Cyclic phosphinamides and phosphonamides, novel series of potent matrix metalloproteinase inhibitors with antitumour activity

Bioorganic & Medicinal Chemistry 11 (2003) 5461–5484

Cyclic Phosphinamides and Phosphonamides,

Novel Series of Potent Matrix Metalloproteinase

Inhibitors with Antitumour Activity

Morten Dahl Sørensen,^a,* Lars K. A. Blæhr,^aMette K. Christensen,^aThomas Høyer,^a

Scilla Latini,^bPernille-Julia V. Hjarnaa^cand Fredrik Bjorkling^a

^aMedicinal Chemistry Research, LEO Pharma, Industriparken 55, DK-2750Ballerup, Denmark

^bDepartment of Biochemistry, LEO Pharma, Industriparken 55, DK-2750Ballerup, Denmark

^cDepartment of Pharmacology, LEO Pharma, Industriparken 55, DK-2750Ballerup, Denmark

Received 14 May 2003; accepted 15 September 2003

Abstract—The design, synthesis, and structure–activity relationship (SAR) of a series of novel nonpeptidic cyclic phosphon- and

phosphinamide-based hydroxamic acids as inhibitors of matrix metalloproteinases MMP-1, MMP-3, and MMP-9 are presented.

Based on modelling studies and X-ray analysis, a model of the binding mode of these novel compounds in the MMP active site was

obtained. This model provided a rational explanation for the observed SAR data, which included a systematic study of diﬀerent S1⁰

directed substituents, zinc-complexing groups, chirality, and variation of the cyclic phosphon- and phosphinamide rings. The in

vivo eﬀect of four compounds in a human ﬁbrosarcoma mouse model (HT1080) was evaluated and compared to that of a reference

compound, Prinomastat. Inhibition of tumour growth was observed for all four compounds.

Introduction

stromelysins (MMP-3, MMP-10, MMP-11), gelatinases

(MMP-2, MMP-9), membrane-type MMPs, and others.

It is still the subject of debate which MMPs are involved

in which diseases. However, some studies suggest that

inhibitors of MMP-13 show potential for reducing car-

tilage loss in osteoarthritis,²while inhibitors of the

gelatinases MMP-2 and MMP-9 may prevent cancer

tumour growth⁵through inhibition of the angiogenetic

process.

Matrix metalloproteinases (MMPs) are a family of zinc-

dependent endoproteinases responsible for degradation

of extracelluar matrix components in tissue remodelling

during pregnancy, wound healing, and angiogenesis.

However, uncontrolled expression and activity of the

MMPs lead to pathological destruction of connective

tissue, and MMPs have been implicated in a number of

diseases such as multiple sclerosis,¹osteoarthritis,²

psoriasis,³and tumor growth and metastasis.⁴Conse-

quently, MMPs are attractive therapeutic targets and con-

siderable eﬀort has been made to discover inhibitors of

MMP activity useful for treatment of the above-mentioned

diseases.

In this paper, we describe the design and synthesis of

novel, potent, and in vivo active cyclic phosphon- and

phosphinamides. The in vitro SAR data of these new

MMP inhibitors are discussed and a molecular model-

ling analysis of their possible binding in the MMP

enzymes is presented. Furthermore, the in vivo eﬀect of

the cyclic phosphon- and phosphinamides in a human

ﬁbrosarcoma mouse model (HT1080) is presented.

More than 20 diﬀerent MMPs have been found to date.

According to their location, substrate preferences, and

structural similarities the MMPs can be divided into ﬁve

groups: collagenases (MMP-1, MMP-8, MMP-13),

Molecular modelling and compound design

One of the ﬁrst low-molecular non-peptidic MMP inhi-

bitors reported was the sulphonamide hydroxamic acid

*Corresponding author: Tel.: +45-72-263525; fax: +45-44-945510;

e-mail: morten.soerensen@leo -p harma.com

doi:10.1016/j.bmc.2003.09.015

5462

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

CGS-27023A (1, Fig. 1).⁶In addition to the investiga-

tion of sulfonamides, MacPherson et al. also showed

that substituting an amide bond for the sulphonamide

bond caused the loss of all activity.⁶Accordingly, it was

speculated that either both sulphonamide oxygens made

crucial H-bonds to the enzyme or the geometry of the

active sulphonamide rotamer was crucial for activity.⁶

The structure of CGS-27023A bound to MMP-1

showed that the zinc atom in the active site was chelated

by the two oxygen atoms of the hydroxamic acid.⁹Fur-

thermore, one of the sulphonamide oxygens formed a

hydrogen bond with the NH of Leu181⁹(MMP-1 num-

bering¹¹). A similar hydrogen bond acceptor is present

in almost all MMP inhibitors and was early on recog-

nised as highly important for inhibitory activity.¹¹One

possible explanation for this critical hydrogen bond is

that the NH of Leu181 (MMP-1) is ‘electrostatic hot’

due to the complexation of the Leu181 carbonyl oxygen

to a nearby structural calcium ion.^11,12Finally, the

methoxyphenyl group was positioned in the S1⁰pocket

of the enzyme. This pocket is a relatively narrow

hydrophobic channel. Several studies have shown that

the S1⁰pocket size diﬀers in the MMPs and is therefore

often described as a ‘selectivity pocket’.^11,13,14Thus,

MMP-1, MMP-7, and MMP-11 have a relatively small

S1⁰pocket whereas MMP-2, MMP-3, and MMP-9 have

a deep S1⁰pocket.

In order to discriminate between these two possible

explanations, Pikul et al. investigated phosphinamide-

based hydroxamic acids (2, Fig. 1).⁷The phosphinamide

bond mimics the geometry and conformational freedom

of the sulphonamide bond but provides only one oxygen

for interaction with the enzyme. The phosphinamide-

based hydroxamic acid compounds are highly potent

MMP inhibitors and it was concluded that only one of

the oxygens and the geometry of the sulphonamide

bond was essential for the activity of CGS-27023A.⁷

A recent study has described the synthesis of novel cyc-

lic phosphonamide-based hydroxamic acids where the

amide nitrogen atom is connected to the carbon a to the

hydroxamic acid (3, Fig. 1); these compounds are also

potent MMP inhibitors.⁸

The three-dimensional structure of the phosphinamide-

based MMP inhibitor 2 bound to MMP-3 revealed the

same binding interactions as those found for CGS-

27023A.^7,10Comparison of the binding of these inhibi-

tors led to the suggestion that CGS-27023A might

experience some unfavourable interactions with MMP

enzymes by placing the sulfonyl oxygen not involved in

a H-bond close to the hydrophobic isobutyl side chain

of Leu164 (MMP-3, Leu81 in MMP-1).⁷The

phosphinamide-based inhibitors had a methyl group at

this position, and the observation that a methyl-to-ethyl

switch resulted in a 6–10-fold decrease of potency

against both MMP-1 and MMP-3 led to the conclusion

that the arrangement of substituents at the phosphorus

center of 2 shown in Figure 1 seemed to be quite opti-

mal.⁷

Structural information of the binding of CGS-27023A

to the active site of the MMPs came from the determi-

nation by NMR of the three-dimensional structure of

CGS-27023A bound to MMP-1 (pdb-codes 3ayk and

4ayk).⁹The information about the bio-active con-

formation of CGS-27023A played a crucial role in our

design of the novel cyclic phosphon- and phosphin-

amide inhibitors presented here. Furthermore, struc-

tural information on the binding of the phosphinamide-

based inhibitors has also been presented.^7,10

In our search for conformationally restrained MMP

inhibitors we speculated that, despite the observations

described above, the MMP enzymes would be able to

accommodate cyclic phosphonamide/phosphinamide-

based hydroxamic acids (4–6, Fig. 1). These compounds

proved highly interesting based on a computational

evaluation. First, a conformational analysis of 4 and 5

[with R1=H, R2=methoxy, and an (R)-conﬁguration

at the phosphorus atom] using the program Macro-

Model revealed that low-energy conformations (approx.

1 kcal/mol above the global minimum) of both cyclic

phosphon- and phosphinamide hydroxamic acids

almost perfectly emulated the bioactive conformation of

CGS-27023A found in the MMP-1 complex (Fig. 2).

Second, superposition of these low-energy conforma-

tions on CGS-27023A in the MMP-1 complex indicated

that the cyclic phosphonamide/phosphinamide ring sys-

tem would not clash signiﬁcantly with the enzyme. The

only steric overlap seemed to be between one of the

protons in the methylene group, which in the cyclophos-

phonamides is adjacent to the ring oxygen, and the a

proton of Asn180 (MMP-1). Consequently, we decided

to further explore the feasibility of cyclic phosphon-

amide/phosphinamide-based hydroxamic acids as MMP

inhibitors.

Figure 1. Structures of MMP inhibitors.

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

5463

Figure 2. Superposition of low-energy conformations of (a) 4 and (b)

5 analogues (atom colours) on the CGS-27023A (shown in cyan) con-

formation found in 3ayk.pdb. The heavy atoms of the hydroxamic

acid moiety, the phosphorous atom, the double bonded oxygen, and

the four-carbon of the aromatic ring were superimposed on the corre-

sponding atoms of CGS-27023A. The RMS values were 0.36 and 0.24

A, respectively.

Chemistry

Synthesis of cyclophosphonamides

The cyclophosponamide hydroxamic acids were pre-

pared by cyclization of N-hydroxyalkyl amino acid

esters (8–17) with phosphonyl dichlorides (18–27), fol-

lowed by conversion of the ester group to a hydroxamic

acid (Scheme 1). Esters 28f–x (R1=H) were converted

directly to the corresponding hydroxamic acids 4f–x by

treatment with hydroxylamine and NaOMe in metha-

nol. However, this method was not suitable for com-

pounds 28a–28e, probably due to steric hindrance.

Instead, these compounds were hydrolyzed to car-

boxylic acids 29 before conversion to hydroxamic acids

4 by the mixed anhydride method.

Cyclophosphonamides 4 prepared from achiral amino-

alcohols 8–12 (R1=H) were prepared as racemates. The

cyclisation of chiral aminoalcohols 13–17, in which

R1¼H, resulted in diastereomeric cyclophosphonamides

4a–e (Table 1) that were separable by ﬂash chromato-

graphy. Except for 4d, these compounds were enantio-

merically pure.

Scheme 1. (a) NMM, CH₂Cl₂; (b) NaOMe, TMSONH₂, MeOH; (c)

NaOH, MeOH; (d) isobutylchloroformate, TMSONH₂, THF.

The aminoalcohols 8–12 with R1=H were prepared by

alkylation of excess o-aminoalcohol with methyl or

ethyl bromoacetate. Amino propanols 13–17 with R1

substituents were prepared from the corresponding

amino acid esters according to Scheme 2. First, the

amino acid ester was protected with a 2-nitrobenzene-

sulfonyl or a benzyl group and subsequently alkylated,

which gave the hydroxypropyl compounds 35–39.

Removal of the protecting group using standard condi-

tions aﬀorded the substituted amino alcohol.

bromides or aryl triﬂates by palladium-catalyzed phos-

phonation.¹⁸

Compound 4q was synthesized via a 4-nitrophenyl

phosphonamide ester 28y intermediate (Scheme 4). The

nitro group was reduced to the amine and subsequently

benzoylated with 4-chlorobenzoylchloride. Finally,

hydroxylaminolysis of the ester gave compound 4q.

Synthesis of cyclophosphinamides

Phosphonyl dichlorides 18–27 were prepared by chlor-

ination of the corresponding alkyl aryl phosphonates

(40–49), preferably with PCl₅¹⁵(Scheme 3). This method

was not suitable for substrates 47 and 48, which under-

went ring-substitution of the bromine atom with chlor-

ine, and for substrate 49, which decomposed to

unidentiﬁable products. In these cases, the alkyl aryl

phosphonate was converted to a bis(trimethylsilyl) aryl

phosphonate¹⁶that was subsequently chlorinated with

oxalyl chloride¹⁷in the presence of a catalytic amount

of DMF. Aryl phosphonates were prepared from aryl

As the phosphonamide hydroxamic acids proved to be

potent MMP inhibitors (vide infra), we then proceeded

to synthesize the corresponding phosphinamides. These

compounds were obtained by a ring-closing metathesis

strategy by procedures described by Gouverneur and

co-workers.¹⁹

Dichloroarylphosphines 50a–c, prepared by standard

methods,²⁰were treated with unsaturated alcohols

(Scheme 5). In the case of allyl alcohol, the resulting

diallyl arylphosphonites underwent a thermal Arbuzov-

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M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

Table 1. In vitro results for a series of six-membered cyclophosphonamide MMP inhibitors: eﬀect of stereochemistry and a substituent

Compd^a

R1

R2

IC₅₀(nM)

MMP-3

MMP-1

500

MMP-9

20

4a

i-Pr

Benzyl

i-Bu

40

25

4b

320

400

16

25

(R,R)-4c

(S,R)-4c

(R,S)-4c

(S,S)-4c

4d^b

63

i-Bu

>10,000

790

7900

>10,000

7900

63

>10,000

10,000

32

i-Bu

Allyl

i-Pr

4e

130

16

0.63

^aThe stereochemistry at the two chiral centres (P,a) is (R,R) unless otherwise speciﬁed.

^bA racemic mixture of the most potent diastereomer.

Analysis of stereochemistry

type rearrangement to yield allyl alkenyl(aryl)phos-

phinates, 51(d,f,h). Esters of higher o-alkenols were

treated with the corresponding bromides to eﬀect rear-

rangement to alkyl alkenyl(aryl)phosphinates, 51(e,g).

In turn, these phosphinates were cleaved with phospho-

rus pentachloride, and amidated with N-allylglycine

ethyl ester, and the resulting phosphinamides 52(d,e,h)

cyclized in the presence of Grubb‘s catalyst to the

phosphinamides 53(d,e,h). Alternatively, the phosphi-

nate 51f,g was cleaved and amidated with allyl amine

yielding phosphinamides 54f,g. After ring closing

metathesis using Grubb’s catalyst, the resulting phos-

phinamides 55f were alkylated with ethyl bromoacetate,

yielding esters 53f. Alkylation prior to RCM was also

feasible as shown by the transformations of alkyl phos-

phinamide 54g to dialkyl phosphinamide 52g and fur-

ther to azaphosphorepene 53g. For the synthesis of

saturated heterocycles, the ring double bond was

hydrogenated using Palladium on charcoal without

adverse ﬁssion of the C–N bond of the cyclic phosphi-

namides 53d–g, (Scheme 6). In all cases, hydroxyl-

aminolysis of the ethyl esters 53 and 56 ﬁnally aﬀorded

hydroxamic acids 6d–h and 5d–g, respectively.

The absolute conﬁguration of the carboxylic precursor

to 4a (29a) was established using X-ray analysis (data

not shown). It was found that both the a carbon and the

phosphorus atom had an (R)-conﬁguration, which

should also apply to 4a. Similar to other studies,^7,8this

structure elucidation was used to assign the stereo-

chemistry of other compounds by characteristic signal

1

patterns in H NMR.

Results and Discussion

In vitro structure–activity relationships

A schematic overview of the in vitro SAR is shown in

Figure 3. In general, the two series (cyclophos-

phonamides and cyclophosphinamides) of MMP inhi-

bitors described in the present work were equally potent

in vitro. However, the SARs of the two series were

slightly diﬀerent. The modelling-based binding mode

proposed for the cyclophosphonamides and cyclophos-

phinamides is shown in Figure 4.

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

5465

Scheme 4. (a) H₂/Pd-C; (b) 4-Cl-benzoylchloride, Et₃N.

Scheme 2. 2-Nitrobenzenesulfonic chloride; Et₃N, CH₂Cl₂; (b)

PhCHO, NaBH₃CN, MeOH; (c) CsCO₃, 3-bromopropanol, DMF; (d)

Allylbromide, DIPEA, DMF; then 9-BBN, THF; H₂O₂, NaOH; (e)

K₂CO₃, PhSH, MeCN, (f) H₂, Pd-C.

Scheme 3. (a) PCl₅(for the preparation of 18–24); (b) TMSCl, Nal;

then (COCl)₂, DMF (cat.) (for the preparation of 25–27).

Scheme 5. (a) (i) CH₂CH(CH₂)_nOH, Et₂O, pyridine; (ii)

CH₂CH(CH₂)_nBr, 130 ^ꢀC, 16 h; (b) (i) PCl₅, DCM; (ii) allylamine,

triethylamine, DCM (c) Grubb’s catalyst, DCM (d) (i) nBuLi, THF;

(ii) Ethyl bromoacetate; (e) (i) PCl₅, DCM; (ii) N-allylglycine ethyl

ester, triethylamine, DCM.

Hydroxamic acids and carboxylic acids were prepared

and tested as zinc binding groups. As expected, the in

vitro potencies of the hydroxamic acids were 100–1000-

fold higher than the corresponding carboxylic acids

(data not shown). Furthermore, N-methylation of the

hydroxamic acid resulted in a decreased potency (data

not shown).

the phosphorus atom is preferred for binding of the

cyclic phosphonamide/phosphinamide-based hydrox-

amic acids to the MMP enzymes.^7,8Furthermore, it is

well known for more bulky substituents, that an (R)-

conﬁguration at the a carbon is important for activ-

ity.^6,8,13,22Stereoisomers with another conﬁguration

than (R,R) were in general 100–1000 times less potent

(Table 1).

Although we did not ﬁnd any carboxylic acids equipo-

tent to their hydroxamic acid counterparts, a recent

study of tetrahydroisoquinoline-3-carboxylates as

MMP-8 inhibitors has indicated that this is sometimes

possible by optimization of the S1⁰-directed sub-

stituent.²¹

Eﬀect of stereochemistry. All compounds presented in

this study have a chiral centre at the phosphorus atom.

Compounds with only this chiral centre were tested as

racemates.

Eﬀect of the phosphorus substituent. Our modelling

studies based primarily on earlier work^6,7indicated that

the aromatic phosphorus substituent (R2, Fig. 1) would

be placed in the S1⁰pocket, generally believed to confer

aﬃnity and selectivity (Fig. 4).

Compounds with two chiral centres (the phosphorus

atom and the carbon atom a to the hydroxamic acid)

were prepared as pure stereoisomers. Determination of

the absolute conﬁguration (see Chemistry section)

revealed that the most potent stereoisomers had the

(R,R)-conﬁguration. This result was in excellent agree-

ment with our modelling studies as well as previous

studies which all indicate that the (R)-conﬁguration at

The in vitro data obtained for our cyclic phosphon-

amide-based hydroxamic acids (Table 2) support our

hypothesis that the aromatic phosphorus substituent is

placed in the S1⁰pocket. Increasing the size of the rela-

tively hydrophobic phosphorus substituent from phenyl

to p-methoxyphenyl to p-phenoxyphenyl (4g, 4h, and 4j)

dramatically increased the aﬃnity towards the deep S1⁰

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M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

pocket enzymes MMP-3 (IC₅₀: 5000, 200, 13 nM) and

MMP-9 (IC₅₀: 1600, 32, 0.40 nM) as well as the selec-

tivity against the short S1⁰pocket MMP-1 (MMP-9/

MMP-1 ratio: 41 for p-methoxyphenyl and 1000 for p-

phenoxyphenyl). These observations correspond very

well with those in other studies,^6,8,21,23strongly sug-

gesting that the increase in aﬃnity and selectivity is

indeed due to improved enzyme–ligand interactions

between the phosphorus substituent and the deep and

relatively narrow hydrophobic S1⁰pocket.

An increased aﬃnity towards MMP-1, although less

dramatic, was also observed when increasing the phos-

phorus substituent from p-methoxyphenyl (IC₅₀: 1300

nM, 4h) to p-phenoxyphenyl (IC₅₀: 400 nM, 4j). This

was somewhat surprizing as MMP-1 has a short S1⁰

pocket which one might expect would not be able to

Scheme 6. (a) H₂, 5% Pd on C, EtOH; (b) NH₂OTMS, KOH, MeOH,

0 ^ꢀC.

Figure 3. An overview of structure–activity relationships of the cyclic phosphon- and phosphinamides.

Figure 4. (a) Schematic view of the expected binding mode of the cyclic phosphon- and phosphinamides in MMP-3; (b) docking model of the MMP-

3-4u complex. Using the same atoms as in Figure 2, a low-energy conformation of the cyclic phosphonamide inhibitor 4u was superimposed on the

conformation of 2 found in the MMP-3–2 complex. (1b3d.pdb). The RMS value was 0.37 A. A MOLCAD surface of the residues surrounding the

inhibitor was generated using Sybyl 6.9. The position of the catalytic zinc is indicated by the magenta part of the surface.

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

5467

Table 2. In vitro results for a series of seven-membered cyclophos-

phonamide MMP inhibitors: eﬀect of P-substituent (R2)

Eﬀect of ring size. In the case of larger P1⁰substituents

(p-phenoxyphenyl and p-bromo-biphenyl) the optimal

ring size for the cyclic phosphonamide was a seven-

membered ring with six- and eight-membered rings

somewhat less potent (Table 3). For the smaller P1⁰

substituent p-methoxyphenyl, seven- and eight-mem-

bered rings were essentially equipotent and six- and

nine-membered somewhat less potent.

Compd

R2

IC₅₀(nM)

MMP-3

MMP-1

MMP-9

We speculated that the variation in activity with varying

ring size may be due to additional hydrophobic interac-

tions between the ring and residues Val163 and Asn162

(MMP-3, numbering^7,8) forming part of the S2⁰/S3⁰site

and/or less steric overlap due to increased ring ﬂex-

ibility. However, the reason for the apparent preference

for seven-membered rings in the phosphonamide series

is diﬃcult to pinpoint.

1

7

4f

160

79

4000

40

6.3

200

79

5.0

79

4g

4h

4i

5000

200

79

1600

32

1300

6300

Eﬀect of the ꢀ substituent. As mentioned above, an (R)-

conﬁguration was important a to the hydroxamic acid;

inversion of the stereochemistry resulted in a 200–400-

fold reduction in potency (Table 1). The optimal sub-

stituents with respect to in vitro activity were isopropyl,

allyl, isobutyl, and benzyl.

63

4j

400

13

0.40

1.0

2.5

4.0

160

20

The (R)-a substitutions increased the potency roughly

by a factor of 10–50 in the compounds containing a 6-

membered phosphonamide ring. Interestingly, (R)-a

substitutions in compounds containing a seven-mem-

bered phosphonamide ring only resulted in a more

modest increase in potency (data not shown).

4k

4l

400

40

400

32

4m

4n

4o

4p

4q

1600

40

The cyclophosphinamides. In the phosphinamide series,

the inﬂuence of ring size was not the same for saturated

and unsaturated cyclophosphinamides (Table 4). For

saturated compounds, seven- and eight-membered rings

appeared to be almost equipotent, and somewhat more

potent than six-membered rings, as observed for the

saturated cyclophosphonamides. In contrast, unsatu-

rated cyclophosphinamides with six-membered rings

gave the most in vitro potent compounds, seven- and

eight-membered rings being up to 10-fold less potent

(compare 6f with 6g). The most potent cyclophos-

phinamides seem to be those with an unsaturated six-

membered ring. One possible explanation could be that

the unsaturated six-membered ring does not contain the

methylene CH₂group which could clash with the a pro-

ton of Asn180 (MMP-1).

>10,000

200

50

79

100

10

accommodate the p-phenoxyphenyl substituent. How-

ever, similar observations have been reported.⁸The

superposition of low-energy conformations of 4j on

CGS-27023A bound to MMP-1 indicates that the S1⁰

pocket of MMP-1 is in fact able to accommodate the p-

phenoxyphenyl substituent even without undergoing the

signiﬁcant rearrangement of the S1⁰loop proposed for

some MMP-1 inhibitors with extended P1⁰substituents

— a rearrangement which results in a more open MMP-

1 S1⁰pocket by repositioning the Arg which normally

blocks the S1⁰pocket.^11,13It should be noted that the p-

phenoxyphenyl substituent is quite unique in this

respect as other biphenyls (4n, 4o, 4p, and 4q) are not

tolerated in the MMP-1 S1⁰pocket (Table 2).

The eﬀect of the aromatic phosphorus substituent was

the same as for the cyclic phosphonamides. No cyclo-

phosphinamides with an a substituent were prepared.

In vivo antitumour activity

The most potent compounds in vitro had a p-phenoxy-

phenyl as the P1⁰substituent. However, based on in

vitro pharmacokinetic analyses we were concerned

about the metabolic stability of the p-phenoxyphenyl.

Accordingly, compounds where the 4-position of the p-

phenoxyphenyl group, the most likely point of meta-

bolic attack, was blocked by a halogen, were tested in

an in vivo antitumor model. Introduction of this halo-

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M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

Table 3. In vitro results for a series of cyclophosphonamide MMP inhibitors: eﬀect of ring size

Compd

R2

n

IC₅₀(nM)

MMP-3

MMP-1

MMP-9

630

4r

4h

4s

4t

1

2

3

4

1

2

3

1

2

3

>10,000

790

200

100

320

40

1300

2000

32

3200

160

4.0

0.40

4.0

100

63

4u

4j

1600

400

13

4v

4w

4i

790

32

>10,000

6300

130

79

4x

>10,000

500

320

gen did indeed increase the stability of the compounds

in the in vitro pharmacokinetic assay (data not shown).

The compounds were tested in a human ﬁbrosarcoma

mouse model (HT1080) (see Experimental). The mice were

treated therapeutically for 14–16 days with 50 mg/kg/day

ip daily starting day 3 to 6 after tumour inoculation. The

compounds were well tolerated by the animals, and there

were no signs of weight loss or other adverse eﬀects.

Good tumour growth inhibition was observed for both the

cyclophosphonamides (4k, 4l, and 4e) and the cyclophos-

phinamide (6h) as well as for the reference compound Pri-

nomastat (7, AG3340) (Table 5). The tumour growth

curves for 4l and 6h are shown in Figure 5. For compounds

4k, 4l, 6h, and Prinomastat the inhibition of tumour

growth was statistically signiﬁcant (p<0.05, one-way ana-

lysis of variance). Compound 4e also reduced tumour

growth, however without reaching statistical signiﬁcance.

The in vitro activities of the compounds tested in the

HT1080 cancer model are also summarised in Table 5.

Figure 5. Tumour growth inhibitory eﬀect in a human ﬁbrosarcoma

mouse model (HT1080). Comparison of the eﬀect of a cyclopho-

sphonamide 4l and a cyclophosphinamide 6h to a control group. The

tumour sizes are mean values. The mice were dosed 50 mg/kg/d ip for

2 weeks from day 5. The tumour reduction is given in Table 5.

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

5469

The tumour growth inhibition of 4k, 4l, 4e, and 6h was

comparable to that of the reference compound Prino-

mastat. However, although the compounds showed a

clear tendency to reduce tumour growth, they did not

stop the tumour growth entirely. This may be explained

by the somewhat short T_1/2(0.7–1.6 h) for all the com-

pounds and/or tumour growth which is not dependent

on MMP activity.

with conventional cytostatics to beneﬁt from the anti-

angiogenic mechanism of the MMP inhibitors.

Conclusion

The design and synthesis of novel cyclic phosphon- and

phosphinamide-based hydroxamic acids were described.

It was shown that these compounds were very potent

(nM) MMP inhibitors in vitro. Variation of the phos-

phorus S1⁰directed substituent produced compounds

with altered selectivity for the MMPs reported herein.

Modelling studies and X-ray analysis revealed that an

(R)-conﬁguration at the phosphorus atom and at the a

The observation that tumour growth does not stop

entirely suggests that administration of MMP inhibitors

to patients in earlier stages would be more eﬃcient. It

could also be interesting to combine MMP inhibitors

Table 4. In vitro results for a series of cyclophosphinamide MMP inhibitors

Compd

R2

Satd.

n

IC₅₀(nM)

MMP-3

MMP-1

10,000

MMP-9

13

5f

Yes

No

Yes

No

Yes

No

Yes

No

1

2

1

3

160

20

6f

2500

3200

0.63

1.3

6h

5g

6g

5d

6d

5e

6e

32

4000

40

5.0

7900

100

3200

320

200

1000

10

>10,000

1300

320

160

1300

Table 5. In vitro and in vivo results for compounds tested in the HT1080 mice cancer model

Compd

IC₅₀(nM)

MMP-3

T_1/2(ip) h

Tumour growth inhibition

(%)

Statistical signiﬁcance

MMP-1

MMP-9

4k

4l

6h

4e

7

400

3200

130

79

40

32

16

6.3

1.0

2.5

1.3

0.63

5.0

1.0

1.3

0.7

1.2

1.6

ꢁ43

ꢁ48

ꢁ29

ꢁ33

ꢁ38

p<0.05

p>0.05

p<0.05

5470

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

carbon, when substituted, was essential for activity. The

inﬂuence of ring size on potency was explored. We

found that seven-membered cyclophosphonamides and

unsaturated six-membered cyclophosphinamides pro-

vided the most potent inhibitors. A model of the bind-

ing mode of these novel compounds in the active site of

MMPs was presented. Finally, four compounds were

tested in a human ﬁbrosarcoma mouse model (HT1080).

The compounds all showed antitumour activity in this

cancer model, the eﬀects being comparable to that of

Prinomastat. This suggested that these novel cyclic

phosphon- and phosphinamide-based MMP inhibitors

may have potential for the treatment of cancer.

3.0 mmol) and isobutyl chloroformate (0.4 mL, 3.0

mmol) were then added with stirring. After stirring

overnight at ꢁ10 ^ꢀC, O-trimethylsilyl hydroxylamine

(0.4 mL, 3.2 mmol) was added, and the mixture was left

at ꢁ10 ^ꢀC for 5 h. The mixture was then acidiﬁed with

4 M acetic acid and extracted with EtOAc/H₂O. The

aqueous layer was extracted with EtOAc, and the com-

bined organic layers were washed with brine, dried

(MgSO₄) and concentrated. The residue was puriﬁed by

chromatography (typically chloroform/methanol/NH₃

(25%) 90:10:1) or crystallisation to aﬀord the hydrox-

amic acid 4.

General procedure B: formation of hydroxamic acids

from esters

Experimental

To a solution of ester 28 (0.20 mmol) in dry methanol (2

mL) was added O-trimethylsilyl-hydroxylamine (72 mL,

0.60 mmol) and sodium methoxide (1.4 M, 214 mL, 0.30

mmol). After stirring at room temperature for 1 h, the

solution was acidiﬁed with 4 M AcOH to pH 4, con-

centrated under reduced pressure and extracted with

EtOAc/H₂O. The aqueous layer was extracted with

EtOAc, and the combined organic layers were washed

with brine, dried (MgSO₄) and concentrated under

reduced pressure. The residue was puriﬁed by chroma-

tography (typically chloroform/methanol/NH₃(25%

aq.) 90:10:1) or crystallisation to aﬀord the hydroxamic

acid 4.

Computational procedures

All calculations were performed on a Silicon Graphics

O₂R10000 workstation. The conformational analyses

were carried out using the Monte Carlo (Mcrlo) routine

of MacroModel (Schrodinger Inc.). The structures in

the conformational analyses were energy-minimized

with the truncated Newton conjugate gradient (TNCG)

method using the MMFF94s force ﬁeld (default con-

vergence criteria). The solvent was set to water using the

SLVNT command. The hydroxamic acid was proto-

nated in the conformational analyses. The resulting

structures were visualized with Sybyl (Tripos Inc.).

Superposition of the cyclic phosphon- and phosphina-

mides on the conformation of CGS-27023A found in

3ayk.pdb⁹was done using the Fit Atoms feature in

Sybyl. All visual inspection of the novel compounds

positioned in the MMP active site was done within

Sybyl.

(2R,ꢀR)-N-Hydroxy-(4-methoxyphenyl)-ꢀ-(1-methyle-

thyl)-2-oxo-1,3,2-oxazaphosphorinane-3-acetamide (4a).

Prepared from 29a using procedure A in 19% yield

after chromatography and recrystallization in acetone,

mp 183.5 ^ꢀC (dec.). H NMR (CD₃OD) d 7.60 (m, 2H),

1

7.06 (m, 2H), 4.36 (m, 1H), 4.11 (m, 1H), 3.85 (s, 3H),

3.71 (m, 1H), 3.42 (m, 2H), 2.30 (m, 1H), 2.11 (m, 1H),

1.86 (m, 1H), 1.12 (d, 3H), 0.96–0.88 (d, 3H). ¹³C NMR

(DMSO-d₆) d 167.2 (d), 161.4 (d), 132.6 (d), 122.8 (d),

114.1 (d), 67.3 (d), 60.1 (d), 55.1, 41.5, 26.5 (d), 26.2 (d),

19.5, 19.0. HRMS calcd for C₁₅H₂₄N₂O₅P (M+H)

343.1423, found 343.1428.

Chemistry

Phenylphosphonic

dichloride

and

4-methoxy-

phenylphosphonic dichloride were obtained from TCI.

Drying of solvents was eﬀected by adding oven dried

molecular sieves to commercial, dry solvents. ‘Diaster-

eomer 1’; refers to the less polar of two possible dia-

stereomers as determined by TLC. ‘Diastereomer 2’;

refers to the more polar diastereomer. All melting points

are uncorrected. NMR spectra were recorded at

300 MHz for ¹H and 75 MHz for ¹³C in the solvent

speciﬁed. Chemical shift values (d, in ppm) are quoted

relative to internal tetramethylsilane (d=0 ppm) or

deuteriochloroform d=76.81 ppm). The mass spectra

were obtained on a Micromass LCT time-of-ﬂight mass

spectrometer operating in either positive or negative ion

electrospray mode, with a cone voltage of 30 V. The

instrument was operated at a resolution of 5000

FWHM. All spectra were recorded at the Department

of Spectroscopy at LEO Pharma.

(2R,ꢀR)-N-Hydroxy-2-(4-methoxyphenyl)-2-oxo-ꢀ-phe-

nylmethyl-1,3,2-oxazaphosphorinane-3-acetamide (4b).

Prepared from 29b using procedure A in 15% yield as

a white solid (crystallized from methanol). ¹H NMR

(CD₃OD) d 7.64 (m, 2H), 7.32–7.15 (m, 5H), 7.04 (m,

2H), 4.21 (m, 2H), 4.10 (m, 1H), 3.84 (s, 3H), 3.83 (m,

1H), 3.43 (m, 1H), 3.31 (m, 1H), 3.06 (dd, 1H), 1.87 (m,

2H). HRMS calcd for C₁₉H₂₄N₂O₅P (M+H) 391.1423,

found 391.1440.

(2R,ꢀR)-N-Hydroxy-2-(4-methoxyphenyl)-ꢀ-(2-methyl-

propyl)-2-oxo-1,3,2-oxazaphosphorinane-3-acetamide

[(R,R)-4c]. Prepared from (R,R)-29c using procedure A

in 48% yield. ¹H NMR (DMSO-d₆) d 11.0–9.7 (bs, 1H),

9.2–8.6 (bs, 1H), 7.56 (m, 2H), 7.02 (m, 2H), 4.25 (m,

1H), 4.07 (m, 1H), 3.92 (m, 1H), 3.81 (s, 3H), 3.60 (m,

1H), 3.12 (m, 1H), 1.90–1.35 (m, 5H), 0.93 (m, 6H). ¹³C

NMR (DMSO-d₆) d 168.0, 161.6, 132.8, 122.4, 114.1,

67.2, 55.2, 52.1, 41.1, 26.7, 24.0, 22.8, 21.9. HRMS calcd

for C₁₆H₂₅N₂O₅P (M+H) 357.1579, found 357.1592.

General procedure A: formation of hydroxamic acids

from carboxylic acids

A solution of carboxylic acid 29 (2.9 mmol) in THF (45

mL) was cooled to ꢁ10 ^ꢀC under argon. NMM (0.3 mL,

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

5471

(2S,ꢀR)-N-Hydroxy-2-(4-methoxyphenyl)-ꢀ-(2-methyl-

propyl)-2-oxo-1,3,2-oxazaphosphorinane-3-acetamide

[(S,R)-4c]. Prepared from (S,R)-29c using procedure A

(ꢂ)-N-Hydroxy-2-(4-biphenylyl)-2-oxo-1,3,2-oxazapho-

sphorepane-3-acetamide (4f). Prepared from 28f using

procedure B in 55% yield as a colourless foam. ¹H

NMR (DMSO-d₆) d 11.0–8.5 (bs, 2H), 7.91 (m, 2H),

7.75 (m, 2H), 7.71 (m, 2H), 7.50 (m, 2H), 7.41 (m, 1H),

4.39 (m, 1H), 4.09 (m, 1H), 4.86 (dd, 1H), 3.58 (dd, 1H),

2.93 (m, 2H), 1.90–1.50 (m, 4H). ¹³C NMR (DMSO-d₆)

d 166.8, 142.9, 139.4, 131.7, 130.2, 129.1, 128.1, 126.9,

126.5, 64.8, 46.5, 29.0, 25.9. HRMS calcd for

C₁₈H₂₁N₂O₄P (M+H) 361.1317, found 361.1293.

1

in 76% yield as a white solid. H NMR (DMSO-d₆) d

11.0–9.8 (bs, 1H), 9.5–8.6 (bs, 1H), 7.67 (m, 2H), 7.05

(m, 2H), 4.25 (m, 2H), 3.81 (s, 3H), 3.66 (m, 1H), 3.48

(m, 1H), 3.11 (m, 1H), 2.00 (m, 1H), 1.91 (m, 1H), 1.51

(m, 1H), 1.28 (m, 2H), 0.68 (d, 3H), 0.52 (d, 3H). ¹³C

NMR (DMSO-d₆) d 168.1, 162.0, 133.5, 121.9, 113.9,

66.5, 55.3, 52.0, 40.1, 37.3, 25.8, 23.7, 22.3, 21.8. HRMS

calcd for C₁₆H₂₅N₂O₅P (M-H) 355.1423, found

355.1439.

(ꢂ)-N-Hydroxy-2-oxo-2-phenyl-1,3,2-oxazaphosphore-

pane-3-acetamide (4g). Prepared from 28g using proce-

dure B in 65% yield as a colourless oil. ¹H NMR

(DMSO-d₆) d 11.0–8.5 (bs, 2H), 7.83 (m, 2H), 7.49 (m,

3H), 4.37 (m, 1H), 4.06 (m, 1H), 3.83 (dd, 1H), 3.56 (dd,

1H), 2.91 (m, 2H), 1.85–1.45 (m, 4H). ¹³C NMR

(DMSO-d₆) d 166.7, 131.5, 131.3, 131.0, 128.2, 64.7,

46.5, 46.4, 29.0, 25.9. HRMS calcd for C₁₂H₁₇N₂O₄P

(MꢁH) 283.0848, found 283.0879.

(2R,ꢀS)-N-Hydroxy-2-(4-methoxyphenyl)-ꢀ-(2-methyl-

propyl)-2-oxo-1,3,2-oxazaphosphorinane-3-acetamide

[(R,S)-4c]. Prepared from (R,S)-29c using procedure A

1

in 63% yield as a white solid. H NMR (DMSO-d₆) d

11.0–9.7 (s, 1H), 9.2–8.6 (s, 1H), 7.55 (m, 2H), 7.02 (m,

2H), 4.25 (m, 1H), 4.07 (m, 1H), 3.92 (m, 1H), 3.81 (s,

3H), 3.61 (m, 1H), 3.12 (m, 1H), 1.83 (m, 1H), 1.66 (m,

3H), 1.42 (m, 1H), 0.93 (m, 6H). ¹³C NMR (DMSO-d₆)

d 168.1, 161.5, 132.8, 122.5, 114.1, 67.2, 55.2, 52.1, 41.1,

26.8, 24.0, 22.8, 21.9. HRMS calcd for C₁₆H₂₅N₂O₅P

(M+H) 357.1579, found 357.1585.

(ꢂ)-N-Hydroxy-2-(4-methoxyphenyl)-2-oxo-1,3,2-oxaza-

phosphorepane-3-acetamide (4h). Prepared from 28h

using procedure B in 47% yield as a white solid. H

1

NMR (DMSO-d₆) d 8.5–11.0 (bs, 2H), 7.75 (m, 2H),

7.00 (m, 2H), 4.33 (m, 1H), 4.05 (m, 1H), 3.80 (m, 3H),

3.80 (m, 1H), 3.55 (dd, 1H), 2.88 (m, 2H), 1.85–1.50 (m,

4H). ¹³C NMR (DMSO-d₆) d 166.7, 161.5, 132.8, 122.7,

113.6, 64.4, 55.1, 46.4, 46.3, 28.9, 25.8. HRMS calcd for

C₁₃H₁₉N₂O₅P (MꢁH) 313.0954, found 313.0969.

(2S,ꢀS)-N-Hydroxy-2-(4-methoxyphenyl)-ꢀ-(2-methyl-

propyl)-2-oxo-1,3,2-oxazaphosphorinane-3-acetamide

[(S,S)-4c]. Prepared from (S,S)-29c using procedure A

in 73% yield. ¹H NMR (DMSO-d₆) d 11.0–9.8 (bs, 1H),

9.6–8.5 (bs, 1H), 7.67 (m, 2H), 7.06 (m, 2H), 4.25 (m,

2H), 3.81 (s, 3H), 3.66 (m, 1H), 3.46 (m, 1H), 3.11 (m,

1H), 2.00 (m, 1H), 1.90 (m, 1H), 1.51 (m, 1H), 1.40–1.15

(m, 2H), 0.68 (d, 3H), 0.52 (d, 3H). ¹³C NMR (DMSO-

d₆) d 168.1, 162.0, 133.5, 122.0, 113.9, 66.5, 55.3, 52.0,

40.1, 37.3, 25.8, 23.7, 22.3, 21.8. HRMS calcd for

C₁₆H₂₅N₂O₅P (M+H) 357.1579, found 357.1592.

(ꢂ)-N-Hydroxy-2-(4⁰-bromo-4-biphenylyl)-2-oxo - 1,3,2 -

oxazaphosphorepane-3-acetamide (4i). Prepared from

28i using procedure B in 49% yield as a white solid. H

1

NMR (DMSO-d₆) d 11.0–9.8 (s, 1H), 9.3–8.7 (s, 1H),

7.91 (m, 2H), 7.76 (m, 2H), 7.68 (m, 4H), 4.39 (m, 1H),

4.10 (m, 1H), 3.86 (dd, 1H), 3.57 (dd, 1H), 2.92 (m, 2H),

1.90–1.50 (m, 4H). ¹³C NMR (DMSO-d₆) d 166.6,

141.5, 138.4, 131.8, 131.7, 130.6, 128.9, 126.3, 121.6,

64.7, 46.4, 46.3, 28.9, 25.8. HRMS calcd for

C₁₈H₂₀BrN₂O₄P (M+H) 439.0422, found 439.0446.

(2R^*,

nyl)-2-oxo-1,3,2-oxazaphosphorinane-3-acetamide (4d).

ꢀR)-(ꢂ)-N-Hydroxy-ꢀ-allyl-2-(4-methoxyphe-

1

Prepared from 29d using procedure A in 53% yield. H

NMR (DMSO-d₆) d 11.0–9.9 (s, 1H), 9.3–8.7 (s, 1H),

7.56 (m, 2H), 7.03 (m, 2H), 7.76 (m, 1H), 5.13 (m, 2H),

4.24 (m, 1H), 4.11 (m, 1H), 3.94 (m, 1H), 3.81 (s, 3H),

3.61 (m, 1H), 3.14 (m, 1H), 2.50 (m, 2H), 1.87 (m, 1H),

1.69 (m, 1H). ¹³C NMR (DMSO-d₆) d 167.4, 161.6,

134.4, 132.8, 122.5, 117.4, 114.1, 67.1, 55.2, 53.6, 41.1,

34.2, 26.6. HRMS calcd for C₁₅H₂₁N₂O₅P (M+H)

341.1266, found 341.1256.

(ꢂ)-N-Hydroxy-2-oxo-2-(4-phenoxyphenyl)-1,3,2-oxaza-

phosphorepane-3-acetamide (4j). Prepared from 28j

1

using procedure B in 60% yield as a white solid. H

NMR (DMSO-d₆) d 11.0–10.1 (bs, 1H), 9.3–8.5 (bs,

1H), 7.82 (m, 2H), 7.44 (m, 2H), 7.22 (m, 1H), 7.10 (m,

2H), 7.03 (m, 2H), 4.35 (m, 1H), 4.05 (m, 1H), 3.82 (m,

1H), 3.54 (m, 1H), 2.91 (m, 2H), 1.88–1.43 (m, 4H).

(2R,ꢀR)-N-Hydroxy-2-[4-(4-chlorophenyloxy)-phenyl]-ꢀ

-(2-methylethyl)-2-oxo-1,3,2-oxazaphosphorinane-3-acet-

amide (4e). Prepared from 29e using procedure A in

26% yield after chromatography. Recrystallized from

methanol to aﬀord a slightly coloured solid, mp 188.5–

(ꢂ)-N-Hydroxy-2-[4-(4-chlorophenoxy)-phenyl]-2-oxo-

1,3,2-oxazaphosphorepane-3-acet-amide (4k). Prepared

from 28k using procedure B in 83% as a white solid. ¹H

NMR (DMSO-d₆) d 11.0–9.8 (s, 1H), 9.2–8.7 (s, 1H),

7.84 (m, 2H), 7.48 (m, 2H), 7.13 (m, 2H), 7.06 (m, 2H),

4.35 (m, 1H), 4.06 (m, 1H), 3.82 (dd, 1H), 3.54 (dd, 1H),

2.91 (m, 2H), 1.90–1.50 (m, 4H). ¹³C NMR (DMSO-d₆)

d 166.6, 159.0, 154.4, 133.3, 130.0, 128.0, 126.1, 121.2,

117.5, 64.6, 46.4, 28.9, 25.8. HRMS calcd for

C₁₈H₂₀ClN₂O₅P (M+H) 411.0876, found 411.0879.

190 ^ꢀC (dec.). H NMR (DMSO-d₆) d 10.64 (bs, 1H),

1

8.81 (s, 1H), 7.58 (m, 2H), 7.49 (m, 2H), 7.14 (m, 2H),

7.07 (m, 2H), 4.28 (m, 1H), 3.97 (m, 1H), 3.66 (m, 1H),

3.52 (m, 1H), 3.14 (m, 1H), 2.16 (m, 1H), 1.94 (m, 1H),

1.70 (m, 1H), 1.01 (d, 3H), 0.82 (d, 3H). ¹³C NMR

(DMSO-d₆) d 167.0 (d), 159.1 (d), 154.2, 133.1 (d),

130.0, 128.2, 126.3 (d), 121.4, 117.8 (d), 67.5 (d), 60.0,

41.5, 26.4 (d), 26.1, 19.5, 19.0. HRMS calcd for

C₂₀H₂₅ClN₂O₅P (M+H) 439.1189, found 439,1176.

(ꢂ)-N-Hydroxy-2-[4-(4-bromophenoxy)-phenyl]-2-oxo-

1,3,2 - oxazaphosphorepane - 3 - acetamide (4l). Prepared

5472

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

from 28l using procedure B in 78% yield as a white solid

1

(ꢂ)-N-Hydroxy-2-(4-methoxyphenyl)-2-oxo-1,3,2-oxaza-

phosphorinane - 3 - acetamide (4r). Prepared from 28r

using procedure B in 43% yield as a white solid. H

NMR (DMSO-d₆) d 11.0–8.0 (m, 2H), 7.71 (m, 2H),

7.04 (m, 2H), 4.30 (m, 1H), 4.06 (m, 1H), 3.82 (s, 3H),

3.46 (m, 2H), 3.21 (m, 2H), 2.01 (m, 1H), 1.85 (m, 1H).

HRMS calcd for C₁₂H₁₈N₂O₅P (M+H) 301.0953,

found 301.0927.

(recrystallized from methanol). H NMR (DMSO-d₆) d

1

11.5–8.0 (bs, 2H), 7.83 (m, 2H), 7.60 (m, 2H), 7.07 (m,

4H), 4.35 (m, 1H), 4.04 (m, 1H), 3.80 (m, 1H), 3.55 (dd,

1H), 2.91 (m, 2H), 1.90–1.50 (m, 4H). ¹³C NMR

(DMSO-d₆) d 166.6, 158.9, 154.9, 133.3, 132.9, 126.2,

121.5, 117.6, 116.0, 64.6, 46.4, 28.9, 25.8. HRMS

calcd for C₁₈H₂₁BrN₂O₅P (M+H) 455.0371, found

455.0392.

(ꢂ)-N-Hydroxy-2-(4-methoxyphenyl)-2-oxo-1,3,2-oxaza-

phosphorocane-3-acetamide (4s). Prepared from 28s

using procedure B in 69% yield as a white solid. H

NMR (DMSO-d₆) d 11.0–9.9 (bs, 1H), 9.2–8.7 (bs, 1H),

7.82 (m, 2H), 7.00 (m, 2H), 4.22 (m, 1H), 3.89 (m, 2H),

3.80 (s, 3H), 3.42 (dd, 1H), 2.91 (m, 2H), 1.90–1.20 (m,

6H). ¹³C NMR (DMSO-d₆) d 166.5, 161.6, 133.1, 123.0,

113.6, 65.3, 55.3, 44.7, 44.6, 28.1, 24.6, 24.1. HRMS

calcd for C₁₄H₂₁N₂O₅P (M+H) 329.1266, found

329.1254.

(ꢂ)-N-Hydroxy-2-[4-(4-triﬂuormethylphenoxy)-phenyl]-

1

2-oxo-1,3,2-oxazaphosphorepa-ne-3-acetamide (4m).

Prepared from 28m using procedure B in 69% yield as

1

an oﬀ-white solid. H NMR (DMSO-d₆) d 11.5–8.0 (bs,

2H), 7.90 (m, 2H), 7.78 (m, 2H), 7.24 (m, 2H), 7.18 (m,

2H), 4.37 (m, 1H), 4.07 (m, 1H), 3.84 (dd, 1H), 3.56 (dd,

1H), 2.92 (m, 2H), 1.90–1.50 (m, 4H). ¹³C NMR

(DMSO-d₆) d 166.6, 159.2, 157.8, 133.4, 127.5, 127.2,

124.1, 124.1, 118.9, 118.8, 64.7, 46.4, 28.9, 25.8. HRMS

calcd for C₁₉H₂₁F₃N₂O₅P (M+H) 445.1140, found

445.1139.

(ꢂ)-N-Hydroxy-2-(4-methoxyphenyl)-2-oxo-1,3,2-oxaza-

phosphoronane-3-acetamide (4t). Prepared from 28t

1

(ꢂ)-N-Hydroxy-2-benzylphenyl-2-oxo-1,3,2-oxazapho-

sphorepane-3-acetamide (4n). Prepared from 28n using

procedure B in 71% yield as a colorless foam. ¹H NMR

(DMSO-d₆) d 9.95 (bs, 1H), 9.02 (bs, 1H), 7.73 (m, 2H),

7.40–7.15 (m, 7H), 4.34 (m, 1H), 4.04 (m, 1H), 3.98 (s,

2H), 3.80 (dd, 1H), 3.53 (dd, 1H), 2.88 (m, 2H), 1.90–

1.40 (m, 4H).

using procedure B in 66% yield as a white solid. H

NMR (DMSO-d₆) d 11.0–9.8 (s, 1H), 9.3–8.6 (s, 1H),

7.90 (m, 2H), 7.01 (m, 2H), 4.43 (m, 1H), 4.01 (m, 1H),

3.84 (m, 1H), 3.80 (s, 3H), 3.39 (dd, 1H), 2.86 (m, 1H),

2.61 (m, 1H), 1.90–1.60 (m, 2H), 1.55–1.05 (m, 6H). ¹³C

NMR (DMSO-d₆) d 166.4, 161.6, 133.5, 122.4, 113.7,

59.6, 55.3, 44.0, 41.5, 27.3, 23.9, 18.5, 17.9. HRMS calcd

for C₁₅H₂₃N₂O₅P (MꢁH) 341.1267, found 341.1267.

(ꢂ)-N-Hydroxy-2-(4-chlorobenzoylphenyl)-2-oxo-1,3,2-

oxazaphosphorepane-3-acetamide (4o). Prepared from

28o using procedure A in 64% yield as a white solid. ¹H

NMR (DMSO-d₆) d 11.0-9.5 (bs, 1H), 9.5–8.5 (bs, 1H),

8.01 (m, 2H), 7.84–7.75 (m, 4H), 7.65 (m, 2H), 4.42 (m,

1H), 4.12 (m, 1H), 3.87 (dd, 1H), 3.59 (dd, 1H), 2.93 (m,

2H), 1.93–1.50 (m, 4H). ¹³C NMR (DMSO-d₆) d 194.9,

167.2, 139.5, 138.6, 136.4 (d), 135.8, 132.2, 131.8 (d),

129.6 (d), 129.4, 65.6 (d), 47.1, 29.5, 26.5. HRMS calcd

for C₁₉H₂₁ClN₂O₅P (M+H) 423.0876, found 423.0890.

(ꢂ)-N-Hydroxy-2-oxo-2-(4-phenoxyphenyl)-1,3,2-oxaza-

phosphorinane-3-acetamide (4u). Prepared from 28u

using procedure B in 39% yield as a white solid. H

1

NMR (DMSO-d₆) d 10.5–8.5 (m, 2H), 7.78 (m, 2H),

7.45 (m, 2H), 7.23 (m, 1H), 7.11 (m, 2H), 7.05 (m, 2H),

4.32 (m, 1H). ¹³C NMR (CD₃OD) d 168.6 (d), 163.2,

156.9, 135.4 (d), 131.3, 125.9, 124.0 (d), 121.3, 118.7 (d),

68.9 (d), 49.9–48.1 (2*CH₂), 27.1 (d). HRMS calcd for

C₁₇H₂₀N₂O₅P (M+H) 363.1110, found 363.1087.

(ꢂ)-N-Hydroxy-2-oxo-2-(4-phenylmethoxyphenyl)-1,3,2-

oxazaphosphorepane-3-acetamide (4p). Prepared from

28p using procedure B in 79% yield as a colorless oil. ¹H

NMR (DMSO-d₆) d 11.0–9.8 (bs, 1H), 9.2–8.6 (bs, 1H),

7.75 (m, 2H), 7.40 (m, 5H), 7.08 (m, 2H), 5.16 (s, 2H),

4.32 (m, 1H), 4.03 (m, 1H), 3.80 (dd, 1H), 3.53 (dd, 1H),

2.89 (m, 2H), 1.90-1.45 (m, 4H). ¹³C NMR (DMSO-d₆)

d 166.7, 160.5, 136.6, 132.8, 128.4, 127.8, 127.6, 123.0,

114.4, 69.2, 64.5, 46.3, 28.9, 25.7. HRMS calcd for

C₁₉H₂₃N₂O₅P (M+H) 391.1423, found 391.1429.

(ꢂ)-N-Hydroxy-2-oxo-2-(4-phenoxyphenyl)-1,3,2-oxaza-

phosphorocane-3-acetamide (4v). Prepared from 28v

using procedure B in 32% yield as a colourless foam. ¹H

NMR (DMSO-d₆) d 10.1 (bs, 1H), 8.89 (bs, 1H), 7.89

(m, 2H), 7.44 (m, 2H), 7.22 (m, 1H), 7.09 (m, 2H), 7.02

(m, 2H), 4.23 (m, 1H), 4.04–3.80 (m, 2H), 3.43 (m, 1H),

2.94 (m, 2H), 1.89–1.20 (m, 6H).

(ꢂ)-N-Hydroxy-2-(4⁰-bromo-4-biphenylyl)-2-oxo-1,3,2-

oxazaphosphorinane-3-acetamide (4w). Prepared from

28w using procedure B in 62% yield as a colourless oil.

¹H NMR (DMSO-d₆) d 10.4 (bs, 1H), 9.0 (bs, 1H),

7.93–7.74 (m, 4H), 7.69 (bs, 4H), 4.36 (m, 1H), 4.13 (m,

1H), 3.69–3.13 (m, 4H), 2.06 (m, 1H), 1.86 (m, 1H).

HRMS calcd for C₁₇H₁₉BrN₂O₄P (M+H) 425.0266,

found 425.0267.

(ꢂ)-N-Hydroxy-2-N-(4-chlorobenzoyl)-4-aminophenyl-2-

oxo-1,3,2-oxazaphosphorepa-ne-3-acetamide (4q). Pre-

pared from 28q using procedure B in 58% yield as a

colorless foam. ¹H NMR (DMSO-d₆) d 11.0–9.5 (s, 1H),

9.5–8.1 (bs, 1H), 8.00 (m, 2H), 7.95–7.75 (m, 4H), 7.62

(m, 2H), 4.37 (m, 1H), 4.09 (m, 1H), 3.81 (dd, 1H), 3.57

(dd, 1H), 2.91 (m, 2H), 1.85-1.50 (m, 4H). ¹³C NMR

(DMSO-d₆) d 166.7, 164.6, 141.6, 136.5, 133.3, 131.7,

129.6, 128.4, 126.1, 119.5, 64.6, 46.5, 46.3, 28.9, 25.8.

HRMS calcd for C₁₉H₂₂ClN₃O₅P (M+H) 438.0985,

found 438.1010.

(ꢂ)-N-Hydroxy-2-(4⁰-bromo-4-biphenylyl)-2-oxo-1,3,2-

oxazaphosphorocane-3-acetamide (4x). Prepared from

28x using procedure B in 50% yield as a colourless oil.

MS calcd for C₁₉H₂₂BrN₂O₄P 452.05, Found

[MꢁH]^ꢁ=451.

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

5473

General procedure C: preparation of N-(hydroxyalkyl)-

glycine ethyl- or methyl esters

methyl ester (35 mmol) in DMF (160 mL). The reaction

mixture heated to 60 ^ꢀC then 3-bromopropanol (4,75

mL, 52.5 mmol) was added dropwise with stirring. The

mixture was heated overnight, was then diluted with

water and extracted three times with ethyl acetate. The

combined organic phases were washed with calcium

chloride solution (3 mol/l), water, brine, dried (MgSO₄)

and concentrated. The resulting N-(3-hydroxypropyl)-

N-(2-nitrophenylsulfonyl)-amino acid methyl ester was

puriﬁed by chromatography eluting with ethyl acetate–

light petroleum.

To an ice-cooled solution of the aminoalcohol (100

mmol) in dry THF (200 mL) was added methyl or ethyl

bromoacetate (16.7 mmol) with stirring. After stirring

for 4 h, the mixture was concentrated and puriﬁed by

ﬂash chromatography (methanol or ethanol in di-

chloromethane).

N-(3-Hydroxypropyl)-glycine ethyl ester (8).²⁴Prepared

from 3-amino-1-propanol and ethyl bromoacetate in

72% yield as a colourless oil, following procedure C. ¹H

NMR (DMSO-d₆) d 4.09 (q, 2H), 3.44 (t, 2H), 3.28 (s,

2H), 2.55 (t, 2H), 2.5–3.5 (bs, 2H), 1.53 (m, 2H), 1.19 (t,

3H). ¹³C NMR (DMSO-d₆) d 172.1, 59.8, 59.3, 50.4,

46.1, 32.6, 14.1.

Potassium carbonate (3.6 g, 26 mmol) was added to a

solution of the resulting N-(3-hydroxypropyl)-N-(2-

nitrophenylsulfonyl)-amino acid methyl ester (8 mmol)

and phenylthiol (1 mL, 9 mmol) in acetonitrile (40 mL).

The reaction mixture was stirred at room temperature

overnight, then reduced under vacuum and the residue

taken up in diethyl ether. Hydrochloric acid solution (1

M) was added and the mixture stirred for 10 min. The

organic layer was removed and washed with water, the

combined aqueous layers were washed with diethyl

ether followed by basiﬁcation with solid potassium

carbonate. The product was extracted from the basic

aqueous layer with DCM (several washes), the com-

bined DCM phases were dried and concentrated. The

resulting N-(3-hydroxypropyl)-amino acid methyl ester

was puriﬁed by chromatography eluting with methanol

in DCM.

N-(4-Hydroxybutyl)-glycine ethyl ester (9). Prepared

from 4-amino-1-butanol and ethyl bromoacetate in

89% yield as a colourless oil, following procedure C. ¹H

NMR (CDCl₃) d 3.74 (s, 3H), 3.60 (t, 2H), 3.41 (s, 2H),

3.14 (bs, 2H), 2.66 (t, 2H), 1.64 (m, 4H). ¹³C NMR

(CDCl₃) d 172.5, 62.5, 51.9, 50.2, 49.3, 31.6, 27.8.

N-(4-Hydroxybutyl)-glycine methyl ester (10). Prepared

from 4-amino-1-butanol and methyl bromoacetate in 65%

yield as a colourless oil, following procedure C. ¹H NMR

(CDCl₃) d 4.20 (q, 2H), 3.61 (t, 2H), 3.39 (s, 2H), 3.02 (bs,

2H), 2.67 (t, 2H), 1.64 (m, 4H), 1.28 (t, 3H). ¹³C NMR

(CDCl₃) d 172.0, 62.6, 60.9, 50.4, 49.4, 31.8, 28.0, 14.2.

N-(3-hydroxypropyl)-^L-leucine methyl ester (13).²⁷Pre-

pared from l-leucine methyl ester hydrochoride.

according to procedure D, with the following inter-

mediates.

N-(5-Hydroxypentyl)-glycine methyl ester (11).²⁵Prepared

from 5-amino-1-pentanol and methyl bromoacetate in

88% yield as a colourless oil, following procedure C. ¹H

NMR (CDCl₃) d 3.73 (s, 3H), 3.63 (t, 2H), 3.41 (s, 2H),

2.62 (t, 2H), 1.97 (bs, 2H), 1.66–1.34 (m, 6H). ¹³C NMR

d 173.3, 62.9, 52.1, 51.1, 49.8, 32.8, 30.0, 23.7.

N-(2-nitrophenylsulfonyl)-^L-leucine methyl ester (30).

Yield: 92%.

N-(3-hydroxypropyl)-N-(2-nitrophenylsulfonyl)- ^L-leucine

methyl ester (35). Yield: 45%.

N-(6-Hydroxyhexyl)-glycine ethyl ester (12). Prepared

from 6-amino-1-hexanol and ethyl bromoacetate in

82% yield as a colorless oil, following procedure C. H

NMR (CDCl₃) d 4.19 (q, 2H), 3.62 (t, 2H), 3.38 (s, 2H),

2.60 (t, 2H), 1.93 (bs, 2H), 1.54 (m, 4H), 1.37 (m, 4H),

1.28 (t, 3H). ¹³C NMR (CDCl₃) d 172.6, 62.7, 60.8, 50.9,

49.5, 32.7, 29.9, 27.0, 25.6, 14.2.

1

N-(3-hydroxypropyl)-^L-leucine methyl ester (13). Yield:

1

44%. H NMR (CDCl₃) d 3.80 (t, 2H), 3.73 (s, 3H),

3.31 (t, 1H), 2.93 (m, 1H), 2.93 (bs, 2H), 2.59 (m, 1H),

1.69 (m, 3H), 1.46 (m, 2H), 0.92 (d, 3H), 0.91 (d, 3H).

¹³C NMR (CDCl₃) d 176.0, 64.0, 59.9, 51.8, 48.1, 42.7,

31.0, 24.9, 22.8, 22.1.

General procedure D: preparation of N-(3-hydroxy-

propyl)-amino acid methyl esters 13–15

N-(3-hydroxypropyl)-^D-leucine methyl ester (14). Pre-

pared from d-leucine methyl ester hydrochoride,

according to procedure D, with the following inter-

mediates

The syntheses were based on the method described in

the literature.²⁶2-Nitrosulfonyl chloride (13.9 g, 63

mmol) was added with stirring to an ice-cooled solution

of amino acid methyl ester hydrochloride (63 mmol)

and triethylamine (20 mL, 143 mmol) in DCM (500

mL). The mixture was allowed to warm to room

temperature and stirred overnight. The resulting solu-

tion was washed with water, dried and concentrated.

The resulting N-(2-nitrophenylsulfonyl)-amino acid

methyl ester was used without puriﬁcation.

N-(2-nitrophenylsulfonyl)-^D-leucine methyl ester (31).

Yield: 85%

N-(3-hydroxypropyl)-N-(2-nitrophenylsulfonyl)-^D-leucine

methyl ester (36). Yield: 52%

N-(3-hydroxypropyl)-^D-leucine methyl ester (14). Yield:

1

60%. H NMR (CDCl₃) d 3.80 (t, 2H), 3.73 (s, 3H),

3.31 (t, 1H), 2.95 (m, 1H), 2.64 (bs, 2H), 2.59 (m, 1H),

1.69 (m, 3H), 1.46 (m, 2H), 0.92 (d, 3H), 0.91 (d, 3H).

Cesium carbonate (12,35 g, 35 mmol) was added to a

solution of the N-(2-nitrophenylsulfonyl)-amino acid

5474

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

¹³C NMR (CDCl₃) d 176.0, 64.1, 59.9, 51.7, 48.2, 42.7,

31.1, 24.9, 22.8, 22.1.

solution was cooled to 0 ^ꢀC and quenched with 40

mL water. After stirring another 15 min, aqueous

NaOH (20%, 30 mL) was added dropwise followed

by aqueous H₂O₂(30%, 21 mL). The mixture was

stirred for 1.5 h at 0 ^ꢀC, then neutralized with 4 M

HCl and concentrated under reduced pressure. Water

was added to the residue, and the mixture was

extracted ﬁve times with EtOAc. Combined extracts

were washed with brine, dried over MgSO₄and con-

centrated under reduced pressure to give 17.1 g of 38 as

a colourless oil. ¹H NMR (CDCl₃) d 7.38–7.20 (m, 5H),

4.07 (d, 1H), 3.74 (s, 3H), 3.63 (m, 2H), 3.29 (d, 1H), 3.05

(d, 1H), 2.82 (m, 1H), 2.56 (bs, 1H), 2.44 (m, 1H), 2.16

(m, 1H), 1.87 (m, 1H), 1.56 (m, 1H), 1.03 (d, 3H), 0.87

(d, 3H). ¹³C NMR (CDCl₃) d 172.4, 139.4, 128.9, 128.4,

127.2, 69.1, 62.3, 54.9, 50.7, 48.8, 29.5, 27.4, 20.1, 19.6.

N-(3-hydroxypropyl)-(^{D, L)}-allylglycine methyl ester (15).

Prepared from d,l-allylglycine methyl ester hydro-

choride, according to procedure D, with the following

intermediates.

N-(2-nitrophenylsulfonyl)-(^D,L)-allylglycine methyl ester

(32). Yield: 82%

N-(3-hydroxypropyl)-N-(2-nitrophenylsulfonyl)-(^D,L)-al-

lylglycine methyl ester (37). Yield: 25%.

N-(3-hydroxypropyl)-(^D,L)-allylglycine methyl ester (15).

1

Yield: 20%. Compound 15: H NMR (CDCl₃) d 5.73

(m, 1H), 5.13 (m, 1H), 5.08 (m, 1H), 3.79 (t, 2H), 3.73

(s, 3H), 3.37 (m, 1H), 2.94 (m, 1H), 2.64 (m, 1H), 2.55

(bs, 2H), 2.40 (m, 2H), 1.69 (m, 2H). ¹³C NMR (CDCl₃)

d 174.7, 133.2, 118.5, 64.0, 60.8, 51.8, 48.0, 37.7, 31.0.

N-Benzyl-N-(3-hydroxypropyl)-^D-phenylalanine methyl

ester (39). In a procedure similar to the above, 34 was

converted to 39 in 38% yield as a colourless oil. ¹H

NMR (CDCl₃) d 7.34–7.14 (m, 8H), 7.10 (m, 2H), 3.93

(d, 1H), 3.75 (dd, 1H), 3.67 (s, 3H), 3.63–3.55 (m, 3H),

3.5–2.0 (bs, 1H), 3.15 (dd, 1H), 2.91 (m, 2H)(, 2.72 (m,

1H), 1.85–1.50 (m, 2H). ¹³C NMR (CDCl₃) d 172.5,

138.6, 138.0, 129.2, 129.0, 128.4, 128.4, 127.3, 126.5,

63.5, 62.6, 55.2, 51.4, 49.1, 34.9, 29.2.

N-Benzyl-^D-valine methyl ester (33). Benzaldehyde (12.7

g, 119 mmol) was added to an ice-cooled mixture of

methyl-d-valinate hydrochloride (20.0 g, 119 mmol) and

NaBH₃CN (7.48 g, 119 mmol) in 350 mL methanol and

stirred overnight at rt. The reaction mixture was cooled

in an ice-bath, acidiﬁed to pH 1 with concentrated HCl

and stirred for 3.5 h at 0 ^ꢀC. The solvent was removed in

vacuo and water was added to the residue. The pH was

adjusted to 9.3 with saturated Na₂CO₃, and the mixture

was extracted three times with EtOAc. The combined

organic layers were washed with brine, dried over

MgSO₄, and concentrated under reduced pressure,

aﬀording 21.2 g of crude product, which was puriﬁed by

ﬂash chromatography to give 16.1 g (61%) N-benzyl-d-

valine methyl ester (33) as a colorless oil. ¹H NMR

(CDCl₃) d 7.37–7.18 (m, 5H), 3.83 (d, 1H), 3.71 (s, 3H),

3.58 (d, 1H), 3.02 (d, 1H), 1.92 (m, 1H), 1.80 (s, 1H), 0.95

(d, 3H), 0.93 (d, 3H). ¹³C NMR (CDCl₃) d 175.8, 140.1,

128.3, 128.2, 127.0, 66.6, 52.6, 51.4, 31.7, 19.3, 18.7.

N-(3-Hydroxypropyl)-^D-valine methyl ester (16). A solu-

tion of crude 38 (17.1 g) in 120 mL ethanol was hydro-

genated over Pd on carbon (10%) for 2.5 h. The catalyst

was ﬁltered oﬀ through Celite, and the ﬁltrate was con-

centrated to an oil. Puriﬁcation by ﬂash chromatography

1

gave 6.4 g (68%) of 16 as a colourless oil. H NMR

(CDCl₃) d 3.81 (t, 2H), 3.73 (s, 3H), 3.5–2.0 (bs, 2H),

3.05 (d, 1H), 2.98 (m, 1H), 2.55 (m, 1H), 1.92 (m, 1H), 1.69

(m, 2H), 0.94 (d, 3H), 0.93 (d, 3H). ¹³C NMR (CDCl₃) d

175.1, 67.4, 64.3, 51.6, 48.8, 31.6, 30.9, 19.4, 18.6.

N-(3-Hydroxypropyl)-^D-phenylalanine methyl ester (17).

In a procedure similar to the above, 39 was converted

1

to 17 in 32% yield as a colorless oil. H NMR (CDCl₃)

N-Benzyl-^D-phenylalanine methyl ester (34). In a proce-

dure similar to the above, d-phenylalanine methyl ester

was converted to 34 in 81% yield as a colourless oil. H

d 7.30 (m, 1H), 7.27 (m, 2H), 7.15 (m, 2H), 3.71 (m,

2H), 3.68 (s, 3H), 3.54 (m, 1H), 2.92 (m, 3H), 2.58 (m,

1H), 1.64 (m, 2H), 3.20–1.60 (bs, 2H).

1

NMR (CDCl₃) d 7.30–7.10 (m, 10H), 3.81 (d, 1H), 3.64 (s,

3H), 3.63 (d, 1H), 3.54 (t, 1H), 2.96 (m, 2H), 1.77 (bs, 1H).

General procedure E: preparation of phosphonyl dichlorides

N-Benzyl-N-(3-hydroxypropyl)-^D-valine methyl ester

(38). A solution of 33 (16.1 g, 72.8 mmol), allyl bro-

mide (17.6 g, 145.5 mmol) and diisopropylethylamine

(18.8 g, 145.5 mmol) in dry DMSO (75 mL) was heated

overnight at 40 ^ꢀC. The reaction mixture was diluted

with water and extracted three times with ethylacetate.

The combined extracts were washed with water and

brine, dried over MgSO₄and concentrated under

reduced pressure, aﬀording 19.7 g of crude N-benzyl-N-

allyl-d-valine methyl ester. Puriﬁcation by ﬂash chro-

matography gave 13.1 g (69%) of a colorless oil.

The diethyl aryl phosphonate was added neat to ice-

cooled PCl₅(2.2 equivalents) under an argon atmo-

sphere over 15 min while stirring. After complete

addition, the suspension was heated to 110–120 ^ꢀC for

4–5 h. During this period, a clear solution was

obtained. Volatiles were removed in vacuo, and the

phosphonyl dichloride was puriﬁed by Kugelrohr dis-

tillation or used immediately without puriﬁcation (not

distillable)

General procedure F: preparation of phosphonyl dichlorides

A solution of N-benzyl-N-allyl-d-valine methyl ester

(13 g, 50 mmol) in 40 mL dry THF was added to an

ice-cooled solution of 9-BBN (149 mL, 0.5 M in

THF) under argon. After stirring at rt for 4.5 h, the

Chlorotrimethylsilane (8.54 mmol) was added dropwise

to a solution of diethyl aryl phosphonate (4.27 mmol)

and NaI (8.54 mmol) in dry CH₃CN (4 mL). The

resulting dark suspension was heated at 40 ^ꢀC for 45

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

5475

min. The precipitated NaCl was ﬁltered oﬀ, and the ﬁl-

trate was concentrated in vacuo to remove excess

chlorotrimethylsilane. The residue was dissolved in dry

CH₂Cl₂(5 mL) containing a few drops of DMF, and

oxalylchloride (12.81 mmol) was added dropwise (gas

evolution). After complete addition, the reaction mix-

ture was stirred at rt for 1 h. The solvent and other

volatiles were removed in vacuo, and the residue was

either puriﬁed by Kugelrohr distillation or used imme-

diately without puriﬁcation (not distillable).

general procedure F. Used directly without further

puriﬁcation.

General procedure G: formation of esters 28

A solution of amino alcohol (8–17) (0.42 mmol) and

NMM (0.85 mmol) in dry CH₂Cl₂(20 mL) was cooled

to 0 ^ꢀC under argon, and

a solution phospho-

nyldichloride (18–27) (0.42 mmol) in dry CH₂Cl₂(10

mL) was added. The mixture was stirred at 0 ^ꢀC for 1 h,

then at rt overnight. After quenching with water, the

aqueous phase was extracted with EtOAc. The com-

bined organic layers were washed with brine, dried

(MgSO₄) and concentrated under reduced pressure. The

residue was puriﬁed by chromatography (chloroform/

methanol) to aﬀord the target compounds 28.

4-Biphenyl phosphonic dichloride (18). Prepared from 40

in quantitative yield (crude) using general procedure E.

1

Used without further puriﬁcation. H NMR (CDCl₃) d

8.04 (m, 2H), 7.78 (m, 2H), 7.62 (m, 2H), 7.47 (m, 3H).

MS (EI+): m/z 270 (M⁺), 235, 152.

4-Phenoxyphenyl phosphonic dichloride (19). Prepared

from 41 using general procedure E in 72% yield. Dis-

Methyl (2R,ꢀR)-2-(4-methoxyphenyl)-ꢀ-(1-methylethyl)-

2-oxo-1,3,2-oxazaphosphorinane-3-acetate (diastereomer

1) (28a). Prepared from 4-methoxyphenylphosphonic

dichloride and 16 in 30% yield as a colourless oil,

according to procedure G. ¹H NMR (CDCl₃) d 7.64 (m,

2H), 6.94 (m, 2H), 4.44 (m, 1H), 4.20 (m, 1H), 3.85 (t,

1H), 3.84 (s, 3H), 3.45 (s, 3H), 3.43 (m, 2H), 2.16 (m,

2H), 1.96 (m, 1H), 1.14 (d, 3H), 0.90 (d, 3H).

tilled at 200 ^ꢀC/0.08 mmHg. H NMR (CDCl₃) d 7.90

1

(m, 2H), 7.44 (m, 2H), 7.26 (m, 1H), 7.15–7.00 (m, 4H).

MS (EI+) m/z 286 (M⁺), 251, 169, 139, 77.

4-(4-Chlorophenoxy)phenyl phosphonic dichloride (20).

Prepared from 42 using _ꢀgeneral procedure E in 83%

yield. Distilled at 200–225 C/0.08–0.10 mmHg. ¹H NMR

(CDCl₃) d 7.91 (m, 2H), 7.39 (m, 2H), 7.12–7.00 (m, 4H).

MS (EI+): m/z 320 (M⁺), 285, 175, 168, 139, 111, 99.

Methyl (2R,ꢀR)-2-(4-methoxyphenyl)-2-oxo-ꢀ-phenylme-

thyl-1,3,2-oxazaphosphorinane-3-acetate (diastereomer 1)

(28b). Prepared from 4-methoxyphenylphosphonic

dichloride and 17 in 33% yield as a colourless oil,

according to procedure G. ¹H NMR (CDCl₃) d 7.73 (m,

2H), 7.35–7.15 (m, 5H), 6.93 (m, 2H), 4.59 (m, 1H),

4.30–4.00 (m, 2H), 3.83 (s, 3H), 3.50 (s, 3H), 3.50–3.25

(m, 3H), 3.14 (dd, 1H), 1.87 (m, 2H). ¹³C NMR

(CDCl₃) d 172.2, 162.5, 136.8, 133.8, 129.4, 128.4, 126.7,

121.7, 113.9, 66.2, 58.5, 55.3, 51.6, 41.1, 36.3, 26.7.

4-Nitrophenyl phosphonic dichloride (21). Prepared from

43 using general procedure E. Distilled at 175–190 ^ꢀC/

0.06 mmHg. (Flaky solid at rt). MS (EI+): m/z 239

(M⁺), 204, 193.

4-(Phenylmethyl)phenyl phosphonic dichloride (22). Pre-

pared from 44 using general procedure E in 88% yield.

Distilled at 250 ^ꢀC/0.08 mmHg. ¹H NMR (CDCl₃) d

7.88 (m, 2H), 7.45–7.13 (m, 7H), 4.07 (s, 2H). ¹³C NMR

(CDCl₃) d 148.7 (d), 139.1, 132.1 (d), 130.6 (d), 129.6

(d), 129.0, 128.8, 126.7, 42.0.

Methyl (2R,ꢀR)-2-(4-methoxyphenyl)-ꢀ-(2-methylpropyl)-

2-oxo-1,3,2-oxazaphosphorinane-3-acetate (diastereomer 1)

[(R,R)-28c]. Prepared from 4-methoxyphenylpho-

sphonic dichloride and 14 in 42% yield as a colourless

oil, according to procedure G. ¹H NMR (CDCl₃) d 7.75

(m, 2H), 6.95 (m, 2H), 4.43 (m, 1H), 4.34 (m, 1H), 4.17

(m, 1H), 3.84 (s, 3H), 3.56 (s, 3H), 3.37 (m, 2H), 2.10–

1.55 (m, 5H), 1.03 (d, 3H), 0.97 (d, 3H).

4-(4-Triﬂuoromethylphenoxy)phenyl phosphonic dichlor-

ide (23). Prepared from 45 using general procedure E in

90% yield. Distilled at 250 ^ꢀC/ 0.07 mmHg. H NMR

1

(CDCl₃) d 7.96 (m, 2H), 7.69 (m, 2H), 7.15 (m, 4H). MS

(EI+): m/z 354 (M⁺), 335, 319, 236.

Methyl (2S,ꢀR)-2-(4-methoxyphenyl)-ꢀ-(2-methylpropyl)-

2-oxo-1,3,2-oxazaphosphorinane-3-acetate (diastereomer

2) [(S,R)-28c]. Prepared from 4-methoxyphenylpho-

sphonic dichloride and 14 in 39% yield as a colorless

oil, according to procedure G. ¹H NMR (CDCl₃) d 7.73

(m, 2H), 6.95 (m, 2H), 4.56 (m, 1H), 4.19 (m, 2H), 3.84

(s, 3H), 3.74 (s, 3H), 3.54 (m, 1H), 3.20 (m, 1H), 2.17

(m, 1H), 2.02 (m, 1H), 1.56 (m, 2H), 1.36 (m, 1H), 0.79

(d, 3H), 0.63 (d, 3H). ¹³C NMR (CDCl₃) d 173.7, 162.5,

133.8, 122.2, 113.9, 66.0, 55.4, 55.3, 52.0, 40.4, 38.5,

26.5, 24.5, 22.8, 21.5.

4-(Phenylmethoxy)phenyl phosphonic dichloride (24).

Prepared from 46 using general procedure E in 31%

yield. Distilled at 225–250 ^ꢀC/0.09 mmHg. MS (EI⁺):

m/z 300 (M⁺), 265, 210, 175, 91.

4-(4-Bromophenoxy)phenyl phosphonic dichloride (25).

Prepared from 47 using general procedure F in 60%

yield. Distilled at 225–250 ^ꢀC/0.06–0.08 mmHg. MS

(EI+): m/z 364 (M⁺), 329.

4⁰-Bromo-4-biphenyl phosphonic dichloride (26). Pre-

pared from 48 in quantitative yield (crude) using general

procedure F. Used directly without further puriﬁcation.

Methyl (2R,ꢀS)-2-(4-methoxyphenyl)-ꢀ-(2-methylpropyl)-

2-oxo-1,3,2-oxazaphosphorinane-3-acetate (diastereomer

1 ) R[(,S)-28c]. Prepared from 4-methoxyphenylpho-

sphonic dichloride and 13 in 33% yield as a colourless

oil, according to procedure G. ¹H NMR (CDCl₃) d

4-(4-Chlorobenzoyl)phenyl phosphonic dichloride (27).

Prepared from 49 in quantitative yield (crude) using

5476

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

7.75 (m, 2H), 6.95 (m, 2H), 4.38 (m, 2H), 4.17 (m,

1H), 3.84 (s, 3H), 3.56 (s, 3H), 3.38 (m, 2H), 2.10–

1.55 (m, 5H), 1.02 (d, 3H), 0.97 (d, 3H). ¹³C NMR

(CDCl₃) d 173.5, 162.5, 133.8, 121.7, 113.9, 66.7,

55.7, 55.3, 51.6, 41.2, 38.6, 26.9, 24.7, 23.1, 21.7.

phenylphosphonic dichloride and 10 in 60% yield as a

colourless oil, according to procedure G. ¹H NMR

(CDCl₃) d 7.84 (m, 2H), 6.95 (m, 2H), 4.54 (m, 1H),

4.35 (m, 1H), 4.12 (m, 1H), 3.83 (s, 3H), 3.69 (s, 3H),

3.90–3.70 (m, 1H), 3.15 (m, 1H), 2.91 (m, 1H), 1.90–1.60

(m, 4H). ¹³C NMR (CDCl₃) d 171.9, 162.3, 133.2, 122.0,

113.8, 65.2, 55.3, 51.9, 48.4, 47.4, 29.5, 26.5.

Methyl (2S,ꢀS)-2-(4-methoxyphenyl)-ꢀ-(2-methylpropyl)-

2-oxo-1,3,2-oxazaphosphorinane-3-acetate (diastereomer

2) [(S,S)-28c]. Prepared from 4-methoxyphenylpho-

sphonic dichloride and 13 in 26% yield as a colourless

oil, according to procedure G. ¹H NMR (CDCl₃) d 7.74

(m, 2H), 6.94 (m, 2H), 4.56 (m, 1H), 4.20 (m, 2H), 3.85

(s, 3H), 3.74 (s, 3H), 3.55 (m, 1H), 3.19 (m, 1H), 2.17

(m, 1H), 2.03 (m, 1H), 1.54 (m, 2H), 1.37 (m, 1H), 0.78

(d, 3H), 0.63 (d, 3H). ¹³C NMR (CDCl₃) d 173.7, 162.5,

133.9, 122.2, 113.9, 66.0, 55.4, 55.3, 52.0, 40.4, 38.5,

26.5, 24.5, 22.8, 21.5.

(ꢂ)-Ethyl 2-(4⁰-bromo-4-biphenylyl)-2-oxo-1,3,2-oxaza-

phosphorepane-3-acetate (28i). Prepared from 26 and 9

in 50% yield as a colourless oil, according to procedure

1

G. H NMR (CDCl₃) d 7.98 (m, 2H), 7.62 (m, 2H),

7.57 (m, 2H), 7.46 (m, 2H), 4.58 (m, 1H), 4.38 (m, 1H),

4.20 (m, 1H), 4.16 (q, 2H), 3.85 (dd, 1H), 3.20 (m, 1H),

2.95 (m, 1H), 2.0–1.6 (m, 4H), 1.24 (t, 3H). ¹³C NMR

(CDCl₃) d 171.3, 143.1, 139.2, 132.0, 131.9, 129.8,

128.8, 126.8, 122.3, 65.4, 61.0, 48.6, 47.4, 29.5, 26.6,

14.2.

(ꢂ)-Methyl

(2R*,ꢀR)-ꢀ-allyl-2-(4-methoxyphenyl)-2-

oxo-1,3,2-oxazaphosphorinane-3-acetate (diastereomers

1) (28d). Prepared from 4-methoxyphenylphosphonic

dichloride and 15 in 35% yield as a colourless oil,

according to procedure G. ¹H NMR (CDCl₃) d 7.75 (m,

2H), 6.95 (m, 2H), 5.91 (m, 1H), 5.15 (m, 2H), 4.41 (m,

2H), 4.15 (m, 1H), 3.84 (s, 3H), 3.59 (s, 3H), 3.36 (m,

2H), 2.80–2.50 (m, 2H), 2.15–1.80 (m, 2H). ¹³C NMR

(CDCl₃) d 172.3, 162.5, 134.1, 133.8, 121.8, 117.9, 113.9,

66.7, 57.0, 55.3, 51.7, 41.3, 34.4, 26.7.

(ꢂ)-Methyl 2-oxo-2-(4-phenoxyphenyl)-1,3,2-oxazaphos-

phorepane-3-acetate (28j). Prepared from 19 and 10 in

60% yield as a colourless oil, according to procedure G.

¹H NMR (CDCl₃) d 7.87 (m, 2H), 7.37 (m, 2H), 7.16

(m, 1H), 7.03 (m,4H), 4.54 (m, 1H), 4.37 (m, 1H), 4.15

(m, 1H), 3,85 (dd, 1H), 3.70 (s, 3H), 3.16 (m, 1H), 2.94

(m, 1H), 1.77 (m, 4H).

(ꢂ)-Ethyl 2-[4-(4-chlorophenoxy)-phenyl]-2-oxo-1,3,2-

oxazaphosphorepane-3-acetamide (28k). Prepared from

20 and 9 in 90% yield as a colourless oil, according to

Methyl (2R,ꢀR)-2-[4-(4-chlorophenoxy)-phenyl]-ꢀ-(2-

methylethyl)-2-oxo-1,3,2-oxazaphosphorinane-3-acetate

1

procedure G. H NMR (CDCl₃) d 7.89 (m, 2H), 7.32

(diastereomer 1) (28e). Prepared from 9c and 16 in 29%

1

(m, 2H), 7.01 (m, 2H), 6.97 (m, 2H), 4.52 (m, 1H), 4.37

(m, 1H), 4.16 (m, 1H), 4.16 (q, 2H), 3.82 (dd, 1H), 3.15

(m, 1H), 2.94 (m, 1H), 1.95–1.60 (m, 4H), 1.24 (t, 3H).

¹³C NMR (CDCl₃) d 171.3, 160.1, 154.6, 133.5, 130.0,

129.3, 125.1, 121.1, 117.8, 65.3, 61.0, 48.4, 47.3, 29.5,

26.5, 14.2.

yield as a colourless oil, according to procedure G. H

NMR (CDCl₃) d 7.68 (m, 2H), 7.33 (d, 2H), 7.01 (m,

2H), 6.97 (m, 2H), 4.46 (m, 1H), 4.22 (m, 1H), 3.88

(t, 1H), 3.47 (s, 3H), 3.42 (m, 2H), 2.17 (m, 2H),

1.96 (m, 1H), 1.14 (d, 3H), 0.92 (d, 3H). ¹³C NMR

(CDCl₃) d 172.3, 160.3, 154.5, 133.6, 130.0, 129.5,

125.5, 121.1, 117.8, 66.8, 63.1, 51.1, 41.0, 27.2, 26.7,

19.4, 19.3.

(ꢂ)-Ethyl 2-[4-(4-bromophenoxy)-phenyl]-2-oxo-1,3,2-

oxazaphosphorepane-3-acetate (28l). Prepared from 25

and 9 in 84% yield as a colourless oil, according to

1

(ꢂ)-Methyl 2-(4-biphenylyl)-2-oxo-1,3,2-oxazaphosphor-

epane-3-acetate (28f). Prepared from 9a and 10 in 48%

procedure G. H NMR (CDCl₃) d 7.90 (m, 2H), 7.47

(m, 2H), 7.01 (m, 2H), 6.92 (m, 2H), 4.54 (m, 1H), 4.38

(m, 1H), 4.16 (m, 1H), 4.16 (q, 2H), 3.82 (dd, 1H), 3.14

(m, 1H), 2.94 (m, 1H), 1.95–1.60 (m, 4H), 1.24 (t, 3H).

¹³C NMR (CDCl₃) d 171.4, 160.0, 155.3, 133.5, 132.9,

125.2, 121.4, 117.9, 116.7, 65.3, 61.0, 48.4, 47.3, 29.5,

26.5, 14.2.

1

yield as a colourless oil, according to procedure G. H

NMR (CDCl₃) d 7.97 (m, 2H), 7.67 (m, 2H), 7.60 (m,

2H), 7.45 (m, 2H), 7.37 (m, 1H), 4.59 (m, 1H), 4.38 (dd,

1H), 4.18 (m, 1H), 3.89 (dd, 1H), 3.71 (s, 3H), 3,21 (m,

1H), 2.95 (m, 1H), 1.95–1.60 (m, 4H). ¹³C NMR

(CDCl₃) d 171.9, 144.4, 140.3, 131.8, 129.2, 128.9, 127.9,

127.3, 127.0, 65.3, 51.9, 48.5, 47.5, 29.5, 26.6.

(ꢂ)-Ethyl 2-oxo-2-[4-(4-triﬂuormethylphenoxy)-phenyl]-

1,3,2-oxazaphosphorepane-3-acetate (28m). Prepared

from 23 and 9 in 88% yield as a colourless oil, accord-

(ꢂ)-Ethyl 2-oxo-2-phenyl-1,3,2-oxazaphosphorepane-3-

acetate (28g). Prepared from phenylphosphonic

dichloride and 9 in 68% yield as a colourless oil,

according to procedure G. ¹H NMR (CDCl₃) d 7.92 (m,

2H), 7.46 (m, 3H), 4.57 (m, 1H), 4.35 (m, 1H), 4.17 (m,

1H), 4.15 (q, 2H), 3.85 (dd, 1H), 3.15 (m, 1H), 2.93 (m,

1H), 1.90–1.60 (m, 4H), 1.22 (t, 3H). ¹³C NMR (CDCl₃)

d 171.3, 131.6, 131.3, 130.6, 128.3, 65.3, 61.0, 48.6, 47.4,

29.5, 26.5, 14.2.

1

ing to procedure G. H NMR (CDCl₃) d 7.94 (m, 2H),

7.61 (m, 2H), 7.09 (m, 4H), 4.55 (m, 1H), 4.39 (m, 1H),

4.17 (m, 1H), 4.16 (q, 2H), 3.83 (dd, 1H), 3.16 (m, 1H),

2.95 (m, 1H), 2.00–1.55 (m, 4H), 1.24 (t, 3H). ¹³C NMR

(CDCl₃) d 171.4, 159.3, 159.0, 133.6, 127.3, 126.2, 119.0,

118.9, 65.4, 61.1, 48.4, 47.4, 29.5, 26.5, 14.2.

(ꢂ)-Ethyl 2-oxo-2-[4-(phenylmethyl)phenyl-1,3,2-oxaza-

phosphorepane-3-acetate (28n). Prepared from 22 and 9

in 44% yield as a colorless oil, according to procedure

(ꢂ)-Methyl 2-(4-methoxyphenyl)-2-oxo-1,3,2-oxazapho-

sphorepane-3-acetate (28h). Prepared from 4-methoxy-

1

G. H NMR (CDCl₃) d 7.82 (m, 2H), 7.40–7.10 (m,

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

5477

7H), 4.55 (m, 1H), 4.33 (m, 1H), 4.23–4.06 (m, 1H), 4.13

(q, 2H), 4.00 (s, 2H), 3.83 (dd, 1H), 3.14 (m, 1H), 2.92

(m, 1H), 2.00–1.55 (m, 4H), 1.20 (t, 3H). ¹³C NMR

(CDCl₃) d 171.1 (d), 144.7 (d), 140.0, 131.3 (d), 128.7,

128.6 (d), 128.3, 126.0, 65.0 (d), 60.7, 48.3 (d), 47.2 (d),

41.7, 29.2, 26.3, 13.9.

(ꢂ)-Ethyl 2-(4-methoxyphenyl)-2-oxo-1,3,2-oxazaphos-

phoronane-3-acetate (28t). Prepared from 4-methoxy-

phenylphosphonic dichloride and 12 in 7% yield as a

colourless oil, according to procedure G. ¹H NMR

(CDCl₃) d 7.94 (m, 2H), 6.95 (m, 2H), 4.55 (m, 2H),

4.19 (m, 2H), 4.01 (m, 1H), 3.84 (s, 3H), 3.70 (dd, 1H),

3.10 (m, 1H), 3.81 (m, 1H), 2.00–1.40 (m, 8H), 1.28 (t,

3H). ¹³C NMR (CDCl₃) d 171.2, 162.3, 133.8, 121.7,

113.8, 60.9, 60.5, 55.3, 45.9, 42.3, 27.7, 24.3, 19.1, 18.5,

14.2.

(ꢂ)-Ethyl 2-(4-chlorobenzoylphenyl)-2-oxo-1,3,2-oxaza-

phosphorepane-3-acetate (28o). Prepared from 27 and 9

in 59% yield as a colourless oil, according to procedure

G. ¹H NMR (CDCl₃) d 8.06 (m, 2H), 7.81 (m, 2H), 7.75

(m, 2H), 7.48 (m, 2H), 4.59 (m, 1H), 4.39 (m, 1H), 4.20

(m, 1H), 4.17 (q, 2H), 3.86 (dd, 1H), 3.15 (m, 1H), 2.96

(m, 1H), 1.95-1.60 (m, 4H), 1.25 (t, 3H).

(ꢂ)-Ethyl 2-oxo-2-(4-phenoxyphenyl)-1,3,2-oxazaphos-

phorinane-3-acetate (28u). Prepared from 19 and 8 in

44% yield as a colourless oil, according to procedure G.

¹H NMR (CDCl₃) d 7.85 (m, 2H), 7.38 (m, 2H), 7.18

(m, 1H), 7.04 (m, 4H), 4.50 (m, 1H), 4.23 (m, 1H), 4.14

(q, 2H), 4.02 (dd,1H), 3.68 (dd, 1H), 3.41 (m, 1H), 3.28

(m, 1H), 2.12 (m, 1H), 2.03 (m, 1H), 1.23 (t, 3H).

(ꢂ)-Ethyl 2-oxo-2-(4-phenylmethoxyphenyl)-1,3,2-oxa-

zaphosphorepane-3-acetate (28p). Prepared from 24 and

9 in 38% yield as a colourless oil, according to procedure

G. ¹H NMR (CDCl₃) d 7.85 (m, 2H), 7.37 (m, 5H), 7.01

(m, 2H), 5.10 (s, 2H), 4.53 (m, 1H), 4.34 (m, 1H), 4.14 (m,

1H), 4.14 (q, 2H), 3.82 (dd, 1H), 3.14 (m, 1H), 2.92 (m,

1H), 1.95–1.60 (m, 4H), 1.22 (t, 3H). ¹³C NMR (CDCl₃)

d 171.4, 161.4, 136.5, 133.2, 128.7, 128.1, 127.5, 122.4,

114.7, 70.0, 65.2, 61.0, 48.6, 47.4, 29.5, 26.5, 14.2.

(ꢂ)-Methyl 2-oxo-2-(4-phenoxyphenyl)-1,3,2-oxazaphos-

phorocane-3-acetate (28v). Prepared from 19 and 11 in

12% yield as a colourless oil, according to procedure G.

¹H NMR (CDCl₃) d 7.92 (m, 2H), 7.36 (m, 2H), 7.16

(m, 1H), 7.03 (m, 4H), 4.47 (m, 2H), 4.05 (m, 1H), 3.72

(m, 1H), 3.69 (s, 3H), 3.19 (m, 1H), 3.02 (m, 1H), 2.10–

1.35 (m, 6H). ¹³C NMR (CDCl₃) d 171.6, 160.6, 155.9,

133.5, 129.9, 124.7, 124.2, 119.9, 117.6, 66.1, 51.8, 46.0,

45.4, 28.6, 25.1, 24.4.

(ꢂ)-Ethyl 2-N-(4-chlorobenzoyl)-4-aminophenyl-2-oxo-

1,3,2-oxazaphosphorepane-3-acetate (28q). A solution of

28y in methanol was hydrogenated with Pd on carbon

(10%) for 1 h at atm pressure. Filtration and concen-

tration aﬀorded ethyl 2-(4-aminophenyl)-2-oxo-1,3,2-

oxazaphosphorepane-3-acetate. This crude aniline was

dissolved in CH₂Cl₂with 3 equivalents triethylamine,

the solution was cooled to 0 ^ꢀC, and 2 equivalents of 4-

chlorobenzoylchloride were added neat. After stirring

overnight at rt, the mixture was concentrated under

reduced pressure, redissolved in ethylacetat and washed

with water and brine. After ﬁltration and evaporation,

the title compound was puriﬁed by ﬂash chromato-

graphy. Yield: 61% (colourless oil). ¹H NMR (CDCl₃) d

9.49 (s, 1H), 7.92 (m, 2H), 7.71 (m, 4H), 7.36 (m, 2H),

4.50 (m, 1H), 4.25–4.00 (m, 2H), 4.12 (q, 2H), 3.83 (dd,

1H), 3.07 (m, 1H), 2.87 (m, 1H), 1.90–1.55 (m, 4H), 1.21

(t, 3H). ¹³C NMR (CDCl₃) d 171.2, 165.3, 141.6, 138.0,

133.1, 132.0, 129.2, 128.7, 125.3, 120.3, 65.4, 61.1, 48.8,

47.4, 29.4, 26.5, 14.2.

(ꢂ)-Ethyl 2-(4⁰-bromo-4-biphenylyl)-2-oxo-1,3,2-oxaza-

phosphorinane-3-acetate (28w). Prepared from 26 and 8

in 62% yield as a colourless oil, according to procedure

1

G. H NMR (CDCl₃) d 7.95 (m, 2H), 7.72–7.42 (m,

6H), 4.53 (m, 1H), 4.26 (m, 1H), 4.15 (q, 2H), 4.06 (dd,

1H), 3.74 (dd, 1H), 3.47 (m, 1H), 3.31 (m, 1H), 2.16 (m,

1H), 2.03 (m, 1H), 1.23 (t, 3H).

(ꢂ)-Methyl 2-(4⁰-bromo-4-biphenylyl)-2-oxo-1,3,2-oxaz-

aphosphorocane-3-acetate (28x). Prepared from 25 and

11 in 30% yield as a colourless oil, according to proce-

dure G. ¹H NMR (CDCl₃) d 8.02 (m, 2H), 7.63 (m, 2H),

7.58 (m, 2H), 7.47 (m, 2H), 4.50 (m, 2H), 4.07 (m, 1H),

3.77 (m, 1H), 3.71 (s, 3H), 3.23 (m, 1H), 3.05 (m, 1H),

2.00–1.33 (m, 6H).

(ꢂ)-Ethyl 2-oxo-2-(4-nitrophenyl)-1,3,2-oxazaphosphor-

epane-3-acetate (28y). Prepared from 21 and 9 in 49%

yield as a white solid, according to procedure G. H

NMR (CDCl₃) d 8.28 (m, 2H), 8.12 (m, 2H), 4.59 (m,

1H), 4.40 (m, 1H), 4.30–4.10 (m, 1H), 4.17 (q, 2H), 3.82

(dd, 1H), 3.10 (m, 1H), 2.94 (m, 1H), 2.00–1.65 (m, 4H),

1.25 (t, 3H). ¹³C NMR (CDCl₃) d 171.1, 149.8, 138.0,

132.6, 123.1, 65.9, 61.3, 48.4, 47.4, 29.4, 26.6, 14.2.

(ꢂ)-Ethyl 2-(4-methoxyphenyl)-2-oxo-1,3,2-oxazaphos-

phorinane-3-acetate (28r). Prepared from 4-methoxy-

phenyl phosphonic dichloride and 8, according to

1

procedure G. H NMR (CDCl₃) d 7.83 (m, 2H), 6.96

(m, 2H), 4.49 (m, 1H), 4.30–4.05 (m, 3H), 3.98 (dd, 1H),

3.84 (s, 3H), 3.65 (dd, 1H), 3.41 (m, 1H), 3.28 (m, 1H),

2.07 (m, 2H), 1.23 (t, 3H).

(ꢂ)-Methyl 2-(4-methoxyphenyl)-2-oxo-1,3,2-oxazapho-

sphorocane-3-acetate (28s). Prepared from 4-methox-

yphenylphosphonic dichloride and 11 in 41% yield as a

colourless oil, according to procedure G. ¹H NMR

(CDCl₃) d 7.89 (m, 2H), 6.95 (m, 2H), 4.46 (m, 2H),

4.03 (m, 1H), 3.84 (s, 3H), 3.73 (dd, 1H), 3.69 (s, 3H),

3.16 (m, 1H), 3.00 (m, 1H), 1.95–1.65 (m, 6H). ¹³C

NMR (CDCl₃) d 171.7, 162.2, 133.3, 122.2, 113.7, 66.0,

55.3, 51.8, 46.2, 45.4, 28.7, 25.2, 24.4.

General procedure H: formation of carboxylic acids 29

from the esters 28

A solution of ester 28 (0.26 mmol) in methanol (2 mL)

and aqueous sodium hydroxide (2 M, 2 mL) was stirred

overnight at rt, acidiﬁed with 4 M AcOH and extracted

with EtOAc/H₂O. The aqueous phase was extracted

with EtOAc, and the combined organic layers were

washed with brine, dried (MgSO₄) and concentrated

5478

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

under reduced pressure. The residue was puriﬁed by

chromatography (chloroform/methanol/acetic acid

80:20:1) or by crystallization to aﬀord the carboxylic

acid 29.

(ꢂ)-ꢀ-Allyl-2-(4-methoxyphenyl)-2-oxo-1,3,2-oxazapho-

sphorinane-3-acetic acid (29d). Prepared from 28d in

78% yield as a white solid, according to procedure H.

¹H NMR (CD₃OD) d 7.78 (m, 2H), 7.02 (m, 2H), 5.95

(m, 1H), 5.20 (m, 1H), 5.12 (m, 1H), 4.38 (m, 1H), 4.20

(m, 2H), 3.85 (s, 3H), 3.38 (m, 2H), 2.66 (m, 2H), 2.06

(m, 1H), 1.91 (m, 1H).

(2R,ꢀR)-2-(4-Methoxyphenyl)-ꢀ-(1-methylethyl)-2-oxo-

1,3,2-oxazaphosphorinane-3-acetic acid (29a). Prepared

from 28a in 91% yield as a crude, white solid, according

to procedure H. Recrystallized in acetone/ether. ¹H

NMR (CD₃OD) d 7.72 (m, 2H), 7.07 (m, 2H), 4.46 (m,

1H), 4.27 (m, 1H), 3.89 (s, 3H), 3.73 (t, 1H), 3.67–3.36

(m, 2H), 2.21 (m, 2H), 1.98 (m, 1H), 1.16 (d, 3H), 1.02

(d, 3H). ¹³C NMR (CD₃OD) 174.5 (d), 164.4 (d), 134.6

(d), 122.6 (d), 115.3 (d), 68.9 (d), 65.0 (d), 55.9, 42.6,

28.3 (d), 27.9 (d), 20.1, 19.8.

(2R,ꢀR)-2-[4-(4-Chlorophenoxy)-phenyl]-ꢀ-(2-methyle-

thyl)-2-oxo-1,3,2-oxazaphosphori-nane-3-acetic acid (29e).

Prepared from 28e in 70% yield as a white solid,

1

according to procedure H. H NMR (CD₃OD) d 7.75

(m, 2H), 7.39 (m, 2H), 7.05 (m, 4H), 4.43 (m, 1H), 4.25

(m, 1H), 3.67 (t, 1H), 3.60–3.30 (m, 2H), 2.30–2.05 (m,

2H), 1.95 (m, 1H), 1.10 (d, 3H), 0.97 (d, 3H). ¹³C

NMR (CD₃OD) d 175.4, 162.0, 156.0, 135.0, 131.2,

130.6, 126.2, 122.4, 119.0, 69.1, 65.7, 42.8, 28.3, 27.9,

20.1, 20.0.

(2R,ꢀR)-2-(4-Methoxyphenyl)-2-oxo-ꢀ-phenylmethyl-

1,3,2-oxazaphosphorinane-3-acetic acid (29b). Prepared

from 28b in 46% yield as a white solid, according to

procedure H. ¹H NMR (CD₃OD) d 7.79 (m, 2H),

7.40–7.20 (m, 5H), 7.04 (m, 2H), 4.50 (m, 1H), 4.17 (m,

2H), 3.87 (s, 3H), 3.60–3.30 (m, 3H), 3.14 (dd, 1H), 1.91

(m, 2H). ¹³C NMR (CD₃OD) d 164.8, 139.2, 135.3,

130.9, 129.8, 128.0, 122.7, 115.5, 68.8, 56.3, 43.3, 37.9,

28.1.

General procedure I: preparation of diethyl aryl phos-

phonates 40–49

Diethyl aryl phosphonates were prepared according to

literature starting from aryl bromides or aryl triﬂates.^18a

All aryl phosphonates were puriﬁed by column chro-

matography with ethyl acetate as the eluent.

(2R,ꢀR)-2-(4-Methoxyphenyl)-ꢀ-(2-methylpropyl)-2-

oxo-1,3,2-oxazaphosphorinane-3-acetic acid (R,R)-29c.

Prepared from (R,R)-28c in 90% as a white solid,

Diethyl 4-biphenyl phosphonate (40). Prepared from

bromobiphenyl in 76% yield as a colourless oil,

according to procedure I. H NMR (CDCl₃) d 7.88 (m,

1

according to procedure H. H NMR (CD₃OD) d 7.82

(m, 2H), 7.07 (m, 2H), 4.46 (m, 1H), 4.23 (m, 2H), 3.89

(s, 3H), 3.43 (m, 2H), 2.04 (m, 2H), 1.84 (m, 2H), 1.69

(m, 1H), 1.05 (d, 3H), 1.03 (d, 3H).

2H), 7.68 (m, 2H), 7.61 (m, 2H), 7.50–7.35 (m, 3H), 4.15

(m, 4H), 1.35 (t, 6H). ¹³C NMR (CDCl₃) d 145.2 (d),

140.0, 132.3 (d), 129.0, 127.6 (d), 127.3, 127.0 (d), 125.7,

62.2 (d), 16.4 (d).

(2S,ꢀR)-2-(4-Methoxyphenyl)-ꢀ-(2-methylpropyl)-2-

oxo-1,3,2-oxazaphosphorinane-3-acetic acid ((S,R)-29c).

Prepared from (S,R)-28c in 98% yield as a white solid,

Diethyl 4-phenoxyphenyl phosphonate (41). Prepared

from 4-phenoxyphenyl triﬂuoromethanesulfonate in

87% yield as a colourless oil, according to procedure I.

¹H NMR (CDCl₃) d 7.77 (m, 2H), 7.38 (m, 2H), 7.19 (m,

1H), 7.05 (m, 2H), 7.01 (m, 2H), 4.11 (m, 4H), 1.31 (t,

6H). ¹³C NMR (CDCl₃) d 161.4 (d), 155.5, 133.9 (d),

130.0, 124.6, 121.8 (d), 120.1, 117.6 (d), 62.2 (d), 16.3

(d).

1

according to procedure H. H NMR (CD₃OD) d 7.79

(m, 2H), 7.09 (m, 2H), 4.58 (m, 1H), 4.36 (m, 1H), 4.01

(m, 1H), 3.90 (s, 3H), 3.57 (m, 1H), 3.31 (m, 1H), 2.27

(m, 1H), 2.14 (m, 1H), 1.62 (m, 2H), 1.42 (m, 1H), 0.82

(d, 3H), 0.59 (d, 3H).

(2R,ꢀS)-2-(4-Methoxyphenyl)-ꢀ-(2-methylpropyl)-2-

oxo-1,3,2-oxazaphosphorinane-3-acetic acid ((R,S)-29c).

Prepared from (R,S)-28c in 76% yield as a white solid,

Diethyl 4-(4-chlorophenoxy)-phenyl phosphonate (42).

Prepared from 4-bromo-4⁰-chloro phenyl ether in 75–

90% yield as a colourless oil, according to procedure I.

¹H NMR (CDCl₃) d 7.77 (d, 2H), 7.35 (m, 2H), 7.02 (m,

2H), 6.99 (m, 2H), 4.12 (m, 4H), 1.33 (t, 6H). ¹³C NMR

(CDCl₃) d 160.9 (d), 154.2, 134.0 (d), 130.1, 129.7, 122.6

(d), 121.3, 117.7 (d), 62.1 (d), 16.4 (d).

1

according to procedure H. H NMR (DMSO-d₆) d 7.67

(m, 2H), 7.03 (m, 2H), 4.28 (m, 1H), 4.10 (m, 2H),

3.81 (s, 3H), 3.23 (m, 2H), 2.00–1.60 (m, 5H), 0.94

(m, 6H). ¹³C NMR (DMSO-d₆) d 174.0, 161.8, 133.2,

122.5, 114.1, 67.3, 55.2, 55.2, 41.1, 38.1, 26.6, 24.1,

23.2, 21.4.

Diethyl 4-nitrophenyl phosphonate (43). Prepared from

1-bromo-4-nitrobenzene in 96% yield as a colourless oil,

(2S,ꢀS)-2-(4-Methoxyphenyl)-ꢀ -(2-methylpropyl)-2-

oxo-1,3,2-oxazaphosphorinane-3-acetic acid [(S,S]-29c).

Prepared from (S,S)-28c in 80% yield as a white solid,

1

according to procedure I. H NMR (CDCl₃) d 8.31 (m,

2H), 8.01 (m, 2H), 4.18 (m, 4H), 1.35 (t, 6H). ¹³C NMR

(CDCl₃) d 150.3 (d), 135.9 (d), 133.0 (d), 123.4 (d), 62.8

(d), 16.4 (d).

1

according to procedure H. H NMR (DMSO-d₆) d 7.64

(m, 2H), 7.05 (m, 2H), 4.36 (m, 1H), 4.20 (m, 1H), 3.90

(m, 1H), 3.81 (s, 3H), 3.37 (m, 1H), 3.12 (m, 1H), 2.10

(m, 1H), 1.95 (m, 1H), 1.75–1.20 (m, 3H), 0.75 (d, 3H),

0.51 (d, 3H). ¹³C NMR (DMSO-d₆) d 174.1, 163.1,

162.0, 133.4, 122.8, 114.0, 66.4, 55.4, 54.9, 40.3, 37.6,

25.8, 24.0, 22.8, 21.0.

Diethyl 4-(phenylmethyl)phenyl phosphonate (44). Pre-

pared from phenyl-(4-bromophenyl)methane in 85%

1

yield as a colourless oil, according to procedure I. H

NMR (CDCl₃) d 7.72 (m, 2H), 7.35–7.14 (m, 7H), 4.10

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

5479

1

(m, 4H), 4.02 (s, 2H), 1.31 (t, 6H). ¹³C NMR (CDCl₃) d

145.9 (d), 140.0, 132.0 (d), 129.0, 129.0 (d), 128.6, 126.4,

126.0 (d), 62.0 (d), 42.0, 16.3 (d).

mmHg was suﬃciently pure for the following step. H

NMR (CDCl₃) d 7.82 (m, 2H), 6.98 (m, 2H), 4.09 (q,

2H), 1.44 (t, 3H). ¹³C NMR (CDCl₃) d 162.8, 132.3 (d),

131.4 (d), 114.9 (d), 63.8, 14.6.

Diethyl 4-triﬂuoromethylphenoxyphenyl phosphonate

(45). Prepared from 4-(4-triﬂuoromethylphenoxy)-phe-

nyltriﬂuoromethanesulfonate in 51% yield as a colour-

Dichloro-(4-phenoxyphenyl)phosphine (50b). Prepared

from phenyl ether by dichlorophosphinylation with

aluminium chloride in 9% yield as an oil distilling at

150 ^ꢀC/0.05 mmHg (Kugelrohr oven). ¹H NMR

(CDCl₃) d 7.83 (m, 2H), 7.40 (m, 2H), 7.20 (m, 1H),

7.12–7.02 (m, 4H). ¹³C NMR (CDCl₃) d 161.9, 155.3,

133.8 (d), 132.4 (d), 130.1, 124.8, 120.3, 117.8 (d).

1

less oil, according to procedure I. H NMR (CDCl₃) d

7.83 (m, 2H), 7.63 (m, 2H), 7.12 (m, 2H), 7.09 (m, 2H),

4.14 (m, 4H), 1.34 (t, 6H). ¹³C NMR (CDCl₃) d 159.8

(d), 158.9, 134.1 (d), 127.4 (q), 126.3 (q), 124.0 (q), 23.7

(d), 119.3, 118.7 (d), 62.2 (d), 16.4 (d).

Diethyl 4-(phenylmethoxy)-phenyl phosphonate (46).

Prepared from 4-(phenylmethoxy)-phenyltriﬂuoro-

methanesulfonate in 85% yield as a colourless oil,

Dichloro-(4-(4-chlorophenoxy)-phenyl)-phosphine (50c).

Prepared by dichlorophosphinylation with aluminium

chloride of 4-chlorophenyl phenyl ether in 14% yield as

an oil of bp: 125–135 ^ꢀC/0.05 mmHg. ¹H NMR (CDCl₃)

d 7.85 (m, 2H), 7.35 (m, 2H), 7.05 (m, 2H), 7.00 (m,

2H). ¹³C NMR (CDCl₃) d 161.3, 153.9, 132.4 (d), 130.1,

129.9, 121.5, 117.9 (d).

1

according to procedure I. H NMR (CDCl₃) d 7.75 (m,

2H), 7.46–7.29 (m, 5H), 7.03 (m, 2H), 5.09 (s, 2H), 4.09

(m, 4H), 1.30 (t, 6H). ¹³C NMR (CDCl₃) d 162.1 (d),

136.2, 133.8 (d), 128.7, 128.2, 127.5, 119.8 (d), 114.9 (d),

70.0, 62.0 (d), 16.3 (d).

General procedure K: allyl/Arbuzov sequence

Diethyl 4-(4-bromophenoxy)-phenyl phosphonate (47).

Prepared from 4-bromophenyl ether in 49% yield as a

colourless oil, according to procedure I. ¹H NMR

(CDCl₃) d 7.77 (m, 2H), 7.49 (m, 2H), 7.02 (m, 2H),

6.94 (m, 2H), 4.12 (m, 4H), 1.33 (t, 6H). ¹³C NMR

(CDCl₃) d 160.8 (d), 154.8, 134.0 (d), 133.1, 122.7 (d),

121.7, 117.7 (d), 117.2, 62.1 (d), 16.4 (d).

The (aryl)dichlorophosphine was dissolved at 0.25 M in

dry diethyl ether under argon and ice cooling. Pyridine

(2.2 molar equivalents) was added, followed by allyl

alcohol (2.2 molar equivalents). After 2 h at room tem-

perature, the mixture was ﬁltered under minimal expo-

sure to air, the ﬁlter washed with additional dry diethyl

ether, and the ﬁltrate concentrated in vacuo. Solvent

residues were removed in high vacuum. The residue was

heated at 130 ^ꢀC overnight and chromatographed on

silica gel in a gradient of ethyl acetate in toluene rising

from 0 to 100%. Fractions containing product were

identiﬁed by TLC (iodine vapour), combined, and con-

centrated in vacuo, yielding the pure product as an oil.

Diethyl 4⁰-bromo-4-biphenyl phosphonate (48). Prepared

from 4,4⁰-dibromobiphenyl in 41% yield as a colourless

1

oil, according to procedure I. H NMR (CDCl₃) d 7.88

(m, 2H), 7.64 (m, 2H), 7.59 (m, 2H), 7.47 (m, 2H), 4.15

(m, 4H), 1.34 (t, 6H). ¹³C NMR (CDCl₃) d 143.9 (d),

138.9, 132.4 (d), 132.1, 128.9, 127.0 (d), 127.6 (d), 122.6,

62.2 (d), 16.4 (d).

(ꢂ)-Allyl Allyl-(4-ethoxyphenyl)-phosphinate (51d). Pre-

pared from 50a by the allyl/Arbuzov sequence in 53%

yield as an oil. H NMR (CDCl₃) d 7.69 (m, 2H), 6.96

(m, 2H), 5.91 (m, 1H), 5.77 (m, 1H), 5.32 (m, 1H), 5.20 (m,

1H), 5.17–5.00 (m, 2H), 4.53 (m, 1H), 4.30 (m, 1H), 4.08

(q, 2H), 2.78 (m, 2H), 1.44 (t, 3H). ¹³C NMR (CDCl₃) d

162.3 (d), 133.8 (d), 133.3 (d), 127.4 (d), 121.1 (d), 120.3

(d), 117.5, 114.5 (d), 64.9 (d), 63.6, 36.2 (d), 14.7.

Diethyl 4-(4-benzoyl)-phenyl phosphonate (49). Prepared

from 4-(chlorobenzoyl)-phenyltriﬂuoromethanesulfon-

ate in 88% yield as a colourless oil, according to proce-

1

dure I. H NMR (CDCl₃) d 7.95 (m, 2H), 7.83 (m, 2H),

7.76 (m, 2H), 7.48 (m, 2H), 4.16 (m, 4H), 1.36 (t, 6H). ¹³C

NMR (CDCl₃) d 194.7, 140.6 (d), 139.6, 135.1, 132.9 (d),

131.8 (d), 131.5, 129.5 (d), 128.9, 62.5 (d), 16.4 (d).

(ꢂ)-Allyl allyl(phenoxyphenyl)phosphinate (51f). Pre-

pared from 50b by the allyl/Arbuzov sequence in 70%

yield as an oil. H NMR (CDCl₃) d 7.72 (m, 2H), 7.40

(m, 2H), 7.20 (m, 1H), 7.12–7.00 (m 4H), 5.92 (m, 1H),

5.77 (m, 1H), 5.33 (m, 1H), 5.21 (m, 1H), 5.21–5.05 (m,

2H), 4.56 (m, 1H), 4.33 (m, 1H), 2.87–2.73 (m, 2H). ¹³C

NMR (CDCl₃) d 161.5 (d), 155.4, 133.9 (d), 133.1 (d),

130.1, 127.2 (d), 124.6, 123.6 (d), 120.5 (d), 120.2, 117.7,

117.5 (d), 65.0 (d), 36.2 (d).

General procedure J: dichlorophosphinylation

1

The aromatic compound was heated at reﬂux with tri-

chlorophosphine (1 molar equivalent) and either alumi-

nium chloride (0.1 molar equivalent) or tin(IV)chloride

(0.1 molar equivalent) for 3 days under a stream of

escaping argon. Unreacted trichlorophosphine was

removed by distillation at atmospheric pressure. The

crude product was distilled quickly at 0.05 mmHg, and

the distillate carefully redistilled at 0.05 mmHg to give a

lower boiling fraction of unreacted aromatic, and the

desired dichloroarylphosphine as the higher boiling

product.

(ꢂ)-Allyl allyl(4-(4-chlorophenoxy)phenyl)phosphinate

(51h). Prepared from 50c by the allyl/Arbuzov

1

sequence in 42% yield as an oil. H NMR (CDCl₃) d

7.73 (m, 2H), 7.35 (m, 2H), 7.10–6.97 (m, 4H), 5.92 (m,

1H), 5.77 (m, 1H), 5.33 (m, 1H), 5.22 (m, 1H), 5.20–5.02

(m, 2H), 4.56 (m, 1H), 4.33 (m, 1H), 2.80 (dd, 2H). ¹³C

NMR (CDCl₃) d 161.0 (d), 154.1, 134.0 (d), 133.1 (d),

Dichloro-(4-ethoxyphenyl)phosphine (50a). Prepared

from fenethole by dichlorophosphinylation with tin(IV)

chloride in 40% yield. The oil collected at 70–8 ^ꢀC/0.05

5480

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

130.1, 129.8, 127.1 (d), 124.2 (d), 121.4, 120.5 (d), 117.7,

117.6 (d), 65.0 (d), 36.2 (d).

171.2 (d), 162.0 (d), 134.2 (d), 133.9 (d), 128.3 (d), 122.0

(d), 120.0 (d), 118.8, 114.5 (d), 63.6, 60.9, 50.2 (d), 46.4

(d), 35.0 (d), 14.7, 14.2.

(ꢂ)-But-3-enyl but-3-enyl(4-phenoxyphenyl)phosphinate

(51g). The aryl-dichloro-phosphine 50b was dissolved

at 0.25 M in dry diethyl ether under argon and ice cool-

ing. Pyridine (2.2 molar equivalents) was added, fol-

lowed by but-3-enol (2.2 molar equivalents). After two h

at room temperature, the mixture was ﬁltered under

minimal exposure to air, the ﬁlter washed with addi-

tional dry diethyl ether, and the ﬁltrate concentrated in

vacuo. Solvent residues were removed in high vacuum.

4-bromobut-1-ene (1 molar equivalent) was added, the

solution heated at 120 ^ꢀC overnight and chromato-

graphed on silica gel in a gradient of ethyl acetate in

toluene rising from 0 to 100%. Fractions containing

product were identiﬁed by TLC (iodine vapour), com-

bined, and concentrated in vacuo, yielding the pure 3-

(ꢂ)-N-Allyl-N-ethoxycarbonylmethyl-4-ethoxyphenyl(pent-

4-enyl)phosphinamide (52e). Prepared from 51e by ami-

dation with N-allyl-glycine ethyl ester in 35% yield as

1

an oil. H NMR (CDCl₃) d 7.77 (m, 2H), 6.96 (m, 2H),

5.80–5.61 (m, 2H), 5.20–5.08 (m, 2H), 5.04–4.92 (m,

2H), 4.15 (q, 2H), 4.08 (q, 2H), 3.83 (dd, 1H), 3.70 (dd,

1H), 3.57 (m, 2H), 2.16–1.40 (m, 6H), 1.43 (t, 3H), 1.25 (t,

3H). ¹³C NMR (CDCl₃) d 171.2 (d), 161.9 (d), 137.5, 134.3

(d), 133.7 (d), 122.6 (d), 118.6, 115.6, 114.5 (d), 63.6, 60.9,

50.1 (d), 46.3 (d), 34.7 (d), 27.6 (d), 21.4 (d), 14.7, 14.2.

(ꢂ)-N-Allyl-allyl(4-phenoxyphenyl)phosphinamide (54f).

Prepared by amidation of 51f with allylamine in 82%

1

yield as an oil. H NMR (CDCl₃) d 7.77 (m, 2H), 7.39

1

butenyl aryl(3-butenyl)phosphinate as an oil. H NMR

(m, 2H), 7.19 (m, 1H), 7.10–6.99 (m, 4H), 6.00–5.75 (m,

2H), 5.27–5.05 (m, 4H), 3.68–3.36 (m, 2H), 2.90–2.65

(m, 2H). ¹³C NMR (CDCl₃) d 161.2, 155.6, 136.4 (d),

134.4 (d), 130.0, 128.5 (d), 124.5 (d), 124.5, 120.2, 120.1

(d), 117.6 (d), 115.8, 42.7, 36.8 (d).

(CDCl₃) d 7.71 (m, 2H), 7.40 (m, 2H), 7.20 (m, 1H), 7.11–

6.99 (m, 4H), 5.89–5.70 (m, 2H), 5.20–4.92 (m, 4H), 4.08

(m, 1H), 3.82 (m, 1H), 2.56–2.17 (m, 4H), 2.11–1.80 (m,

2H). ¹³C NMR (CDCl₃) d 161.4 (d), 155.5, 137.3 (d),

133.8, 133.7 (d), 130.1, 124.6, 124.2 (d), 120.2, 117.7 (d),

117.5, 115.0, 63.4 (d), 35.0 (d), 29.2 (d), 25.9 (d).

(ꢂ)-N-Allyl-but-3-enyl(4-phenoxyphenyl)phosphinamide

(54g). Prepared from 51g by amidation with allylamine

1

(ꢂ)-Pent-4-enyl 4-ethoxyphenyl(pent-4-enyl)phosphinate

(51e). In a procedure similar to the above, aryl-

dichlorophosphine 50a was treated ﬁrst with pent-4-enol

and then with 5-bromopent-1-ene to aﬀord the pure 4-

pentenyl aryl(4-pentenyl)phosphinate 51e in 66% yield.

¹H NMR (CDCl₃) d 7.67 (m, 2H), 6.96 (m, 2H), 5.85–

5.63 (m, 2H), 5.07–4.90 (m, 4H), 4.09 (q, 2H), 4.00 (m,

1H), 3.74 (m, 1H), 2.20–2.02 (m, 4H), 2.00–1.50 (m,

6H), 1.44 (t, 3H). ¹³C NMR (CDCl₃) d 162.1 (d), 137.5,

137.5, 133.5 (d), 121.9 (d), 115.6, 115.2, 114.6 (d), 63.6,

63.5 (d), 34.5 (d), 29.9 (d), 29.8, 29.2 (d), 21.1 (d), 14.7.

in 67% yield as an oil. H NMR (CDCl₃) d 7.77 (m,

2H), 7.39 (m, 2H), 7.19 (m, 1H), 7.11–6.99 (m, 4H),

5.98–5.71 (m, 2H), 5.55 (m, 1H), 5.10 (m, 1H), 5.03 (m,

1H), 4.97 (m, 1H), 3.73–3.37 (m, 2H), 2.74 (q, 1H), 2.37

(m, 1H), 2.21 (m, 1H), 2.11–1.86 (m, 2H). ¹³C NMR

(CDCl₃) d 161.2 (d), 155.6, 137.5 (d), 136.4 (d), 134.2

(d), 130.0, 124.9 (d), 124.5, 120.1, 117.7 (d), 115.8,

115.2, 42.9, 29.3 (d), 26.4 (d).

(ꢂ)-N-Allyl-N-(ethoxycarbonyl)methyl-allyl(4-(4-chloro-

phenoxy)phenyl)phosphinamide (52h). Prepared from 51h

by amidation with N-allyl-glycine ethyl ester in 85%

1

yield as an oil. H NMR (CDCl₃) d 7.86 (m, 2H), 7.35

General procedure L: amidation

(m, 2H), 7.03 (m, 2H), 7.00 (m, 2H), 5.93–5.63 (m, 2H),

5.22–5.08 (m, 4H), 4.17 (q, 2H), 3.88 (dd, 1H), 3.78 (dd,

1H), 3.60 (m, 2H), 2.97 (m, 2H), 1.26 (t, 3H). ¹³C NMR

(CDCl₃) d 171.1 (d), 160.7 (d), 154.2, 134.1 (d), 133.9

(d), 130.1, 129.7, 128.0 (d), 125.5 (d), 121.3, 120.2 (d),

119.0, 117.6 (d), 61.0, 50.2 (d), 46.3 (d), 34.9 (d), 14.2.

The alkenyl arylakenylphosphinate was dissolved under

argon in dry dichloromethane (to 0.5 M), and phos-

phorpentachloride (1.05 molar equivalents) was added.

After stirring at room temperature for 3 h, volatiles

were removed in vacuo, and a high vacuum (0.04

mmHg) was applied for 15 min. The residue was redis-

solved under argon in dry dichloromethane (to 0.5 M),

and the allylic amine (2 molar equivalents) was added,

followed by triethylamine (2 molar equivalents). After 3

h, the mixture was evaporated onto silica gel (1 g of

silica gel per 100 mg of product) and eluted with a gra-

dient of methanol in ethyl acetate rising from 0% to

10%. Fractions containing product were identiﬁed by

TLC (iodine vapour), and combined. Removal of sol-

vents in vacuo yielded the pure product as an oil.

General procedure M: ring closing metathesis

The diene was dissolved to 0.01 M in dichloromethane,

and Grubb’s catalyst, benzylidene-bis-(tricyclohexylpho-

sphine)-dichloro-ruthenium (0,02 molar equivalents),

was added. When the reaction had gone to completion

(within 2 h at reﬂux), silica gel (1 g silica gel per 100 mg

product) was added, volatiles removed in vacuo, and the

residue eluted in a gradient of methanol in ethyl ace-

tate rising from 0 to 10%. Fractions containing pro-

duct were identiﬁed by TLC (iodine vapour),

combined, and concentrated in vacuo to give the pure

product as an oil.

(ꢂ)-N-Allyl-N-ethoxycarbonylmethyl-allyl(4-ethoxyphe-

nyl)-phosphinamide (52d). Prepared from 51d by amida-

tion with N-allyl-glycine ethyl ester in 62% yield as an

1

oil. H NMR (CDCl₃) d 7.80 (m, 2H), 6.96 (m, 2H),

5.92–5.63 (m, 2H), 5.20–5.06 (m, 4H), 4.16 (q, 2H), 4.08

(q, 2H), 3.86 (dd, 1H), 3.71 (dd, 1H), 3.59 (m, 2H), 2.96

(m, 2H), 1.43 (t, 3H), 1.26 (t, 3H). ¹³C NMR (CDCl₃) d

(ꢂ)-Ethyl

2-(4-ethoxyphenyl)-2-oxo-azaphosphorin-4-

ene-1-acetate (53d). Prepared from 52d by ring closing

1

metathesis in 82% yield as an oil. H NMR (CDCl₃) d

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

5481

7.76 (m, 2H), 6.93 (m, 2H), 5.90–5.70 (m, 2H), 4.18–4.00

(m, 2H), 4.07 (q, 2H), 3.89 (m, 2H), 3.85 (dd, 1H), 3.61

(dd, 1H), 2.73–2.60 (m, 2H), 1.42 (t, 3H), 1.19 (t, 3H).

¹³C NMR (CDCl₃) d 170.2 (d), 161.9 (d), 133.4 (d),

126.4 (d), 123.6 (d), 120.0 (d), 114.5 (d), 63.6, 60.9, 50.2,

48.3 (d), 28.2 (d), 14.7, 14.1.

5.23–4.92 (m, 4H), 4.16 (q, 2H), 3.79 (m, 2H), 3.60 (m,

2H), 2.50–2.00 (m, 4H), 1.25 (t, 3H). ¹³C NMR (CDCl₃)

d 171.1 (d), 161.1 (d), 155.6, 137.5 (d), 134.0 (d), 133.8

(d), 130.0, 125.1 (d), 124.5, 120.1, 118.8, 117.7 (d),

115.0, 61.0, 50.0 (d), 46.3 (d), 27.5 (d), 26.2, 14.2.

(ꢂ)-Ethyl 2-oxo-2-(4-phenoxyphenyl)-azaphosphorin-4-

ene-1-acetate (53f). Prepared by alkylation of 55f as an

oil. H NMR (CDCl₃) d 7.79 (m, 2H), 7.38 (m, 2H),

7.18 (m, 1H), 7.10–6.96 (m, 4H), 6.00–5.67 (m, 2H),

4.24–4.06 (m, 3H), 3.91 (m, 1H), 3.89 (dd, 1H), 3.65 (dd,

1H), 2.74–2.62 (m, 2H), 1.20 (t, 3H). ¹³C NMR (CDCl₃)

d 170.1 (d), 161.0 (d), 155.6, 133.4 (d), 130.0, 126.5 (d),

126.5 (d), 124.5, 120.1, 119.9 (d), 117.6 (d), 61.0, 50.3,

48.2 (d), 28.2 (d), 14.1.

(ꢂ)-Ethyl 2-(4-ethoxyphenyl)-2-oxo-azaphosphoroc-6-

ene-1-acetate (53e). Prepared from 52e by ring closing

metathesis in 35% yield as an oil. H NMR (CDCl₃) d

7.84 (m, 2H), 6.94 (m, 2H), 5.76 (m, 1H), 5.55 (m, 1H),

4.28 (m, 1H), 4.16 (q, 2H), 4.06 (q, 2H), 3.76–3.64 (m,

2H), 3.54 (dd, 1H), 2.65 (m, 1H), 2.30–1.60 (m, 5H), 1.42

(t, 3H), 1.25 (t, 3H). ¹³C NMR (CDCl₃) d 171.6 (d), 161.7

(d), 132.8 (d), 129.2, 128.0, 124.7 (d), 114.5 (d), 63.5, 60.9,

47.4 (d), 47.3 (d), 25.6 (d), 24.0 (d), 21.5 (d), 14.7, 14.2.

1

General procedure O: hydrogenation

(ꢂ)-2-Oxo-2-(4-phenoxyphenyl)-azaphosphorin-4-ene (55f).

Prepared by ring closing metathesis of 54f in 87% yield.

¹H NMR (CDCl₃) d 7.77 (m, 2H), 7.38 (m, 2H), 7.17

(m, 1H), 7.10–6.96 (m, 4H), 5.97–5.69 (m, 2H), 4.07 (m,

1H), 3.84 (m, 1H), 3.13 (bs, 1H), 2.83–2.44 (m, 2H). ¹³C

NMR (CDCl₃) d 160.9 (d), 155.7, 133.3 (d), 130.0, 127.5

(d), 126.4 (d), 124.4, 120.0 (d), 120.0, 117.7 (d), 43.2 (d),

27.1 (d).

The cyclic oleﬁn, 70 mg, was shaken in ethyl acetate (2

mL) with 10% palladium on carbon (30 mg) under

hydrogen at 1 atm, until the calculated amount of

hydrogen had been absorbed. Filtration through a pad

of Celite and removal of solvent yielded the corre-

sponding saturated product in a pure state.

(ꢂ)-Ethyl 2-(4-ethoxyphenyl)-2-oxo-azaphosphorinane-

1-acetate (56d). Prepared from 53d by hydrogenation in

51% yield as an oil. H NMR (CDCl₃) d 7.81 (m, 2H),

6.95 (m, 2H), 4.20–4.00 (m, 2H), 4.08 (q, 2H), 3.63 (dd,

1H), 3.37 (dd, 1H), 3.35 (m, 1H), 3.12 (m, 1H), 2.20–

1.80 (m, 6H), 1.43 (t, 3H), 1.21 (t, 3H). ¹³C NMR

(CDCl₃) d 170.5 (d), 162.1 (d), 134.2 (d), 122.2 (d), 114.6

(d), 63.6, 60.9, 49.3, 48.9, 28.8 (d), 26.7 (d), 20.7 (d),

14.7, 14.2.

(ꢂ)-Ethyl 2-oxo-2-(4-phenoxyphenyl)-azaphosphorep-5-

ene-1-acetate (53g). Prepared from 52g by ring closing

metathesis in 21% yield as an oil. H NMR (CDCl₃) d

7.88 (m, 2H), 7.38 (m, 2H), 7.18 (m, 1H), 7.10–7.00 (m,

4H), 5.87–5.68 (m, 2H), 4.26–4.07 (m, 3H), 3.83–3.54

(m, 3H), 2.85–2.20 (m, 4H), 1.25 (t, 3H). ¹³C NMR

(CDCl₃) d 171.2 (d), 161.0 (d), 155.7, 133.5 (d), 131.7,

130.0, 127.1, 124.4, 120.0, 117.9 (d), 61.0, 47.5 (d), 44.9

(d), 27.9 (d), 22.0 (d), 14.2.

1

(ꢂ)-Ethyl 2-(4-ethoxyphenyl)-2-oxo-azaphosphorocane-

1-acetate (56e). Prepared from 53e by hydrogenation in

51% yield as an oil. H NMR (CDCl₃) d 7.82 (m, 2H),

6.93 (m, 2H), 4.23 (m, 1H), 4.08 (q, 2H), 4.06 (q, 2H),

3.54 (dd, 1H), 3.47–3.14 (m, 2H), 2.29–1.15(m, 10H),

1.42 (t, 3H), 1.19 (t, 3H). ¹³C NMR (CDCl₃) d 171.2 (d),

161.6 (d), 133.2 (d), 125.3 (d), 114.3 (d), 63.5, 60.8, 45.6

(d), 44.5 (d), 28.5 (d), 26.4, 26.1, 23.3, 22.3 (d), 14.7, 14.1.

(ꢂ)-Ethyl 2-(4-(4-chlorophenoxy)-phenyl)-2-oxo-azapho-

sphorin-4-ene-1-acetate (53h). Prepared from 52h by

ring closing metathesis in 67% yield as an oil. ¹H NMR

(CDCl₃) d 7.80 (m, 2H), 7.34 (m, 2H), 7.05–6.95 (m,

4H), 5.90–5.70 (m, 2H), 4.11 (m, 2H), 4.02–3.80 (m,

3H), 3.65 (dd, 1H), 2.68 (m, 2H), 1.20 (t, 3H). ¹³C NMR

(CDCl₃) d 170.1 (d), 160.5 (d), 154.3, 133.5 (d), 130.0,

128.1, 127.3 (d), 126.5 (d), 121.3, 119.9 (d), 117.7 (d),

61.0, 50.4, 48.1, 28.1 (d), 14.1.

1

(ꢂ)-Ethyl 2-oxo-2-(4-phenoxyphenyl)-azaphosphorinane-

1-acetate (56f). Prepared by hydrogenation of 53f in

1

practically quantitative yield. H NMR (CDCl₃) d 7.84

General procedure N: alkylation

(m, 2H), 7.39 (m, 2H), 7.19 (m, 1H), 7.10–6.97 (m, 4H),

4.12 (m, 2H), 3.67 (dd, 1H), 3.42 (dd, 1H), 3.36 (m, 1H),

3.14 (m, 1H), 2.20–1.77 (m, 6H), 1.22 (t, 3H).

To the phosphonamide suspended at 0 ^ꢀC in dry THF at

0.5 M was added butyl lithium (1.15 molar equivalents of a

1.6 M solution in hexanes). The clear solution obtained

was cooled to ꢁ78 ^ꢀC, ethyl bromoacetate (1.6 molar

equivalents) was added, and the reaction mixture left to

reach room temperature. The next day, aqueous work up

with phosphate buﬀer at pH 7 and ethyl acetate followed

by chromatography in a gradient of ethyl acetate in hexane

rising from 0 to 10% aﬀorded the pure product as an oil.

(ꢂ)-2-Oxo-2-(4-phenoxyphenyl)-azaphosphorepane-1-

acetate (56g). Prepared from 53g by hydrogenation in

1

quantitative yield as an oil. H NMR (CDCl₃) d 7.85

(m, 2H), 7.38 (m, 2H), 7.17 (m, 1H), 7.10–6.97 (m, 4H),

4.12 (q, 2H), 4.02 (dd, 1H), 3.62 (dd, 1H), 3.37 (m, 1H),

3.16 (m, 1H), 2.34–1.50 (m, 8H), 1.21 (t, 3H).

(ꢂ)-N-Allyl-N-(ethoxycarbonyl)methyl-but-3-enyl(4-phe-

noxyphenyl)phosphinamide (52g). Prepared from 54g by

alkylation with ethyl bromoacetate in 59% yield as an

oil. H NMR (CDCl₃) d 7.83 (m, 2H), 7.39 (m, 2H),

7.19 (m, 1H), 7.11–6.97 (m, 4H), 5.90–5.63 (m, 2H),

General procedure P: hydroxaminolysis

To the ethyl ester, dissolved at 0.7 M in dry methanol at

0 ^ꢀC, was added O-trimethylsilyl hydroxylamine (2

molar equivalents), followed by potassium hydroxide

1

5482

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

monohydrate (2 molar equivalents) dissolved at 1 M in

dry methanol. When TLC indicated complete conver-

sion, aqueous work up with phosphate buﬀer at pH 2

and ethyl acetate, and drying with magnesium sulphate,

followed by addition of silica gel (1 g of silica gel per

100 mg of crude product), removal of volatiles in vacuo,

and elution with a gradient of methanol in chloroform

rising from 0 to 15%, aﬀorded fractions containing the

hydroxamic acid (identiﬁed on TLC plates with fer-

richloride spray), which were combined, and con-

centrated in vacuo to give the pure hydroxamic acid.

(ꢂ)-N-Hydroxy-2-(4-ethoxyphenyl)-2-oxo-azaphosphoroc-

6-ene-1-acetamide (6e). Prepared from 53e by hydro-

xylaminolysis in 48% yield as an oil. ¹H NMR (DMSO-

d₆) d 11.5–9.5 (bs, 1H), 9.5–8.5 (bs, 1H), 7.82 (m, 2H),

6.99 (m, 2H), 5.63 (m, 2H), 4.08 (q, 2H), 3.75–3.40 (m,

3H), 3.18 (dd, 1H), 2.60–1.50 (m, 6H), 1.34 (t, 3H). ¹³C

NMR (DMSO-d₆) d 166.6, 160.8, 132.6, 128.8, 128.7,

125.2, 114.1, 63.1, 46.1, 45.7, 24.9, 23.6, 21.3, 14.4.

HRMS Calcd. for C₁₆H₂₄N₂O₄P (M+H) 339.1473,

found 339.1486.

(ꢂ) - N - Hydroxy - 2 - oxo - 2 - (4 - phenoxyphenyl) - azapho-

sphorin-4-ene-1-acetamide (6f). Prepared by hydro-

xaminolysis of 53f as an oil. ¹H NMR (CD₃CN) d 11.0–

8.0 (bs, 2H), 7.69 (m, 2H), 7.41 (m, 2H), 7.21 (m, 1H),

7.04 (m, 4H), 5.76 (m, 2H), 3.90 (m, 1H), 3.68 (m, 2H),

3.24 (m, 1H), 2.62 (m, 2H). ¹³C NMR (CD₃CN) d

167.5, 162.1, 156.6, 134.5, 131.2, 127.6, 126.6, 125.7,

121.0, 120.4, 118.7, 51.5, 49.7, 28.0. HRMS calcd for

C₁₈H₂₀N₂O₄P (M+H) 359.1160, found 359.1155.

(ꢂ)-N-Hydroxy-2-(4-ethoxyphenyl)-2-oxo-azaphosphori-

nane-1-acetamide (5d). Prepared from 56d by hydroxyl-

aminolysis in 51% yield as an oil. ¹H NMR (DMSO-d₆)

d 11.5–9.5 (bs, 1H), 9.5–8.0 (bs, 1H), 7.76 (m, 2H), 7.04

(m, 2H), 4.09 (q, 2H), 3.60-3.10 (m, 3H), 2.97 (m, 1H),

2.10–1.65 (m, 6H), 1.34 (t, 3H). ¹³C NMR (DMSO-d₆) d

165.5, 161.3, 133.7, 122.3, 114.5, 63.2, 49.2, 48.6, 27.3,

25.7, 20.4, 14.4. HRMS calcd for C₁₄H₂₂N₂O₄P (M+H)

313.1317, found 313.1316.

(ꢂ) - N - Hydroxy - 2 - oxo - 2 - (4 - phenoxyphenyl) - azapho-

sphorep-5-ene-1-acetamide (6g). Prepared from 53g by

hydroxylaminolysis in 15% yield as an oil. H NMR

(DMSO-d₆) d 11.5–9.5 (bs, 1H), 9.5–8.5 (bs, 1H), 7.87 (m,

2H), 7.45 (m, 2H), 7.22 (m, 1H), 7.09 (m, 4H), 5.74 (m,

2H), 3.75–3.50 (m, 3H), 3.26 (dd, 1H), 2.60–2.00 (m, 4H).

¹³C NMR (DMSO-d₆) d 166.5, 159.8, 155.2, 133.3, 131.1,

130.2, 127.6, 127.2, 124.4, 119.6, 117.4, 46.2, 44.0, 26.5,

21.5. HRMS calcd for C₁₉H₂₀N₂O₄P (MꢁH) 371.1161,

found 371.1170.

(ꢂ)-N-Hydroxy-2-(4-ethoxyphenyl)-2-oxo-azaphosphor-

ocane-1-acetamide (5e). Prepared from 56e by

hydroxylaminolysis in 65% yield as an oil. H NMR

(DMSO-d₆) d 11.0–9.5 (bs, 1H), 9.5–8.5 (bs, 1H), 7.74 (m,

2H), 6.99 (m, 2H), 4.07 (q, 2H), 3.55 (m, 1H), 3.40–3.10

(m, 3H), 2.24 (m, 1H), 2.00-1.20 (m, 9H), 1.34 (t, 3H). ¹³C

NMR (DMSO-d₆) d 166.7, 160.7, 132.9, 125.4, 114.1, 63.1,

45.3, 44.7, 27.3, 26.0, 25.5, 23.1, 22.1, 14.5. HRMS Calcd.

for C₁₆H₂₆N₂O₄P (M+H) 341.1630, found 341.1611.

1

(ꢂ)-N-hydroxy-2-oxo-2-(4-phenoxyphenyl)-azaphosphor-

inane-1-acetamide (5f). Prepared by hydroxylaminolysis

of 56f as an oil. ¹H NMR (CD₃CN) d 7.71 (m, 2H), 7.37

(m, 2H), 7.17 (m, 1H), 7.00 (m, 4H), 3.40 (dd, 1H), 3.24

(m, 1H), 3.19 (t, 1H), 3.00 (m, 1H), 2.00–1.65 (m, 6H).

¹³C NMR (CD₃CN) d 167.3, 162.2, 156.4, 135.0, 131.0,

125.5, 125.3, 120.8, 118.6, 51.7, 50.2, 28.3, 26.8, 21.1.

HRMS calcd for C₁₈H₂₂₂N₂O₄P (M+H) 361.1317,

found 361.1300.

(ꢂ)-N-Hydroxy-2-(4-(4-chlorophenoxy)-phenyl-2-oxo-

azaphosphorin-4-ene-1-acetamide (6h). Prepared from

53h by hydroxaminolysis in 30% yield after recrystalli-

zation from methanol/ethyl acetate. Mp: 161.5–162.5 ^ꢀC

1

(dec.) H NMR (DMSO-d₆) d 11.0–9.5 (s, 1H), 9.5–8.5

(s, 1H), 7.75 (m, 2H), 7.48 (m, 2H), 7.12 (m, 2H), 7.07 (m,

2H), 5.73 (m, 2H), 3.77 (m, 2H), 3.51 (dd, 1H), 3.30 (m,

1H), 2.85–2.40 (m, 2H). ¹³C NMR (DMSO-d₆) d 165.5,

159.3, 154.2, 133.1, 130.0, 128.2, 127.8, 126.4, 121.4, 119.7,

117.6, 50.2, 47.0, 27.0. Anal. calcd for C₁₈H₁₈Cl₂N₂O₄P:

C, 55.04; H, 4.62. Found: C, 55.09; H, 4.66.

(ꢂ)-N-Hydroxy-2-oxo-2-(4-phenoxyphenyl)-azapho-

sphorepane-1-acetamide (5g). Prepared from 56g by

hydroxaminolysis in 40% yield as an oil. ¹H NMR

(DMSO-d₆) d 11.5–9.5 (bs, 1H), 9.5–8.5 (bs, 1H), 7.82

(m, 2H), 7.45 (m, 2H), 7.22 (m, 1H), 7.10 (m, 2H), 7.04

(m, 2H), 3.50 (dd, 1H), 3.40–3.00 (m, 3H), 2.06 (m, 2H),

1.90–1.40 (m, 6H). ¹³C NMR (DMSO-d₆) d 166.7,

159.7, 155.2, 133.5, 130.2, 127.8, 124.3, 119.7, 117.3,

48.8, 47.8, 30.0, 29.3, 28.7, 20.4. HRMS Calcd. for

C₁₉H₂₄N₂O₄P (M+H) 375.1473, found 375.1496.

Enzyme assays

MMP-1 and MMP-9, derived from human dermal

ﬁbroblasts, were obtained from Chemicon (CA, USA).

MMP-3, derived from human synovial ﬁbroblasts, was

purchased from Biogenesis (England, UK). The metal-

loproteinases were activated immediately before the

assay by incubation with amino phenyl mercuric acetate

(APMA) (Sigma, MO, USA), as recommended by the

producers, and were diluted in buﬀer (50 mM Tris, 200

mM NaCl, 5 mM CaCl₂, 1 mM ZnCl₂, pH 7.5 for MMP-

3 and MMP-9, or 50 mM Tris, 200 mM NaCl, 100 mM

CaCl₂, 1 mM ZnCl₂, pH 7.5, for MMP-1). The ﬁnal con-

centration of the enzyme was 190 ng/mL (MMP-1), 330

ng/mL (MMP-3), and 50 ng/mL (MMP-9).

(ꢂ) - N - Hydroxy - 2 - (4 - ethoxyphenyl) - 2 - oxo - azapho-

sphorin-4-ene-1-acetamide (6d). Prepared from 53d by

hydroxaminolysis in 40% yield as an oil. ¹H NMR

(DMSO-d₆) d 11.5–9.5 (bs, 1H), 9.5–8.5 (bs, 1H), 7.65

(m, 2H), 7.01 (m, 2H), 5.73 (m, 2H), 4.08 (q, 2H), 3.90–

3.60 (m, 2H), 3.48 (dd, 1H), 3.25 (dd, 1H), 2.68 (m, 1H),

2.50 (m, 1H), 1.33 (t, 3H). ¹³C NMR (DMSO-d₆) d

165.6, 161.0, 132.7, 126.3, 123.9, 119.8, 114.3, 63.2, 50.1,

47.3, 27.1, 14.4. HRMS calcd for C₁₄H₂₀N₂O₄P (M+H)

311.1160, found 311.1182.

The compounds were tested for their inhibitory proper-

ties against each metalloproteinase with a ﬂuorogenic

assay, by measuring the ﬂuorescent products formed by

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

5483

the cleavage of the substrate in the presence of diﬀerent

concentrations of the inhibitors.

Experiments. Female athymic NMRI nu/nu mice 6

weeks of age were purchased from M&B (Ry, Den-

mark). The mice were allowed an acclimatization period

of 1–3 weeks before start of the experiments. The nude

mice were housed in Scantainers under semi-sterile con-

ditions where light was controlled on a 12 h light–dark

cycle. They were given free access to food (Altromin

1324), sterilized by irradiation and ﬁltered (0.2 mM)

drinking water. All handling of the mice was performed

in a laminar ﬂow bench. The bodyweight of the animals

was recorded twice weekly.

In the assay with MMP-9, ﬂuorescein-conjugated gela-

tine (Molecular Probes, OR, USA), which is so heavily

labelled that the ﬂuorescence is quenched in the

uncleaved substrate, was diluted in buﬀer to the ﬁnal con-

centration of 50 mg/mL. The Dnp-Pro-b-cyclohexyl-Ala-

Gly - Cys(Me)- His - Ala - Lys(N-Me-Abz)-NH₂peptide

(Bachem, Switzerland), which is quenched by 2,4-dini-

trophenyl (Dnp), was diluted in buﬀer to the ﬁnal con-

centration of 50 mM in the assays with MMP-1 and

MMP-3.

HT 1080 xenografts

The formation of free ﬂuorescein from the uncleaved,

quenched substrate was monitored by excitation at 485

nm and emission at 530 nm. The formation of the free

N-methyl-antranilyl moiety (N-Me-Abz) was monitored

by excitation at 350 nm and emission at 450 nm. The

maximal enzymatic activity measured in the absence of

inhibitors was approximately 10000 units in all assays,

as determined after a total incubation time of 3 h for

MMP-3, and 24 h for MMP-1 and MMP-9.

NMRI nu/nu mice were injected with a single cell sus-

pension of 2ꢃ10⁶cells in 0.2 mL of medium sub-

cutaneously in each ﬂank and tumour size was

measured twice weekly using a digital caliper. The

tumor area was used as expression of tumor size and

was calculated from two perpendicular dimensions (d₁

& d₂). The test compounds were given intraperitoneally

for 2 weeks starting day 3–6, when the tumour size was

22–24 mm². There were no signs of weight loss (data not

shown) or other adverse eﬀects after 2 weeks of dosing.

Test compounds

Mice were sacriﬁced at the end of the treatment or pre-

viously in case of (a) large tumours, (b) ulceration of

tumours or (c) for humane reasons.

For the enzyme assays, the compounds were dissolved

in DMSO at the concentration of 10 mM and were

stored at ꢁ20 ^ꢀC. For the experiments, the compounds

were diluted in buﬀer to the concentrations of 0.1 nM

to 10 mM. The ﬁnal concentration of DMSO was 0.1%

v/v.

Statistical methods

Tumour area (A) was calculated as the product of the

two diameters:

Statistical methods

A ¼ d₁Â d₂

For the enzyme assays, the data are means of at least

two independent experiments. The IC₅₀values were

calculated from the dose–response curves for each

inhibitor.

For mice with only one tumour, the area of this tumour

at each day of observation was calculated and used in

the statistical analysis. For mice with both left and right

ﬂank tumours the average of the two tumour areas was

calculated and used in the statistical analysis. For each

animal, an individual curve of the tumour areas

observed during the experiment was plotted against

time. The trend was an increasing growth rate of the

tumours. In order to ensure an approximate linear cor-

respondence between tumour data and time, the obser-

vations were transformed by taking the square root of

the tumour areas. As a summary measure of the repe-

ated measurements of tumour areas during the experi-

ment, the estimated slope of the individual curve of the

square root transformed data was used. The slope was

interpreted to be a measure of tumour growth and was

estimated using linear regression analysis. Comparison

with controls was expressed as T/C% and calculated as

follows:

In vivo experiments

For dilution of the test compounds, a mixture of 2%

carboxymethylcellulose and demineralised water was

used.

Cells

HT 1080 is a human ﬁbrosarcoma cell line that over-

expresses MMP. The cells are semi-adherent and were

cultured in Dulbecco’s modiﬁed Eagle Medium supple-

mented with 100 IU of penicillin/streptomycin, sodium

bicarbonate, glucose and 10% FBS. Medium was chan-

ged twice a week. Before inoculation, cells were har-

vested from sub-conﬂuent monolayers. The viability of

the HT 1080 cells was between 60 and 80%, determined

by the eosin exclusion test.

½A=B À 1ꢀ^ꢁ100 where

A=(overall mean tumour size at baseline+mean trend

of treatment*intended days of treatment)

B =(overall mean tumour size at baseline+mean trend

of control*intended days of treatment)

Animal models

Animals and animal welfare. All of the animal experi-

ments were conducted according to the guidelines and

ethical standards of the Danish Committee for Animal

5484

M. D. Sørensen et al. / Bioorg. Med. Chem. 11 (2003) 5461–5484

Overall mean tumour size at baseline=pooling all

treatments this is the mean of all baseline values. For

each mouse the baseline value is calculated as the square

root of the (mean) area of the tumour(s).

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Acknowledgements

The authors wish to thank Mr. Jens B. Jensen, Mrs.

Rikke H. Schaarup, Mrs. Birte Thoke-Jensen, Mrs.

Liselotte B. Gurzulidis, Mrs. Birte A. Ewertsen, Mrs.

Alette S. G. Ebbesen, and Mrs. Gitte B. Høstbo for

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Article Doi

Cyclic phosphinamides and phosphonamides, novel series of potent matrix metalloproteinase inhibitors with antitumour activity

DOI: 10.1016/j.bmc.2003.09.015

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