100

series LC/MSD, using automated Agilent HPLC sys-

tems, with a gradient of 10–90% B in A over 10 min on

drous 2-propanol (390 mL) under

a

nitrogen

ꢀ

Phenomenex Columbus C₈, 5 lm, 300 A, 50 · 2.0 mm at

0.4 mL/min. Semipreparative HPLC puriﬁcation was

atmosphere. Powdered sodium cyanide (2.6 g, 53 mmol)

was then added in one portion and the mixture stirred

for 2 h at ambient temperature. The mixture was then

rapidly ﬁltered through anhydrous sodium sulfate and

evaporated in vacuo. Flash chromatography over silica

gel (200 g) using ethyl acetate/heptane (3:7) as eluent

gave crude 5-benzyl-2,3-dimethyl-6-oxo-morpholine-3-

carbonitrile (2.7 g). Recrystallisation (diethyl ether–

heptane) gave crystalline (2S,3S,5S)-20 (1.05 g), TLC

ꢀ

performed on Phenomenex Jupiter C₄, 5 lm, 300 A,

250 · 10 mm, using a gradient of 10–90% B in A over

25 min at 4 mL/min on automated Agilent systems with

215 and/or 254 nm UV detection. Flash column puriﬁ-

cation was performed on silica gel 60 (Merck 9385).

Multipins (polyamide 1.2 or 10 lmol loadings, see

www.mimotopes.com) were used for the solid-phase

2912

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

1

(single UV spot, R_f¼ 0:38, 50% ethyl acetate in hep-

tane), analytical HPLC R_t¼ 15:94 min, HPLC-MS

(single main UV peak with R_t¼ 7:53 min, 245.1

[MþH]^þ, 267.1 [MþNa]^þ); ¹H NMR (400 MHz,

CDCl₃): d 1.35 (d, J ¼ 6:50 Hz, 2-CH₃3H), 1.48 (s, 3-

CH₃, 3H,), 3.01 (dd, J ¼ 9:10 and 13.80 Hz, CH₂Ph,

1H), 3.48 (dd, J ¼ 3:55 and 13.85 Hz, CH₂Ph, 1H), 4.17

(dd, J ¼ 3:60 and 9.10 Hz, 5-H, 1H), 4.57 (q,

J ¼ 6:50 Hz, 2-H, 1H), 7.29–7.42 (m, C₆H₅, 5H) (lit.¹⁵).

ethyl acetate in heptane); H NMR (400 MHz, CDCl₃):

d 1.59 (d, J ¼ 6:30 Hz, 5-CH₃, 3H), 3.86 (s, COCH₃3H),

4.53 (d, J ¼ 7:50 Hz, 4-H, 1H), 5.02–5.09 (m, 5-H, 1H),

7.45–7.57 (m, phenyl CH, 3H) and 8.03–8.05 (m, phenyl

CH, 2H); analytical HPLC R_t¼ 10:33 min, HPLC-MS

(single main UV peak with R_t¼ 5:76 min, 220 [MþH]^þ)

(lit.¹⁸).

4.1.5. (4R,5R)-4,5-Dimethyl-2-phenyl-4,5-dihydrooxaz-

ole-4-carboxylic acid methyl ester (23). A solution of 21

(8.0 g, 36.5 mmol) in anhydrous tetrahydrofuran

(43 mL) was added to a solution of lithium diisoprop-

ylamine (20.2 mL, 2 M solution in heptanes and tetra-

hydrofuran) in anhydrous tetrahydrofuran/hexane

(243 mL, 10:1) at ꢁ78 ꢁC under an atmosphere of

nitrogen. After 1 h of stirring at ꢁ78 ꢁC iodomethane

(5.70 mL, 91.74 mmol) was added. The mixture was

stirred for a further 4 h at ꢁ78 ꢁC and then allowed to

warm to ambient temperature over 18 h. Saturated

aqueous ammonium chloride solution (200 mL) was

then added, followed by heptane (300 mL). The phases

were separated and the organic phase washed with

brine, dried (Na₂SO₄) and evaporated in vacuo to give a

residue. Flash chromatography over silica (300 g) using

ethyl acetate/heptane (3:7) as the eluent gave 23 (7.5 g,

88%), TLC (single UV spot, R_f¼ 0:49, 50% EtOAc in

heptane); ¹H NMR (400 MHz, CDCl₃): d 1.07 (d,

J ¼ 6:55 Hz, 5-CH₃, 3H), 1.38 (s, 4-CH₃, 3H), 3.46 (s,

COCH₃, 3H), 4.27 (q, J ¼ 6:55 Hz, 5-H, 1H), 7.12–7.24

(m, phenyl CH, 3H) and 7.69–7.72 (m, phenyl CH, 2H);

analytical HPLC R_t¼ 10:99 min, HPLC-MS (single

main peak, 234 [MþH]^þ) (lit.¹⁸).

4.1.3. (2R,3S)-2-Amino-3-hydroxy-2-methylbutyric acid

(16). To a solution of 20 (1.38 g, 5.63 mmol) in anhy-

drous diethyl ether (120 mL) was added tert-butylhy-

pochlorite (1.26 mL, 11.25 mmol) at 0 ꢁC under a

nitrogen atmosphere. The solution was stirred at ambi-

ent temperature for 2 h, then triethylamine (1.26 mL)

was added at 0 ꢁC. The mixture was then allowed to

warm to ambient temperature and stirred for 20 h. The

resulting suspension was ﬁltered and the solid residue

washed with diethyl ether (20 mL). Water (100 mL) was

then added to the ﬁltrate and the product extracted into

ether (50 mL · 3). The combined ethereal layers were

dried (MgSO₄) and evaporated in vacuo to give a resi-

due (1.9 g). Concentrated hydrochloric acid (70 mL) was

then added to the residue at 0 ꢁC. The mixture was

stirred for 1 h at 0 ꢁC then at ambient temperature for

24 h. The mixture was then transferred to a sealed

pressure tube and heated at 80 ꢁC for 2 days. The mix-

ture was then cooled and extracted with diethyl ether

(50 mL · 3). The aqueous layer was then concentrated

under reduced pressure then puriﬁed over Dowex^â

50W · 4 (activated with 0.01 M hydrochloric acid).

Elution with water (until the eluent approached ꢀ pH 7),

followed by ammonium hydroxide (1 M) gave impure 16

(555 mg). Recrystallisation from H₂O(trace)/EtOH/Et₂O

gave 16 (250 mg, 33%), TLC (single UV spot, R_f¼ 0:27,

4:1:1 n-BuOH/H₂O/AcOH); ¹H NMR (400 MHz, D₂O):

d 0.92 (d, J ¼ 6:65 Hz, CHCH₃, 3H), 1.06 (s, 2-CH₃,

3H), 3.85 (q, J ¼ 6:60 Hz, 3-H, 1H); HPLC-MS (single

main UV peak with R_t¼ 0:45 min, 134 [MþH]^þ) (lit.¹⁵).

4.1.6. (2R,3R)-2-Amino-3-hydroxy-2-methylbutyric acid

(15). Hydrochloric acid (6 M, 10 mL) was added to 23

(1.12 g, 4.81 mmol), and the mixture was then heated

under reﬂux for 18 h. The mixture was cooled to ambi-

ent temperature then water (7 mL) added, followed by

diethyl ether (14 mL). The phases were thoroughly

mixed and then separated. The aqueous phase was

concentrated under reduced pressure then puriﬁed over

Dowex^â50W · 4 (activated with 0.01 M HCl). Elution

with water (100 mL), followed by 1 M NH₄OH gave 15

(640 mg, 99%). Recrystallisation from H₂O (trace)/

EtOH/Et₂O gave (2R,3R)-15, TLC (single UV spot,

R_f¼ 0:27, 4:1:1 N-BuOH/H₂O/AcOH); ¹H NMR

(400 MHz, D₂O): d 0.84 (d, J ¼ 6:60 Hz, CHCH₃, 3H),

1.13 (s, 2-CH₃, 3H), 3.69 (q, J ¼ 6:60 Hz, 3-H, 1H);

HPLC-MS (single main UV peak with R_t¼ 0:45 min,

134 [MþH]^þ) (lit.¹⁸).

4.1.4. (4S,5R)-5-Methyl-2-phenyl-4,5-dihydrooxazole-4-

carboxylic acid methyl ester (21). Saturated aqueous

sodium hydrogen carbonate (100 mL) was added to a

solution of ethyl benzimidate hydrochloride (4 g,

21.25 mmol) in diethyl ether (100 mL). The phases were

vigorously mixed and then separated. The ethereal layer

was dried (Na₂SO₄) and evaporated in vacuo to give a

crude residue. The residue was then dissolved in diethyl

ether (8.5 mL) and then added to a solution of L-threo-

nine methyl ester hydrochloride 22 (2.1 g, 12.4 mmol) in

water (1.5 mL). The mixture was stirred for 20 h then

water (20 mL) added. The mixture was then extracted

with diethyl ether (40 mL · 3) and the combined ethereal

layers washed with brine (20 mL), dried (Na₂SO₄) and

evaporated in vacuo to give a residue. The excess ethyl

benzimidate was removed by distillation at 100 ꢁC under

reduced pressure (ꢂ0.5 mbar). The remaining material

was puriﬁed by ﬂash chromatographed on silica gel

(300 g) using ethyl acetate/heptane (1:3) as eluent to give

21 (1.83 g, 67%), TLC (single UV spot, R_f¼ 0:47, 50%

4.1.7.

(2R,3R)-2-(9H-Fluoren-9-ylmethoxycarbonyl-

amino)-3-hydroxy-2-methylbutyric acid (12a). Amino

acid 15 (1.54 g, 11.6 mmol) was added to a vigorously

stirred solution of sodium carbonate (3.05 g, 28.9 mmol)

in water (66 mL) at 0 ꢁC. 1,4-Dioxan (32 mL) was then

added, providing an opaque mobile mixture. 9-Fluoren-

ylmethyl chloroformate (3.13 g, 12.16 mmol) in 1,4-di-

oxan (35 mL) was then added over 40 min. The mixture

was then allowed to warm to ambient temperature over

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

2913

30 min. Water (300 mL) was then added, and the reac-

tion mixture washed with chloroform (2 · 250 mL) and

the combined organic layers discarded. The aqueous

phase was acidiﬁed with 1 M HCl (ꢀpH 2), providing a

thick opaque mixture. The acidiﬁed aqueous mixture

was extracted with chloroform (3 · 250 mL) and the

combined organic phase dried (Na₂SO₄) and evaporated

in vacuo to give a residue (4.2 g). Recrystallisation (from

ethyl acetate/heptane) gave 12a (3.8 g, 92%), TLC (single

UV spot, R_f¼; 20% MeOH in CHCl₃); ¹H NMR

(400 MHz, CDCl₃): d 1.17 (d, J ¼ 5:60 Hz, CHCH₃,

3H), 1.63 (s, 2-CH₃, 3H), 4.20–4.30 (m, 3-H and ﬂu-

orenyl CHCH₂O, 2H), 4.44 (br, CHCH₂O, 2H), 6.09 (s,

NH, 1H), 7.31–7.35 (m, 2 · ﬂuorenyl CH, 2H), 7.40–7.43

(m, 2 · ﬂuorenyl CH, 2H), 7.60 (d, J ¼ 7:45 Hz, 2 · ﬂu-

orenyl CH, 2H) and 7.78 (d, J ¼ 7:50 Hz, 2 · ﬂuorenyl

CH, 2H); ¹³C NMR (100 MHz, CDCl₃): d 18.6

(CHCH₃), 20.4 (2-CH₃), 47.5 (CHCH₂O), 64.0 (C-2),

67.6 (CH₂O), 71.6 (C-3), 120.5, 125.4, 127.5, 128.2

(8 · ﬂuorenyl CH), 141.7, 144.0 (4 · ﬂuorenyl quaternary

C), 157.0 (OCON) and 176.4 (C-1); analytical HPLC

R_t¼ 16:09 min, HPLC-MS (single main peak, 356

[MþH]^þ, 378 [MþNa]^þ). Anal. Calcd for C₂₀H₂₁NO₅:

C, 67.59; H, 5.96; N, 3.94. Found: C, 67.25; H, 6.00; N,

3.95. Exact mass calcd for C₂₀H₂₁NO₅Na: 378.1312,

found 378.1327, d þ4.11 ppm.

HPLC-MS (single main peak, 370 [MþH]^þ, 392

[MþNa]^þ), followed by 9a (220 mg, 11%), HPLC-MS

(main UV peak with R_t¼ 8:84 min, 352.2 [MꢁN₂þH]^þ,

374.2 [MꢁN₂þNa]^þ).

4.1.9. (2R,3R)-(2,3-Dimethyl-4-oxo-tetrahydro-furan-3-

yl)-carbamic acid 9H-ﬂuoren-9-ylmethyl ester (6). A

solution of lithium chloride (247 mg, 5.7 mmol) in water

(1.5 mL) and acetic acid (6.0 mL) was added to 9a

(220 mg, 0.57 mmol). A gas evolved and after 1 h, chlo-

roform (75 mL) was added and the organic phase

washed with saturated aqueous sodium hydrogen

carbonate (2 · 70 mL) and brine (70 mL). The chloro-

form layer was dried (Na₂SO₄) and evaporated in vacuo

to give a residue (220 mg). Flash chromatography of the

residue over silica (35 g) using ethyl acetate/heptane (1:4)

as the eluent gave 6 (100 mg, 50%), TLC (single UV

spot, R_f¼ 0:72, ethyl acetate/heptane, 1:1); ¹H NMR

(400 MHz, CDCl₃): d 1.19 (s, 3-CH₃, 3H), 1.29 (d,

J ¼ 6:25 Hz, 2-CH₃, 3H), 4.19–4.23 (m, 5-H₂and ﬂu-

orenyl CHCH₂O, 3H), 4.40 (d, J ¼ 6:60 Hz, CHCH₂O,

2H), 4.48 (q, J ¼ 6:20 Hz, 2-H, 1H), 4.97 (s, NH, 1H),

7.31–7.70 (m, ﬂuorenyl CH, 8H); ¹³C NMR (100 MHz,

CDCl₃): d 14.5 (2-CH₃), 16.3 (3-CH₃), 47.5 (CHCH₂O),

61.4 (C-2 and C-3), 67.3 (CHCH₂O), 70.7 (C-5), 120.4,

125.4, 127.5, 128.2 (8 · ﬂuorenyl CH), 141.7, 143.8 and

144.2 (4 · ﬂuorenyl quaternary C), 155.1 (OCON) and

214.1 (C-4); analytical HPLC R_t¼ 18:21 min, HPLC-

MS (single main peak, 352 [MþH]^þ, 374 [MþNa]^þ).

Anal. Calcd for C₂₁H₂₁NO₄0.1EtOAc: C, 70.05; H,

6.33; N, 3.59. Found: C, 70.35; H, 5.95; N, 3.91. Exact

mass calcd for C₂₁H₂₂NO₄: 352.1543, found 352.1554, d

þ3.03 ppm.

4.1.8. (1R,1⁰R)-[3-Diazo-1-(1-hydroxyethyl)-1-methyl-2-

oxopropyl]carbamic acid 9H-ﬂuoren-9-ylmethyl ester

(9a). The acid 12a (1.77 g, 5.0 mmol) was dissolved

with stirring in anhydrous dichloromethane (30 mL) and

tetrahydrofuran (10 mL). The reaction was ﬂushed with

nitrogen and cooled to ꢁ15 ꢁC. iso-Butylchloroformate

(0.74 mL, 5.44 mmol) in anhydrous dichloromethane

(5 mL) and N-methylmorpholine (1.03 mL, 10.0 mmol)

in anhydrous dichloromethane (5 mL) were added

simultaneously in 1 mL aliquots over 15 min. Etheral

diazomethane [generated from addition of diazald^â

(4.7 g, ꢂ15 mmol) in diethyl ether (75 mL) onto sodium

hydroxide (5.25 g) in water (7.5 mL)/ethanol (15 mL) at

60 ꢁC] was added to the activated amino acid solution at

ꢁ20 ꢁC. The mixture was then allowed to warm to

ambient temperature and stirred for 20 h. A few drops of

acetic acid were added to the mixture. tert-Butyl methyl

ether (100 mL) was then added to the mixture. The

ethereal layers were then washed with water (3 · 75 mL),

dried (Na₂SO₄) and the solvents removed under reduced

pressure to give a yellow residue (2 g). Flash chroma-

tography of the residue over silica (100 g) using gradient

elution with ethyl acetate/heptane in the ratios of (1:3) to

(1:0) gave an unidentiﬁed fraction (236 mg), followed by

an unstable unidentiﬁed product (790 mg), followed by

(2R,3R)-2-(9H-ﬂuoren-9-ylmethoxycarbonylamino)-3-hy-

droxy-2-methylbutyric acid methyl ester (344 mg, 18%);

¹H NMR (400 MHz, CDCl₃): d 1.10 (d, J ¼ 5:90 Hz,

CHCH₃3H), 1.61 (s, 2-CH₃, 3H), 3.69 (s, OCH₃, 3H),

3.90–4.08 (br, 3-H, 1H), 4.23 (t, J ¼ 6:80 Hz, ﬂuorenyl

CHCH₂O, 1H), 4.69 (br s, CHCH₂O, 2H), 5.95 (s, NH,

1H), 7.31–7.35 (m, 2 · ﬂuorenyl CH, 2H), 7.42 (t,

J ¼ 7:40Hz, 2 · ﬂuorenyl CH, 2H), 7.60 (d, J ¼ 7:40 Hz,

2 · ﬂuorenyl CH, 2H) and 7.68 (d, J ¼ 7:40 Hz, 2 · ﬂu-

orenyl CH, 2H); analytical HPLC R_t¼ 17:47 min,

4.1.10.

(2R,3R)-2-(9H-Fluoren-9-ylmethoxycarbonyl-

amino)-3-hydroxy-2-methylbutyric acid allyl ester (12c).

(a) A solution of 12a (1.92 g, 5.41 mmol) and trica-

prylmethylammonium chloride (2.19 g, 5.41 mmol) in

dichloromethane (8 mL) was added to a stirred solution

of sodium hydrogen carbonate (454 mg, 5.41 mmol) in

water (8 mL), then allyl bromide (0.47 mL, 5.41 mmol)

was added in one portion. The biphasic mixture was

vigorously stirred for 5 days then diluted with water

(80 mL) and the product extracted into dichloromethane

(3 · 100 mL). The combined organic layers were dried

(Na₂SO₄) and evaporated in vacuo to give a residue

(4.9 g). Flash chromatography of the residue over silica

gel (200 g) using ethyl acetate/heptane (3:7) as the eluent

gave 12c (2.02 g, 94%), TLC (single UV spot, R_f¼ 0:48,

50% ethyl acetate in heptane); ¹H NMR (400 MHz,

CDCl₃): d 1.13 (d, J ¼ 5:00 Hz, CHCH₃, 3H), 1.61 (s,

2-CH₃, 3H), 4.10–4.30 (m, OH, 3-H and ﬂuorenyl

CHCH₂O, 3H), 4.35–4.55 (m, ﬂuorenyl CHCH₂O, 2H),

4.68 (br s, OCH₂CHCH₂, 2H), 5.25–5.43 (m,

OCH₂CHCH₂2H),

5.83–6.09

(m,

NH

and

OCH₂CHCH₂, 2H), 7.30–7.39 (m, 2 · ﬂuorenyl CH,

2H), 7.40–7.50 (m, 2 · ﬂuorenyl CH, 2H), 7.62 (d,

J ¼ 7:45 Hz, 2 · ﬂuorenyl CH, 2H) and 7.79 (d,

J ¼ 7:55 Hz, 2 · ﬂuorenyl CH, 2H); ¹³C NMR

(100 MHz, CDCl₃): d 18.9 (CHCH₃), 20.6 (2-CH₃), 47.8

(CHCH₂O), 64.6 (C-2), 67.3 (OCH₂CHCH₂), 67.7

2914

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

(CHCH₂O), 71.8 (C-3), 120.0 (OCH₂CHCH₂), 120.7,

125.7, 127.8, 128.3 and 128.5 (8 · ﬂuorenyl CH), 131.8

(OCH₂CHCH₂), 142.0 and 144.3 (4 · ﬂuorenyl quater-

nary C), 156.8 (OCON) and 173.9 (C-1); analytical

HPLC R_t¼ 18:33 min, HPLC-MS (single main UV

peak, 396.2 [MþH]^þand 418.2 [MþNa]^þ).

128.1 (8 · ﬂuorenyl CH), 132.2 (OCH₂CHCH₂), 141.7,

144.4 and 144.5 (4 · ﬂuorenyl quaternary C), 155.6

(OCON) and 172.5 (C-1); analytical HPLC

R_t¼ 23:30 min, HPLC-MS (single main UV peak, 452.3

[MþH]^þand 474.2 [MþNa]^þ); Exact mass calcd for

C₂₇H₃₄NO₅: 452.2431, found 452.2447, d þ3.40 ppm,

followed by recovered 12c (260 mg, 17%).

(b) Toluene (50 mL) was added to 12a (1.01 g,

2.84 mmol), followed by allyl alcohol (3.74 mL) and

para-toluenesulfonic acid (710 mg). The mixture was

reﬂuxed using Dean–Stark conditions for 1.5 h. Dilute

hydrochloric acid (1 M, 100 mL) was added and the

mixture extracted with ethyl acetate (3 · 100 mL). The

combined organics were dried (Na₂SO₄) and evaporated

in vacuo to give a residue. Flash chromatography of the

residue over silica gel (250 g) using ethyl acetate/heptane

(1:9) as the eluent gave a mixture of uncharacterised

products (400 mg), followed by 12c (22 mg, 2%), fol-

lowed by 4,5-dimethyl-2-oxo-oxazolidine-4-carboxylic

acid allyl ester 24 (476 mg, 84%), TLC (single spot,

R_f¼ 0:20, 50% ethyl acetate in heptane); ¹H NMR

(400 MHz, CDCl₃): d 1.27 (d, J ¼ 6:55 Hz, 5-CH₃, 3H),

1.50 (s, 4-CH₃, 3H), 4.38 (q, CHCH₃, 1H), 4.55–4.65 (m,

OCH₂CHCH₂, 2H), 5.20–5.33 (m, OCH₂CHCH₂, 2H),

5.78–5.92 (m, OCH₂CHCH₂, 1H) and 5.99 (s, NH, 1H);

HPLC-MS (single main peak, 200.1 [MþH]^þ, 399.2

[2MþH]^þand 421.2 [2MþNa]^þ); Exact mass calcd for

C₉H₁₃NO₄: 200.0917, found 200.0926, d þ4.56 ppm.

4.1.12. (2R,3R)-3-tert-Butoxy-2-(9H-ﬂuoren-9-ylmeth-

oxycarbonylamino)-2-methyl butyric acid (12b). Tetraki-

striphenylphosphine palladium(0) (69 mg, 0.06 mmol),

dichloromethane (35 mL) then phenyltrihydrosilane

(0.72 mL, 5.84 mmol) were added consecutively to 12d

(1.2 g, 2.66 mmol) under nitrogen. The mixture was

stirred for 1 h then saturated aqueous sodium hydrogen

carbonate (100 mL) added and the mixture extracted

with chloroform (100 mL · 3). The combined organics

were washed with dilute aqueous hydrochloric acid

(0.01 M, 100 mL), dried (Na₂SO₄) and evaporated in

vacuo to give a residue. Flash chromatography of the

absorbed residue over silica gel (100 g) using ethyl ace-

tate/heptane (2:3), followed by (1:1), followed by (7:3) as

the eluent gave 12b (1.07 g, 98%), TLC (single UV spot,

R_f¼ 0:32, 50% ethyl acetate in heptane); ¹H NMR

(400 MHz, CDCl₃): d 1.00–1.25 (m, CHCH₃and

C(CH₃)₃, 12H), 1.49 (s, 2-CH₃, 3H), 4.01–4.19 (m, ﬂu-

orenyl CHCH₂O and CHCH₂O, 3H), 4.22–4.45 (m, 3-

H, 1H), 5.59 (s, NH, 1H), 7.31–7.33 (m, 2 · ﬂuorenyl

CH, 2H), 7.37–7.41 (m, 2 · ﬂuorenyl CH, 2H), 7.55–7.60

(m, 2 · ﬂuorenyl CH, 2H) and 7.75 (d, J ¼ 7:55 Hz,

2 · ﬂuorenyl CH, 2H); ¹³C NMR (100 MHz, CDCl₃): d

18.7 (CHCH₃), 21.5 (2-CH₃), 29.1 (C(CH₃)₃), 47.6

(CHCH₂O), 60.9 (C(CH₃)₃), 64.2 (C-2), 67.2 (CH₂O),

71.1 (C-3), 120.4, 125.4, 127.5, 128.1 (8 · ﬂuorenyl CH),

134.58, 141.7, 144.2 (4 · ﬂuorenyl quaternary C), 156.3

(OCON) and 175.5 (C-1); analytical HPLC

R_t¼ 19:90 min, HPLC-MS (single main peak, 434.2

[MþNa]^þ). Anal. Calcd for C₂₄H₂₉NO₅0.07CDCl₃: C,

65.85; H, 6.60; N, 3.16. Found: C, 66.04; H, 6.47; N,

2.66; Exact mass calcd for C₂₄H₃₀NO₅: 412.2118, found

412.2136, d þ4.16 ppm.

4.1.11. (2R,3R)-3-tert-Butoxy-2-(9H-ﬂuoren-9-ylmeth-

oxycarbonylamino)-2-methyl butyric acid allyl ester

(12d). A stirred solution of 12c (1.55 g, 3.92 mmol) in

dichloromethane (18 mL) was cooled in a pressure vessel

to ꢁ78 ꢁC then isobutylene gas (ꢂ10 mL) condensed into

the solution. Concentrated sulfuric acid (89 lL) was

added then the pressure vessel sealed. The mixture was

stirred at ambient temperature for 3 days then cooled to

ꢁ78 ꢁC. N-Methylmorpholine (187 lL) was added then

the unsealed pressure vessel allowed to warm to ambient

temperature. The mixture was diluted with saturated

aqueous sodium hydrogen carbonate solution (100 mL)

then the product extracted into dichloromethane

(2 · 100 mL). The combined dichloromethane layers

were washed with brine (40 mL), dried (Na₂SO₄) and

evaporated in vacuo to give a residue. Flash chroma-

tography of the residue over silica gel (150 g) using ethyl

acetate/heptane (1:3) as the eluent gave 12d (1.28 g,

72%), TLC (single UV spot, R_f¼ 0:70, 50% ethyl acetate

4.1.13. (4R,4⁰R)-4-(1-tert-Butoxyethyl)-4-methyl-oxazoli-

dine-2,5-dione (25). The acid 12b (1.01 g, 2.46 mmol) was

dissolved with stirring in anhydrous dichloromethane

(16 mL) and tetrahydrofuran (6 mL). The reaction was

ﬂushed with nitrogen and cooled to ꢁ15 ꢁC. iso-Butyl-

chloroformate (0.35 mL, 2.71 mmol) in anhydrous

dichloromethane (5 mL) and N-methylmorpholine

(0.54 mL, 4.92 mmol) in anhydrous dichloromethane

(5 mL) were added simultaneously in portions over

15 min. The mixture was stirred at ꢁ15 ꢁC for 30 min

(>95% activation was observed by HPLC-MS and

analytical HPLC). Etheral diazomethane [generated

from addition of diazald^â(2.11 g, ꢂ9.8 mmol) in diethyl

ether (43 mL) onto sodium hydroxide (3.0 g) in water

(4.3 mL)/ethanol (8.7 mL) at 60 ꢁC] was added to the

activated amino acid solution at ꢁ15 ꢁC. The mixture

was then allowed to warm to ambient temperature and

stirred for 20 h. A few drops of acetic acid were added to

the mixture. Ethyl acetate (150 mL) was then added to

1

in heptane); H NMR (400 MHz, CDCl₃): d 0.95–1.28

(m, CHCH₃and C(CH₃)₃, 12H), 1.55 (br, 2-CH₃, 3H),

3.85–4.90 (m, 3-H, ﬂuorenyl CHCH₂O and ﬂuorenyl

CHCH₂O, 4H), 4.56 (br, OCH₂CHCH₂, 2H), 5.10–5.13

(m, OCH₂CHCH₂, 1H), 5.27 (br d, J ¼ 17:15 Hz,

OCH₂CHCH₂, 1H), 5.70–5.89 (m, NH and

OCH₂CHCH₂, 1H), 7.21–7.27 (m, 2 · ﬂuorenyl CH,

2H), 7.29–7.36 (m, 2 · ﬂuorenyl CH, 2H), 7.50–7.59 (m,

2 · ﬂuorenyl CH, 2H) and 7.68 (d, J ¼ 7:50 Hz, 2 · ﬂu-

orenyl CH, 2H); ¹³C NMR (100 MHz, CDCl₃): d 19.1

(CHCH₃), 20.7 (2-CH₃), 29.11 (C(CH₃)₃), 47.6

(CHCH₂O), 64.5 (C-2), 66.4 (OCH₂CHCH₂), 66.9

(CHCH₂O), 71.6 (C-3), 74.8 (C(CH₃)₃), 118.6

(OCH₂CHCH₂), 120.4, 125.5, 125.6, 127.4, 127.5 and

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

2915

the mixture. The organics were then washed with satu-

rated aqueous sodium hydrogen carbonate (80 mL),

saturated aqueous ammonium chloride (80 mL), dried

(Na₂SO₄) and the solvents removed under reduced

pressure to give a yellow residue (1.2 g). Flash chroma-

tography of the residue over silica (100 g) using gradient

elution with ethyl acetate/heptane in the ratios of (1:9) to

(1:4) gave 9-methylene-9-ﬂuorene (160 mg, 36%), TLC

(single UV spot, R_f¼ 0:82, 20% ethyl acetate in hep-

tane), HPLC-MS (single main UV peak, 179.1

[MþH]^þ), followed by an unidentiﬁed fraction (345 mg),

followed by 25 (325 mg, 61%), TLC (single spot,

R_f¼ 0:10, 20% ethyl acetate in heptane); ¹H NMR

(400 MHz, CDCl₃): d 1.21 (s, C(CH₃)₃, 9H), 1.23 (d,

J ¼ 6:15 Hz, 4⁰-CH₃, 3H), 1.52 (s, 4-CH₃, 3H), 3.78 (q,

J ¼ 6:20 Hz, CHCH₃, 1H) and 6.39 (br s, NH, 1H); ¹³C

NMR (100 MHz, CDCl₃): d 18.8 (4⁰-CH₃), 22.3 (4-CH₃),

29.6 (C(CH₃)₃), 68.5 (HNCCH₃), 71.8 (OCH), 76.1

(OCCH₃), 153.2 (OCON) and 172.4 (C₂); HPLC-MS

(single main peak, 216.1 [MþH]^þand 453.1 [2MþNa]^þ);

Exact mass calcd for C₁₀H₁₇NO₄Na: 238.1050, found

238.1058, d þ3.35 ppm.

(m, 3-H, 1H), 4.25 (t, J ¼ 6:85 Hz, ﬂuorenyl CHCH₂O,

1H), 4.38–4.42 (m, ﬂuorenyl CHCH₂O, 2H), 4.67 (br,

OCH₂CHCH₂, 2H), 5.24 (dd, J ¼ 1:15 and 10.45 Hz,

OCH₂CHCH₂, 1H), 5.35 (m, OCH₂CHCH₂, 1H), 5.52

(s, NH, 1H), 5.87–5.95 (m, OCH₂CHCH₂, 1H), 7.31–

7.35 (m, 2 · ﬂuorenyl CH, 2H), 7.40–7.43 (m, 2 · ﬂu-

orenyl CH, 2H), 7.60 (d, J ¼ 7:40 Hz, 2 · ﬂuorenyl CH,

2H) and 7.78 (d, J ¼ 7:50 Hz, 2 · ﬂuorenyl CH, 2H);

analytical HPLC R_t¼ 18:42 min, HPLC-MS (single

main peak, 396.2 [MþH]^þ, 418.2 [MþNa]^þ). Anal.

Calcd for C₂₃H₂₅NO₅0.06EtOAc: C, 68.94; H, 6.54; N,

3.33. Found: C, 68.56; H, 6.24; N, 3.30; Exact mass

calcd for C₂₃H₂₆NO₅: 396.1805, found 396.1802, d

ꢁ0.10 ppm.

(c)

A stirred solution of (2R,3S)-2-(9H-ﬂuoren-9-

ylmethoxycarbonylamino)-3-hydroxy-2-methylbutyric acid

allyl ester 13c (312 mg, 0.79 mmol) in dichloromethane

(10 mL) was cooled in a pressure vessel to ꢁ78 ꢁC then

isobutylene gas (ꢂ8 mL) condensed into the solution.

Concentrated sulfuric acid (27 lL) was added then the

pressure vessel sealed. The mixture was stirred at

ambient temperature for 3 days then cooled to ꢁ78 ꢁC.

N-Methylmorpholine (56 lL) was added then the un-

sealed pressure vessel allowed to warm to ambient

temperature. The mixture was diluted with saturated

aqueous sodium hydrogen carbonate solution (100 mL)

then the product extracted into dichloromethane

(3 · 100 mL). The combined dichloromethane layers

were washed with brine (50 mL), dried (Na₂SO₄) and

evaporated in vacuo to give a residue. Flash chroma-

tography of the residue over silica gel (35 g) using ethyl

acetate/heptane (3:7) as the eluent gave (2R,3S)-3-tert-

butoxy-2-(9H-ﬂuoren-9-ylmethoxycarbonylamino)-2-

methyl butyric acid allyl ester 13d (310 mg, 87%), TLC

(single UV spot, R_f¼ 0:70, 50% ethyl acetate in hep-

tane); ¹H NMR (400 MHz, CDCl₃): d 1.16–1.25 (m,

CHCH₃and C(CH₃)₃, 12H), 1.62 (s, 2-CH₃, 3H), 3.95–

4.05 (m, 3-H, 1H), 4.23–4.43 (m, ﬂuorenyl CHCH₂O

and ﬂuorenyl CHCH₂O, 3H), 4.55–4.71 (m,

OCH₂CHCH₂, 2H), 5.21 (dd, J ¼ 1:20, 2.50 Hz,

4.1.14. (2R,3S)-3-tert-Butoxy-2-(9H-ﬂuoren-9-ylmeth-

oxycarbonylamino)-2-methyl butyric acid (13b).

(a) Amino acid 16 (288 mg, 2.17 mmol) was added to a

vigorously stirred solution of sodium carbonate (570 mg,

5.41 mmol) in water (12 mL) at 0 ꢁC. 1,4-Dioxan (6 mL)

was then added, providing an opaque mobile mixture. 9-

Fluorenylmethyl chloroformate (585 mg, 2.27 mmol) in

1,4-dioxan (6 mL) was then added over 40 min. The

mixture was then allowed to warm to ambient temper-

ature over 40 min. Water (100 mL) was then added, the

reaction mixture washed with chloroform (100 mL) and

the organic layer discarded. The aqueous phase was

acidiﬁed with 1 M HCl (ꢀpH 2), providing a thick

opaque mixture. The acidiﬁed aqueous mixture was

extracted with chloroform (5 · 80 mL) and the combined

organic phase dried (Na₂SO₄) and evaporated in vacuo

to give 13a (616 mg, 80%), TLC (single UV spot,

R_f¼ 0:24, 20% methanol in chloroform), analytical

HPLC R_t¼ 15:54 min, HPLC-MS (single main UV peak

with R_t¼ 7:70 min, 356.2 [MþH]^þ, 378.1 [MþNa]^þ).

OCH₂CHCH₂,

1H),

5.34

(d,

J ¼ 17:15 Hz,

OCH₂CHCH₂, 1H), 5.72 (s, NH, 1H), 5.86–5.95 (m,

OCH₂CHCH₂, 1H), 7.31–7.35 (m, 2 · ﬂuorenyl CH,

2H), 7.40–7.43 (m, 2 · ﬂuorenyl CH, 2H), 7.62–7.70 (m,

2 · ﬂuorenyl CH, 2H) and 7.78 (d, J ¼ 7:50 Hz, 2 · ﬂu-

orenyl CH, 2H); analytical HPLC R_t¼ 23:59 min,

HPLC-MS (single main peak, 474.1 [MþNa]^þ); Exact

mass calcd for C₂₇H₃₄NO₅: 452.2431, found 452.2425, d

ꢁ1.39 ppm.

(b) A solution of acid 13a (616 mg, 1.74 mmol) and tri-

caprylmethylammonium chloride (701 mg, 1.74 mmol)

in dichloromethane (2.6 mL) was added to a stirred

solution of sodium hydrogen carbonate (146 mg,

1.74 mmol) in water (2.6 mL), then allyl bromide

(0.15 mL, 1.74 mmol) was added in one portion. The

biphasic mixture was vigorously stirred for 5 days then

diluted with water (40 mL) and the product extracted

into dichloromethane (3 · 80 mL). The combined or-

ganic layers were dried (Na₂SO₄) and evaporated in

vacuo to give a residue (1.1 g). Flash chromatography of

the residue over silica gel (200 g) using ethyl acetate/

heptane (3:7) as the eluent gave (2R,3S)-2-(9H-ﬂuoren-

9-ylmethoxycarbonylamino)-3-hydroxy-2-methyl but-

yric acid allyl ester 13c (360 mg, 52%), TLC (single UV

spot, R_f¼ 0:48, 50% ethyl acetate in heptane); ¹H NMR

(400 MHz, CDCl₃): d 1.21 (d, J ¼ 6:20 Hz, CHCH₃,

3H), 1.52 (s, 2-CH₃, 3H), 3.38 (br s, OH, 1H), 3.47–4.16

(d) Tetrakistriphenylphosphine palladium(0) (17.25 mg,

0.015 mmol), dichloromethane (10 mL) then phen-

yltrihydrosilane (0.18 mL, 1.33 mmol) were added con-

secutively to (2R,3S)-3-tert-butoxy-2-(9H-ﬂuoren-9-

ylmethoxycarbonyl amino)-2-methyl butyric acid allyl

ester 13d (300 mg, 0.665 mmol) under nitrogen. The

mixture was stirred for 90 min then absorbed onto silica

gel (2 g). Flash chromatography of the absorbed residue

over silica gel (35 g) using ethyl acetate/heptane (2:3),

followed by (1:1) as the eluent gave 13b (140 mg, 51%),

TLC (single UV spot, R_f¼ 0:33, 50% ethyl acetate in

heptane); ¹H NMR (400 MHz, CDCl₃): d 1.19 (d,

2916

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

J ¼ 6:10 Hz, CHCH₃, 3H), 1.32 (s, C(CH₃)₃, 9H), 1.69

(s, 2-CH₃, 3H), 4.23 (t, J ¼ 7:05 Hz, ﬂuorenyl CHCH₂O,

1H), 4.37 (d, J ¼ 6:95 Hz, ﬂuorenyl CHCH₂O, 2H),

4.61–4.65 (m, 3-H, 1H), 6.12 (s, NH, 1H), 7.31–7.34 (m,

2 · ﬂuorenyl CH, 2H), 7.39–7.43 (m, 2 · ﬂuorenyl CH,

2H), 7.59–7.62 (m, 2 · ﬂuorenyl CH, 2H) and 7.77 (d,

J ¼ 7:50 Hz, 2 · ﬂuorenyl CH, 2H); ¹³C NMR

(100 MHz, CDCl₃): d 18.3 (CHCH₃), 21.3 (2-CH₃), 28.7

(C(CH₃)₃), 47.2 (CHCH₂O), 60.5 (C(CH₃)₃), 62.7 (C-2),

66.7 (CH₂O), 69.5 (C-3), 120.1, 125.2, 127.2, 127.8

(8 · ﬂuorenyl CH), 141.4, 143.8 (4 · ﬂuorenyl quaternary

C), 154.6 (OCON) and 174.8 (C-1); analytical HPLC

R_t¼ 20:20 min, HPLC-MS (single main peak, 434.1

[MþNa]^þ).

125.4, 125.7, 127.3, 128.0, 128.1 (8 · ﬂuorenyl CH),

141.4, 141.5, 143.0, 143.6 (4 · ﬂuorenyl quaternary C),

157.6 (OCON) and 178.8 (C-5); analytical HPLC

R_t¼ 21:15 min and HPLC-MS (single main peak with

R_t¼ 11:65 min, 434.2 [MþH₂OþNa]^þ). Anal. Calcd for

C₂₄H₂₇NO₄0.13DCM: C, 65.57; H, 6.33; N, 3.10.

Found: C, 65.78; H, 6.69; N, 3.16; Exact mass calcd for

C₂₇H₂₇NO₄NaÆH₂O: 434.1938, found 434.1948, d

þ2.22 ppm, followed by a mixed fraction containing 26,

10b [TLC UV spot, R_f¼ 0:75, 50% ethyl acetate in

heptane] and (2S,3R)-3-tert-butoxy-2-(9H-ﬂuoren-9-yl-

methoxycarbonylamino)-2-methyl butyric acid methyl

ester (35 mg).

4.1.17. (2S,3R)-(2,3-Dimethyl-4-oxo-tetrahydrofuran-3-

yl)carbamic acid 9H-ﬂuoren-9-ylmethyl ester (7). A

solution of lithium chloride (21.7 mg, 0.51 mmol) in

water (0.25 mL) and acetic acid (1 mL) was added to the

above mixed fractions containing 10b (35 mg). A gas

evolved and after 1 h, chloroform (40 mL) was added

and the organic phase washed with saturated aqueous

sodium hydrogen carbonate (20 mL). The chloroform

layer was dried (Na₂SO₄) and evaporated in vacuo to

give a residue (40 mg). Flash chromatography of the

residue over silica (20 g) using ethyl acetate/heptane

(1:4), followed by (1:1) as the eluent gave the methyl

ester of 7 (10 mg, 7.4% from 13b), TLC (single UV spot,

R_f¼ 0:73, 50% ethyl acetate in heptane); ¹H NMR

(400 MHz, CDCl₃): d 1.20–1.21 (br, CHCH₃and

C(CH₃)₃, 12H), 1.57 (s, 2-CH₃, 3H), 3.72 (br, OCH₃3H),

3.96 (br d, J ¼ 5:35 Hz, 3-H, 1H), 4.21–4.31 (m, ﬂu-

orenyl CHCH₂O and ﬂuorenyl CHCH₂O, 2H), 4.39–

4.44 (m, ﬂuorenyl CHCH₂O, 1H), 5.68 (s, NH, 1H),

7.30–7.34 (m, 2 · ﬂuorenyl CH, 2H), 7.39–7.42 (m,

2 · ﬂuorenyl CH, 2H), 7.60–7.65 (m, 2 · ﬂuorenyl CH,

2H) and 7.77 (d, J ¼ 7:55 Hz, 2 · ﬂuorenyl CH, 2H);

analytical HPLC R_t¼ 23:01 min, HPLC-MS (single

main peak, 448.2 [MþNa]^þ), followed by 7 (6 mg, 5.4%

from 13b), TLC (single UV spot, R_f¼ 0:57, ethyl ace-

4.1.15. (1R,2S)-(2-tert-Butoxy-1-ﬂuorocarbonyl-1-meth-

ylpropyl)-carbamic acid 9H-ﬂuoren-9-ylmethyl ester

(13e). Pyridine (47 lL, 0.55 mmol) then cyanuric ﬂuo-

ride (63 lL, 0.71 mmol) were added consecutively at 0 ꢁC

to a stirred solution of 13b (130 mg, 0.316 mmol) in di-

chloromethane (8 mL) under nitrogen. The suspension

was slowly warmed to ambient temperature and stirred

for 20 h. Crushed ice (ꢂ10 mL) and ice-chilled water

(10 mL) were added, then the product was extracted into

dichloromethane (40 mL). The dichloromethane layer

was dried (MgSO₄) and evaporated in vacuo to give 13e

(138 mg), TLC (single UV spot, R_f¼ 0:75, 50% ethyl

acetate in heptane), HPLC-MS (single main UV peak

with R_t¼ 11:66 min, 436.1 [MþNa]^þ).

4.1.16. (1R,1⁰S)-[1-(1-tert-Butoxyethyl)-3-diazo-1-meth-

yl-2-oxopropyl]carbamic acid 9H-ﬂuoren-9-ylmethyl ester

(10b). Ethereal diazomethane [generated from diazald^â

(ꢂ15 mmol) addition in diethyl ether (75 mL) to sodium

hydroxide (5.25 g) in water (7.5 mL)/ethanol (15 mL) at

60 ꢁC and dried over potassium hydroxide pellets] was

added to

a

stirred solution of 13e (138 mg,

ꢂ0.316 mmol) in dichloromethane (3 mL) at 0 ꢁC. The

mixture was then allowed to warm to ambient temper-

ature and stirred for 20 h. A few drops of acetic acid

were added to quench any excess diazomethane then the

solution was stirred for 5 min before adding tert-butyl

methyl ether (100 mL). The ethereal layer was washed

with saturated aqueous sodium hydrogen carbonate

(2 · 50 mL), dried (Na₂SO₄) and evaporated in vacuo to

give a residue. Flash chromatography of the residue

over silica gel (35 g) using ethyl acetate/heptane (1:19) as

the eluent gave 4-(1S-tert-butoxyethyl)-2-(9H-ﬂuoren-9-

ylmethoxy)-4R-methyl-4H-oxazol-5-one 26 (55 mg,

45%), TLC (single UV spot, R_f¼ 0:77, 50% ethyl acetate

in heptane); ¹H NMR (400 MHz, CDCl₃): d 1.13 (s,

C(CH₃)₃, 9H), 1.25 (d, J ¼ 6:10 Hz, CHCH₃, 3H), 1.35

(s, 4-CH₃, 3H), 3.81 (q, J ¼ 6:10 Hz, CHCH₃, 1H), 4.41

(dd, ﬂuorenyl CHCH₂O, 1H), 4.50 (m, ﬂuorenyl

CHCH₂O, 1H), 4.80 (dd, ﬂuorenyl CHCH₂O, 1H),

7.31–7.35 (m, 2 · ﬂuorenyl CH, 2H), 7.41–7.45 (m,

2 · ﬂuorenyl CH, 2H), 7.67 (d, J ¼ 7:50 Hz, 2 · ﬂuorenyl

CH, 2H) and 7.79 (d, J ¼ 7:55 Hz, 2 · ﬂuorenyl CH,

2H); ¹³C NMR (100 MHz, CDCl₃): d 17.3 (CHCH₃),

20.5 (4-CH₃), 28.8 (C(CH₃)₃), 46.3 (CHCH₂O), 71.1 (C-

4), 72.1 (CH₂O), 74.4 (CHCH₃), 75.6 (C(CH₃)₃), 120.2,

1

tate/heptane 1:1); H NMR (400 MHz, CDCl₃): d 1.12

(d, J ¼ 6:25 Hz, 2-CH₃, 3H), 1.50 (s, 3-CH₃, 3H), 4.05

(d, J ¼ 17:85 Hz, 5-H, 1H), 4.16–4.24 (m, 5-H and ﬂu-

orenyl CHCH₂O, 2H), 4.38–4.48 (m, CHCH₂O, 2H),

4.50–4.65 (br, 2-H, 1H), 4.84 (s, NH, 1H), 7.31–7.33 (m,

2 · ﬂuorenyl CH, 2H), 7.39–7.43 (m, 2 · ﬂuorenyl CH,

2H), 7.58 (d, J ¼ 7:40 Hz, 2 · ﬂuorenyl CH, 2H) and

7.78 (d, J ¼ 7:40 Hz, 2 · ﬂuorenyl CH, 2H); analytical

HPLC R_t¼ 18:22 min, HPLC-MS (single main UV peak

with R_t¼ 8:92 min, 352 [MþH]^þ, 374 [MþNa]^þ), fol-

lowed by recovered 13b (8 mg, 6.2%).

4.1.18. (2S)-2-tert-Butoxycarbonylamino-3-phenylprop-

ionic acid 2-oxo-cyclopentyl ester (29). (a) A solution

of cyclopentanone (11.6 mL, 130 mmol) in methanol

(250 mL) was added dropwise at 0 ꢁC over 20 min to a

stirred solution of potassium hydroxide (85% tech.,

22.1 g, 335 mmol) in methanol (75 mL). The mixture was

stirred at 0 ꢁC for 30 min then 2-iodosylbenzoic acid

(36.45 g, 138 mmol) was added in portions over 1 h. The

mixture was allowed to warm to ambient temperature

over 4 h then stirred at ambient temperature for 20 h.

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

2917

The majority of solvent was removed in vacuo then the

product was extracted into dichloromethane (400 mL).

The extracts were washed with water (2 · 250 mL), dried

(Na₂SO₄) and the solvent removed in vacuo to leave 2,2-

dimethoxycyclopentanol 27 as a colourless oil (11.98 g)

(lit.¹⁶).

hydrogen carbonate solution (750 mL). The mixture was

diluted with water (750 mL), extracted with diethyl ether

(3 · 500 mL), washed with a saturated aqueous sodium

chloride solution (350 mL), dried (MgSO₄) and the sol-

vents removed in vacuo to give a brown oil (10.05 g).

Flash chromatography of the residue over silica using

ethyl acetate/heptane (1:4), followed by (2:3) as the

eluent gave 30 as a white solid (4.54 g, 36%). TLC (single

(b) 4-(Dimethylamino)pyridine (1.0 g, 8.2 mmol) was

added at 0 ꢁC to a stirred suspension of 2,2-dimeth-

oxycyclopentanol 27 (11.98 g, 82 mmol), (S)-2-tert-but-

yloxycarbonylamino-3-phenylpropionic acid (23.9 g,

90.3 mmol) and 1-[3-(dimethylamino)propyl]-3-ethyl-

carbodiimide hydrochloride (23.6 g, 123.1 mmol) in di-

chloromethane (500 mL). The mixture was stirred at

0 ꢁC for 4 h, then it was washed with water (2 · 300 mL)

and a saturated aqueous sodium chloride solution

(200 mL), dried (Na₂SO₄) and the solvent removed in

vacuo to give 28 as a yellow oil (36.0 g) (lit.¹⁶).

1

UV spot, R_f¼ 0:45, 25% ethyl acetate in heptane); H

NMR (400 MHz, CDCl₃): d 1.70–2.29 (m, (CH₂)₃and

NH, 7H), 2.84 (dd, J ¼ 9:85 and 14.30 Hz, CH₂Ph, 1H),

3.52 (dd, J ¼ 3:60 and 14.35 Hz, CH₂Ph, 1H), 3.90 (dd,

J ¼ 3:65 and 9.80 Hz, CHNH, 1H), 4.74 (dd, J ¼ 6:50

and 6.80 Hz, CHO, 1H), 7.15–7.32 (m, C₆H₅, 5H);

analytical HPLC R_t¼ 14:521 min, HPLC-MS (single

main peak, 257.2 [MþH]^þ, 279 [MþNa]^þ) (lit.¹⁶).

4.1.20. (1R,2S)-1-Amino-2-hydroxycyclopentanecarboxy-

lic acid (17b). (a) tert-Butylhypochlorite (4.0 mL,

35.4 mmol) was added dropwise under nitrogen at 0 ꢁC

over 2 min to a stirred suspension of 30 (4.53 g,

17.7 mmol) in diethyl ether (350 mL). The mixture was

stirred at 0 ꢁC for 140 min then triethylamine (7.4 mL,

53 mmol) was added dropwise over 30 min. The resulting

suspension was stirred at 0 ꢁC for 3 h then at ambient

temperature for 23 h. Insoluble materials were removed

by ﬁltration and then the ﬁltrate was concentrated in

vacuo to give a residue. Flash chromatography of the

residue over silica using ethyl acetate/heptane (3:7) as

the eluent gave 31 (3.3 g), TLC (single UV spot,

R_f¼ 0:3, 30% ethyl acetate in heptane); analytical

HPLC R_t¼ 16:07 min.

(c) para-Toluenesulfonic acid monohydrate (1.7 g,

9.2 mmol) was added to a stirred solution of 28 (36 g,

91.6 mmol) in wet acetone (450 mL) at ambient tem-

perature. The solution was stirred for 3 days then water

(600 mL) and saturated aqueous sodium hydrogen car-

bonate solution (200 mL) were added, then the product

was extracted into ethyl acetate (600 mL). The aqueous

phase was extracted with ethyl acetate (2 · 400 mL) and

the combined ethyl acetate solutions were washed with a

saturated

aqueous

sodium

chloride

solution

(2 · 150 mL), dried (Na₂SO₄) and the solvent removed in

vacuo to give a residue (18.05 g). Flash chromatography

of the residue over silica using ethyl acetate/heptane

(2:1), followed by (3:1) as the eluent gave 29 as a col-

ourless oil (18.05 g, 40% from cyclopentanone), TLC

(single UV spot, R_f¼ 0:25, 30% ethyl acetate in hep-

(b) Concentrated hydrochloric acid (190 mL) was added

to 31 (3.3 g) at 0 ꢁC. The suspension was allowed to

warm to ambient temperature over 2 h then stirred for a

further 20 h. The reaction mixture was partitioned

equally between six pressure vessels that were sealed

then heated at 100 ꢁC for 26 h then allowed to cool to

ambient temperature. The reaction mixtures were

recombined then washed with diethyl ether (2 · 200 mL)

and the aqueous phase concentrated in vacuo to leave a

residue. Chromatography of the residue over Dowex^â

50W · 4-200 ion-exchange resin using 0.01 M hydro-

chloric acid, water and then 1.0 M aqueous ammonium

hydroxide solution gave 17b after freeze drying, as a

light brown solid (1.73 g, 67% from 30); ¹H NMR

(400 MHz, D₂O): d 1.50–1.90 (m, (CH₂)₂, 4H), 2.16–2.25

(m, CH₂, 2H), 4.36 (t, J ¼ 7:65 Hz, CHOH, 1H); ¹³C

NMR (100 MHz, D₂O): d 21.6 (CH₂CH₂CH₂), 33.5 and

35.0 (CH₂CH₂CH₂, 69.7 (CNH₂), 77.5 (CHOH), 178.3

(CH₂H); HPLC-MS (single main peak, 146.1 [MþH]^þ)

(lit.¹⁶).

1

tane); H NMR (400 MHz, CDCl₃): d 1.42 (s, 3 · CH₃,

9H), 1.79–2.48 (m, (CH₂)₃, 6H), 3.06–3.28 (m, CH₂Ph,

2H), 4.60–5.20 (m, CHO, CHN and NH, 3H), 7.17–7.36

(m, C₆H₅, 5H); analytical HPLC with main broad peak

R_t¼ 17:9–19:2 min, HPLC-MS (main broad peak, 248.1

[MꢁBocþ2H]^þ, 370.2 [MþNa]^þ, 717.3 [2MþNa]^þ)

(lit.¹⁶).

4.1.19.

(3S,4aR,7aS)-3-Benzyl-2-oxo-hexahydrocyclo-

penta[1,4]oxazine-4a-carbonitrile (30). Triﬂuoroacetic

acid (75 mL) was added dropwise at 0 ꢁC over 1 h to a

stirred solution of 29 (17.05 g, 49.1 mmol) in dichlo-

romethane (250 mL). The mixture was stirred at 0 ꢁC for

75 min then the majority of solvent was removed in

vacuo. Toluene (75 mL) was added to the residue then

the solvent was removed in vacuo to give an oil, which

was dissolved in acetonitrile (700 mL). Magnesium sul-

fate (29.5 g) and then sodium acetate (20.1 g) were added

to the stirred solution. The resulting suspension was

stirred for 1.5 h then the solids were removed by ﬁltra-

tion and the solvents removed in vacuo to give a residue.

The residue was dissolved in propan-2-ol (650 mL) then

stirred under nitrogen. Trimethylsilyl cyanide (13.1 mL,

98.4 mmol) was added dropwise over 15 min and then

zinc chloride (49 mL, 1 M solution in diethyl ether) was

added over 40 min. The mixture was stirred for 18 h then

cautiously added to a saturated aqueous sodium

4.1.21.

(1R,2S)-1-(9H-Fluoren-9-ylmethoxycarbonyl-

amino)-2-hydroxycyclopentanecarboxylic acid allyl ester

(14c). (a) To a stirred solution of sodium carbonate

(1.0 g, 9.7 mmol) in water/1,4-dioxan (2:1, 45 mL) was

added 17b (0.67 g, 4.6 mmol) at 0 ꢁC. A solution of 9-

ﬂuorenylmethyl chloroformate (1.25 g, 4.85 mmol) in

1,4-dioxan (15 mL) was added dropwise over 30 min.

2918

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

The resultant suspension was stirred for 75 min at 0 ꢁC

then at ambient temperature for 45 min. Water (200 mL)

was added then the cloudy solution washed with chlo-

roform (2 · 140 mL). Chloroform (100 mL) was added

and the mixture acidiﬁed with 1 M hydrochloric acid

(pH ꢀ 2). The chloroform layer was separated then the

(OCH₂CHCH₂), 141.7, 144.2, 144.3 (4 · ﬂuorenyl qua-

ternary C), 156.7 (OCON), 173.6 (CH₂CH₂CHCH₂);

analytical HPLC R_t¼ 20:37 min (major) and

R_t¼ 19:71 min (minor) and HPLC-MS (main peak,

408.1 [MþH]^þ, 430.1 [MþNa]^þ; minor peak, 408.1

[MþH]^þ, 430.1 [MþNa]^þ). Anal. Calcd for C₂₄H₂₅NO₅

0.15DCM: C, 62.25; H, 5.61; N, 2.92. Found: C, 62.08;

H, 5.60; N, 3.02; Exact mass calcd for C₂₄H₂₆NO₅:

408.1805, found 408.1807, d þ0.03 ppm.

aqueous

layer

re-extracted

with

chloroform

(2 · 100 mL). The chloroform extracts (obtained from

the acidiﬁed aqueous layer) were combined then dried

(Na₂SO₄) and the solvent removed in vacuo to give a

colourless oil to which heptane (100 mL) was added

before storing at ꢁ80 ꢁC for 16 h. The solvent was rap-

idly decanted from the oily residue, which was washed

with heptane (5 mL) then remaining solvents removed in

vacuo to give (1R,2S)-1-(9H-ﬂuoren-9-ylmethoxycar-

bonylamino)-2-hydroxycyclopentanecarboxylic acid as

an oil (1.27 g, 75%), TLC (main UV spot, R_f¼ 0:20,

together with minor UV spot, R_f¼ 0:15, 20% MeOH in

4.1.22.

(1R,2R)-1-(9H-Fluoren-9-ylmethoxycarbonyl-

amino)-2-hydroxycyclopentanecarboxylic acid allyl ester

(14d). (a) A solution of dimethyl sulfoxide (0.224 mL,

3.15 mmol) in dichloromethane (1.0 mL) was added

under nitrogen to a stirred solution of oxalyl chloride

(0.132 mL, 1.51 mmol) in dichloromethane (2.5 mL) at

ꢁ70 ꢁC over 20 min. The mixture was stirred for 10 min

then a solution of 14c (0.535 g, 1.31 mmol) in dichlo-

romethane (3 mL) added over 20 min. The mixture was

stirred for 10 min then triethylamine (0.92 mL,

6.57 mmol) added dropwise over 5 min. The cooling

bath was removed and stirring continued for 45 min at

ambient temperature. A saturated aqueous ammonium

chloride solution (50 mL) was added then the product

extracted into diethyl ether (2 · 50 mL). The combined

ethereal layers were washed with water (25 mL), dried

(MgSO₄) and the solvent was removed in vacuo to give a

residue (520 mg). Flash chromatography of the residue

over silica using ethyl acetate/heptane (1:3) as the eluent

gave (1R)-1-(9H-ﬂuoren-9-ylmethoxycarbonylamino)-2-

oxocyclopentanecarboxylic acid allyl ester as a colour-

less oil (0.43 g, 81%), TLC (single UV spot, R_f¼ 0:30,

30% ethyl acetate in heptane); ¹H NMR (400 MHz,

CDCl₃): d 2.15–2.23 (m, CH₂CH₂CH₂, 2H), 2.46–2.70

(m, CH₂CH₂CH₂, 4H), 4.21 (t, J ¼ 7:15 Hz, CHCH₂O,

1H), 4.35 (d, J ¼ 7:10 Hz, CHCH₂O, 2H), 4.66 (br s,

OCH₂CHCH₂, 2H), 5.26–5.35 (m, OCH₂CHCH₂, 2H),

5.80–5.91 (m, OCH₂CHCH₂, 1H), 6.14 (br s, NH, 1H),

7.27–7.35 (m, Fmoc H-2 and H-7, 2H), 7.36–7.41 (m,

Fmoc H-3 and H-6, 2H), 7.54–7.60 (Fmoc H-1 and H-8,

2H aromatic), 7.74–7.77 (Fmoc H-4 and H-5, 2H aro-

1

CHCl₃); H NMR (400 MHz, CDCl₃): d 1.60–2.16 (m,

CH₂CH₂CH₂, 5H), 2.35 (m, CH₂CH₂CH₂, 1H), 4.10 (br

s, OH, 1H), 4.24 (m, ﬂuorenyl CHCH₂O, 1H), 4.36–4.57

(m, ﬂuorenyl CHCH₂O and CHOH, 3H), 5.93 (s, NH,

1H), 7.28–7.33 (m, 2 · ﬂuorenyl CH, 2H), 7.34–7.41 (m,

2 · ﬂuorenyl CH, 2H), 7.54–7.62 (m, 2 · ﬂuorenyl CH,

2H), 7.72–7.79 (m, 2 · ﬂuorenyl CH, 2H), analytical

HPLC R_t¼ 17:17 min (major), R_t¼ 16:80 min (minor)

and HPLC-MS (main UV peak with R_t¼ 7:84 min,

368.1 [MþH]^þ, 390.1 [MþNa]^þ, minor UV peak with

R_t¼ 7:646 min, 368.1 [MþH]^þ, 390.1 [MþNa]^þ). Anal.

Calcd for C₂₁H₂₁NO₅0.25EtOAc: C, 65.11; H, 6.61; N,

2.86. Found: C, 64.72; H, 6.28; N, 3.22; Exact mass

calcd for C₂₁H₂₂NO₅: 368.1492, found 368.1509, d

þ4.62 ppm.

(b) A solution of (1R,2S)-1-(9H-ﬂuoren-9-ylmethoxy-

carbonylamino)-2-hydroxy cyclopentanecarboxylic acid

(1.75 g, 4.8 mmol, prepared as above) and tricaprylme-

thylammonium chloride (1.93 g, 4.8 mmol) in dichlo-

romethane (14 mL) was added to a stirred solution of

sodium hydrogen carbonate (0.4 g, 4.8 mmol) in water

(14 mL). Allyl bromide (1.44 mL, 16.7 mmol) was added

and the biphasic mixture stirred for 20 h then diluted

with water (50 mL). The mixture was extracted with

dichloromethane (2 · 50 mL), dried (MgSO₄) and the

solvents removed in vacuo to give a residue. Flash

chromatography of the residue over silica using ethyl

acetate/heptane (3:10), followed by (7:20) as the eluent

gave 14c as a colourless oil (1.32 g, 68%), TLC (single

matic); ¹³C NMR (100 MHz, CDCl₃):

d

19.2

(CH₂CH₂CH₂), 34.5 and 36.9 (CH₂CH₂CH₂), 47.4

(Fmoc C-9), 67.4 and 67.7 (Fmoc CH₂and

OCH₂CHCH₂),

67.85

(CCO₂CH₂),

119.9

(OCH₂CHCH₂), 120.4, 125.5, 127.5, 128.2 (8 · ﬂuorenyl

CH), 131.1 (OCH₂CHCH₂), 141.7, 144.0, 144.2

(4 · ﬂuorenyl quaternary C), 155.25 (OCON), 169.25

(C₂CH₂CHCH₂), 211.3 (CCOCH₂CH₂); analytical

HPLC with main peak R_t¼ 19:99 min, HPLC-MS

(single UV peak with R_t¼ 10:13 min, 406.1 [MþH]^þ,

428.1 [MþNa]^þ).

1

UV spot, R_f¼ 0:25, 25% ethyl acetate in heptane); H

NMR (400 MHz, CDCl₃): d 1.60–2.63 (m, CH₂CH₂CH₂

and OH, 7H), 4.8–4.27 (m, CHCH₂O, 1H), 4.29–

4.48 (m, CHCH₂O and CHOH, 3H), 4.57–4.66

(br s, OCH₂CHCH₂, 2H), 5.22 (dd, J ¼ 1:05 and

10.45 Hz, OCH₂CHCH₂, 1H), 5.29 (d, J ¼ 9:65 Hz,

OCH₂CHCH₂, 1H), 5.77 (br s, NH, 1H), 5.82–5.94 (m,

OCH₂CHCH₂, 1H), 7.27–7.32 (m, 2 · ﬂuorenyl CH,

2H), 7.36–7.41 (m, 2 · ﬂuorenyl CH, 2H), 7.55–7.62 (m,

2 · ﬂuorenyl CH, 2H), 7.74–7.77 (m, 2 · ﬂuorenyl CH,

(b) Sodium borohydride (39 mg, 1.04 mmol) was added

in one portion to a stirred solution of (1R)-1-(9H-ﬂuo-

ren-9-ylmethoxycarbonylamino)-2-oxocyclopentanecarb-

oxylic acid allyl ester (0.42 g, 1.04 mmol) in methanol

(6 mL) at 0 ꢁC. The mixture was stirred for 10 min then

the solvents removed in vacuo to give a residue. Water

(20 mL) and dichloromethane (20 mL) were added and

the mixture was then acidiﬁed with 1 M hydrochloric

acid (pH ꢀ 1.5). The dichloromethane layer was col-

2H); ¹³C NMR (100 MHz, CDCl₃):

d

20.4

(CH₂CH₂CH₂), 32.5, 32.3 and 34.2 (C₂CH₂CH₂), 47.6,

47.5 (CH₂CHO), 66.5 (CH₂CHO), 67.3 (OC₂CHCH₂),

76.0 (CCO₂CH₂), 77.8 (CHOH), 119.0 (OCH₂CHCH₂),

120.4, 125.5, 127.5, 128.1 (8 · ﬂuorenyl CH), 132.1

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

2919

lected then the aqueous layer extracted with dichlo-

romethane (20 mL). The combined dichloromethane

layers were washed with aqueous saturated sodium

chloride solution (20 mL). The aqueous saturated so-

dium chloride solution was extracted with dichlorome-

thane (10 mL) then the dichloromethane layers were

combined then dried (MgSO₄) and the solvent removed

in vacuo to give a residue (420 mg). Flash chromatog-

raphy of the residue over silica using ethyl acetate/hep-

tane (3:7), followed by (7:13) as the eluent gave 14d as a

colourless oil (288 mg, 68%), TLC (single UV spot,

R_f¼ 0:25, 25% ethyl acetate in heptane), analytical

HPLC R_t¼ 19:68 min (major), R_t¼ 20:32 min (minor)

and HPLC-MS (main UV peak with R_t¼ 9:08 min,

408.1 [MþH]^þ, 430.0 [MþNa]^þ; minor UV peak with

R_t¼ 9:451 min, 408.1 [MþH]^þ, 430.0 [MþNa]^þ). para-

Toluenesulfonic acid monohydrate (30 mg, 0.16 mmol)

was added to a solution of the oil (230 mg) in toluene

(12 mL) then the mixture heated at 100 ꢁC for 75 min.

The solvents were removed in vacuo to give a residue.

Flash chromatography as above gave 14d as a colourless

oil (200 mg, 87%). TLC (single UV spot, R_f¼ 0:25, 25%

layer was extracted with chloroform (20 mL) then the

combined organic layers were washed with hydrochloric

acid (50 mL, 0.1 M) and aqueous saturated sodium

chloride solution then dried (MgSO₄) and the solvent

removed in vacuo to give a residue (860 mg). Flash

chromatography of the residue over silica using ethyl

acetate/heptane (1:4), followed by (1:1) as the eluent

gave

(1R,2R)-1-(9H-ﬂuoren-9-ylmethoxycarbonyla-

mino)-2-hydroxycyclopentanecarboxylic acid allyl ester

(14d) (100 mg, 9%) and (3aR,6aS)-2-oxo-hexahydrocy-

clopentaoxazole-3a-carboxylic acid allyl ester 32

(270 mg, 49%) as a colourless oil. TLC (single non-UV

1

active spot, R_f¼ 0:1, 25% ethyl acetate in heptane); H

NMR (400 MHz, CDCl₃):

d

1.88–2.20 (m,

CH₂CH₂CH₂, 6H), 4.67–4.95 (m, CH₂O, 2H), 5.09–5.13

(m, H-6a, 1H), 5.30 (dd, J ¼ 10:40, 1.10 Hz,

CH₂CH@CH₂, 1H), 5.34 (dd, J ¼ 17:20, 1.40 Hz,

CH₂CH@CH₂, 1H), 5.81–5.98 (m, CH₂CH@CH₂, 1H),

6.30 (br s, NH, 1H); ¹³C NMR (100 MHz, CDCl₃): d

23.9 (CH₂CH₂CH₂), 34.7 and 39.5 (CH₂CH₂CH₂), 67.0

(CHCH₂O), 70.4 (C-3a), 85.6 (C-6a), 119.9

(CH₂@CHCH₂), 131.4 (CH₂@CHCH₂), 158.8 (C-2),

172.2 (CH₂CH₂CH@CH₂); HPLC-MS (single non-UV

active peak with R_t¼ 4:9 min, 212.1 [MþH]^þ, 423.2

[2MþH]^þ, 445.2 [2MþNa]^þ). Anal. Calcd for

C₁₀H₁₃NO₄: C, 56.86; H, 6.20; N, 6.63. Found: C, 56.80;

H, 6.26; N, 6.54; Exact mass calcd for C₁₀H₁₃NO₄Na:

234.0737, found 234.0748, d þ4.82 ppm.

1

ethyl acetate in heptane); H NMR (400 MHz, CDCl₃):

d 1.72–2.44 (m, CH₂CH₂CH₂, 6H), 4.12–4.19 (m,

CHOH, 1H), 4.27 (t, J ¼ 6:55 Hz, ﬂuorenyl CHCH₂O,

1H), 4.37–4.55 (m, ﬂuorenyl CHCH₂O and OH, 3H),

4.60–4.75 (br s, OCH₂CHCH₂, 2H), 5.25 (d,

J ¼ 10:45 Hz, OCH₂CHCH₂, 1H), 5.30–5.39 (m,

OCH₂CHCH₂and NH, 2H), 5.83–5.96 (m,

OCH₂CHCH₂, 1H), 7.29–7.35 (Fmoc H-2 and H-7, 2H),

7.39–7.44 (Fmoc H-3 and H-6, 2H), 7.58–7.65 (Fmoc H-

1 and H-8, 2H), 7.77–7.80 (Fmoc H-4 and H-5, 2H); ¹³C

NMR (100 MHz, CDCl₃): d 20.9 (CH₂CH₂CH₂), 32.5

and 35.6 (CH₂CH₂CH₂), 47.5 (Fmoc C-9), 66.55 and

67.4 (CHCH₂O and OC₂CHCH₂), 69.4 (CCO₂CH₂),

80.5 (CHOH), 119.0 (OC₂CHCH₂), 120.4 (Fmoc C-4

and C-5), 125.4 (Fmoc C-1 and C-8), 127.5, 127.45

(Fmoc C-2 and C-7), 128.1, 128.2 (Fmoc C-3 and C-6),

132.1 (OCH₂CHCH₂), 141.7 (Fmoc C-4⁰and C-5⁰),

144.0, 144.2 (Fmoc C-1⁰and C-8⁰), 156.9 (OCON), 173.0

(CH₂CH₂CHCH₂); analytical HPLC single UV peak

with R_t¼ 18:14 min, HPLC-MS (single UV peak with

R_t¼ 9:14 min, 408.1 [MþH]^þ, 430.1 [MþNa]^þ). Anal.

Calcd for C₂₄H₂₅NO₅0.17DCM: C, 61.12; H, 5.53; N,

2.86. Found: C, 61.10; H, 5.52; N, 3.20; Exact mass

calcd for C₂₄H₂₆NO₅: 408.1805, found 408.1823, d

þ4.31 ppm.

4.1.24. (1R,2R)-2-tert-Butoxy-1-(9H-ﬂuoren-9-ylmeth-

oxycarbonylamino)cyclopentanecarboxylic acid allyl ester

(14e) . A stirred solution of (1R,2R)-1-(9H-ﬂuoren-9-yl-

methoxycarbonylamino)-2-hydroxycyclopentanecarbox-

ylic acid allyl ester 14d (360 mg, 0.88 mmol) in

dichloromethane (5 mL) was cooled in a pressure vessel

to ꢁ70 ꢁC then isobutylene gas (ꢂ3 mL) condensed into

the solution. Concentrated sulfuric acid (25 lL) was

added then the pressure vessel sealed. The mixture was

stirred at ambient temperature for 20 h then cooled to

ꢁ70 ꢁC. N-Methylmorpholine (50 lL) was added then

the unsealed pressure vessel allowed to warm to ambient

temperature. The mixture was diluted with saturated

aqueous sodium hydrogen carbonate solution (50 mL)

and water (25 mL) then the product extracted into di-

chloromethane (50 mL then 2 · 25 mL). The combined

dichloromethane layers were washed with saturated

aqueous sodium chloride solution (25 mL), dried

(Na₂SO₄) and the solvent removed in vacuo. The residue

(370 mg) was puriﬁed by ﬂash chromatography over

silica gel eluting with a gradient of heptane/ethyl acetate

4:1 to 7:3. Appropriate fractions were combined and the

solvents removed in vacuo to leave 14e as a colourless

oil (305 mg, 75%). TLC (single UV spot, R_f¼ 0:50,

4.1.23. (3aR,6aS)-2-Oxo-hexahydrocyclopentaoxazole-

3a-carboxylic acid allyl ester (32). para-Toluenesulfonic

acid monohydrate (0.49 g, 2.59 mmol) was added to a

stirred solution of (1R,2S)-1-(9H-ﬂuoren-9-ylmethoxy-

carbonylamino)-2-hydroxycyclopentanecarboxylic acid

(0.95 g, 2.59 mmol) and allyl alcohol (3 mL, 44.2 mmol)

in toluene (10 mL). The solution was heated at reﬂux in

a Dean and Stark apparatus for 15 min after which time

an additional amount of allyl alcohol (3 mL, 44.2 mmol)

was added. Heating at reﬂux was continued for 45 min

then the mixture allowed to cool to ambient tempera-

ture. The products were extracted into chloroform

(50 mL) then washed with saturated aqueous sodium

hydrogen carbonate solution (50 mL). The aqueous

heptane/ethyl

acetate

2:1),

analytical

HPLC

R_t¼ 22:623 min, HPLC-MS (single UV peak with

t

R_t¼ 11:611 min,

408.1

[Mꢁ Buþ2H]^þ,

486.1

[MþNa]^þ). ¹H NMR (400 MHz, CDCl₃): d 1.06 (s,

C(CH₃)₃, 9H), 1.80–2.41 (m, CH₂CH₂CH₂, 6H), 4.21 (t,

J ¼ 6:80 Hz, Fmoc H-9, 1H), 4.26–4.50 (m, Fmoc CH₂

and H-2, 3H), 4.58–4.74 (br s, OCH₂CHCH₂, 1H), 5.21

(d, J ¼ 10:40 Hz, OCH₂CHCH₂, 1H), 5.35 (d,

J ¼ 17:20 Hz, OCH₂CHCH₂, 1H), 5.84–6.01 (m,

2920

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

CH₂CH@CH₂and NH, 2H), 7.26–7.30 (Fmoc H-2 and

H-7, 2H), 7.37–7.39 (Fmoc H-3 and H-6, 2H), 7.57–7.61

(Fmoc H-1 and H-8, 2H), 7.70–7.77 (Fmoc H-4 and

H-5, 2H); ¹³C NMR (100 MHz, CDCl₃): d 21.7,

21.5 (CH₂CH₂CH₂), 33.0 and 33.8 (CH₂CH₂CH₂),

47.6 (Fmoc C-9), 66.5 and 66.9 (Fmoc CH₂

and OCH₂CHCH₂), 70.1 and 74.0 (CCO₂CH₂

ꢁ15 ꢁC. The mixture was then allowed to warm to

ambient temperature and stirred for 24 h. Acetic acid

(0.4 mL) was added to the mixture followed by tert-butyl

methyl ether (25 mL). The organic layer was washed

with water (3 · 10 mL), dried (Na₂SO₄) and the solvent

removed in vacuo. The residue (53 mg) was puriﬁed by

ﬂash chromatography over silica gel eluting with a

gradient of heptane/ethyl acetate 7:3 to 2:1. Appropriate

fractions were combined and the solvents removed in

vacuo to leave (1R,2R)-(7-oxo-6-oxabicyclo[3.2.0]hept-

yl) carbamic acid 9H-ﬂuoren-9-ylmethyl ester (33) as a

white solid (23 mg, 41%). Analytical HPLC R_t¼

19:891 min, HPLC-MS (main UV peak with R_t¼

9:349 min, 372.0 [MþNa]^þ.

and

OC(CH₃)₃),

79.5

(CHOC(CH₃)₃),

118.5

(OCH₂CHCH₂), 120.4 (Fmoc C-4 and C-5), 125.5

(Fmoc C-1 and C-8), 127.5 (Fmoc C-2 and C-7), 128.1

(Fmoc C-3 and C-6), 132.4 (OCH₂CHCH₂), 141.7

(Fmoc C-4⁰and C-5⁰), 144.3 (Fmoc C-1⁰and C-8⁰), 155.1

(OCON), 173.0 (CO₂CH₂CHCH₂). Anal. Calcd for

CH₂₈H₃₃NO₅0.07DCM: C, 68.46; H, 6.84; N, 2.81.

Found: C, 68.90; H, 7.17; N, 2.86; Exact mass calcd for

C₂₈H₃₄NO₅: 486.2251, found 486.2254, d þ0.06 ppm.

4.1.27. (1R,2R)-2-tert-Butoxy-1-(9H-ﬂuoren-9-ylmeth-

oxycarbonylamino)cyclopentanecarboxylic acid (14b).

4.1.25.

amino)-2-hydroxycyclopentanecarboxylic acid (14a).

Tetrakistriphenylphosphine palladium(0) (5 mg,

(1R,2R)-1-(9H-Fluoren-9-ylmethoxycarbonyl-

Tetrakistriphenylphosphine

palladium(0)

(15 mg,

0.013 mmol), dichloromethane (5 mL) then phen-

yltrihydrosilane (153 lL, 1.24 mmol) were added con-

secutively to 14e (288 mg, 0.62 mmol) under nitrogen.

The mixture was stirred for 45 min then 0.01 M hydro-

chloric acid (30 mL) added and the product extracted

into chloroform (1 · 20 mL then 1 · 10 mL). The com-

bined chloroform layers were dried (Na₂SO₄) and the

solvent removed in vacuo. The residue (460 mg) was

puriﬁed by ﬂash chromatography over silica gel eluting

with a gradient of heptane/ethyl acetate 2:1 to 1:3.

Appropriate fractions were combined and the solvents

removed in vacuo to leave 14b as a colourless oil

(205 mg, 78%). TLC (single UV spot, R_f¼ 0:25,

0.004 mmol), dichloromethane (2 mL) then phen-

yltrihydrosilane (55 lL, 0.44 mmol) were added consec-

utively to 14d (90 mg, 0.22 mmol) under nitrogen. The

mixture was stirred for 90 min then diluted with chlo-

roform (5 mL) and then extracted with aqueous satu-

rated sodium hydrogen carbonate solution (5 mL). The

aqueous layer was then extracted with chloroform

(3 · 5 mL), then chloroform (5 mL) added and the bi-

phasic mixture acidiﬁed to pH ¼ 1 using 1 M hydro-

chloric acid. The chloroform layer was separated then

the aqueous layer re-extracted with chloroform

(2 · 5 mL). The combined chloroform extracts (obtained

from the acidiﬁed aqueous layer) were dried (Na₂SO₄)

and the solvent removed in vacuo to leave the product

(14a) as an oil (40 mg). An additional 25 mg of 14a was

obtained by extracting initial chloroform washings with

aqueous saturated sodium hydrogen carbonate solution

(5 mL), which was then washed with chloroform

(2 · 5 mL) before acidifying to pH ¼ 1 using 1 M

hydrochloric acid and extracting the product into chlo-

roform (3 · 5 mL) and drying with (Na₂SO₄). The two

extracts were combined then used without further

puriﬁcation (80% total yield). Analytical HPLC

R_t¼ 16:786 min, HPLC-MS (main UV peak with

R_t¼ 7:760 min, 368.1 [MþH]^þ, 390.0 [MþNa]^þ, 757.1

[2MþNa]^þ).

heptane/ethyl

acetate

1:2),

analytical

HPLC

R_t¼ 19:539 min, HPLC-MS (single UV peak with

t

R_t¼ 9:850 min, 368.1 [Mꢁ Buþ2H]^þ, 446.1 [MþNa]^þ).

¹H NMR (400 MHz, CDCl₃): d 1.22 (s, C(CH₃)₃, 9H),

1.77–2.37 (m, CH₂CH₂CH₂, 6H), 4.22 (t, J ¼ 6:80 Hz,

Fmoc H-9, 1H), 4.27–4.36 (m, Fmoc CH₂, 2H), 4.65 (br

s, H-2, 1H), 5.39 (br s, NH, 1H), 7.27–7.35 (Fmoc H-2

and H-7, 2H), 7.36–7.41 (Fmoc H-3 and H-6, 2H), 7.56–

7.61 (Fmoc H-1 and H-8, 2H), 7.73–7.76 (Fmoc H-4 and

H-5, 2H); ¹³C NMR (100 MHz, CDCl₃): d 21.5

(CH₂CH₂CH₂), 28.8 (C(CH₃)₃), 33.7 and 35.3

(C₂CH₂CH₂), 47.6 (Fmoc C-9), 67.2 (Fmoc CH₂), 68.8

(CCO₂CH₂), 78.6 (CHOC(CH₃)₃), 120.4 (Fmoc C-4 and

C-5), 125.5 (Fmoc C-1 and C-8), 127.5 (Fmoc C-2 and

C-7), 128.1, 128.1 (Fmoc C-3 and C-6), 141.7, 141.7

(Fmoc C-4⁰and C-5⁰), 144.0, 144.4 (Fmoc C-1⁰and C-

8⁰), 156.1 (OCON), 174.9 (C₂H). Anal. Calcd for

C₂₅H₂₉NO₅0.05EtOAc: C, 70.08; H, 7.01; N, 3.14.

Found: C, 69.89; H, 6.88; N, 3.08; Exact mass calcd for

C₂₅H₂₉NO₅Na: 446.1938, found 446.1960, d þ4.84 ppm.

4.1.26. Attempted preparation of diazoketone 11a: for-

mation of (1R,2R)-(7-oxo-6-oxabicyclo[3.2.0]heptyl)car-

bamic acid 9H-ﬂuoren-9-ylmethyl ester (33). N-

Methylmorpholine (36 lL, 0.33 mmol) and iso-butyl-

chloroformate (23 lL, 0.167 mmol) in dichloromethane

(0.5 mL) were added simultaneously in portions over

15 min to a stirred solution of (1R,2R)-1-(9H-ﬂuoren-9-

ylmethoxycarbonylamino)-2-hydroxycyclopentanecarb-

oxylic acid 14a (60 mg, 0.16 mmol) in dichloromethane

(1.0 mL) at ꢁ15 ꢁC. The mixture was stirred at ꢁ15 ꢁC

for 45 min then etheral diazomethane [approximately

2 mmol generated from diazald^âand sodium hydroxide

in water/ethanol (l:2) at 60 ꢁC in diethyl ether (ꢂ10 mL)]

was added to the activated amino acid solution at

4.1.28. (1R,2R)-(2-tert-Butoxy-1-ﬂuorocarbonylcyclopent-

yl)carbamic acid 9H-ﬂuoren-9-ylmethyl ester (14f).

Pyridine (53 lL, 0.66 mmol) then cyanuric ﬂuoride

(71 lL, 0.85 mmol) were added consecutively at 0 ꢁC to a

stirred solution of (1R,2R)-2-tert-butoxy-1-(9H-ﬂuoren-

9-ylmethoxycarbonylamino)-2-hydroxycyclopentanecarb-

oxylic acid 14b (159 mg, 0.38 mmol) in dichloromethane

(5 mL) under nitrogen. The suspension was stirred for

30 min at 0 ꢁC then for 5 h at ambient temperature.

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

2921

Crushed ice (ꢂ10 mL) and ice-chilled water (10 mL)

were added, then the product was extracted into di-

chloromethane (20 mL). The dichloromethane layer was

dried (MgSO₄) and the solvent removed in vacuo to

leave 14f as a pale brown oil (115 mg, 72%), which was

used without further puriﬁcation. TLC (single UV spot,

R_f¼ 0:45, heptane/ethyl acetate 2:1), analytical HPLC

main UV peak with R_t¼ 23:93 min, HPLC-MS (main

R_t¼ 9:208 min, 364.0 [MþH]^þ, 386.0 [MþNa]^þ); ¹H

NMR (400 MHz, CDCl₃):

d

1.55–2.19 (m,

CH₂CH₂CH₂, 6H), 4.15 (d, J ¼ 16:75 Hz, H-2, 1H),

4.19 (t, J ¼ 6:65 Hz, Fmoc H-9, 1H), 4.31 (d,

J ¼ 16:80 Hz, H-2, 1H), 4.36–4.44 (m, Fmoc CH₂, 2H),

4.74 and 4.97 (each br s, H-6a and NH, 1H), 7.29–7.36

(Fmoc H-2 and H-7, 2H), 7.38–7.44 (Fmoc H-3 and H-

6, 2H), 7.53–7.61 (Fmoc H-1 and H-8, 2H), 7.74–7.80

(Fmoc H-4 and H-5, 2H); ¹³C NMR (100 MHz, CDCl₃):

d 24 (CH₂CH₂CH₂), 33 and 37 (CH₂CH₂CH₂), 48

(Fmoc C-9), 68 (Fmoc CH₂), 70 (C-3a), 72 (C-2), 87 (C-

6a), 120 (Fmoc C-4 and C-5), 125 (Fmoc C-1 and C-8),

127 (Fmoc C-2 and C-7), 128 (Fmoc C-3 and C-6), 142

(Fmoc C-4⁰and C-5⁰), 144 (Fmoc C-1⁰and C-8⁰), 156

(OCON), 215 (C-3). Anal. Calcd for C₂₂H₂₁NO₄: C,

72.71; H, 5.82; N, 3.85. Found: C, 72.68; H, 5.88; N,

3.70; Exact mass calcd for C₂₂H₂₂NO₅: 364.1543, found

364.1531, d ꢁ3.33 ppm.

t

UV peak with R_t¼ 11:439 min, 370.1 [Mꢁ Buþ2H]^þ,

448.1 [MþNa]^þ).

4.1.29. (3aR,6aR)-(3-Oxo-hexahydrocyclopenta[b]furan-

3a-yl)-carbamic acid 9H-ﬂuoren-9-ylmethyl ester (8).

(a) Ethereal diazomethane [generated from diazald^â

(0.94 g, ꢂ3 mmol) addition in diethyl ether (15 mL) to

sodium hydroxide (1.05 g) in water (1.5 mL)/ethanol

(3.0 mL) at 60 ꢁC] was added to a stirred solution of

(1R,2R)-(2-tert-butoxy-1-ﬂuorocarbonylcyclopentyl)carb-

amic acid 9H-ﬂuoren-9-ylmethyl ester 14f (115 mg,

0.27 mmol) in dichloromethane (2 mL) at 0 ꢁC. The

solution was stirred for 20 min at 0 ꢁC then at ambient

temperature for 20 h. Acetic acid (0.6 mL) was added

then the solution was stirred for 5 min before adding

tert-butyl methyl ether (50 mL). The ethereal layer was

washed with saturated aqueous sodium hydrogen car-

bonate solution (40 mL) then water (2 · 30 mL), dried

(Na₂SO₄) and the solvent removed in vacuo. The residue

(130 mg) was puriﬁed by ﬂash chromatography over

silica gel eluting with a gradient of heptane/ethyl acetate

4:1 to 1:3. Appropriate fractions were combined and the

solvents removed in vacuo to leave a 4:1 mixture

of (5R,6R)-6-tert-butoxy-2-(9H-ﬂuoren-9-ylmethoxy)-3-

oxa-1-azaspiro[4.4]non-1-en-4-one 34 and (1R,2R)-2-

tert-butoxy-1-(9H-ﬂuoren-9-ylmethoxycarbonylamino)cy-

clopentanecarboxylic acid methyl ester (70 mg) (14b

where R³¼ OMe) together with (1R,2R)-[2-tert-butoxy-

1-(2-diazoacetyl)cyclopentyl]carbamic acid 9H-ﬂuoren-

9-ylmethyl ester 11b (16 mg, 13%) as an oil. Data for

11b: TLC (single UV spot, R_f¼ 0:50, heptane/ethyl

acetate 2:1), analytical HPLC main UV peak with

R_t¼ 18:927 min, HPLC-MS (main UV peak with

4.2. Solid-phase chemistry

4.2.1. Preparation of (2R,3S)-(2,3-dimethyl-4-oxo-

tetrahydrofuran-3-yl)carbamic acid 9H-ﬂuoren-9-yl-

methyl ester––linker construct (35a). 4-[(Hydrazinocar-

bonyl) amino]methylcyclohexanecarboxylic acid triﬂu-

oroacetate (74 mg, 0.223 mmol, 1 equiv)²⁷and sodium

acetate trihydrate (46 mg, 0.334 mmol, 1.5 equiv) were

added to (2R,3R)-(2,3-dimethyl-4-oxotetrahydrofuran-

3-yl)carbamic acid 9H-ﬂuoren-9-ylmethyl ester (6)

(78 mg, 0.223 mmol, 1 equiv), followed by ethanol

(3.90 mL) and water (0.56 mL). The mixture was heated

for 3 days at 86 ꢁC then allowed to cool to ambient

temperature and diluted with chloroform (50 mL). The

chloroform layer was washed with dilute aqueous

hydrochloric acid (pH 3, 2 · 30 mL), brine (25 mL), dried

(Na₂SO₄) and evaporated in vacuo to give (35a) as a

white solid (80 mg). Analytical HPLC indicated one

main peak at R_t¼ 16:99 min and a minor peak at R_t¼

17:39 min (mixture of E- and Z-isomers), HPLC-MS

(main UV peaks with R_t¼ 8:02 and 8.30 min, 549

[MþH]^þ.

t

R_t¼ 9:130 min, 364.1 [MꢁN₂ꢁ Buþ2H]^þ, 386.0

t

[MꢁN₂ꢁ BuþHþNa]^þ.

4.2.2. Preparation of (2S,3S)-(2,3-dimethyl-4-oxo-

tetrahydrofuran-3-yl)carbamic acid 9H-ﬂuoren-9-yl-

methyl ester––linker construct (36a). 4-[(Hydra-

zinocarbonyl) amino]methylcyclohexanecarboxylic acid

triﬂuoroacetate (35 mg, 0.107 mmol, 7.5 equiv)^27;28and

sodium acetate trihydrate (22 mg, 0.1605 mmol,

7.5 equiv) were added to (2S,3R)-(2,3-dimethyl-4-oxo-

tetrahydrofuran-3-yl)carbamic acid 9H-ﬂuoren-9-yl-

methyl ester (7) (8 mg, 0.0214 mmol, 1 equiv), followed

by ethanol (1.86 mL) and water (0.27 mL). The mixture

was heated for 2 days at 86 ꢁC then allowed to cool to

ambient temperature and diluted with chloroform

(50 mL). The chloroform layer was washed with dilute

aqueous hydrochloric acid (pH 3, 2 · 30 mL), brine

(25 mL), dried (Na₂SO₄) and evaporated in vacuo to

give (36a) as a white solid (10 mg). Analytical HPLC

indicated one main peak at R_t¼ 17:53 min and a minor

peak at R_t¼ 17:88 min (mixture of E- and Z-isomers),

(b) A solution of lithium chloride (15 mg, 0.36 mmol) in

acetic acid: water (4:1, 1.0 mL) was added to 11b (16 mg,

0.036 mmol). The solution was stirred for 2.5 h then

chloroform (25 mL) and saturated aqueous sodium

hydrogen carbonate solution (25 mL) was added. The

chloroform layer washed with saturated aqueous so-

dium hydrogen carbonate solution (25 mL), saturated

aqueous sodium chloride solution (25 mL), dried

(Na₂SO₄) and the solvent removed in vacuo. The residue

(16 mg) was puriﬁed by ﬂash chromatography over silica

gel eluting with a gradient of heptane/ethyl acetate 17:5

to 3:1. Appropriate fractions were combined and the

solvents removed in vacuo to leave (3aR,6aR)-(3-oxo-

hexahydrocyclopenta[b]furan-3a-yl)carbamic acid 9H-

ﬂuoren-9-ylmethyl ester 8 (11.0 mg, 83%) as a white

solid. TLC (single UV spot, R_f¼ 0:3, heptane/ethyl

acetate 2:1), analytical HPLC main UV peak with

R_t¼ 18:872 and HPLC-MS (main UV peak with

2922

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

HPLC-MS (main UV peaks with R_t¼ 8:37 and

8.98 min, 549 [MþH]^þ.

4.3.2. (2R,3R)-Thiophene-3-carboxylic acid [2-cyclo-

hexyl-1S-(2,3-dimethyl-4-oxo-tetrahydrofuran-3-ylcarba-

moyl)ethyl]amide (38b). HPLC R_t¼ 17:03 min (84%),

HPLC-MS 393.2 [MþH]^þ, 807.3 [2MþNa]^þ; Exact

mass calcd for C₂₀H₂₉N₂O₄S: 393.1843, found 393.1854,

d þ2.87 ppm.

4.2.3. Preparation of (3aR,6aR)-(3-oxo-hexahydrocyclo-

penta[b]furan-3a-yl)carbamic acid 9H-ﬂuoren-9-ylmethyl

ester––linker

construct

(37a).

4-[(Hydrazinocar-

bonyl)amino]methylcyclohexanecarboxylic acid triﬂuo-

roacetate (23.6 mg, 0.072 mmol, 1 equiv)²⁷and sodium

acetate trihydrate (14.6 mg, 0.107 mmol, 1.5 equiv)

were added to (3aR,6aR)-(3-oxohexahydrocyclo-

penta[b]furan-3a-yl)carbamic acid 9H-ﬂuoren-9-yl-

methyl ester (8) (26.0 mg, 0.072 mmol, 1 equiv), followed

by ethanol (1.75 mL) and water (0.25 mL). The mixture

was heated for 24 h at 86 ꢁC then allowed to cool to

ambient temperature and diluted with chloroform

(35 mL). The chloroform layer was washed with dilute

aqueous hydrochloric acid (pH 3, 2 · 15 mL), brine

(15 mL), dried (Na₂SO₄) and evaporated in vacuo to

give (37a) as a colourless gum (40.8 mg). Analytical

HPLC indicated one main peak at R_t¼ 17:57 min and a

minor peak at R_t¼ 18:08 min (mixture of E- and Z-

isomers), HPLC-MS (main UV peaks with R_t¼ 8:45

and 9.07 min, 561 [MþH]^þ, 1121 [2MþH]^þ.

4.3.3. (2R,3R)-Benzo[b]thiophene-2-carboxylic acid [1S-

(2,3-dimethyl-4-oxo-tetra-hydrofuran-3-ylcarbamoyl)-3-

methylbutyl]amide (38c). HPLC R_t¼ 17:68 min (93%),

HPLC-MS 403.2 [MþH]^þ, 827.3 [2MþNa]^þ; Exact

mass calcd for C₂₁H₂₆N₂O₄SNa: 425.1505, found

425.1521, d þ3.64 ppm.

4.3.4. (2R,3R)-3-Bromo-N-[1S-(2,3-dimethyl-4-oxo-tetra-

hydrofuran-3-ylcarbamoyl)-2-(4-hydroxphenyl)ethyl]benz-

amide (38d). HPLC R_t¼ 14:90 min (87%), HPLC-MS

475.3/477.3 [MþH]^þ; Exact mass calcd for

C₂₂H₂₄N₂O₅Br: 475.0863, found 475.0875, d þ2.48 ppm.

4.3.5.

(2R,3R)-3-Aminomethyl-N-[1S-(2,3-dimethyl-4-

oxo-tetrahydrofuran-3-ylcarbamoyl)-3-methylbutyl]benz-

amide (38e). HPLC R_t¼ 9:81 min (92%), HPLC-MS

376.2 [MþH]^þ, 398.2 [MþNa]^þ, 773.4 [2MþNa]^þ; Ex-

act mass calcd for C₂₀H₃₀N₃O₄: 376.2231, found

376.2233, d þ0.06 ppm.

4.3. Solid-phase protocols

Example inhibitors (38–40) were prepared from con-

structs (35a–37a) by solid-phase assembly techniques

utilising multipins (see www.mimotopes.com).^29;32In

general, 1.2 lmol gears (GEXXOGAP) were used to

provide small scale crude examples for preliminary

screening, whilst 10 lmol crowns (SPMDINOF) were

used for scale-up synthesis and puriﬁcation of selected

examples.³⁴Constructs (35a–37a), 3 equiv w.r.t. solid-

phase surface loading, were coupled overnight onto

1.2 lmol gears and 10 lmol crowns using standard

HBTU, HOBt and NMM activation to provide loaded

constructs (35b–37b). Constructs (35b–37b) were then

utilised in standard rounds of washing, Fmoc depro-

tection and coupling, followed by acidolytic cleavage to

give crude inhibitors (38–40).^10a;bExamples derived

from 10 lmol crowns were puriﬁed by semipreparative

HPLC (see General methods) and appropriate fractions

combined and lyophilised into pre-tared glass vials.

Puriﬁed analogues were then weighed and a volume of

dimethylsulfoxide added as appropriate to give 10 mM

stock solutions used for general storage and inhibition

assays.

4.3.6. (2R,3R)-4-tert-Butyl-N-[1S-(2,3-dimethyl-4-oxo-

tetrahydrofuran-3-ylcarbamoyl)-2-(4-hydroxyphenyl)ethyl]-

benzamide (38f). HPLC R_t¼ 16:2 min (90%), HPLC-MS

453.2 [MþH]^þ, 475.2 [MþNa]^þ, 927.3 [2MþNa]^þ; Ex-

act mass calcd for C₂₆H₃₂N₂O₅Na: 475.2203, found

475.2196, d ꢁ1.58 ppm.

4.3.7. (2S,3R)-N-[1S-(2,3-Dimethyl-4-oxo-tetrahydrofu-

ran-3-ylcarbamoyl)-3-methyl

butyl]-4-(4-methylpipera-

zin-1-yl)benzamide (39a). HPLC R_t¼ 9:90 min (95%),

HPLC-MS 445.2 [MþH]^þ.

4.3.8. (2S,3R)-Thiophene-3-carboxylic acid [2-cyclo-

hexyl-1S-(2,3-dimethyl-4-oxo-tetrahydrofuran-3-ylcarba-

moyl)ethyl]amide (39b). HPLC R_t¼ 16:85 min (92%),

HPLC-MS 393.2 [MþH]^þ; Exact mass calcd for

C₂₀H₂₈N₂O₄SNa: 415.1662, found 415.1663,

d

þ0.02 ppm.

4.3.9. (2S,3R)-3-Bromo-N-[1S-(2,3-dimethyl-4-oxo-tetra-

hydrofuran-3-ylcarbamoyl)-2-(4-hydroxphenyl)ethyl]benz-

amide (39d). HPLC R_t¼ 14:31 min (89%), HPLC-MS

475.1/477.1 [MþH]^þ; Exact mass calcd for

Each puriﬁed analogue was analysed giving the follow-

ing characterisation data:

C₂₂H₂₃N₂O₅BrNa: 497.0683, found 497.0680,

d

ꢁ0.05 ppm.

4.3.1. (2R,3R)-N-[1S-(2,3-Dimethyl-4-oxo-tetrahydrofu-

ran-3-ylcarbamoyl)-3-methyl butyl]-4-(4-methylpiperazin-

1-yl)benzamide (38a)

. HPLC R_t¼ 10:73 min (94%),

HPLC-MS 445.3 [MþH]^þ, 911.5 [2MþNa]^þ; Exact

mass calcd for C₂₄H₃₇N₄O₄: 445.2809, found 445.2794,

d ꢁ3.40 ppm.

4.3.10. (2S,3R)-3-Aminomethyl-N-[1S-(2,3-dimethyl-4-

oxo-tetrahydrofuran-3-ylcarbamoyl)-3-methylbutyl]benz-

amide (39e). HPLC R_t¼ 9:56 min (93%), HPLC-MS

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

2923

376.2 [MþH]^þ, 398.2 [MþNa]^þ; Exact mass calcd for

C₂₀H₃₀N₃O₄: 376.2231, found 376.2234, d þ0.08 ppm.

based on literature precedent (Table 1; Ref. 1 and ref-

erences cited therein) and modiﬁed as required to suit

local assay protocols.^10b

4.3.11. (2S,3R)-4-tert-Butyl-N-[1S-(2,3-dimethyl-4-oxo-

tetrahydrofuran-3-ylcarbamoyl)-2-(4-hydroxyphenyl)ethyl]-

benzamide (39f). HPLC R_t¼ 17:13 min (92%), HPLC-

MS 453.2 [MþH]^þ; Exact mass calcd for

4.5. Measurement of inhibitor on-rates and oﬀ-rates

The observed rates of reaction for the association of

compound with enzyme (k_on) and for the dissociation of

compound from enzyme (k_off) were analysed as previ-

ously described.³⁶Bovine cathepsin S (Merck Bio-

sciences) was assayed in 10 mM bis-tri-propane; pH 6.5

containing 1 mM calcium chloride, 1 mM EDTA and

5 mM 2-mercaptoethanol employing 50 lM (equal to

K_M^app) Boc-Val-Leu-Lys-AMC (Bachem) as the substrate.

For measurements of the association rates, assays were

carried out by addition of various concentrations of

inhibitor to assay buﬀer containing substrate and initi-

ated by the addition of enzyme. For the measurements

of dissociation rates, pre-incubated enzyme plus inhibi-

tor were diluted at least 20-fold into assay buﬀer con-

taining substrate. During the course of the assay less

than 10% of the substrate was consumed and the ob-

served rates corrected for substrate kinetics.

C₂₆H₃₂N₂O₅Na: 475.2203, found 475.2192,

d

ꢁ2.51 ppm.

4.3.12. (3aR,6aR)-N-[3-Methyl-1S-(3-oxo-hexahydrocy-

clopenta[b]furan-3a-ylcarbamoyl)butyl]-4-(4-methylpipera-

zin-1-yl)benzamide (40a). HPLC R_t¼ 11.23 min (95%)

HPLC-MS 457.3 [MþH]^þ, 935.3 [2MþNa]^þ; Exact

mass calcd for C₂₅H₃₇N₄O₄: 457.2809, found 457.2828, d

þ4.17 ppm.

4.3.13. (3aR,6aR)-Thiophene-3-carboxylic acid [2-cyclo-

hexyl-1S-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcar-

bamoyl)ethyl]amide (40b). HPLC R_t¼ 17:52 min (93%)

HPLC-MS 405.2 [MþH]^þ, 831.3 [2MþNa]^þ; Exact

mass calcd for C₂₁H₂₈N₂O₄SNa: 427.1662, found

427.1681, d þ4.41 ppm.

Acknowledgements

4.3.14. (3aR,6aR)-Benzo[b]thiophene-2-carboxylic acid

[3-methyl-1S-(3-oxo-hexahydrocyclopenta[b]furan-3a-ylcarba-

moyl)butyl]amide (40c). HPLC R_t¼ 15:02 min (93%),

HPLC-MS 415.1 [MþH]^þ, 851.2 [2MþNa]^þ; Exact

mass calcd for C₂₂H₂₇N₂O₄S: 415.1686, found 415.1707,

d þ4.93 ppm.

The authors wish to thank Dr. Chris Urch for his help in

editing this manuscript and Mr. Alan Dickerson, Mr.

Paul Skelton and Mr. Brian Crysell of Cambridge

University Chemistry Department for elemental analy-

sis, high resolution mass spectrometry and NMR analysis.

4.3.15. (3aR,6aR)-3-Bromo-N-[2-(4-hydroxyphenyl)-1-(3-

oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)-ethyl]benz-

amide (40d). HPLC R_t¼ 15:25–15:60 min (86%) HPLC-

MS 487.1/489.1 [MþH]^þ; Exact mass calcd for

C₂₃H₂₄N₂O₅Br: 487.0863, found 487.0881, d þ3.68 ppm.

References and notes

1. (a) Otto, H.-H.; Schirmeister, T. Chem. Rev. 1997, 97, 133;

(b) Hernandez, A. A.; Roush, W. R. Curr. Opin. Chem.

Biol. 2002, 6, 459; (c) Veber, D. F.; Thompson, S. K. Curr.

Opin. Drug Disc. Dev. 2000, 3, 362–369; (d) Leung-Toung,

R.; Li, W.; Tam, T. F.; Karimian, K. Curr. Med. Chem.

2002, 9, 979.

4.3.16.

(3aR,6aR)-3-Aminomethyl-N-[3-methyl-1S-(3-

oxo-hexahydrocyclopenta[b]furan-3a-ylcarbamoyl)butyl]benz-

amide (40e). HPLC R_t¼ 10:87 min (89%) HPLC-MS

388.2 [MþH]^þ, 410.2 [MþNa]^þ, 775.4 [2MþH]^þ; Exact

mass calcd for C₂₁H₃₀N₃O₄: 388.2231, found 388.2225, d

ꢁ0.86 ppm.

€

2. (a) Lecaille, F.; Kaleta, J.; Bromme, D. Chem. Rev. 2002,

102, 4459; (b) Chapman, H. A.; Riese, R. J.; Shi, G.-P.

Annu. Rev. Physiol. 1997, 59, 63.

3. Barrett, A. J.; Rawlings, N. D.; Woessner, J. F. Handbook

of Proteolytic Enzymes; Academic: New York, 1998.

€

4. For example, see: (a) Bromme, D.; Kaleta, J. Curr. Pharm.

Des. 2002, 8, 1639–1658; (b) Kim, W.; Kang, K. Expert

Opin. Ther. Patents 2002, 12(3), 419.

4.4. Assays for cysteinyl proteinase activity

5. In mid 2002, GlaxoSmithKline announced the advance-

ment of SB-462795, a cyclic peptidomimetic ketone

inhibitor of cathepsin K, into phase I clinical trials for

osteoporosis. In autumn 2002, Novartis announced the

advancement of AAE-581, an inhibitor of cathepsin K,

into phase I clinical trials progressing to phase IIb by

autumn 2003.

6. (a) Marquis, R. W.; Ru, Y.; Zeng, J.; Lee Trout, R. E.;

LoCastro, S. M.; Gribble, A. D.; Witherington, J.;

Fenwick, A. E.; Garnier, B.; Tomaszek, T.; Tew, D.;

Hemling, M. E.; Quinn, C. J.; Smith, W. W.; Zhao, B.;

McQueney, M. S.; Janson, C. A.; D’Alessio, K.; Veber, D.

Stock solutions of substrate or inhibitor were made up

to 10 mM in 100% dimethylsulfoxide (DMSO) (Rath-

burns, Glasgow, UK) and diluted as appropriately re-

quired. In all cases the DMSO concentration in the

assays was maintained at less than 1% (v/v). The equi-

librium inhibition constants (K_i^ss) for each compound

were measured under steady-state conditions monitor-

ing enzyme activity as a function of inhibitor concen-

tration. The values were calculated on the assumption of

pure competitive behaviour.³⁵Assay protocols were

2924

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

F. J. Med. Chem. 2001, 44, 725, and references cited

12. The literature cathepsin K X-ray crystal structures that

show inhibitors bound in a retro mode span the whole

active site from S2/S3 to S2⁰/S3⁰. These examples are

therefore able to retain the major elements of the

nonprime hydrogen-bonding pattern that are present in

inhibitors bound in the S2 and S3 pockets only.^6b

13. The conclusion for R and S a-methyl-3-amidopyrrolidin-

4-ones may be diﬀerent since here the retro binding modes,

which in a-methyl-3-amidotetrahydrofuran-4-ones 3a and

4a generally show no obvious clash with the proteinase,

will be able to span the entire active site and may retain the

major elements of the nonprime hydrogen-bonding

pattern.

14. The conclusion for R and S a,b-dimethyl-3-amidopyrroli-

din-4-ones may be diﬀerent since here the retro binding

modes will have substituents that span the entire active site

and may retain the major elements of the nonprime

hydrogen-bonding pattern.

15. Moon, S. H.; Ohfune, Y. J. Am. Chem. Soc. 1994, 116,

7405.

16. Ohfune, Y.; Nanba, K.; Takada, I.; Kan, T.; Horikawa,

M.; Nakajima, T. Chirality 1997, 9, 459–462.

therein; (b) Marquis, R. W.; Ru, Y.; LoCastro, S. M.;

Zeng, J.; Yamashita, D. S.; Oh, H.-J.; Erhard, K. F.;

Davis, L. D.; Tomaszek, T. A.; Tew, D.; Salyers, K.;

Proksch, J.; Ward, K.; Smith, B.; Levy, M.; Cummings,

M. D.; Haltiwanger, R. C.; Trescher, G.; Wang, B.;

Hemling, M. E.; Quinn, C. J.; Cheng, H.-Y.; Lin, F.;

Smith, W. W.; Janson, C. A.; Zhao, B.; McQueney, M. S.;

D’Alessio, K.; Lee, C.-P.; Marzulli, A.; Dodds, R. A.;

Blake, S.; Hwang, S.-M.; James, I. E.; Gress, C. J.;

Bradley, B. R.; Lark, M. W.; Gowen, M.; Veber, D. F. J.

Med. Chem. 2001, 44, 1380, and references cited therein.

7. Fenwick, A. E.; Gribble, A. D.; Ife, R. J.; Stevens, N.;

Witherington, J. Bioorg. Med. Chem. Lett. 2001, 11, 199.

8. See: Quibell, M.; Taylor, S. Patent WO 00/69855. It has

been suggested that the addition of a substituent to the b-

carbon (C-2) of a 3-amidotetrahydrofuran-4-one 2a pro-

vides improved conﬁgurational stability at the a-carbon

(C-3). However, any inhibitors that retain the a-proton,

may still potentially undergo enolisation and hence loss of

chiral integrity.

9. In-house molecular modelling was performed using Web-

Lab ViewerPro (http://www.accelrys.com). The literature

cathepsin K X-ray crystal structures 1mem (nonprime

orientations) and 1au0 (prime and nonprime orientations)

were used as templates to examine the general overall

spatial ﬁt and potential binding orientations of a-methyl-

3-amidotetrahydrofuran-4-ones 3a and 4a. The existing

inhibitors bound within 1mem and 1au0 were modiﬁed

into the desired monocyclic compounds 3a and 4a by

simple draw and clean commands whilst retaining as close

as possible the main thread of the inhibitor backbone. A

hemithioketal tetrahedral intermediate formed between

the cyclic ketone carbonyl and the active site Cys²⁵was

built to examine the products of both re and si stereofacial

addition of the thiolate. As part of the assessment,

calculations were performed on the isolated hemithioketal

tetrahedral intermediate extracted from the predicted

bound complex, to ascertain whether the binding model

contained a reasonable energy inhibitor conformation.

Conformer energy calculations were performed using

CAChe (Conﬂex molecular mechanics engine, CAChe

Group, Fujitsu). Additionally, the full modelling process

considering each possible binding orientation was repeated

using the literature cruzain X-ray structure 1aim and the

mammalian cathepsin S X-ray structure 1ms6. Virtually

identical conclusions concerning the preferred binding

orientations described herein for the cathepsin K 1mem

and 1au0 structures were found when substituting with the

1aim and 1ms6 structures.

17. Ohfune et al.¹⁵report that the prolonged exposure of a 1:4

mixture of 19:20 to 1 equiv TFA in 2-propanol gives an

equilibrated mixture of 19:20 in a ratio of 9:1, which upon

recrystallisation gives enantiopure 19.

18. Seebach, D.; Aebi, J. D.; Gander-Coquoz, M.; Naef, R.

Helv. Chim. Acta 1987, 70, 1194.

19. High yield preparation of the a-diazomethylketones of all

four isomers of Fmoc-Thr and the cis isomers of Fmoc-3-

hydroxyproline and Fmoc-3-hydroxypipecolic acid, with

and without side chain tert-butyl b-hydroxyl protection,

have been reported, see: (a) Quibell, M. Patent WO 02/

57248; (b) Quibell, M. Patent WO 02/57270.

20. Nicolaou, K. C.; Baran, P. S.; Zong, Y.-L.; Choi, H.-S.;

Fong, K. C.; He, Y.; Won, W. H. Org. Lett. 1999, 1, 883–

886.

21. Friedrich-Bochnitschek, S. H.; Waldmann, H.; Kunz, H.

J. Org. Chem. 1989, 54, 751.

22. Dessolin, M.; Guillerez, M.-G.; Thieriet, N.; Guibe, F.;

Loﬀet, A. Tetrahedron Lett. 1995, 36(32), 5741.

23. Urethane Na-protected a,a-disubstituted amino acids are

known to undergo base-catalysed 4H-oxazol-5-one for-

mation in order to relieve the strain imposed by the

sterically conjested groups. It is interesting to speculate

that diazomethane acting as a base could promote b-

elimination of dibenzofulvene from the ﬂuorenylmethyl

ether of the 4H-oxazol-5-one, which followed by tauto-

merisation could give N-carboxyanhydride 25.

24. Fiammengo, R.; Licini, G.; Nicotra, A.; Modena, G.;

Pasquato, L.; Scrimin, P.; Broxterman, Q. B.; Kaptein, B.

J. Org. Chem. 2001, 66, 5905–5910, and references cited

therein.

25. For a study on the Mitsunobu inversion of 3-hydroxy-

proline see: Kolodziej, S. A.; Marshall, G. R. Int. J. Pept.

Prot. Res. 1996, 48, 274.

26. (a) James, W. Tetrahedron 1999, 55, 4855; (b) Fenwick, A.

E.; Garnier, B.; Gribble, A. D.; Ife, R. J.; Rawlings, A. D.;

Witherington, J. Bioorg. Med. Chem. Lett. 2001, 11, 195;

(c) Huang, L.; Ellman, J. A. Bioorg. Med. Chem. Lett.

2002, 12, 2993; (d) Huang, L.; Lee, A.; Ellman, J. A. J.

Med. Chem. 2002, 45, 676.

27. Murphy, A. M.; Dagnino, R.; Vallar, P. L.; Trippe, A. J.;

Sherman, S. L.; Lumpkin, R. H.; Tamura, S. Y.; Webb, T.

R. J. Am. Chem. Soc. 1992, 114, 3156.

28. (a) The formation of linker constructs 35a–37a was

signiﬁcantly more diﬃcult than for the equivalent non-a-

alkylated monocycles that are formed quantitatively in

1 ﬁ 2 h (see Ref. 10b). We have discovered that hindered

10. See: (a) Quibell, M.; Ramjee, M. K. Patent WO 02/57246;

(b) Quibell, M. Patent WO 02/57249.

11. Predictions of likely binding conformations were based

solely upon comparison of the four possible re/si/prime-

side/nonprime-side complexes for each a-stereochemistry.

Major considerations in the development of a ranking for

each possibility were the identiﬁcation of a reasonable

energy conformer for the inhibitor that retained a good

hydrogen-bonding network to the proteinase along with

the absence of spatial clashes. The overall goodness of ﬁt

was assessed using the scoring functions generated by the

software package ‘GOLD 2.1’ (Cambridge Crystallo-

graphic Data Centre) performing the calculations on the

tetrahedral intermediates. It is worthwhile to note that the

analysis of each possibility was warranted since three out

of the four possible combinations have subsequently been

reported in X-ray complexes of various 3-amido-

tetrahydrofuran-4-ones 2a and 3-amidopyrrolidin-4-ones

2b.^6b

J. Watts et al. / Bioorg. Med. Chem. 12 (2004) 2903–2925

2925

ketones may be more readily converted to linker con-

structs through the use of up to 7.5 equiv linker during

reﬂux. Excess linker may then be removed by extraction

with pH 3 HCl prior to solid-phase synthesis; (b) Cleavage

of non-a-alkylated monocycles and 4-acyltetrahydrof-

uro[3,2-b]pyrrol-3-one bicycles has been shown to be

essentially quantitative within 2 h (see Refs. 10b and 19).

29. For a thorough description of general multipin techniques

see: Grabowska, U.; Rizzo, A.; Farnell, K.; Quibell, M.

J. Comb. Chem. 2000, 2(5), 475.

30. For an example of typical coupling eﬃciencies to a,a-

dialkylated amino acids see: Fu, Y.; Hammer, R. P. Org.

Lett. 2002, 4(2), 237.

31. For comparison, the equivalent analogues with achiral

unsubstituted monocyclic scaﬀold 2a give inhibitors with

K_i< 250 nM against particular proteinases as follows:

(a) Cat. K, CPB, cruzain; (b) Cat. S; (c) Cat. K, cruzain;

(d) Cat. L; (e) All; (f) Cruzain. However, due to the a-

lability of scaﬀold 2a in typical inhibition assay buﬀers,

values for the individual 3S and 3R isomers are diﬃcult to

ascertain with certainty.⁷

32. Atherton, E.; Sheppard, R. C. Solid Phase Peptide

Synthesis A Practical Approach; IRL: Oxford, UK, 1989.

33. Meldal, M.; Breddam, K. Anal. Biochem. 1991, 195, 141.

34. In general, the quality of crude examples was >80% as

judged by analytical HPLC. In addition, a comparison of

the K_idata for crude and semiprep puriﬁed examples

indicated that the crude analogues provided a good basis

for SAR and potency analysis.

35. Cornish-Bowden, A. Fundamentals of Enzyme Kinetics;

Portland Ltd: London, 1995.

36. (a) Morrison, J. F.; Walsh, C. T. Adv. Enzymol. Relat.

Areas Mol. Biol. 1988, 61, 201; (b) Tian, W. X.; Tsou, C.

L. Biochemistry 1982, 21(5), 1028.

Article Doi

Functionalised 2,3-dimethyl-3-aminotetrahydrofuran-4-one and N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)acylamide based scaffolds: Synthesis and cysteinyl proteinase inhibition

DOI: 10.1016/j.bmc.2004.03.042

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2004.03.042

Full text of DOI:10.1016/j.bmc.2004.03.042

Products guided by the article

R&D Labs maybe for 443761-48-6

Relevant to this article

Hot Product