The synthesis of novel bisphosphonates as inhibitors of phosphoglycerate kinase (3-PGK)

View Article Online / Journal Homepage / Table of Contents for this issue

The synthesis of novel bisphosphonates as inhibitors of

phosphoglycerate kinase (3-PGK)

Neil A. Caplan,^aChristopher I. Pogson,^b† David J. Hayes^band G. Michael Blackburn^a*

1

^aChemistry Department, University of Sheﬃeld, Brook Hill, Sheﬃeld, UK S3 7HF

^bGlaxo-Wellcome, Stevenage, Hertfordshire, UK

Received (in Cambridge, UK) 10th August 1999, Accepted 22nd November 1999

A series of conformationally-restrained analogues of 1,3-bisphospho--glyceric acid (1,3-BPG) 1 has been

synthesised for use as inhibitors of 3-PGK (E.C. 2.7.2.3). These compounds have non-scissile phosphonate

linkages and also incorporate α-halogen substituents to make them isopolar and isosteric mimics of the natural

substrate. A monocyclic aryl core between the two phosphoryl centres provides both a rigid framework linking

these moieties and loci for further substitution. The compounds were tested against human 3-PGK and found to be

good competitive inhibitors. α-Fluorination of the phosphonic acids increased the aﬃnity for the enzyme into the

3

4

submicromolar range. Correlation of IC₅₀data with pK_aand pK_avalues indicates that the acidity of the phosphoryl

group exerts a strong inﬂuence on protein binding.

the face of the N-terminal domain and that the nucleotide is

Introduction

bound to the face of the C-terminal domain. The fact that the

The glycolytic pathway converts glucose into pyruvate and is

two binding sites are too far apart for direct phosphoryl trans-

the major source of energy in the citric acid cycle. Phospho-

fer to occur led to the proposal by Blake⁸that 3-PGK under-

glycerate kinase, 3-PGK, is the sixth enzyme in the pathway

goes a conformational change on binding of both substrates

and converts 1,3-bisphospho--glyceric acid, 1,3-BPG 1, into

in a mechanism termed ‘hinge-bending’. The resultant ‘closed’

structure of 3-PGK was recently identiﬁed by Bernstein et al.⁹

in a crystal structure that shows hinge-bending by 3-PGK only

when two substrates, ADP and 3-PGA, are bound. Modelling

based on conformational change locates the substrates ATP

and 3-PGA some 6 Å apart, close enough for direct phosphoryl

transfer to occur.

We have sought in this work to probe the inﬂuence of

anionic charge on this conformational change and on the aﬃn-

ity of analogues of 1,3-BPG for 3-PGK primarily through

the production of a series of conformationally restrained

bisphosphonic acids whose acidities are modulated through

α-halogenation. Syntheses of α-ﬂuorophosphonates have

usually employed one of three general strategies: the reaction

of diethylaminosulfur triﬂuoride, DAST, with α-hydroxy or

α-ketophosphonate esters,^10,11the ﬂuorination of phosphonate

11

anions by “F^ϩ” donors such as FClO₃and, more recently,

N-ﬂuorobisbenzenesulfonimide (Accuﬂuor^TM, NFSi),¹²and

alkylation/acylation processes employing ﬂuorohalomethyl-

phosphonate esters.^13–16Each of these methods has found

application in the present work.

3-phospho--glyceric acid (3-PGA) with concomitant conver-

sion of ADP into ATP. There is a loop in the glycolytic pathway

Results and discussion

Analogue design

at this point whereby 1 is converted into 2,3-bisphospho--

glyceric acid, 2,3-BPG, by 2,3-bisphosphoglycerate mutase

(2,3-BPGM) which is then further converted into 3-PGA in a

process that also generates ATP. The active site of the mutase

enzyme is known to be smaller than that of 3-PGK.^1,2Thus,

3-PGK is a signiﬁcant therapeutic target, especially where there

is a desire to impede glycolysis without shutting down the

pathway completely. Simple modiﬁcation of the routes used for

the synthesis of these analogues could lead to bulkier bisphos-

phonates capable of selective inhibition of 3-PGK but not of

2,3-BPGM.

Systematic design of inhibitors of 3-PGK has generally focused

on analogues closely isosteric to the natural substrate^17,18and in

which the enzyme-labile phosphate linkage has been replaced

by a non-scissile P–C bond. The mismatch resulting from

replacement of the bridging oxygen with a methylene group has

frequently been corrected by α-ﬂuorination or α-chlorination

of the phosphonic acid and much evidence has suggested that

replacements of this nature maintain both their isosteric and

isoelectronic character compared to the parent phosphate

monoester.^19–25

3-PGK is a bilobal enzyme and has been crystallised from a

wide variety of sources.^3–7It is known that 3-PGA is bound to

In order to incorporate speciﬁc features into the design, we

embarked on the following approach. We focused on bisphos-

phonates with a separation of the two phosphoryl centres close

† Current address: Meadowbank Associates, Meadowbank House,

Church Rd, Oﬀham, West Malling, Kent, UK, ME19 5NY.

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

This journal is © The Royal Society of Chemistry 2000

421

View Article Online

to that in the extended conformation of 1 and also having

α-halogenation to provide closer mimicry to the polarity of

1,3-BPG. Secondly, we wished to restrict the large number of

potential conformational states of pentane-1,5-bisphosphon-

ates by introducing a rigid core. Finally, we sought analogues

incorporating sites where additional functionality could be

added without signiﬁcant modiﬁcation to the synthetic route.

Such functionality inter alia could change the polarity of the

bisphosphonates, increase their gross volume, and/or provide

additional hydrogen bonding capacity. These considerations

are satisﬁed through the use of a 1,3- or 1,4-disubstituted

aromatic ring as a spacer, e.g. benzene or pyridine 4a,b. Other

studies have described good inhibition of 3-PGK by analogues

of 1,3-BPG which place the phosphorus centres somewhat

closer together than in the natural substrate.^17,18We there-

fore also sought to synthesise bisphosphonates with four-

and six-atom spacers between the phosphoryl centres (e.g. 2

and 3) to compare with those having a ﬁve-carbon spacer

(e.g. 4).

of diisopropyl bromocadmium diﬂuoromethylphosphonate

with 1,4-diiodobenzene¹⁴was employed giving 9 in 65% yield

(Scheme 2). The esters 8 and 9 were smoothly de-esteriﬁed with

bromotrimethylsilane (for 8a and 9) or iodotrimethylsilane²⁷

(8b, where TMSBr did not work) to give the corresponding

bisphosphonic acids 4a,b and 3 respectively (Scheme 2).

The bis-α-hydroxymethylenephosphonates were prepared

from the corresponding dialdehydes by H-phosphonate addi-

tion (Scheme 3). Isophthaldehyde 10a and terephthaldehyde

Synthesis of symmetrical bisphosphonates

The bis(bromomethyl) compounds 5a–c (Scheme 1) were con-

Scheme 3 Reagents and conditions: i, (EtO)₂PHO–NEt₃, rt or 100 ЊC,

10 min to 36 h, 77–92%; ii, DAST, rt, 10 min, 80–89%; iii, SOCl₂, rt,

2–8 h, 25–68%; iv, TMSBr, rt, 10–20 h then MeOH, 57–85%.

10c were commercially available while pyridine-2,6-dicarb-

oxaldehyde 10b was prepared from commercially available

pyridine-2,6-dimethanol as described.²⁸Each of the dialde-

hydes was phosphorylated using a typical Michaelis–Becker

procedure with diethyl phosphonate and triethylamine to give

the products in excellent yield. Compound 11a was formed by

heating 10a for 17 h. Compound 11b was generated from 10b

after stirring for 36 h at rt (Scheme 3). In the case of aldehyde

10c, the reaction was extremely rapid and gave 11c as a solid

product within 10 min.

Fluorination and chlorination of hydroxyphosphonates 11a

and 11c were achieved in good to excellent yields using diethyl-

aminosulfur triﬂuoride (DAST) and thionyl chloride respec-

tively giving 12a,c and 13a,c respectively. Attempts to ﬂuorinate

the pyridyl compound 11b with DAST failed as also did the use

of a wide variety of alternative ﬂuorinating agents, which either

led to decomposition or complete recovery of starting material

11b. Compound 12b was therefore isolated from the mixture of

products formed in the ﬂuorination of dilithio diethyl 2,6-bis-

[(diethoxyphosphoryl)methyl]pyridine 6b through the use of

limiting NFSi (Scheme 2).^29,30

Scheme 1 Reagents and conditions: i, (EtO)₃P, 120 ЊC, 12–15 h, 61–

82%; ii, 6 M HCl, 110 ЊC, 12 h, 71–77%; iii, TMSBr, rt, 24 h then

MeOH, 61%.

densed with triethyl phosphite using an Arbuzov reaction to

give the corresponding bismethylenephosphonates 6a–c. The

ester products were isolated in good yield, 6a and 6b as oils

and 6c as a white solid. De-esteriﬁcation was achieved either

using hot 6 M HCl (for 6a,c) or bromotrimethylsilane²⁶fol-

lowed by methanolysis of the intermediate tetrakistrimethylsilyl

ester (6b) to give the corresponding bisphosphonic acids 7a,b,c

respectively.

Compounds 6a and 6b were ﬂuorinated with excess NFSi

using the procedure described by Taylor and co-workers¹²in

yields of ca. 60% to give the corresponding bisdiﬂuoromethyl-

enephosphonates 8a,b (Scheme 2). Attempts to ﬂuorinate 6c

by this procedure failed. However, the Cu()-catalysed reaction

Both oxalyl and thionyl chlorides failed to convert 11b into

13b in satisfactory yield under a wide range of conditions and

only small amounts of the desired product mixed with decom-

position products were produced. The best yield (25%) of 13b

was achieved using excess thionyl chloride at room temperature.

Likewise, reactions of 11b with reagents for N-oxidation gave

similar mixtures of decomposition products. It is noteworthy

that the α-chloro- and α-ﬂuoro-bisphosphonates are themselves

perfectly stable species. As before, all of the esters 11, 12, and 13

were smoothly de-esteriﬁed by the use of bromotrimethylsilane

followed by methanolysis to give the corresponding bisphos-

phonic acids 14, 15 and 16 respectively.

To support an exploration of the tolerance of the active site

of 3-PGK to additional steric features in potential inhibitors, a

series of bisphosphonates was prepared with one, two, or three

methyl substituents on the aromatic ring. Compounds 18, 20,

22, 24, 26, and 28 were prepared from the commercially avail-

able bis(bromo and chloromethyl)aromatics by the same routes

as for compounds 4 and 7.

Scheme 2 Reagents and conditions: i, 6a,b added to NaHMDS,

Ϫ78 ЊC, 1 h; ii, N-ﬂuorobisbenzenesulfonimide (NFSi), Ϫ78 ЊC, 2 h,

59–60%; iii, TMSBr, rt, 72 h then MeOH, 65–73%; iv, TMSI, rt, 12 h

then MeOH, 74%; v, NaHMDS–THF, Ϫ78 ЊC then NFSi (1.0 equiv.),

Ϫ78 ЊC, 1 h; vi, (ⁱPrO)₂P(O)CF₂CdBr, Cu()Br, rt, 12 h, 65%.

422

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

View Article Online

achieved using a modiﬁcation of Berlin’s procedure.³¹These

products were reduced to the corresponding α-hydroxy-

phosphonates 31a,c with sodium borohydride using Shahak’s

method.³²A standard Arbuzov reaction gave the unsym-

metrical α-hydroxybisphosphonates 32a,c. α-Fluorination

was achieved using DAST to give 33a,c in 90% yield while

α-chlorination with thionyl chloride in reﬂuxing DCM gave

34a,c with yields of 50–62% (93–95% based on unrecovered

starting material). Use of more vigorous conditions led to

increased side-product formation while milder conditions gave

lower product yields.

For synthesis of α-diﬂuorinated unsymmetrical bisphos-

phonates, the α-ketophosphonates 30a,c were ﬂuorinated using

DAST in good yield to give 35a,c. The Arbuzov reaction failed

for 35a and therefore a Michaelis–Becker procedure with

dialkyl phosphonate and sodium hydride was used. The prod-

uct bisphosphonates 36a,c were formed in 90% yield, based

on 50% recovery of starting material. As before, each of the

unsymmetrical bisphosphonate esters 32, 33, 34, and 36 were

smoothly de-esteriﬁed with TMSBr followed by methanolysis

to give the corresponding bisphosphonic acids 37, 38, 39, and

40 respectively.

Unsymmetrical bisphosphonates

These compounds were generally prepared by procedures

similar to those used for the symmetrical bisphosphonates

(Scheme 4). The need for bifunctional starting materials having

Arenebisphosphonates

The synthesis of these compounds called for the co-formation

of arene and benzyl phosphonates and the palladium-catalysed

coupling used by Hirao et al.^33,34was chosen. 3-(Bromomethyl)-

bromobenzene was reacted with triethyl phosphite in a stand-

ard Arbuzov reaction to give 41 and the product converted into

the arylphosphonate 42 in 68% yield using palladium-catalysed

coupling (Scheme 5). The tetraethyl ester 42 was eﬃciently con-

verted into the bisphosphonic acid 43 with TMSBr as before.

Methylenephosphonate 42 was ﬂuorinated¹²in 53% yield to

give 44, which was converted into the free bisphosphonic acid 2

using TMSBr. By contrast to 41, its α-ﬂuoro-derivative did not

undergo the palladium-catalysed reaction so the route was

modiﬁed by reversing the sequence of introduction of the two

phosphonates. The commercially available dioxolane 45 pro-

vided an ideal starting material for this modiﬁed route and gave

46 in good yield using the palladium-catalysed coupling con-

ditions. The aldehyde function was unmasked under mild

conditions³⁵to give 47 and then phosphonate addition gave

48 in 53% yield. α-Fluorination and chlorination gave 49 and

50 respectively by usual procedures in good yields and the

bisphosphonate esters 48, 49, and 50 were converted into the

corresponding free phosphonic acids 51, 52, and 53 respectively

using TMSBr (Scheme 5).

Other bisphosphonates

As an example of a bisphosphonate with the phosphoryl

centres closer together than in the aromatic bisphosphonates,

54 was synthesised from methallyl chloride (3-chloro-2-methyl-

propene) (Scheme 5). The starting material was condensed with

triethyl phosphite in a typical Arbuzov reaction and the prod-

uct de-esteriﬁed as usual with TMSBr to give the free acid 55.

Biological results and discussion

With a wide range of bisphosphonates available (Fig. 1), testing

was undertaken to determine their mode of inhibition and

activity against the enzyme and to relate these data to the

physical characteristics of these ligands for 3-PGK.

Scheme 4 Reagents and conditions: i, (RO)₃P, 0 ЊC, 5 h, 90–92%; ii,

NaBH₄, 0 ЊC, 3 h, 50–77%; iii, (RO)₃P, 120 ЊC, 36 h, 61–75%; iv, DAST,

rt, 10 min, X = F, 90–91%; v, SOCl₂, 40 ЊC, 12 h, X = Cl, 93–95%; vi,

DAST, rt, 14 h, 73–76%; vii, (RO)₂PONa, 0 ЊC to rt, 12 h, 90–94%; viii,

TMSBr, rt, 10–20 h then MeOH, 56–75%.

Determination of 3-PGK inhibitory activity. 3-PGK can be

assayed using either the forward or the reverse reaction (Fig. 2).

The forward assay system detects ATP formed in a coupled

reaction with ﬁreﬂy luciferase (FFL)³⁶whereby the action of

FFL on luciferin and ATP yields oxyluciferin and light (Fig.

2A). The reduction in light emission in the presence of an

diﬀerential electrophilic reactivity at two sites was met by

3- and 4-(chloromethyl)benzoyl chlorides 29a,c. Phosphoryl-

ation of 29a,c to give the α-ketophosphonates 30a,c was

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

423

View Article Online

Table 1 K_ivalues for selected bisphosphonate inhibition of 3-PGK

a

Compound

K_m^a,b/µM

K_i/µM

IC₅₀^a,c/µM

7a

7b

14a

14b

15a

16b

71 7.1

120 15

80 10

75 8.5

156 17

0.69 0.12

0.33 0.05

200

182

150

163

1.3

77

73 7.5

95

74 15

84

9

7

8

1.34

^aAt 37 ЊC in 0.1 M NaCl, pH 7.1. ^bK_mfor 1,3-BPG. ^cIC₅₀values 5%.

Results average of duplicate runs.

Fig. 1 Bisphosphonates synthesized as inhibitors of 3-PGK.

and to evaluate K_i(Table 1) and graphed as Lineweaver–Burk

plots. In all cases, the quality of ﬁt of the data (not shown)

using analysis for competitive inhibition was clearly superior to

ﬁts based on uncompetitive or non-competitive inhibition.

Scheme 5 Reagents and conditions: i, (EtO)₂PHO, NEt₃, Pd(PPh₃)₄,

95 ЊC, 12 h, 68%; ii, 42 added to NaHMDS, Ϫ78 ЊC, 1 h then NFBS,

Ϫ78 ЊC, 2 h, 53%; iii, 15% H₂SO₄on silica, rt, 4 h, 90%; iv, (EtO)₂PHO,

NEt₃, rt, 36 h, 53%; v, DAST, rt, 10 min, X = F, 93%; vi, xs. SOCl₂, 40 ЊC,

8 h, X = Cl, 61%; vii, TMSBr, rt, 10–20 h, 58–81%; viii, (EtO)₃P, 120 ЊC,

17 h, 40%.

Determination of pK_avalues. pK_aValues were determined for

22 of the bisphosphonic acids by potentiometric titration in the

range 11 > pH > 3.5. This range covers the observed values of

3

4

pK_aand pK_aand of the pyridinium–NH (in the case of com-

pounds 4b, 7b and 14b). Data were analysed using a multiple

pK_aanalysis programme written for Kaleidagraph^TM. Experi-

mental pK_avalues so obtained were corrected for statistical

factors^38–40to take into account the diﬀerences in acidity

between bisphosphonates having two equivalent phosphonic

acid functions and those with non-equivalent phosphonic acids.

For non-equivalent bisphosphonic acids, the assignment of

inhibitor enables determination of IC₅₀values for inhibitors.

α-Halogenated bisphosphonates proved strong inhibitors of

3-PGK and were tested at 1 µM concentration while bis-α-

hydroxy- and bismethylenephosphonates were used at 10 µM.

Kinetic analysis. The kinetics of inhibition of 3-PGK were

determined by assaying the conversion of 3-PGA into 1,3-BPG,

which is coupled to formation of glyceraldehyde 3-phosphate

(GA3P) using GA3P dehydrogenase (GAPDH) (Fig. 2B), and

monitored at 340 nm for formation of NADH.³⁷A represen-

tative group of the above bisphosphonates was selected for

kinetic analysis, namely compounds 7a,b, 14a,b, 15a, and 16b.

Concentrations of inhibitors at 0.3, 1.0, and 2.5 times IC₅₀were

used in kinetic runs for a range of six concentrations of 3-PGA

spanning its K_mfor 3-PGA (~150 µM). The data were analysed

computationally to identify the nature of the enzyme inhibition

3

4

observed pK_avalues as pK_aand pK_arespectively was based

on previous results for α-substituted bisphosphonic acids

(Table 2).^18,21,41,42

Discussion of results

Comparisons between diﬀerent inhibitors based on their IC₅₀

and pK_avalues support a number of conclusions about the

requirements of the active site of 3-PGK for inhibitor structure.

First, all of the compounds evaluated by K_idetermination

are competitive inhibitors of the enzyme, the best showing sub-

424

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

View Article Online

Table 2 IC₅₀values for inhibitors

IC₅₀^a/µM

pK_a, pK_a

Compound

IC₅₀^a/µM

pK_a, pK_a

3

4

3

4

Compound

2

1.0

0.98

0.96

1.17

200

182

193

150

163

181

1.3

1.33

1.15

1.0

1.34

1.08

99

5.16, 6.90

4.82, 5.60

22

73

0.84

78

147

68.8

89

7.24, 7.78

5.33, 5.92

7.40, 8.16

3

24

4a

26

28

4b

5.18, 5.30

7.11, 7.71

6.70, 8.49

7.03, 7.49

6.78, 7.23

6.57, 7.58

6a

37a

37c

38a

38c

39a

39c

40a

40c

43

6.98, 7.29

6b

6c

8.35

14a

14b

14c

15a

15b

15c

16a

16b

16c

18

10.7

8.0

9.4

1.8

1.98

138

102

16

17

6.30, 7.72

6.01, 6.37

6.02, 6.37

5.04, 5.84

5.27, 6.63

6.47, 7.61

6.55, 7.46

5.80, 6.99

51

52

53

55

87

6.45, 7.45

20

0.88

^aIC₅₀values are 5%. ^bpK_avalues were determined at 37 0.1 ЊC in 0.152 M NaCl and are accurate to 0.05.

Fig. 2 Assay of the forward (A) and reverse (B) reactions catalysed by 3-PGK.

micromolar IC₅₀values (Table 1). The values of the inhibition

compounds with a separation of phosphoryl centres greater

constants K_ifor the seven compounds determined correlate well

with the corresponding values for IC₅₀(Table 1).⁴³All of these

values were determined at high sulfate concentration (40 mM)

in order to achieve Michaelis–Menten kinetic behaviour for

3-PGK.³⁷

than that of the natural substrate, notably in the series of

p-substituted unsymmetrical p-xylylbisphosphonates (37c–40c).

This kinase can, however, show good binding to inhibitors with

fewer atoms linking these phosphoryl centres (the series 2, 52,

53) though there appears to be a lower limit to this tolerance

(55).

Secondly, there appears to be a clear rank order of inhibitor

strength as a function of the phosphonic acid α-substituent

that is apparently independent of changes in ring-orientation

and further substitution patterns of: –CH₂P < –CH(OH)P Ӷ

–CHClP ≈ –CHFP < –CF₂P though diﬀerences are sometimes

small for the last three groups. It is evident that α-hydroxylation

of the phosphonic acids does not materially enhance their aﬃn-

ity for 3-PGK (Table 2, entries 7a and 14a, 7b and 14b, 43 and

51, etc.). By contrast, α-halogenation always improves aﬃnity

of the bisphosphonic acids for 3-PGK, usually by one to two

orders of magnitude and in the best cases leading to submicro-

molar values for K_iand IC₅₀. This shows the value of isopolar

mimicry of the phosphate linkage of the natural substrate. In

quantitative terms, there is a modest correlation of IC₅₀with

Lastly, the unsymmetrical benzenephosphonic acid inhibi-

tors with one α-halo- and especially one α,α-diﬂuoromethylene-

phosphonate substituent show activity at least as good as their

bismethylenephosphonate counterparts with two such α-halo-

methylene centres. This observation is borne out by comparison

of the pairs 16a and 39a, 15a and 38a, and 4 and 40a. This

analysis shows that 3-PGK reads out the charge on each phos-

phonic acid group independently, a feature that has yet to

emerge from studies on α-ﬂuorinated arylalkylbisphosphonates

with other enzymes, notably protein tyrosine phosphatases.⁴⁴

Further studies will be necessary to determine whether these

unsymmetrical inhibitors bind in one particular orientation to

the basic patch of 3-PGK, though such behaviour has been

detected in work on some unsymmetrical azapentane-1,5-

bisphosphonates.^18,42It may well emerge that appropriately

designed inhibitors for 3-PGK (and perhaps other kinases) may

diﬀerentiate between phosphonate mimicry of the transferable

phosphate and the static one at C-3 of 3-PGA.

3

pK_a(R = 0.87, Fig. 3a) and a similarly modest relationship

4

with pK_a(R = 0.85) over the range of 21 bisphosphonates thus

evaluated and with slopes close to unity for both curves. What is

most clearly shown by these data is that to achieve high aﬃnity

for 3-PGK, the inhibitors must have a third phosphonic acid

3

4

dissociation, pK_a, below 6.5 (Fig. 3a) and a fourth one, pK_a,

below 7 (cf. 2, 38c, 52, 53) (Fig. 3b).

In conclusion, the present results provide one of the clearest

cut examples yet observed of the value of α-ﬂuorination of

phosphonic acids to improve their aﬃnity for proteins as non-

hydrolysable enzyme inhibitors or substrate analogues of

phosphates and demonstrate that sub-micromolar inhibition

can be achieved for highly polar, water-soluble inhibitors of

kinases.

Thirdly, it appears that 3-PGK can tolerate bisphosphonates

with additional steric components (e.g. the series 18, 20, 22, 24,

26, 28) where in some cases strain must force the two phos-

phoryl functions to lie outside the plane of the benzene ring

(notably 26). Also, 3-PGK shows little discrimination against

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

425

View Article Online

the DS90 data station. Electrospray (ES) mass spectra were

recorded on a Fisons V.G. Platform. R_fvalues of the products

were obtained by thin layer chromatography (TLC) using silica

gel plates (Merck Art. 5554 DC-Alufolien Kieselgel 60 F₂₅₄) and

spots were detected by UV light or by dipping the plate in either

alkaline potassium permanganate or an ethanolic solution of

phosphomolybdic acid with development at 120 ЊC.

Except where stated all intermediates were puriﬁed by ﬂash

column chromatography on Kieselgel 60, 230–400 mesh (Merck

9385) or Chromatographiegel 560, 40–63 mesh (Chemische

Fabrik Uetikon CH-8707).

Lineweaver–Burk plots were determined using Origin^TMand

this programme was also used to calculate K_ivalues and error

factors for the kinetic data. pH Titration proﬁles were obtained

using a Radiometer (Copenhagen) TTT80 Titrator with

ABU880 Autoburette and the pK_avalues obtained from the

data using software written for Kaleidagraph^TM

.

(3-Phosphonophenyl)diﬂuoromethylphosphonic acid (2)

Diethyl [3-(diethoxyphosphoryl)phenyl]diﬂuoromethylphos-

phonate 44 (0.22 g, 0.53 mmol) was dissolved in DCM (20 ml)

under nitrogen and TMSBr (0.58 g, 3.70 mmol, 7 eq., 0.49 ml)

added via syringe. ³¹P NMR spectroscopy showed the reaction

to be complete after 22 h and the reaction was quenched with

methanol (10 ml) and the solvent was removed under reduced

pressure. The residue was co-evaporated with methanol (4

volumes) to yield the crude free acid (0.13 g). The crude free

acid was dissolved in methanol, cyclohexylamine (2 eq.) added,

and the solvent was removed under reduced pressure and the

product puriﬁed by recrystallisation from methanol, with

acetone to give the biscyclohexylammonium salt of the title

compound as a white solid (0.22 g, 80%, mp >275 ЊC); δ_H(D₂O)

1.15–1.95 (22 H, m, cyclohexyl-H), 7.50–7.55 (1 H, m, ArH),

7.70–7.85 (3 H, m, 3 ArH); δ_P(D₂O) 4.65 (t, J_FP102), 12.7 (s);

δ_F(D₂O) Ϫ110.7 (d, J_FP103); δ_C(D₂O) 23.8, 24.2, 30.3, 50.1,

126.3, 128.3, 130.9, 134.0, 136.3.

Fig. 3 Graph of log(IC₅₀) vs. pK_afor bisphosphonate inhibitors of

3-PGK.

1,4-Bis(phosphonodiﬂuoromethyl)benzene (3)

1,4-Bis[(diisopropoxyphosphoryl)diﬂuoromethyl]benzene

9

(0.17 g, 0.33 mmol) was deprotected as for 2 with TMSBr (0.35

g, 2.31 mmol, 0.31 ml). The reaction was stirred at rt and fol-

lowed by ³¹P NMR spectroscopy. After 96 h the reaction was

complete and was worked up as usual to yield the crude free

acid (0.10 g). Salt formation and puriﬁcation as for 2 gave the

biscyclohexylammonium salt of the title compound as a white

solid (0.11 g, 65%, mp >275 ЊC) (Found: C 44.4, H 6.05, N 5.1.

C₂₀H₃₄F₄N₂O₆P₂(salt) requires C 44.7, H 6.4, N 5.2%);

δ_H(D₂O) 1.25–1.95 (22 H, m, cyclohexyl-H), 7.30, 7.75 (2 H

each, 2 d, J 8.0, 2 ArH each); δ_P(D₂O) 5.21 (t, J_PF92.5);

δ_F(d₄-MeOH) Ϫ109.4 (d, J_PF91.8); free acid: δ_C(D₂O) 128.3,

134.0.

Experimental

Tetrahydrofuran (THF) was reﬂuxed over sodium and benzo-

phenone and distilled when the mixture turned blue. Dichloro-

methane and toluene were distilled oﬀ calcium hydride and

toluene was stored over calcium hydride. Unless otherwise

stated, petrol refers to petroleum ether bp 40–60 ЊC. Melting

points were measured on a Koﬂer hot stage micro-melting

point apparatus and are uncorrected. IR spectra were recorded

on a Perkin-Elmer 457 grating infra-red spectrometer. NMR

spectra were recorded on a Bruker AC-250 spectrometer

sample changer with a quadronuclear probe. All spectra

are reported using the δ scale in parts per million. Proton

chemical shifts are accurate to ϩ0.01 ppm. Coupling constant

J values are recorded in Hz and refer to vicinal proton–proton

coupling (³J_H,H) unless otherwise stated. Phosphorus, ﬂuorine

and carbon chemical shifts are accurate to ϩ0.1 ppm. Proton

spectra have been calibrated by either external tetramethyl-

silane or via internal solvent reference except in cases where

D₂O has been used as the solvent. In this case 2,2-dimethyl-2-

silapentane-5-sulfonate was used as the reference sample.

Phosphorus NMR spectra were recorded at 101 MHz and are

referenced downﬁeld from external 85% H₃PO₄. Fluorine NMR

spectra were recorded at 235 MHz and are referenced downﬁeld

from external CFCl₃. Carbon NMR spectra were recorded at

62.9 MHz. Mass spectra were obtained on a Kratos MS80 mass

spectrometer in conjunction with a Kratos DS90 data station

using chemical ionisation with ammonia as the reagent gas (CI)

or electron impact (EI). Fast atom bombardment (FAB) spectra

were obtained on a Kratos MS80RF machine together with

1,3-Bis(phosphonodiﬂuoromethyl)benzene (4a)

1,3-Bis[(diethoxyphosphoryl)diﬂuoromethyl]benzene 8a (0.31

g, 0.70 mmol) was de-esteriﬁed as for 2 with TMSBr (0.75 g, 4.87

mmol, 0.64 ml). Salt formation and puriﬁcation as for 2 gave

the biscyclohexylammonium salt of the title compound as a

white solid (0.27 g, 73%, mp >275 ЊC); δ_H(D₂O) 1.05–2.00

(22 H, m, cyclohexyl-H), 7.55–7.65 (4 H, m, ArH); δ_P(D₂O)

5.07 (t, J_PF103); δ_F(D₂O) Ϫ110.1 (d, J_PF105); δ_C(D₂O) 23.8,

24.2, 34.1, 50.2, 128.3, 128.8, 130.1, 134.0.

2,6-Bis(phosphonodiﬂuoromethyl)pyridine (4b)

2,6-Bis[(diethoxyphosphoryl)diﬂuoromethyl]pyridine 8b (0.49

g, 1.08 mmol) was dissolved in DCM (20 ml) under argon.

Iodotrimethylsilane (1.52 g, 7.58 mmol, 7 eq., 1.08 ml) was

added via syringe and the reaction followed by ³¹P NMR spec-

troscopy until de-esteriﬁcation was complete then the reaction

426

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

View Article Online

was quenched by dropwise addition of methanol. Solvent was

removed under reduced pressure and the residue co-evaporated

with methanol (6 vol). The resulting yellow solid was dissolved

in water (15 ml) and the solution neutralised with 0.5 M NaOH.

The product was lyophilised and the title compound isolated as

a white solid (0.22 g, 64%, mp 268 ЊC) (Found: C 42.5, H 5.9,

N 8.1. C₁₉H₃₃F₄N₃O₆P₂(salt) requires C 42.4, H 6.2, N 7.8%);

δ_H(D₂O) 7.70–7.75 (2 H, m, 2 ArH), 7.95–8.05 (1 H, m, ArH);

δ_P(D₂O) 3.53 (t, J_PF95.3); δ_F(D₂O) Ϫ114.3 (d, J_FP96.9);

δ_C(D₂O) 117.5 (dt, J_CP182, J_CF260), 123.4, 139.3, 152.4.

the tetrasodium salt of the title compound, isolated as a glassy

white solid. The remaining crude free acid (1.20 g) was lyo-

philised, dissolved in methanol, cyclohexylamine (2 eq.) added,

and solvent removed under reduced pressure to yield a white

solid. This was crystallised by precipitation from methanol with

acetone. The biscyclohexylammonium salt of the title com-

pound was isolated as a white solid (1.33 g, 71%, mp >275 ЊC)

(Found: C 51.6, H 8.3, N 5.85. C₂₀H₃₈N₂O₆P₂(salt) requires C

51.7, H 8.25, N 6.0%); free acid: δ_H(D₂O) 3.15 (4 H, d, J_HP25.5,

2 PCH₂–), 7.05–7.30 (4 H, m, ArH); δ_P(D₂O) 25.8 (s); δ_C(D₂O)

34.1 (d, J_CP131), 127.9, 128.9, 130.7, 132.9; m/z (ES^Ϫ) 267

(100%, MH^ϩ), 185 (63, M Ϫ PO₃Et₂), 105 (48, M Ϫ 2PO₃Et₂).

1,3-Bis[(diethoxyphosphoryl)methyl]benzene⁴⁵(6a)

1,3-Bis(bromomethyl)benzene 5a (5.00 g, 18.94 mmol) and tri-

ethyl phosphite (4.62 g, 39.78 mmol, 4.81 ml) were stirred

together at 120 ЊC overnight under a nitrogen atmosphere.

The reaction was cooled and the volatiles removed in vacuo. The

crude product was puriﬁed by ﬂash column chromatography

to yield the title compound as a colourless oil (5.94 g, 80%);

R_f0.22 (DCM–MeOH, 95:5) (Found: M^ϩ378.135. C₁₆H₂₈O₆P₂

2,6-Bis(phosphonomethyl)pyridine (7b)

2,6-Bis[(diethoxyphosphoryl)methyl]pyridine 6b (1.25 g, 3.30

mmol) was deprotected as for 2 with TMSBr to aﬀord the crude

product as an oﬀ-white solid (0.57 g). Salt formation and puriﬁ-

cation as for 2 gave the biscyclohexylammonium salt of the title

compound as a white solid (0.94 g, 61%, mp >275 ЊC) (Found:

C 48.9, H 7.9, N 8.85. C₁₉H₃₈N₃O₆P₂(salt) requires C 48.9,

H 8.2, N 9.0%. Found: M^ϩ268.014. C₇H₁₂NO₆P₂requires MH

268.014); free acid: δ_H(D₂O) 3.50 (4 H, d, J_HP24.7, 2 PCH₂–),

7.65–7.75 (1 H, m, ArH), 8.30–8.35 (2 H, m, ArH); δ_P(D₂O)

14.4 (s); δ_C(D₂O) 34.1 (d, J_CP124), 117.5, 125.6, 138.1; m/z

(FAB^Ϫ) 266 (100%, M Ϫ 1); m/z (FAB^ϩ) 268 (67%, MH^ϩ), 367

(25, M ϩ C₆H₁₁NH₃^ϩ), 467 (M ϩ 2C₆H₁₁NH₃^ϩ).

requires M 378.136); ν_max/cm^Ϫ1(ﬁlm) 2909 (C–H), 1559 (C᎐C),

᎐

1213 (P᎐O), 1097 (P–O–C); δ (CDCl₃) 1.25 (12 H, t, J 6.1,

᎐

H

4 CH₃CH₂–), 3.10 (4 H, d, J_HP20.0, 2 PCH₂–), 3.95–4.05 (8 H,

m, 4 CH₃CH₂–), 7.05–7.15 (4 H, m, ArH); δ_P(CDCl₃) 26.7 (s);

δ_C(CDCl₃) 16.0, 16.1, 33.2 (d, J_CP125), 61.7, 61.8, 128.3, 128.7,

130.7, 131.5; m/z (EI^ϩ) 379 (100%, MH^ϩ), 351 (12, M Ϫ C₂H₄),

323 (6, M Ϫ 2C₂H₄), 295 (8, M Ϫ 3C₂H₄), 267 (13, M Ϫ

4C₂H₄), 185 (26, M Ϫ 4C₂H₄Ϫ PO₃H₂).

1,4-Bis(phosphonomethyl)benzene⁴⁵

2,6-Bis[(diethoxyphosphoryl)methyl]pyridine (6b)

1,4-Bis[(diethoxyphosphoryl)methyl]benzene 6c (2.00 g, 5.29

mmol) was dissolved in 6 M hydrochloric acid (50 ml) and

heated at reﬂux overnight then solvent removed under reduced

pressure. Co-evaporation of the residue with water (5 × 50 ml)

furnished the title compound as a white solid (1.08 g, 77%,

mp 269–70 ЊC, lit.⁴⁶268–271 ЊC); δ_H(d₄-MeOH) 3.05 (4 H, d,

J_HP15.9, PCH₂–), 7.25 (4 H, s, 4 ArH); δ_P(d₄-MeOH) 24.8 (s);

δ_C(d₄-MeOH) 33.2 (d, J_CP134), 129.4, 131.1.

2,6-Bis(bromomethyl)pyridine 5b (1.52 g, 5.75 mmol) was dis-

solved in toluene (20 ml) and triethyl phosphite (1.40 g, 12.07

mmol, 2.00 ml) added via syringe. The reaction was heated at

reﬂux for 15 h. Solvent was removed in vacuo and the crude

product puriﬁed by ﬂash column chromatography to give the

title compound as a pale yellow liquid (1.33 g, 61%); R_f0.45

(DCM–MeOH, 90:10); ν_max/cm^Ϫ1(ﬁlm) 2909 (C–H), 1576

(C᎐C), 1222 (P᎐O), 1097 (P–O–C) (Found: M^ϩ379.132.

᎐

C₁₅H₂₇NO₆P₂requires 379.131); δ_H(CDCl₃) 1.25 (12 H, t, J 6.2,

4 CH₃CH₂–), 3.35 (4 H, d, J_HP21.5, 2 PCH₂–), 4.45–4.60 (8 H,

m, J 6.2, 4 CH₃CH₂–), 7.20–7.25 (2 H, m, ArH), 7.50–7.60 (1 H,

m, ArH); δ_P(CDCl₃) 25.5 (s); δ_C(CDCl₃) 16.1, 16.2, 36.6 (d, J_CP

135), 61.9, 62.0, 118.2, 122.2, 136.7; m/z (EI^ϩ) 379 (55%, M^ϩ),

323 (10, M Ϫ 2C₂H₄).

1,3-Bis[(diethoxyphosphoryl)diﬂuoromethyl]benzene (8a)

Sodium hexamethyldisilazide (NaHMDS) (8.00 mmol, 1.0 M

solution in THF, 8.00 ml) was added to THF (50 ml) under

an inert atmosphere and the solution cooled to Ϫ78 ЊC. 1,3-

Bis[(diethyoxyphosphoryl)methyl]benzene 6a (0.55 g, 1.45

mmol) in THF (15 ml) was added dropwise at this temperature

over 5 min and the resulting dark green solution stirred for 1 h.

A solution of N-ﬂuorobisbenzenesulfonimide (3.35 g, 10.62

mmol) in THF (10 ml) was added to the reaction during 5 min

at Ϫ78 ЊC and the resulting solution stirred at Ϫ78 ЊC for

2 h. The solution was allowed to warm to Ϫ20 ЊC and then

quenched with 0.01 M hydrochloric acid (50 ml). Volatiles were

removed under reduced pressure and the residue extracted with

ethyl acetate (3 × 100 ml). The combined organic extracts were

washed with 5% sodium bicarbonate (50 ml) and brine (50 ml),

dried over MgSO₄, ﬁltered, and solvent removed under reduced

pressure. The crude material was puriﬁed by ﬂash column

chromatography to yield the title compound as a yellow oil (0.42

g, 65%); R_f0.15 (DCM–MeOH, 98:2) (Found: M^ϩ451.106.

C₁₆H₂₅F₄O₆P₂requires 451.107); δ_H(CDCl₃) 1.25 (12 H, t, J 6.3,

4 CH₃CH₂–), 4.10–4.25 (8 H, m, 4 CH₃CH₂–), 7.50–7.55, 7.75–

7.85 (2 H each, 2 m, 2 ArH); δ_P(CDCl₃) 6.20 (t, J_PF113);

δ_F(CDCl₃) Ϫ108.2 (t, J_PF116); δ_C(CDCl₃), 16.1, 16.2, 64.9,

65.0, 117.7 (dt, J_CP218, J_CF263), 126.6, 128.1, 128.8, 133.2 (dt,

²J_CP12.4, ²J_CF21.0, C-CF₂P); m/z (EI^ϩ) 451 (100%, MH^ϩ), 423

(8, M Ϫ C₂H₄), 395 (5, M Ϫ 2C₂H₄).

1,4-Bis[(diethoxyphosphoryl)methyl]benzene⁴⁵(6c)

1,4-Bis(bromomethyl)benzene 5c (10.00 g, 37.89 mmol) and tri-

ethyl phosphite (13.22 g, 79.58 mmol, 2.1 eq., 13.65 ml) were

stirred together under an inert atmosphere at 120 ЊC for 15 h

and volatiles were then removed in vacuo. The title compound

was isolated as a white crystalline solid by ﬁltration and was

used without further puriﬁcation (11.74 g, 82%); R_f0.10

(DCM–MeOH, 95:5) (Found: M^ϩ378.135. C₁₆H₂₈O₆P₂

requires 378.136); ν_max/cm^Ϫ1(Nujol mull) 2909 (C–H), 1654

(C᎐C), 1247 (P᎐O), 1025 (P–O–C); δ (CDCl₃) 1.05 (12 H, t,

᎐

H

J 6.0, 4 CH₃CH₂–), 2.90 (4 H, d, J_HP20.5, PCH₂–), 3.75–

3.85 (8 H, m, 4 CH₃CH₂–), 7.05 (4 H, s, 4 ArH); δ_P(CDCl₃) 27.1

(s); δ_C(CDCl₃) 16.0, 16.1, 33.2 (d, J_CP130), 61.7, 61.8, 128.9,

131.7; m/z (EI) 378 (15%, M^ϩ), 241 (76, M Ϫ PO₃Et₂), 213

(10, M Ϫ PO₃Et₂Ϫ C₂H₄), 185 (27, M Ϫ PO₃Et₂Ϫ 2C₂H₄), 104

(100, M Ϫ 2PO₃Et₂).

1,3-Bis(phosphonomethyl)benzene (7a)

1,3-Bis[(diethoxyphosphoryl)methyl]benzene 6a (2.00 g, 5.29

mmol) was dissolved in 6 M hydrochloric acid (30 ml) and the

mixture heated to reﬂux for 18 h. Solvents were removed under

reduced pressure and the product co-evaporated with 5 vol

water. Some of this was dissolved in water (5 ml) and titrated to

pH 7.5 with 1 M NaOH. Lyophilisation of this solution yielded

2,6-Bis[(diethoxyphosphoryl)diﬂuoromethyl]pyridine (8b)

The title compound was made by the same method as 8a using

2,6-bis[(diethoxyphosphoryl)methyl]pyridine 6b (0.31 g, 0.81

mmol), NaHMDS (4.47 mmol, 1.0 M solution in THF, 4.47 ml)

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

427

View Article Online

and NFSi (2.13 g, 6.77 mmol). The crude material was puriﬁed

by ﬂash column chromatography (EtOAc–MeOH, 98:2) to give

the title compound as a colourless oil (0.22 g, 59%); R_f0.25

(DCM–MeOH, 95:5) (Found: M^ϩ451.095. C₁₅H₂₃F₄NO₆P₂

water (50 ml), dried over MgSO₄, ﬁltered, and volatiles removed

under reduced pressure. The crude product was puriﬁed by ﬂash

column chromatography and the title compound isolated as a

pale yellow oil (9.13 g, 86%); R_f0.37 (DCM–MeOH, 90:10);

requires 451.094); ν_max/cm^Ϫ1(ﬁlm) 2967 (C–H), 1614 (C᎐C),

ν_max/cm^Ϫ1(ﬁlm) 3382 (O–H), 2985 (C–H), 1641 (C᎐C), 1595

᎐

1280 (P᎐O), 1040 (P–O–C); δ (CDCl₃) 1.10 (12 H, t, J 6.3,

(C᎐C), 1257 (P᎐O), 1026 (P–O–C); δ (CDCl ) 1.20–1.35 (12 H,

᎐ ᎐

H 3

᎐

H

4 CH₃CH₂–), 4.00–4.10 (8 H, m, 4 CH₃CH₂–), 7.25–7.30 (1 H,

m, ArH), 7.60–7.65 (2 H, m, ArH); δ_P(CDCl₃) 5.03 (t, J_PF107);

δ_F(CDCl₃) Ϫ111.5 (t, J_PF109); δ_C(CDCl₃) 15.7, 15.8, 65.2,

65.3, 115.7 (dt, J_CP224, J_CF266), 128.5, 132.6, 151.1 (dt,

m, 4 CH₃CH₂–), 4.00–4.15 (8 H, m, 4 CH₃CH₂–), 5.10 (2 H, 2 d,

J_HP8.0, 2 PCH(OH)–), 7.45–7.55 (2 H, m, ArH), 7.70–7.80

(1 H, m, ArH); δ_P(CDCl₃) 20.1, 20.2 (2 s); δ_C(CDCl₃) 16.3,

16.4, 63.0, 63.3, 70.6 (d, J_CP159), 121.3, 137.3, 154.0; m/z (EI^ϩ)

412 (72%, MH^ϩ), 396 (25, M Ϫ OH), 380 (10, M Ϫ 2OH), 274

(98, M Ϫ PO₃Et₂), 136 (68, M Ϫ 2PO₃Et₂).

2

²J_CP14.3, J_CF24.8, C-CF₂P); m/z (EI^ϩ) 451 (100%, M^ϩ),

423 (27, M Ϫ C₂H₄), 315 (45%, M Ϫ PO₃Et₂)^ϩ, 178 (49, M Ϫ

2PO₃Et₂)^ϩ.

1,4-Bis[(diethoxyphosphoryl)hydroxymethyl]benzene⁴⁶(11c)

1,4-Bis[(diisopropoxyphosphoryl)diﬂuoromethyl]benzene¹⁴(9)

Terephthalaldehyde 10c (10.00 g, 74.55 mmol) was placed in a

ﬂask under nitrogen and diethyl phosphonate (20.54 g, 149.11

mmol, 19.20 ml) added with stirring at rt. Triethylamine (7.54 g,

74.55 mmol, 10.40 ml) was added and an exothermic reaction

occurred. After 10 min the mixture solidiﬁed, was allowed

to cool, suspended in DCM (100 ml), ﬁltered, and the solid

washed with DCM (50 ml) to aﬀord the title compound as

a white solid (28.19 g, 92%, mp 207–208 ЊC, lit.⁴⁷204–205 ЊC;

R_f0.25 (DCM–MeOH, 90:10); ν_max/cm^Ϫ1(Nujol mull) 3246

Diisopropyl bromodiﬂuoromethanephosphonate (2.98 g, 10.10

mmol, 3.33 eq.) was dissolved in dry DMF (25 ml) under an

inert atmosphere and acid-washed cadmium (1.25 g, 11.11

mmol, 3.67 eq.) added with cooling. The suspension was stirred

at rt for 2 h and excess metal allowed to settle. The organo-

metallic was transferred to a fresh ﬂask under argon, the

residue washed with DMF (10 ml) which was added to the

solution. 1,4-Diiodobenzene (1.00 g, 3.03 mmol) in DMF (10

ml) and copper() chloride (0.70 g, 7.07 mmol, 2.67 eq.) were

added at rt and the reaction was stirred overnight. Ether (100

ml) was added, the solution ﬁltered, and the ﬁltrate washed

with ether. The ether layer was washed with ammonium

chloride and water, dried over Na₂SO₄, ﬁltered and solvent was

removed under reduced pressure. The crude product was

puriﬁed by ﬂash column chromatography to yield the title

compound as a pale yellow oil (2.74 g, 60%); R_f0.59 (DCM);

ν_max/cm^Ϫ1(ﬁlm) 2989 (C–H), 1641 (C᎐C), 1266 (P᎐O), 1001

(O–H), 2924 (C–H), 1616 (C᎐C), 1228 (P᎐O), 1020 (P–O–C);

᎐

δ_H(CDCl₃) 1.25 (12 H, t, J 6.3, 4 CH₃CH₂–), 3.95–4.10 (8 H,

m, 4 CH₃CH₂–), 5.00 (2 H, d, J_HP10.1, PCH₂–), 7.50 (4 H, s,

4 ArH); δ_P(CDCl₃) 21.6 (s); δ_C(CDCl₃) 16.1, 16.2, 62.2, 74.1

(d, J_CP162), 126.4, 135.4.

1,3-Bis[(diethoxyphosphoryl)ﬂuoromethyl]benzene (12a)

1,3-Bis[(diethoxyphosphoryl)hydroxymethyl]benzene 11a (2.07

g, 5.05 mmol) was dissolved in DCM (40 ml) under argon and

DAST (2.45 g, 15.15 mmol, 2.01 ml) was added via syringe. The

reaction was stirred for 15 min, cooled to 0 ЊC, and quenched

with sodium bicarbonate solution. The layers were separated

and the aqueous layer washed with DCM (2 × 50 ml). The

combined organics were washed with water (2 × 50 ml) and

brine (50 ml), dried over MgSO₄, ﬁltered, and the solvent was

removed under reduced pressure to give the title compound

as a yellow liquid (1.32 g, 80%); R_f0.50 (DCM–MeOH, 90:10)

(Found: M^ϩ414.116. C₁₆H₂₆P₂O₆F₂requires 414.117);

δ_H(CDCl₃) 1.20–1.30 (12 H, t, J 6.2, 4 CH₃CH₂–), 3.90–

4.10 (8 H, m, 4 CH₃CH₂–), 5.65 (2 H, dd, J_HP7.5, J_HF43.5,

2 PCHF–), 7.40–7.55 (4 H, m, ArH); δ_P(CDCl₃) 14.6, 15.4 (2 d,

J_PF83.3); δ_F(CDCl₃) Ϫ201.3, Ϫ201.8 (2 d); H-coupled Ϫ201.3,

Ϫ201.9 (2 dd, J_PF83.3, J_HF43); δ_C(CDCl₃) 16.2, 16.3, 62.4,

62.5, 88.7 (dd, J_CP164, J_CF181), 125.1, 126.7, 128.0, 134.3; m/z

(EI^ϩ) 414 (51%, M^ϩ), 137 (100, PO₃Et₂), 108 (70, PO₃HEt),

81 (52, PO₃Et₂).

᎐

(P–O–C); δ_H(CDCl₃) 1.20, 1.30 (12 H each, 2 d, J 6.0, 2

(CH₃)₂CH– each), 4.70–4.75 (4 H, m, 4 (CH₃)₂CH–), 7.70 (4 H,

d, J 8.3, 4 ArH); δ_P(CDCl₃) 4.42 (t, J_PF115); δ_F(CDCl₃) Ϫ105.2

(t, J_PF112); δ_C(CDCl₃) 23.6, 24.2, 74.0, 74.1, 117.6 (dt, J_CP218,

J_CF263) 128.1, 132.3 (dt, ²J_CP14.3, ²J_CF22.8, C–CF₂P), 137.5.

1,3-Bis[(diethoxyphosphoryl)hydroxymethyl]benzene (11a)

Isophthalaldehyde 10a (3.00 g, 22.82 mmol) and diethyl phos-

phonate (6.32 g, 45.85 mmol, 5.90 ml) were stirred together in

a dry ﬂask under an inert atmosphere. Triethylamine (2.32 g,

22.8 mmol) was added with cooling and the reaction was then

heated and stirred at 100 ЊC for 17 h. The reaction was cooled,

diluted with water (100 ml), and extracted with DCM (2 × 50

ml). The combined organics were washed with 1 M hydro-

chloric acid (50 ml) and brine (50 ml), dried over MgSO₄, ﬁl-

tered, and the solvent was removed under reduced pressure. The

title compound was isolated as a viscous colourless oil after ﬂash

column chromatography (DCM–MeOH, 90:10) (7.02 g, 77%);

R_f0.43 (DCM–MeOH, 90:10) (Found: M^ϩ411.134. C₁₆H₂₉-

P₂O₈requires 411.134); ν_max/cm^Ϫ1(ﬁlm) 3411 (O–H), 2987

2,6-Bis[(diethoxyphosphoryl)ﬂuoromethyl]pyridine (12b)

This compound was prepared from 6b by a similar reaction as

for 8b but using 1.0 equivalent of NFSi and chromatography of

the crude product mixture on silica (eluent EtOAc); R_f0.46

(EtOAc–MeOH, 90:10) (Found: M^ϩ415.114. C₁₅H₂₅F₂NO₆P₂

requires 415.113); δ_H(CDCl₃) 1.25 (12 H, t, J 6.2, 4 CH₃CH₂–),

4.00–4.05 (8 H, m, 4 CH₃CH₂–), 5.95 (2 H, dd, J_HP9.2, J_HF43.6,

2 PCHF–), 7.60–7.65 (2 H, m, ArH), 7.90–7.95 (1 H, m, ArH);

δ_P(CDCl₃) 13.7 (d, J_PF79.8); δ_F(CDCl₃) Ϫ206.2 (d); H-coupled

Ϫ206.3 (dd, J_PF80.7, J_HF43.6); δ_C(CDCl₃) 16.0, 16.1, 64.0,

(C–H), 1658 (C᎐C), 1266 (P᎐O), 1028 (P–O–C); δ (CDCl₃)

᎐

H

1.10–1.20 (12 H, m, 4 CH₃CH₂–), 2.60 (2 H, br, 2 –OH), 3.80–

4.05 (8 H, m, 4 CH₃CH₂–), 4.90 (2 H, d, J_HP9.5, 2 PCH(OH)–),

7.10–7.30 (4 H, m, ArH); δ_P(CDCl₃) 21.89, 21.95 (2 s);

δ_C(CDCl₃) 16.4, 16.5, 68.1, 68.2, 71.1 (d, J_CP161), 126.1, 126.9,

128.0, 128.1, 137.2; m/z (ES^ϩ) 411 (100%, MH^ϩ), 433 (67,

M ϩ Na), 821 (23, 2M ϩ H), 843 (66, 2M ϩ Na).

2

2,6-Bis[(diethoxyphosphoryl)hydroxymethyl]pyridine (11b)

64.2, 89.1 (dd, J_CP166, J_CF186), 128.1, 134.0, 152.7 (d, J_CF

20.2); m/z (EI^ϩ) 415 (86%, M^ϩ), 141 (100, M Ϫ 2PO₃Et₂).

2,6-Pyridinedicarbaldehyde⁴⁶10b (3.49 g, 26.23 mmol) and

diethyl phosphonate (7.25 g, 52.46 mmol, 6.80 ml) were stirred

together in DCM (50 ml) under nitrogen. Triethylamine (2.65 g,

26.23 mmol, 3.65 ml) was added via syringe and the reaction

stirred at rt for 36 h then water (100 ml) was added. The layers

were separated and the aqueous layer extracted with DCM

(2 × 50 ml). The combined organic extracts were washed with

1,4-Bis[(diethoxyphosphoryl)ﬂuoromethyl]benzene (12c)

1,4-Bis[(diethoxyphosphoryl)hydroxymethyl]benzene 11c (0.33

g, 0.57 mmol) was suspended in DCM (30 ml) under an inert

atmosphere and DAST (0.37 g, 2.27 mmol, 4 eq., 0.30 ml)

added via syringe, the resulting solution stirred for 10 min at rt

428

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

View Article Online

and then quenched with aqueous NaHCO₃. The reaction was

worked up as usual and the crude product isolated as a yellow

oil. Puriﬁcation by ﬂash column chromatography aﬀorded the

title compound as a colourless oil (0.30 g, 89%); R_f0.24 (DCM–

MeOH, 90:10) (Found: 414.117. C₁₆H₂₆F₂O₆P₂requires M

Salt formation and puriﬁcation as for 2 gave the biscyclohexyl-

ammonium salt of the title compound as a white solid (mp

>275 ЊC) (Found: C 47.5, H 7.4, N 5.45. C₂₀H₃₈N₂O₈P₂(salt)

requires C 47.75, H 7.7, N 5.65%); δ_H(D₂O) 5.00 (2 H, d,

J_HP11.0, 2 PCH(OH)–), 7.40–7.50 (3 H, m, ArH), 7.50–7.55

(1 H, m, ArH); δ_P(D₂O) 19.5 (s); δ_C(D₂O) 69.7 (d, J_CP159),

125.0, 126.4, 128.2, 136.3; m/z (ES^ϩ) 299 (100%, MH^ϩ), 281

(7, M Ϫ OH), 597 (20, 2M).

414.117); ν_max/cm^Ϫ1(Nujol mull) 2984 (C–H), 1629 (C᎐C),

᎐

1265 (P᎐O), 1030 (P–O–C); δ (CDCl₃) 1.20–1.35 (12 H, m,

᎐

H

4 CH₃CH₂–), 3.95–4.05 (8 H, m, 4 CH₃CH₂–), 5.60 (2 H, dd, J_HP

7.2, J_HF45.8, 2 PCHF–), 7.50–7.60 (4 H, m, 4 ArH); δ_P(CDCl₃)

15.0 (d, J_FP88.8); δ_F(CDCl₃) Ϫ202.3; H-coupled Ϫ202.2 (J_FP

88.8, J_HF45.8); δ_C(CDCl₃) 16.2, 16.3, 63.4, 63.8, 89.9 (dd, J_CP

169, J_CF183), 126.7, 134.1; m/z (EI^ϩ) 414 (5%, M^ϩ).

2,6-Bis(phosphonohydroxymethyl)pyridine (14b)

2,6-Bis[(diethoxyphosphoryl)hydroxymethyl]pyridine 11b (0.70

g, 1.65 mmol) was deprotected as for 2 with TMSBr (1.77 g,

11.56 mmol, 7 eq., 1.50 ml). Salt formation and puriﬁcation as

for 2 gave the biscyclohexylammonium salt of the title com-

pound as a white solid (0.58 g, 68%, mp >275 ЊC) (Found:

C 45.9, H 7.7, N 8.1. C₁₉H₃₈N₃O₈P₂(salt) requires C 45.75,

H 7.7, N 8.4%); free acid: δ_H(D₂O) 3.50 (2 H, d, J_HP11.7,

PCH(OH)–), 7.75–7.90 (2 H, m, H_3,5), 7.50–7.60 (1 H, m, H₄);

δ_P(D₂O) 14.0 (s); Biscyclohexylammonium salt: δ_C(D₂O) 23.7,

24.2, 30.3, 50.1, 70.9 (d, J_CP154), 121.4, 139.9, 155.8; m/z (EI^ϩ)

300 (38%, MH^ϩ), 399 (22, M ϩ C₆H₁₁NH₃^ϩ).

1,3-Bis[(diethoxyphosphoryl)chloromethyl]benzene (13a)

1,3-Bis[(diethoxyphosphoryl)hydroxymethyl]benzene 11a (6.44

g, 15.08 mmol) was stirred with thionyl chloride (20 ml) at rt for

8 h then volatiles removed under reduced pressure. The residue

was dissolved in DCM (50 ml) and washed with water (2 × 100

ml). The combined organics were dried over MgSO₄, ﬁltered,

and the solvent was removed under reduced pressure. The crude

product was puriﬁed by ﬂash column chromatography (DCM–

MeOH, 98:2) to give the title compound as a white, crystalline

solid (4.73 g, 68%); R_f0.50 (DCM–MeOH, 90:10); ν_max/cm^Ϫ1

1,4-Bis(phosphonohydroxymethyl)benzene (14c)

(ﬁlm) 2984, 2937 (C–H), 1654 (C᎐C), 1266 (P᎐O), 1001 (P–

᎐

1,4-Bis[(diethoxyphosphoryl)hydroxymethyl]benzene 11c (2.01

g, 4.88 mmol) was deprotected as for 2 with TMSBr (5.23 g,

34.15 mmol, 7 eq., 4.50 ml). Salt formation and puriﬁcation as

for 2 gave the biscyclohexylammonium salt of the title com-

pound as a white solid (1.46 g, 60%, mp >275 ЊC) (Found:

C 45.1, H 7.95, N 5.3. C₂₀H₄₂N₂O₁₀P₂(salt ϩ 2 H₂O) requires C

45.4, H 7.7, N 5.6); δ_H(d₄-MeOH) 1.10–2.00 (22 H, m,

cyclohexyl-H), 5.10 (1 H, d, J_HP9.8 Hz, PCH(OH)–), 7.10 (4 H,

s, ArH); δ_P(d₄-MeOH) 17.1 (s); δ_C(d₄-MeOH) 23.8, 24.3, 30.3,

50.2, 91.6 (d, J_CP153), 126.9, 138.5.

OEt), 737 (C–Cl); δ_H(CDCl₃) 1.15–1.30 (12 H, t, J 6.2, 4 CH₃-

CH₂–), 3.90–4.10 (8 H, m, 4 CH₃CH₂–), 4.80 (2 H, d, J_HP8.5,

2 PCHCl–), 7.30–7.60 (4 H, m, ArH); δ_P(CDCl₃) 15.4, 15.6

(2 s); δ_C(CDCl₃) 16.1, 16.2, 53.7 (d, J_CP161), 62.4, 62.5, 128.8,

129.6, 129.6, 134.9; m/z (FAB^ϩ) 445 (26%, M^ϩ), 447 (25,

M ϩ 4), 335 (100, M Ϫ 4C₂H₄), 301 (82, M Ϫ 4C₂H₄Ϫ Cl).

2,6-Bis[(diethoxyphosphoryl)chloromethyl]pyridine (13b)

2,6-Bis[(diethoxyphosphoryl)hydroxymethyl]pyridine

11b

(1.00 g, 2.36 mmol) was dissolved in thionyl chloride (10 ml),

the reaction stirred for 8 h at rt, then excess thionyl chloride

removed under reduced pressure. The residue was dissolved in

DCM (50 ml) and washed with water (2 × 100 ml), the com-

bined organics dried over MgSO₄, ﬁltered, and solvent removed

under reduced pressure. The crude product was puriﬁed by ﬂash

column chromatography (DCM–MeOH, 95:5) to give the title

compound as a yellow oil (0.26 g, 24%); δ_H(CDCl₃) 1.15–1.30

(12 H, m, 4 CH₃CH₂–), 4.05–4.15 (8 H, m, 4 CH₃CH₂), 4.80

(2 H, 2 d, J_HP9.0, 2 PCHCl–), 7.30–7.40 (2 H, m, ArH),

7.55–7.70 (1 H, m, ArH); δ_P(CDCl₃) 16.0, 16.2 (2 s); δ_C(CDCl₃)

16.1, 16.3, 53.7 (d, J_CP165), 62.0, 62.2, 128.6, 129.6, 134.3;

m/z (EI^ϩ) 441 (32%, MH^ϩ), 304 (37, M Ϫ PO₃Et₂), 166 (86,

M Ϫ 2PO₃Et₂).

1,3-Bis(phosphonoﬂuoromethyl)benzene (15a)

1,3-Bis[(diethoxyphosphoryl)ﬂuoromethyl]benzene 12a (0.67 g,

1.62 mmol) was deprotected as for 2 with TMSBr (1.74 g, 11.35

mmol, 1.50 ml) to yield the title compound as an oﬀ-white solid

(0.42 g, 85%). Salt formation and puriﬁcation as for 2 gave the

biscyclohexylammonium salt of the title compound as a white

solid (Found: C 47.95, H 7.2, N 5.6. C₂₀H₃₆F₂N₂O₆P₂(salt)

requires C 48.0, H 7.25, N 5.6%); free acid: δ_H(D₂O) 5.65 (2 H,

dd, J_HP7.5, J_HF48.1, 2 PCHF–), 7.30–7.35 (4 H, m, ArH);

δ_P(D₂O) 12.7 (d, J_PF82.2); δ_F(D₂O) Ϫ201.6 (d); H-coupled

Ϫ201.6 (dd, J_PF83.3, J_HF43.0); δ_C(D₂O) 23.8, 24.3, 30.3, 50.3,

91.9 (dd, J_CP156, J_CF176), 124.3, 126.5, 128.7, 135.7; m/z (EI^ϩ)

301 (100%, MH^ϩ).

1,4-Bis[(diethoxyphosphoryl)chloromethyl]benzene (13c)

2,6-Bis(phosphonoﬂuoromethyl)pyridine (15b)

1,4-Bis[(diethoxyphosphoryl)hydroxymethyl]benzene 11c (4.83

g, 11.78 mmol, 2.1 eq., 1.80 ml) was reacted with thionyl chlor-

ide (2.95 g, 24.75 mmol) as above to furnish the title compound

as a white solid (3.42 g, 65%); R_f0.41 (DCM–MeOH, 90:10)

(Found: C 43.3, H 5.85, Cl 15.45. C₁₆H₂₆Cl₂O₆P₂requires C

43.0, H 5.9, Cl 15.7%. Found: M^ϩ446.058. C₁₆H₂₆Cl₂O₆P₂

requires M 446.058); ν_max/cm^Ϫ1(Nujol mull) 2924 (C–H), 1612

2,6-Bis[(diethoxyphosphoryl)ﬂuoromethyl]pyridine 12b (0.29 g,

0.64 mmol) was de-esteriﬁed as for 2 with TMSBr (0.69 g, 4.51

mmol, 0.59 ml) to yield the title compound as a brown solid

(0.19 g). Salt formation and puriﬁcation as for 2 gave the bis-

cyclohexylammonium salt of the title compound as a white

solid (0.22 g, 65%, mp >275 ЊC); δ_H(D₂O) 1.05–1.95 (22 H, m,

cyclohexyl-H), 5.75 (2 H, dd, J_HP9.6, J_HF44.7, 2 PCHF–), 7.45–

7.55 (2 H, m, ArH), 7.95–8.00 (1 H, m, ArH); δ_P(D₂O) 8.7 (d,

J_PF70.0); δ_C(D₂O) 23.7, 24.4, 30.2, 50.1, 88.7 (dd, J_CP164, J_CF

183), 128.0, 134.0, 152.9.

(C᎐C), 1254 (P᎐O), 1039 (P–O–C); δ_H(CDCl₃) 1.15, 1.30 (6 H

᎐

each, 2 t, J 6.4, 4 CH₃CH₂–), 4.00–4.10 (8 H, m, 4 CH₃CH₂–),

4.90 (2 H, d, J_HP13.7, PCH₂–), 7.50 (4 H, s, 4 ArH); δ_P(CDCl₃)

17.3 (s); δ_C(CDCl₃, d₄-MeOH to dissolve) 16.3, 16.3, 53.1 (d,

J_CP160), 64.0, 64.2, 129.1, 135.0; m/z (EI^ϩ) 446 (36%, M^ϩ), 411

(35, M Ϫ Cl), 309 (25, M Ϫ PO₃Et₂), 172 (57, M Ϫ 2PO₃Et₂Ϫ

Cl), 138 (100, HPO₃Et₂).

1,4-Bis(phosphonoﬂuoromethyl)benzene (15c)

1,4-Bis[(diethoxyphosphoryl)ﬂuoromethyl]benzene 12c (0.20 g,

0.47 mmol) was de-esteriﬁed as for 2 with TMSBr (0.51 g, 3.30

mmol, 7 eq., 0.44 ml). Salt formation and puriﬁcation as for 2

gave the biscyclohexylammonium salt of the title compound as

a white solid (0.15 g, 64%, mp >275 ЊC) (Found: C 44.7, H 7.8,

N 5.3. C₂₀H₄₀F₂N₂O₈P₂(salt ϩ 2 H₂O); requires C 44.8, H 7.5,

1,3-Bis(phosphonohydroxymethyl)benzene (14a)

1,3-Bis[(diethoxyphosphoryl)hydroxymethyl]benzene 11a (2.53

g, 4.88 mmol) was deprotected as for 2 with TMSBr (5.23 g,

34.65 mmol, 4.52 ml) to yield the title compound (1.20 g, 74%).

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

429

View Article Online

N 5.2%); δ_H(D₂O) 1.10–2.00 (22 H, m, cyclohexyl-H), 5.60 (1

H, dd, J_HP7.4, J_HF45.4, PCHF–), 7.30–7.35 (4 H, m, ArH); δ_P

(D₂O) 11.2 (d, J_FP79.5); δ_F(D₂O) Ϫ200.8 (d, J_FP79.5); H-

coupled Ϫ201.0 (dd, J_FP79.1, J_HF45.2); δ_C(D₂O) 23.8, 24.3,

30.3, 50.2, 115.3 (dd, J_CP171, J_CF184), 126.4, 136.1.

a white solid (1.22 g, 77%, mp >275 ЊC) (Found: C 52.46, H

8.21, N 5.83. C₂₁H₄₀N₂O₆P₂(salt) requires C 52.69, H 8.43, N

5.86%) (Found: M^ϩ281.033. C₉H₁₅O₆P₂requires M 281.034);

biscyclohexylammonium salt: δ_H(D₂O) 1.50–2.00 (22 H, m,

cyclohexyl-H), 2.25 (3 H, s, Me), 2.85 (4 H, d, J_HP23.5 Hz,

2 PCH₂–), 6.95–7.00 (3 H, m, ArH); δ_P(D₂O) 20.6 (s); free acid:

δ_C(D₂O) 20.0, 33.5 (d, J_CP132), 127.4, 128.8, 131.4, 137.7; m/z

(FAB^ϩ) 281 (38%, MH^ϩ), 479 (M Ϫ 1 ϩ 2C₆H₁₁NH₃^ϩ), 578

(M Ϫ 2 ϩ 3C₆H₁₁NH₃^ϩ), 677 (M Ϫ 3 ϩ 4C₆H₁₁NH₃^ϩ).

1,3-Bis(phosphonochloromethyl)benzene (16a)

1,3-Bis[(diethoxyphosphoryl)chloromethyl]benzene 13a (0.59

g, 1.80 mmol) was deprotected as for 2 with TMSBr (1.26 g,

8.25 mmol, 1.10 ml) to yield the title compound as a white

solid (0.53 g). Salt formation and puriﬁcation as for 2 gave the

biscyclohexylammonium salt of the title compound as a white

solid (0.36 g, 57%, mp >275 ЊC) (Found: C 45.6, H 6.85, N 4.9,

Cl 13.2. C₂₀H₃₆Cl₂N₂O₆P₂(salt) requires C 45.2, H 6.8, N 5.2, Cl

13.1%); δ_H(D₂O) 1.50–2.05 (22 H, m, cyclohexyl-H), 5.00 (2 H,

d, J_HP12.3, 2 PCHCl–), 7.45–7.60 (4 H, m, ArH); δ_P(D₂O) 12.8

(s); δ_C(D₂O) 23.8, 24.3, 30.3, 50.2, 72.1 (d, J_CP152), 125.6,

126.6, 128.2, 138.0.

3,5-Bis[(diethoxyphosphoryl)diﬂuoromethyl]toluene (19)

The title compound was made by the same method as 8a using

tetraethyl 3,5-bis(phosphonomethyl)toluene 17 (0.38 g, 9.73

mmol), NaHMDS (5.36 mmol, 1.0 M solution in THF, 5.36

ml), and NFSi (2.25 g, 7.12 mmol). The crude material was

puriﬁed by ﬂash column chromatography (DCM–MeOH, 98:2

to 95:5) to give a colourless oil (1.22 g, 63%); R_f0.13 (DCM–

MeOH, 95:5); δ_H(CDCl₃) 1.25 (12 H, t, J 6.3 Hz, 4 CH₃CH₂–),

2.35 (3 H, s, Me), 4.05–4.15 (8 H, m, 4 CH₃CH₂–), 7.45–7.55

(2 H, m, ArH), 7.85–7.90 (1 H, m, ArH); δ_P(CDCl₃) 6.3 (t,

J_PF112); δ_F(CDCl₃) Ϫ108.9 (t, J_PF114); δ_C(CDCl₃) 15.8, 15.9,

20.9, 65.0, 65.1, 117.1 (dt, J_CP219, J_CF263), 125.8, 128.6,

2,6-Bis(phosphonochloromethyl)pyridine (16b)

2,6-Bis[(diethoxyphosphoryl)chloromethyl]pyridine 13b (0.21

g, 0.42 mmol) was de-esteriﬁed as for 2 with TMSBr (0.45 g,

2.96 mmol, 0.39 ml) to yield the crude free acid (0.11 g). Salt

formation and puriﬁcation as for 2 gave the biscyclohexyl-

ammonium salt of the title compound as a white solid (0.13 g,

69%, mp >275 ЊC); δ_H(D₂O) 1.10–2.00 (22 H, m, cyclohexyl-

H), 4.80 (2 H, d, J_HP9.1, 2 PCHCl–), 7.30–7.35 (2 H, m, ArH),

7.45–7.50 (1 H, m, ArH); δ_P(D₂O) 17.0 (s); δ_C(D₂O) 23.8, 24.2,

30.3, 50.3, 72.1 (d, J_CP153), 125.7, 126.4, 139.0; m/z (ES^ϩ) 336

(27%, MH^ϩ).

2

132.8 (dt, J_CP14.3, J_CF20.9), 139.1; m/z (FAB^ϩ) 465 (100%,

MH^ϩ).

3,5-Bis(phosphonodiﬂuoromethyl)toluene (20)

3,5-Bis[(diethoxyphosphoryl)diﬂuoromethyl]toluene 19 (0.30 g,

0.65 mmol) was de-esteriﬁed as for 2 with TMSBr (0.23 g,

1.47 mmol, 0.20 ml) to yield the crude free acid (0.18 g). Salt

formation and puriﬁcation as for 2 gave the biscyclohexyl-

ammonium salt of the title compound as a white solid (0.20 g,

70%, mp >275 ЊC); δ_H(D₂O) 1.05–2.00 (22 H, m, cyclohexyl-

H), 2.20 (3 H, s, Me), 7.45–7.50 (2 H, m, 2 ArH), 7.75–7.80

(1 H, m, ArH); δ_P(D₂O) 5.00 (t, J_PF104); δ_F(d₄-MeOH) Ϫ106.2

(d, J_PF104); δ_C(D₂O) 20.6, 24.3, 24.8, 30.2, 50.3, 125.7, 129.4,

133.3, 138.2.

1,4-Bis(phosphonochloromethyl)benzene (16c)

1,4-Bis[(diethoxyphosphoryl)chloromethyl]benzene 13c (1.30 g,

2.90 mmol) was deprotected as for 2 with TMSBr (3.11 g, 20.33

mmol, 7 eq., 2.68 ml) and the crude free acid isolated as usual

(1.22 g). Salt formation and puriﬁcation as for 2 gave the

biscyclohexylammonium salt of the title compound as a white

solid (0.82 g, 70%, mp >275 ЊC); δ_H(d₄-MeOH) 1.00–2.00

(22 H, m, cyclohexyl-H), 4.20 (2 H, d, J_HP7.5, 2 PCHCl–),

7.20 (4 H, s, ArH); δ_P(d₄-MeOH) 13.1 (s); δ_C(d₄-MeOH) 25.4,

25.9, 31.9, 51.7, 54.9 (d, J_CP159), 131.3, 140.0.

1,5-Dimethyl-2,4-bis[(diethoxyphosphoryl)methyl]benzene (21)

1,5-Bis(chloromethyl)-2,4-dimethylbenzene (5.00 g, 24.63

mmol) and triethyl phosphite (5.72 g, 49.26 mmol, 5.90 ml)

were stirred together at 120 ЊC under nitrogen for 27 h and

volatiles removed in vacuo. The crude yellow oil was freed from

the corresponding monophosphonate by ﬂash column chrom-

atography (DCM–MeOH, 95:5) to furnish the title compound

as a pale yellow oil (9.13 g, 91%); R_f0.30 (DCM–MeOH,

90:10); ν_max/cm^Ϫ1(ﬁlm) 2981 (C–H), 1618 (C᎐C), 1250 (P᎐O),

3,5-Bis[(diethoxyphosphoryl)methyl]toluene (17)

3,5-Bis(bromomethyl)toluene (10.00 g, 36.23 mmol) and tri-

ethyl phosphite (12.04 g, 72.47 mmol, 12.43 ml) were stirred

together at 120 ЊC overnight under nitrogen then volatiles

removed in vacuo. The crude product was puriﬁed by ﬂash

column chromatography (DCM–MeOH, 95:5) to yield the title

compound as a colourless oil (12.57 g, 89%); R_f0.22 (DCM–

᎐

1029 (P–O–C) (Found: M^ϩ406.167. C₁₈H₃₂O₆P₂requires M

406.167); δ_H(CDCl₃) 1.20 (12 H, t, J 5.5, 4 CH₃CH₂–), 2.25

(6 H, s, 2 Me), 3.05 (4 H, d, J_HP21.2, 2 PCH₂–), 3.85–4.00 (8 H,

m, 4 CH₃CH₂–), 6.90 (1 H, s, ArH), 7.05 (1 H, s, ArH);

δ_P(CDCl₃) 27.4 (s); δ_C(CDCl₃) 16.3, 16.4, 19.4, 19.5, 30.5 (d,

J_CP139), 61.8, 62.0, 127.4, 127.5, 132.5, 135.7; m/z (EI^ϩ) 406

(97%, M^ϩ), 378 (38, M^ϩϪ C₂H₄).

MeOH, 90:10); ν_max/cm^Ϫ1(ﬁlm) 2982, 2910 (C–H), 1604 (C᎐C),

᎐

1249 (P᎐O), 1027 (P–O–C) (Found: M^ϩ393.158. C₁₇H₃₀O₆P₂

᎐

requires M 393.159); δ_H(CDCl₃) 1.00 (12 H, t, J 6.4, 4 CH₃-

CH₂–), 2.15 (3 H, s, Me), 2.85 (4 H, d, J_HP27.0, 2 PCH₂–), 3.70–

3.85 (8 H, m, 4 CH₃CH₂–), 6.70–6.80 (4 H, m, ArH); δ_P(CDCl₃)

26.6 (s); δ_C(CDCl₃) 16.1, 16.2, 21.0, 33.2 (d, J_CP137), 61.8, 62.0,

128.0, 129.0, 131.5, 137.8; m/z (FAB^ϩ) 393 (87%, MH^ϩ), 365

(32, M Ϫ C₂H₄), 337 (17, M Ϫ 2C₂H₄), 309 (15, M Ϫ 3C₂H₄),

281 (36, M Ϫ 4C₂H₄), 199 (56, M Ϫ 4C₂H₄Ϫ PO₃H₂), 119 (100,

M Ϫ 4C₂H₄Ϫ 2PO₃H₂).

1,5-Dimethyl-2,4-bis(phosphonomethyl)benzene (22)

1,5-Dimethyl-2,4-bis[(diethoxyphosphoryl)methyl]benzene 21

(2.00 g, 4.93 mmol) was dissolved in 6 M hydrochloric acid (25

ml) and the mixture heated at reﬂux for 30 h then volatiles

removed under reduced pressure and the product co-evaporated

with 5 vol water to give the crude free acid (mp 257 ЊC). Salt

formation and puriﬁcation as for 2 gave the biscyclohexyl-

ammonium salt of the title compound as a white solid (1.09 g,

75%, mp >275 ЊC) (Found: M^ϩC 51.01, H 7.71, N 5.80.

C₂₂H₃₈N₂O₆P₂(salt) requires C 50.82, H 7.79, N 5.74%); free

acid: δ_H(D₂O) 2.25 (6 H, s, 2 Me), 3.15 (4 H, d, J_HP21.3,

2 PCH₂–), 6.95 (1 H, s, ArH), 7.05 (1 H, s, ArH); δ_P(D₂O) 27.5

(s); δ_C(D₂O) 19.3, 30.6 (d, J_CP133), 127.8, 128.1, 130.8, 133.2;

m/z (ES) 295 (8%, MH^ϩ), 394 (7, Mϩ C₆H₁₁NH₃^ϩ).

3,5-Bis(phosphonomethyl)toluene (18)

3,5-Bis[(diethoxyphosphoryl)methyl]toluene 17 (1.73 g, 4.41

mmol) was deprotected as for 2 with TMSBr (4.73 g,

30.89 mmol, 4.08 ml) to aﬀord the crude product as an oﬀ-

white solid (0.97 g). Salt formation and puriﬁcation as for 2

gave the biscyclohexylammonium salt of the title compound as

430

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

View Article Online

1,5-Dimethyl-2,4-bis[(diethoxyphosphoryl)diﬂuoromethyl]-

benzene (23)

(P–O–C) (Found: M^ϩ396.126; C₁₆H₂₇FO₆P₂requires M

396.127); δ_H(CDCl₃) 1.15–1.25 (12 H, t, J 6.0, 4 CH₃CH₂–),

3.15 (4 H, d, J_HP24.5, 2 PCH₂–), 3.85–4.05 (8 H, m, 4 CH₃-

CH₂–), 6.95–7.05 (1 H, m, ArH), 7.10–7.20 (2 H, m, ArH);

δ_P(CDCl₃) 25.4 (s); δ_F(CDCl₃) Ϫ121.8 (s), H-coupled Ϫ121.9

The title compound was made by the same method as for 8a

using 1,5-dimethyl-2,4-bis[(diethoxyphosphoryl)methyl]benz-

ene 21 (0.20 g, 0.50 mmol), NaHMDS (2.75 mmol, 1.0 M

solution in THF, 2.75 ml), and NFSi (1.15 g, 3.65 mmol). The

crude product was puriﬁed by ﬂash column chromatography

(DCM to DCM–MeOH, 99:1) to give a colourless oil (0.14 g,

59%); R_f0.35 (DCM–MeOH, 90:10) (Found: M^ϩ479.138.

C₁₈H₂₉F₄O₆P₂requires M 479.138); δ_H(CDCl₃) 1.20 (12 H, t,

J 6.2, 4 CH₃CH₂–), 2.40 (6 H, s, 2 Me), 4.00–4.15 (8 H, m, 4

CH₃CH₂–), 7.40–7.45 (1 H, m, ArH), 7.70–7.75 (1 H, d, J 2.1,

ArH); δ_P(CDCl₃) 6.94 (t, J_PF117); δ_F(CDCl₃) Ϫ110.8 (t, J_PF

116); δ_C(CDCl₃) 16.2, 16.3, 20.3, 64.8, 65.0, 117.5 (dt, J_CP219,

J_CF256), 126.9, 128.9, 136.3, 140.3; m/z (FAB^ϩ) 478 (100%,

MH^ϩ), 451 (6, M Ϫ C₂H₄), 341 (10, M Ϫ PO₃Et₂), 319 (37,

M Ϫ PO₃Et₂Ϫ C₂H₄).

4

(t, J_HF5.0); δ_C(CDCl₃) 16.2, 16.2, 26.5 (dd, J_CP140, J_CF

3.0), 61.7, 61.8, 121.5, 123.7, 130.4, 156.7 (dt, J_CF246, ⁴J_CP7.6,

ArC–F); m/z (EI^ϩ) 396 (77%, M^ϩ), 260 (77, M Ϫ PO₃Et₂), 122

(77, M Ϫ 2PO₃Et₂).

1-Fluoro-2,6-bis(phosphonomethyl)benzene (28)

1-Fluoro-2,6-bis[(diethoxyphosphoryl)methyl]benzene 27 (1.38

g, 3.50 mmol) was deprotected as for 2 with TMSBr (3.75 g,

24.46 mmol, 3.23 ml) to yield the title compound as a pale yel-

low solid (0.68 g, 69%). Salt formation and puriﬁcation as for 2

gave the biscyclohexylammonium salt of the title compound as

a white solid (Found: C 50.07, H 7.91, N 5.66. C₂₀H₃₇FN₂O₆P₂

(salt) requires C 49.77, H 7.73, N 5.81%) (Found: M^ϩ285.009.

C₈H₁₂P₂O₆F requires M 285.009); free acid: δ_H(D₂O) 3.05 (4 H,

d, J_HP25.2, 2 PCH₂–), 6.90–7.00 (1 H, m, ArH), 7.05–7.20 (2 H,

m, ArH); δ_P(D₂O) 24.6 (s); δ_F(D₂O) Ϫ121.2 (s), H-coupled

1,5-Dimethyl-2,4-bis(phosphonodiﬂuoromethyl)benzene (24)

1,3-Dimethyl-2,4-bis[(diethoxyphosphoryl)diﬂuoromethyl]-

benzene 23 (0.35 g, 0.27 mmol) was deprotected as for 2 with

TMSBr (0.13 g, 1.91 mmol, 0.25 ml) to yield the crude free acid

(0.20 g). Salt formation and puriﬁcation as for 2 gave the bis-

cyclohexylammonium salt of the title compound as a white

solid (0.29 g, 71%, mp >275 ЊC); δ_H(D₂O) 1.05–1.95 (22 H, m,

cyclohexyl-H), 2.40 (6 H, s, 2 Me), 7.00, 7.50 (1 H each, 2 s, 1

ArH each); δ_P(D₂O) 5.39 (t, J_PF97.1); δ_F(d₄-MeOH) Ϫ100.3 (d,

J_PF95.9); δ_C(D₂O) 19.8, 23.7, 24.2, 30.3, 50.1, 118.1 (dt, J_CP

215, J_CF252), 125.6, 135.2, 138.3.

4

Ϫ121.0 (s); δ_C(d₄-MeOH) 28.2 (dd, J_CP136, J_CF4.0), 121.5,

4

125.3, 131.8, 158.4 (dt, J_CF192, J_CP5.2, ArC–F); m/z (FAB^ϩ)

285 (100%, MH^ϩ).

Diethyl 3-chloromethylbenzoylphosphonate (30a)

3-Chloromethylbenzoyl chloride 29a (5.00 g, 26.45 mmol) was

dissolved in dry DCM under nitrogen at 0 ЊC and triethyl phos-

phite (4.40 g, 26.45 mmol, 4.54 ml) added dropwise over 2 h.

The reaction was stirred at rt for 2 h then allowed to stand over-

night. Volatiles were removed in vacuo and the resulting crude

pale oil used without puriﬁcation (7.07 g, 92%); δ_H(CDCl₃) 1.30

(6 H, t, J 6.5, 2 CH₃CH₂–), 4.15–4.25 (4 H, m, 2 CH₃CH₂–),

4.55 (2 H, s, CH₂Cl), 7.40–7.45 (1 H, m, H⁵), 7.50–7.55 (1 H, m,

H⁶), 8.15–8.20 (1 H, m, H^2,4); δ_P(CDCl₃) Ϫ1.41 (s); δ_C(CDCl₃)

16.1, 16.2, 45.2, 63.8, 63.9, 129.2, 129.4, 131.2, 134.7, 136.3,

138.4, 197.1.

1,3,5-Trimethyl-2,4-bis[(diethoxyphosphoryl)methyl]benzene

(25)

1,3-Bis(chloromethyl)-2,4,6-trimethylbenzene (10.00 g, 23.81

mmol) and triethyl phosphite (7.91 g, 47.52 mmol, 16.00 ml)

were stirred together at 120 ЊC overnight, volatiles removed

in vacuo, and the title compound was obtained as a pale yellow

oil which was used without further puriﬁcation (15.49 g, 80%);

R_f0.10 (DCM–MeOH, 95:5); ν_max/cm^Ϫ1(ﬁlm) 2907 (C–H),

1654 (C᎐C), 1193 (P᎐O), 1002 (P–O–C); δ (CDCl ) 1.20 (12 H,

Diisopropyl 4-chloromethylbenzoylphosphonate (30c)

᎐

H

3

t, J 6.2, 4 CH₃CH₂–), 2.25 (6 H, s, 2 Me), 2.40 (3 H, s, Me), 3.20

(4 H, d, J_HP23.0, 2 PCH₂–), 3.80–3.90 (8 H, m, 4 CH₃CH₂–),

6.75 (1 H, s, ArH); δ_P(CDCl₃) 28.0 (s); δ_C(CDCl₃) 16.2, 16.3,

19.2, 19.3, 20.7, 28.6 (d, J_CP139), 61.7, 61.8, 126.8, 130.2, 135.9,

136.7; m/z (FAB^ϩ) 421 (99%, MH^ϩ), 393 (6, M Ϫ C₂H₄).

4-Chloromethylbenzoyl chloride 29c (25.00 g, 132.25 mmol)

and triisopropyl phosphonate (27.54 g, 132.25 mmol, 32.60 ml)

were reacted in the same way as for 30a. The title compound

was recovered as a yellow oil (34.61 g, 90%); δ_H(CDCl₃)

1.25–1.35 (12 H, m, 2 (CH₃)₂CH–), 4.50 (2 H, s, CH₂Cl),

4.60–4.75 (2 H, m, 2 (CH₃)₂CH–), 7.40–7.50 (2 H, m, ArH),

8.15–8.20 (2 H, m, ArH); δ_P(CDCl₃) Ϫ2.42 (s); δ_C(CDCl₃) 23.6,

23.7, 23.9, 24.0, 45.2, 73.2, 73.3, 128.8, 130.3, 135.0, 143.8,

196.3.

1,3,5-Trimethyl-2,4-bis(phosphonomethyl)benzene (26)

1,3,5-Trimethyl-2,4-bis[(diethoxyphosphoryl)methyl]benzene

25 (2.00 g, 4.76 mmol) was dissolved in 6 M hydrochloric

acid (30 ml) and the mixture heated at reﬂux for 20 h. Volatiles

were removed in vacuo and the residue co-evaporated with 5 vol

water to give the free acid of the title compound as a white solid

(0.98 g, 67%, mp >275 ЊC) (Found: M^ϩC 54.32, H 8.42, N 5.93.

C₂₃H₄₁N₂O₆P₂(salt) requires C 54.52, H 8.76, N 5.53%);

δ_H(D₂O) 2.35 (6 H, s, 2 Me), 2.40 (3 H, s, Me), 3.25 (4 H, d, J_HP

26.5, 2 PCH₂–), 6.85 (1 H, m, ArH); δ_P(D₂O) 27.5 (s); δ_C(D₂O)

19.0, 20.4, 29.8 (d, J_CP134), 126.6, 130.7, 133.0, 137.5; m/z

(ESϩ) 306 (100%, M^ϩ).

Diethyl (3Ј-chloromethylphenyl)hydroxymethylphosphonate

(31a)

Diethyl 3-chloromethylbenzoylphosphonate 30a (2.00 g, 6.68

mmol) was dissolved in DCM (100 ml) and ice-cold water (100

ml) added. Sodium borohydride (0.65 g, 17.20 mmol, 2.5 eq.)

added in portions over 5 min at 0 ЊC then the mixture was

stirred for 3 h at rt. The reaction was partitioned, the aqueous

layer extracted with DCM (50 ml), and the combined organics

washed with water (100 ml), dried over MgSO₄, ﬁltered, and

solvents removed in vacuo. The title compound was isolated by

ﬂash column chromatography as a colourless oil (1.55 g, 77%);

R_f0.19 (DCM–MeOH, 95:5) (Found: M^ϩ282.063. C₁₂H₁₈-

ClO₄P requires M 282.063); δ_H(CDCl₃) 1.20 (6 H, t, 2 CH₃-

CH₂–, J 6.4), 3.95–4.05 (4 H, m, 2 CH₃CH₂–), 4.55 (2 H, s,

CH₂Cl), 5.00 (1 H, d, J_HP11.6, PCH(OH)–), 7.20–7.45 (4 H, m,

ArH); δ_P(CDCl₃) 19.7 (s); δ_C(CDCl₃) 16.2, 16.3, 45.2, 63.5,

63.6, 70.3 (d, J_CP160), 126.9, 127.2, 128.0, 128.4, 137.3, 137.7;

m/z (EI^ϩ) 292 (42%, M^ϩ).

1-Fluoro-2,6-bis[(diethoxyphosphoryl)methyl]benzene (27)

1,3-Bis(bromomethyl)-2-ﬂuorobenzene (2.50 g, 8.87 mmol)

and triethyl phosphite (2.95 g, 17.73 mmol, 3.0 ml) were

stirred together in a dry ﬂask under nitrogen then heated to

120 ЊC for 24 h. The ﬂask was cooled and volatiles removed in

vacuo at 40 ЊC. The crude product was puriﬁed by ﬂash column

chromatography to yield the title compound as a colourless

oil (2.80 g, 80%); R_f0.28 (DCM–MeOH, 90:10); ν_max/cm^Ϫ1

(ﬁlm) 2987 (C–H), 1630 (C᎐C), 1266 (P᎐O), 1167 (C–F), 1029

᎐

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

431

View Article Online

Diisopropyl (4Ј-chloromethylphenyl)hydroxymethylphosphonate

(31c)

(d); H-coupled Ϫ200.3 (dd, J_PF84.8, J_HF34.5); δ_C(CDCl₃) 16.3,

16.4, 33.6 (d, J_CP138), 62.0, 62.3, 89.2 (dd, J_CP169, J_CF184),

125.3, 127.9, 128.7, 130.5, 131.2, 133.1; m/z (EI^ϩ) 396 (100%,

M^ϩ), 260 (70, M Ϫ PO₃Et₂), 123 (55, M Ϫ 2PO₃Et₂).

Diisopropyl 4-chloromethylbenzoylphosphonate 30c (20.47 g,

64.26 mmol) and sodium borohydride (6.08 g, 160.65 mmol)

were reacted under the same conditions as for 31a. The product

was puriﬁed by ﬂash column chromatography (gradient elution

from DCM–light petrol, 80:20 to DCM–MeOH, 95:5) to yield

the title compound as an oﬀ-white solid (10.51 g, 50%, mp 81–

83 ЊC); R_f0.07 (DCM–MeOH, 95:5) (Found: M^ϩ320.094.

C₁₄H₂₂ClO₄P requires M 320.094); δ_H(CDCl₃) 1.20–1.35 (12 H,

m, 2 (CH₃)₂CH–), 4.20 (2 H, s, CH₂Cl), 4.55–4.65 (2 H, m,

2 (CH₃)₂CH–), 4.95 (1 H, d, J_HP9.3, PCH(OH)–), 7.45 (2 H, dd,

J_HHЈ8.2, J_HHЈ2.2, ArH), 7.55 (2 H, dd, J_HHЈ8.2, J_HHЈ2.2, ArH);

δ_P(CDCl₃) 19.9 (s); δ_C(CDCl₃) 23.6, 23.8, 24.0, 24.2, 46.0, 70.5

(d, J_CP140), 127.5, 128.4, 137.0, 137.2; m/z (FAB^ϩ) 320 (5%,

M^ϩ).

Diisopropyl 4-[(diisopropoxyphosphoryl)methylphenyl]ﬂuoro-

methylphosphonate (33c)

Diisopropyl

4-[(diisopropoxyphosphoryl)methylphenyl]-

hydroxymethylphosphonate 33c (0.73 g, 1.63 mmol) and DAST

(0.53 g, 3.26 mmol, 2 eq., 0.43 ml) were reacted as for 33a.

Usual work-up gave the crude product as an orange oil, was

puriﬁed by ﬂash column chromatography (gradient elution,

DCM to DCM–MeOH, 98:2) to give the title compound as a

white crystalline solid (0.87 g, 90%); R_f0.20 (DCM–MeOH,

95:5) (Found: C 53.2, H 7.8. C₂₀H₃₅FO₆P₂requires C 53.1, H

7.8; M^ϩ452.189. C₂₀H₃₅FO₆P₂requires M 452.190); δ_H(CDCl₃)

1.10–1.25 (24 H, m, 4 (CH₃)₂CH–), 3.05 (2 H, d, J_HP21.7,

PCH₂–), 4.45–4.60 (4 H, m, 4 (CH₃)₂CH–), 5.50 (1 H, dd, J_HP

7.6, J_HF34.8, PCHF–), 7.20–7.30 (4 H, m, ArH); δ_P(CDCl₃)

13.8 (d, J_PF86.0), 24.6 (s); δ_F(CDCl₃) Ϫ200.5 (d); H-coupled

Ϫ200.6 (dd, J_PF85.0, J_HF7.6); δ_C(CDCl₃) 23.5, 23.6, 23.8, 23.9,

34.4 (d, J_CP139), 70.3, 70.4, 71.8, 71.9, 89.2 (dd, J_CP172, J_CF

183), 126.8, 129.7, 131.5, 132.8; m/z (EI^ϩ) 452 (60%, M^ϩ),

287 (49%, M Ϫ PO₃Prⁱ₂), 284 (77%, M Ϫ 4C₃H₆), 204 (77%,

M Ϫ PO₃Prⁱ₂Ϫ 2C₃H₆), 122 (100%, M Ϫ 2PO₃Prⁱ₂).

Diethyl 3-[(diethoxyphosphoryl)methylphenyl]hydroxymethyl-

phosphonate (32a)

Diethyl

(3Ј-chloromethylphenyl)hydroxymethylphosphonate

31a (5.99 g, 20.56 mmol) and triethyl phosphite (3.59 g, 21.59

mmol, 1.05 eq., 3.70 ml) were stirred together at 120 ЊC for

36 h and the reaction cooled. The crude product was puriﬁed

by ﬂash column chromatography (gradient elution, DCM to

DCM–MeOH, 95:5) to yield the title compound as a pale yel-

low oil (6.06 g, 75%); R_f0.46 (DCM–MeOH, 90:10) (Found:

M^ϩ394.131. C₁₆H₂₈O₇P₂requires M 394.131); ν_max/cm^Ϫ1(ﬁlm)

Diethyl 3-[(diethoxyphosphoryl)methylphenyl]chloromethyl-

phosphonate (34a)

2984, 2910 (C–H), 1607 (C᎐C), 1243 (P᎐O), 1027 (P–O–C);

᎐

δ_H(CDCl₃) 1.20, 1.25 (6 H each, 2 t, J 6.4, 2 CH₃CH₂– each),

2.20 (1 H, br, –OH), 3.10 (1 H, d, J_HP16.4, PCH(OH)–), 3.95–

4.10 (8 H, m, 4 CH₃CH₂–), 7.15–7.30 (4 H, m, ArH); δ_P(CDCl₃)

21.8, 26.8 (2 s); δ_C(CDCl₃) 16.2, 16.3, 33.5 (d, J_CP138), 62.8,

63.4, 70.2 (d, J_CP161), 125.7, 128.4, 130.4, 131.1, 131.2, 137.8;

m/z (EI^ϩ) 394 (16%, MH^ϩ), 378 (97, M Ϫ OH), 257 (77,

M Ϫ PO₃Et₂), 242 (98, M Ϫ PO₃Et₂Ϫ OH).

Diethyl 3-[(diethoxyphosphoryl)methylphenyl]hydroxymethyl-

phosphonate 32a (1.30 g, 3.29 mmol) was dissolved in DCM

(15 ml) under argon, thionyl chloride (5 ml) added, and the

reaction reﬂuxed for 14 h. After this time, TLC showed the

reaction was still incomplete but considerable side-product

formation was observed. The reaction was cooled and volatiles

removed in vacuo. The resulting yellow oil was puriﬁed by ﬂash

column chromatography to aﬀord the title compound as a pale

yellow oil (0.80 g, 95%, 62% conversion); R_f0.48 (DCM–

MeOH, 95:5) (Found: M^ϩ412.099. C₁₆H₂₇ClO₆P₂requires M

412.097); ν_max/cm^Ϫ1(ﬁlm) 2981 (C–H), 1654 (C᎐C), 1252 (P᎐O),

Diisopropyl 4-[(diisopropoxyphosphoryl)methylphenyl]hydroxy-

methylphosphonate (32c)

᎐

Diisopropyl (4Ј-chloromethylphenyl)hydroxymethylphosphon-

ate 31c (6.15 g, 21.03 mmol) and triisopropyl phosphite (4.60 g,

22.08 mmol, 1.05 eq., 5.45 ml) were reacted as for 32a and the

title compound was isolated by ﬂash column chromatography

as a white solid (5.28 g, 61%); R_f0.11 (DCM–MeOH, 95:5)

(Found: M^ϩ450.194. C₂₀H₃₆O₇P₂requires M 450.194); δ_H

(CDCl₃) 1.15, 1.25 (12 H each, 2 s, 2 (CH₃)₂CH– each), 3.05 (2

H, d, J_HP21.7, PCH₂–), 4.50–4.60 (4 H, m, 2 (CH₃)₂CH– each),

4.90 (1 H, d, J_HP10.3, PCH(OH)–), 7.25 (2 H, dd, J_HHЈ6.4, J_HHЈ

2.1, ArH), 7.35 (2 H, d, J_HHЈ6.4, J_HHЈ2.1, ArH); δ_P(CDCl₃)

24.5, 20.2 (2 s); δ_C(CDCl₃) 23.7, 24.1, 34.5 (d, J_CP139), 69.5 (d,

J_CP162), 127.2, 129.7, 131.7, 135.3; m/z (EI^ϩ) 450 (10%, M^ϩ),

285 (52, M Ϫ PO₃Prⁱ₂), 243 (20, M Ϫ PO₃Prⁱ₂Ϫ C₃H₆), 201

(100, M Ϫ PO₃Prⁱ₂Ϫ 2C₃H₆), 120 (75, M Ϫ 2PO₃Prⁱ₂).

1024 (P–O–C); δ_H(CDCl₃) 1.15, 1.20 (6 H each, 2 t, J 6.4,

2 CH₃CH₂– each), 3.15 (2 H, d, J_HP21.3, PCH₂–), 3.95–4.05,

4.10–4.20 (4 H each, 2 m, 2 CH₃CH₂– each), 4.85 (1 H, d, J_HP

14.4, PCH(OH)–), 7.25–7.30 (4 H, m, ArH); δ_P(CDCl₃) 17.6,

26.3 (2 s); δ_C(CDCl₃) 16.2, 16.3, 33.6 (d, J_CP137.8), 53.5 (d, J_CP

158.9), 62.1, 62.2, 64.0, 64.1, 127.5, 128.8, 130.3, 130.5, 132.2,

134.5; m/z (EI^ϩ) 412 (100%, M^ϩ), 276 (58, M Ϫ PO₃Et₂).

Diisopropyl 4-[(diisopropoxyphosphoryl)methylphenyl]chloro-

methylphosphonate (34c)

Diisopropyl

4-[(diisopropoxyphosphoryl)methylphenyl]-

hydroxymethylphosphonate 32c (2.13 g, 4.72 mmol) was dis-

solved in DCM (20 ml) and thionyl chloride (5.61 g, 47.20

mmol, 10 eq., 3.44 ml) added slowly via syringe. The reaction

was reﬂuxed for 12 h then volatiles removed in vacuo. The title

compound was separated from ~50% starting material by ﬂash

column chromatography, aﬀording a clear oil (1.03 g, 93%,

50% conversion); R_f0.44 (DCM–MeOH, 90:10); δ_H(CDCl₃)

1.20, 1.30 (12 H each, 2 d, J 6.2, 2 (CH₃)₂CH– each), 3.05 (2 H,

d, J 22.0, PCH₂–), 4.50–4.60 (4 H, m, 4 (CH₃)₂CH–), 4.75 (1 H,

d, J_HP14.4, PCHCl–), 7.25, 7.40 (2 H each, 2 d, J 7.4, ArH);

δ_P(CDCl₃) 15.8, 24.3 (2 s); δ_C(CDCl₃) 23.6, 23.8, 34.5 (d, J_CP

139), 53.7 (d, J_CP162), 70.5, 72.5, 128.4, 129.1, 131.4, 133.0;

m/z (EI^ϩ) 468 (54, M^ϩ), 300 (100, M Ϫ PO₃Et₂).

Diethyl 3-[(diethoxyphosphoryl)methylphenyl]ﬂuoromethyl-

phosphonate (33a)

Diethyl 3-[(diethoxyphosphoryl)methylphenyl]hydroxymethyl-

phosphonate 32a (0.17 g, 0.45 mmol) was dissolved in DCM (20

ml) under argon and DAST (0.12 g, 0.76 mmol, 1.7 eq., 0.10 ml)

was added via syringe. The reaction was stirred for 10 min then

cooled to 0 ЊC and quenched with aqueous sodium bicarbonate.

After usual work-up the title compound was isolated as a yellow

liquid (0.16 g, 91%); R_f0.31 (DCM–MeOH, 90:10) (Found:

M^ϩ396.127. C₁₆H₂₇FO₆P₂requires 396.127); ν_max/cm^Ϫ1(ﬁlm)

2984, 2912 (C–H), 1645 (C᎐C), 1253 (P᎐O), 1027 (P–O–C);

᎐

Diethyl 3-(chloromethylphenyl)diﬂuoromethylphosphonate (35a)

δ_H(CDCl₃) 1.20, 1.25 (6 H each, 2 t, J 6.1, 2 CH₃CH₂– each),

3.05 (2 H, d, J_HP16.9, PCH₂–), 3.85–4.05 (8 H, m, 4 CH₃CH₂–),

5.70 (1 H, dd, J_HP6.1, J_HF34.5, PCHF–), 7.25–7.35 (4 H, m,

ArH); δ_P(CDCl₃) 15.4 (d, J_PF84.3), 26.3 (s); δ_F(CDCl₃) Ϫ200.5

Diethyl 3-chloromethylbenzoylphosphonate 30a (1.00 g, 3.44

mmol) was dissolved in DCM (15 ml) under an inert atmos-

phere. DAST (1.66 g, 10.32 mmol, 3 eq., 1.40 ml) was added via

432

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

View Article Online

syringe at 0 ЊC, the reaction stirred overnight at rt, then

quenched by dropwise addition of aqueous NaHCO₃. The reac-

tion was worked up as usual and the crude product puriﬁed by

ﬂash column chromatography (DCM–MeOH, 98:2) to give the

title compound as a pale orange oil (1.55 g, 76%); R_f0.36

(DCM–MeOH, 95:5) (Found: M^ϩ313.057. C₁₂H₁₇ClFO₃P

34.2 (d, J_CP139), 70.6, 73.7, 117.9 (dt, J_CP221, J_CF263), 126.5,

129.7, 133.0 (ddt, ²J_CP13.8, ²J_CF22.1, ⁴J_CP3.8), 134.9; m/z (EI^ϩ)

470 (44%, M^ϩ), 428 (7, M Ϫ C₃H₆), 386 (10, M Ϫ C₃H₆), 306

(97, M Ϫ PO₃Prⁱ₂), 264 (62, M Ϫ PO₃Prⁱ₂Ϫ C₃H₆), 222 (87

M Ϫ PO₃Prⁱ₂Ϫ 2C₃H₆), 140 (88, M Ϫ 2PO₃Prⁱ₂).

requires M 313.057); ν_max/cm^Ϫ1(ﬁlm) 2985 (C–H), 1611 (C᎐C),

3-(Phosphonomethylphenyl)hydroxymethylphosphonic acid (37a)

᎐

1265 (P᎐O), 1183 (C–F), 1023 (P–O–C); δ_H(CDCl₃) 1.25 (6 H,

᎐

Diethyl 3-[(diethoxyphosphoryl)methylphenyl]hydroxymethyl-

phosphonate 32a (0.19 g, 0.48 mmol) was deprotected as for 2

with TMSBr (0.59 g, 3.82 mmol, 8 eq., 0.50 ml) and the free

acid was isolated as usual (0.11 g). Salt formation and puriﬁ-

cation as for 2 gave the biscyclohexylammonium salt of the title

compound as a white solid (0.18 g, 56%, mp >275 ЊC) (Found:

C 49.9, H 8.2, N 5.5. C₂₀H₃₈N₂O₇P₂(salt) requires C 50.0, H

8.0, N 5.8%; Found: M^ϩ382.118. C₁₄H₂₆NO₇P₂requires M

382.118); δ_H(D₂O) 1.10–2.00 (22 H, m, cyclohexyl-H), 2.90

(2 H, d, J_HP16.4, PCH₂–), 4.60 (1 H, d, J_HP14.0, PCH(OH)–),

7.05–7.25 (4 H, m, ArH); δ_P(D₂O) 15.8, 19.6 (2 s); δ_C(D₂O)

23.8, 24.2, 30.3, 36.5 (d, J_CP124), 50.2, 73.5 (d, J_CP144), 124.4,

127.8, 128.0, 131.4, 136.5, 140.7; m/z (FAB^ϩ) 283 (25%, MH^ϩ),

382 (29, M ϩ C₆H₁₁NH₃^ϩ).

t, 2 CH₃CH₂–), 3.95–4.10 (4 H, m, 2 CH₃CH₂–), 4.55 (2 H, s,

CH₂Cl), 7.40–7.50 (4 H, m, ArH); δ_P(CDCl₃) 6.41 (t, J_PF111.9);

δ_F(CDCl₃) Ϫ109.5 (d, J_FP112); δ_C(CDCl₃) 16.2. 16.3, 45.5,

64.8, 64.9, 117.8 (dt, J_CP218, J_CF263), 126.3, 127.7, 128.9,

129.2, 133.0, 137.9; m/z (FAB^ϩ) 313 (30%, MH^ϩ), 175 (27,

M Ϫ PO₃Et₂), 137 (25, PO₃Et₂).

Diisopropyl 4-(chloromethylphenyl)diﬂuoromethylphosphonate

(35c)

Diisopropyl 4-chloromethylbenzoylphosphonate 30c (0.85 g,

2.67 mmol) and DAST (1.29 g, 8.01 mmol, 3 eq., 1.06 ml) were

reacted under the same conditions as for 35a. The product was

puriﬁed by ﬂash column chromatography (DCM–MeOH,

98:2), aﬀording the title compound as a yellow oil (0.67 g,

73%) (Found: M^ϩ340.080. C₁₄H₂₀F₂O₃P requires M 340.081);

δ_H(CDCl₃) 1.15, 1.20 (6 H each, 2 s, (CH₃)₂CH– each), 4.60

(2 H, s, CH₂Cl), 4.70–4.80, 4.85–4.90 (1 H each, 2 m, (CH₃)₂-

CH– each), 7.45 (2 H, d, J 7.7, ArH), 7.55 (2 H, d, J 7.7, ArH);

δ_P(CDCl₃) 4.81 (t, J_FP116); δ_F(CDCl₃) Ϫ109.3 (d, J_PF117);

δ_C(CDCl₃) 23.5, 23.6, 24.1, 24.2, 45.4, 73.8, 74.0, 118.2 (dt, J_CP

220, J_CF263), 126.7, 128.5, 133.0, 140.0; m/z (EI^ϩ) 340 (9%,

M^ϩ), 256 (18, M Ϫ 2C₃H₆), 175 (67, M Ϫ PO₃Prⁱ₂), 140 (100,

M Ϫ Cl Ϫ PO₃Prⁱ₂).

4-(Phosphonomethylphenyl)hydroxymethylphosphonic acid (37c)

Diisopropyl

4-[(diisopropoxyphosphoryl)methylphenyl]-

hydroxymethylphosphonate 32c (0.62 g, 1.36 mmol) was

de-esteriﬁed as for 2 with TMSBr (1.67 g, 10.90 mmol, 8 eq.,

1.45 ml) and the free acid was isolated as usual (0.42 g). Salt

formation and puriﬁcation as for 2 gave the biscyclohexyl-

ammonium salt of the title compound as a white solid (0.57 g,

66%, mp >275 ЊC) (Found: C 49.7, H 8.25, N 5.8. C₂₀H₃₈N₂-

O₇P₂(salt) requires C 50.0, H 8.0, N 5.8%); δ_H(D₂O) 1.20–2.00

(22 H, m, cyclohexyl-H), 2.90 (2 H, d, J_HP20.8, PCH₂–), 4.80

(1 H, d, J_HP9.7, PCH(OH)–), 7.15–7.30 (4 H, m, ArH); δ_P

(D₂O) 21.2, 17.1 (2 s); δ_C(D₂O) 23.8, 24.2, 30.3, 35.7 (d, J_CP

137), 50.3, 72.0 (d, J_CP128), 126.9, 129.2, 131.0, 134.4; m/z

(FAB^ϩ) 283 (100%, MH^ϩ), 201 (87, M Ϫ PO₃H₂).

Diethyl 3-[(diethoxyphosphoryl)methylphenyl]diﬂuoromethyl-

phosphonate (36a)

Sodium hydride (0.08 g, 2.80 mmol, 90% powder) was sus-

pended in dry THF under nitrogen and diethyl phosphonate

(0.43 g, 3.08 mmol, 1.1 eq., 0.40 ml) added dropwise at 0 ЊC

then diethyl 3-(chloromethylphenyl)diﬂuoromethylphosphon-

ate 35a (1.00 g, 2.80 mmol) in dry THF (15 ml) was added

dropwise over 5 min. The reaction was stirred overnight at rt

then quenched with ethanol. After standard work-up the crude

product was puriﬁed (~50%) by ﬂash column chromatography

(DCM–MeOH, 98:2) to yield the title compound as a pale yel-

low oil (0.62 g, 94%, 50% conversion); R_f0.23 (DCM–MeOH,

3-(Phosphonomethylphenyl)ﬂuoromethylphosphonic acid (38a)

Diethyl

3-[(diethoxyphosphoryl)methylphenyl]ﬂuoromethyl-

phosphonate 33a (0.07 g, 0.18 mmol) was deprotected as for 2

with TMSBr (0.19 g, 1.24 mmol, 7 eq., 0.17 ml) and the free

acid isolated as usual (0.05 g). Salt formation and puriﬁcation

as for 2 gave the biscyclohexylammonium salt of the title com-

pound as a white solid (0.05 g, 62%, mp >300 ЊC) (Found: C

50.0, H 7.4, N 6.1. C₂₀H₃₇FN₂O₆P₂(salt) requires C 49.8, H

7.4, N 6.1%. Found: M^ϩ384.114. C₁₄H₂₅FNO₆P₂requires M

384.114); δ_H(D₂O) 1.10–2.00 (22 H, m, cyclohexyl-H), 3.00

(2 H, d, J_HP16.4, PCH₂–), 5.50 (1 H, dd, J_HP7.0, J_HF39.1,

PCHF–), 7.15–7.40 (4 H, m, ArH); δ_P(D₂O) 11.1 (d, J_PF73.0),

20.3 (s); δ_F(D₂O) Ϫ193.4 (d); H-coupled Ϫ193.6 (dd, J_HF34.2,

J_PF73.0); δ_C(D₂O) 23.8, 24.3, 30.3, 35.9 (d, J_CP127), 50.2, 91.6

(dd, J_CP169, J_CF184), 124.2, 127.5, 128.2, 129.1, 134.1, 135.9;

m/z (FAB^ϩ) 285 (27%, M^ϩ), 384 (92, M ϩ C₆H₁₁NH₃^ϩ).

95:5); ν_max/cm^Ϫ1(ﬁlm) 2986, 2913 (C–H), 1653 (C᎐C), 1273

᎐

(P᎐O), 1024 (P–O–C); δ (CDCl ) 1.18, 1.25 (6 H each, 2 t, J 7.1,

᎐

H

3

2 CH₃CH₂– each), 3.15 (2 H, d, J_HP21.6, PCH₂–), 3.80–4.00,

4.10–4.15 (4 H each, 2 m, 2 CH₃CH₂– each), 7.30–7.45 (4 H, m,

ArH); δ_P(CDCl₃) 6.65 (t, J_PF116), 25.9; δ_F(CDCl₃) Ϫ108.9 (d,

J_FP114.9); δ_C(CDCl₃) 16.2. 16.3, 33.1 (d, J_CP138), 62.1, 62.2,

64.7, 64.8, 117.9 (dt, J_CP217, J_CF263), 127.4, 127.5, 128.6,

132.1, 132.4, 132.9.

Diisopropyl 4-[(diisopropoxyphosphoryl)methylphenyl]diﬂuoro-

methylphosphonate (36c)

4-(Phosphonomethylphenyl)ﬂuoromethylphosphonic acid (38c)

Diisopropyl 4-(chloromethylphenyl)diﬂuoromethylphosphon-

ate 35c (0.53 g, 1.57 mmol) and diisopropyl phosphonate (0.29

g, 1.72 mmol, 1.1 eq., 0.29 ml) and sodium hydride (0.04 g, 1.72

mmol, 90% powder) were reacted as for 36a. The title compound

was isolated by ﬂash column chromatography (DCM–MeOH,

98:2) as a colourless oil (0.34 g, 90%, 52% conversion); R_f0.14

(DCM–MeOH, 98:2) (Found: M^ϩ470.181. C₂₀H₃₄F₂O₆P₂

Diisopropyl 4-[(diisopropoxyphosphoryl)methylphenyl]ﬂuoro-

methylphosphonate 33c (0.26 g, 0.56 mmol) was deesteriﬁed as

for 2 with TMSBr (0.61 g, 3.95 mmol, 7 eq., 0.52 ml) and the

free acid isolated as usual (0.18 g). Salt formation and puriﬁ-

cation as for 2 gave the biscyclohexylammonium salt of the title

compound as a white solid (0.12 g, 63%, mp >300 ЊC) (Found:

C 49.5, H 8.1, N 5.8. C₂₀H₃₇FNO₆P₂(salt) requires C 49.8, H

7.4, N 6.1%); δ_H(D₂O) 1.20–2.00 (22 H, m, cyclohexyl-H), 3.00

(2 H, d, J_HP21.3, PCH₂–), 5.80 (1 H, d, J_HP6.8, J_HF42.0,

PCHF–), 7.30, 7.40 (2 H each, 2 d, J 7.1, 2 ArH each); δ_P(D₂O)

11.5 (d, J_FP72.6), 20.9 (s); δ_F(D₂O) Ϫ195.2 (d); H-coupled

Ϫ195.3 (dd, J_PF74.1, J_HF42.6); δ_C(D₂O) 23.8, 24.3, 30.2, 50.2,

128.3, 130.6, 134.0, 135.9.

requires M 470.180); ν_max/cm^Ϫ1(ﬁlm) 2923 (C–H), 1618 (C᎐C),

᎐

1265 (P᎐O), 1024 (P–O–C); δ (CDCl₃) 1.05–1.25 (24 H, m,

᎐

H

4 (CH₃)₂CH–), 3.05 (2 H, d, J_HP22.0, PCH₂–), 4.50–4.55, 4.60–

4.65 (2 H each, 2 m, 2 (CH₃)₂CH– each), 7.25 (2 H, d, J 7.9,

ArH), 7.45 (2 H, d, J 7.7, ArH); δ_P(CDCl₃) 4.93 (t, J_FP119),

24.1 (s); δ_F(CDCl₃) Ϫ109.1 (d, J_PF118); δ_C(CDCl₃) 23.7, 23.9,

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

433

View Article Online

3-(Phosphonomethylphenyl)chloromethylphosphonic acid (39a)

61.3, 62.1, 122.3, 128.4, 129.9, 132.6, 134.0; m/z (EI^ϩ) 306

(43%, MH^ϩ), 250 (35, M Ϫ C₂H₄), 227 (41, M Ϫ Br), 171 (87,

M Ϫ C₂H₄Ϫ Br).

Diethyl 3-[(diethoxyphosphoryl)methylphenyl]chloromethyl-

phosphonate 34a (0.31 g, 0.75 mmol) was deprotected as for 2

with TMSBr (0.81 g, 5.26 mmol, 7 eq., 0.70 ml) and the free

acid isolated as usual (0.26 g). Salt formation and puriﬁcation

as for 2 gave the biscyclohexylammonium salt of the title com-

pound as a white solid (0.27 g, 71%, mp >300 ЊC) (Found: C

48.0, H 7.8, N 5.4, Cl 6.9. C₁₄H₂₅ClNO₆P₂(salt) requires C 48.2,

H 7.5, N 5.6, Cl 7.0%. Found: M^ϩ384.114. C₁₄H₂₅ClNO₆P₂

requires M 384.114); δ_H(D₂O) 1.25–2.00 (22 H, m, cyclohexyl-

H), 3.00 (2 H, d, J_HP19.8, PCH₂–), 4.85 (1 H, d, J_HP11.3,

PCHCl–), 7.20–7.35 (4 H, m, ArH); δ_P(D₂O) 12.9, 20.8 (2 s);

δ_C(D₂O) 23.8, 24.2, 30.3, 35.8 (d, J_CP128), 50.3, 56.8 (d, J_CP

141), 126.2, 128.4, 129.3, 129.3, 135.5, 137.7; m/z (FAB^ϩ) 301

(50%, MH^ϩ), 400 (50, M ϩ C₆H₁₁NH₃^ϩ).

Diethyl [3-(diethoxyphosphoryl)phenyl]methylphosphonate (42)

Diethyl (3-bromophenylmethyl)phosphonate 41 (1.00 g, 3.26

mmol) and diethyl phosphonate (0.50 g, 3.58 mmol) were

placed in a 5 ml reaction vessel under argon and triethylamine

(0.36 g, 3.58 ml) added via syringe. Tetrakis(triphenylphos-

phine)palladium(0) (190 mg, 0.16 mmol, 0.05 eq.) was added

and the reaction heated at 100 ЊC for 22 h. The reaction mixture

was ﬁltered through Celite (ether–hexanes, 50:50 as eluent) and

solvent removed in vacuo. The crude product was puriﬁed by

ﬂash column chromatography (DCM–MeOH, 98:2), furnish-

ing the title compound as a yellow liquid (0.80 g, 67%); R_f0.19

(DCM–MeOH, 90:10) (Found: M^ϩ364.120. C₁₅H₂₆O₆P₂

requires M 364.121); ν_max/cm^Ϫ1(ﬁlm) 2984 (C–H), 1630 (C᎐C),

4-(Phosphonomethylphenyl)chloromethylphosphonic acid (39c)

᎐

1246 (P᎐O), 1026 (P–O–C); δ (CDCl ) 1.20, 1.30 (6 H each, 2 t,

᎐

H

3

Diisopropyl 4-[(diisopropoxyphosphoryl)methylphenyl]chloro-

methylphosphonate 34c (0.44 g, 0.93 mmol) was deprotected as

for 2 with TMSBr (1.00 g, 6.54 mmol, 7 eq., 0.86 ml) and the

free acid isolated as usual (0.29 g). Salt formation and puriﬁ-

cation as for 2 gave the biscyclohexylammonium salt of the title

compound as a white solid (0.28 g, 60%) (Found: C 47.7, H 7.4,

N 5.5, Cl 6.9. C₂₀H₃₇ClN₂O₆P₂(salt) requires C 48.3, H 7.5, N

5.6, Cl 7.0%); δ_H(D₂O) 1.15–2.00 (22 H, m, cyclohexyl-H), 3.00

(2 H, d, J_HP20.8, PCH₂–), 4.90 (1 H, d, J_HP12.5, PCHCl–),

7.35, 7.45 (2 H each, 2 d, J 7.7, 2 ArH each); δ_P(D₂O) 13.4, 17.3

(2 s); δ_C(D₂O) 23.8, 24.2, 30.3, 34.8 (d, J_CP136), 50.3, 58.4 (d,

J_CP143), 126.8, 128.1, 134.5, 136.8.

J 6.1, 2 CH₃CH₂– each), 3.15 (2 H, d, J_HP18.5, PCH₂–), 3.95–

4.05, 4.05–4.15 (4 H each, 2 m, 2 CH₃CH₂– each), 7.45–7.60

(4 H, m, ArH); δ_P(CDCl₃) 18.8, 25.9 (2 s); δ_C(CDCl₃) 16.2,

16.3, 33.5 (d, J_CP128), 62.0, 127.2, 128.8, 130.1, 132.1, 132.4,

133.6; m/z (EI^ϩ) 364 (90%, M^ϩ), 228 (100, M Ϫ PO₃Et₂), 200

(37, M Ϫ PO₃Et₂Ϫ C₂H₄), 172 (37, M Ϫ PO₃Et₂Ϫ 2C₂H₄).

(3-Phosphonophenyl)methylphosphonic acid (43)

Diethyl [3-(diethoxyphosphoryl)phenyl]methylphosphonate 42

(0.45 g, 1.24 mmol) was deprotected as for 2 with TMSBr (1.33

g, 8.66 mmol, 1.20 ml) to yield the free acid as an oﬀ-white solid

(0.26 g, mp 270 ЊC). Salt formation and puriﬁcation as for 2

gave the biscyclohexylammonium salt of the title compound as a

white solid (0.32 g, 58%, mp >275 ЊC) (Found: C 50.5, H 8.05,

N 5.8. C₁₉H₃₆N₂O₆P₂(salt) requires C 50.7, H 8.05, N 6.2%); free

acid: δ_H(D₂O) 3.00 (2 H, d, J_HP19.4, PCH₂–), 7.50–7.65 (4 H,

m, ArH); δ_P(D₂O) 17.0, 25.0 (2 s); biscyclohexylammonium

salt: δ_C(D₂O) 23.7, 24.2, 30.2, 35.8 (d, J_CP127), 50.1, 127.6,

128.3, 130.8, 131.5, 131.9, 135.6.

3-(Phosphonomethylphenyl)diﬂuoromethylphosphonic acid (40a)

Diethyl 3-[(diethoxyphosphoryl)methylphenyl]diﬂuoromethyl-

phosphonate 36a (0.38 g, 0.92 mmol) was de-esteriﬁed as for 2

with TMSBr (0.99 g, 6.42 mmol, 7 eq., 0.85 ml). Salt formation

and puriﬁcation as for 2 gave the biscyclohexylammonium salt

of the title compound as a white solid (0.23 g, 57%) (Found:

C 47.75, H 7.55, N 5.45. C₂₀H₃₆FN₂O₆P₂(salt) requires C 48.0,

H 7.3, N 5.6%; Found: M^ϩ402.104. C₁₄H₂₄FNO₆P₂requires

402.105); δ_H(D₂O) 1.25–2.00 (22 H, m, cyclohexyl-H), 2.95

(2 H, d, J_HP21.7, PCH₂–), 7.40–7.50 (4 H, m, 4 ArH); δ_P(D₂O)

4.88 (t, J_FP102), 20.4; δ_F(D₂O) Ϫ109.9 (d, J_FP102); δ_C(D₂O)

23.7, 24.2, 30.2, 50.0, 128.2, 128.3, 128.8, 130.1, 133.1, 134.0;

m/z (FAB^ϩ) 303 (29%, MH^ϩ), 402 (97, M ϩ C₆H₁₁NH₃^ϩ).

Diethyl [3-(diethoxyphosphoryl)phenyl]diﬂuoromethylphosphon-

ate (44)

NaHMDS (1.89 mmol, 1.0 M solution in THF, 1.89 ml) and

diethyl [3-(diethoxyphosphoryl)phenyl]methylphosphonate 42

(0.25 g, 0.69 mmol) were reacted as for 8a at Ϫ78 ЊC for 1 h then

NFSi (0.79 g, 2.52 mmol) added dropwise and the reaction

stirred for 2 h at Ϫ78 ЊC. After work-up, the crude product was

puriﬁed by ﬂash column chromatography to give the title com-

pound as a colourless oil (0.15 g, 53%); R_f0.15 (DCM–MeOH,

95:5); ν_max/cm^Ϫ1(ﬁlm) 2985 (C–H), 1629 (C᎐C), 1265 (P᎐O),

4-(Phosphonomethylphenyl)diﬂuoromethylphosphonic acid (40c)

Diisopropyl

4-[(diisopropoxyphosphoryl)methylphenyl]-

diﬂuoromethylphosphonate 36c (0.05 g, 0.11 mmol) was

deprotected as for 2 with TMSBr (0.11 g, 0.74 mmol, 7 eq., 0.10

ml). Salt formation and puriﬁcation as for 2 gave the biscyclo-

hexylammonium salt of the title compound as a white solid

(0.04 g, 75%) (Found: C 48.15, H 7.4, N 5.3. C₂₀H₃₆F₂N₂O₆P₂

(salt) requires C 48.0, H 7.25, N 5.6%); δ_H(D₂O) 1.25–2.00 (22

H, m, H-cyclohexyl), 3.15 (2 H, d, J_HP22.7, PCH₂–), 7.25, 7.45

(2 H each, 2 d, J 7.1, 2 ArH each); δ_P(D₂O) 4.30 (t, J_FP113),

26.0 (s); δ_F(D₂O) Ϫ111.2 (d, J_FP112); δ_C(D₂O) 35.5 (d, 136),

126.1, 129.6.

᎐

1032 (P–O–C); δ_H(CDCl₃) 1.20 (12 H, t, J 6.1, 4 CH₃CH₂–),

4.00–4.15 (8 H, m, 4 CH₃CH₂–), 7.55–7.70 (4 H, m, ArH);

δ_P(CDCl₃) 6.21 (t, J_PF96.2), 17.5 (s); δ_F(CDCl₃) Ϫ114.8 (d,

J_PF96.5); δ_C(CDCl₃) 16.2, 16.3, 63.3, 63.5, 117.4 (dt, J_CP218,

J_CF268), 128.8, 129.2, 130.1, 131.5, 131.8, 133.3.

Diethyl 3-(dioxolan-2Ј-yl)phenylphosphonate (46)

Diethyl phosphonate (1.20 g, 8.69 mmol, 1.1 eq., 1.12 ml) and

triethylamine (0.88 g, 8.69 mmol, 1.21 ml) were added via

syringe to 2-(3Ј-bromophenyl)-1,3-dioxolane 45 (1.81 g, 7.90

mmol) under argon. Tetrakis(triphenylphosphine) palladium(0)

(0.46 g, 0.40 mmol, 0.05 eq.) was added and the reaction heated

to 95 ЊC for 12 h. The crude product was ﬁltered through

Celite^TM(ether–hexane, 50:50 as eluent) and volatiles evapor-

ated to yield the crude product as a deep yellow oil, puriﬁed

by ﬂash column chromatography (DCM–MeOH, 98:2) to

give the title compound as a yellow oil (1.54 g, 68%); R_f0.46

(DCM–MeOH, 90:10); ν_max/cm^Ϫ1(ﬁlm) 2983, 2901 (C–H),

Diethyl (3-bromophenylmethyl)phosphonate (41)

3-Bromobenzyl bromide (10.19 g, 40.77 mmol) and triethyl

phosphite (4.74 g, 40.77 mmol, 7.10 ml) were stirred together in

a dry ﬂask under argon and heated at 120 ЊC for 14 h. The crude

product was puriﬁed by ﬂash column chromatography (DCM–

MeOH, 95:5), aﬀording the title compound as a colourless

liquid (11.19 g, 90%); R_f0.30 (DCM–MeOH, 95:5); δ_H(CDCl₃)

1.20 (6 H, t, J 6.3, 2 CH₃CH₂–), 3.05 (2 H, d, J_HP27.0, PCH₂–),

3.90–4.05 (4 H, m, 2 CH₃CH₂–), 7.10–7.45 (4 H, m, ArH);

δ_P(CDCl₃) 25.7 (s); δ_C(CDCl₃) 16.0, 16.1, 33.1 (d, J_CP139),

1646 (Ar C᎐C), 1250 (P᎐O), 1023 (P–O–C); δ (CDCl₃) 1.27

᎐

H

(6 H, t, J 6.2, CH₃CH₂–), 3.95–4.10 (8 H, m, CH₃CH₂– and

434

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

View Article Online

–OCH₂CH₂– overlapping), 5.80 (1 H, s, CH(OR)), 7.30–7.90

(4 H, m, ArH); δ_P(CDCl₃) 18.8 (s); δ_C(CDCl₃) 16.2, 16.3, 62.1,

62.2, 65.3 (–OCH₂CH₂–), 103.0 (CH(OR)), 128.6, 127.0, 130.0,

131.9, 132.5, 138.3; m/z (EI^ϩ) 285 (48%, M Ϫ 1), 241 (10,

M Ϫ CH₂CH₂O–), 158 (M Ϫ 1 Ϫ PO₃Et₂).

with thionyl chloride (5 ml) under argon as for 34c and worked

up as usual. The resulting yellow oil was puriﬁed from excess

starting material by ﬂash column chromatography to aﬀord the

title compound as a pale yellow oil (0.41 g, 61% conversion); R_f

0.20 (DCM–MeOH, 95:5) (Found: M^ϩ398.082. C₁₅H₂₅ClO₆P₂

requires M 398.082); δ_H(CDCl₃) 1.25 (12 H, t, J 7.0, 4 CH₃-

CH₂–), 3.95–4.10 (8 H, m, 4 CH₃CH₂–), 4.85 (1 H, d, J_HP14.3,

PCHCl–), 7.40–7.45 (1 H, m, ArH), 7.70–7.85 (3 H, m, ArH);

δ_P(CDCl₃) 16.9, 18.5 (s); δ_C(CDCl₃) 16.1, 16.2, 53.4 (d, J_CP

158), 62.7, 62.8, 127.3, 128.7, 128.9, 132.1, 132.8, 134.6; m/z

(EI^ϩ) 398 (30%, M^ϩ), 370 (12, M Ϫ C₂H₄), 363 (19, M Ϫ Cl),

342 (11, M Ϫ 2C₂H₄), 314 (12, M Ϫ 3 C₂H₄), 286 (32, M Ϫ

4C₂H₄).

Diethyl (3-carbonylphenyl)phosphonate (47)

Aqueous 15% sulfuric acid (0.35 ml) was adsorbed onto silica

gel (3.50 g) suspended in DCM (10 ml). Diethyl 3-(dioxolan-2Ј-

yl)-phenylphosphonate 46 (0.40 g) was then added in DCM (10

ml) and the reaction stirred for 24 h at rt. The mixture was

ﬁltered, washed with DCM (100 ml), and solvent removed in

vacuo. The crude product was puriﬁed by ﬂash column chrom-

atography to aﬀord the title compound as a yellow oil (0.30 g,

90%); R_f0.28 (EtOAc–petrol, 70:30) (Found: M^ϩ242.070. C₁₁-

H₁₅O₄P requires M 242.071); ν_max/cm^Ϫ1(ﬁlm) 2983, 2899 (C–H),

(3-Phosphonophenyl)hydroxymethylphosphonic acid (51)

Diethyl [3-(diethoxyphosphoryl)phenyl]hydroxymethylphos-

phonate 48 (0.37 g, 0.98 mmol) was deprotected as for 2 with

TMSBr (1.20 g, 7.80 mmol, 8 eq., 1.03 ml) and the free acid

isolated as usual (0.26 g). Salt formation and puriﬁcation as for

2 gave the biscyclohexylammonium salt of the title compound as

a white solid (0.21 g, 80%, mp >275 ЊC) (Found: C 48.4, H 8.0,

N 5.9. C₁₉H₃₆N₂O₇P₂(salt) requires C 48.9, H 7.8, N 6.0%); M^ϩ

368.103. C₁₃H₂₄NO₇P₂requires M 368.103); δ_H(D₂O) 1.20–2.00

(22 H, m, cyclohexyl-H), 4.50 (1 H, d, J_HP9.7, PCH(OH)–),

7.05–7.25 (4 H, m, 4 ArH); δ_P(D₂O) 12.9, 15.8 (2 s); δ_C(D₂O)

23.7, 24.2, 30.2, 71.0 (d, J_CP143), 128.0, 128.6, 129.9, 131.9,

134.1, 137.7; m/z (FAB^ϩ) 267 (27%, M Ϫ 1), 368 (M ϩ C₆H₁₁-

NH₃^ϩ), 187 (77, M Ϫ PO₃H₂).

1712 (CHO), 1646 (C᎐C), 1250 (P᎐O), 1023 (P–O–C);

᎐

δ_H(CDCl₃) 1.25 (6 H, t, J 6.7, 2 CH₃CH₂–), 4.00–4.20 (4 H, m,

2 CH₃CH₂–), 7.50–7.60 (1 H, m, H⁵), 7.95–8.05 (2 H, m, H^4,6),

8.25 (1 H, ddt, J_HH0.6, J_HH1.5, J_HP12.8, H²), 10.0 (1 H, s,

CHO); δ_P(CDCl₃) 17.4 (s); δ_C(CDCl₃) 16.3, 16.4, 62.5, 62.6,

128.4, 129.3, 132.0, 132.6, 133.4, 137.3, 191.3; m/z (EI^ϩ) 242

(27%, M^ϩ), 214 (15, M Ϫ C₂H₄), 187 (32, M Ϫ 2C₂H₄), 105

(M Ϫ PO₃Et₂).

5

4,6

Diethyl [3-(diethoxyphosphoryl)phenyl]hydroxymethylphosphon-

ate (48)

Triethylamine (0.05 g, 0.52 mmol, 0.5 eq., 0.07 ml) was added to

diethyl (3-carbonylphenyl)phosphonate 47 (0.25 g, 1.03 mmol)

and diethyl phosphonate (0.14 g, 1.03 mmol, 0.13 ml) in dry

DCM (20 ml) and the reaction stirred overnight. A second ali-

quot of triethylamine was then added and a third aliquot after

4 h and volatiles removed in vacuo. The crude mixture was puri-

ﬁed by ﬂash column chromatography (EtOAc–petrol, 50:50)

to yield the title compound as a colourless oil (0.25 g, 53%); R_f

0.16 (EtOAc–petrol, 70:30) (Found: M^ϩ380.116. C₁₅H₂₆O₇P₂

(3-Phosphonophenyl)ﬂuoromethylphosphonic acid (52)

Diethyl

[3-(diethoxyphosphoryl)phosphonophenyl]ﬂuoro-

methylphosphonate 49 (0.31 g, 0.82 mmol) was deprotected as

for 2 with TMSBr (0.87 g, 5.71 mmol, 7 eq., 0.75 ml) and the

free acid isolated as usual (0.25 g). Salt formation and puriﬁ-

cation as for 2 gave the biscyclohexylammonium salt of the title

compound as a white solid (0.17 g, 78%, mp >275 ЊC) (Found:

C 48.4, H 7.3, N 5.95. C₁₉H₃₅FN₂O₆P₂(salt) requires C 48.7,

H 7.5, N 6.0%. Found: M^ϩ370.098. C₁₃H₂₄FNO₆P₂requires

M 370.099); δ_H(D₂O) 1.15–2.00 (22 H, m, cyclohexyl-H), 5.70

(1 H, d, J_HP7.9, J_HF44.8, PCHF–), 7.25–7.50 (4 H, m, ArH);

δ_P(D₂O) 11.0 (d, J_FP75.8), 15.8 (s); δ_F(D₂O) Ϫ198.4 (d);

H-coupled (dd, J_FP75.0, J_HF45.2); δ_C(D₂O) 23.7, 24.2, 30.3,

50.2, 92.5 (dd, J_CP153, J_CF176), 128.0, 128.2, 129.7, 129.9,

134.1, 136.5; m/z (FAB^ϩ) 370 (72%, M ϩ C₆H₁₁NH₃^ϩ).

requires M 380.115); ν_max/cm^Ϫ1(ﬁlm) 2980 (C–H), 1631 (C᎐C),

᎐

1262 (P᎐O), 1032 (P–O–C); δ (CDCl ) 1.20, 1.25 (6 H each, 2 t,

᎐

H

3

J 7.0, 2 CH₃CH₂– each), 2.70 (1 H, br, –OH), 3.90–4.10 (8 H, m,

4 CH₃CH₂–), 5.05 (1 H, d, J_HP11.6, PCH(OH)–), 7.30–7.40

(1 H, m, H⁵), 7.60–7.70 (2 H, m, H^4,6), 7.80–7.85 (1 H, app. d,

H²); δ_P(CDCl₃) 18.2, 20.4 (2 s); δ_C(CDCl₃) 16.2, 16.3, 63.4

(d, J_CP122), 64.4, 64.5, 128.1, 128.8, 131.2, 131.4, 135.3, 137.1;

m/z (EI^ϩ) 380 (14%, M^ϩ), 243 (100, M Ϫ PO₃Et₂).

Diethyl [3-(diethoxyphosphoryl)phenyl]ﬂuoromethylphosphonate

(3-Phosphonophenyl)chloromethylphosphonic acid (53)

(49)

Diethyl

[3-(diethoxyphosphoryl)phenyl]chloromethylphos-

Diethyl [3-(diethoxyphosphoryl)phenyl]hydroxymethylphos-

phonic acid 48 (0.36 g, 0.94 mmol) in DCM (20 ml) under argon

was treated with DAST (0.31 g, 1.87 mmol, 0.25 ml) and the

reaction stirred at rt for 10 min then worked up as usual. The

resulting pale yellow oil was puriﬁed by ﬂash column chrom-

atography (DCM–MeOH, 95:5) to aﬀord the title compound as

a pale yellow oil (0.34 g, 93%); R_f0.63 (DCM–MeOH, 90:10)

phonate 50 (0.16 g, 0.41 mmol) was deprotected as for 2 with

TMSBr (0.44 g, 2.85 mmol, 7 eq., 0.38 ml) and the free acid

isolated as usual (0.13 g). Salt formation and puriﬁcation as for

2 gave the biscyclohexylammonium salt of the title compound as

a white solid (0.15 g, 79%, mp >275 ЊC) (Found: C 46.9, H 7.7,

N 5.5, Cl 7.0. C₁₉H₃₅ClN₂O₆P₂(salt) requires C 47.1, H 7.3, N

5.8, Cl 7.3%. Found: M^ϩ386.086. C₁₃H₂₄ClNO₆P₂requires M

386.069); δ_H(CDCl₃) 1.20–2.00 (22 H, m, cyclohexyl-H), 4.95

(1 H, d, J_HP12.2, PCHCl–), 7.35–7.40 (1 H, m, ArH), 7.50–7.60

(3 H, m, 3 ArH); δ_P(CDCl₃) 13.0, 16.8 (2 s); δ_C(CDCl₃) 23.7,

24.2, 30.2, 50.3, 53.7 (d, J_CP161), 126.3, 128.2, 130.9, 133.6,

134.0, 136.3; m/z (FAB^ϩ) 386 (37%, M ϩ C₆H₁₁NH₃^ϩ), 253 (87,

M Ϫ Cl).

(Found: M^ϩ382.111. C₁₅H₂₅FO₆P₂requires M 382.111); ν_max

cm^Ϫ1(ﬁlm) 2985 (C–H), 1629 (C᎐C), 1265 (P᎐O), 1032 (P–O–

/

᎐

C); δ_H(CDCl₃) 1.25, 1.30 (6 H each, 2 t, J 6.3, 2 CH₃CH₂–

each), 4.00–4.15 (8 H, m, 4 CH₃CH₂–), 5.65 (1 H, dd, J_HP10.5,

J_HF39.5, PCHF–), 7.55–7.65 (3 H, m, ArH), 7.70–7.85 (1 H, m,

ArH); δ_P(CDCl₃) 14.7 (d, J_PF83.3), 18.1 (s); δ_F(CDCl₃) Ϫ202.5

(d); H-coupled Ϫ202.6 (dd, J_PF83.3, J_HF39.5); δ_C(CDCl₃) 16.3,

16.4, 63.5, 63.8, 89.0 (dd, J_CP169, J_CF185), 128.7, 129.7, 130.6,

132.3, 132.5, 133.6; m/z (EI^ϩ) 382 (71%, M^ϩ), 354 (22,

M Ϫ C₂H₄).

Tetraethyl 2-methylenepropane-1,3-bisphosphonate (54)

Methallyl dichloride (2.00 g, 16.00 mmol) was placed in a dry

ﬂask under nitrogen and triethyl phosphite (5.32 g, 32.02 mmol,

5.90 ml) added. The reaction was heated at 120 ЊC for 17 h

and the resultant pale yellow liquid puriﬁed by ﬂash column

chromatography from its monophosphonate to aﬀord the title

compound as a colourless liquid (2.10 g, 40%); R_f0.12 (DCM–

MeOH, 98:2) (Found: M^ϩ329.129, C₁₂H₂₇O₆P₂requires M

Diethyl [3-(diethoxyphosphoryl)phenyl]chloromethylphosphon-

ate (50)

Diethyl [3-(diethoxyphosphoryl)phenyl]hydroxymethylphos-

phonate 48 (0.65 g, 1.71 mmol) in DCM (15 ml) was treated

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

435

View Article Online

329.128); ν_max/cm^Ϫ1(ﬁlm) 2909 (C–H), 1645 (C᎐C), 1250 (P᎐O),

80 mM), and 3-PGA (100 µl, 60 mM) added. These were

᎐

1064 (P–O–C); δ_H(CDCl₃) 1.30 (12 H, t, J 6.2, 4 CH₃CH₂–),

2.70 (4 H, d, J_HP18.5, 2 PCH₂–), 4.00–4.10 (8 H, m, 4 CH₃-

allowed to reach room temperature over 5 min (stock solutions

kept at 0 ЊC) and then dithiothreitol (5 µl, 1.0 mM) and ATP

(100 µl, 60 mM) added. After a further 5 min, NADH (20 µl,

12 mM), glyceraldehyde 3-phosphate dehydrogenase (20 µl,

100 u per ml) and the 3-PGK (20 µl) under test added. The

cuvettes were immediately analysed in the spectrophotometer

at 25 ЊC. From the rates of reaction the enzyme activity was

calculated and found to be 12.5 u per ml.

CH₂–), 5.10 (2 H, t, J 5.5, C᎐CH ); δ (CDCl₃) 26.9 (s);

᎐

2

P

δ_C(CDCl₃) 16.2, 16.3, 33.7 (d, J_CP107), 61.8, 61.9, 119.4, 130.8;

m/z (EI^ϩ) 328 (30%, M^ϩ), 191 (98, M^ϩϪ PO₃Et₂), 163 (45,

M Ϫ PO₃Et₂Ϫ C₂H₄), 135 (38, M^ϩϪ PO₃Et₂Ϫ 2C₂H₄).

2-Methylenepropane-1,3-bisphosphonic acid (55)

Tetraethyl 2-methylenepropane-1,3-bisphosphonate 54 (1.69 g,

5.61 mmol) was deprotected as for 2 with TMSBr (5.23 g, 36.63

mmol, 4.80 ml) to yield the free acid as an oﬀ-white solid (0.91

g). Salt formation and puriﬁcation as for 2 gave the biscyclo-

hexylammonium salt of the title compound as a white solid

(1.50 g, 81%, mp >275 ЊC); δ_H(D₂O) 2.55 (4 H, d, 2 PCH₂–,

Estimation of inhibitor activity

Each of the inhibitors under test (10 µl, 10 mM solutions)

was used in duplicate in the above assay at a 3-PGA concen-

tration chosen to give a V_maxrate (100 µl, 6 mM solution)

with 3-PGK (20 µl, 5 u per ml solution). These results were

compared to the enzyme reaction in the absence of inhibitor

and from these a percentage inhibition result calculated for

each. From these results an estimated IC₅₀value was calcu-

lated for each.

J 23.6), 4.95 (2 H, t, J 4.5, C᎐CH ); δ (D O) 20.4 (s); δ (D O)

᎐

2

P

2

C

2

31.3 (d, J_CP132), 119.2, 136.7.

Biological experimental

1,3-Bisphospho-D-glyceric acid⁴⁷(1)

Determination of K_ivalues for inhibitors

-Glyceraldehyde 3-phosphate (100 mg) in water (3 ml) was

neutralised with 1 M NaOH. Phosphate solution (3.4 ml, 0.5

M) and NAD^ϩsolution (2.3 ml, 0.004 M) were added to this

and the volume brought to 24 ml with water. GAPDH (300

units) and ADH (680 units) were added and then acetaldehyde

(3 × 0.23 ml) in 3 aliquots after 0, 2, and 4 min. After 25 min at

18 ЊC, the reaction was acidiﬁed with H₂SO₄(4 ml, 1 M) and

poured immediately into acetone at 0 ЊC (300 ml). The 1,3-BPG

precipitate was collected by centrifugation, washed once with

cold acetone, and dissolved in cold water (18.7 ml). To this

solution, a solution of strychnine hydrochloride (18.7 ml, 0.1

M) was added and the solution left in the cold room overnight

to crystallise. The salt was collected by ﬁltration, washed with

cold water (3 ml) and then dissolved in cold water (10 ml). The

strychnine was removed by extraction with cold chloroform

(8 × 15 ml), with enough NaOH (0.2 M) added each time to

keep the pH slightly alkaline.

Solutions of inhibitor were made up at 0.3, 1.0, and 2.5 times

the IC₅₀values determined for the inhibitor above. Solutions of

20, 50, 100, 150, 200, and 400 µM 3-PGA were also made up.

These were used in duplicated 6 × 3 experiments in 36 one ml

cuvettes in the back reaction assay.⁴⁸TRISCl solution (pH 7.4,

625 µl, 100 mM stock solution), magnesium sulfate (100 µl,

80 mM), and 3-PGA (100 µl, 6 mM) were added. These

were allowed to reach room temperature over 5 min and then

dithiothreitol (5 µl, 1.0 mM) and ATP (100 µl, 60 mM) added.

After a further 5 min, NADH (20 µl, 12 mM), glyceraldehyde

3-phosphate dehydrogenase (20 µl, 100 u per ml) and 3-PGK

(20 µl, 5 u per ml) were added. The cuvettes were immediately

analysed in the spectrophotometer at 25 ЊC. The results were

analysed graphically using Origin^TM

.

ATP-free adenosine diphosphate (ADP)⁴⁹

The strychnine-free solution was placed in a vacuum desic-

cator over silica to remove dissolved chloroform and aliquots of

the solution frozen and stored at Ϫ80 ЊC.

Commercial ADP (100 mg) was dissolved in TRIS buﬀer (6 ml,

0.1 M, pH 7.4), potassium chloride (6 ml, 50 mM), magnesium

acetate (6 ml, 10 mM), and glucose (6 ml, 0.16 mM). Hexo-

kinase (25 units) was added to this solution and the reaction

was incubated for 30 min at rt. The solution was stored at

Ϫ20 ЊC and when needed 50 ml was acidiﬁed with HCl (50 ml,

1 M) and the pH brought to 7.5 with saturated TRIS buﬀer.

Aliquots of the solution were frozen and stored at Ϫ20 ЊC.

Isolation of 3-PGK⁴⁸

Whole human blood (100 ml) was centrifuged at 15 krpm for 20

min at 0 ЊC and the supernatant removed. The red blood cells

were washed twice with two volumes of physiological (0.9%)

sodium chloride and two volumes of water added to haemolyse.

The solution was dialysed against ten volumes of water for

3 h at 0 ЊC and again overnight. After this time, the solution

was removed from the dialysis tubing and cooled to Ϫ60 ЊC.

Ethanol–chloroform (2:1, 50 ml) was added at this temperature

all at once and the pH adjusted to 7.0 with 1 M KOH. This

solution was stirred in an ice-bath for 20 min and then centri-

fuged at 15 krpm for 20 min at 0 ЊC. The precipitate was dis-

carded and 2.5 volumes of ethanol added slowly to the resulting

solution at Ϫ25 ЊC. The temperature was held below 0 ЊC and

after 20 min the precipitate was collected by centrifugation at

15 krpm for 15 min at 0 ЊC. The precipitate was dissolved in

Na–K phosphate buﬀer (15 ml, 10 mM) and the solution

dialysed overnight in the cold against the same buﬀer.

Determination of IC₅₀values by forward reaction assay

For the forward reaction assay, triethanolamine (570 µl, 100

mM), water (335 ml), inhibitor (10 µl, 2, 20, or 200 µM), mag-

nesium sulfate (20 µl, 80 mM) previously prepared ADP (35 µl,

3 mM), and Ap₅A (10 µl, 125 µM) added. 3-PGK (10 µl, 5 mu

per ml) and 1,3-BPG (1, 10 µl, 200 µM) were added and the

cuvette incubated at 25 ЊC for 8 min. After this time, the reac-

tion was stopped by boiling at 70 ЊC for 4 min and the cuvettes

stored at Ϫ20 ЊC for further analysis. The luminometer was

calibrated with solutions of commercial ATP from 100 to 500

mM strength. In a 1 ml cuvette, potassium dihydrogen phos-

phate solution (333 µl, 10 mM), magnesium sulfate (333 µl, 80

mM), and sodium arsenate (333 µl, 100 mM) were placed. The

ATP solution (10 µl) was added and, after mixing, the ﬁreﬂy

luciferase/luciferin solution (30 µl) was added. This had been

made up from the commercial vials by adding 10 ml cold water

and centrifuging at 150 rpm for 10 min and was stored at 0 ЊC

during the analysis then discarded. The cuvettes were unfrozen

and a sample (10 µl) used in the luminometric analysis as

detailed above. By comparison with the reaction performed

without inhibitor, a % inhibition result could be calculated and

hence the IC₅₀for each inhibitor.

After analysis, glycerol was added to the buﬀered 3-PGK

solution to give a 3-PGK solution in 40% glycerol and 60%

buﬀer and this was stored at Ϫ20 ЊC.

Assay of PGK activity

The isolated enzyme solution was assayed⁴⁸spectrophoto-

metrically at high 3-PGA concentration to determine the activ-

ity. Three 1 ml cuvettes were taken and TRISCl solution (pH

7.4, 635 µl, 100 mM stock solution), magnesium sulfate (100 µl,

436

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

View Article Online

19 G. M. Blackburn, Chem. Ind., 1981, 134.

20 R. D. Chambers, R. Jaouhari and D. O’Hagan, J. Chem. Soc.,

Chem. Commun., 1988, 1169.

Protocol for pH titrations

The bisphosphonic acid (15 mg) was dissolved in NaCl (5 ml,

0.152 M) and incubated in a water bath at 25 ЊC. The sample

was adjusted to pH 11.5 (4 M NaOH) and titrated against HCl

(0.5 M) across the pH range under study.

21 P. C. Crofts and G. M. Kosolapoﬀ, J. Am. Chem. Soc., 1953, 75,

5738.

22 J. Kraut, Acta Crystallogr., 1961, 14, 1146.

23 D. O’Hagan, J. Nieschalk, A. S. Batsanov and J. A. K. Howard,

Tetrahedron, 1996, 52, 165.

24 Y. Okaya, Acta Crystallogr., 1966, 20, 712.

25 G. R. Thatcher and A. Campbell, J. Org. Chem., 1993, 58, 2272.

26 C. E. McKenna, M. T. Higa, N. H. Cheung and M.-C. McKenna,

Tetrahedron Lett., 1977, 155.

27 G. M. Blackburn and D. Ingleson, J. Chem. Soc., Chem. Commun.,

1978, 870.

Acknowledgements

We thank the EPSRC for a CASE studentship (to NAC), for

continued support from Glaxo-Wellcome plc, and Dr A. Poss

and Dr G. Shia (Allied Signal Co, Buﬀalo, NY) for generous

provision of Accuﬂuor^TM(NFSi).

28 E. P. Papadopoulos, A. Jarrar and C. H. Issidorides, J. Org. Chem.,

1966, 31, 615.

29 B. Iorga, F. Eymery and P. Savignac, Tetrahedron Lett., 1998, 39,

3693.

30 B. Iorga, F. Eymery and P. Savignac, Tetrahedron, 1999, 55, 2671.

31 K. D. Berlin and H. A. Taylor, J. Am. Chem. Soc., 1964, 86, 3862.

32 I. Shahak, E. D. Bergman and R. C. Matthewman, Isr. J. Chem.,

1966, 4, 225.

References

1 L. F. Hass, W. K. Kappel, K. B. Miller and R. L. Engel, Curr. Top.

Cell. Regul., 1978, 14, 75.

2 L. F. Hass, W. K. Kappel, K. B. Miller and R. L. Engel, J. Biol.

Chem., 1978, 253, 77.

3 T. N. Bryant, H. C. Watson and P. L. Wendell, Nature (London),

1974, 247, 14.

4 H. C. Watson, N. P. C. Walker, P. J. Shaw, T. N. Bryant, P. L.

Wendell, L. A. Fothergill, R. E. Perkins, S. C. Conroy, M. J.

Dobson, M. F. Tuite, A. J. Kingsman and S. M. Kingsman, EMBO

J., 1982, 1, 1635.

5 R. D. Banks, C. C. F. Blake, P. R. Evans, R. Haser, D. W. Rice,

G. W. Hardy, M. Merrett and A. W. Phillips, Nature (London), 1979,

279, 773.

6 G. J. Davies, S. J. Gamblin and J. Littlechild, Acta Crystallogr.,

Sect. D, 1994, 50, 202.

7 K. Harlos, M. Vas and C. F. Blake, Proteins: Struct., Funct., Genet.,

1992, 12, 133.

8 C. Blake, D. Rice and F. Cohen, Int. J. Pept. Protein Res., 1986, 27,

443.

9 W. G. J. Hol, B. E. Bernstein and P. A. M. Michels, Nature (London),

1997, 385, 275.

10 G. M. Blackburn, D. A. England and F. Kolkmann, J. Chem. Soc.,

Chem. Commun., 1981, 930.

11 J. Wrobel and A. Dietrich, Tetrahedron Lett., 1993, 34, 3543.

12 E. Diﬀerding, R. O. Duthaler, A. Krieger, G. M. Ruegg and C.

Schmit, Synlett, 1991, 6, 395; D. Taylor, N. Dinaut, A. N. Thadani

and Z. Huang, Tetrahedron Lett., 1996, 37, 8089.

13 G. M. Blackburn, T. D. Perrée, A. Rashid, C. Bisbal and B. Lebleu,

Chem. Scr., 1986, 26, 21.

14 D. J. Burton and W. Qui, Tetrahedron Lett., 1996, 37, 2745.

15 D. J. Burton and L. G. Sprague, J. Org. Chem., 1989, 54, 613.

16 X. Zhang, W. M. Qui and D. J. Burton, Tetrahedron Lett., 1999, 40,

2681.

33 T. Hirao, T. Masunaga, N. Yamada, Y. Ohshiro and T. Agawa,

Synthesis, 1981, 56.

34 T. Hirao, T. Masunaga, N. Yamada, Y. Ohshiro and T. Agawa, Bull.

Chem. Soc. Jpn., 1982, 55, 909.

35 J. Conia, F. Huel, A. Lechavellier and M. Pellet, Synthesis, 1978, 63.

36 P. E. Stanley and S. G. Williams, Anal. Biochem., 1969, 29, 381.

37 R. K. Scopes, The Enzymes, Academic Press, New York, 1973,

vol. VIII.

38 S. W. Benson, J. Am. Chem. Soc., 1958, 80, 5151.

39 D. Bishop and K. J. Laidler, J. Chem. Phys., 1965, 42, 1688.

40 R. P. Bell and P. G. Evans, Philos. Trans. R. Soc. London A, 1966,

291, 297.

41 F. W. Bennett, H. J. Emeléus and R. N. Haszeldine, J. Chem. Soc.,

1954, 3598.

42 D. L. Jakeman, A. I. Ivory, M. P. Williamson and G. M. Blackburn,

J. Med. Chem., 1998, 41, 4439.

43 R. A. Copeland, D. Lombardo, J. Giannaras and C. P. Decicco,

Bioorg. Med. Chem. Lett., 1995, 5, 1947.

44 Q. Wang, Z. Huang, C. Ramachandran, A. Dinaut and S. Taylor,

Bioorg. Med. Chem. Lett., 1998, 8, 345.

45 L. Ernst, Org. Magn. Reson., 1977, 9, 35.

46 A. N. Pudovik, I. V. Gur’yanova and Z. G. Zimin, J. Gen. Chem.

USSR (Engl. Transl.), 1967, 37, 1979.

47 E. Negelein, Methods Enzymol., 1957, 3, 216.

48 A. Yoshida and S. Watanabe, J. Biol. Chem., 1972, 247, 440.

49 C. I. Pogson, S. V. Gurnah and S. A. Smith, Int. J. Biochem., 1979,

10, 995.

17 Y.-W. Li and L. D. Byers, Biochim. Biophys. Acta, 1993, 1164, 17.

18 G. M. Blackburn, A. J. Ivory, D. L. Jakeman and M. P. Williamson,

Bioorg. Med. Chem. Lett., 1994, 4, 2573.

Paper a906507e

J. Chem. Soc., Perkin Trans. 1, 2000, 421–437

437

Article Doi

The synthesis of novel bisphosphonates as inhibitors of phosphoglycerate kinase (3-PGK)

DOI: 10.1039/a906507e

Source and publish data:

Authors:

Article abstract of DOI:10.1039/a906507e

Full text of DOI:10.1039/a906507e

Products guided by the article

R&D Labs maybe for 263164-83-6

Relevant to this article

Hot Product