Framework-reactive siderophore analogs as potential cell-selective drugs. Design and syntheses of trimelamol-based iron chelators

Article abstract of DOI:10.1021/jo9600621

Currently, the role of DNA-directed alkylating agents as potential anticancer/antimicrobial drugs is of wide interest. Most of the alkylating agents used clinically as drugs damage DNA in cells without specificity, and this can lead to undesired toxicity problems. Minimizing serum residence time by targeting the drug to select pathogens or organs might diminish the effects of nonselective reactivity. This paper describes the syntheses and preliminary studies of analogs of siderophores (microbial iron chelators) 2 and 20 that incorporate centers within the siderophore framework capable of generating potent electrophiles (iminium ions), hopefully after directed cellular recognition and uptake. Formation of N-aminals from trimelamol (3) and substituted hydroxamic acid 4 or 5 was critical for the design and synthesis of the targets. In preliminary biological testing, compound 2, a trimelamol-based siderophore analog, was active against Escherichia coli X580, illustrating the therapeutic potential of this new type of siderophore-mediated drug design and delivery.

Full text of DOI:10.1021/jo9600621

4120
J . Org. Chem. 1996, 61, 4120-4124
F r a m ew or k -Rea ctive Sid er op h or e An a logs a s P oten tia l
Cell-Selective Dr u gs. Design a n d Syn th eses of Tr im ela m ol-Ba sed
Ir on Ch ela tor s
Savithri Ramurthy and Marvin J . Miller*
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556
Received J anuary 9, 1996X
Currently, the role of DNA-directed alkylating agents as potential anticancer/ antimicrobial drugs
is of wide interest. Most of the alkylating agents used clinically as drugs damage DNA in cells
without specificity, and this can lead to undesired toxicity problems. Minimizing serum residence
time by targeting the drug to select pathogens or organs might diminish the effects of nonselective
reactivity. This paper describes the syntheses and preliminary studies of analogs of siderophores
(microbial iron chelators) 2 and 20 that incorporate centers within the siderophore framework
capable of generating potent electrophiles (iminium ions), hopefully after directed cellular recognition
and uptake. Formation of N-aminals from trimelamol (3) and substituted hydroxamic acid 4 or 5
was critical for the design and synthesis of the targets. In preliminary biological testing, compound
2, a trimelamol-based siderophore analog, was active against Escherichia coli X580, illustrating
the therapeutic potential of this new type of siderophore-mediated drug design and delivery.
In tr od u ction
Drugs that might be useful in vivo are often therapeu-
tically ineffective because of their inability to permeate
a targeted cell. One possible solution to this problem is
conjugation of drugs to siderophores1 to promote active
transport of drugs into specific cells2 (Figure 1). Sidero-
phores are low molecular weight, organism selective,
iron-sequestering agents. They tightly bind and solubi-
lize ionic forms of iron and are recognized and actively
transported into cells where, by modification, iron is
released for intracellular use.2 Microbes that recognize
the iron-binding component of properly designed sidero-
phore-drug conjugates as an iron delivery agent as-
similate the conjugate and in effect commit suicide, since
the attached drug is lethal to them. This alternate mode
of drug delivery may lead to the development of a whole
new class of microbe-selective drugs on the basis of the
active transport of an essential nutrient. Siderophore
conjugation also may rejuvenate known drugs which have
previously relied on passive diffusion and to which
resistance has now developed. Our laboratory has com-
pleted the first total synthesis of various siderophores
including aerobactin,3 arthrobactin,4 schizokinen,5 my-
cobactin,6 and foroxymithine,7 and all of the components
of pseudobactin and several analogs.8 The existence of
albomycin,9 salmycin10 and related natural compounds
that incorporate both a siderophore component and a
toxic agent prompted attempts to prepare mimics by
conjugation of siderophores directly to antimicrobial
F igu r e 1.
F igu r e 2.
agents.10 Recently, an extensive research program has
been developed in our laboratory toward the design and
synthesis of siderophores attached to a variety of drugs,
including, for example, carbacephalosporins, a potent
class of â-lactam antibiotics.11,12 One carbacephalosporin
adduct with the siderophore component in the form of
hydroxamates is shown in Figure 2. Detailed biological
studies of this first generation siderophore-drug conju-
gate demonstrated the feasibility of microbial iron trans-
port-mediated drug delivery.11 Herein, we describe the
design, syntheses, and preliminary studies of a poten-
tially new class of siderophore-based antimicrobial agents
that, rather than containing a separate known drug,
incorporate within their framework functionality capable
of generating reactive species (iminium ions in this case),
hopefully after microbial recognition and assimilation.
Intracellular reaction of the delivered and generated
reactive agents are anticipated to be lethal to the
undesired and targeted cell, thus effecting the develop-
ment of new types of potential drugs.
X Abstract published in Advance ACS Abstracts, May 1, 1996.
(1) Winkelman, G. Handbook of Microbial Iron Chelates; CRC: Boca
Raton, FL, 1991.
(2) Hider, R. C. Struct. Bonding (Berlin) 1990, 58, 25.
(3) Maurer, P. J .; Miller, M. J . J . Am. Chem. Soc. 1982, 104, 3096.
(4) Lee, B. H.; Miller, M. J . J . Org. Chem. 1983, 48, 24.
(5) Maurer, P. J .; Miller, M. J . J . Org. Chem. 1981, 46, 2835.
(6) Maurer, P. J .; Miller, M. J . J . Am. Chem. Soc. 1983, 105, 240.
(7) Dolence, E. K.; Miller, M. J . J . Org. Chem. 1991, 56, 492.
(8) (a) Kolasa, T.; Miller, M. J . J . Org. Chem. 1990, 55, 4246. (b)
Kolasa, T.; Miller, M. J . J . Org. Chem. 1990, 55, 1711. (c) Okonya, J .
F.; Kolasa, T.; Miller, M. J . J . Org. Chem. 1995, 60, 1932.
(9) Benz, G.; Schroder, T.; Kurz, J .; Wunsche, C.; Karl, W.; Steffens,
G.; Pfitzner, J .; Schmidt. D. Angew. Chem., Int. Ed. Engl. 1982, 21,
527.
The design of our first framework-reactive siderophore
analogs (1 and 2) was based on trimelamol (3). Tri-
(10) Ve´rtesy, L.; Aretz, W.; Fehlaber, H.; Kogler. H. Helv. Chim. Acta
1995, 78, 46.
(11) Malouin, F.; Miller, M. J . Acc. Chem. Res. 1993, 26, 241.
(12) Ghosh, M.; Miller, M. J . J . Org. Chem. 1994, 59, 1020.
S0022-3263(96)00062-X CCC: $12.00 © 1996 American Chemical Society

Trimelamol-Based Iron Chelators
J . Org. Chem., Vol. 61, No. 12, 1996 4121
Sch em e 1
F igu r e 3.
F igu r e 4.
melamol is a nitrogen mustard that is used extensively
in cancer chemotherapy.13 The antitumor activity of the
nitrogen mustards is similar to alkylating agents which
have the ability to cross-link the two strands of DNA.
Mattes et al. have shown that the nitrogen mustard class
of alkylating agents alkylate the most negative site (the
N7 position) of guanine.14 As mentioned earlier, most of
the alkylating agents used clinically damage DNA in cells
without specificity.15 In our case, it is expected that after
rapid active-transport-facilitated assimilation, the tri-
melamol-like framework of the siderophore analog may
generate a highly reactive iminium ion, which might
react as an alkylating agent within the targeted cell and
cause damage (Figure 3). Another reason for choosing
trimelamol as the core structure was that it resembles
cyanurates, and we have previously shown that synthetic
cyanurate-based siderophore analogs (Figure 4) are
remarkably effective microbial iron transport agents that
are recognized and assimilated by a number of mi-
crobes.16,17 As shown in Scheme 1, the key to the
synthesis of our desired trimelamol-containing sidero-
phore analogs (1 and 2) involved coupling trimelamol (3)
to the appropriately substituted hydroxamates 4 or 5.
Our first objective was construction of the substituted
hydroxylamines 4 and 5 which were needed for attach-
ment of trimelamol through an N-aminal linkage to give
forms of 1 and 2. The reaction sequence for the conver-
sion of N-((trichloroethoxy)carbonyl-O-benzylhydroxyl-
amine (9)16 to the substituted hydroxamate 4 is repre-
sented in Scheme 2. 3-Bromopropanol (10) was silylated
to provide 7. The bromo group was subsequently dis-
placed by the protected hydroxylamine 9 to afford the
substituted hydroxamate 11 in 45% yield. Interestingly,
attempted removal of the silyl group in the presence of
tetrabutylammonium fluoride (TBAF)18 in THF resulted
in the loss of the N-(trichloroethoxy)carbonyl (Troc) group
Sch em e 2
as well. At this stage, as we eventually wanted to
incorporate an N-acetyl group to mimic the hydroxamate
portion of many natural siderophores, we tried reductive
acetylation of 11 in the presence of zinc, acetic anhydride,
and acetic acid and obtained the desired fully protected
hydroxamate 12.19 Subsequent removal of the TBDMS
group provided the corresponding free alcohol 4 in 86%
yield.
The synthesis of homologous fragment 5 started with
commercially available 4-bromobutyl acetate (13) (Scheme
3). N-Troc-O-benzylhydroxylamine (9) was alkylated
with 13 in the presence of sodium hydride in DMF to
provide the desired hydroxamino acetate 14 in 66% yield.
Again, the Troc group was removed using zinc and acetic
acid in the presence of acetic anhydride to afford 15 in
93% yield. Finally, the O-acetyl group was removed by
subjecting the compound to hydrolysis in aqueous metha-
nol and K2CO3 to provide alcohol 5.
(13) Cumber, A. J .; Ross, W. C. J . Chem. Biol. Interact. 1977, 17,
349.
(14) Mattes, W. B.; Hartley, J . A.; Kohn, K. W. Nucleic Acids Res.
1986, 14, 2971.
(15) Gibson, N. W. In Cancer Chemotherapy: Concepts, Clinical
Investigation and Therapeutic Advances; Muggia, F. M., Ed.; Kluwer
Academic: Boston, 1988.
(16) Lee, B. H.; Miller, M. J .; Prody, C. A. J . Med. Chem. 1985, 28,
323.
(17) Neilands, J . B.; Altin, C. L. Biochemistry 1968, 7, 734.
(18) Corey, E. J .; Venkateswaralu, A. J . Am. Chem. Soc. 1972, 94,
6190.
(19) J ust, G.; Grozinger, K. Synthesis 1976, 457.

4122 J . Org. Chem., Vol. 61, No. 12, 1996
Ramurthy and Miller
Sch em e 3
Sch em e 6
Sch em e 4
each was attempted. Treatment of 18 with 10% Pd/C in
methanol under a hydrogen atmosphere resulted in rapid
deprotection. However, the resulting hydroxamic acid 1
was unstable and decomposition to the corresponding
alcohol and trimelamol dominated, perhaps indicating
that designed hydroxamic acid assisted intramolecular
cleavage of the aminal to generate an iminium ion
(Figure 3) was too facile in this case. Two solutions to
this problem were then considered: first, development
of a prodrug analog to delay release of the free hydrox-
amate and, second, alteration of the chain length (n in
Figure 2) between the hydroxamate and the trimelamol
unit. Thus, we turned our attention to the synthesis of
peracetylated prodrug 20 rather than the free hydrox-
amic acids. Catalytic reduction with hydrogen and 10%
Pd/C in ethyl acetate containing acetic anhydride and
sodium acetate provided the desired product 20 in 82%
yield (Scheme 6). In contrast, we did not encounter any
problem in the removal of the benzyl group from the
chain-extended analog 19 during hydrogenolysis in ethyl
acetate and deprotected siderophore 2 was obtained in
85% yield. Thus, the first total syntheses of trimelamol
siderophore analogs were achieved.
Sch em e 5
With protected hydroxamates 4 and 5 in hand, we
focused our attention on the synthesis of trimelamol (3)
(Scheme 4). The procedure described by Cumber et al.13
was slightly modified. Cyanuric chloride was allowed to
react with methylamine hydrochloride in an autoclave
with diisopropylethylamine in a mixture of water and
dioxane at 95 °C to afford 6 in 62% yield. Next, 6 was
condensed with formaldehyde to provide trimelamol (3)
in 87% yield.
After the successful synthesis of these subunits, we
encountered some problems during their attempted
elaboration. The condensation reaction between 3 and
4 or between 3 and 5 was complicated by the formation
of not only the mixture of mono- and disubstituted
trimelamol deriviatives in low yields but also unreacted
starting materials. After considerable investigation, the
desired trisubstituted, protected condensation products
(18 and 19) were obtained by the simple reaction of 3
with alcohols 4 and 5, respectively, for 3 days in DMF in
the presence of catalytic p-toluenesulfonic acid (Scheme
5).
Each of the final deprotected synthetic siderophore
analogs 2 and 20 has been subjected to antimicrobial
activity assays with Escherichia coli X580 and Candida
albicans in Luria broth cultures. Compound 2 was
active, showing considerable delay in growth similar to
our previous siderophore conjugates,11 against E. coli
X580 at a concentration of 5 µM, providing preliminary
indication that its iron complex is recognized by outer
membrane receptors and transported into the cell where
it either exerts toxic action or reacts during the transport
process by generation of electrophilic iminium ions as
designed. As a result of this promising preliminary
bioassay, the siderophore analogs have been submitted
for detailed broad spectrum screening, which will be
reported later.
Exp er im en ta l Section
After preparation of the protected forms (18 and 19)
of 1 and 2, reductive removal of the benzyl groups from
Instruments and general methods used have been described
earlier.20 Solvents used were dried and purified by standard

Trimelamol-Based Iron Chelators
J . Org. Chem., Vol. 61, No. 12, 1996 4123
methods.21 The term “dried” refers to the drying of an organic
layer over magnesium or sodium sulfate. All reactions were
performed under a nitrogen or argon atmosphere. The purity
of the final compounds was confirmed by using the standard
HPLC analysis.
stirred overnight at rt. The excess volatiles were removed in
vacuo. Purification by chromatography on silica gel using
ethyl acetate (Rf ) 0.3) provided 0.80 g of substituted alcohol
4 (86%) as a clear oil: IR (neat) 3400, 2960, 1650, 1400, 1100
1
cm-1; H NMR (500 MHz, CDCl3) δ 7.39 (s, 5H), 4.82 (s, 2H),
3.77-3.73 (t, J ) 6.3 Hz, 2H), 3.54-3.49, (t, J ) 6.0 Hz, 2H),
3.00 (br s, 1H), 2.00 (s, 3H), 1.82-1.74 (quintet, J ) 6.0 Hz ,
2H); 13C NMR (125 MHz, CDCl3) δ 172.8, 133.9, 128.9, 128.7,
1-((ter t-Bu tyld im eth ylsilyl)oxy)-3-br om op r op a n e (7).
To a solution of 3-bromopropanol (10, 2.50 g, 17.9 mmol) in
methylene chloride (100 mL) were added tert-butyldimethyl-
silyl chloride (2.90 g, 19.8 mmol), 4-(N,N- dimethylamino)-
pyridine (DMAP) (1.10 g, 8.90 mmol), and triethylamine (1.90
mL, 19.8 mmol). The reaction was stirred overnight at rt and
quenched with a saturated ammonium chloride solution (15
mL). The organic layer was separated, washed with brine (15
mL), sodium bicarbonate (15 mL), and water (15 mL), dried,
filtered, and concentrated in vacuo. The crude product was
subjected to column chromatography on silica gel using 2:1
hexanes/ethyl acetate (Rf ) 0.8) to afford 3.00 g (66%) of
substituted alkane 7 as a colorless oil: IR (neat) 2960, 2860,
128.4, 76.0, 58.3, 41.6, 29.5, 19.9; HRMS (FAB) calcd for C12H17
-
NO3 (MH+) 224.1287, found 224.1290. Anal. Calcd for
C12H18NO3: C, 64.55; H, 7.67; N, 6.27. Found: C, 64.35; H,
7.5; N, 6.23.
4-[((Tr ich lor oet h oxy)ca r b on yl)(b en zyloxy)a m in o]-1-
a cetoxybu ta n e (14). Sodium hydride (0.240 g, 60% disper-
sion in mineral oil, 5.8 mmol) was washed with dry hexanes
(2 × 2 mL) and suspended in DMF (5 mL). A solution of
N-((trichloroethoxy)carbonyl)-O-benzylhydroxylamine (9, 1.30
g, 4.50 mmol) in DMF (5 mL) was added to the mixture at 0
°C, and the solution was stirred for 45 min at rt. 4-Bromobutyl
acetate (13, 1.10g, 5.80 mmol) in DMF (5 mL) was added to
the reaction mixture at 0 °C, and the solution was stirred
overnight at rt. Cold water was added to the mixture, and
the solution was extracted with ethyl acetate (3 × 25 mL). The
combined organic extracts were washed with brine (5 mL) and
water (5 mL), dried, filtered, and evaporated. Column chro-
matography of the crude residue on silica gel using 6:1
hexanes/ether (Rf ) 0.55 in 4:1 hexanes/ether) afforded 1.20 g
(67%) of substituted hydroxamate 14 as a clear oil: IR (neat)
1
1250 cm-1; H NMR (300 MHz, CDCl3) δ 3.63 (t, J ) 5.5 Hz,
2H), 3.41 (t, J ) 6.0 Hz, 2H), 1.9 (m, 2H), 0.79 (s, 9H), 0.03 (s,
6H); 13C NMR (80 MHz, CDCl3) δ 35.4, 30.5, 30.1, 25.70, 18.1;
HRMS (FAB) calcd for C19H31NO4SiCl3 (MH+) 254.7032, found
254.7038.
3-[((Tr ich lor oet h oxy)ca r b on yl)(b en zyloxy)a m in o]-1-
((ter t-bu tyld im eth ylsilyl)oxy)p r op a n e (11). Sodium hy-
dride (250 mg, 60% dispersion in mineral oil, 5.9 mmol) was
washed with dry hexanes (2 × 2 mL) and suspended in DMF
(5 mL). A solution of N-((trichloroethoxy)carbonyl)-O-benzyl-
hydroxylamine (9,16 1.30 g, 4.50 mmol) in DMF (5 mL) was
added to the mixture at 0 °C, and the solution was stirred for
45 min at rt. 1-((tert-Butyldimethylsilyl)oxy)-3-bromopropane
(7, 1.50 g, 5.90 mmol) in DMF (5 mL) was added to the reaction
mixture at 0 °C, and the solution was stirred overnight at rt.
Cold water was added to the mixture, and the resulting
solution was extracted with ethyl acetate (3 × 25 mL). The
combined organic extracts were washed with water, dried,
filtered, and evaporated. Column chromatography of the crude
residue on silica gel using 6:1 hexanes/ether (Rf ) 0.7 in 2:1
hexanes/ether) afforded 0.964 g (45%) of TBDMS-protected
hydroxamate 11 as a clear oil: IR (neat) 2940, 2860, 1720,
1250, 1100 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.45-7.35 (m,
5H), 4.90 (s, 2H), 4.84 (s, 2H), 3.67 (t, J ) 6.0 Hz, 2H), 3.64 (t,
J ) 7.0 Hz, 2H), 1.87 (quintet, J ) 7.5 Hz, 2H), 0.91 (s, 9H),
0.08 (s, 3H), 0.04 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 155.0,
134.8, 129.43, 128.7, 128.4, 95.0, 74.9, 60.2, 47.1, 30.1, 25.78,
18.2; HRMS (FAB) calcd for C19H31NO4SiCl3 (MH+) 470.2016,
found 470.2018.
1
2980, 1740, 1240 cm-1; H NMR (300 MHz, CDCl3) δ 7.40-
7.30 (m, 5H), 4.90 (s, 2H), 4.80 (s, 2H), 4.06-4.01 (t, J ) 6.0
Hz, 2H), 3.54-3.50 (t, J ) 6.9 Hz, 2H), 2.02 (s, 3H), 1.73-
1.60 (m, 4H); 13C NMR (80 MHz, CDCl3) δ 171.0, 155.1, 134.8,
129.4, 128.8, 128.5, 95.2, 76.8, 75.1, 63.8, 49.3, 25.7, 23.5, 20.9;
HRMS (FAB) calcd for C16H21NO5NCl3 (MH+) 412.0485, found
412.0506. Anal. Calcd for C16H20NO5Cl3: C, 46.57; H, 4.88;
N, 3.39. Found: C, 46.39; H, 4.99; N, 3.3.
4-[Acetyl(ben zyloxy)a m in o]-1-a cetoxybu ta n e (15). To
a solution of 14 (1.20 g, 2.90 mmol) in THF (15 mL) and acetic
acid (10 mL) were added zinc dust (2.80 g, 43.6 mmol) and
acetic anhydride (4.1 mL, 43.6 mmol). The mixture was stirred
for 2 h at rt and diluted with THF (40 mL). The reaction
mixture was filtered, and the excess volatiles were removed
in vacuo. The mixture was taken up in ethyl acetate (50 mL),
washed with a saturated sodium bicarbonate solution (3 × 10
mL), brine (15 mL), and water (15 mL), dried, filtered,
evaporated, and purified by column chromatography on silica
gel with 2:1 hexanes/ethyl acetate (Rf ) 0.2) to provide 0.750
g (93%) of protected hydroxamate 15 as a clear oil: 1H NMR
(300 MHz, CDCl3) δ 7.40-7.30 (m, 5H), 4.80 (s, 2H), 4.07-
4.03 (t, J ) 6.3 Hz, 2H), 3.68-3.64 (t, J ) 6.6 Hz, 3H), 2.10 (s,
3H), 2.02 (s, 3H), 1.70-1.61(m, 4H); 13C NMR (80 MHz, CDCl3)
δ 172.2, 170.9, 134.3, 128.9, 128.6, 76.1, 63.7, 44.8, 25.7, 23.3,
3-[Acetyl(ben zyloxy)a m in o]-1-((t-bu tyld im eth ylsilyl)-
oxy)p r op a n e (12). To a solution of 11 (0.195 g, 0.414 mmol)
in THF (2 mL) and acetic acid (2 mL) were added zinc dust
(0.405 g, 6.2 mmol) and acetic anhydride (0.585 mL, 6.2 mmol).
The mixture was stirred for 2 h at rt and diluted with THF
(10 mL). The reaction mixture was filtered, and the excess
volatiles were removed in vacuo. The mixture was taken up
in ethyl acetate (20 mL), washed with a saturated sodium
bicarbonate solution (3 × 10 mL), water (5 mL), and brine (5
mL), dried, filtered, evaporated, and purified by chromatog-
raphy on silica gel with 4:1 hexanes/ethyl acetate (Rf ) 0.5 in
2:1 hexanes/ethyl acetate) to provide hydroxamate 12 as an
20.7, 20.3; MS (CI) m/ z 280 (MH+). Anal. Calcd for C15H21
-
NO4: C, 64.50; H, 7.58; N, 5.01. Found: C, 64.36; H, 7.39; N,
4.99.
4-[Acetyl(ben zyloxy)a m in o]-1-bu ta n ol (5). 4-[Acetyl-
(benzyloxy)amino]-1-acetoxybutane (15, 0.600 g, 2.15 mmol)
was dissolved in a mixture of methanol (7 mL) and water (1
mL). A potassium carbonate solution (1 N, 4 mL, 4.30 mmol)
was added, and the solution was stirred overnight at rt. The
methanol was removed. The resulting aqueous solution was
acidified to pH ) 2 with 1 N HCl and extracted with ethyl
acetate (3 × 20 mL). The combined organic extracts were
washed with water (5 mL) and brine (5 mL), dried, and
evaporated to afford 0.462 g (92%) (Rf ) 0.2 in ethyl acetate)
of substituted alcohol 5 as a clear oil: 1H NMR (300 MHz,
CDCl3) δ 7.29 (m, 5H), 4.70 (s, 2H), 3.59 (br s, 1H), 3.55-3.50
(t, J ) 6.6 Hz, 4H), 1.99 (s, 3H), 1.70-1.60 (quintet, J ) 6.9
Hz, 2H), 1.51-1.42 (quintet, J ) 6.6 Hz, 2H); 13C NMR (80
MHz, CDCl3) δ 172.3, 134.3, 129.1, 128.3, 128.2, 76.15, 61.7,
45.0, 29.6, 23.4, 20.3; HRMS (FAB) calcd for C13H20NO3 (MH+)
238.1443, found 238.1440. Anal. Calcd for C13H19NO3: C,
65.80; H, 8.07; N, 5.90. Found: C,65.96; H, 7.94; N, 6.04.
N2,N4,N6-Tr im eth ylm ela m in e (6). To a mixture of cya-
nuric chloride (16, 2.00 g, 10.8 mmol) and methylamine
hydrochloride (17, 5.90 g, 86.7 mmol) in a mixture of dioxane
oil: IR (neat) 2970, 1660, 1100 cm-1 1H NMR (500 MHz,
;
CDCl3) δ 7.33-7.29 (m, 5H), 4.81 (s, 2H), 3.73-3.70 (t, J )
6.0 Hz, 2H), 2.07 (s, 3H), 1.84-1.80 (m, 2H), 0.86 (s, 9H), 0.02
(s, 6H); 13C NMR (125 MHz, CDCl3) δ 170.8, 133.1, 129.4,
128.9, 128.7, 76.7, 58.1, 47.0, 40.9, 29.5, 29.3, 19.8, 18.0; MS
(CI) m/ z 290 (MH+). Anal. Calcd for C18H34O3NSi: C, 63.48;
H, 10.06; N, 4.11. Found: C, 63.29; H, 9.88; N, 4.11.
3-[Acetyl(ben zyloxy)a m in o]-1-p r op a n ol (4). 3-[Acetyl-
(benzyloxy)amino]-1-((t-butyldimethylsilyl)oxy)propane (12, 0.139
g, 0.414 mmol) was dissolved in THF (5 mL). Tetrabutylam-
monium fluoride (TBAF) in THF (1.24 mL of 1 M solution,
1.24 mmol) was added to the mixture, and the solution was
(20) Teng, M.; Miller, M. J . J . Am. Chem. Soc. 1993, 115, 548
(21) Gordon, A. J .; Ford, R. A. The Chemist’s Companionsa Hand-
book of Practical Data, Techniques, and References; J ohn Wiley and
Sons: New York, 1972; p 408.

4124 J . Org. Chem., Vol. 61, No. 12, 1996
Ramurthy and Miller
(100 mL) and water (10 mL) was added diisopropylethylamine
(24.0 mL, 130 mmol). The mixture was heated at 95 °C in a
ZipperClave autoclave for 8 h and stirred overnight at rt.
Excess volatiles were removed in vacuo, and the residue was
diluted with ethyl acetate (200 mL). The mixture was washed
with brine (50 mL) and water (50 mL). The separated organic
layer was dried and concentrated in vacuo. Column chroma-
tography of the crude on silica gel with 2:1 hexanes/ethyl
acetate provided 1.12 g of 616 (62%) as a pale yellow solid: 1H
NMR (500 MHz, CDCl3) δ 5.40-6.00 (br s, 3H), 2.84 (s, 9H);
13C NMR (125 MHz, CDCl3) δ 166.2, 28.0; HRMS (EI) m/ z
calcd for C6H12N6: 168.1123, found 168.1119.
6H), 3.15-2.90 (br s, 9H), 2.10 (s, 9H), 2.05 (br s, 9H), 1.88-
1.82 (br s, 6H); 13C NMR (125 MHz, CDCl3) δ 170.9, 168.4,
166.0, 77.9, 65.4, 45.4, 33.1, 27.3, 20.2, 18.4; HRMS (FAB) calcd
for C30H51N9O12Na (M + Na) 752.3555, Found 752.3585.
Tr is(N-((((N′-a cetyl-O-ben zylh yd r oxa m in o)bu tyl)oxy)-
m eth yl-N-m eth yla m in o) Cya n u r a te (19). To a solution of
trimelamol (3, 0.040 g, 0.149 mmol) in DMF (2 mL) were added
5 (0.100 g, 0.448 mmol), p-toluenesulfonic acid (0.024 g, 0.149
mmol), and 4 Å molecular sieves (0.100 g), and the reaction
was stirred for 48 h at rt. The mixture was diluted with ethyl
acetate (20 mL), filtered, washed with sodium bicarbonate
solution (10%, 15 mL), water (10 mL), and brine (10 mL), and
dried. The volatiles were removed in vacuo, and the residue
was chromatographed on silica gel with 5:2.5:1 methylene
chloride/ethyl acetate/ethanol (Rf ) 0.5) to provide 0.055 g of
19 (40%) as a clear oil: 1H NMR (500 MHz, CDCl3) δ 7.29 (s,
15H), 5.15-5.00 (br s, 6H), 4.80 (s, 6H), 3.72-3.52 (br s, 6H),
3.55-3.40 (br s, 6H), 3.15-2.90 (br s, 9H), 2.05 (s, 9H), 1.88-
1.82 (br s, 12H); 13C NMR (125 MHz, CDCl3) δ 171.9, 165.8,
134.2, 129.2, 128.8, 128.6, 75.9, 66.9, 44.9, 32.6, 23.6, 20.2;
HRMS (FAB) calcd for C48H70N9O9 (MH+) 916.5297, found
916.5295.
Tr im ela m ol
[N2,N4,N6-Tr im et h yl-N2,N4,N6-t r im et h -
ylolm ela m in e] (3). To a suspension of 6 (2.00 g, 11.8 mmol)
in potassium carbonate solution (0.200 g in 7 mL of water)
was added aqueous formaldehyde (33%, 3.2 mL). The mixture
was heated slightly for 5 min and was left stirring overnight
at rt. The resulting precipitate was collected by filtration,
washed with cold water, and dried to give 2.02 g of trimelamol
(3) (72%) as white crystals: mp 128-131 °C (lit.16 mp 128-
1
131 °C); H NMR (500 MHz, CD3OD) δ 5.11 (s, 6Η), 3.15 (s,
9Η); 13C NMR (125 MHz, CD3OD) δ 167.1, 72.8, 33.7; HRMS
(FAB) calcd for C9H19N6O3 (MH+) 259.1519, found 259.1511.
Tr is(N-((((N′-acetyl-O-ben zylh ydr oxam in o)pr opyl)oxy)-
m eth yl)-N-m eth yla m in o) Cya n u r a te (18). To a solution
of trimelamol (3, 0.040 g, 0.149 mmol) in DMF (2 mL) were
added 4 (0.100 g, 0.448 mmol) p-toluenesulfonic acid (0.024 g,
0.149 mmol), and 4 Å molecular sieves (0.100 g), and the
reaction was stirred for 48 h at rt. The mixture was diluted
with ethyl acetate (20 mL), filtered, washed with a sodium
bicarbonate solution (10%, 15 mL), water (10 mL), and brine
(10 mL), and dried. The volatiles were removed in vacuo. The
crude product was purified on silica gel by column chroma-
tography with 5:2.5:1 methylene chloride/ethyl acetate/ethanol
(Rf ) 0.6) to provide 0.060 g of protected siderophore 18 (46%)
Tr is(N-((((N′-a cet ylh yd r oxa m in o)b u t yl)oxy)m et h yl)-
N-m et h yla m in o) Cya n u r a t e (2). To a solution of tris(N-
((((N′-acetyl-O-benzylhydroxamino)butyl)oxy)methyl-N-meth-
ylamino) cyanurate (19, 0.018 g, 0.12 mmol) in ethyl acetate
(2 mL) were added powderd sodium acetate (0.075 g) and Pd/C
and Pd(OH)2 (0.002 g, 0.008 mmol) . The reaction was stirred
overnight at rt under an atmosphere of hydrogen (1 atm). The
reaction mixture was filtered through a pad of Celite, and the
volatiles were removed in vacuo to provide 0.008 g of 2 (85%)
as a clean oil. The reaction was reproducible on larger scale
(0.250 g). For 2: HPLC tR 7.5 min (15% 2-propanol in
1
methylene chloride); H NMR (500 MHz, CDCl3) δ 5.20-5.15
(br s, 6H), 3.72-3.52 (br s, 6H), 3.55-3.40 (br s, 6H), 3.15-
2.90 (br s, 9H), 2.05 (br s, 9H), 1.88-1.82 (br s, 12H); 13C NMR
(125 MHz, CDCl3) δ 168.0, 166.0, 77.9, 67.3, 47.3, 33.1, 27.3,
20.2, 18.4; MS (FAB) m/ z 664 (M + H2O).
as a clear oil: IR (neat) 2965, 2870, 1670, 1540, 1200 cm-1
;
1H NMR (500 MHz, CDCl3) δ 7.39-7.25 (s, 15H), 5.15-5.00
(br s, 6H), 3.72-3.52 (br s, 6H), 3.55-3.40 (br s, 6H), 3.15-
2.90 (br s, 9H), 2.05 (s, 9H), 1.88-1.82 (br s, 6H); 13C NMR
(125 MHz, CDCl3) δ 172.2, 166.0, 134.4, 129.1, 128.8, 128.6,
77.8, 76.1, 65.0, 42.8, 32.9, 27.2, 20.5; HRMS (FAB) calcd for
C45H64N9O9 (MH+) 874.4827, found 874.4832.
Ack n ow led gm en t. We gratefully acknowledge sup-
port from NIH for this research and the Lizzadro
Magnetic Resonance Research Center at Notre Dame
for the NMR facilities. We also sincerely appreciate the
preliminary biological tests performed at Eli Lilly and
Co., Indianapolis, IN.
Tr is(N -((((N ′,O-d ia ce t ylh yd r oxa m in o)p r op yl)oxy)-
m eth yl)-N-m eth yla m in o) Cya n u r a te (20). To a solution
of 18 (0.020 g, 0.11 mmol) in ethyl acetate (2 mL) were added
acetic anhydride (0.5 mL), sodium acetate (0.250 g), and 10%
Pd/C (0.002 g, 0.016 mmol). The reaction mixture was stirred
at rt and atmospheric pressure under hydrogen for 4 h. The
catalyst was removed by filtration through a pad of Celite, and
the solvent was evaporated to afford 0.012 g of siderophore
analog 20 (95%) as a colorless oil. The reaction also was
reproducible on larger scale (0.250 g). For 20: HPLC tR 7.4
min (15% 2-propanol in methylene chloride); IR (neat) 3500,
Su p p or tin g In for m a tion Ava ila ble: HPLC traces of 2
and 20 and 1H and 13C NMR spectra of 2, 7, 11, 18, 19, and
20 (14 pages). This material is contained in libraries on
microfiche, immediately follows this article in the microfilm
version of the journal, and can be ordered from the ACS; see
any current masthead page for ordering information.
1
2970, 2870, 1670, 1540, 1400; H NMR (500 MHz, CDCl3) δ
5.15-5.00 (br s, 6H), 3.72-3.52 (br s, 6H), 3.55-3.40 (br s,
J O9600621