Synthesis and antituberculosis activity of C-phosphonate analogues of decaprenolphosphoarabinose, a key intermediate in the biosynthesis of mycobacterial arabinogalactan and lipoarabinomannan.

Article abstract of DOI:10.1021/jo026247r

The cell wall complex in mycobacteria, including the human pathogen Mycobacterium tuberculosis, is comprised in large part of two polysaccharides that contain a significant number of arabinofuranose residues. Both polysaccharides are assembled by a family of arabinosyltransferases that use decaprenolphosphoarabinose (3) as the donor species. In this paper, we describe the synthesis of a panel of C-phosphonate analogues of 3, which were designed to inhibit these arabinosyltransferases and thus block the biosynthesis of mycobacterial cell wall polysaccharides. A number of routes were explored for the preparation of the targets. The successful approach involved the synthesis of a protected C-phosphonate allyl ester 16, which was then coupled to an alkene via an olefin cross metathesis reaction. Subsequent reduction of the alkene with diimide and deprotection afforded the targets. Screening of these compounds in vitro against Mycobacterium tuberculosis revealed that one of the compounds, 15f, possessed antituberculosis activity, with an MIC value of 3.13 microg/mL.

Full text of DOI:10.1021/jo026247r

Syn th esis a n d An titu ber cu losis Activity of C-P h osp h on a te
An a logu es of Deca p r en olp h osp h oa r a bin ose, a Key In ter m ed ia te in
th e Biosyn th esis of Mycoba cter ia l Ar a bin oga la cta n a n d
Lip oa r a bin om a n n a n
Charla A. Centrone and Todd L. Lowary*
Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210
lowary.2@osu.edu
Received J uly 26, 2002
The cell wall complex in mycobacteria, including the human pathogen Mycobacterium tuberculosis,
is comprised in large part of two polysaccharides that contain a significant number of arabino-
furanose residues. Both polysaccharides are assembled by a family of arabinosyltransferases that
use decaprenolphosphoarabinose (3) as the donor species. In this paper, we describe the synthesis
of a panel of C-phosphonate analogues of 3, which were designed to inhibit these arabinosyltrans-
ferases and thus block the biosynthesis of mycobacterial cell wall polysaccharides. A number of
routes were explored for the preparation of the targets. The successful approach involved the
synthesis of a protected C-phosphonate allyl ester 16, which was then coupled to an alkene via an
olefin cross metathesis reaction. Subsequent reduction of the alkene with diimide and deprotection
afforded the targets. Screening of these compounds in vitro against Mycobacterium tuberculosis
revealed that one of the compounds, 15f, possessed antituberculosis activity, with an MIC value of
3.13 µg/mL.
In tr od u ction
Our interest in mycobacterial arabinosyltransferases
(AraT’s) originated from studies that validated these
enzymes as suitable targets for drug action. Previous
reports6 have demonstrated that ethambutol, an antibi-
otic used to treat tuberculosis, acts by inhibiting the
AraT’s involved in AG and LAM biosynthesis.
Mycobacteria, including the human pathogens Myco-
bacterium tuberculosis and Mycobacterium leprae, pos-
sess an extraordinarily thick and complicated cell wall
structure that provides the organism with a great deal
of protection from its environment.1 The viability of the
organism is dependent upon its ability to synthesize an
intact cell wall and, consequently, inhibitors of the
enzymes that are involved in the biosynthesis of various
cell wall components are potential antimycobacterial
agents.2 Indeed, some of the antibiotics currently used
to treat tuberculosis (e.g., ethambutol and isoniazid) act
by blocking cell wall assembly.3
Key structural components of the mycobacterial cell
wall are two polysaccharides, arabinogalactan (AG) and
lipoarabinomannan (LAM), which are unusual in that all
of the arabinose and galactose residues exist in the
furanose ring form. Glycoconjugates that contain galacto-
furanose and arabinofuranose are xenobiotic to mammals
and, hence, the enzymes that are involved in the biosyn-
thesis of these glycans are ideal targets for drug action.4
Over the past few years, we have carried out synthetic
and conformational investigations directed ultimately at
the development of inhibitors of the glycosyltransferases
that assemble the arabinan portions of AG and LAM.5
The structure of the arabinan in AG and LAM is
essentially identical. The glycan core consists of a linear
R-(1f5) linked chain of arabinofuranose residues, with
periodic R-(1f3)-linked branch points from which ad-
ditional R-(1f5) linked chains are attached. Attached to
this core arabinan, at the nonreducing termini of each
linear chain, is the hexasaccharide 1 (Chart 1). The
biosynthesis of this arabinan is postulated to involve a
family of AraT’s that produce â-(1f2), R-(1f3), and
R-(1f5) arabinofuranosyl linkages. A prototypical AraT-
catalyzed reaction (Figure 1) involves the coupling of an
oligosaccharide acceptor (e.g., 2), with decaprenolphos-
phoarabinose (DPA, 3)7 to afford an elongated oligosac-
charide, 4.8
(4) (a) Besra, G. S.; Brennan, P. J . J . Pharm. Pharmacol. 1997, 49
(Suppl. 1), 25. (b) Crick, D. C.; Mahapatra, S.; Brennan, P. J .
Glycobiology 2001, 11, 107R.
(5) (a) Ayers, J . D.; Lowary, T. L.; Morehouse, C. B.; Besra, G. S.
Bioorg. Med. Chem. Lett. 1998, 8, 437. (b) Subramaniam, V.; Lowary,
T. L. Tetrahedron 1999, 55, 5965. (c) Callam, C. S.; Lowary, T. L. J .
Org. Chem. 2001, 67, 8961. (d) Yin, H.; D’Souza, F. W.; Lowary, T. L.
J . Org. Chem. 2002, 67, 892. (e) McGurk, P.; Chang, G. X.; Lowary, T.
L.; McNeil, M.; Field, R. A. Tetrahedron Lett. 2001, 42, 2231.
(6) (a) Mikusova´, K.; Slayden, R. A.; Besra, G. S.; Brennan, P. J .
Antimicrob. Agents Chemother. 1995, 39, 2484. (b) Deng, L.; Mikusova´,
K.; Robuck, K. G.; Scherman, M.; Brennan, P. J .; McNeil, M. R.
Antimicrob. Agents Chemother. 1995, 39, 694. (c) Khoo, K.-H.; Douglas,
E.; Azadi, P.; Inamine, J . M.; Besra, G. S.; Mikusova´, K.; Brennan, P.
J .; Chatterjee, D. J . Biol. Chem. 1996, 271, 28682-28690.
(1) (a) Lowary, T. L. Mycobacterial Cell Wall Components. In
Glycoscience: Chemistry and Chemical Biology; Fraser-Reid, B., Tat-
suta, K., Thiem, J ., Eds.; Springer-Verlag: Berlin, Germany, 2001; pp
2005-2080. (b) Brennan, P. J .; Nikaido, H. Annu. Rev. Biochem. 1995,
64, 29.
(2) Barry, C. E., III Biochem. Pharmacol. 1997, 54, 1165.
(3) (a) Rastogi, N.; David, H. L. Res. Microbiol. 1993, 144, 133. (b)
Blanchard, J . S. Annu. Rev. Biochem. 1996, 65, 215-239. (c) Young,
D. B.; Duncan, K. Annu. Rev. Microbiol. 1995, 49, 641-673.
10.1021/jo026247r CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/08/2002
8862
J . Org. Chem. 2002, 67, 8862-8870

C-Phosphonate Analogues of Decaprenolphosphoarabinose
F IGURE 1. Prototypical arabinosyltransferase-catalyzed reaction.
CHART 1
CHART 2
We describe here the synthesis of a panel of C-
phosphonate-based DPA analogues. It was our expecta-
tion that replacement of the glycosidic oxygen in 3 with
a methylene group would provide compounds (5, Chart
2) that would be bound by the AraT’s, but that could not
turn over. These C-phosphonates would therefore block
the assembly of mycobacterial arabinan and thus prevent
growth of the organism. Our goal was not to prepare
compounds that contained the long isoprenoid chain
present in 3. Rather, we endeavored to synthesize
molecules in which simple long-chain linear alkyl groups
replace this lipid.
In designing potential inhibitors of these AraT’s, logical
first choices are analogues of the two substrates recog-
nized by the enzyme. The majority of our previous work
in this area has focused on synthesizing analogues of the
acceptor oligosaccharides.5a-d This approach has also
been used widely in developing inhibitors that are specific
for particular human glycosyltransferases.9 Donor ana-
logues have been less widely studied because a single
glycosyl donor is usually a substrate for a number of
glycosyltransferases, and these compounds are therefore
expected to lack specificity for a particular enzyme. In
bacterial systems, this lack of specificity with regard to
the donor can be exploited. Because 3 is a substrate for
more than one AraT, analogues of this compound would
be expected to block a number of biosynthetic steps and,
in turn, be especially potent antimycobacterials.
Resu lts a n d Discu ssion
In itia l Ap p r oa ch es. Although a number of C-phos-
phonate analogues of glycosyl phosphates (e.g., 6, Chart
2) have been synthesized,10 far fewer reports have
described the preparation of the corresponding phospho-
nate ester derivatives (e.g., 5).11 We initially explored the
possibility of synthesizing these compounds from iodide
7 (Figure 2A) via a route that involves the formation of
an H-phosphonate,12 esterification with the appropriate
alcohol,13 oxidation of the H-phosphonate,14 and subse-
quent deprotection. Thus (Scheme 1), 7 was prepared15
and then treated with bis(trimethylsilyl)phosphonite.12
However, despite a number of attempts under different
reaction conditions, only unreacted 7 was isolated fol-
lowing the reaction. We therefore explored an approach
in which the carbon-phosphorus bond was installed via
an Michaelis-Arbuzov reaction16 (Figure 2B). The long-
(7) Biosynthesized from 5-phosphoribose pyrophosphate (pRpp): (a)
Scherman, M. S.; Kalbe-Bournonville, L.; Bush, D.; Xin, Y.; Deng, L.;
McNeil, M. J . Biol. Chem. 1996, 271, 29652. (b) Scherman, M.; Weston,
A.; Duncan, K.; Whittington, A.; Upton, R.; Deng, L.; Comber, R.;
Friedrich, J . E.; McNeil, M. J . Bacteriol. 1995, 177, 7125.
(8) Incorporation of arabinose into AG has been shown through the
use of radiolabeled
3 and mycobacterial membrane extracts. In
addition, incubation of small oligosaccharide substrates with 3, in the
presence of a mycobacterial membrane preparation, led to the forma-
tion of an oligosaccharide with additional â-(1f2) and R-(1f5) linkages
(Lee, R. E.; Brennan, P. J .; Besra, G. S. Glycobiology 1997, 7, 1121).
The lack of R-(1f3) linkages was attributed to either instability or
absence of R-(1f3) AraT activity in the membrane preparation, or the
possibility that this enzyme recognizes oligosaccharide substrates
larger than those investigated. It is also conceivable that another
activated donor (e.g., a sugar nucleotide) is used by this AraT. The
presence of UDP-Araf in mycobacteria has been reported (Singh, S.;
Hogan S. E. Microbios 1994, 77, 217), but incorporation of this donor
into arabinan has not been demonstrated.
(10) Nicotra, F. Synthesis of Glycosyl Phosphate Mimics. In Car-
bohydrate Mimics; Chapleur, Y., Ed.; Wiley-VCH: Weinheim, Ger-
many, 1998.
(11) (a) Brooks, G.; Edwards, P. D.; Hatto, J . D. I.; Smale, T. C.
Southgate, R. Tetrahedron 1995, 29, 7999. (b) Qiao, L.; Vederas, J . C.
J . Org. Chem. 1993, 58, 3480.
(12) (a) Boyd, E. A.; Regan, A. C.; J ames, K. Tetrahedron Lett. 1994,
35, 4223. (b) Boyd, E. A.; Regan, A. C.; J ames, K. Tetrahedron Lett.
1992, 33, 813.
(13) Malachowski, W. P.; Coward, J . K. J . Org. Chem. 1994, 59,
7625.
(14) Lindh, I.; Stawinski, J . J . Org. Chem. 1989, 54, 1338.
(15) Freeman, F.; Robarge, K. D. Carbohydr. Res. 1987, 171, 1.
(9) Qian, X.; Palcic, M. M. Carbohydr. Chem. Biol. 2000, 3, 293.
J . Org. Chem, Vol. 67, No. 25, 2002 8863

Centrone and Lowary
F IGURE 2. Retrosynthesis of 5.
SCHEME 1a
SCHEME 3a
a
Conditions: (a) (TMSO)2PH, CH2Cl2, 40 °C; (b) H2O.
SCHEME 2a
a
Conditions: (a) THF, -78 °C, 93%; (b) TMSOTf, Et3SiH,
CH2Cl2, 95%.
deprotection of the ethyl groups using TMSI and TMSBr
was explored. We were unsuccessful, however, in produc-
ing 10 in good yield as the cleavage of the ethyl groups
was also accompanied by loss of one or more of the benzyl
groups. It was extremely difficult to separate the reaction
products and we therefore abandoned this approach.
We next explored a route starting from lactone 1118
(Figure 2C). To assess the viability of this approach
(Scheme 3), 11 was reacted with 1219 to provide the
adduct 1320 in 93% yield. Deoxygenation of 13 with
triethylsilane and trimethylsilyltrifluoromethanesul-
a
Conditions: (a) (EtO)3P, 156 °C, 91%; (b) TMSBr or TMSI.
chain alkyl moiety was to be introduced via an esterifi-
cation reaction following the selective cleavage of the
ethyl groups. With this route in mind (Scheme 2),
reaction of 7 with triethyl phosphite at 156 °C (reflux)
provided the diethyl phosphonate derivative 917 in 91%
yield. Next, the conversion of 9 to 10 via the selective
(17) Maryanoff, B. E.; Nortey, S. O.; Inners, R. R.; Campbell, S. A.;
Reitz, A. B.; Liotta, D. Carbohydr. Res. 1987, 171, 259.
(18) Csuk, R.; Doerr, P. J . Carbohydr. Chem. 1995, 14, 35.
(19) Teulade, M. P.; Savignac, P.; Aboujaoude, E. E.; Collignon, N.
J . Organomet. Chem. 1986, 312, 283.
(20) Nicotra, F.; Panza, L.; Russo, G.; Senaldi, A.; Burlini, N.;
Tortora, P. J . Chem. Soc., Chem. Commun. 1990, 1396.
(16) Bhattacharya, A. K.; Thyagarajan, G. Chem. Rev. 1981, 81, 415.
8864 J . Org. Chem., Vol. 67, No. 25, 2002

C-Phosphonate Analogues of Decaprenolphosphoarabinose
F IGURE 3. Retrosynthesis of 15.
SCHEME 4a
F IGURE 4. Proposed formation of 16 from 24 via Mitsunobu
cyclization.
double bond and deprotection would afford the targets.
Phosphonate 16 can be accessed from 19 and the pro-
tected L-xylose derivative 18.24
P r ep a r a tion of P h osp h on a te 19. For the synthesis
of 19 we employed methodology developed by Gagne and
co-workers (Scheme 4).25 Dimethyl methylphosphonate
(20) was converted to the corresponding dibenzyl ester
2126 in 90% yield by reaction with benzyl acetate and
potassium tert-butoxide. Subsequent transesterification
of 21 with allyl acetate and potassium tert-butoxide under
controlled conditions afforded 22 in 47% yield. The
synthesis of 22 from the commercially available dichlo-
ride 23 was also possible, but the yield of the product
was lower than the route from 20. Lithiation of 22 with
n-BuLi in THF afforded 19.
Syn th esis of 16 fr om 18 a n d 19. On the basis of pre-
vious investigations,27 we envisioned that C-phosphonate
16 could be obtained via a Mitsunobu reaction of acyclic
diol 24 (Figure 4). In this earlier study, diols of this
general type were shown to undergo Mitsunobu cycliza-
tion with inversion of the stereochemistry at C-5, pre-
sumably via formation of a phosphonium ion such as 25.
The synthesis of 16 is illustrated in Scheme 5. Reaction
of the L-xylose derivative 1824 with 19 afforded a 90%
yield of ketose 26, which was subsequently reduced, in
87% yield, with sodium borohydride. This reaction was
only marginally stereoselective affording the two possible
diastereomeric products (24 and 27) as a 2:1 mixture,
which was impossible to separate. Other reducing agents
a
Conditions: (a) BnOAc, t-BuOK, THF, rt, 90%; (b) AllOAc,
t-BuOK, THF, rt, 47%; (c) BnOH, pyridine, then AllOH 0 °C, 33%;
(d) n-BuLi, THF, -78 °C.
fonate as the Lewis acid21 provided a deoxygenated
product in nearly quantitative yield. Unfortunately, the
product produced from this reaction was shown to be 14,
which possessed the incorrect stereochemistry at the
“anomeric” center.22
Having been unsuccessful in the synthesis of 5 via
what we considered to be the most direct routes, a
number of other approaches were explored, also without
success. Therefore, we slightly modified the structure of
the targets and developed a successful route for their
synthesis. The retrosynthesis for this approach is pro-
vided in Figure 3. The modified targets (15) differ from
5 in that an oxygen atom is present in the long alkyl
chain. We anticipated that this modification would not
significantly influence the inhibitory potential of 15
relative to 5. In this approach, the long-chain alkyl group
was to be introduced via an olefin cross metathesis
reaction23 of 16 and 17. Subsequent reduction of the
(21) Dondoni, A.; Marra, A.; Pasti, C. Tetrahedron: Asymmetry 2000,
11, 305.
(22) The stereochemistry was proven by comparison of the 1H, 13C,
and 31P spectral data previously reported for 14: McClard, R. W.;
Tsimikas, S.; Schriver, K. E. Arch. Biochem. Biophys. 1986, 245, 282.
(23) Blackwell, H. E.; O’Leary, D. J .; Chatterjee, A. K.; Washen-
felder, R. A.; Bussmann, D. A.; Grubbs, R. H. J . Am. Chem. Soc. 2000
122, 58.
(24) Meng, Q.; Hesse, M. Helv. Chim. Acta 1991, 74, 445.
(25) Kissling, R. M.; Gagne, M. R. J . Org. Chem. 1999, 64, 1585.
(26) Campbell, M. M.; Carruthers, N. I.; Mickel, S. J . Tetrahedron
1982, 38, 2513.
(27) Perksy, R.; Albeck, A. J . Org. Chem. 2000, 65, 3775.
J . Org. Chem, Vol. 67, No. 25, 2002 8865

Centrone and Lowary
SCHEME 5 a
a
Conditions: (a) 19, THF, -78 °C, 90%; (b) NaBH4, THF, rt, 87%; (c) benzaldehyde dimethyl acetal, p-TsOH, CH3CN, 75% from 26
(combined 28A and 28B); (d) 80% AcOH, 50 °C, 93%; (e) 80% AcOH, 50 °C, 81%; (f) Ph3P, DIAD, THF, rt, 90%; (g) Ph3P, DIAD, THF, rt,
72%.
SCHEME 6 a
SCHEME 7 a
a
Conditions: (a) NaH, RI, DMF, rt.
buten-1,4-diol (30) under standard conditions (Scheme
7). Each was then reacted with 16 in the presence 20 mol
% of the Grubbs catalyst (31,28 Scheme 8) in refluxing
dichloromethane, which provided the cross metathesis
products 32 in 51-66% yield as a mixture of cis/trans
isomers. Subsequent reduction of the alkene was achieved
by reaction with diimide to afford the products 33 in
74-95% yield. We also explored the use of (Ph3P)3RhCl/
H2 and Raney Nickel/H2 to carry out this reduction, but
found the diimide reaction gave better yields of the
products.29 The final targets 15 were obtained by cleavage
of the benzyl groups by hydrogenation. Following chro-
matography on Iatrobeads,30 the products were obtained
in low to modest yield (20-68%). We are unsure as to
why the yield of the deprotection step is relatively poor
in some cases. TLC of the crude reaction mixtures
indicated that in some cases a significant amount of very
polar material was formed in addition to the desired
product. Previous work with C-phosphonate analogues
of arabinofuranosyl pyrophosphates (e.g., 32, Figure 5)
a
Conditions: (a) H2, Pd/C, AcOH; then TMSBr, CH2Cl2, rt, 81%;
(b) H2, Pd/C, AcOH; then TMSBr, CH2Cl2, rt, 75%.
were explored to carry out this reduction (e.g., L-Selec-
tride and (+)-diisopinocamphenylborane), but with no
improvement in stereoselectivity. To separate the dia-
stereomers, the mixture was treated with benzaldehyde
dimethyl acetal and p-TsOH, which provided all four
possible diastereomeric benzylidene derivatives 28A and
28B. These were separated by chromatography and the
acetal protecting group was cleaved under acidic condi-
tions to provide 24 and 27 as pure compounds. Each of
the diols was cyclized upon treatment with triphenylphos-
phine and diisopropyl-azodicarboxylate (DIAD) affording
16 and its stereoisomer 29, in 90 and 72% yields,
respectively. The stereochemistry of 16 was proven by
complete deprotection, which afforded a product (6) that
was identical with that obtained by total deprotection of
9 (Scheme 6).
(28) Schwab, P.; France, M. B.; Ziller, J . W.; Grubbs, R. H. Angew.
Chem., Int. Ed. Engl. 1995, 34, 2039.
(29) We attempted simultaneous reduction of the alkene and hy-
drogenolysis of the benzyl ethers upon reaction with H2, Pd/C, but were
unsuccessful. Under these conditions, decomposition of the substrate
was observed, which we attribute to the formation of a palladium-π-
allyl complex from the allyl phosphonate.
Syn th esis of 17 a n d Cr oss Meta th esis Rea ction s.
The synthesis of the targets required as a key step an
olefin cross metathesis reaction between 16 and a panel
of alkenes with the general structure 17. The synthesis
of these alkenes was achieved by alkylation of cis-2-
(30) Iatrobeads refers to a beaded silica gel 6RS-8060, which is
manufactured by Iatron Laboratories (Tokyo).
8866 J . Org. Chem., Vol. 67, No. 25, 2002

C-Phosphonate Analogues of Decaprenolphosphoarabinose
SCHEME 8 a
a
Conditions: (a) 31, 17, CH2Cl2, 40 °C, 51-66%; (b) KOC(O)NdNC(O)OK, AcOH, CH3OH, 40 °C, 74-95%; (c) H2, Pd/C, CH3OH,
20-68%.
In conclusion, we report here the synthesis of six
C-phosphonate analogues of decaprenolphosphoarabi-
nose, 3. These compounds were synthesized via a route
that had as a key step an olefin cross metathesis between
allyl C-phosphonate 16 and an alkene 17. One of the six
compounds was shown to prevent growth of mycobacteria
in an in vitro assay. Screening of the compounds as
inhibitors of mycobacterial arabinosyltransferases is
currently in progress.
Exp er im en ta l Section
Gen er a l. Solvents were distilled from the appropriate
drying agents before use. Unless stated otherwise, all reactions
were carried out at room temperature under
pressure of argon and were monitored by TLC on silica gel 60
254. Spots were detected under UV light or by charring with
a positive
F
10% H2SO4 in ethanol. Solvents were evaporated under
reduced pressure and below 40 °C (bath). Organic solutions
of crude products were dried over anhydrous Na2SO4. Unless
otherwise indicated, column chromatography was performed
on silica gel 60 (40-60 µM). The ratio between silica gel and
crude product ranged from 100 to 50:1 (w/w). Optical rotations
F IGURE 5. Formation of phostone 33 from 32 (see ref 32).
demonstrated31 the formation of phostone 33. The forma-
tion of a compound analogous to 33 is also possible here,
although we could not detect the presence of such a
species in the eluants obtained by washing the Iatrobead
column with 3:1 dichloromethane:methanol.
Scr een in g of F in a l Com p ou n d s a s An titu ber cu -
losis Agen ts. Compounds 15a -f were tested in vitro for
the ability to prevent growth of Mycobacterium tubercu-
losis strain H37Rv (ATCC 27294) using the Alamar Blue
Microplate assay.32 Only one of the analogues, 15f,
possessed activity, and was shown to have an MIC of 3.13
µg/mL. It appears, therefore, that a fairly lengthy alkyl
chain is required for compounds of this type to possess
antituberculosis activity.
1
were measured at 21 ( 2 °C. H NMR spectra were recorded
at 250, 400, or 500 MHz, and chemical shifts are referenced
to either TMS (0.0, CDCl3) or CD3OH (4.78, CD3OD). 13C NMR
spectra were recorded at 62.5, 100, or 125 MHz and chemical
shifts are referenced to CDCl3 (77.00, CDCl3) or CH3OH (49.00,
CD3OD). 31P NMR spectra were recorded at 101, 162, or 202
MHz and chemical shifts are referenced to external phosphoric
acid (0.0, CDCl3, CD3OD). Elemental analyses were performed
by Atlantic Microlab Inc., Norcross, GA. Electrospray mass
spectra were recorded on samples suspended in mixtures of
THF and CH3OH with added trifluoroacetic acid or NaCl.
Analytical data for all new compounds (1H NMR, 13C NMR,
HRMS, elemental analysis, [R]D) are provided in the Support-
ing Information.
2,5-An h yd r o-D-glu cityl P h osp h on ic Acid (6). Phospho-
nate 917 (2.7 g, 4.87 mmol) was dissolved in glacial HOAc (10
mL). Palladium (10% on activated carbon, 500 mg) was added
and the reaction mixture was stirred under hydrogen overnight
at atmospheric pressure. The solid was then filtered off and
(31) McClard, R. W.; Witte, J . F. Bioorg. Chem. 1990, 18, 165.
(32) Collins, L. A.; Franzblau, S. G. Antimicrob. Agents. Chemother.
1997, 41, 1004.
J . Org. Chem, Vol. 67, No. 25, 2002 8867

Centrone and Lowary
the filtrate was concentrated to a clear residue that was
purified by chromatography (9:1, CH2Cl2:CH3OH) affording
1-diethyl-2,5-anhydroglucityl phosphonate (1.13 g, 83%): Rf
0.33 (9:1, CH2Cl2:CH3OH). The diethyl phosphonate from
above was dissolved in CH2Cl2 (5 mL). Bromotrimethylsilane
(1.7 mL, 12.9 mmol) was added and the reaction mixture
stirred overnight. Methanol (1 mL) was added to the orange
reaction mixture and the solvent was evaporated to give a
crude oil. The oil was purified by ion-exchange chromatography
(AG 1-X8 resin) and was eluted with a 0.2 M solution of
triethylammonium bicarbonate. After evaporation of the elu-
ant, the clear oil was dissolved in CH3OH (5 mL) and treated
with Amberlite H+ resin. The resin was then filtered to afford
6 (853 mg, 91%) as a clear oil. Compound 6 was also prepared
(in 81% yield) by subjecting phosphonate 16 to these reaction
conditions.
1-(Bu tyl-4′-O-h ep tyl)-2,5-a n h yd r o-D-glu cityl P h osp h o-
n a te (15a ). Phosphonate 33a (95 mg, 0.125 mmol) was
dissolved in a mixture of CH3OH and AcOH (5 mL:10 µL).
Palladium (10% on activated carbon, 22 mg) was added and
the reaction mixture was stirred under hydrogen overnight
at atmospheric pressure. After filtering off the solid and
evaporating the solvent, the residual oil was purified by
chromatography on Iatrobeads (CH2Cl2 f 3:1, CH2Cl2:CH3-
OH). The pure product was then redissolved in water and then
lyophilized to give 15a (13 mg, 26%) as an off-white solid.
1-(Bu t yl-4′-O-oct yl)-2,5-a n h yd r o-D-glu cit yl P h osp h o-
n a te (15b). Phosphonate 33b (132 mg, 0.171 mmol) was
hydrogenated as described for 33a . Purification of the product
was done as outlined for 15a to provide 15b (33 mg, 47%) as
an off-white solid.
1-(Bu tyl-4′-O-n on yl)-2,5-a n h yd r o-D-glu cityl P h osp h o-
n a te (15c). Phosphonate 33c (190 mg, 0.254 mmol) was
hydrogenated as described for 33a . Purification of the product
was done as outlined for 15a to provide 15c (56 mg, 54%) as
an off-white solid.
1-(Bu tyl-4′-O-d ecyl)-2,5-a n h yd r o-D-glu cityl P h osp h o-
n a te (15d ). Phosphonate 33d (99 mg, 0.124 mmol) was
hydrogenated as described for 33a . Purification of the product
was done as outlined for 15a to provide 15d (11 mg, 20%) as
an off-white solid.
cis-2-Bu ten yl-1,4-d ioctyl Eth er (17b). The preparation
of 17b was achieved by the reaction of 30 (500 mg, 5.67 mmol)
and 1-iodooctane (4.1 g, 17.01 mmol) as described for the
preparation of 17a . The product was purified by chromatog-
raphy (8:1, hexane:EtOAc) to afford 17b (1.63 g, 92%) as a clear
oil.
cis-2-Bu ten yl-1,4-d in on yl Eth er (17c). The preparation
of 17c was achieved by the reaction of 30 (500 mg, 5.67 mmol)
and 1-iodononane (4.3 g, 17.01 mmol) as described for the
preparation of 17a . The product was purified by chromatog-
raphy (9:1, hexane:EtOAc) to afford 17c (1.60 mg, 83%) as a
clear oil.
cis-2-Bu ten yl-1,4-d id ecyl Eth er (17d ). The preparation
of 17d was achieved by the reaction of 30 (535 mg, 6.07 mmol)
and 1-iododecane (6.5 g, 6.07 mmol) as described for the
preparation of 17a . The product was purified by chromatog-
raphy (10:1, hexane:EtOAc) to afford 17d (2.0 g, 91%) as a
clear oil.
cis-2-Bu ten yl-1,4-d id od ecyl Eth er (17e). The preparation
of 17e was achieved by the reaction of 30 (500 mg, 5.67 mmol)
and 1-iodododecane (6.72 g, 22.68 mmol) as described for the
preparation of 17a . The product was purified by chromatog-
raphy (12:1, hexane:EtOAc) to afford 17e (2.39 g, 99%) as a
clear oil.
cis-2-Bu ten yl-1,4-d ih exa d ecyl Eth er (17f). The prepara-
tion of 17f was achieved by the reaction of 30 (500 mg, 5.67
mmol) and 1-iodohexadecane (8.0 g, 22.68 mmol) as described
for the preparation of 17f. The product was purified by
chromatography (12:1, hexane:EtOAc) to afford 17e (1.6 g,
52%) as a clear oil.
Allylben zyl Meth ylp h osp h on a te (22). THF (40 mL) was
added to a stirring solution of 2125 (36.7 g, 0.133 mol) and allyl
acetate (28.7 mL, 0.266 mol). A 1 M solution of potassium tert-
butoxide (20 mL) was prepared and then added in 5-mL
aliquots approximately every 15 min. The reaction mixture
was then neutralized with AcOH and diluted with EtOAc (100
mL). The organic layer was washed with water (100 mL)
followed by brine (100 mL) and then dried over Na2SO4.
Evaporation of the solvent provided a thick yellow liquid. The
product was distilled at 135 °C (1 mmHg) affording 22 (14.1
g, 47%) as a clear liquid.
1-(Allylben zyl)-3,4,6-tr i-O-ben zyl-D-glu cityl P h osp h o-
n a te (24). Benzylidene 28 (559 mg, 0.761 mmol) was dissolved
in an 80:20 mixture of AcOH/H2O (20 mL) and heated at 50
°C overnight. After being cooled to room temperature, the
reaction mixture was diluted with CH2Cl2 (30 mL) and the
product was extracted from the aqueous layer. The organic
layer was dried over Na2SO4 and the solvent was evaporated.
The residue was purified by chromatography (1:3, hexane:
EtOAc) to give 24 (456 mg, 93%) as a clear oil.
1-(Allylben zylp h osp h in yl)-1-d eoxy-3,4,6-tr i-O-(ben zyl)-
D-fr u ctofu r a n ose (26). Phosphonate 22 (1.5 g, 6.7 mmol) was
dissolved in 5 mL of THF and the mixture was stirred at -78
°C. n-BuLi (11.7 mL of a 1.6 M solution in hexane) was added
and, after 5 min, a solution of lactone 1824 (4.9 g, 11.7 mmol)
in THF (10 mL) was added in one aliquot. After 1 h, the
solution was removed from the dry ice bath and stirred for
another 30 min. The reaction mixture was then neutralized
with AcOH and diluted with EtOAc (100 mL). The organic
layer was washed with water (75 mL) followed by brine (75
mL) and then dried over Na2SO4. The solvent was evaporated
and the residue was purified by chromatography (1:1, hexane:
EtOAc) to give 26 (6.8 g, 90%) as a clear oil.
1-(Allylben zyl)-3,4,6-tr i-O-ben zyl-D-m a n n ityl P h osp h o-
n a te (27). Benzylidene 29 (336 mg, 0.457 mmol) was dissolved
in an 80:20 mixture of AcOH/H2O (10 mL) and heated at 40
°C for 3 h. After being cooled to room temperature, the reaction
mixture was diluted with CH2Cl2 (30 mL) and the product was
extracted from the aqueous layer. The organic layer was dried
over Na2SO4 and the solvent was evaporated. The residue was
purified by chromatography (1:3, hexane:EtOAc) to give 27
(240 mg, 81%) as a clear oil.
1-(Bu t yl-4′-O-d od ecyl)-2,5-a n h yd r o-D-glu cit yl P h os-
p h on a te (15e). Phosphonate 33e (132 mg, 0.159 mmol) was
hydrogenated as described for 33a . Purification of the product
was done as outlined for 15a to provide 15e (51 mg, 68%) as
an off-white solid.
1-(Bu tyl-4′-O-h exa d ecyl)-2,5-a n h yd r o-D-glu cityl P h os-
p h on a te (15f). Phosphonate 33f (122 mg, 0.138 mmol) was
hydrogenated as described for 33a . Purification of the product
was done as outlined for 15a (except that a gradient of CH2-
Cl2 f CH2Cl2:CH3OH, 5:1 was used in the chromatography)
to provide 15f (38 mg, 53%) as an off-white solid.
1-(Allylben zyl)-3,4,6-tr i-O-ben zyl-2,5-a n h yd r o-D-glu ci-
tyl P h osp h on a te (16). Acyclic phosphonate 24 (638 mg, 0.987
mmol) was dissolved in THF (12 mL) and stirred before Ph3P
(311 mg, 1.19 mmol) and diisopropylazodicarboxylate (0.39 mL,
1.97 mmol) were added. The reaction mixture was stirred for
2 h and then the solvent was evaporated to give a clear yellow
residue that was purified by chromatography (2:1, hexane:
EtOAc) to yield 16 (547 mg, 90%) as a clear oil.
cis-2-Bu ten yl-1,4-d ih ep tyl Eth er (17a ). Sodium hydride
(874 mg, 36.4 mmol) was added to a solution of cis-2-buten-
1,4-diol (30, 803 mg, 9.1 mmol) in DMF (20 mL). After ∼10
min, 1-iodoheptane (6.0 mL, 36.4 mmol) was added and the
reaction mixture was stirred for 1 h. Water (20 mL) was added
to dissolve the salt precipitate and the solution was diluted
with hexane (75 mL). The organic layer was separated, washed
with brine (20 mL), and dried over Na2SO4. After the solvent
was evaporated, the yellowish residue was purified by chro-
matography (hexane f 6:1, hexane:EtOAc) to afford cis-2-
butenyl-1,4-diheptyl ether (2.48 g, 96%) as a clear liquid.
8868 J . Org. Chem., Vol. 67, No. 25, 2002

C-Phosphonate Analogues of Decaprenolphosphoarabinose
1-(Allylben zyl)-3,4,6-tr i-O-ben zyl-2,5-O-ben zylid en e-D-
glu cityl P h osp h on a te (28A) a n d 1-(Allylben zyl)-3,4,6-tr i-
O-b en zyl-2,5-O-b en zylid en e-D-m a n n it yl P h osp h on a t e
(28B). Sodium borohydride (963 mg, 25.5 mmol) was added
to a solution of lactol 26 (13.7 g, 21.2 mmol) in THF (200 mL)
and the reaction mixture was stirred for 2 h. Upon completion,
the reaction mixture was neutralized with AcOH, diluted with
EtOAc (100 mL), and washed with water (100 mL) followed
by brine (100 mL). The organic layer was dried over Na2SO4
and the solvent was evaporated affording a crude reduction
product (14.6 g, 87%), which was a 2:1 mixture of 24 and 27.
This crude material was used in the next step without any
further purification. Benzaldehyde dimethyl acetal (9.5 mL,
63.6 mmol) and p-toluenesulfonic acid (193 mg, 1.01 mmol)
were added to a solution of the crude diol in CH3CN (60 mL)
and the mixture stirred overnight. The solvent was evaporated
and the residue, which was a mixture of 4 diastereomers, was
purified by chromatography (hexane:EtOAc, 1:1) to afford the
major diasteromers 28A (5.9 g, 38% over both steps) and 28B
(5.8 g, 37% over both steps) as clear oils. It was possible to
obtain more of these products by resubjecting the minor
diastereomers and mixed fractions from the column to the
reaction.
clear oil. The product was a mixture of cis and trans isom-
ers.
1-[Ben zyl((E)-2′-b u t en yl-4′-O-h exa d ecyl)]-3,4,6-t r i-O-
ben zyl-2,5-a n h yd r o-D-glu cityl P h osp h on a te (32f). The
preparation of 32f was achieved by the reaction of 16 (218 mg,
0.347 mmol) and 17f (746 mg, 1.39 mmol) as described for the
preparation of 32a . The product was purified by chromatog-
raphy (2:1, hexane:EtOAc) to afford 32c (188 mg, 62%) as a
clear oil. The product was a mixture of cis and trans iso-
mers.
1-[Ben zyl(bu tyl-4′-O-h ep tyl)]-3,4,6-tr i-O-ben zyl-2,5-a n -
h yd r o-D-glu cityl P h osp h on a te (33a ). Dipotassium azodi-
carboxylate (DAPA, 20 mg, 0.123 mmol) was added to a
solution of 32a (149 mg, 0.197 mmol) in methanol (10 mL).
Glacial HOAc (10 µL) was added and the reaction mixture was
heated to 40 °C open to the atmosphere. As the yellow color of
the solution started to fade, more DAPA (20 mg) and HOAc
(10 µL) were added. This addition procedure was repeated
several times over the course of 10 h, and the reaction progress
was monitored by NMR. Upon completion of the reaction, the
reaction mixture was cooled and then a saturated aqueous
solution of NaHCO3 (15 mL) was added. The product was then
extracted into CH2Cl2 (3 × 25 mL) and the organic layer dried
over Na2SO4. After evaporation of the solvent, the clear residue
was purified by chromatography (2:1, hexane:EtOAc) to give
33a (130 mg, 87%) as a clear oil.
1-(Allylben zyl)-3,4,6-tr i-O-ben zyl-2,5-a n h yd r o-D-m a n -
n ityl P h osp h on a te (29). Acyclic phosphonate 27 (240 mg,
0.371 mmol) was dissolved in THF (5 mL) and stirred.
Triphenylphosphine (107 mg, 0.408 mmol) was added, followed
by diisopropyl azodicarboxylate (0.15 mL, 0.742 mmol), and
the reaction mixture was stirred for 2 h. The solvent was then
evaporated to give a clear yellow residue that was purified by
chromatography (1:1, hexane:EtOAc) to afford 29 (168 mg,
72%) as a clear oil.
1-[Ben zyl(b u t yl-4′-O-oct yl)]-3,4,6-t r i-O-b en zyl-2,5-a n -
h yd r o-D-glu cityl P h osp h on a te (33b). Alkene 32b (149 mg,
0.193 mmol) was converted to 33b as described for the
preparation of 33a . Purification by chromatography (2:1,
hexane:EtOAc) afforded the product (132 mg, 89%) as a clear
oil.
1-[Ben zyl(bu tyl-4′-O-n on yl)]-3,4,6-tr i-O-ben zyl-2,5-a n -
h yd r o-D-glu cityl P h osp h on a te (33c). Alkene 32c (135 mg,
0.172 mmol) was converted to 33c as described for the
preparation of 33a . Purification by chromatography (2:1,
hexane:EtOAc) afforded the product (100 mg, 74%) as a clear
oil.
1-[Ben zyl(b u t yl-4′-O-d ecyl)]-3,4,6-t r i-O-b en zyl-2,5-a n -
h yd r o-D-glu cityl P h osp h on a te (33d ). Alkene 32d (143 mg,
0.179 mmol) was converted to 33d as described for the
preparation of 33a . Purification by chromatography (2:1,
hexane:EtOAc) afforded the product (122 mg, 85%) as a clear
oil.
1-[Ben zyl((E)-2′-bu ten yl-4′-O-h eptyl)]-3,4,6-tr i-O-ben zyl-
2,5-a n h yd r o-D-glu cityl P h osp h on a te (32a ). Alkene 17a
(366 mg, 1.29 mmol) was added to a solution of 16 (269 mg,
0.43 mmol) in dry CH2Cl2 (10 mL). The Grubbs’ catalyst 3128
(70 mg, 20 mol %) was added and the reaction mixture was
heated at reflux for 3 h. After the mixture was cooled to room
temperature, Pb(OAc)4 (114 mg, 0.26 mmol) was added and
the mixture was stirred overnight. The solvent was evaporated
and the black residue was purified by chromatography (2:1,
hexane:EtOAc) to give 32a (179 mg, 55%) as a clear oil. The
product was a mixture of cis and trans isomers.
1-[Ben zyl((E)-2′-bu ten yl-4′-O-octyl)]-3,4,6-tr i-O-ben zyl-
2,5-a n h yd r o-D-glu cityl P h osp h on a te (32b). The prepara-
tion of 32b was achieved by the reaction of 16 (210 mg, 0.334
mmol) and 17b (313 mg, 1.00 mmol) as described for the
preparation of 32a . The product was purified by chromatog-
raphy (2:1, hexane:EtOAc) to afford 32b (163 mg, 63%) as a
clear oil. The product was a mixture of cis and trans isomers.
1-[Ben zyl(b u t yl-4′-O-d od ecyl)]-3,4,6-t r i-O-b en zyl-2,5-
a n h yd r o-D-glu cityl P h osp h on a te (33e). Alkene 32e (175
mg, 0.212 mmol) was converted to 33e as described for the
preparation of 33a . Purification by chromatography (2:1,
hexane:EtOAc) affords the product (150 mg, 86%) as a clear
oil.
1-[Ben zyl(bu tyl-4′-O-h exa d ecyl)]-3,4,6-tr i-O-ben zyl-2,5-
a n h yd r o-D-glu cityl P h osp h on a te (33f). Alkene 32f (158 mg,
0.179 mmol) was converted to 33f as described for the
preparation of 33a . Purification by chromatography (2:1,
hexane:EtOAc) afforded the product (150 mg, 95%) as a clear
oil.
Mea su r em en t of An titu ber cu losis Activity of 15a -f.
Measurement of the antituberculosis activity of the target
compounds was carried out as previously reported using the
fluorescence-based Alamar Blue Microplate assay.32 All com-
pounds were initially tested against Mycobacterium tubercu-
losis strain H37Rv (ATCC 27294) at a concentration of 6.25
µg/mL. At this concentration, only 15f inhibited the growth of
the bacteria. The MIC for 15f was determined by testing this
compound at lower concentrations of the compound; the MIC
is defined as the lowest concentration producing a 90%
reduction in fluorescence relative to controls.
1-[Ben zyl((E)-2′-bu ten yl-4′-O-n on yl)]-3,4,6-tr i-O-ben zyl-
2,5-a n h yd r o-D-glu cityl P h osp h on a te (32c). The prepara-
tion of 32c was achieved by the reaction of 16 (235 mg, 0.373
mmol) and 17c (508 mg, 1.49 mmol) as described for the
preparation of 32a . The product was purified by chromatog-
raphy (2:1, hexane:EtOAc) to afford 32c (168 mg, 57%) as a
clear oil. The product was a mixture of cis and trans isomers.
1-[Ben zyl((E)-2′-bu ten yl-4′-O-d ecyl)]-3,4,6-tr i-O-ben zyl-
2,5-a n h yd r o-D-glu cityl P h osp h on a te (32d ). The prepara-
tion of 32d was achieved by the reaction of 16 (268 mg, 0.426
mmol) and 17d (628 mg, 1.70 mmol) as described for the
preparation of 32a . The product was purified by chromatog-
raphy (2:1, hexane:EtOAc) to afford 32c (174 mg, 51%) as a
clear oil. The product was a mixture of cis and trans isomers.
1-[Ben zyl((E)-2′-bu ten yl-4′-O-d od ecyl)]-3,4,6-tr i-O-ben -
zyl-2,5-a n h yd r o-D-glu cityl P h osp h on a te (32e). The prepa-
ration of 32e was achieved by the reaction of 16 (237 mg, 0.377
mmol) and 17e (640 mg, 1.51 mmol) as described for the
preparation of 32a . The product was purified by chromatog-
raphy (2:1, hexane:EtOAc) to afford 32c (205 mg, 66%) as a
Ackn owledgm en t. The National Institutes of Health
(AI44045-01) supported this work. C.A.C. is a recipient
of a GAANN fellowship from the U.S. Department of
J . Org. Chem, Vol. 67, No. 25, 2002 8869

Centrone and Lowary
Su p p or tin g In for m a tion Ava ila ble: 1H, 13C, and 31P
NMR spectra for all new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
Education. Antimycobacterial data were provided by the
Tuberculosis Antimicrobial Acquisition and Coordinat-
ing Facility (TAACF) through a research and develop-
ment contract with the U.S. National Institute of
Allergy and Infectious Diseases.
J O026247R
8870 J . Org. Chem., Vol. 67, No. 25, 2002