Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases

Article

pubs.acs.org/jmc

Synthesis and Kinetic Evaluation of Cyclophostin and Cyclipostins

Phosphonate Analogs As Selective and Potent Inhibitors of Microbial

Lipases

Vanessa Point,^†,§Raj K. Malla,^‡,§Sadia Diomande,^†Benjamin P. Martin,^‡Vincent Delorme,^†

Frederic Carriere,^†Stephane Canaan,^†Nigam P. Rath,^‡Christopher D. Spilling,*^,‡

and Jean−Franco̧

is Cavalier*^,†

^†CNRS - Aix-Marseille Universite,

13402 Marseille cedex 20, France

́

Enzymologie Interfaciale et Physiologie de la Lipolyse, UMR 7282, 31 chemin Joseph Aiguier,

^‡Department of Chemistry and Biochemistry, University of MissouriSt. Louis, 1 University Boulevard, St. Louis, Missouri 63121,

United States

S

* Supporting Information

ABSTRACT: A new series of customizable diastereomeric cis-

and trans-monocyclic enol-phosphonate analogs to Cyclo-

phostin and Cyclipostins were synthesized. Their potencies

and mechanisms of inhibition toward six representative

lipolytic enzymes belonging to distinct lipase families were

examined. With mammalian gastric and pancreatic lipases no

inhibition occurred with any of the compounds tested.

Conversely, Fusarium solani Cutinase and lipases from

Mycobacterium tuberculosis (Rv0183 and LipY) were all fully

inactivated. The best inhibitors displayed a cis conformation

(H and OMe) and exhibited higher inhibitory activities than

the lipase inhibitor Orlistat toward the same enzymes. Our results have revealed that chemical group at the γ-carbon of the

phosphonate ring strongly impacts the inhibitory eﬃciency, leading to a signiﬁcant improvement in selectivity toward a target

lipase over another. The powerful and selective inhibition of microbial (fungal and mycobacterial) lipases suggests that these

seven-membered monocyclic enol-phosphonates should provide useful leads for the development of novel and highly selective

antimicrobial agents.

attractive leads for developing speciﬁc treatments toward

various organisms involving lipolytic enzymes as essential

metabolic enzymes or virulence factors.^13,14Phosphonate

analogs of biologically active phosphates have been shown to

be extremely useful tools in investigating mechanistic details of

various enzymatic systems.^15,16Their success is usually

attributed to the nonhydrolyzability of a P−C bond

(phosphonate analog) when compared to the P−O bond of

the corresponding phosphate leading to an enhanced

compound lifetime in vivo.^17,18Moreover, phosphonates are

known to mimic in both their charge distribution and geometry

the ﬁrst transition state occurring during enzymatic carboxy-

lester hydrolysis, with the formation of an irreversible covalent

bond between the nucleophilic Oγ of the active site serine and

the phosphorus atom.¹⁹

INTRODUCTION

■

Lipases and their inhibitors have potential applications in the

ﬁelds of chemistry,¹biotechnology,²and medicine. In the latter

domain, for instance, inhibiting digestive lipases to reduce fat

absorption has become the main pharmacological approach to

the treatment of obesity during the past decade.³The best

example is the FDA-approved antiobesity drug Orlistat

(marketed as Xenical by Roche and Alli by GlaxoSmithKline),

which reacts with the nucleophilic serine residue from the

catalytic triad of pancreatic and gastric lipases and inhibits these

enzymes within the gastrointestinal tract.^4,5Orlistat is also

known to inhibit various other enzymes, like fatty acid

synthase,⁶providing this compound with potential applications

in the treatment of Alzheimer’s disease,⁷type II diabetes,⁸and

antitumoral⁶and antimycobacterial⁹⁻¹¹activities. However, this

lack of selectivity could also lead to undesirable side eﬀects,

which might aﬀect its current clinical use.

Cyclophostin (Scheme 1), a bicyclic enol-phosphate isolated

from a fermentation solution of Streptomyces lavendulae (strain

NK901093),²⁰was identiﬁed as an irreversible inhibitor of

acetylcholinesterase (AChE) with IC₅₀values in the nanomolar

In this context, designing and synthesizing selective

inhibitors of various animal and microbial lipases represent

attractive and useful probes to reveal the catalytic mechanisms

of these lipases, and especially to better understand speciﬁc

enzyme−substrate interactions.¹²Such inhibitors also represent

Received: September 6, 2012

Published: October 24, 2012

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Scheme 1. Rationale for Synthesis of Cyclophostin and Cyclipostins Analogs, And Related Structures Investigated:

Cyclophostin Molecule (1)²¹and Phosphonate Analogs (2);¹⁵Monocyclic Phosphonate Analogs to Either Cyclophostin (3−

4,¹⁶5−10) or Cyclipostins (11−12)

range.^16,20,21The unusual bicyclic enol-phosphate moiety is also

found in a second family of structurally related natural

products, named the Cyclipostins²²(Scheme 1). The

Cyclipostins possess a core structure similar to that of

Cyclophostin but are phosphate esters of long chain lipophilic

alcohols of various lengths and structures. All identiﬁed

Cyclipostins have been described to be potent inhibitors of

hormone-sensitive lipase (HSL)²²and have also been reported

to inhibit the growth of various mycobacteria including

Mycobacterium smegmatis, Mycobacterium phlei, Nocardia

abcessus, and Corynebacterium diphteriae.²³The minimum

inhibitory concentrations (MICs) obtained were similar or

even lower than those of the well-known antibiotics Rifampicin

or Penicillin G. These recent results strongly suggest that

Cyclophostin and Cyclipostins compounds can inhibit serine

hydrolases produced by these organisms, including mycobacte-

rial lipases.

In the present article, we report the synthesis and ﬁrst

mechanistic study of a new series of cyclic enol phosphonate

analogs of both Cyclophostin and Cyclipostins (Scheme 1) as

potential inhibitors of lipases. Three mammalian digestive

lipases (human pancreatic lipase, HPL; dog gastric lipase, DGL;

and guinea pig pancreatic lipase-related protein 2, GPLRP2)

and three microbial lipases (Fusarium solani Cutinase,

Mycobacterium tuberculosis Rv0183 and LipY) were chosen as

representative enzyme targets. HPL and DGL, the main lipases

involved in the digestion of dietary lipids, are sn-1,3-

regioselective and sn-3-stereoselective lipases, respectively,

acting on both triacylglycerols and diacylglycerols.²⁴GPLRP2

belongs to the pancreatic lipase gene family,²⁵but diﬀers from

HPL by its kinetic and structural properties.²⁶More precisely,

the lid domain controlling the access to the active site, as in the

case of HPL and DGL,²⁷is missing in GPLRP2 and its catalytic

serine is therefore easily accessible to the solvent.²⁵GPLRP2

exhibits lipase, phospholipase A1, and galactolipase activ-

ities,²⁸⁻³⁰and its main physiological function is the hydrolysis

of dietary galactolipids²⁹and phospholipids.²⁵Among the three

microbial lipases investigated, Cutinase is able to hydrolyze a

wide range of substrates (fatty acid esters, triacylglycerols, and

phospholipids) and possesses, like GPLRP2, the advantage to

exhibit an active site always accessible to the solvent.³¹⁻³³

Mycobacterium tuberculosis Rv0183 is a monoglyceride lipase

implicated in the architecture of the membrane and in the

degradation of extracellular monoacylglycerols,^14,34,35while

LipY is a triacylglycerol lipase belonging to the HSL family,

involved in triacylglycerol degradation during persistence³⁶and

in host lipid degradation during reactivation of the bacteria.³⁷

Both LipY and Rv0183, which may contribute to the growth

and pathogenicity of Mycobacterium tuberculosis, therefore

appear as potential drug targets against tuberculosis.

These six lipases were then chosen as representative models

belonging to diﬀerent lipase families (Table 1). They have

distinct structural properties (with or without a lid controlling

the access to the active site for instance) and display various

substrate speciﬁcities. Such diversity, covering a wide range of

lipolytic enzymes, will then allow us to access structure−activity

relationships between these targeted enzymes and the

diastereoisomeric cyclic enol-phosphonate inhibitors analogous

to Cyclipostins and Cyclophostin.

In this context, the inhibitory power and selectivity of this

new series of organophosphorous compounds have been

evaluated on the basis of the following structural properties:

(1) eﬀect of removal of conjugation between the enol-

phosphonate bond and the ester functional group; (2)

requirement of the formal lactone ring; (3) eﬀect of substituent

R²at the C-5 carbon atom; and (4) eﬀect of substituent R¹at

the phosphorus atom (Scheme 1). To reach this goal, new

synthetic routes were developed in order to easily provide a

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library of enol-phosphonate compounds bearing diﬀerent

chemical groups.

Table 1. Biochemical Properties of the Six Lipases Tested

Lipase gene

family

Presence of a

Enzyme

DGL

“lid”

Main substrates

RESULTS

■

Acid lipase

Yes (PDB ID: Triacylglycerols

a

1K8Q)

Diacylglycerols

Synthesis of Cyclophostin and Cyclipostins Phospho-

nate Analogs. The syntheses of racemic Cyclophostin 1,²¹

racemic bicyclic phosphonate analogs 2(α)/2(β),¹⁵and

unsubstituted monocyclic analogs 3 and 4¹⁶have already

been reported. In general, the methods employed in the

synthesis of 2(α)/2(β) and 4 were adapted to the synthesis of

the novel substituted monocyclic phosphonate analogs 5−10.

The precursor carbonates 13a−13c were prepared following

literature methods³⁸(Scheme 2A). Additional carbonates 13d−

13g were prepared by the cross metathesis reaction of the

parent acrolein-derived carbonate 13a with terminal alkenes

using Grubbs' second generation catalyst with copper(I) iodide

as the cocatalyst.^38,39The carbonates 13d−13g were formed in

good yields (70−79%) as a mixture of E and Z isomers in ratios

HPL

Pancreatic lipase Yes (PDB ID: Triacylglycerols

b

1LPB)

Diacylglycerols

GPLRP2 Pancreatic lipase No (PDB ID: Triacylglycerols

c

1GPL)

Phospholipids

Galactolipids

Cutinase Fungal lipase

No (PDB ID: Cutin Triacylglycerols

d

1XZL)

Phospholipids

Galactolipids

Rv0183

LipY

Monoglyceride

lipase

Structural data Monoacylglycerols

unavailable

Hormone-

sensitive lipase

Structural data Triacylglycerols

unavailable

a

b

c

d

See ref 44. See ref 45. See ref 25. See ref 31.

Scheme 2. Synthesis of Novel Monocyclic Enol-Phosphonate Analogs of Either Cyclophostin (5−10) or Cyclipostins (11−12)

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varying from 9:1 to 14:1. Palladium(0)-catalyzed reactions of

the carbonates 13c−13g with methyl acetoacetate gave the

vinyl phosphonates 14c−g (Scheme 2A) in good yields (70−

80%). The products were formed as diastereoisomeric mixtures,

but with exclusively E alkene geometry. However, the reaction

of carbonate 13b produced both the vinyl phosphonate 14b

and the allyl phosphonate 15, which is the result of the

migration of the double bond into conjugation with the phenyl

group. Interestingly, spectroscopic analysis (NMR and IR) and

X-ray crystallography showed that the allyl phosphonate 15

exists predominantly in the enol-form of β-keto ester. Both

vinyl 14b and allyl phosphonates 15 were taken on to the next

step since, after hydrogenation, they should yield the same alkyl

phosphonate.

The treatment of a methanolic solution of the vinyl

phosphonates 14b−g (or 15) with hydrogen gas over 10%

palladium on carbon gave saturated phosphonates 16a−g in

quantitative yields (Scheme 2B). As expected, hydrogenation of

either 14 or 15 gave the saturated phosphonate 16b. The

phosphonates 16b−16g were monodemethylated using sodium

iodide (1.1 equiv) in reﬂuxing acetonitrile to give the

corresponding sodium salts, which were subsequently con-

verted to monophosphonic acids 17a−g by treatment with

Amberlite IR-120H resin in methanol. The monophosphonic

acids 17a−g were cyclized, without further puriﬁcation, using a

combination of EDC, HOBt, and Hunig’s base (i-Pr NEt) to

̈

2

Figure 1. Projection view of 10(α) with 30% thermal ellipsoids

(disorder atoms omitted for clarity). This crystal structure has been

deposited at the Cambridge Crystallographic Data Centre and

allocated the deposition number CCDC 873939.

give the substituted monocyclic phosphonates 5−10 as

diastereoisomeric mixtures in 45−59% yields over the three

steps. As previously demonstrated in the synthesis of the

racemic bicyclic phosphonate analogs of Cyclophostin 2(α)

and 2(β),¹⁵compounds 5 to 10 were best described by the

relationship between the OMe on phosphorus and the H-

substituent on the C-5 carbon atom as being either in a trans

(α-isomer) or cis (β-isomer) relationship. The two racemic

diastereoisomers (α and β) were further separated by careful

silica gel column chromatography (Scheme 2B). A single crystal

X-ray diﬀraction structure of 10(α) was further obtained at an

edge resolution of ∼0.97 Å (Tables S1 to S5) thus conﬁrming

these stereochemical assignments (trans-α and cis-β). As shown

in Figure 1, the obtained structure clearly exhibits a trans

relationship between the methoxy (numbered O2−C25) and

the hydrogen atom located on the carbon numbered C3.

The initial approach to forming long chain phosphonate

esters (Scheme 2C) from the monocyclic phosphonate 4

involved demethylation and then realkylation in three separate

reaction steps. Thus, the phosphonate 4 was treated with NaI in

acetone to give the sodium salt, which was protonated by

treatment with Amberlite IR-120H in methanol. The

phosphonic acid was then realkylated using potassium

carbonate, 18-Crown-6, and octadecyl triﬂate⁴⁰to give

phosphonate 12 in 49% yield over three steps. However, we

have discovered more recently that a trans-esteriﬁcation

reaction of the phosphonate methyl ester 4 (and others)

could be performed in a single pot reaction.²¹The phosphonate

4 was then treated with a 5-fold excess of hexadecyl bromide in

toluene with a catalytic amount of nBu₄NI (TBAI) (5 mol %)

leading to the hexadecyl phosphonate 11 in 86% yields. Final

products 1−12 were fully analyzed and characterized by NMR,

HRMS, and HPLC.

toward HPL, DGL, GPLRP2, Cutinase, Rv0183, and LipY. The

pH-stat technique⁴¹was used to measure lipase activities and

quantify the inhibitory power, deﬁned here as the inhibitor

molar excess leading to 50% lipase inhibition (x_I50value).⁴²The

lipase stereopreference was also assessed through the

Diastereoselectivity Index (DI)⁴³between the two diaster-

eoisomers trans-(α) and cis-(β) as deﬁned in eq 1 (see

Experimental Section).

Inhibition Results Using Cyclic Enol-Phosphorus Com-

pounds. A signiﬁcant inhibition was obtained with the three

microbial lipases tested (Table 2). Cutinase was strongly

inactivated by most of the organophosphorous compounds, and

the eﬃciency of the inhibition was found to be dependent on

the chemical structure of each inhibitor. First, synthetic racemic

Cyclophostin 1 (x_I50= 0.96) was 4- to 10-fold more potent

than its two homologous phosphonates 2(α) (x_I50= 4.49) and

2(β) (x_I50= 8.77). With these two latter compounds, one can

also notice that Cutinase exhibited a slight stereopreference for

the trans isomer 2(α) (DI = 32.3%). Regarding the monocyclic

enol-phosphonates analogous to Cyclophostin molecules,

compounds 3 and 5, bearing respectively an alcohol instead

of an ester function and a phenyl group at the C-5 position of

the oxaphosphepine cycle, did not inhibit Cutinase. The

following classiﬁcation based on the decreasing inhibitory

potency was then obtained: 10(β) (x_I50= 1.18) > 4 (x_I50

1.39) ≅ 6(β) (x_I50= 1.42) ≥ 7(β) (x_I50= 1.98) ≅ 8(β) (x_I50

=

2.00) > 9(β) (x_I50= 2.54). In all cases x_I50values were

indicative of a very potent inhibition. In addition, Cutinase

showed a strong stereopreference for the (β) isomers having a

cis conformation. The higher diastereoselectivity (DI > 70.0%)

was then found for compounds 6(β), 7(β), and 10(β) bearing

C5, C10, and C16 aliphatic alkyl chains, respectively. Finally,

the Cyclipostin analogs 11 (x_I50= 0.56) and 12 (x_I50= 0.95) as

Lipase Inhibition. The 18 new chiral organophosphorus

compounds synthesized (i.e., compounds 1, 3 to 5, 11 and 12,

as well as trans-(α) and cis-(β) isomers of 2 and 6 to 10;

Scheme 1) were further tested for their inhibitory activity

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were found to be the Cyclophostin enol-phosphonate analogs

7(β) (x_I50= 1.13) and 9(β) (x_I50= 1.32).

Table 2. Inhibition of Cutinase, Rv0183 and LipY by Cyclic

Enol-Phosphorous Compounds and Orlistat after 30-min

Incubation Period

a

Compounds 1, 2, 4, and 6 exhibited very weak inhibitory

properties toward LipY, with 14% to 36% inhibition at a high x_I

value of 40. No inhibition of LipY was obtained with

compounds 3 and 5. Conversely, LipY displayed an

unambiguous chemopreference for the most lipophilic

phosphonates 7(β) (x_I50= 2.81), 8(β) (x_I50= 3.46), 9(β)

(x_I50= 0.60), and 10(β) (x_I50= 0.77), all bearing medium to

long alkyl chain lengths. Moreover, and as expected for a

member of the HSL family,²²LipY was strongly inactivated by

the two monocyclic enol-phosphonates 11 (x_I50= 0.83) and 12

(x_I50= 0.59) analogous to Cyclipostins. Interestingly, no

signiﬁcant diﬀerences in x_I50values were observed between the

Cyclophostin analogs 9(β) and 10(β) (mean x_I50= 0.69

0.08), and phosphonates 11 and 12 (mean x_I50= 0.71 0.12).

Such a result suggests that the presence of a long C16−C18

lipophilic alkyl chain is essential for high inhibition to take

place, whatever its positioning: either on the C-5 carbon atom

of the oxaphosphepine cycle (i.e., in 9(β) and 10(β)) or on the

phosphorus atom (i.e., in 11 and 12). As in the case of Cutinase

and Rv0183, LipY exhibited a high diastereoselectivity (28.0 ≤

DI ≤ 81.3%) for the cis-(β) isomers. The best inhibitors of

LipY were then Cyclipostin analogs 11 (x_I50= 0.83) and 12

(x_I50= 0.59) as well as Cyclophostin analogs 9(β) (x_I50= 0.60)

and 10(β) (x_I50= 0.77).

Cutinase

x_I50DI

Rv0183

LipY

compd

x_I50

DI

x_I50

DI

1

0.96

4.49

8.77

>100

1.39

>100

26.2

1.42

14.6

1.98

3.63

2.00

4.43

2.54

7.26

1.18

0.56

0.95

2.52

−

5.22

41.6

43.2

NI

−

>100

NI

−

2(α)

2(β)

3

32.3%

1.9%

NA

−

4

30.5

16.2

3.79

2.70

3.57

1.13

2.44

5.23

>100

1.32

>100

3.15

NI

>100

NI

5

6(α)

6(β)

7(α)

7(β)

8(α)

8(β)

9(α)

9(β)

10(α)

10(β)

11

>100

25.8

2.81

6.14

3.46

3.47

0.60

7.47

0.77

0.83

0.59

7.07

89.7%

76.2%

29.0%

27.2%

72.1%

16.7%

51.9%

36.3%

>97.0%

NA

80.3%

28.0%

70.5%

81.3%

>93.0%

−

12

NI

Orlistat

>200

a

x_I50values were determined as described in Experimental Section.

The inﬂuence of the incubation time on the levels of

inhibition of Cutinase, Rv0183, and LipY by inhibitors 1, 7(β),

and 11 was then investigated. As depicted in Figure 2, the

Diastereoselectivity Index (DI) between trans-(α) and cis-(β) isomers

was calculated for compounds 2 and 6 to 10 using eq 1. Results are

expressed as mean values of at least two independent assays (CV% <

5.0%). NA = not applicable. NI = no signiﬁcant inhibition observed.

well as Cyclophostin 1 (x_I50= 0.96) were found to be the best

inhibitors of Cutinase. Based on the absence of inhibition

observed with compounds 3 and 5 (x_I50> 100), a free or cyclic

(lactone) ester group appears to therefore be required to

provide the enol-phosphonates with inhibitory activity.

Concerning Rv0183, the monoglyceride lipase from

Mycobacterium tuberculosis,³⁴Cyclophostin 1 displayed a x_I50

value of 5.22 which was nearly 8-fold lower than that of its

homologous phosphonates 2(α, β) (mean x_I50= 42.4 0.8).

Low lipophilic compounds 2(α, β), 4, and 5 also exhibited

rather weak inhibitory power with x_I50values ranging from 16.2

to 43.2. Compound 3, which has an alcohol instead of an ester

function, was completely inactive toward this lipase. In contrast,

the more lipophilic enol-phosphonates 6 to 10 strongly

inhibited Rv0183, with x_I50values in the 1.1 to 5.2 range.

Surprisingly, Cyclipostins analogs 11 and 12 were not

inhibitors of Rv0183, suggesting that the positioning of the

C16−C18 alkyl chains on the phosphorus atom may be

responsible for this lack of inhibition. Rv0183 displayed a low

diastereoselectivity (DI = 17−52%) between trans-(α) and cis-

(β) isomers when using compounds 6 to 8, suggesting that

both conformations where similarly recognized when short

(C5) and medium (C10−C12) alkyl chain lengths were present

at the C-5 carbon atom. By contrast, only cis-(β) isomers of

compounds 9 and 10 bearing long (C16−C18) alkyl chains

were found to be active against the Rv0183 monoglyceride

lipase. These ﬁndings highlight the fact that the ester function is

necessary but not suﬃcient to inhibit Rv0183. This

monoglyceride lipase therefore displayed a strong chemo-

preference for lipophilic Cyclophostin enol-phosphonate

analogs and a high stereopreference for the cis-(β) isomers

bearing long C16−C18 alkyl chain length. The best inhibitors

Figure 2. Eﬀect of the incubation time on the inhibition level of

◆

□

●

Cutinase ( ), Rv0183 ( ), and LipY ( ) by compounds 1, 7(β), and

11, respectively. Each enzyme was preincubated with the correspond-

ing phosphonate compound at a ﬁxed inhibitor molar excess x_I= 4,

and the residual activity was measured over a 60-min period at various

time intervals using the pH-stat technique. Results are expressed as

mean values of at least two independent assays (CV% < 5.0%).

residual activity of all three enzymes decreased rapidly and

reached a plateau value at around 6.0% residual lipase activity

after approximately 30 min of incubation. From these inhibition

curves, the values of the half-inactivation time (t_1/2) were

determined and found to be 0.18 min for 1 acting on Cutinase,

1.44 min for 7(β) acting on Rv0183, and 0.16 min for 11 acting

on LipY. Such values thus reﬂect an extremely high rate of

inhibition of each lipase by the corresponding phosphonate

compounds.

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In order to compare the inhibitory activity of these

organophosphorous compounds with that of Orlistat, the

same kinetic parameters were also measured with this inhibitor

and each of the tested lipases. The inhibitory eﬀects of Orlistat

toward Cutinase (x_I50= 2.52), LipY (x_I50= 7.07), and Rv0183

(x_I50> 200) was found to be 2.6- to 177-fold lower than that of

pine ring adopted a nonproductive orientation. The phospho-

rus atom of 7(β) was indeed found in an opposite and

unfavorable position preventing the occurrence of any reactions

with the catalytic serine ([Ser-Oγ/P] distances >6.0 Å) and

thus the formation of a covalent bond.

Mass Spectrometry Analysis of Inhibitor-Modiﬁed En-

zymes. To investigate whether the inhibition with cyclic enol-

phosphonates occurring with microbial lipases is due to the

formation of a covalent bond with the catalytic serine, mass

spectrometry analyses were further conducted on Cutinase,

Rv0183, and LipY inhibited by compounds 4, 7(β), and 11,

respectively. Samples of the lipase−inhibitor complexes were

then subjected to MALDI-TOF mass spectrometry analyses.

The obtained m/z mass values are summarized in Table 3.

compounds 1 (x_I50= 0.96), 11 (x_I50= 0.83), and 7(β) (x_I50

=

1.13) acting on the same lipases, respectively. Conversely, while

Orlistat is a potent inhibitor of mammalian lipases, i.e. HPL

(x_I50= 18.4), GPLRP2 (x_I50= 0.53), and DGL (x_I50= 5.60),

these lipases are not inhibited by all the enol-phosphorus

compounds tested, even by using a high inhibitor molar excess

(x_I= 200) and after 2 h of incubation. To further investigate

such an absence of inhibition experimentally observed with

these cyclic enol-phosphorus compounds, in silico molecular

docking experiments were conducted on 7(β) with the

reported crystal structures of DGL,⁴⁴HPL,⁴⁵and GPLRP2²⁵

(Figure 3). In all cases, the best scoring positions obtained (i.e.

lowest energy complex) showed that the reactive oxaphosphe-

Table 3. Mass Spectrometry Analysis of Lipases Inhibited by

a

Monocyclic Enol-Phosphonate Inhibitors

m/z native lipase,

m/z inhibited

lipase, (2)

mass modiﬁcations,

(2)−(1)

species

(1)

Cutinase

Rv0183

LipY

22 187.9 Da

30 177.4 Da

47 145.6 Da

22 420.8 Da

30 494.2 Da

47 589.4 Da

+232.9 Da

+376.8 Da

+443.8 Da

a

Cutinase, Rv0183 and LipY were preincubated for 30 min at 25 °C

with compound 4 (M_w, 234.2 Da), 7(β) (M_w, 374.4 Da), and 11 (M_w,

444.3 Da) respectively, at a x_Ivalue of 40, prior to MALDI-TOF mass

spectrometry analysis.

In the presence of an inhibitor, a shift in the molecular mass

of each inhibited protein was observed, corresponding to the

molecular mass of the inhibitor and reﬂecting the covalent

binding of 4 (M_w, 234.2 Da) to Cutinase, of 7(β) (M_w, 374.4

Da) to Rv0183, as well as of 11 (M_w, 444.3 Da) to LipY.

Moreover, in order to conﬁrm the mechanism of action of these

phosphonate compounds and consequently their speciﬁc

covalent binding via a nucleophilic attack of the catalytic serine

residue to the phosphorus center, as initially argued with

human AChE,¹⁶tryptic digestion of the Cutinase-4 complex

was performed. The peptide mass ﬁngerprint (PMF)⁴⁶was

then established by MALDI-TOF mass spectrometry. Peptide

C¹⁰⁹-R¹³⁸(C¹⁰⁹PDATLIAGGYS¹²⁰QGAALAAASIEDLD-

SAIR¹³⁸) containing the catalytic Ser¹²⁰residue was detected

in the native Cutinase as a multiplet at a molecular mass of

around 2977.5 Da (Figure 4A). This catalytic peptide appeared

concomitantly with a second multiplet peptide (V¹⁶⁹-R¹⁹⁶

)

detected at a molecular mass of around 2974.5 Da. When

Cutinase was complexed with compound 4, a mass increase of

+234.14 Da was obtained for the catalytic peptide only (Figure

4B), while multiplet peptide V¹⁶⁹-R¹⁹⁶remained unchanged.

The disappearance of 2977.5 Da peptide in the inhibited lipase

followed by the appearance of a new one at m/z of 3211.7 Da

conﬁrmed that a covalent bond had been formed between the

enol-phosphonate 4 and the catalytic serine residue of Cutinase,

in accordance with the reaction mechanism depicted in Figure

4C.

Figure 3. In silico molecular docking of compound 7(β) into

crystallographic structures of (A) DGL (PDB: 1K8Q), (B) HPL

(PDB: 1LPB), and (C) GPLRP2 (PDB: 1GPL) in a van der Waals

surface representation. Hydrophobic residues (alanine, leucine,

isoleucine, valine, tryptophan, tyrosine, phenylalanine, proline, and

methionine) are highlighted in white. The side chain of the catalytic

serine residue is shown as sticks and colored in magenta. Compound

7(β) is represented in atom color-code sticks (carbon, yellow;

phosphorus, orange; oxygen, red). Structures were drawn using the

DISCUSSION

■

Cyclophostin and the Cyclipostins are known to be very potent

natural organophosphate-based inhibitors of AChE²⁰and

HSL,²²respectively. Variation of the nature of the alkyl group

at the phosphate center, methyl vs long lipophilic alkyl groups,

leads to a dramatic switch of the enzyme selectivity by these

organophosphates. This particular fact provides a unique

PyMOL Molecular Graphics System (Version 1.3, Schrodinger, LLC).

̈

The [Ser-Oγ/P] calculated distances are indicated in blue.

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Figure 4. Peptide mass ﬁngerprint spectra of the digested Cutinase, before (upper panels, A) and after (lower panels, B) a 30-min incubation period

with compound 4 at an inhibitor molar excess x_I= 40; and (C) the corresponding conﬁrmed reaction mechanism with the active site Ser¹²⁰. (A and

B ) Z o o m o n t h e r e g i o n o f i n t e r e s t . ( L e f t p a n e l s ) R e g i o n o f t h e u n m o d i ﬁ e d p e p t i d e C ^{1 0 9}− R ^{1 3 8}

(C¹⁰⁹PDATLIAGGYS¹²⁰QGAALAAASIEDLDSAIR¹³⁸) containing the catalytic Ser¹²⁰and detected at 2977.52 Da. This multiplet peak is overlaid

with a ﬁrst one corresponding to peptide V¹⁶⁹−R¹⁹⁶, detected at 2974.51 Da. (Right panels) Region in which a new peptide was expected to occur

resulting from the covalent binding of compound 4 to the catalytic serine. Mass shifts were calculated as the diﬀerence between the experimental m/z

of the modiﬁed peptide and the theoretical m/z of the unmodiﬁed catalytic peptide. The expected exact mass shift is +234.07 Da.

opportunity to investigate the enzyme selectivity of this class of

compounds or their analogs, based on structure modiﬁcation of

the parent molecule. In that way, phosphonates have garnered a

reputation for being excellent structural mimics of natural

phosphates. It is also well-known that, for phosphonate

compounds, the mode of inhibition involves the formation of

a nonhydrolyzable covalent bond between the phosphorus and

the catalytic Oγ serine residue of the enzyme active site.¹⁹For

this purpose, novel synthetic routes were proposed and a full

library of new monocyclic enol-phosphonate compounds,

analogous to either Cyclophostin or Cyclipostins (Schemes 1

and 2), has been synthesized.

the methoxy) (Scheme 3). Consequently, the isomer 18α with

the downﬁeld shift (26.6 ppm) had the phosphonate methoxy

and the 5-hydrogen substituent trans to each other.

From these experimental observations, combined with the

obtained single crystal X-ray diﬀraction structure of 10(α)

(Figure 1), it was then possible to assign without any ambiguity

the trans-α and cis-β relative stereochemistry of the monocyclic

phosphonates 5−10.

Scheme 3. Relative Stereochemistry of the Monocyclic

Phosphonates 5−10

The strategy applied, involving palladium(0)-catalyzed

addition of methyl acetoacetate to phosphono allylic carbonates

and selective monodemethylation and cyclization, allowed for

the custom synthesis of monocyclic phosphonates substituted

at the phosphorus atom or at the C-5 position. The monocyclic

phosphonates 6−10 were formed as mixtures of diaster-

eoisomers which were further easily separated and puriﬁed. The

fast eluting (α) isomer had a signal in the ³¹P NMR spectrum at

26.8 ppm, whereas the slower eluting (β) isomer had a signal at

24.2 ppm, consistent with all examples (6−10) studied. We had

earlier demonstrated in the synthesis of the racemic bicyclic

phosphonate analogs of cyclophostin 2(α) and 2(β)¹⁵that the

monocyclic phosphonate isomer 18β with the upﬁeld shift

(24.2 ppm) had the phosphonate methoxy and the 5-hydrogen

in a cis conformation (5-alkyl substituent is therefore trans to

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Overall, the chemical pathways developed here make possible

the synthesis of large libraries of fully customizable enol-

phosphorus compounds with controlled cis or trans diaster-

eochemistry.

selectivity exerted by the three lipases. Indeed, although the

best monocyclic phosphonate inhibitors all displayed a cis

conformation (i.e. (β) isomers), the group on the γ-carbon of

the phosphonate ring signiﬁcantly aﬀects and modiﬁes the

biological activity of the monocyclic analogs. Such structural

changes lead to more selective inhibitors toward the microbial

lipases tested. Rv0183 indeed reacts selectively with Cyclo-

phostin analogs bearing medium to long alkyl chains, while

LipY is only fully inactivated by highly lipophilic compounds.

Cutinase, however, is found to be less selective since it reacts

with most of the organophosphorous compounds without any

real chemopreference (Table 2). Such a high and stringent

chemoselectivity exerted by Rv0183 and LipY, or conversely

the lower one in the case of Cutinase, is consistent with the

respective substrate speciﬁcity of these three lipolytic

enzymes.^{32−34,36,37}

Comparisons with the potent but nonselective lipase

inhibitor Orlistat were very instructive. Indeed, the inhibitory

power exerted by compounds 11, 12, and 7(β) on Cutinase,

LipY, and Rv0183, respectively, was 4.5 to 176 times higher

than that of Orlistat acting on the same lipases. Moreover, with

x_I50values as low as 0.56 for 11 acting on Cutinase, which is

close to the lowest achievable value (the theoretical minimum

value being 0.50), these phosphorus compounds react in a

quasi-stoichiometric way with these enzymes. By contrast, the

enol-phosphonates had no eﬀects on HPL, DGL, and GPLRP2,

while these mammalian lipases were strongly inhibited by

Orlistat. In silico molecular docking experiments with 7(β)

suggest that these seven-membered ring phosphorus molecules

can enter the active site of HPL, DGL, and GPLRP2 but cannot

interact with the catalytic serine residue. In each case, the

lipophilic alkyl chains of the best scoring positions of 7(β)

would be stabilized by hydrophobic residues located near the

active site (Figure 3, white color). In this binding mode,

however, the reactive oxaphosphepine ring of 7(β) would adopt

a nonproductive orientation: the phosphorus atom is indeed

found in the opposite position making it impossible to react

with the catalytic serine.

Based on their structural properties previously depicted in

Scheme 1, these organophosphorous compounds have been

tested for their abilities to inhibit six lipolytic enzymes,

including mammalian digestive lipases (HPL, DGL, and

GPLRP2) and three microbial lipases (Cutinase, Rv0183, and

LipY). In view of the speciﬁc mechanism of action of these

lipolytic enzymes,²⁷the Michaelis−Menten−Henri model no

longer applies⁴⁷and therefore the K_m, K_iand IC₅₀values often

estimated for lipases are irrelevant when insoluble substrates

and/or inhibitors are involved. The use of more appropriate

kinetic constants such as the inhibitor molar excess leading to

50% lipase inhibition, i.e. x_I50value,⁴²is thus recommended

when assessing the inhibitory potency of insoluble inhibitors,

such as these cyclic enol-phosphorus derivatives. Thereby, an

x_I50value of 0.5 is synonymous with a 1:1 stoichiometric ratio

between the inhibitor and the lipase and is therefore the highest

level of inhibitory activity that can be achieved. Regarding lipase

stereopreference, this parameter has also been studied on the

basis of the Diastereoselectivity Index (DI)⁴³between the two

diastereoisomers trans-(α) and cis-(β) (see eq 1 in the

Experimental Section).

Regarding the three microbial lipases, Cutinase, Rv0183 and

LipY were all inhibited by the cyclic enol-phosphorus

compounds. The fused butyrolactone did not appear to be

necessary for activity since the monocyclic diastereomers

showed similar or even better x_I50values than those of the

bicycles 1 and 2(α, β). However, the presence of ester

functionality in conjugation with the enol-phosphate or

phosphonate bond was found essential to the biological

activity. This eﬀect was well illustrated when comparing 3

with 4. These two compounds only diﬀer at the exocyclic

functionality: the alcohol in 3 is a poor leaving group and 3 is

an extremely poor inhibitor, while compound 4 with the ester

group is more potent. This phenomenon, which depicts the

electron-withdrawing contribution of the ester moiety in

increasing the leaving group characteristics of the enol from

the P-center, is in line with mass spectrometry analyses

conﬁrming that the inhibition is due to phosphorylation of the

catalytic serine residue for each enzyme, as depicted in Figure

4C. Such ﬁndings demonstrate that the enol-phosphonate really

acts as a leaving group on reaction with the active site serine,

leading to the formation of a stoichiometric covalent enzyme−

inhibitor complex. The fact that comparable results had also

been observed when testing the inhibitory properties of the

phosphonate analogs of Cyclophostin (i.e., compounds 1 to 4)

against the houseﬂy AChE (CSMA strain) therefore suggests a

similar mode of action of this enzyme.^15,16,21The trans-2(α)

diastereoisomer of bicyclic phosphonate analogs of Cyclo-

phostin was also found to be almost 10 times more potent than

the cis-2(β) stereoisomer when tested against human AChE.¹⁵

Based on these previous experimental kinetic data, it was

suggested that the diastereopreference could be due to some

speciﬁc initial contacts between the inhibitor and the active site

amino acid residues, which ultimately led to a successful

inhibition.¹⁶In the present study, LipY was barely aﬀected by

the bicyclic molecules 1 and 2(α, β), while Rv0183 did not

discriminate between the two conformations (DI = 1.9%) and

Cutinase exhibited a slight stereopreference (DI = 32.3%) for

the trans isomer 2(β). These results illustrate the diﬀerence in

Taken together, all these ﬁndings suggest that this series of

enol-phosphonates 6−12 may be selective inhibitors of these

families of microbial lipolytic enzymes, i.e., Cutinase, mono-

glyceride lipase, and hormone sensitive lipase families.

It has been shown recently that the use of Orlistat could

signiﬁcantly reduce the growth of Mycobacterium smegmatis,¹⁰

a

nonpathogen Mycobacterium species, and of Mycobacterium

bovis BCG⁹which is closely related to the pathogen

Mycobacterium tuberculosis. In addition, some recent papers

pointed out the fact that Orlistat could also target potentially

essential enzymes involved either in the mycobacteria cell wall

biosynthesis such as the Cutinase-like Rv3802c¹¹or in the

hydrolysis of stored triacylglycerols during persistence and

reactivation phases such as LipY.³⁶Fighting tuberculosis is still

challenging, not only in third world countries but also in the so-

called industrialized world, due to the impressive recrudescence

of this major infectious disease. In that context, our

encouraging results on the selective inhibition of Cutinase as

well as of pure microbial lipolytic enzymes from Mycobacterium

tuberculosis suggest that this new class of phosphonate analogs

of Cyclophostin and Cyclipostins could be potential scaﬀolds

and may provide useful leads for the further development of

more selective antimicrobial agents, including antimycobacterial

compounds. In particular, thanks to our synthetic routes, we

have clearly showed that modulation of the lipophilicity and/or

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(3H, s), 3.8 (d, 3H, J_HP= 10.6 Hz), 2.08 (2H, ddd, J_HH= 13.9, 6.6 Hz,

J_HP= 3.4 Hz), 1.37 (2H, t, J_HH= 6.7 Hz), 1.25 (16H, br s), 0.87 (3H, t,

J_HH= 6.6 Hz); ¹³C NMR (CDCl₃) δ 155.0 (d, J_CP= 9.9 Hz), 139.4 (d,

J_CP= 12.5 Hz), 120.3 (d, J_CP= 3.8 Hz), 73.3 (d, J_CP= 170.2 Hz), 55.5,

functionalization at the C-5 carbon atom or at the phosphorus

can be exploited to either attenuate or increase the aﬃnity of

one inhibitor to target a speciﬁc microbial lipase.

54.0 (d, J_CP= 7.2 Hz), 32.6, 32.1, 29.8, 29.6, 29.5, 29.3, 28.8 (d, J_CP

=

CONCLUSION

2.4 Hz), 22.9, 14.3; ³¹P NMR (CDCl₃) δ 20.8, 20.5 (E:Z = 10:1);

HRMS (FAB, NBA, MH⁺) calcd for C₁₇H₃₄O₆P: 365.2092, found

365.2103.

■

This work provided the ﬁrst fundamental premise for the

understanding of the structure−activity relationships of

monocyclic enol-phosphonate analogs in connection with the

inhibition of several lipases having distinct substrate speciﬁc-

ities. Moreover, the major interest in this new family of

phosphonate inhibitors is that their chemical structure is

ﬂexible at will. The strategy developed here, involving

palladium(0)-catalyzed addition of methyl acetoacetate to

phosphono allylic carbonates followed by selective mono-

demethylation and cyclization, allows for the synthesis of a large

variety of fully customizable monocyclic enol-phosphonates

substituted at the C-5 position. Furthermore, the methyl

phosphonate ester can be easily trans-esteriﬁed to give long

chain ester compounds analogous to Cyclipostins. This

ﬂexibility makes it possible to design selective inhibitors for a

given enzyme. In order to shed more light on the inﬂuence of

the chirality at both phosphorus and carbon centers on lipase

inhibition, further experiments are currently underway and will

be reported in due course.

( )-(E)-1-(Dimethoxyphosphoryl)pentadec-2-en-1-yl Methyl Car-

bonate (13e). To a solution of carbonate 13a (1.12 g, 5.00 mmol) and

1-tetradecene (12.7 mL, 50.0 mmol) in dry CH₂Cl₂(10 mL) was

added Grubbs' second generation catalyst (0.21 g, 0.25 mmol)

followed by CuI (0.07 g, 0.35 mmol) to give 13e (1.5 g, 76%). IR

1

(neat, NaCl) 1755 cm⁻¹; H NMR (CDCl₃) δ 5.95 (1H, ddt, J_HH

15.2, 6.7 Hz, J_HP= 3.8 Hz), 5.55 (1H, ddd, J_HH= 15.2, 7.6 Hz, J_HP

5.2 Hz), 5.45 (1H, dd, J_HH= 7.8 Hz, J_HP= 12.4 Hz), 3.81 (d, 3H, J_HP

=

10.6 Hz), 3.81 (3H, s), 3.80 (d, 3H, J_HP= 10.6 Hz), 2.08 (2H, ddd, J_HH

= 13.9, 6.7 Hz, J_HP= 3.5 Hz), 1. 37 (2H, t, J_HH= 6.7), 1.24 (18H, br s),

0.87 (3H, t, J_HH= 6.6 Hz); ¹³C NMR (CDCl₃) δ 154.9 (d, J_CP= 9.8

Hz), 139.3 (d, J_CP= 12.5 Hz), 120.3 (d, J_CP= 3.8 Hz), 73.2 (d, J_CP

=

170.3 Hz), 55.5, 54.0 (d, J_CP= 7.2 Hz), 32.6, 32.1, 29.9, 29.8, 29.7,

29.6, 29.5, 29.4, 29.3, 28.8 (d, J_CP= 2.4 Hz), 22.9, 14.3; ³¹P NMR

(CDCl₃) δ 20.9, 20.5 (E:Z = 8:1); HRMS (FAB, NBA, MH⁺) calcd for

C₁₉H₃₇O₆PNa: 415.2225, found 415.2227.

( )-(E)-1-(Dimethoxyphosphoryl)nonadec-2-en-1-yl Methyl Car-

bonate (13f). To a solution of carbonate 13a (1.12 g, 5.00 mmol) and

1-octadecene (8.0 mL, 25 mmol) in dry CH₂Cl₂(10 mL) was added

Grubbs' second generation catalyst (0.21 g, 0.25 mmol) followed by

CuI (0.07 g, 0.35 mmol) to give 13f (1.57g, 70%). IR (neat, NaCl),

1755 cm⁻¹; ¹H NMR (CDCl₃) δ 5.96 (1H, ddt, J_HH= 14.6, 7.1 Hz, J_HP

= 3.8 Hz), 5.55 (1H, ddd, J_HH= 7.7, 15.2 Hz, J_HP= 5.2 Hz), 5.44

EXPERIMENTAL SECTION

■

ChemistrySynthesis of Compounds 1 to 12. General

Experimental. All reactions were carried out in oven-dried glassware

under an atmosphere of argon unless otherwise noted. H, ¹³C, and

1

(1H,dd, J_HH= 7.8 Hz, J_HP= 12.3 Hz), 3.82 (3H, s), 3.81 (d, 3H, J_HP

=

³¹P NMR spectra were recorded at 300, 75, and 121 MHz respectively.

¹H NMR spectra are referenced to CDCl₃(7.27 ppm), ¹³C NMR

spectra are referenced to the center line of CDCl₃(77.23 ppm) and

³¹P NMR spectra are referenced to external H₃PO₄. Coupling

constants, J, are reported in Hz. Analytical thin-layer chromatography

10.6 Hz), 3.81 (d, 3H, J_HP= 10.6 Hz), 2.1 (2H, ddd, J_HH= 7, 13.7 Hz,

J_HP= 3.2 Hz), 1.37 (2H, t, J_HH= 6.5 Hz), 1.25 (26H, br s), 0.87 (3H, t,

J_HH= 6.44 Hz); ¹³C NMR (CDCl₃) δ 155 (d, J_CP= 9.8 Hz), 139.3 (d,

J_CP= 12.4 Hz), 120.3 (d, J_CP= 3.8 Hz), 73.3 (d, J_CP= 170.3 Hz), 55.5,

54.0 (d, J_CP= 7.3 Hz), 32.6, 32,1, 29.9, 29.8, 29.7, 29.6, 29.5, 29.3,

28.8, 28.7, 22.9, 14.4; ³¹P NMR (CDCl₃) δ 20.8, 20.5 (E:Z = 10:1);

HRMS (FAB, NBA, MNa⁺) calcd for C₂₃H₄₅O₆PNa: 471.2851, found

471.2860.

( )-(E)-1-(Dimethoxyphosphoryl)henicos-2-en-1-yl Methyl Car-

bonate (13g). To a solution of carbonate 13a (0.67 g, 3 mmol)

and 1-eicocene (4.21 mL, 15 mmol) in dry CH₂Cl₂(6 mL) was added

Grubbs' second generation catalyst (0.13 g, 0.15 mmol) followed by

CuI (0.04 g, 0.21 mmol) to give 13g (1.1 g, 70%). IR (neat, NaCl)

1755 cm⁻¹; ¹H NMR (CDCl₃) δ 5.97 (1H, ddt, J_HH= 14.9, 6.7 Hz, J_HP

= 3.8 Hz), 5.56 (1H, ddd, J_HH= 15.1, 7.7 Hz, J_HP= 5.3 Hz), 5.46 (1H,

dd, J_HH= 7.8 Hz, J_HP= 12.2 Hz), 3.82 (3H, s), 3.81 (d, 3H, J_HP= 10.6

(TLC) analyses were performed on silica gel plates 60PF₂₅₄

.

Visualization was accomplished with UV light, KMnO₄solution, or

iodine. The syntheses of racemic 1,²¹racemic 2(α) and 2(β),¹⁵3, and

4¹⁶as well as carbonates 13a, 13b, and 13c³⁸have already been

reported. Target compounds were puriﬁed by chromatography and

have purities of ≥95% as determined by HPLC analysis conducted on

an Agilent 1100 system, using a Zorbax Eclipse XBD-C18 column.

Elution was performed at a ﬂow rate of 2 mL·min⁻¹using a gradient of

H₂O/TFA/CH₃CN 95:0.1:5 to H₂O/TFA/CH₃CN 5:0.1:95 over 6

min or using a gradient of H₂O/TFA/CH₃CN 95:0.1:5 to 100%

CH₃CN over 5 min followed by a hold at 100% CH₃CN for 3 min.

The compounds were detected using a diode array and ELS. The

Hz), 3.81 (d, 3H, J_HP= 10.6 Hz), 2.1 (2H, J_HH= 6.7, 13.7 Hz, J_HP

=

3.4 Hz), 1.39 (2H, t, J_HH= 6.7 Hz), 1.26 (30 H, br s), 0.88 (3H, t, J_HH

= 6.7 Hz); ¹³C NMR (CDCl₃) δ 155.0 (d, J_CP= 9.8 Hz), 139.4 (d, J_CP

= 12.5 Hz), 120.3 (d, J_CP= 3.8 Hz), 73.3 (d, J_CP= 170.3 Hz), 55.5,

54.0 (d, J_CP= 7.1 Hz), 32.6, 32,1, 30, 29.9, 29.8, 29.6, 29.5, 29.3, 28.8

(d, J_CP= 2.4 Hz), 22.9, 14.4; ³¹P NMR (CDCl₃) δ 20.8, 20.5 (E:Z =

10:1); HRMS (FAB, NBA, MH⁺) calcd for C₂₅H₄₉O₆PNa: 499.3164,

found 499.3170.

General Procedure for Synthesis of Vinyl Phosphonates 14. To a

stirring solution of carbonate 13 (1 mmol) and methyl acetoacetate (3

mmol) in dry THF (2.5 mL) was added Pd₂(dba)₃(0.02 mmol)

followed by dppe (0.5 mmol), and the resulting mixture was stirred at

room temperature for 3−4 min. The septum was replaced by a

condenser connected to an argon bubbler, and the reaction ﬂask was

placed in an oil bath preheated to 45 °C. After completion of the

reaction (TLC and ³¹P NMR analysis), the reaction mixture was

partitioned between brine and EtOAc. The organic layer was collected,

and the aqueous layer was extracted with additional EtOAc. The

combined organic layers were dried over anhydrous Na₂SO₄and

evaporated under reduced pressure to give the crude product.

Puriﬁcation of the crude by column chromatography (SiO₂, 20−70%

EtOAc in hexanes) gave the vinyl phosphonate 14.

HPLC data were supported by careful analysis of the H, ¹³C, and

1

particularly the ³¹P NMR spectra.

General Procedure for Synthesis of the Carbonates 13 via Cross

Metathesis Reaction.³⁹To a solution of carbonate 13a (1.0 mmol)

and alkene (5.0−10.0 mmol) in CH₂Cl₂(2 mL) was added Grubbs'

second generation catalyst (5 mol %) followed by CuI (7 mol %).

After stirring the solution for 2 min at room temperature, the septum

was replaced by a condenser connected to an argon bubbler and the

reaction ﬂask was placed in an oil bath preheated to 45 °C. After

completion of the reaction (TLC and ³¹P NMR analysis), the solvent

was removed under reduced pressure to give the crude product.

Puriﬁcation of the crude product by column chromatography (SiO₂,

20−50% EtOAc in hexanes) gave the carbonate 13 (E:Z ≥ 10:1).

( )-(E)-1-(Dimethoxyphosphoryl)tridec-2-en-1-yl Methyl Carbo-

nate (13d). To a solution of carbonate 13a (1.12 g, 5.00 mmol) and 1-

dodecene (11.1 mL, 50.0 mmol) in dry CH₂Cl₂(10 mL) was added

Grubbs' second generation catalyst (0.21 g, 0.25 mmol) followed by

CuI (0.07 g, 0.35 mmol) to give 13d (1.43 g, 79%). IR (neat, NaCl)

1756 cm⁻¹; ¹H NMR (CDCl₃) δ 5.97 (1H, ddt, J_HH= 15.2, 6.7 Hz, J_HP

= 3.7 Hz), 5.55 (1H, ddd, J_HH= 15.2, 7.6 Hz, J_HP= 5.2 Hz), 5.45 (1H,

dd, J_HH= 8.0 Hz, J_HP= 12.4 Hz), 3.81 (d, 3H, J_HP= 10.6 Hz), 3.81

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( )-(E)-Methyl 2-Acetyl-5-(dimethoxyphosphoryl)-3-phenylpent-

4-enoate (14b) and (2Z,3Z)-Methyl 5-(Dimethoxyphosphoryl)-2-(1-

hydroxyethylidene)-3-phenylpent-3-enoate (15). To a solution of

carbonate 13b (0.75 g, 2.5 mmol) and methyl acetoacetate (0.87 mL,

7.6 mmol) in THF (6 mL) were added Pd₂(dba)₃(0.05 g, 0.05 mmol)

and dppe (0.05 g, 0.13 mmol) to give 14b as 1:1 diastereoisomeric

mixture (0.32 g, 46%). Pale yellow solid; IR (neat), 1742, 1714 cm⁻¹;

¹H NMR (CDCl₃) δ 7.26 (m, 5H), 6.73 (1H, m), 5.75 (1H, m), 4.28

(CDCl₃) δ 201.5, 168.7 (168.6), 153.5 (d, J_CP= 4.4 Hz), 118.9 (185)

(d, J_CP= 184.8 Hz), 63.9 (63.8), 52.6 (52.7), 52.5 (d, J_CP= 5.6 Hz),

44.3 (44), 32.1, 31.9 (31.8), 30.3 (30.1), 29.9, 29.8, 29.7, 29.6, 29.5,

27.3 (27.2), 22.9, 14.3; ³¹P NMR (CDCl₃) δ 20.7, 20.6; HRMS (FAB,

MH⁺) calcd for C₂₂H₄₂O₆P: 433.2718, found 433.2720.

( )-(E)-Methyl 2-Acetyl-3-(2-(dimethoxyphosphoryl)vinyl)-

nonadecanoate (14f). To a stirring solution of carbonate 13f (0.45

g, 1.0 mmol) and methyl acetoacetate (0.64 mL, 3.0 mmol) in dry

THF (2 mL) was added Pd₂(dba)₃(0.02g, 0.02 mmol) followed by

dppe (0.02g, 0.05 mmol) to give 14f as a 1:1 mixture of

diastereoisomers (0.36 g, 72%). Waxy solid; IR (neat, NaCl), 1743,

1637 cm⁻¹; ¹H NMR (CDCl₃) δ 6.59 (0.5H, ddd, J_HH= 17.1, 9.5 Hz,

J_HP= 21.5 Hz), 6.45 (0.5H, ddd, J_HH= 17.1, 9.4 Hz, J_HP= 21.5 Hz),

(1H, m), 4.1(1H, d, J_HH= 11.27 Hz), 3.66 (6H, m). 3.61 (3H, s), 2.14

(3H, s); ¹³C NMR (CDCl₃) δ 200.4 (200.3), 167.7 (167.3), 152.3

(152.26) (d, J_CC= 5.2 Hz), 137.9 (137.6), 129.1 (128.9), 128.2

(128.1), 128.1, 127.8 (127.6), 117.8 (117.5) (d, J_CP= 185.4 Hz), 64.1

(63.5), 52.7 (52.5), 52.3 (d, J_CP= 5.7 Hz), 49.3 (48.9) (d, J_CP= 22.2

Hz), 30.4 (30.1); ³¹P NMR (CDCl₃) δ 20.87, 20.1 ppm; HRMS (FAB,

NBA, MH⁺) calcd for C₁₆H₂₁O₆P: 340.1153, found 341.1157 and 15

5.72 (0.5H, dd, J_HH= 17.1 Hz, J_HP= 21.5 Hz), 5.65 (0.5H, dd, J_HH

=

17.1 Hz, J_HP= 21.5 Hz), 3.71 (9H, m), 3.54 (1H, 2d, J_HH= 9.4 and 9.2

Hz), 2.97 (1H, 2q, J_HH= 9.1 and 9.3 Hz), 2.21 (1.5H, s), 2.15 (1.5H,

s), 1.22 (30H, br s), 0.85 (3H, t, J_HH= 6.66 Hz); ¹³C NMR (CDCl₃) δ

201.3, 168.6 (168.5), 153.4 (153.3) (d, J_CP= 8.4 Hz), 118.8 (118.6)

(d, J_CP= 184.3 Hz), 63.8 (63.7), 52.7 (52.5), 52.7 (d, J_CP= 5.6 Hz),

44.2 (43.9) (d, J_CP= 2.2 Hz), 32.1, 31.8 (31.7), 30.2 (30), 29.9, 29.8,

29.7, 29.6, 29.5, 29.4, 27.2 (27.1), 22.8, 14.3; ³¹P NMR (CDCl₃) δ

20.7, 20.6; HRMS (FAB, NBA, MH⁺) calcd. for C₂₆H₅₀O₆P: 485.3345,

found 489.3340.

1

(0.34 g, 47%). White solid; IR (neat) 1646, 1611 cm⁻¹; H NMR

(CDCl₃) δ 13.02, (1H, s), 7.37−7.25 (5H, m), 6.18 (1H, J_HH= 7.6

Hz), 3.76 (6H, d, J = 10.87 Hz). 3.71 (3H, s), 2.7 (2H, dd, J_HH= 7.6

Hz, J_Hp= 22.6 Hz), 1.88 (3H, s); ¹³C NMR (CDCl₃) δ 175.5, 172.9,

140.7, 138.2, 128.9, 128.0, 126.3, 121.1, 99.4, 53.0 (d, J_CP= 11.43 Hz),

52.2, 27.9 (d, J_CP= 139.88 Hz), 19.8; ³¹P NMR (CDCl₃) δ 30.46;

HRMS (FAB, NBA, MH⁺) calcd for C₁₆H₂₂O₆P: 340.1153, found

341.1150.

( )-(E)-Methyl 2-Acetyl-3-(2-(dimethoxyphosphoryl)vinyl)-

henicosanoate (14g). To a stirring solution of carbonate 13g (0.48

g, 1.0 mmol) and methyl acetoacetate (0.33 mL, 3.0 mmol) in dry

THF (2 mL) was added Pd₂(dba)₃(0.02 g, 0.02 mmol) followed by

dppe (0.02 g, 0.05 mmol) to give 14g as a 1:1 mixture of

diastereoisomers (0.35 g, 70%). Pale yellow oil; IR (neat, NaCl)

( )-(E)-Methyl 2-Acetyl-3-(2-(dimethoxyphosphoryl)vinyl)-

octanoate (14c). To a solution of carbonate 14c (0.29 g, 1.0

mmol) and methyl acetoacetate (0.33 mL, 3.0 mmol) in dry THF (2

mL) were added Pd₂(dba)₃(0.02 g, 0.02 mmol) and dppe (0.02 g,

0.05 mmol) to give 14c as a 1:1 mixture of diastereoisomers (1.17 g,

70%). Pale yellow oil; IR (neat, NaCl) 1743, 1717; ¹H NMR (CDCl₃)

δ 6.55 (0.5H, ddd, J_HH= 17.13, 9.5 Hz, J_HP= 21.5 Hz), 6.45 (0.5H,

ddd, J_HH= 17.13, 9.5 Hz, J_HP= 21.5 Hz), 5.68 (0.5H, dd, J_HH= 17.13

Hz, J_HP= 21.5 Hz), 5.61 (0.5H, ddd, J_HH= 17.13 Hz, J_HP= 21.5 Hz),

3.67 (9H, m), 3.51 (1H, 2d, J_HH= 9.4, 9.3 Hz), 2.95 (1H, 2q, J_HH= 9.4

and 9.4 Hz), 2.21 (1.5H, s), 2.15 (1.5H, s), 1.28 (8H, m); 0.83 (3H, t,

J_HH= 6.44 Hz); ¹³C NMR (CDCl₃) δ 201.3, 168.6 (168.4), 153.4

(153.3) (d, J_CP= 4.6 Hz), 118.8 (118.7) (d, J_CP=184.5 Hz), 63.8

(63.7) (d, J_CP= 1.0 Hz), 52.7 (52.5), 52.4 (d, J_CP= 5.6 Hz), 44.0

(43.9) (d, J_CP= 21.8 Hz), 31.8 (31.6), 31.5 (d, J_CP= 1.3 Hz), 30.1

(29.9), 26.8 (26.7), 22.5, 14.0; ³¹P NMR (CDCl₃) δ 20.7, 20.6; HRMS

(FAB, NBA, MH⁺) calcd for C₁₅H₂₇O₆PNa: 357.1442, found

357.1450.

1

1742, 1713 cm⁻¹; H NMR (CDCl₃) δ 6.56 (0.5H, ddd, J_HH= 17.1,

9.5 Hz, J_HP= 21.4 Hz), 6.45 (0.5H, ddd, J_HH= 17.1, 9.5 Hz, J_HP= 21.5

Hz), 5.67 (0.5H, dd, J_HH= 17.1 Hz, J_HP= 21.5 Hz), 5.60 (0.5H, dd,

J_HH= 17.1 Hz, J_HP= 21.5 Hz), 3.67 (9H, m), 3.54 (1H, 2d, J_HH= 9.4

and 9.2 Hz), 2.97 (1H, 2q, J_HH= 9.4 and 9.2 Hz), 2.21 (1.5H, s), 2.15

(1.5H, s), 1.21 (34H, br s), 0.84 (3H, t, J_HH= 6.6 Hz); ¹³C NMR

(CDCl₃) δ 201.3, 168.6 (168.4), 153.4 (153.3) (d, J_CP= 4.6 Hz), 118.8

(118.6) (d, J_CP= 184.6 Hz), 63.8 (63.7), 52.7 (52.5), 52.4 (d, J_CP= 5.5

Hz), 44.2 (43.9) (d, J_CP= 2.3 Hz), 32.0, 31.8 (31.7), 30.2 (30), 29.9,

29.8, 29.7, 29.6, 29.5, 29.4, 27.2 (27.1), 22.8, 14.3; ³¹P NMR (CDCl₃)

δ 20.7, 20.6; HRMS (FAB, NBA, MH⁺) calcd for C₂₆H₅₀O₆P:

485.3345, found 489.3340.

General Procedure for Hydrogenation of Vinyl Phosphonate. To

a solution of vinyl phosphonate 14 (1 mmol) in methanol (3 mL) was

added 10% palladium on charcoal (0.07 g). The solution was ﬁrst

ﬂushed with argon and then with hydrogen gas. A reservoir of

hydrogen was provided by a balloon. After completion of the reaction

(³¹P NMR analysis), the reaction mixture was ﬁltered through Celite

which was washed with additional CH₂Cl₂. The solvent was

evaporated under reduced pressure to give the saturated phosphonate

16.

( )-(E)-Methyl 2-Acetyl-3-(2-(dimethoxyphosphoryl)vinyl)-

tridecanoate (14d). To the stirring solution of carbonate 13d

(0.182 g, 0.50 mmol) and methyl acetoacetate (0.17 mL, 1.5 mmol) in

dry THF (1 mL) was added Pd₂(dba)₃(0.01 g, 0.01 mmol) followed

by dppe (0.10 g, 0.025 mmol) to give 14d as a 1:1 mixture of

diastereoisomers (0.16 g, 78%). Pale yellow oil; IR (neat, NaCl), 1743,

1719 cm⁻¹; ¹H NMR (CDCl₃) δ 6.59 (0.5H, ddd, J_HH= 17.1, 9.6 Hz,

J_HP= 21.5 Hz), 6.49 (0.5H, ddd, J_HH= 17.1, 9.6 Hz, J_HP= 21.5 Hz),

5.7 (0.5H, dd, J_HH= 17.1 Hz, J_HP= 21.5 Hz), 5.65 (0.5H, dd, J_HH

=

17.1 Hz, J_HP= 21.5 Hz), 3.67 (9H, m), 3.54 (1H, 2d, J_HH= 9.4 and 9.2

Hz), 2.98 (1H, 2q, J_HH= 9.3 Hz), 2.24 (1.6H, s), 2.18 (1.4H, s), 1.23

(30H, br s), 0.87 (3H, t, J_HH= 6.7 Hz); ¹³C NMR (CDCl₃) δ 201.5,

168.7 (168.5), 153.5 (d, J_CP= 9.1 Hz), 118.8 (d, J_CP= 171.7 Hz), 63.9

(63.8), 52.8 (52.7), 52.5 (d, J_CP= 5.4 Hz), 44.3 (44), 32.1, 31.9 (31.8),

30.3 (30.1), 29.8, 29.6, 29.5, 27.3 (27.20), 22.9, 14.3; ³¹P NMR

(CDCl₃) δ 20.7, 20.6; HRMS (FAB, NBA, MH⁺) calcd for

C₂₀H₃₈O₆P: 405.2406, found 405.2403.

( )-Methyl 2-Acetyl-5-(dimethoxyphosphoryl)-3-phenylpenta-

noate (16b). To a solution of 14b (or 15) (0.29 g, 0.84 mmol) in

methanol (3 mL) were added palladium on charcoal (0.09 g) and

hydrogen gas to give the saturated phosphonate 14b as a 1:1 mixture

of diastereoisomers (0.30 g, 100%). Colorless oil; IR (neat, NaCl)

1

1745, 1717 cm⁻¹; H NMR (CDCl₃) δ 7.34 (m, 5H), 4.28(1H, m),

4.1(1H, d, J_HH= 11.27 Hz), 3.66 (6H, m), 3.61 (3H, s), 2.14 (3H, s);

¹³C NMR (CDCl₃) δ 201.8, 168.8 (168.2), 139.5 (139.2), 129.1,

( )-(E)-Methyl 2-Acetyl-3-(2-(dimethoxyphosphoryl)vinyl)-

pentadecanoate (14e). To a stirring solution of carbonate 13e

(1.26 g, 3.20 mmol) and methyl acetoacetate (1.05 mL, 9.60 mmol) in

dry THF (6.5 mL) was added Pd₂(dba)₃(0.06 g, 0.64 mmol) followed

by dppe (0.06 g, 0.16 mmol) to give 14e as a 1:1 mixture of

diastereoisomers (1.13 g, 80%). Pale yellow oil; IR (neat, NaCl) 1743,

1719 cm⁻¹; ¹H NMR (CDCl₃) δ 6.59 (0.5H, ddd, J_HH= 17.1, 9.5 Hz,

J_HP= 21.5 Hz), 6.49 (0.5H, ddd, J_HH= 17.1, 9.5 Hz, J_HP= 21.5 Hz),

128.8, 128.4, 127.7, 127.6, 66.6 (65.8), 52.8, 52.5 (d, J_CP= 6.5 Hz),

46.1 (45.6) (d, J_CP= 19.1 Hz), 30.3 (30.0), 26.9 (26.8) (d, J_CP= 16.4

Hz), 23.5 (23.4), 21.6 (d, J_CP= 8.7 Hz); ³¹P NMR (CDCl₃) δ 34.6

ppm; HRMS (FAB, MH⁺) calcd for C₁₆H₂₄O₆P: 343.1310, found

343.1311.

( )-Methyl 2-Acetyl-3-(2-(dimethoxyphosphoryl)ethyl)octanoate

(16c). To a solution of 14c (0.650 g, 1.96 mmol) in methanol (5 mL)

were added palladium on charcoal (0.21 g) and hydrogen gas to give

the saturated phosphonate 16c as a 1:1 mixture of diastereoisomers

5.72 (0.5H, dd, J_HH= 17.1 Hz, J_HP= 21.5 Hz), 5.65 (0.5H, dd, J_HH

=

1

17.1 Hz, J_HP= 21.5 Hz), 3.71 (9H, m), 3.5 (1H, 2d, J_HH= 9.4 and 9.2

Hz), 2.98 (1H, 2q, J_HH= 9.1 and 9.3 Hz, J_HH= 1.6 Hz), 2.21 (1.5H, s),

2.15 (1.5H, s), 1.22 (22H, br s), 0.88 (3H, t, J_HH= 6.7 Hz); ¹³C NMR

(0.65 g, 100%). Colorless oil; IR (neat, NaCl) 1739, 1716 cm⁻¹; H

NMR (CDCl₃) δ 3.73 (9H, m), 3.45 (1H, d, J_HH= 8.3 Hz), 2.26 (1H,

br s), 2.23 (4H, s), 1.67 (m, 4H), 1.25 (br, 8H), 0.87 (t, 3H, J_HH= 6.7

10213

dx.doi.org/10.1021/jm301216x | J. Med. Chem. 2012, 55, 10204−10219

Journal of Medicinal Chemistry

Article

Hz); ¹³C NMR (CDCl₃) δ 202.9, 169.7 (169.69), 63.2(63.1), 52.6,

52.5 (d, J_CP= 6.5 Hz), 38.1 (d, J_CP= 17.2a Hz), 32.0 (d, J_CP= 7.1 Hz),

30.6 (30.4), 29.6, 26.1, 22.7, 14.2; ³¹P NMR (CDCl₃) δ 35.0, 34.9;

HRMS (FAB, NBA, MH⁺) calcd for C₁₅H₂₉O₆P: 336.1780, found

337.1779.

organic layer was separated and dried over anhydrous Na₂SO₄and

then evaporated under reduced pressure to give the crude product.

Puriﬁcation of the crude product by chromatography (SiO₂, 10−60%

EtOAc in hexanes) gave the product as a mixture of diastereomers

which were separated by further careful column chromatography.

( )-Methyl 2-Methoxy-7-methyl-5-phenyl-2,3,4,5-tetrahydro-1,2-

oxaphosphepine-6-carboxylate 2-Oxide (5(α) and 5(β)). A solution

of phosphonate 16b (0.5 g, 1.46 mmol) in acetonitrile (5 mL) was

treated as described above to give the monocyclic phosphonate as a

1:1 mixture of diastereoisomers (0.28 g, 52%). Further chromato-

graphic separation of the diastereoisomers gave 5(α) (95:5 ratio with

5(β)). IR (neat, NaCl) 1719 cm⁻¹; ¹H NMR (CDCl₃) δ 7.27(m, 5H),

4.28 (1H, t, J_HH= 7 Hz), 3.86 (3H, d, J_HP= 11.15 Hz), 3.51 (3H, s),

2.25 (3H, s), 2.19 (1H, m), 2.05 (1H, m), 1.98 (1H, t, J_HH= 6 Hz);

¹³C NMR (CDCl₃) δ 168.97, (168.78), 155.65 (d, J_CP= 7.28 Hz),

(154.66); 141.35, (141.12); (129.04), 129.00; (128.10), 127.91;

(127.25), 127.15; 121.44 (d, J_CP= 5.1 Hz), 53.07 (d, J_CP= 6.9 Hz),

52.71 (d, J_CP= 6.9 Hz), 52.33, 45.78 (45.63), 28.53 (d, J_CP= 7.04 Hz),

23.16 (d, J_CP= 133.1 Hz), 23.04 (d, J_CP= 133.1 Hz), 22 (21.76); ³¹P

NMR (CDCl₃) δ 30.1; HRMS (EI⁺) calcd for C₁₅H₁₉O₅P: 310.0970,

found 310.0975.

( )-Methyl 2-Methoxy-7-methyl-5-pentyl-2,3,4,5-tetrahydro-1,2-

oxaphosphepine-6-carboxylate 2-Oxide (6(α) and 6(β)). A solution

of 16c (0.52 g, 1.55 mmol) in acetonitrile (5 mL) was treated as

described above to give the monocyclic phosphonates 6 as a 1:1.25

mixture of diastereoisomers (0.27 g, 56%). Further chromatographic

separation of the diastereoisomers gave 6(α) as a pale yellow oil. IR

(neat, NaCl) 1717 cm⁻¹; ¹H NMR (CDCl₃) δ 3.82 (d, 3H, J_HP= 11.18

Hz), 3.73 (3H, s), 2.97 (1H, m), 2.22 (3H, d, J_HH= 1.28 Hz), 2.12−

1.81 (4H, m), 1.61 (1H, m), 1.46 (1H, m), 1.25 (6H, br s), 0.87 (3H,

t, J_HH= 6.5 Hz); ¹³C NMR (CDCl₃) δ 169.34, 156.1 (d, J_CP= 7.28

Hz), 123.55 (d, J_CP= 5.1 Hz), 52.39 (d, J_CP= 7.1 Hz), 52.26, 37.63,

32.21, 31.1, 27.7, 25.31 (d, J_CP= 6.8 Hz), 22.93, 22.3 (d, J_CP= 134.3

Hz), 21.67, 14.44; ³¹P NMR (CDCl₃) δ 26.8; HRMS (FAB, NBA,

MH⁺) calcd for C14H₂₆O₅P: 305.1517, found 305.1523; and 6(β) as a

pale yellow oil. IR (neat, NaCl) 1717 cm⁻¹; ¹H NMR (CDCl₃) δ 3.82

(3H, d, J_HP= 11.18 Hz), 3.75 (3H, s), 2.88 (1H, m), 2.18 (3H, d, J_HH

= 1.3 Hz), 2.14−1.87 (4H, m), 1.63 (1H, m), 1.47 (1H, m), 1.25 (6H,

br s), 0.87 (3H, t, J_HH= 6.5 Hz); ¹³C NMR (CDCl₃) δ 169.51, 155.27

(d, J_CP= 9.3 Hz), 123.44 (d, J_CP= 4.73 Hz), 52.95 (d, J_CP= 6.67 Hz),

52.32, 37.5, 32.13, 31.2, 30.1, 27.65, 25.51 (d, J_CP= 7.91 Hz), 22.91,

22.64 (d, J_CP= 133.33 Hz), 21.51 (d, J_CP= 1.87 Hz), 14.43; ³¹P NMR

(CDCl₃) δ 24.2; HRMS (FAB, NBA, MH⁺) calcd for C₁₄H₂₆O₅P:

305.1517, found 305.1523.

( )-Methyl 5-Decyl-2-methoxy-7-methyl-2,3,4,5-tetrahydro-1,2-

oxaphosphepine-6-carboxylate 2-Oxide (7(α) and 7(β)). A solution

of alkyl phosphonate 16d (0.47 g, 1.16 mmol) in acetonitrile (3 mL)

was treated as described above to give the monocyclic phosphonates 7

as a 1:1.25 mixture of diastereoisomers (0.21 g, 46%). Further

chromatographic separation of the diastereoisomers gave 7(α) as pale

yellow oil. IR (neat, NaCl) 1719 cm⁻¹; ¹H NMR (CDCl₃) δ 3.82 (3H,

d, J_HP= 11.2 Hz), 3.75 (3H, s), 2.99 (1H, m), 2.23 (3H, d, J_HH= 1.6

Hz), 2.14 −1.87 (4H, m), 1.62 (1H, m), 1.48 (1H, m), 1.25 (16H, br

s), 0.88 (3H, t, J_HH= 6.7 Hz); ¹³C NMR (CDCl₃) δ 169.2 (d, J_CP= 1.9

Hz), 155.9 (d, J_CP= 7.3 Hz), 123.4 (d, J_CP= 5.3 Hz), 52.2 (d, J_CP= 7.1

(

)-Methyl 2-Acetyl-3-(2-(dimethoxyphosphoryl)ethyl)-

tridecanoate (16d). To the solution of 14d (0.44 g, 1.08 mmol) in

methanol (5 mL) were added palladium on charcoal (0.01 g) and

hydrogen gas to give the saturated phosphonate 16d as a 1:1 mixture

of diastereoisomers (0.44 g, 100%). Colorless oil; IR (neat, NaCl)

1

1737, 1712 cm⁻¹; H NMR (CDCl₃) δ 3.73 (6H, d, J_HD= 10.8 Hz),

3.73 (3 H, app d), 3.45(1H, d, J_HH= 8.3 Hz), 2.26 (1H, br s), 2.23

(3H, s), 1.76−1.6 (4H, m), 1.25 (18 H, br s), 0.88 (3H, t, J_HH= 6.44

Hz); ¹³C NMR (CDCl₃) δ 202.9, 169.7, 63.2, 52.6, 52.5, 38.2 (38),

32.1, 30.5 (d, J_CP= 10.5 Hz), 30, 29.9, 29.8, 29.7, 29.6, 29.5, 26.5, 22.9,

14.3; ³¹P NMR (CDCl₃) δ 35.0, 34.9; HRMS (FAB⁺) calcd for

C₂₀H₃₉O₆PNa: 429.2381, found 429.2351.

(

)-Methyl 2-Acetyl-3-(2-(dimethoxyphosphoryl)ethyl)-

pentadecanoate (16e). To a solution of 14e (0.33 g, 0.757 mmol)

in methanol (4 mL) were added palladium on charcoal (0.08 g) and

hydrogen gas to give the saturated phosphonate 16e as a 1:1 mixture

of diastereoisomers (0.33 g, 100%). Colorless oil; IR (neat, NaCl)

1736, 1712 cm⁻¹; ¹H NMR (CDCl₃) δ 3.73 (6H, J_HP= 10.8 Hz), 3.73

(3H, app d), 3.46 (1H, d, J_HH= 8.3 Hz), 2.27 (1H, br s), 2.23 (3H, s),

1.77−1.6 (4H, m), 1.25 (20 H, br s), 0.88 (3H, t, J_HH= 6.7 Hz); ¹³C

NMR (CDCl₃) δ 203, 169.7, 63.2, 52.6 (d, J_CP= 7.1 Hz), 52.5, 38.2

(38), 32.1, 30.6 (d, J_CP= 10.6 Hz), 30, 29.9, 29.8, 29.7, 29.6, 29.5, 26.5,

22.9 14.3; ³¹P NMR (CDCl₃) δ 35.0, 34.9; HRMS (FAB, NBA, MH⁺)

calcd for C₂₂H₄₄O₆P: 435.2875, found 435.2868.

(

)-Methyl 2-Acetyl-3-(2-(dimethoxyphosphoryl)ethyl)-

nonadecanoate (16f). To a solution of 14f (0.22 g, 0.45 mmol) in

methanol (3 mL) were added palladium on charcoal (0.05 g) and

hydrogen gas to give the saturated phosphonate 16f as a 1:1 mixture of

diastereoisomers (0.22 g, 100%). Colorless oil; IR (neat, NaCl) 1736,

1718 cm⁻¹; ¹H NMR (CDCl₃) 3.72 (6H, d, J_HP= 10.7 Hz), 3.71 (3H,

app d), 3.44 (1H, d, J_HH= 8.9 Hz), 2.24 (1H, br s), 2.21 (3H, s),

1.73−1.58 (4H, m), 1.23 (30H, br s), 0.86 (3H, t, J_HH= 6.7 Hz); ¹³

C

NMR (CDCl₃) δ 203, 169.7, 63.1, 52.6 (d, J_CP= 7.1 Hz), 52.5, 38.3,

38, 32.1, 30.5 (d, J_CP= 10.2 Hz), 29.9, 29.7, 29.6, 29.5, 26.5, 23.5, 22.9,

20.7, 14.3; ³¹P NMR (CDCl₃) δ 35, 34.9; HRMS (FAB, NBA, MH⁺)

calcd for C₂₆H₅₂O₆P: 491.3501, found 491.3507.

(

)-Methyl 2-Acetyl-3-(2-(dimethoxyphosphoryl)ethyl)-

henicosanoate (16g). To a solution of 14g (0.28 g, 0.55 mmol) in

methanol (5 mL) were added palladium on charcoal (0.06 g, 0.06

mmol) and hydrogen gas to give the saturated phosphonate 16g as a

1:1 mixture of diastereoisomers (0.29 g, 100%). Colorless oil; IR (neat,

1

NaCl) 1735, 1712 cm⁻¹; H NMR (CDCl₃) 3.73 (6H, d, J_HP= 10.8

Hz), 3.73 (3H, app d), 3.46 (1H, d, J_HH= 8.3 Hz), 2.26 (1H, br s),

2.23 (3H, s), 1.78−1.55 (4H, m), 1.25 (34H, br s), 0.88 (3H, t, J_HH

=

6.7 Hz); ¹³C NMR (CDCl₃) δ 203, 169.7, 63.2, 52.6 (d, J_CP= 6.2 Hz),

52.5, 38.2 (38), 32.1, 30.5, 29.9, 29.8, 29.7, 29.6, 26.5, 22.9, 14.3; ³¹P

NMR (CDCl₃) δ 35.0, 34.9; HRMS (FAB, NBA, MH⁺) calcd for

C₂₆H₅₇O₆PNa: 519.3814, found 519.3818.

General Procedure for the Synthesis of Monocylic Phosphonate

Analogs (5(α, β)−10(α, β)). To a solution of alkyl phosphonate 16 (1

mmol) in acetonitrile (2.6 mL) was added NaI (1.1 equiv), and the

resulting mixture was heated at reﬂux. After completion of the reaction

(³¹P NMR analysis), the solvent was removed under reduced pressure

to give monosodium salt as a solid. The sodium salt was suspended in

methanol, Amberlite IR-120H resin (prewashed with methanol) was

added, and the resulting mixture was shaken in an orbit shaker. After

completion of the reaction (³¹P NMR analysis), the resin was removed

by ﬁltration and washed with additional methanol. The solvent was

removed under reduced pressure to give free monophosphonic acid as

a red viscous liquid. The monophosphonic acid (1 mmol) was

dissolved in freshly distilled CH₂Cl₂(6 mL), and EDC (1.5 mmol)

and HOBt (1.5 mmol) were added, followed by Hunig’s base (i-

Pr₂NEt) (1.5 mmol). After the completion of the reaction (³¹P NMR

analysis), the reaction mixture was diluted with additional CH₂Cl₂and

the resulting solution was washed with saturated NaHCO₃. The

Hz), 52.1, 37.4, 32.1, 30.9, 29.9, 29.8, 29.7, 29.5, 27.9, 25.1 (d, J_CP

=

6.9 Hz), 22.9, 22.1 (d, J_CP= 134.4 Hz), 21.51, 14.3; ³¹P NMR

(CDCl₃) δ 26.8; HRMS (FAB, NBA, MNa⁺) calcd for C₁₉H₃₅O₅PNa:

397.2119, found 397.2125; and 7(β) also as pale yellow oil. IR (neat,

1

NaCl) 1719 cm⁻¹; H NMR (CDCl₃) δ 3.83 (3H, d, J_HP= 11.1 Hz),

3.76 (3H, s), 2.88 (1H, m), 2.18 (3H, d, J_HH= 1.3 Hz), 2.26−1.87

(4H, m), 1.63 (1H, m), 1.45 (1H, m), 1.25 (16H, br s), 0.88 (3H, t,

J_HH= 6.7 Hz); ¹³C NMR (CDCl₃) δ 169.3 (d, J_CP= 1.7 Hz), 155.1 (d,

J_CP= 9.4 Hz), 123.3 (d, J_CP= 4.6 Hz), 52.8 (d, J_CP= 6.7 Hz), 52.1,

37.3, 32.1, 31.1, 29.9, 29.8, 29.7, 29.5, 27.8, 25.3 (d, J_CP= 7.8 Hz),

22.9, 22.5 (d, J_CP= 133.4 Hz), 21.3 (d, J_CP= 1.9 Hz), 14.3; ³¹P NMR

(CDCl₃) δ 24.2; HRMS (FAB, NBA, MH⁺) calcd for C₁₉H₃₅O₅PNa:

397.2119, found 397.2115.

( )-Methyl 5-Dodecyl-2-methoxy-7-methyl-2,3,4,5-tetrahydro-

1,2-oxaphosphepine-6-carboxylate 2-Oxide (8(α) and 8(β)). To a

10214

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Journal of Medicinal Chemistry

Article

solution of alkyl phosphonate 16e (0.32 g, 0.74 mmol) in acetonitrile

(2 mL) was treated as described above to give the monocyclic

phosphonates 8 as a 1:1.25 mixture of diastereomers (0.14 g, 45%)

which were further separated by column chromatography to give 8(α)

as a pale yellow oil. IR (neat, NaCl) 1719 cm⁻¹; ¹H NMR (CDCl₃) δ

3.83 (3H, d, J_HP= 11.2 Hz), 3.75 (3H, s), 2.99 (1H, m), 2.23 (3H, d,

J_HH= 1.6 Hz), 2.14−1.87 (4H, m), 1.63 (1H, m), 1.47 (1H, m), 1.25

(20H, br s), 0.89 (3H, t, J_HH= 6.7 Hz); ¹³C NMR (CDCl₃) δ 169.2,

155.9 (d, J_CP= 7.1 Hz), 123.4 (d, J_CP= 5.3 Hz), 52.2 (d, J_CP= 7.8 Hz),

52.1, 37.4, 32.1, 30.9, 29.9, 29.8, 29.7, 29.6, 27.9, 25.1 (d, J_CP= 6.8

Hz), 22.9, 22.6 (d, J_CP= 134.3 Hz), 21.5, 14.3; ³¹P NMR (CDCl₃) δ

26.8; HRMS (FAB, NBA, MH⁺) calcd for C₂₁H₄₀O₅P: 403.2613,

found 403.2613; and 8(β) as pale yellow oil. IR (neat, NaCl) 1719

cm⁻¹; ¹H NMR (CDCl₃) δ 3.83 (3H, d, J_HP= 11.2 Hz), 3.76 (3H, s),

2.88 (1H, m), 2.19 (3H, d, J_HH= 1.2 Hz), 2.14 −1.87 (4H, m), 1.63

(1H, m), 1.47 (1H, m), 1.25 (6H, br s), 0.87 (3H, t, J_HH= 6.5 Hz);

Methyl 7-Methyl-2-(hexadecyloxy)-2,3,4,5-tetrahydro-1,2-

oxaphosphepine-6-carboxylate 2-Oxide (11). To a solution of

monocyclic phosphonate analog 4 (0.024 g, 0.10 mmol) and 1-

bromohexadecane (0.15 g, 0.50 mmol) in dry toluene (0.5 mL) was

added nBu₄NI (TBAI) (0.003 mmol, 0.002 g, 5 mol %) at room

temperature. The mixture was heated to reﬂux. After completion of the

reaction (TLC and ³¹P NMR analysis), the solvent was removed under

reduced pressure and the crude product was puriﬁed by column

chromatography (SiO₂, 10−30% EtOAc in hexane) to give

phosphonate analog 11 (0.037g, 86%) as a waxy solid. IR (neat,

1

NaCl), 1717 cm⁻¹; H NMR (CDCl₃) δ 4.12 (2H, m), 3.73 (3H, s),

2.65 (1H, m), 2.47 (1H, m), 2.31 (3H, s), 2.18−1.89 (4H, m), 1.68

(2H, p, J_HH= 6.9 Hz), 1.37−1.24 (26H, br s), 0.86 (3H, t, J_HH= 6.6

Hz); ¹³C NMR (CDCl₃) δ 168.22 (d, J_CP= 1.7 Hz), 159.43 (d, J_CP

=

7.8 Hz), 119.20 (d, J_CP= 4.6 Hz), 66.24 (d, J_CP= 7.0 Hz), 52.00,

32.08, 30.57 (d, J_CP= 6.0 Hz), 29.85, 29.81, 29.80, 29.71, 29.66, 29.52,

27.68, 26.42, 26.78 (d, J_CP= 133.6 Hz), 26.41 (d, J_CP= 2.7 Hz), 25.61,

22.85, 21.29 21.67 (d, J_CP= 7.5 Hz), 21.12 (d, J_CP= 1.6 Hz), 14.44;

³¹P NMR (CDCl₃) δ 23.1; HRMS (FAB, NBA, MH⁺) calcd for

¹³C NMR (CDCl₃) δ 169.4, 155.1 (d, J_CP= 9.4 Hz), 123.3 (d, J_CP

=

4.3 Hz), 52.8 (d, J_CP= 6.7 Hz), 52.2, 37.3, 32.1, 31.0, 29.9, 29.8, 29.7,

29.6, 27.8, 25.3 (d, J_CP= 7.7 Hz), 22.9, 22.4 (d, J_CP= 133.5 Hz), 21.3

(d, J_CP= 2.0 Hz), 14.3; ³¹P NMR (CDCl₃) δ 24.2; HRMS (FAB, NBA,

MNa⁺) calcd for C₂₁H₃₉O₅PNa: 425.2439, found 425.2427.

( )-Methyl 5-Hexadecyl-2-methoxy-7-methyl-2,3,4,5-tetrahydro-

1,2-oxaphosphepine-6-carboxylate 2-Oxide (9(α) and 9(β)). A

solution of alkyl phosphonate 16f (1.05 g, 2.14 mmol) in acetonitrile

(5 mL) was treated as described above to give the monocyclic

phosphonates 9 as a 1:1.25 mixture of diastereomers (0.45 g, 46%)

which were further separated by column chromatography to give 9(α)

as a white solid. IR (neat, NaCl) 1720 cm⁻¹; ¹H NMR (CDCl₃) δ 3.82

C₂₄H₄₆O₅P: 445.3083, found 445.3076.

Methyl 7-Methyl-2-(octadecyloxy)-2,3,4,5-tetrahydro-1,2-

oxaphosphepine-6-carboxylate 2-Oxide (12). The monocyclic

phosphonate 4 (0.064 g, 0.26 mmol) was dissolved in dry acetone (0.5

mL), and NaI (0.044 g, 0.29 mmol) was added. The yellow solution

was heated at reﬂux overnight. After completion of the reaction (³¹P

NMR analysis) the solvent was removed under reduced pressure. The

dark orange residue was dissolved in dry methanol (10 mL), and

prerinsed Amberlite IR-120H resin (0.320 g) was added. The mixture

was shaken on an orbital shaker for 45 min, ﬁltered, and concentrated

under reduced pressure to yield a light orange oil. The oil was

dissolved in dry CH₂Cl₂(0.20 mL), and then potassium carbonate

(0.040 g, 0.29 mmol) and 18-crown-6 (0.002 g, 0.08 mmol) were

added, followed by the addition of n-octadecyl triﬂate (0.149 g, 0.369

mmol)⁴⁰in dry CH₂Cl₂(0.30 mL) dropwise. After 48 h, the reaction

was quenched with deionized water and extracted three times with

CH₂Cl₂. The combined organic extracts was dried over Na₂SO₄and

concentrated under reduced pressure. The crude product was puriﬁed

by column chromatography (SiO₂, 7−15% EtOAc in hexanes) to give

12 (0.061 g, 49%) as a white waxy solid. IR (neat, ATR), 1712, 1646

cm⁻¹; ¹H NMR (CDCl₃) δ 4.13 (2H, m), 3.72 (3H, s), 2.65 (1H, m),

2.47 (1H, m), 2.30 (3H, s), 2.20−1.83 (4H, m), 1.67 (2H, p, J_HH= 6.8

Hz), 1.30−1.16 (28H, br s), 0.85 (3H, t, J_HH= 6.6 Hz); ¹³C NMR

(CDCl₃) δ 168.19, 159.414 (d, J_CP= 7.8 Hz), 119.18 (d, J_CP= 4.6 Hz),

66.22 (d, J_CP= 7.2 Hz), 51.98, 32.07, 30.56 (d, J_CP= 6.2), 29.84, 29.81,

29.78, 29.70, 29.65, 29.51, 29.28, 26.78 (d, J_CP= 134.3 Hz), 26.39 (d,

J_CP= 2.7 Hz), 25.61, 22.84, 21.33, 21.12, 14.27; ³¹P NMR (CDCl₃) δ

23.7; HRMS (FAB, NBA, MH⁺) calcd for C₂₆H₄₉O₅P: 473.3396,

found 473.3385.

X-ray Structure Determination of Compound 10(α). Crystals of

appropriate dimension were obtained by slow evaporation of acetone.

A crystal with approximate dimensions 0.01 × 0.03 × 0.16 mm³was

mounted on a Mitgen cryoloop in a random orientation. Intensity data

were collected at 150 K on a D8 goniostat at a 70 mm crystal-to-

detector distance equipped with a Bruker APEXII CCD detector at

Beamline 11.3.1 at the Advanced Light Source (Lawrence Berkeley

National Laboratory) using synchrotron radiation tuned to λ = 1.2399

Å. For data collection, frames were measured for a duration of 1 s for

low angle data and 3 s for high angle data at 0.3° intervals of ω with a

maximum 2θ value of ∼60°. The data frames were collected using the

program APEX2 and processed using the program SAINT routine

within APEX2 (Bruker Analytical X-ray, Madison, WI, 2010). The data

were corrected for absorption and beam corrections based on the

multiscan technique as implemented in SADABS (Bruker Analytical X-

ray, Madison, WI, 2010). Crystal data and intensity data collection

parameters are listed in Table 4. Structure solution and reﬁnement

were carried out using the SHELXTL-PLUS software package.⁴⁸The

structure was solved by direct methods and reﬁned successfully in the

space group P2₁/c. Full matrix least-squares reﬁnement was carried out

by minimizing Σw(F_o²− F_c²)². The non-hydrogen atoms were reﬁned

anisotropically to convergence. All hydrogen atoms were treated using

(3H, d, J_HP= 11.2 Hz), 3.74 (3H, s), 2.98 (1H, m), 2.23 (3H, d, J_HH

=

1.5 Hz), 2.14 − 1.88 (4H, m), 1.62 (1H, m), 1.45 (1H, m), 1.25 (30

H, br s), 0.88 (3H, t, J_HH= 6.7 Hz); ¹³C NMR (CDCl₃) δ 169.2 (d, J_CP

= 1.8 Hz), 155.9 (d, J_CP= 7.3 Hz), 123.4 (d, J_CP= 5.3 Hz), 52.2 (d, J_CP

= 7.1 Hz), 52.1, 37.4 (d, J_CP= 1.5 Hz), 32.1, 30.9, 30.0, 29.9, 29.8,

29.7, 29.6, 27.9, 25.1 (d, J_CP= 6.8 Hz), 22.9, 22.1 (d, J_CP= 134.4 Hz),

21.5 (d, J_CP= 1.3 Hz), 14.3; ³¹P NMR (CDCl₃) δ 26.8; HRMS (FAB,

NBA, MH⁺) calcd for C₂₅H₄₈O₅P: 459.3239, found 459.3239; and

9(β) as white solid. IR (neat, NaCl) 1720 cm⁻¹; ¹H NMR (CDCl₃) δ

3.82 (3H, d, J_HP= 11.1 Hz), 3.76 (3H, s), 2.88 (1H, m), 2.18 (3H, s),

2.11(1H, m), 1.99 (2H, m), 1.89 (1H, m), 1.63 (1H, m), 1.49 (1H,

m), 1.25 (30H, br s), 0.89 (3H, t, J_HH= 6.6 Hz); ¹³C NMR (CDCl₃) δ

169.3 (d, J_CP= 1.7 Hz), 155.1 (d, J_CP= 9.5 Hz), 123.3 (d, J_CP= 4.6

Hz), 52.8 (d, J_CP= 6.7 Hz), 52.2, 37.3, 32.1, 31.0, 30.0, 29.9, 29.8, 29.7,

29.6, 29.3, 27.8, 25.3 (d, J_CP= 7.9 Hz), 22.9, 22.4 (d, J_CP= 133.4 Hz),

21.3 (d, J_CP= 1.9 Hz), 14.3; ³¹P NMR (CDCl₃) δ 24.1; HRMS (FAB,

NBA, MH⁺) calcd for C₂₅H₄₈O₅P: 459.3239, found 459.3244.

( )-Methyl 2-Methoxy-7-methyl-5-octadecyl-2,3,4,5-tetrahydro-

1,2-oxaphosphepine-6-carboxylate 2-Oxide (10(α) and 10(β)). A

solution of alkyl phosphonate 9g (0.29 g, 0.56 mmol) in acetonitrile (2

mL) was treated as described above to give the monocyclic

phosphonates 10 as a 1:1.25 mixture of diastereomers (0.16 g, 59%)

which were further separated by column chromatography to give

10(α) as a white solid. IR (neat, NaCl) 1719 cm⁻¹; ¹H NMR (CDCl₃)

δ 3.83 (3H, d, J_HP= 11.18 Hz), 3.73 (3H, s), 2.99 (1H, m), 2.24 (3H,

d, J_HH= 1.6 Hz), 2.14−1.88 (4H, m), 1.63 (1H, m), 1.47 (1H, m),

1.26 (32 H, br s), 0.89 (3H, t, J_HH= 6.7 Hz); ¹³C NMR (CDCl₃) δ

169.2, 155.9 (d, J_CP= 7.5 Hz), 123.4 (d, J_CP= 5.3 Hz), 52.2 (d, J_CP

=

7.1 Hz), 52.1, 37.4, 32.1, 30.9, 30.0, 29.9, 29.8, 29.7, 27.9, 25.5 (d, J_CP

= 6.9 Hz), 22.9, 22.1 (d, J_CP= 134.5 Hz), 21.5, 14.3; ³¹P NMR

(CDCl₃) δ 26.8; HRMS (FAB, NBA, MH⁺) calcd for C₂₇H₅₁O₅PNa:

509.3371, found 509.3375; and 10(β) as a white solid. IR (neat, NaCl)

1

1718 cm⁻¹; H NMR (CDCl₃) δ 3.83 (3H, d, J_HP= 11.1 Hz), 3.76

(3H, s), 2.88 (1H, m), 2.19 (3H, d, J_HH= 1.2 Hz), 2.11 (1H, m), 2.01

(2H, m), 1.89 (1H, m)1.64 (2H, m), 1.50 (2H, m), 1.26 (26H, br s),

0.88 (3H, t, J_HH= 6.6 Hz); ¹³C NMR (CDCl₃) δ 169.3, 155.1 (d, J_CP

=

9.3 Hz), 123.3 (d, J_CP= 4.7 Hz), 52.8 (d, J_CP= 6.7 Hz), 52.2, 37.3,

32.1, 31.0, 30, 29.9, 29.7, 29.6, 27.8, 25.3 (d, J_CP= 8 Hz), 22.9, 22.4 (d,

J_CP= 133.3 Hz), 21.3 (d, J_CP= 2.1 Hz), 14.3; ³¹P NMR (CDCl₃) δ

24.2; HRMS (FAB, MH⁺) calcd for C₂₇H₅₁O₅PNa: 509.3371, found

509.3363.

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to Lauwereys et al.⁵²The lipolytic enzymes from Mycobacterium

tuberculosis, Rv0183 (monoglyceride lipase), and LipY (triacylglycerol

lipase) were produced and puriﬁed as previously reported.^34,53Porcine

pancreatic colipase (i.e., colipase) was puriﬁed from lipid-free porcine

pancreatic powder (Laboratoire Industriel de Biologie - Soisy-sous-

Montmorency, France) according to Fernandez et al.⁵⁴

Table 4. Crystal Data and Structure Reﬁnement for

Compound 10(α)

empirical formula

formula weight

temperature

C₂₇H₅₁O₅P

486.65

150(2) K

Lipase Activity Measurements Using the pH-Stat Technique.

Enzymatic activity was assayed at 37 °C by measuring the amount of

free fatty acid (FFA) released from a mechanically stirred acylglycerol

emulsion using 0.1 N NaOH with a pH-stat (Metrohm 718 STAT

Titrino, Switzerland) adjusted to a ﬁxed end point value.⁴¹Except for

Rv0183 acting on MC18 emulsions, all lipase activities were

determined using TC4 speciﬁc assay emulsions: 0.5 mL of TC4 was

mixed with 14.5 mL of a buﬀer solution. The activities of HPL were

determined using the standard assay solution for pancreatic lipase:⁵⁵

0.3 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM CaCl₂, and 4 mM

NaTDC, in the presence of colipase added at a molar excess.⁵⁶The

assay solution for GPLRP2 was 1 mM Tris-HCl (pH 8.0), 150 mM

NaCl, 5 mM CaCl₂, and 2 mM NaTDC.⁵⁰In the case of DGL, the

assay solution was 150 mM NaCl, 2 mM NaTDC, and 2 μM BSA at

pH 5.5.⁵⁷With Cutinase, the assay solution was 2.5 mM Tris-HCl (pH

8.0), 150 mM NaCl, and 0.25 mM NaTDC. With LipY, the assay

solution was 2.5 mM Tris-HCl (pH 7.5), 300 mM NaCl, and 3 mM

NaTDC. With regards to Rv0183, assays were performed with 100 μL

of MC18 added to 15 mL of 2.5 mM Tris-HCl (pH 8.0), 150 mM

NaCl, and 3 mM NaTDC.³⁴All experiments were at least performed

in duplicate. Activities were expressed as international units: 1 U = 1

μmol of FFA released per minute. The speciﬁc activities of HPL,

GPLRP2, DGL, Cutinase, LipY, and Rv0183, expressed in U per mg of

pure enzyme, were found to be 8000 144, 2270 33, 340 21,

2270 63, 129 2, and 287 7 U/mg, respectively.

wavelength

1.23990 Å

monoclinic

P2₁/c

crystal system

space group

unit cell dimensions

a = 10.4218(10) Å

b = 5.6365(5) Å

α = 90°

β =

91.908(5)°

c = 48.570(5) Å

2851.5(5) Å³

4

1.134 Mg/m³

0.571 mm^‑1

1072

γ = 90°

volume

Z

density (calculated)

absorption coeﬃcient

F(000)

crystal size

0.16 × 0.03 × 0.01 mm³

2.93° to 39.46°

θ range for data

collection

index ranges

−10 ≤ h ≤ 10, −5 ≤ k ≤ 5, −49 ≤

l ≤ 49

111 131

reﬂections collected

independent reﬂections 3094 [R(int) = 0.0813]

completeness to θ =

39.46°

absorption correction

96.1%

semiempirical from equivalents

0.9704 and 0.7626

max and min

transmission

Inhibition Assays. The lipase-inhibitor preincubation method was

used to test, in an aqueous medium and in the absence of substrate,

the possible direct reactions between lipases and potential inhibitors.⁴¹

Each enzyme was then preincubated for 30 min at 25 °C with each

inhibitor (5 mg/mL stock solution in isopropanol) at varying inhibitor

molar excess (x_I= 2, 4, 20, 40, 100). Experiments with HPL were

performed in the presence of a 5-fold molar excess of colipase vs HPL.

Lipase incubations with inhibitors were performed in the presence of 4

mM NaTDC in the case of HPL and DGL;⁵⁸2 mM NaTDC with

GPLRP2 and Cutinase; and in the absence of NaTDC with Rv0183

and LipY. Regarding the inhibition by Orlistat, 2 mM NaTDC was

used in the case of Cutinase, Rv0183, and GPLRP2. During the

inhibition experiments, samples of the incubation medium were

collected after a 30 min incubation period and injected into the pH-

stat vessel in order to measure the residual lipase activity at varying

inhibitor molar excess. The variation in the residual lipase activity was

then used to determine the molar excess of the inhibitor which

reduced the enzyme activity to 50% of its initial value (x_I50).⁴²In

addition, each enzyme was preincubated at 25 °C with the best

inhibitor compounds, at a ﬁxed molar excess of x_I= 4 for 1 h. Then,

the residual enzyme activity was measured at various times in order to

determine the half-inactivation time (t_1/2).⁵⁹The x_Ivalue used in these

latter experiments was chosen so that the average residual activities of

Cutinase, Rv0183, and LipY obtained after 30 min of incubation were

in the 10−15% range. In each case, control experiments were

performed in the absence of inhibitor and with the same volume of

isopropanol. It is worth noting that isopropanol at a ﬁnal volume of

less than 5% has no eﬀect on the enzyme activity.

reﬁnement method

full-matrix least-squares on F²

data/restraints/

parameters

3094/47/320

goodness-of-ﬁt on F²

1.105

ﬁnal R indices [I >

2σ(I)]

R1 = 0.1522, wR2 = 0.3741

R indices (all data)

R1 = 0.1740, wR2 = 0.3822

0.398 and −0.399 e·Å⁻³

largest diﬀ. peak and

hole

the appropriate riding model (AFIX m3). Complete listings of

positional and isotropic displacement coeﬃcients for hydrogen atoms,

anisotropic displacement coeﬃcients for the non-hydrogen atoms are

listed as Supporting Information (Tables S1−S5). The obtained crystal

structure has been deposited at the Cambridge Crystallographic Data

Centre and allocated the deposition number CCDC 873939.

In Vitro Biological Evaluation. Reagents. Orlistat, glyceryl

tributyrate (tributyrin, TC4), 1-oleoyl-rac-glycerol (1-monoolein,

MC18), bovine serum albumin (BSA), sodium taurodeoxycholate

(NaTDC), calcium chloride dihydrate (CaCl₂, 2H₂O), and benzami-

dine, 4-morpholineethanesulfonic acid (MES) were purchased from

Sigma-Aldrich-Fluka Chimie (St-Quentin-Fallavier, France). Sodium

chloride (NaCl) and Tris(hydroxymethyl)aminomethane (Tris) were

purchased from VWR International (Fontenay-sous-Bois, France) and

from Euromedex (Mundolsheim, France), respectively. All organic

solvents were purchased from Carlo Erba Reactifs-SDS (Val de Reuil,

France) and were of HPLC grade. For protein digestion, trypsin of

sequencing grade was purchased from Sigma-Aldrich-Fluka Chimie

(St-Quentin-Fallavier, France).

Lipases. All lipolytic enzymes were produced and puriﬁed to

homogeneity at our laboratory. Recombinant Human pancreatic lipase

(HPL) was produced in the yeast Pichia pastoris and puriﬁed as

described previously.⁴⁹Recombinant guinea pig pancreatic lipase-

related protein 2 (GPLRP2) was expressed in Aspergillus orizae and

puriﬁed as described by Hjorth et al.⁵⁰Recombinant dog gastric lipase

(DGL) was produced in transgenic maize by Meristem Therapeutics

(Clermont-Ferrand, France)⁵¹and puriﬁed as described in Roussel et

al.⁴⁴Fusarium solani pisi Cutinase was produced and puriﬁed according

The lipase stereopreference for the compounds tested was also

studied on the basis of the discrimination between the two

diastereoisomers trans-(α) and cis-(β) of the same compound. A

Diastereoselectivity Index (DI)⁴³was deﬁned by eq 1:

|(x_I)_{β isomer}− (x_I)_{α isomer}

|

50

DI_(%)

=

× 100

(x )_{β isomer}+ (x )_{α isomer}

I₅₀

(1)

Protein Digestion Prior to MALDI-TOF Mass Spectrometry

Analysis. Protein digestion was performed with sequencing grade

trypsin, in line with the manufacturer’s recommendations. Brieﬂy,

10216

dx.doi.org/10.1021/jm301216x | J. Med. Chem. 2012, 55, 10204−10219

Journal of Medicinal Chemistry

Article

samples were treated as follows: 5−50 pmol of native and inhibited

protein were diluted in 15 μL of 100 mM ammonium bicarbonate (pH

8.0) buﬀer and successively treated with 10 mM dithiothreitol (DTT)

for 45 min at 56 °C, and 55 mM iodoacetamide (IAA) for 30 min at

room temperature in the dark. Finally samples were incubated with

0.33 mg/mL trypsin solution in 25 mM ammonium bicarbonate (pH

8) buﬀer, and digestions were performed overnight at 37 °C. The

generated peptides were then dried on a SpeedVac (10 min) and

acidiﬁed with 1 μL of 12.5% TFA in water before being analyzed by

MALDI-TOF.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight

(MALDI-TOF) Mass Spectrometry Analysis. Cutinase, Rv0183, and

LipY were preincubated for 30 min at 25 °C with compounds 4, 7(β),

and 11 (5 mg/mL stock solution in isopropanol), respectively, at an x_I

value of 40 in order to abolish any residual lipolytic activity. In each

case, blank experiments were performed in the absence of inhibitor. A

saturated solution of α-cyano-4-hydroxycinnamic acid in acidiﬁed

water (0.1% triﬂuoroacetic acid, TFA) and acetonitrile (30:70, v/v)

was used as a matrix. Prior to the direct mass analysis of the entire

lipases, aliquots of 1 μL of native and inhibited lipase solutions

(corresponding to 20−100 pmol of protein) were mixed with 1 μL of a

saturated solution of sinapinic acid in acidiﬁed water (0.1% TFA) and

acetonitrile (60:40, v/v) and spotted onto the target. Samples were left

to air-dry at room temperature. MALDI-TOF analyses were performed

on a Bruker Microﬂex II mass spectrometer (Daltonik, Deutchland).

Mass spectra were acquired in the positive ion mode, using the

FlexAnalysis software program (Bruker, Daltonik, Deutchland).

Protein identiﬁcations were carried out using the MASCOT version

2.2.0 search engine (Matrix Science, London, UK) and the NCBI

protein database. Theoretical and experimental peptide masses were

obtained using the BioTools software program (Bruker, Daltonik,

Deutchland).

ACKNOWLEDGMENTS

■

Sadia Diomande and Vincent Delorme were funded by PhD

fellowships from EGIDE and the Ministere de l′Enseignement

Superieur et de la Recherche, respectively. This work was

supported by the CNRS, the Agence Nationale de la Recherche

Francaise (ANR-07-PCVI-0009 and ANR MIEN 2009-00904

̀

́

̧

FOAMY_TUB) and by the LISA Carnot Institute (Convention

ANR n°07-CARN-009-01). The compound synthesis was

supported by Grant Number R01-GM076192 from the

National Institute of General Medical Studies. The authors

would like to thank Dr. R. Lebrun, S. Lignon, and R. Puppo (at

the Proteomics platform of the Institut de Microbiologie de la

́ ́

Mediterranee, Marseille, France) for their precious help and

support with the mass spectrometry experiments. Crystallo-

graphic data were collected through the SCrALS (Service

Crystallography at Advanced Light Source) program at the

Small-Crystal Crystallography Beamline 11.3.1 at the Advanced

Light Source (ALS), Lawrence Berkeley National Laboratory.

The ALS is supported by the U.S. Department of Energy,

Oﬃce of Energy Sciences Materials Sciences Division, under

Contract DE-AC02-05CH11231.

ABBREVIATIONS USED

■

AChE, acetylcholinesterase; DAG, diacylglycerol; DGL, dog

gastric lipase; GPLRP2, guinea pig pancreatic lipase-related

protein 2; HPL, human pancreatic lipase; HSL, hormone-

sensitive lipase; MIC: minimal inhibitory concentration; MAG,

monoacylglycerol; TAG, triacylglycerol; x_I, inhibitor molar

excess related to 1 mol of enzyme; x_I50, inhibitor molar excess

leading to 50% lipase inhibition

Molecular Docking. In silico molecular docking of lipase inhibitors

present in the active site of lipases was performed with the Autodock

Vina program.⁶⁰The PyMOL Molecular Graphics System (version

1.3, Schrodinger, LLC) was used as the working environment with an

̈

in-house version of the AutoDock/Vina PyMOL plugin.⁶¹The X-ray

crystallographic structures of HPL, DGL, and GPLRP2 (PDB entry

codes: 1LPB,⁴⁵1K8Q,⁴⁴and 1GPL,²⁵respectively) available at the

Protein Data Bank were used as receptors. Docking runs were

performed after replacing the catalytic serine by a glycine residue to

enable the ligand (i.e., the inhibitor) to adopt a suitable position

corresponding to the prebound intermediate before the nucleophilic

attack in the lipase active site. The box size used for the various

receptors was chosen to ﬁt the whole active site cleft and to allow

nonconstructive binding positions. The model structure ﬁle was

generated for each inhibitor molecule, and its geometry was reﬁned

using the Avogadro open-source program (Version 1.0.1. http://

avogadro.openmolecules.net/).

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ASSOCIATED CONTENT

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̀

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S

* Supporting Information

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AUTHOR INFORMATION

■

Corresponding Author

*E-mail: jfcavalier@imm.cnrs.fr. Phone: +33 491 164 093 (J.-F.

Cavalier). E-mail address: SpillingC@msx.umsl.edu; Phone:

(314) 516-5437 (C. Spilling).

Author Contributions

^§Both authors should be considered as equal ﬁrst authors.

Notes

The authors declare no competing ﬁnancial interest.

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Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases

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