The preparation of a fully differentiated "multiwarhead" siderophore precursor

Article abstract of DOI:10.1021/jo026391c

A protected, fully differentiated siderophore analogue has been prepared so that "Trojan Horse"-like siderophore drug conjugates with different drugs can be synthesized. The key steps in the synthesis include controlled preparation of an unsymmetrical urea and its conversion to a fully differentiated isocyanurate by reaction with chloro(carbonyl) isocyanate.

Full text of DOI:10.1021/jo026391c

Th e P r ep a r a tion of a F u lly Differ en tia ted
“Mu ltiw a r h ea d ” Sid er op h or e P r ecu r sor
Aaron P. Murray and Marvin J . Miller*
University of Notre Dame, Department of Chemistry and
Biochemistry, 251 Nieuwland Science Hall,
Notre Dame, Indiana 46556
mmiller1@nd.edu
Received September 3, 2002
Abstr a ct: A protected, fully differentiated siderophore
analogue has been prepared so that “Trojan Horse”-like
siderophore drug conjugates with different drugs can be
synthesized. The key steps in the synthesis include con-
trolled preparation of an unsymmetrical urea and its conver-
sion to a fully differentiated isocyanurate by reaction with
chloro(carbonyl) isocyanate.
F IGURE 1. Rhodotorulic acid 1, a natural bis-hydroxamate
siderophore and synthetic trihydroxamate isocyanurate 2.
Specific drug delivery for bacterial and fungal infec-
tions is of considerable interest in this time of growing
resistance to modern chemotherapeutics.1 In particular,
our laboratories have been investigating the use of
“Trojan Horse”-like drug delivery agents,2 whereby known
antimicrobial agents are covalently attached to actively
transported, low molecular weight iron chelators, or
siderophores.2,3 The artificial siderophore 2, a trihydrox-
amate-containing isocyanurate, has been previously syn-
thesized by our research group and mimics the natural
siderophore rhodotorulic acid 1 (Figure 1), but with the
ability to bind iron(III) stoichiometrically.4 Isocyanurate
2 promoted growth of various strains of E. coli, thus
demonstrating its ability to substitute for naturally
occurring siderophores. Furthermore, over the course of
an iron excretion study in a rat model, isocyanurate 2
effectively treated iron overload while being completely
nontoxic.4a
Isocyanurate siderophore 2 was thought to be an ideal
candidate for “Trojan Horse” drug delivery because of its
selective microbial uptake4a and low mammalian toxicity.
By simply replacing the terminal acetyl moieties with
succinyl linkers, the opportunity of conjugating up to
three drugs exists. Toward this end, drug conjugates of
5-fluorouridine (5-FU) have been synthesized and pre-
liminary in vitro testing completed.4 The initial findings
F IGURE 2. Conceptual multiwarhead siderophore-drug
conjugate.
show drug conjugates containing three 5-FU molecules
are more efficient than mono 5-FU drug conjugates, even
at the same effective drug concentration.4d However,
when we embarked on preparing truly “multiwarhead”-
type conjugates containing two or more different drugs,
the need arose to differentiate the arms of the isocyanu-
rate scaffold (Figure 2). Previous methodologies devel-
oped in this laboratory were not compatible with such a
goal, so a new synthetic scheme was required.
Our previous syntheses4a-d of isocyanurate siderophore
drug conjugates did not allow facile differentiation of the
three side chains of the symmetrical isocyanurate. A
typical procedure involved attempted sequential removal
of the identical 2,2,2-trichloroethoxycarbonyl (Troc) groups
on the side chains of the scaffold. Unfortunately, this
method yielded only small quantities of mono-deprotected
material (∼10-15% yields, 75% based on recovered
starting material) and required careful chromatographic
isolation. To circumvent these problems, we designed a
new synthesis of the isocyanurate ring with each side
chain having a different protecting group. The key step
in this construction is the cyclization of chloro(carbonyl)
isocyanate with an appropriate unsymmetrical urea as
shown retrosynthetically in Scheme 1.5
(1) (a) Koshland, D. E., J r. Science 1994, 254, 327 (Editorial). (b)
Travis, J . Science 1994, 254, 360. (c) Davies, J . Science 1994, 254, 375.
(d) Nikaido, H. Science 1994, 254, 382. (e) Spratt, B. G. Science 1994,
254, 388. (f) Review: Chu, D. T. W.; Plattner, J . J .; Katz, L. J . Med.
Chem. 1996, 39, 3853.
(2) For relevant reviews and “Trojan-Horse” delivery concepts, see:
(a) Roosenberg, J . M.; Lin, Y.-M.; Lu, Y.; Miller, M. J . Curr. Med. Chem.
2000, 7, 159. (b) Malouin, F.; Miller, M. J . Acc. Chem. Res. 1993, 26,
241. (c) Miller, M. J .; Malouin, F. The Development of Iron Chelators
for Clinical Use; Bergeron, R. J ., Brittenham, G. M., Eds.; CRC: Ann
Arbor, 1994; p 275. (d) Roosenberg, J . M.; Miller, M. J . J . Org. Chem.
2000, 65, 4833.
(3) Neilands, J . B. Methodology of Siderophores. Structure and
Bonding; Springer-Verlag: Berlin, 1984.
(4) (a) Lee, B. H.; Prody, C. A.; Neilands, J . B.; Miller, M. J . J . Med.
Chem. 1985, 28, 323. (b) Ghosh, M.; Miller, M. J . J . Org. Chem. 1994,
59, 1020. (c) Ghosh, M.; Miller, M. J . Bioorg. Med. Chem. 1995, 3, 1519.
(d) Miller, M. J .; Lu, Y. Bioorg. Med. Chem. 1999, 7, 3035.
As shown in Schemes 2 and 3, the synthesis began with
the appropriately protected O-benzyl hydroxylamine
10.1021/jo026391c CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/23/2002
J . Org. Chem. 2003, 68, 191-194
191

SCHEME 1
SCHEME 3a
SCHEME 2a
a
Reagents: (a) NaH, 1,4-dibromobutane, DMF, 16 h; (b)
Chloro(carbonyl) isocyanate, 2,6-lutidine, CH2Cl2, 1 h; (c) NaH,
NaI, 11, DMF, 24 h.
rearrangement to generate the corresponding isocyanate.
Upon cooling, the reaction mixture was exposed to amine
5, providing the desired unsymmetrical urea 9.
Gratifyingly, cyclization of urea 9 with chloro(carbonyl)
isocyanate in the presence of 2,6-lutidine or 2,4,6-collidine
provided dialkyl isocyanurate 12 in 90% yield. The use
of more basic amines, such as triethylamine, gave poorer
yields. Alkylation of 12 by treatment with NaH followed
by exposure to excess bromide 11 reproducibly afforded
the desired differentially protected isocyanurate 13 in
multigram quantities.
In conclusion, we have developed methodology for
scalable syntheses of a differentially protected trialkyl
isocyanurates which are anticipated to be useful for the
preparation of siderophore analogue conjugates that
contain up to three different drugs. To this end, we are
specifically interested in siderophore drug conjugates that
contain drugs with different targets within microbial
cells. Libraries of “multiwarhead” siderophores can be
envisioned as tools for discovery of new antimicrobial
agents and will be reported in due course.
a
Reagents: (a) NaH, NaI, 4-chlorobutyronitrile, DMF, 70 °C,
5 h; (b) Raney Ni, ammonium hydroxide, H2 (45 psi), MeOH; (c)
5-hydroxypentanoic acid methyl ester, triphenylphosphine, diiso-
propylazodicarboxylate, THF, 8 h; (d) 2 M HCl, dioxane, reflux,
12 h; (e) (i) diphenylphophoryl azide, NEt3, toluene, 4 h, (ii) reflux,
2 h, (iii) 5, NMM, 8 h.
(OBHA) derivatives 3, 6, and 10.6-8 Alkylation of 3 with
4-chlorobutyronitrile provided nitrile 4, which was re-
duced with Raney nickel under a pressurized (45 psi) H2
atmosphere to afford amine 5.9 Reaction of 6 with
5-hydroxypentanoic acid methyl ester under Mitsunobu10
conditions followed by acid hydrolysis of product 7
afforded acid 8 in 59% yield for two steps. Acid 8 was
directly treated with diphenylphosphoryl azide (DPPA)
for 4 h and heated at reflux for 2 h to induce a Curtius
Exp er im en ta l Section
Gen er a l Meth od s. 1H NMR and 13C NMR spectra were
obtained on 300, 500, or 600 MHz spectrometers and were
referenced to TMS or residual CDCl3. Analytical TLC was
carried out using Merck aluminum-backed 0.2 mm silica gel 60
F-254 plates. Column chromatography was conducted using
Merck silica gel 60 (230-400 mesh). All reactions were periodi-
cally monitored by TLC and worked up after the complete
consumption of starting materials unless specified otherwise.
The term “dried” refers to treatment with MgSO4. Solvents were
removed under aspirator pressure on a rotary evaporator. Water
was deionized (DI) prior to use. Anhydrous tetrahydrofuran
(THF) was freshly distilled from sodium and benzophenone.
Dimethylformamide (DMF) was distilled under reduced pressure
(5) Gorbatenko, V. I. Tetrahedron 1993, 49, 3227. After submission
of this manuscript, a synthesis on solid-support of 1,3,5-trisubstituted
isocyanurates was reported by Houghten and co-workers: Yu, Y. P.;
Ostresh, J . M.; Houghten, R. A. J . Comb. Chem. 2002, 4, 484.
(6) (a) For the preparation of Boc OBHA, see: Lee, B. H.; Miller,
M. J . J . Org. Chem. 1983, 48, 24. (b) For an excellent review of related
chemistry relating to protected hydroxylamines, see: Romine, J . L.
Org. Prep. Proced. Int. 1996, 28, 249.
(7) For the preparation of Troc OBHA, see; Lee, B. H.; Gerfen, G.
J .; Miller, M. J . J . Org. Chem. 1984, 49, 2418.
(8) For the preparation of Alloc OBHA, see: Dolence, E. K.; Miller,
M. J . J . Org. Chem. 1991, 56, 492.
(9) Bergeron, R. J .; McManis, J . S. Tetrahedron 1989, 45, 4939.
(10) Mitsunobu, O. Synthesis 1981, 1.
192 J . Org. Chem., Vol. 68, No. 1, 2003

over CaH2 and stored over 4 Å sieves and under argon.
Methylene chloride (CH2Cl2) was distilled over CaH2 and used
directly. All other purchased reagents were of reagent grade
quality and were used without further purification.
to reflux (oil bath temperature 120 °C) in dioxane (15 mL) and
2 M HCl (15 mL) for 16 h and then cooled to room temperature.
The mixture was extracted with EtOAc (4 × 25 mL). The organic
layers were combined, washed with brine, dried, filtered, and
evaporated. The crude acid was isolated by extraction and used
directly in the next reaction. Thus, the residue was dissolved in
Et2O (75 mL) and washed with aqueous 10% Na2CO3 (4 × 50
mL). The organic layer was removed, and the aqueous layers
were combined, acidified with 2 M HCl, and extracted with
EtOAc (4 × 50 mL). The combined organic layers were washed
with brine, dried, filtered, and evaporated to yield acid 8 as a
colorless oil: 2.94 g, 90%. Although used directly, characteriza-
tion data includes: 1H NMR (500 MHz, CDCl3) δ 7.45-7.36 (m,
5H), 4.93 (s, 2H), 4.83 (s, 2H), 3.52 (m, 2H), 2.36 (m, 2H), 1.67
(m, 2H); 13C NMR (125 MHz, CDCl3) δ 179.22, 155.17, 134.84,
129.53, 128.87, 128.57, 95.24, 77.30, 75.15, 49.33, 33.43, 26.30,
4-[N-(ter t-Bu t yloxyca r b on yl)b en zyloxya m in o]b u t yr o-
n itr ile (4). To a solution of 36 (5.58 g, 25.0 mmol) in DMF (50
mL) under argon was added NaH (1.04 g, 60% oil dispersion
(Caution: flammable/ pyrophoric), 26.1 mmol) at 0 °C (ice bath).
After H2 evolution ceased, 4-chlorobutyronitrile (2.35 mL, 26.3
mmol) and NaI (370 mg, 2.5 mmol) were added. The solution
turned to pale yellow and was heated in an oil bath (60-70 °C
external bath temperature). After 5 h, the reaction mixture was
cooled to room temperature and diluted with EtOAc (150 mL)
and water (200 mL). The organic layer was separated, and the
aqueous layer was washed with EtOAc (2 × 100 mL). The
combined organic layers were washed with brine, dried, filtered,
and evaporated to yield a crude reaction product which was
purified by flash chromatography over silica gel (4:1 hexanes/
EtOAc) yielding nitrile 4 as a colorless oil: 5.80 g, 80%; 1H NMR
(300 MHz, CDCl3) δ 7.40-7.35 (m, 5H), 4.84 (s, 2H), 3.52 (t, J
) 6.5 Hz, 2H), 2.32 (t, J ) 7.3 Hz, 2H), 1.88 (m, 2H), 1.52 (s,
9H); 13C NMR (75 MHz, CDCl3) δ 156.31, 135.30, 129.53, 128.74,
128.54, 119.18, 81.96, 76.90, 48.12, 28.28, 23.49, 14.86; IR (neat)
2978, 2247, 1701 cm-1; HRMS (FAB) for C16H22N2O3 (M + H)
calcd 291.171, found 291.169.
21.67; IR (neat) 3435, 2942, 1711 cm-1; HRMS (FAB) for C15H18
-
Cl3NO5 (M + H) calcd 398.0329, found 398.0349.
1-[4-[N -(Tr ich lor oe t h oxyca r b on yl)b e n zyloxya m in o]-
b u t yl]-3-[4-[N-(ter t-b u t yloxyca r b on yl)b en zyloxya m in o]-
bu tyl]u r ea (9). To a solution of triethylamine (0.790 mL, 5.64
mmol) and acid 8 (2.25 g, 5.64 mmol) in toluene (20 mL) was
added diphenylphosphoryl azide (1.28 mL, 5.92 mmol). The
solution was stirred at room temperature for 4 h followed by
heating to reflux (125 °C oil bath temperature) for 2 h. The
reaction mixture was cooled to room temperature, amine 5 (1.66
g, 5.64 mmol) was added in one portion, and the resulting
solution stirred for 8 h. The volatiles were evaporated, and the
residue was taken up in EtOAc (100 mL) and water (100 mL).
The aqueous layer was removed and extracted with EtOAc (2 ×
80 mL). The organic layers were combined and washed with 1
M HCl (100 mL), water (100 mL), saturated NaHCO3 (100 mL),
and brine (100 mL). The organic layer was separated, dried,
filtered, and evaporated. The crude material was purified by
flash chromatography over silica gel (1:1-1:3 hexanes/EtOAc)
to yield 9 as a colorless oil: 2.5360 g, 65%; 1H NMR (600 MHz,
CDCl3) δ 7.44-7.32 (m, 10H), 4.91 (s, 2H),4.82 (s, 2H), 4.80 (s,
2H), 4.47 (t, 1H, J ) 6 Hz), 4.45 (t, 1H, J ) 6 Hz), 3.52 (t, 2H,
J ) 6.9 Hz), 3.42 (t, 2H, J ) 6.6 Hz), 3.12 (m, 4H), 1.62 (m, 4H),
1.49 (s, 9H), 1.45 (m, 4H); 13C NMR (150 MHz, CDCl3) δ 158.18,
156.64, 155.14, 135.54, 134.84, 129.54, 129.42, 128.87, 128.58,
128.48, 95.32, 81.39, 77.24, 76.89, 75.16, 49.30, 48.99, 40.16,
39.99, 28.36, 27.31, 27.20, 24.55, 24.40; IR (neat) 3032, 2938,
1717, 1634, 1571 cm-1; HRMS (FAB) for C31H43Cl3N4O7 (M +
H) calcd 689.2276, found 689.2243.
4-[N-(ter t-Bu tyloxyca r bon yl)ben zyloxya m in o]-1-bu tyl-
a m in e (5). Nitrile 4 (1.89 g, 6.50 mmol) was added to a Parr
bottle containing methanol (16 mL), ammonium hydroxide (5
mL), and Raney nickel (1 mL, Raney 2800 nickel 50% slurry in
H2O). The heterogeneous mixture was cooled in an ice bath, and
gaseous ammonia was bubbled into the mixture for 20 min. The
Parr bottle was placed on the Parr apparatus under a H2
atmosphere (45 psi) for 5 h. The mixture was purged with argon
and filtered (Caution: Ra Ni is pyrophoric when dry and exposed
to air). The biphasic mixture was washed with diethyl ether (100
mL) and 1 M NaOH (50 mL). The aqueous layer was separated,
and the organic layer was washed with brine (50 mL), dried,
filtered, and evaporated. Amine 5 was isolated as a colorless oil
with greater than 95% purity based on 1H NMR and was used
1
without further purification: 1.72 g, 90%; H NMR (300 MHz,
CDCl3) δ 7.40-7.33 (m, 5H), 4.81 (s, 2H), 3.41 (t, J ) 7.35 Hz,
2H), 2.67 (t, J ) 7.05 Hz, 2H), 1.61 (m, 2H), 1.49 (s, 9H), 1.41
(m, 2H), 1.30 (bs, 2H); 13C NMR (75 MHz, CDCl3) δ 156.59,
135.65, 129.37, 128.48, 128.41, 81.24, 76.90, 49.42, 41.59, 30.79,
28.32, 24.42; IR (neat) 3375, 3066, 2927, 2934, 1702, 1588 cm-1
;
HRMS (FAB) for C16H26N2O3 (M + H) calcd 295.202, found
4-[N-(Allyloxyca r b on yl)b en zyloxya m in o]-1-b r om ob u -
ta n e (11). To a solution of 108 (1.95 g, 9.41 mmol) in DMF (30
mL) under argon was added NaH (0.450 g, 11.3 mmol) at 0 °C.
After H2 evolution ceased, 1,4-dibromobutane (2.60 mL, 21.8
mmol) was added, and the resulting solution stirred for 18 h.
The reaction mixture was diluted with EtOAc (100 mL) and
water (200 mL). The organic layer was separated, and the
aqueous layer was washed with EtOAc (2 × 50 mL). The
combined organic layers were washed with brine, dried, filtered,
and evaporated to yield a crude reaction product which was
purified by flash chromatography over silica gel (10:1 hexanes/
EtOAc). Compound 11 was isolated as a colorless oil: 2.58 g,
295.201.
5-[N-(Tr ich lor oeth oxyca r bon yl)ben zyloxya m in o]p en t-
a n oic Acid Meth yl Ester (7). To a solution of 67 (3.24 g, 10.9
mmol), triphenylphosphine (3.12 g, 11.9 mmol), and 5-hydroxy-
pentanoic acid methyl ester11 (1.57 g, 11.9 mmol) in anhydrous
THF (40 mL) was added a solution of diisopropylazodicarboxy-
late (DIAD, 2.34 mL, 11.9 mmol) in THF (10 mL) dropwise over
10 min. The solution was stirred for 8 h before being diluted
with EtOAc (150 mL) and water (200 mL). The organic layer
was removed, and the aqueous layer was washed with EtOAc
(2 × 100 mL). The combined organic layers were washed with
brine, dried, filtered, and evaporated to yield a yellow oil. The
crude product was purified by flash chromatography over silica
gel (4:1 hexanes/EtOAc) after most of the triphenylphosphine
oxide was removed by crystallization from 1:1 Et2O/hexanes.
1
80%; H NMR (600 MHz, CDCl3) δ 7.40-7.35 (m, 5H), 5.97(m,
1H, J ) 17.1, 10.2, and 5.7 Hz), 5.36 (d, 1H, J ) 17.1 Hz), 5.27
(d, 1H, J ) 10.2 Hz), 4.88 (s, 2H), 4.67 (d, 2H, J ) 5.7 Hz), 3.48
(t, 2H, J ) 6.9 Hz) 3.38 (t, 2H, J ) 6.3 Hz), 1.86 (m, 2H), 1.75
(m, 2H); 13C NMR (150 MHz, CDCl3) δ 156.88, 135.24, 132.19,
129.19, 128.47, 128.32, 118.00, 76.99, 66.43, 48.65, 32.98, 29.63,
25.53; IR (neat) 3065, 3032, 2942, 1705, 1648, 1497, 1454, 1398,
1342, 1293, 1242, 1160, 1092, 995, 934, 750, 699 cm-1; HRMS
1
Methyl ester 7 was isolated as a colorless oil: 3.37 g, 75%; H
NMR (600 MHz, CDCl3) δ 7.43-7.32 (m, 5H), 4.91 (s, 2H),4.81
(s, 2H),3.62 (s, 3H), 3.50 (t, J ) 6.3 Hz, 2H), 2.29 (t, J ) 6.75
Hz, 2H), 1.64 (m, 4H); 13C NMR (150 MHz, CDCl3) δ 173.54,
155.00, 134.79, 129.17, 128.64, 128.41, 95.20, 77.12, 75.02, 51.42,
(FAB) for
342.0714.
C15H18 Cl3NO5 (M + H) calcd 342.0705, found
49.23, 33.38, 26.28, 21.84; IR (neat) 3033, 2951,1740, 1720 cm-1
;
HRMS (FAB) for C16H20Cl3NO5 (M + H) calcd 412.0485, found
412.0471.
5-[N-(Tr ich lor oeth oxyca r bon yl)ben zyloxya m in o]p en t-
a n oic Acid (8). Methyl ester 7 (3.37 g, 8.18 mmol) was heated
1-[4-[N -(Tr ich lor oe t h oxyca r b on yl)b e n zyloxya m in o]-
b u t yl]-3-[4-[N-(ter t-b u t yloxyca r b on yl)b en zyloxya m in o]-
bu tyl][1.3.5]tr ia zin a n e-2,4,6-tr ion e (12). To a stirring solution
of urea 9 (2.54 g, 3.67 mmol) in dry CH2Cl2 (40 mL) at 0 °C was
added 2,6-lutidine (0.43 mL, 3.7 mmol) followed by chloro-
(carbonyl) isocyanate (technical grade, 0.30 mL, 3.7 mmol). The
solution turned deep yellow and was allowed to warm to room
(11) Huckstep, M.; Taylor, R. J . K.; Caton, M. P. L. Synthesis 1982,
881.
J . Org. Chem, Vol. 68, No. 1, 2003 193

temperature. After 1 h, the solvent was evaporated, and the
crude yellow oil was directly purified by flash chromatography
over silica gel (2:1 hexanes/EtOAc) to yield 12 as a viscous,
colorless oil: 2.37 g, 90%; 1H NMR (600 MHz, CDCl3) δ 8.67 (s,
1H), 7.44-7.33 (m, 10H), 4.92 (s, 2H), 4.83 (s, 2H), 4.82 (s, 2H),
3.83 (broad t, 4H), 3.54 (bt, 2H), 3.44 (bt, 2H), 1.64 (m, 8H), 1.50
(s, 9H); 13C NMR (150 MHz, CDCl3) δ 156.55, 155.12, 149.49,
148.09, 148.03, 135.61, 134.87, 129.55, 129.42, 128.87, 128.58,
128.54, 128.47, 95.32, 81.41, 77.30, 76.90, 75.18, 49.21, 49.06,
42.17, 42.00, 28.36, 25.08, 24.94, 24.28, 24.15; IR (neat) 3224,
3109, 2952, 1720, 1687, 1457, 1409, 1369, 1288, 1156, 1111, 1049
cm-1; HRMS (FAB) for C33H42Cl3N5O9 (M + H) calcd 758.2126,
found 758.2142.
g, 70%; 1H NMR (300 MHz, CDCl3) δ 7.43-7.33 (m, 15H), 5.97-
(m, 1H, J ) 17.1, 10.2, and 5.7 Hz), 5.36 (d, 1H, J ) 17.1 Hz),
5.27 (d, 1H, J ) 10.2 Hz), 4.92 (s, 2H), 4.85 (s, 2H), 4.83 (s, 2H),
4.81(s, 2H), 4.67 (d, 2H, J ) 5.7 Hz), 3.83 (m, 6H), 3.53 (broad
t, 2H), 3.47 (bt, 2H), 3.43 (bt,2H), 1.66-1.59 (m, 12 H), 1.49 (s,
9H); 13C NMR (125 MHz, CDCl3) δ 157.03, 156.54, 155.08,
148.88, 135.61, 135.39, 134.87, 129.53, 129.41, 129.36, 128.85,
128.63, 128.56, 128.50, 128.45, 118.16, 95.33, 81.33, 77.28, 77.19,
76.9, 75.16, 66.63, 60.37, 49.26, 49.12, 42.65, 42.59, 42.49, 28.36,
25.12, 25.04, 24.97, 24.35, 24.26, 24.22, 21.04; IR (neat) 3032,
2948, 2875, 1689, 1464, 1360, 1286, 1233, 1159, 1113 cm-1
;
HRMS (FAB) for C48H61Cl3N6O12 MS FAB (M + H) found
1021.30.
1-[4-[N -(Tr ich lor oe t h oxyca r b on yl)b e n zyloxya m in o]-
b u t yl]-3-[4-[N-(ter t-b u t yloxyca r b on yl)b en zyloxya m in o]-
bu tyl]-5-[4-[N-(a llyloxyca r bon yl)ben zyloxya m in o]bu tyl]-
[1.3.5]tr ia zin a n e-2,4,6-tr ion e, 13. To a stirring solution of
compound 12 (2.37 g, 3.12 mmol) in DMF (3.5 mL) under argon
at 0 °C was added NaH (60% dispersion in mineral oil, 0.143 g,
3.43 mmol). Upon complete addition of NaH, the solution turned
brown and NaI (90 mg, 0.60 mmol) was added followed by
bromide 11 (1.58 g, 4.61 mmol) in DMF (1.5 mL). The solution
was stirred for 24 h. The disappearance of compound 12 was
monitored by TLC (2:1 hexanes/EtOAc, Rf ) 0.15). The reaction
was quenched with water (80 mL), and the mixture was
extracted with EtOAc (3 × 30 mL). The organic layers were
combined, washed with brine (50 mL), dried, filtered, and
evaporated. The crude product was purified by flash chroma-
tography (7:3 hexanes/EtOAc) to yield 13 as a colorless oil: 2.20
Ack n ow led gm en t. We gratefully acknowledge fi-
nancial support by the NIH (AI30988). We also appreci-
ate the use of the NMR facilities provided by the
Lizzadro Magnetic Center and the mass spectrometry
services (Dr. W. Boggess and Ms. N. Sevova) provided
by the University of Notre Dame. Finally, we appreciate
Maureen Metcalf’s assistance with the manuscript.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra of 4, 5, 7, 9, and 11-13. This material is available
free of charge via the Internet at http://pubs.acs.org.
J O026391C
194 J . Org. Chem., Vol. 68, No. 1, 2003