Tethered bisubstrate derivatives as probes for mechanism and as inhibitors of aminoglycoside 3'-phosphotransferases

Article abstract of DOI:10.1021/jo000589k

Aminoglycoside 3'-phosphotransferases [APH(3')s] phosphorylate aminoglycoside antibiotics, a reaction that inactivates the antibiotics. These enzymes are the primary cause of resistance to aminoglycosides in bacteria. APH(3')-Ia operates by a random-equilibrium BiBi mechanism, whereas APH(3')-IIIa catalyzes its reaction by the Theorell-Chance mechanism, a form of ordered BiBi mechanism. Hence, both substrates have to be present in the active site prior to the transfer of phosphate by both mechanisms. Four bisubstrate analogues, compounds 1 - 4, were designed and synthesized as inhibitors for APH(3')s. These compounds are made of adenosine linked covalently to the 3'-hydroxyl of neamine (an aminoglycoside) via all-methylene tethers of 5 - 8 carbons. The K(i) values measured for these compounds indicated that affinities of APH(3')-Ia and APH(3')-IIa for compounds 2 and 8 (six- and seven-carbon tethers, respectively) were the best, and the inhibition constants for the two were comparable.

Full text of DOI:10.1021/jo000589k

7422
J . Org. Chem. 2000, 65, 7422-7431
Teth er ed Bisu bstr a te Der iva tives a s P r obes for Mech a n ism a n d a s
In h ibitor s of Am in oglycosid e 3′-P h osp h otr a n sfer a ses
Meizheng Liu, J alal Haddad, Eduardo Azucena, Lakshmi P. Kotra, Maria Kirzhner, and
Shahriar Mobashery*
Department of Chemistry and the Institute for Drug Design, Wayne State University,
Detroit, Michigan 48202
som@mobashery.chem.wayne.edu
Received April 18, 2000
Aminoglycoside 3′-phosphotransferases [APH(3′)s] phosphorylate aminoglycoside antibiotics, a
reaction that inactivates the antibiotics. These enzymes are the primary cause of resistance to
aminoglycosides in bacteria. APH(3′)-Ia operates by a random-equilibrium BiBi mechanism, whereas
APH(3′)-IIIa catalyzes its reaction by the Theorell-Chance mechanism, a form of ordered BiBi
mechanism. Hence, both substrates have to be present in the active site prior to the transfer of
phosphate by both mechanisms. Four bisubstrate analogues, compounds 1-4, were designed and
synthesized as inhibitors for APH(3′)s. These compounds are made of adenosine linked covalently
to the 3′-hydroxyl of neamine (an aminoglycoside) via all-methylene tethers of 5-8 carbons. The Ki
values measured for these compounds indicated that affinities of APH(3′)-Ia and APH(3′)-IIa for
compounds 2 and 3 (six- and seven-carbon tethers, respectively) were the best, and the inhibition
constants for the two were comparable.
The most common mechanism for resistance to amino-
combination therapy has been implemented successfully
to counter the effect of â-lactamases, which are resistance
determinants for â-lactam antibiotics.8
glycoside antibiotics is the structural modification of
these antibiotics by aminoglycoside-modifying enzymes.1,2
Among these enzymes, aminoglycoside 3′-phosphotrans-
ferases [APH(3′)s], which catalyze the transfer of the
γ-phosphoryl group of ATP to the 3′-hydroxyl of amino-
glycosides, are most common, and indeed these enzymes
were the cause of the demise of kanamycin treatment in
the clinic.
Aminoglycoside 3′-phosphotransferases types Ia and
IIIa [APH(3′)-Ia and APH(3′)-IIIa] are the most common
members of this family of enzymes in Gram-negative and
Gram-positive bacteria, respectively.1,2 Inhibitors for
these enzymes are highly sought.1 Although there are a
handful of known inhibitors for these enzymes,3-5 none
appear to be exceptionally effective. A combination
therapy of an inhibitor for APH(3′)s and an aminoglyco-
side antibiotic that would otherwise be a substrate for
the resistance enzymes would revive clinical utility of
obsolete drugs such as kanamycin. Such a combination
drug would expand our therapeutic options in the face
of the serious needs in fighting multiply resistant bac-
teria in the clinic.6,7 We hasten to add that such a
Mechanisms of both APH(3′)-Ia and APH(3′)-IIIa have
been investigated. APH(3′)-Ia operates by a random-
equilibrium BiBi mechanism,9 whereas APH(3′)-IIIa
catalyzes its reaction by the Theorell-Chance mecha-
nism, a form of ordered BiBi mechanism.10 Both mech-
anisms stipulate that the two substrates (aminoglycoside
and ATP) should be present in the active site prior to
the transfer of the phosphate group, and they refute the
possibility for a double-displacement ping-pong mecha-
nism for the enzymes. In essence, one would expect that
there should be specific binding sites for both the amino-
glycoside and ATP in the active site, and that the enzyme
would stabilize the transition state for the transfer of
the γ-phosphoryl group from the latter to the former.
Figure 1 depicts an energy-minimized ternary complex
for APH(3′)-IIIa, kanamycin A, and MgATP.
We set out to prepare bisubstrate analogues as inhibi-
tors for APH(3′)s. Such molecules would bind to both
subsites for substrate binding in the enzyme active site
and should give potent inhibition of APH(3′)s. We have
synthesized four conjoint molecules (compounds 1-4)
that incorporate structures of adenosine and neamine (an
aminoglycoside) into one compound (Figure 2).
* To whom correspondence should be addressed:Phone: 313-577-
3924, FAX: 313-577-8822.
(1) Wright, G. D.; Berghuis, A. M.; Mobashery, S. In Resolving the
Antibiotic Paradox: Progress in Understanding Drug Resistance and
Development of New Antibiotics; Rosen, B. P., Mobashery, S., Eds;
Plenum Press: New York, 1998; pp 27-69.
Exp er im en ta l Section
1,1-Dimethoxycyclohexane was purchased from the TCI
America Co. Pyruvate kinase (PK), lactic dehydrogenase (LD),
(2) Shaw, K. J .; Rather, P. N.; Hare, R. S.; Miller, G. H. Microbiol.
Rev. 1993, 57, 138-163.
(3) Roestamadji, J .; Mobashery, S. Bioorg. Med. Chem. Lett. 1998,
8, 3483-3488.
(8) Bush, K.; Mobashery, S. in Resolving the Antibiotic Paradox:
Progress in Understanding Drug Resistance and Development of New
Antibiotics; Rosen, B. P., Mobashery, S., Eds; Plenum Press: New York,
1998; pp 71-98.
(9) Siregar, J . J .; Miroshnikov, K.; Mobashery, S. Biochemistry 1995,
34, 12681-12688.
(10) Wright, G. D.; McKay, G. A. J . Biol. Chem. 1995, 270, 24686-
24692.
(4) Roestamadji, J .; Grapsas, I.; Mobashery, S. J . Am. Chem. Soc.
1995, 117, 80-84.
(5) Daigle, D. M.; McKay, G. A.; Wright, G. D. J . Biol. Chem. 1997,
272, 24755-24758.
(6) Cohn, M. A. Science 1992, 257, 1050-1054.
(7) The World Health Report. Fighting Disease Fostering Develop-
ment; World Health Organization: Geneva, 1996.
10.1021/jo000589k CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/29/2000

Inhibitors of Aminoglycoside 3′-Phosphotransferases
J . Org. Chem., Vol. 65, No. 22, 2000 7423
F igu r e 1. The energy-minimized computational model for the ternary complex of APH(3′)-IIIa: (A) Stereoview of the active site
of APH(3′)-IIIa (Hon, W. C.; McKay, G. A.; Thompson, P. R.; Sweet, R. M.; Yang, D. S.; Wright, G. D.; Berghuis, A. M. Cell 1997,
89, 887-895.) occupied by the substrates kanamycin A and ATP. A portion of the binding sites for kanamycin A and ATP in the
active site are shown as gray surfaces, which are rendered as Connolly water-accessible surfaces. Two magnesium ions, depicted
as gray spheres, are coordinated to the phosphate backbone of ATP. The backbone of the enzyme is depicted as a gray wire, and
kanamycin A, the catalytic residue Asp-190, and ATP are shown as capped-sticks. The 3′-hydroxyl moiety of kanamycin A is
marked by an arrow. A hydrogen bond, depicted as a broken line, is shown between the carboxyl moiety of Asp-190 and the
3-hydroxyl moiety of kanamycin A. A schematic of the interactions observed in the energy-minimized model between kanamycin
A, ATP, Asp-190, and the Mg2+ ions are shown in panel B. The 3′-hydroxyl moiety is shown by an arrow, which forms a hydrogen
bond with the side chain of Asp-190.
Deter m in a tion of Kin etic P a r a m eter s. The kinetic
parameters for inhibition of APH(3′)s by the four inhibitors
(1, 2, 3, and 4) were determined by the coupled spectrophoto-
metric assay described by Goldman and Northrop.12 A typical
assay mixture contained phosphoenol pyruvate (5 mM), mag-
nesium chloride (10 mM), NADH (0.3 mM), lactate dehydro-
genase (21 units), pyruvate kinase (6 units), and the concen-
trations of the inhibitors were varied from 2 µM to 1 mM in
25 mM HEPES buffer at pH 7.4. The final volume for each
assay mixture was 500 µL. The reactions were performed
either at saturating concentration of kanamycin A (200 µM)
F igu r e 2. The chemical structures for the tethered bi-
and varied concentrations of ATP (100 and 200 µM), or at
substrate probes for aminoglycoside 3′-phosphotransferases.
saturating concentration of ATP (800 µM) and varied concen-
The attachement of the tether is to the 3′-hydroxyl of amino-
trations of kanamycin A (30 and 60 µM). The reaction was
glycoside, the site of phosphorylation by APH(3′)s, and to the
initiated by the addition of APH(3′)-Ia or APH(3′)-IIa at final
6′-hydroxyl of adenosine. The tether serves as a surrogate for
the triphosphoryl group of ATP. A somewhat different kind of
tether for a bisubstrate analogue for a fucosyltransferase has
been described previously (Palcic, M. M.; Heerze, L. D.;
Srivastava, O. P.; Hindsgaul, O. J . Biol. Chem. 1989, 262,
17174-17181).
concentrations of 800 and 400 nM, respectively. The change
in optical density at 340 nm was monitored. The Ki values were
calculated from Dixon plots.13
Assessm en t of th e P oten tia l for Slow -Bin d in g In h ibi-
tion . To determine whether 1, 2, 3, or 4 were slow-binding
inhibitors for APH(3′)s, assays were carried out with and
without preincubation of the enzymes with the inhibitors. The
shapes of the reaction progress curves were compared to that
phospho(enol)pyruvate (PEP), ATP, and NADH were pur-
obtained in the absence of inhibitor over a period during which
chased from the Sigma Chemical Co. All other reagents were
the reaction progress was linear in the absence of inhibitor.
purchased from the Aldrich Chemical Co. All calculations were
performed by the MS Excell program. Aminoglycoside 3′-
For reactions without preincubation with inhibitor, the same
components of the spectrophotometric assay described above
were mixed including kanamycin A and each of the inhibitors
phosphotransferase type Ia and IIa were purified according
to the procedures developed by Siregar et al.9,11
(12) Goldman, P. R.; Northrop, D. B. Biochem. Biophys. Res.
Commun. 1976, 69, 230-236.
(13) Dixon, M. 1953 Biochem. J . 1953, 55, 161-170.
(11) Siregar, J . J .; Lerner, S. A.; Mobashery, S. Antimicrob. Agents
Chemother. 1994, 38, 641-647.

7424 J . Org. Chem., Vol. 65, No. 22, 2000
Liu et al.
(1, 2, 3, or 4). The reaction was initiated by the addition of
800 nM of APH(3′)-Ia or 400 nM of APH(3′)-IIa. The reaction
curves at three different inhibitor concentrations (ranging from
2 to 400 µM, depending on their respective Ki values) were
obtained for APH(3′)-Ia and APH(3′)-IIa. In the presence of
preincubation with inhibitor, the enzyme was mixed with each
inhibitor (1, 2, 3, or 4), at concentrations three times their Ki
values, and the mixture was incubated for 20 min, 1 h, and 3
h. The preincubation mixtures contained all the components
of the assay described above, except kanamycin A. The
reactions were then initiated by the addition of kanamycin A
to a final concentration of 50 µM in the case of APH(3′)-Ia,
and to 25 µM for APH(3′)-IIa.
Molecu la r Mod elin g. The X-ray structure of kana-
mycin A was obtained from Cambridge Structural Database
(KNMYSL). The coordinates for the three-dimensional struc-
ture of APH(3′)-IIIa with ADP, and two Mg2+ ions bound in
the active site of the enzyme were kindly provided by Professor
Albert Berghuis.14 The model was constructed from the X-ray
structure, as described below. ADP was modified to ATP by
the addition of the γ-phosphate and kanamycin A was docked
in the active site of the enzyme in such a way that the 3′-
hydroxyl, the site of phosphorylation, approached the terminal
phosphate group of ATP. The resulting complex was energy-
minimized using AMBER 5.0 software (Oxford Molecular,
Inc.).15 For nonstandard residues, MNDO ESP charges were
used, and the energy minimization was performed for 25000
iterations. The energy-minimized complex was visualized and
analyzed using the Sybyl molecular modeling software.16 Other
modeling tasks that are mentioned in this manuscript were
also performed using the Sybyl software.
concentrated in vacuo to afford a crude product, which was
purified by column chromatography (SiO2, 10:1 hexane/ethyl
acetate) to give compound 9 (861 mg, 79%) as a white solid.
mp 101-102 °C; IR (film) 2933, 1697 cm-1 1H NMR (CD3-
;
COCD3, 500 MHz) δ 7.60-6.80 (m, 20 H), 5.45 (m, 1 H), 5.00
(m, 2 H), 4.60 (m, 2 H), 4.50-4.00 (m, 11 H), 3.80 (m, 3 H),
3.40 (m, 2 H), 1.80-1.00 (m, 39 H), 0.95 (s, 9 H), 0.25 (s, 6 H);
13C NMR (CD3COCD3, 125 MHz) δ 156.7, 155.4, 152.0, 141.4,
139.7, 137.1, 128.7-126.2, 111.6, 96.9, 80.4, 79.9, 79.4, 75.7,
72.4, 68.3, 67.8, 61.2, 59.3, 54.2, 52.0, 47.7, 47.6, 45.8, 36.5,
36.4, 36.1, 32.0, 27.9, 27.7, 27.5, 25.7, 24.9, 23.8, 23.7, 18.0,
-3.6, -5.3; MS (FAB, NBA) for C68H94O13N4 (M + Na+) 1226.
1,3,2′,6′-Tetr a -N-ben zyl-3′-O-ter t-bu tyld im eth ylsilyl-6′-
N,4′-O-ca r bon yln ea m in e (10). A stirred solution of com-
pound 9 (100 mg, 0.08 mmol) in a mixed solvent system of
CH2Cl2 (2 mL) and distilled water (0.01 mL) was treated with
trifluoroacetic acid (2 mL) at room temperature for 1 h. The
solvent was evaporated to dryness in vacuo, and the residue
was purified by column chromatography (SiO2, 20:1:0.1 CHCl3/
MeOH/NH4OH,) to afford the title compound (45 mg, 66%) as
a white powder. mp 67-69 °C; IR (film) 3327, 2950, 1710 cm-1
;
1H NMR (CD3OD, 300 MHz) δ 7.42-7.22 (m, 20 H), 5.10 (d, 1
H, J ) 3.3 Hz), 4.38 (d, 2 H, J ) 15.0 Hz), 4.18 (dq, 1 H, J )
3.3, 6.3, 10.2 Hz), 3.99 (dd, 1 H, J ) 8.7, 9.6 Hz), 3.97 (d, 2 H,
J ) 13.3 Hz), 3.83 (d, 1 H, J ) 13.2 Hz), 3.82 (dd, 2 H, J )
9.6, 12.9 Hz), 3.71 (d, 1 H, J ) 12.6 Hz), 3.45 (t, 1 H, J ) 9.0
Hz), 3.28 (t, 1 H, J ) 9.0, 9.3 Hz), 2.96 (t, 1 H, J ) 10.5 Hz),
2.78 (dd, 1 H, J ) 6.6, 10.5 Hz), 2.69 (dd, 1 H, J ) 3.3, 9.9
Hz), 2.60 (m, 1 H), 2.58 (m, 1 H), 2.41 (m, 1 H), 0.92 (s, 9 H),
0.24 (s, 3 H), 0.18 (s, 3 H); 13C NMR (CD3OD, 75 MHz) δ 141.5,
141.3, 139.6, 137.1, 128.8, 128.7, 128.4, 128.3, 128.3, 128.2,
128.1, 127.9, 127.6, 127.2, 126.8, 126.6, 101.5, 89.2, 79.0, 77.1,
76.0, 71.7, 62.7, 62.3, 57.2, 56.5, 53.1, 52.2, 51.0, 48.0, 32.6,
25.7, 18.1, -5.08, -4.37; MS (FAB, NBA) for C47H62O7N4Si
(M+) 823; (M + Na+) 846.
1,3,2′,6′-Tetr a -N-ter t-bu toxyca r bon yl-3′-O-ter t-bu tyld i-
m eth ylsilyl-5,6-O-cycloh exylid en en ea m in e (8). To a mix-
ture of compound 7 (2.25 g, 2.81 mmol) and imidazole (243
mg, 3.57 mmol) in 20 mL of anhydrous N,N-dimethylforma-
mide (DMF) was added tert-butyldimethylsilyl chloride (493
1,3,2′,6′-Tetr a-N-ben zyl-1,3,2′-tr i-N-ter t-bu toxycar bon yl-
6′-N,4′-O-ca r bon yl-5,6-O-cycloh exylid en en ea m in e (11).
To an ice-cold solution of compound 9 (756 mg, 0.63 mmol) in
dry THF (5 mL) was added a 1.0 M solution of tetra-n-
butylammonium fluoride in THF (2.5 mL, 2.5 mmol). The
mixture was stirred for 10 min, quenched with ice-water, and
concentrated to dryness in vacuo. The residue was dissolved
in CH2Cl2, extracted with water and then brine, dried (Na2-
SO4), concentrated, and purified on a column (SiO2, 8:1 hexane/
ethyl acetate) to afford the desired compound 11 as a white
solid (453 mg, 66%). mp 123-125 °C; IR (film) 3405, 2933, 1697
cm-1; 1H NMR (CD3COCD3, 500 MHz) δ 7.55-6.95 (m, 20 H),
5.40 (m, 1 H), 4.96-4.82 (m, 2 H), 4.73-3.14 (m, 16 H), 2.99
(m, 2 H), 2.39 (m, 2 H), 1.72-1.18 (m, 38 H); 13C NMR (CD3-
COCD3, 125 MHz) δ 156.8, 155.6, 155.1, 152.5, 141.6, 139.8,
138.6, 137.2, 137.0, 128.7-126.1, 111.6, 97.1, 81.2, 80.9, 80.2,
80.0, 79.5, 75.7, 72.4, 66.0, 61.9, 61.2, 61.3, 60.3, 58.8, 54.5,
52.7, 47.8, 47.5, 45.8, 36.6, 36.4, 36.2, 31.9, 27.8, 27.7, 27.5,
25.0, 23.8; MS (FAB, NBA) for C62H80N4O13 (M + Na+) 1112.
1,3,2′,6′-Tetr a -N-ben zyl-3′-O-(5”-br om op en tyl)-1,3,2′-tr i-
N-ter t-bu toxycar bon yl-6′-N,4′-O-car bon yl-5,6-O-cycloh ex-
ylid en en ea m in e (12a ). To a stirred solution of compound 11
(926 mg. 0.85 mmol) and 1,5-dibromopentane (2.44 mL, 17.91
mmol) in DMSO (12 mL) was added powdered potassium
hydroxide (238 mg, 4.25 mmol). The resulting mixture was
vigorously stirred at room temperature under a nitrogen
atmosphere for 30 min. The reaction was quenched with cold
water (4 mL), extracted by CH2Cl2 (3 × 50 mL), dried (Na2-
SO4), and concentrated in vacuo to furnish a syrup, which was
purified by column chromatography (SiO2, 3:1 hexane/ethyl
acetate) to give the title compound (971 mg, 92%) as a white
solid. mp 96-97 °C; IR (film) 2975, 2934, 1693 cm-1; 1H NMR
(CD3COCD3, 500 MHz) δ 7.51-6.90 (m, 20 H), 5.45 (m, 1 H),
4.93 (m, 1 H), 4.80-4.02 (m, 9 H), 3.93-3.80 (m, 5 H), 3.54-
3.49 (m, 5 H), 3.37 (m, 1 H), 3.13 (m, 1 H), 1.89 (m, 2 H), 1.61-
1.17 (m, 44 H); 13C NMR (CD3COCD3, 125 MHz) δ 156.6, 155.5,
152.0, 141.2, 141.1, 139.6, 137.0, 128.6-126.0, 111.5, 96.6, 80.3,
80.2, 80.1, 80.1, 79.5, 79.4, 73.6, 72.4, 72.1, 70.9, 61.3, 61.2,
58.7, 57.2, 54.4, 54.3, 52.0, 47.4, 45.7, 36.3, 36.0, 36.0, 34.0,
33.9, 32.6, 32.5, 31.9, 29.3, 27.8, 27.6, 27.5, 24.8, 24.5, 24.4,
mg, 3.27 mmol) at room temperature under
a nitrogen
atmosphere, and the mixture was stirred overnight. The
reaction was quenched by addition of water (3 mL), extracted
with CH2Cl2 (3 × 20 mL), back extracted with water and then
brine, dried over MgSO4, and concentrated to dryness in vacuo.
The residue was purified on a column (SiO2, 2:1 hexane/ethyl
acetate) to furnish compound 8 (1.91 g, 74%) as a pure white
solid. mp 140-142 °C; IR (film) 3451, 3356, 2977, 1721, 1703,
1507 cm-1 1H NMR (CDCl3, 500 MHz) δ 6.28 (s, 1 H, NH),
;
6.20 (d, 1 H, J ) 5.7 Hz, NH), 6.02 (d, 1 H, J ) 8.1 Hz, NH),
5.44 (s, 1 H, NH), 5.12 (s, 1 H, H-1′), 4.88 (d, 1 H, J ) 3.0 Hz,
OH), 3.80-3.46 (m, 9 H), 3.17 (d, 2 H, J ) 9.0 Hz), 1.65-1.28
(m, 47 H), 0.86 (s, 9 H), 0.07 (s, 6 H); 13C NMR (CDCl3, 125
MHz) δ 158.2, 155.4, 155.1, 111.4, 97.7, 81.1, 79.0, 78.3, 77.9,
77.2, 72.9, 71.8, 71.4, 55.7, 50.7, 48.76, 40.9, 37.1, 36.4, 36.2,
28.1, 27.9, 27.8, 27.8, 25.8, 25.0, 24.0, 23.8, 18.2, -4.3, -5.4;
MS (FAB, NBA) 917 (MH+); HRMS (FAB, NBA) calcd for
C
44H81O14N4Si (MH+) 917.5519, found 917.5521.
1,3,2′,6′-Tetr a-N-ben zyl-1,3,2′-tr i-N-ter t-bu toxycar bon yl-
3′-O-ter t-bu tyld im eth ylsilyl-6′-N,4′-O-ca r bon yl-5,6-O-cy-
cloh exylid en en ea m in e (9). To an ice-cold solution of 8 (842
mg, 0.92 mmol) in anhydrous DMF (7 mL) was added sodium
hydride (60% dispersion in mineral oil, 441 mg, 11.03 mmol),
and the mixture was allowed to stir at room temperature under
an atmosphere of nitrogen. After 2 h, benzyl bromide (2.18
mL, 18.33 mmol) was added dropwise over 30 min and stirring
was continued for another 6 h. The reaction was quenched with
a 25% aqueous HOAc solution (2 mL), diluted with water (3
mL), and extracted with CH2Cl2 (3 × 20 mL). The organic layer
was washed with water, dried over MgSO4, filtered, and
(14) Hon, W. C.; McKay, G. A.; Thompson, P. R.; Sweet, R. M.; Yang,
D. S.; Wright, G. D.; Berghuis, A. M. Cell 1997, 89, 887-895.
(15) Case, D. A.; Pearlman, D. A.; Caldwell, J . W.; Cheatham III,
T. E.; Ross, W. S.; Simmerling, C. L.; Darden, T. A.; Merz, K. M.;
Stanton, R. V.; Cheng, A. L.; Vincent, J . J .; Crowley, M.; Ferguson, D.
M.; Radmer, R. J .; Seibel, G. L.; Singh, U. C.; Weiner, P. K.; Kollman,
P. A. 1997, AMBER 5. University of California, San Francisco, CA.
(16) Tripos Associates, St. Louis, MO.

Inhibitors of Aminoglycoside 3′-Phosphotransferases
J . Org. Chem., Vol. 65, No. 22, 2000 7425
23.7; MS (FAB, NBA) 1236, 1238 (M+); HRMS (FAB, NBA)
calcd for C67H89BrN4O13 (M+) 1236.5610, 1238.5610, found
1236.5603, 1238.5556.
1.19 (m, 50 H); 13C NMR (CD3COCD3, 125 MHz) δ 156.11,
155.50, 152.91, 149.44, 141.75, 139.39, 128.52-125.67, 119.53,
113.25, 111.29, 96.27, 90.70, 86.03, 85.89, 84.85, 81.85, 79.47,
78.96, 78.33, 76.79, 75.19, 72.03, 71.85, 71.15, 70.76, 59.01,
57.47, 54.52, 53.89, 51.19, 50.96, 47.43, 46.83, 46.42, 36.33,
36.01, 30.20, 27.85, 27.65, 27.50, 26.68, 24.80, 24.71, 23.67,
22.72; MS (FAB, NBA) 1439 (MH+); HRMS (FAB, NBA) calcd
for C79H108N9O16 (MH+) 1438.7990, found 1438.7987.
1,3,2′,6′-Tetr a -N-ben zyl-3′-O-(6”-br om oh exyl)-1,3,2′-tr i-
N-ter t-bu toxycar bon yl-6′-N,4′-O-car bon yl-5,6-O-cycloh ex-
ylid en en ea m in e (12b). The title compound was prepared
according to the procedure described for 12a from the reaction
of 11 (1.18 g, 1.08 mmol) and 1,6-dibromohexane to afford
compound 12b as a white solid (1.23 g, 91%). mp 90-92 °C;
Com p ou n d 14b. The title compound was prepared accord-
ing to the procedure described for 14a from compound 12b
(800 mg, 0.64 mmol) and 13a (268 mg, 0.65 mmol) to give the
desired compound 14b as a white solid (250 mg, 27%). mp 95-
IR (film) 2973, 2932, 1694 cm-1 1H NMR (CD3COCD3, 500
;
MHz) δ 7.49-6.96 (m, 20 H), 5.43 (m, 1 H), 4.94 (dd, 1 H),
4.80-4.11 (m, 9 H), 3.92 (m, 5 H), 3.50 (m, 3 H), 3.38 (m, 2
H), 3.18 (m, 2 H), 1.87 (m, 2 H), 1.61-1.17 (m, 46 H); 13C NMR
(CD3COCD3, 125 MHz) δ 157.4, 156.2, 152.7, 142.0, 140.4,
137.8, 129.7-126.7, 112.2, 97.3, 81.4, 81.0, 80.9, 80.8, 80.2,
74.3, 73.1, 72.8, 71.8, 62.0, 59.4, 58.0, 55.1, 55.0, 52.7, 48.2,
46.4, 37.0, 36.7, 34.7, 33.5, 30.7, 28.5, 28.2, 28.4, 28.2, 25.6,
25.5, 24.4; MS (FAB, NBA) 1250, 1252 (M+); HRMS (FAB,
NBA) calcd for C68H91BrN4O13 (M+) 1250.5770, 1252.5770,
found 1250.5770, 1252.5780.
1
96 °C; IR (film) 3328, 3184, 2934, 1694, 1639, 1162 cm-1; H
NMR (CD3COCD3, 500 MHz) δ 8.22 (s, 1 H), 8.21 (s, 1 H),
7.47-7.14 (m, 20 H), 6.74 (m, 1 H), 6.20 (s, 1 H), 5.47 (dd, 1
H, J ) 2.5, 5.5 Hz), 5.36 (s, 1 H), 5.06 (d, 1 H, J ) 5.0 Hz),
4.33 (m, 1 H), 4.94-2.91 (m, 25 H), 2.46 (br, 1 H), 1.64-1.19
(m, 52 H); 13C NMR (CD3COCD3, 125 MHz) δ 156.9, 153.5,
150.2, 140.3, 129.2-126.4, 120.4, 113.9, 112.0, 97.1, 91.6, 86.8,
85.2, 82.8, 80.2, 77.3, 75.7, 71.8, 71.4, 59.7, 58.2, 55.2, 54.6,
51.8, 48.0, 47.1, 37.0, 36.7, 31.0, 28.6, 28.4, 28.2, 27.3, 26.4,
25.5, 25.4, 24.4; MS (FAB, NBA) 1453 (MH+); HRMS (FAB,
NBA) calcd for C80H110N9O16 (MH+) 1452.8100, found 1452.8095.
Com p ou n d Test 14c. The title compound was prepared
according to the procedure described for 14a from compound
12c (1.04 g, 0.82 mmol) and 13a (407 mg, 0.98 mmol) to give
the desired compound 14c as a white solid (322 mg, 27%). mp
1,3,2′,6′-Tetr a -N-ben zyl-3′-O-(7”-br om oh ep tyl)-1,3,2′-tr i-
N-ter t-bu toxycar bon yl-6′-N,4′-O-car bon yl-5,6-O-cycloh ex-
ylid en en ea m in e (12c). The title compound was prepared
according to the procedure described for 12a from the reaction
of 11 (1.03 g, 0.95 mmol) and 1,7-dibromoheptane to afford
compound 12c as a white solid (1.06 g, 88%). mp 85-86 °C;
IR (film) 2972, 2931, 1695 cm-1 1H NMR (CD3COCD3, 500
;
MHz) δ 7.48-6.95 (m, 20 H), 5.46 (m, 1 H), 4.99 (m, 1 H), 4.80-
4.09 (m, 9 H), 3.89 (m, 5 H), 3.48 (m, 3 H), 3.37 (m, 2 H), 3.21
(m, 2 H), 1.86 (m, 2 H), 1.63-1.38 (m, 48 H); 13C NMR (CD3-
COCD3, 100 MHz) δ 157.4, 156.2, 155.7, 152.7, 142.0, 140.4
137.8, 129.4-126.7, 112.2, 97.4, 81.4, 81.2, 81.0, 80.9, 80.2,
80.1, 74.3, 73.2, 72.9, 72.3, 71.9, 62.3, 62.0, 59.5, 58.0, 55.2,
52.8, 48.2, 46.5, 37.1, 36.8, 34.7, 34.7, 33.5, 30.9, 29.1, 28.7,
28.5, 28.4, 26.4, 26.3, 25.6, 24.4; MS (FAB, NBA) 1264, 1266
(M+); HRMS (FAB, NBA) calcd for C69H93BrN4O13 (M+)
1264.5920, 1266.5920, found 1264.5926, 1266.5914.
90-92 °C; IR (film) 3327, 3185, 2935, 1693, 1638, 1163 cm-1
;
1H NMR (CD3COCD3, 400 MHz) δ 8.25 (s, 1 H), 8.24 (s, 1 H),
7.48-7.15 (m, 20 H), 6.84 (m, 1 H), 6.23 (d, 1 H, J ) 1.6 Hz),
5.50 (d, 1 H, J ) 2.4 Hz), 5.49 (s, 1 H), 5.08 (dd, 1 H, J ) 2.4,
6.0 Hz), 4.99-2.88 (m, 26 H), 2.48 (br, 1 H), 1.76-1.25 (m, 54
H); 13C NMR (CD3COCD3, 100 MHz) δ 157.8, 157.3, 156.6,
153.8, 150.6, 141.7, 140.7, 129.7-126.8, 120.8, 114.3, 112.4,
97.4, 92.1, 91.2, 85.7, 83.2, 80.6, 80.4, 80.1, 79.5, 77.7, 76.7,
72.2, 72.2, 71.8, 71.6, 71.4, 60.2, 58.7, 55.6, 55.0, 52.4, 52.2,
48.5, 47.9, 47.5, 37.5, 37.1, 34.0, 33.0, 31.5, 30.5, 29.0, 28.8,
1,3,2′,6′-Tetr a -N-ben zyl-3′-O-(8”-br om ooctyl)-1,3,2′-tr i-
N-ter t-bu toxycar bon yl-6′-N,4′-O-car bon yl-5,6-O-cycloh ex-
ylid en en ea m in e (12d ). The title compound was prepared
according to the procedure described for 12a from the reaction
of 11 (0.92 g, 0.83 mmol) and 1,8-dibromooctane to afford
compound 12d as a white solid (1.06 g, 98%). mp 84-86 °C;
28.6, 27.8, 27.0, 26.0, 25.8, 24.8; MS (FAB, NBA) for C81H111-
N9O16 (M+) 1466, (M + Na+) 1489.
Com p ou n d 14d . The title compound was prepared accord-
ing to the procedure described for 14a from compound 12d
(744 mg, 0.58 mmol) and 13a (241 mg, 0.58 mmol) to give the
desired compound 14d as a white solid (290 mg, 34%). mp 89-
1
IR (film) 2971, 2929, 1694 cm-1
;
1H NMR (CD3COCD3, 400
90 °C; IR (film) 3329, 3183, 2932, 1694, 1638, 1164 cm-1; H
MHz) δ 7.67-6.93 (m, 20 H), 5.46 (m, 1 H), 4.95 (m, 1 H), 4.78-
4.08 (m, 9 H), 3.86 (m, 5 H), 3.48 (m, 3 H), 3.36 (m, 2 H), 3.20
(m, 2 H), 1.86 (m, 2 H), 1.63-1.38 (m, 50 H); 13C NMR (CD3-
COCD3, 100 MHz) δ 157.8, 156.7, 156.6, 156.1, 153.1, 153.0,
142.4, 142.3, 140.8 138.2, 138.1, 138.0, 129.8-127.0, 112.6,
97.7, 81.8, 81.4, 81.3, 81.2, 80.6, 77.1, 74.7, 73.5, 73.2, 72.8,
72.4, 62.4, 59.8, 58.4, 55.5, 55.4, 53.1, 48.6, 46.8, 37.4, 37.2,
37.1, 35.2, 33.9, 31.3, 29.7, 29.1, 29.0, 28.8, 28.7, 26.8, 25.9,
24.8, 24.7; MS (FAB, NBA) for C70H95BrN4O13 (M + Na+) 1303.
Com p ou n d 14a . To a mixture of 12a (784 mg, 0.63 mmol),
N6-benzoyl-2′,3′-O-isopropylideneadenosine 13a (266 mg, 0.64
mmol), and tetrabutylammonium iodide (469 mg, 1.26 mmol)
in anhydrous DMSO (10 mL) was added powdered potassium
hydroxide (80 mg, 1.43 mmol). The mixture was vigorously
stirred at room temperature under a nitrogen atmosphere for
1 h, quenched with cold water, and extracted with CH2Cl2 (3
× 30 mL). The organic layer was dried (Na2SO4) and concen-
trated to a syrup in vacuo. The residue [19a , MS (FAB, NBA)
1594 (M + Na+)] was subsequently treated with an equal
volume of 3 M aqueous sodium hydroxide and 1,4-dioxane (1:
1, 3 mL) at 50 °C for 24 h. The mixture was cooled and
extracted with CH2Cl2 (3 × 20 mL). The organic layer was
washed with water and brine, dried over MgSO4, and concen-
trated in vacuo to give a residue, which was purified by column
chromatography (SiO2, 50:1 CHCl3/MeOH) to afford the desired
compound 14a as a white solid (214 mg, 24%). mp 96-97 °C;
NMR (CD3COCD3, 400 MHz) δ 8.35 (s, 1 H), 8.33 (s, 1 H),
7.59-7.26 (m, 20 H,), 6.94 (m, 1 H), 6.32 (d, 1 H, J ) 1.6 Hz),
5.57 (m, 2 H, J ) 2.8, 6.0 Hz), 5.48 (s, 1 H), 5.17 (dd, 1 H, J )
2.4, 6.0 Hz), 5.02-3.03 (m, 26 H), 2.57 (br, 1 H), 1.75-1.30
(m, 56 H); 13C NMR (CD3COCD3, 100 MHz) δ 157.9, 157.4,
156.7, 154.0, 150.6, 142.7, 141.8, 140.8, 129.7-126.9, 120.9,
114.4, 112.5, 97.5, 92.1, 87.2, 85.8, 83.3, 81.7, 80.7, 80.5, 80.2,
77.9, 77.0, 76.5, 73.5, 73.2, 71.9, 71.6, 71.4, 60.9, 60.3, 58.7,
55.7, 55.1, 54.8, 52.6, 52.4, 48.6, 47.6, 37.5, 37.2, 33.2, 31.6,
30.6, 29.1, 28.9, 28.7, 27.9, 27.1, 26.0, 25.9, 24.9; MS (FAB,
NBA) for C83H113N9O16 (M - H+ + Na+) 1502, (M+) 1480.
Com p ou n d 15a . To a solution of 14a (100 mg, 0.07 mmol)
in a mixed solvent of CH2Cl2 (2 mL) and water (0.05 mL) was
added trifluoroacetic acid (2 mL), and the resulting solution
was stirred at room temperature for 1 h. The solvent was
subsequently evaporated to dryness in vacuo, and the residue
was purified by column chromatography (SiO2, 20:1:0.1 CHCl3/
MeOH/NH4OH) to afford compound 15a as a white powder
(65 mg, 92%). mp 77-79 °C; IR (KBr) 3389, 3329, 2924, 1644,
1
1028 cm-1; H NMR (CD3OD, 300 MHz) δ 8.59 (s, 1 H), 8.48
(s, 1 H), 7.51-7.23 (m, 20 H), 6.10 (d, 1 H, J ) 4.2 Hz), 5.95
(d, 1 H, J ) 3.9 Hz), 4.55-3.04 (m, 28 H), 2.77 (m, 1 H), 2.24
(m, 1 H), 1.69-1.28 (m, 6 H); 13C NMR (CD3OD, 300 MHz) δ
159.2, 144.3, 141.8, 130.9, 130.7, 130.5, 130.1, 129.8, 129.4,
129.2, 128.9, 128.7, 103.5, 95.1, 88.9, 84.2, 77.1, 75.8, 75.7, 75.2,
71.4, 71.0, 70.4, 69.7, 57.6, 55.7, 54.7, 54.2, 50.7, 46.3, 29.5,
29.0, 24.1, 22.4; MS (FAB, NBA) for C55H71N9O10 (M+) 1018.
Com p ou n d 15b. The title compound was prepared as
described for 15a from compound 14b (230 mg, 0.16 mmol) to
afford the desired compound 15b (125 mg, 77%). mp 76-77
°C; IR (KBr) 3390, 3331, 2926, 1646, 1029 cm-1; 1H NMR (CD3-
1
IR (film) 3329, 3186, 2934, 1695, 1639, 1162 cm-1; H NMR
(CD3COCD3, 500 MHz) δ 8.26 (s, 1 H), 8.21 (s, 1 H), 7.48-
7.14 (m, 20 H), 6.74 (m, 1 H), 6.22 (s, 1 H), 5.63 (s, 1 H), 5.47
(dd, 1 H, J ) 1.5, 6.5 Hz), 5.42 (s, 1 H), 5.06 (dd, 1 H, J ) 1.5,
5.0 Hz), 4.41 (m, 1 H), 4.80-2.80 (m, 25 H) 2.46 (m, 1 H), 1.64-

7426 J . Org. Chem., Vol. 65, No. 22, 2000
Liu et al.
OD, 500 MHz) δ 8.37 (s, 1 H), 8.18 (s, 1 H), 7.35-7.16 (m, 20
H), 6.05 (d, 1 H, J ) 4.5 Hz), 4.96 (d, 1 H, J ) 3.3 Hz), 4.55 (t,
1 H, J ) 4.5 Hz), 4.34 (t, 1 H, J ) 4.5 Hz), 4.19 (m, 1 H), 3.94-
3.24 (m, 23 H), 2.58 (m, 2 H), 2.47 (m, 1 H), 2.36 (m, 1 H),
1.57 (m, 4 H), 1.36 (m, 4 H); 13C NMR (CD3OD, 125 MHz) δ
153.2, 141.0, 139.9, 139.7, 138.0, 129.0, 129.0, 128.9, 128.8,
128.7, 127.7, 127.6, 127.4, 102.0, 89.3, 89.2, 84.6, 81.7, 77.1,
75.8, 73.6, 73.0, 72.7, 71.9, 71.2, 70.4, 61.4, 56.8, 55.2, 54.2,
53.2, 50.9, 50.3, 50.1, 30.8, 30.4, 30.0, 26.5, 26.4; MS (FAB,
NBA) 1032 (MH+); HRMS (FAB, NBA) calcd for C56H74N9O10
(MH+) 1032.5590, found 1032.5586.
Com p ou n d 15c. The title compound was prepared as
described for 15a from compound 14c (320 mg, 0.20 mmol) to
afford the desired compound 15c (198 mg, 86%). mp 75-76
°C; IR (KBr) 3389, 3330, 2926, 1644, 1029 cm-1; 1H NMR (CD3-
OD, 500 MHz) δ 8.40 (s, 1 H), 8.21 (s, 1 H), 7.37-7.18 (m, 20
H), 6.09 (d, 1 H, J ) 4.4 Hz), 5.07 (d, 1 H, J ) 3.2 Hz), 4.57 (t,
1 H, J ) 4.4 Hz), 4.38 (t, 1 H, J ) 4.8 Hz), 4.23 (m, 1 H), 3.98-
3.32 (m, 23 H), 2.67 (m, 2 H), 2.56 (m, 1 H), 2.46 (m, 1 H),
1.59 (m, 4 H), 1.34 (m, 6 H); 13C NMR (CD3OD, 125 MHz) δ
156.5, 153.2, 149.8, 140.0, 139.8, 139.6, 139.2, 139.1, 129.0,
129.0, 128.8, 127.9, 127.8, 127.6, 119.6, 101.5, 89.4, 88.3, 84.6,
81.6, 77.1, 75.8, 75.6, 73.5, 73.2, 72.4, 72.0, 71.2, 70.4, 61.2,
56.9, 55.4, 53.9, 53.1, 50.8, 50.4, 49.9, 30.8, 30.3, 30.0, 29.7,
26.4; MS (FAB, NBA) 1046 (MH+); HRMS (FAB, NBA) calcd
for C57H76N9O10 (MH+) 1046.5720, found 1046.5703.
Com p ou n d 15d . The title compound was prepared as
described for 15a from compound 14d (290 mg, 0.20 mmol) to
afford the desired compound 15d (197 mg, 95%). mp 74-75
°C; IR (KBr) 3388, 3331, 2927, 1645, 1030 cm-1; 1H NMR (CD3-
OD, 500 MHz) δ 8.37 (s, 1 H), 8.19 (s, 1 H), 7.34-7.16 (m, 20
H), 6.08 (d, 1 H, J ) 4.5 Hz), 5.07 (d, 1 H, J ) 3.5 Hz), 4.56 (t,
1 H, J ) 4.5, 5.0 Hz), 4.35 (t, 1 H, J ) 4.5 Hz), 4.20 (m, 1 H),
3.95-3.31 (m, 23 H), 2.66 (m, 2 H), 2.52 (m, 1 H), 2.38 (m, 1
H), 1.57 (m, 4 H), 1.29 (m, 8 H); 13C NMR (CD3OD, 125 MHz)
δ 156.5, 153.2, 152.8, 149.8, 141.3, 140.0, 139.8, 139.6, 139.0,
138.8, 129.0, 128.9 (3C), 127.9, 127.8 (2C), 127.7, 119.7, 101.4,
89.4, 88.0, 84.7, 81.6, 77.1, 75.8, 75.5, 73.5, 73.2, 72.3, 72.0,
71.2, 70.5, 61.2, 56.9, 55.4, 53.9, 53.1, 50.8, 50.5, 49.8, 30.9,
30.3, 30.1, 29.9, 29.8, 26.4; MS (FAB, NBA) for C58H77N9O10
(M+) 1060, (M + Na+) 1083.
96.7, 88.9, 84.6, 78.4, 77.2, 76.3, 75.1, 74.8, 73.6, 72.7, 72.1,
71.4, 70.6, 70.5, 53.8, 50.8, 49.4, 41.1, 30.3, 29.6, 29.4, 26.2,
25.9; MS (FAB, NBA) 672 (MH+); HRMS (FAB, NBA) calcd
for C28H50N9O10 (MH+) 672.3681, found 672.3678.
Com p ou n d 3. The title compound was prepared as de-
scribed for 1 from compound 15c (187 mg, 0.18 mmol) to give
the desired compound 3 (109 mg, 89%) as a white solid. IR
(KBr) 3419, 2926, 1641, 1576, 1421, 1097, 1048 cm-1; 1H NMR
(D2O, 400 MHz) δ 8.25 (s, 1 H), 8.11 (s, 1 H), 5.96 (d, 1 H, J )
4.4 Hz), 5.87 (d, 1 H, J ) 3.6 Hz), 4.68 (t, 1 H, J ) 4.8 Hz),
4.34 (t, 1 H, J ) 5.2 Hz), 4.18 (m, 1 H), 3.97 (m, 1 H), 3.91 (t,
1 H, J ) 9.6 Hz), 3.84-3.73 (m, 2 H), 3.70-3.57 (m, 3 H), 3.51
(t, 1 H, J ) 10.4, 9.2 Hz), 3.48 (m, 1 H), 3.47 (t, 1 H, J ) 9.2,
8.2 Hz), 3.38 (t, 4 H, J ) 7.2, 5.6 Hz), 3.35 (d, 1 H, J ) 3.6
Hz), 3.27 (m, 1 H), 3.16 (dd, 1 H, J ) 7.6, 13.2 Hz), 2.43 (m, 1
H), 1.83 (m, 1 H), 1.43 (m, 2 H), 1.35 (m, 2 H), 1.19 (m, 1 H),
1.08 (m, 5 H); 13C NMR (D2O, 100 MHz) δ 156.0, 154.9, 152.2,
143.8, 119.5, 99.2, 91.5, 87.2, 80.9, 79.6, 78.7, 77.5, 77.4, 76.0,
75.2, 74.5, 73.9, 73.1, 72.9, 56.3, 53.2, 51.9, 43.5, 32.8, 32.0,
31.8, 28.7, 28.8, 25.3; MS (FAB, NBA) 686 (MH+); HRMS (FAB,
NBA) calcd for C29H52N9O10 (MH+) 686.3837, found 686.3849.
Com p ou n d 4. The title compound was prepared as de-
scribed for 1 from compound 15d (200 mg, 0.19 mmol) to give
the desired compound 4 (85.4 mg, 65%) as a white solid. IR
(KBr) 3403, 2930, 1642, 1576, 1420, 1097, 1047 cm-1; 1H NMR
(D2O, 500 MHz) δ 8.12 (s, 1 H), 7.98 (s, 1 H), 5.85 (d, 1 H, J )
4.0 Hz), 5.85 (d, 1 H, J ) 3.0 Hz), 4.59 (t, 1 H, J ) 4.5 Hz),
4.24 (t, 1 H, J ) 5.0, 4.5 Hz), 4.07 (m, 1 H), 3.85 (m, 1 H), 3.79
(t, 1 H, J ) 10.0, 9.5 Hz), 3.69 (t, 1 H, J ) 9.5, 10.0 Hz), 3.64
(t, 1 H, J ) 7.0, 7.5 Hz), 3.57-3.46 (m, 3 H), 3.42-3.34 (m, 3
H), 3.27 (m, 4 H), 3.24 (d, 1 H, J ) 3.5 Hz), 3.16 (m, 1 H), 3.07
(dd, 1 H, J ) 7.0, 13.5 Hz), 2.32 (m, 1 H), 1.72 (m, 1 H), 1.32
(m, 2 H), 1.22 (m, 2 H), 1.09 (m, 1 H), 0.95 (m, 7 H); 13C NMR
(D2O, 125 MHz) δ 155.7, 152.9, 149.4, 140.6, 96.4, 88.4, 84.3,
78.3, 76.8, 75.8, 74.5, 73.1, 72.3, 71.6, 71.0, 70.2, 69.9, 53.4,
50.4, 49.0, 40.6, 29.9, 29.1, 29.0, 28.9, 25.8, 25.5, 25.0; MS
(FAB, NBA) 700 (MH+); HRMS (FAB, NBA) calcd for C30H54
-
N9O10 (MH+) 700.3994, found 700.3987.
1,3,2′,6′-Tetr a-N-ben zyl-4′-O-ben zyl-1,3,2′,6′-tetr a-N-ter t-
bu toxyca r bon yl-3′-O-ter t-bu tyld im eth ylsilyl-5,6-O-cyclo-
h exylid en en ea m in e (16). To an ice-cold solution of 8 (4.74
g, 5.17 mmol) in anhydrous DMF (40 mL) was added sodium
hydride (60% dispersion in mineral oil, 2.48 g, 0.06 mol), and
benzyl bromide (12.40 mL, 18.33 mmol) was subsequently
added in one portion. The mixture was stirred at room
temperature under a nitrogen atmosphere for 2 h. The reaction
was quenched with a 25% aqueous HOAc solution (5 mL),
diluted with water (20 mL), and extracted with CH2Cl2 (3 ×
50 mL). The organic layer was washed with water, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo to afford
a crude product, which was purified by column chromatogra-
phy (SiO2, 10:1 hexane/ethyl acetate) to give compounds 16
(3.00 g, 48%) and 9 (2.40 g, 34%) as white solids. Compound
16: Rf 0.62 (8:1 hexane/ethyl acetate); mp 104-105 oC; IR
(film) 2931, 1695, 1143 cm-1; 1H NMR (CD3COCD3, 500 MHz)
δ 7.55-7.24 (m, 25 H), 5.51 (m,1 H), 5.04-3.05 (m, 22 H),
1.65-1.19 (m, 47 H), 0.93 (s, 9 H), 0.25 (s, 6 H); 13C NMR (CD3-
COCD3, 125 MHz) δ 156.9, 156.0, 152.8, 142.3, 140.0, 139.4,
129.1, 128.8, 128.7, 128.5, 127.8, 127.4, 127.0, 126.5, 112.1,
95.4, 80.1, 72.3, 70.9, 59.4, 55.1, 48.0, 36.9, 28.5, 28.2, 28.1,
26.6, 26.4, 25.5, 24.4, 18.5, -3.31; MS (FAB, NBA) for C79H110
N4O14Si (M + Na+) 1390.
Com p ou n d 1. To a suspension of 10% palladium on carbon
(10 mg) and 20% palladium hydroxide on carbon (14 mg) in
anhydrous methanol was added a solution of compound 15a
(15 mg, 0.01 mmol) in anhydrous methanol-acetic acid (4 mL,
3:1), and the resulting mixture was stirred under a hydrogen
atmosphere for 48 h. The mixture was filtered through Celite
and the filtrate was concentrated in vacuo to give the title
compound as a white solid (8 mg, 83%). IR (KBr) 3403, 2931,
1
1643, 1573, 1413, 1097, 1048 cm-1; H NMR (D2O, 400 MHz)
δ 8.23 (s, 1 H), 8.07 (s, 1 H), 5.93 (d, 1 H, J ) 4.4 Hz), 5.84 (d,
1 H, J ) 3.6 Hz), 4.63 (t, 1 H, J ) 4.8, 4.8 Hz), 4.31 (t, 1 H, J
) 5.2, 4.8 Hz), 4.18 (m, 1 H), 3.93 (m, 1 H), 3.87 (t, 1 H, J )
10.0 Hz), 3.80-3.73 (m, 2 H), 3.70-3.55 (m, 4 H), 3.49 (t, 1 H,
J ) 9.6, 10.0 Hz), 3.41 (m, 4 H,), 3.31 (dd, 1 H, J ) 3.6, 13.6
Hz), 3.30 (dd, 1 H, J ) 3.6, 10.8 Hz), 3.25 (m, 1 H), 3.12 (dd,
1 H, J ) 7.6, 13.6 Hz), 2.41 (m, 1 H), 1.83 (m, 1 H), 1.45 (m,
4 H), 1.18 (m, 2 H); 13C NMR (D2O, 100 MHz) δ 155.7, 152.7,
149.6, 141.2, 96.8, 88.9, 84.5, 78.5, 77.2, 76.3, 75.0, 74.6, 73.6,
72.5, 72.1, 71.4, 70.6, 64.4, 53.8, 50.8, 49.4, 41.1, 30.1, 29.4,
22.8; MS (FAB, NBA) 658 (MH+); HRMS (FAB, NBA) calcd
for C27H48N9O10 (MH+) 658.3525, found 658.3538.
Com p ou n d 2. The title compound was prepared as de-
scribed for 1 from compound 15b (80 mg, 0.08 mmol) to give
the desired compound 2 (35 mg, 67%) as a white solid. IR (KBr)
3415, 2931, 1641, 1572, 1412, 1097, 1049 cm-1; 1H NMR (D2O,
400 MHz) δ 8.18 (s, 1 H), 8.02 (s, 1 H), 5.90 (d, 1 H, J ) 4.4
Hz), 5.84 (d, 1 H, J ) 3.2 Hz), 4.60 (t, 1 H, J ) 4.4 Hz), 4.29
(t, 1 H, J ) 5.2 Hz), 4.14 (m, 1 H), 3.92 (m, 1 H, J ) 3.2, 7.6
Hz), 3.86 (t, 1 H, J ) 10.4, 9.2 Hz), 3.77 (t, 1 H, J ) 10.0 Hz),
3.70-3.35 (m, 4 H), 3.47 (t, 1 H, J ) 10.0 Hz), 3.44 (m, 1 H),
3.42 (t, 1 H, J ) 9.6 Hz), 3.34 (t, 4 H, J ) 6.4, 5.6 Hz), 3.30 (d,
1 H, J ) 3.6 Hz), 3.22 (m, 1 H), 3.11 (dd, 1 H, J ) 7.6, 13.6
Hz), 2.39 (m, 1 H), 1.81 (m, 1 H), 1.37 (m, 4 H), 1.08 (m, 4 H);
13C NMR (D2O, 100 MHz) δ 155.8, 152.8, 149.6, 141.0, 119.5,
1,3,2′,6′-Tetr a-N-ben zyl-4′-O-ben zyl-1,3,2′,6′-tetr a-N-ter t-
bu toxyca r bon yl-5,6-O-cycloh exylid en en ea m in e (17). To
a solution of compound 16 (5.70 g, 4.73 mmol) in dry THF (50
mL) was added a 1.0 M solution of tetra-n-butylammonium
fluoride in THF (14.6 mL, 14.6 mmol) at 0 °C. The mixture
was stirred for 10 min under a nitrogen atmosphere, quenched
with ice water, and concentrated to dryness in vacuo. The
residue was dissolved in CH2Cl2, extracted with water, then
with brine and purified on a column (SiO2, 9:1 hexane/ethyl
acetate) to afford the desired compound 17 as a white solid
(3.28 g, 63%): Rf 0.48 (6:1 hexane/ethyl acetate); mp 112-
o
114 C; IR (film) 3450, 2975, 2934, 1693 cm-1; 1H NMR (CD3-
COCD3, 500 MHz) δ 7.51-7.10 (m, 25 H), 5.52 (m, 1 H), 4.80-

Inhibitors of Aminoglycoside 3′-Phosphotransferases
J . Org. Chem., Vol. 65, No. 22, 2000 7427
4.69 (m, 6 H), 4.49-4.24 (m, 5 H), 3.57-3.24 (m, 3 H), 2.40
(m, 1 H), 1.67-1.21 (m, 47 H); 13C NMR (CD3COCD3, 125 MHz)
δ 157.6, 156.2, 142.6, 139.9, 129.2-126.5, 112.1, 96.2, 81.8,
80.3, 79.8, 75.6, 75.0, 73.0, 70.3, 61.9, 61.2, 59.7, 55.2, 50.2,
47.4, 46.8, 37.0, 36.9, 32.6, 28.5, 28.4, 28.3, 25.6, 24.4; MS
(FAB, NBA) for C73H96N4O14 (M+) 1253.
to dryness in vacuo. The resulting residue was subsequently
treated with an equal volume of 3 M aqueous sodium hydrox-
ide and 1,4-dioxane (1:1, 2 mL) at 50 °C for 24 h. The mixture
was cooled and extracted with CH2Cl2 (3 × 10 mL). The organic
layer was washed with water and with brine, dried over
MgSO4, concentrated, and purified on a column (SiO2, 50:1
CHCl3/MeOH) to afford 14a as a white solid (55 mg, 48%),
which was identical with the title compound prepared by the
alternative procedure (prepared from 12a and 13a ) in all
respects.
1,3,2′,6′-Tet r a -N-b en zyl-4′-O-Ben zyl-1,3,2′,6′-t et r a -N-
ter t-bu t oxyca r bon yl-3′-O-(5”-b r om op en t yl)-5,6-O-cyclo-
h exylid en en ea m in e (18a ). To a stirred solution of compound
17 (1.50 g, 1.20 mmol) and 1,5-dibromopentane (3.54 mL, 25.32
mmol) in DMSO (15 mL) was added powdered potassium
hydroxide (336 mg, 6.00 mmol). The resulting mixture was
vigorously stirred at room temperature under a nitrogen
atmosphere for 30 min. The reaction was quenched with cold
water (5 mL), extracted with CH2Cl2 (3 × 50 mL), dried (Na2-
SO4), and concentrated in vacuo to furnish a syrup, which was
purified by column chromatography (SiO2, 9:1 hexane/ethyl
acetate) to give the title compound (1.36 g, 81%) as a white
solid: Rf 0.71 (6:1 hexane/ethyl acetate); mp 89-90 °C; IR
Com p ou n d 21a . To a mixture of 18a (1.35 g, 0.96 mmol),
N,6N6-dibenzoyl-2′,3′-O-isopropylideneadenosine 13b (595 mg,
1.16 mmol), and tetrabutylammonium iodide (1.42 g, 3.84
mmol) in anhydrous DMSO (40 mL) was added powdered
potassium hydroxide (270 mg, 4.82 mmol). The mixture was
vigorously stirred at room temperature under a nitrogen
atmosphere for 1 h, quenched with cold water, extracted with
CH2Cl2 (3 × 50 mL), dried, and concentrated in vacuo to give
a residue, which was purified by column chromatography
(SiO2, 70:1 CHCl3/MeOH) to give the desired compound 21a
(801 mg, 48%) as a white solid: Rf 0.44 (50:1 CHCl3/MeOH);
mp 99-100 °C; IR (film) 3303, 2932, 1694, 1163 cm-1; 1H NMR
(CD3COCD3, 400 MHz) δ 8.69 (s, 1 H), 8.49 (s, 1 H), 8.15 (d, 2
H, J ) 7.6 Hz), 7.65 (t, 1 H, J ) 7.2 Hz), 7.56 (t, 2 H, J ) 7.6
Hz), 7.47-7.20 (m, 25 H), 6.35 (d, 1 H, J ) 2.0 Hz), 5.50 (m,
2 H), 5.10 (d, 1 H, J ) 3.6 Hz), 4.94-3.00 (m, 29 H), 1.67-
1.20 (m, 59 H); 13C NMR (CD3COCD3, 100 MHz) δ 166.1, 157.7,
156.6, 156.0, 153.0, 152.9, 151.4, 142.7, 139.0, 135.2, 133.4,
129.6-126.1, 114.5, 112.5, 96.5, 92.1, 87.2, 85.8, 83.2, 82.1,
81.2, 80.5, 80.4, 79.6, 78.6, 77.2, 75.6, 73.2, 72.0, 71.8, 62.6,
60.2, 58.7, 55.5, 50.5, 48.2, 47.6, 42.9, 37.4, 37.3, 33.9, 32.9,
31.6, 31.4, 30.4, 29.0, 28.8, 28.7, 27.8, 25.9, 24.9, 24.8, 23.5;
MS (FAB, NBA) for C98H125N9O19 (M+) 1733, (M + Na+) 1756.
Com p ou n d 21b. The title compound was prepared as
described for 21a from compound 18b (927 mg, 0.65 mmol) to
give compound 21b (736 mg, 64%): Rf 0.46 (50:1 CHCl3/
(film) 2934, 1693 cm-1 1H NMR (CD3COCD3, 500 MHz) δ
;
7.54-7.23 (m, 25 H), 5.51 (m, 1 H), 4.91-3.07 (m, 24 H), 1.79
(m, 2 H), 1.64-1.22 (m, 53 H); 13C NMR (CD3COCD3, 125 MHz)
δ 157.2, 156.2, 142.3, 142.1, 139.7, 129.1-126.5, 112.1, 96.1,
81.7, 80.9, 80.0, 78.2, 76.8, 75.3, 73.2, 72.8, 59.8, 58.2, 55.1,
50.0, 47.7, 47.1, 37.0, 36.8, 34.4, 33.4, 30.3, 28.5, 28.4, 28.2,
25.5, 25.2, 24.4, 24.4; MS (FAB, NBA) for C78H105BrN4O14 (M
+ H+) 1403.
3′-O-(6′′-Br om oh exyl)-4′-O-ben zyl-5,6-O-cycloh exyliden e-
tetr a-N-ben zyl-tetr a-N-ter t-bu toxycar bon yln eam in e (18b).
The title compound was prepared according to the procedure
described for 18a from the reaction of 17 (1.18 g, 1.08 mmol)
and 1,6-dibromohexane to afford compound 18b (1.06 g, 59%)
as a white solid: Rf 0.73 (6:1 hexane/ethyl acetate 6:1); mp
86-88 °C; IR (film) 2933, 1693 cm-1 1H NMR (CD3COCD3,
;
500 MHz) δ 7.47-7.24 (m, 25 H), 5.53 (m, 1 H), 4.93-3.05 (m,
24 H), 1.79 (m, 2 H), 1.66-1.20 (m, 55 H); 13C NMR (CD3-
COCD3, 125 MHz) δ 157.6, 156.2, 155.7, 142.4, 142.1, 140.3,
139.5, 129.2-126.5, 112.1, 96.1, 81.8, 80.8, 80.1, 80.0, 78.2,
76.9, 75.2, 73.1, 72.8, 72.5, 59.9, 58.2, 55.1, 54.8, 50.2, 47.8,
47.2, 37.0, 36.9, 34.6, 33.3, 31.1, 28.7, 28.6, 28.5, 28.3, 25.8,
25.6, 24.5, 24.4; MS (FAB, NBA) for C79H107BrN4O14 (M+) 1416,
(M + H+ + Na+) 1440.
MeOH); mp 93-95 °C; IR (film) 3305, 2934, 1695, 1164 cm-1
;
1H NMR (CD3COCD3, 500 MHz) δ 8.67 (s, 1 H), 8.60 (s, 1 H),
8.15 (d, 2 H, J ) 8.0 Hz), 7.60 (t, 1 H, J ) 7.5 Hz), 7.51 (t, 2
H, J ) 7.5 Hz), 7.47-7.10 (m, 25 H), 6.35 (d, 1 H, J ) 2.0 Hz),
5.48 (m, 2 H), 5.09 (m, 1 H), 4.91-3.00 (m, 29 H), 1.65-1.19
(m, 61 H); 13C NMR (CD3COCD3, 125 MHz) δ 166.1, 157.3,
156.3, 155.7, 152.6, 152.5, 151.0, 143.3, 142.4, 142.2, 140.3,
139.7, 134.8, 133.2, 129.3-125.5, 114.2, 112.2, 96.2, 91.6, 86.8,
85.5, 82.8, 81.72, 80.8, 80.2, 80.1, 79.8, 78.2, 76.8, 75.4, 75.2,
73.1, 72.8, 71.9, 71.5, 62.3, 60.5, 59.9, 58.2, 55.1, 50.2, 47.3,
42.9, 36.9, 33.5, 32.5, 31.6, 31.3, 31.4, 28.7, 28.6, 28.4, 27.6,
26.6, 26.5, 26.2, 25.7, 24.4; MS (FAB, NBA) for C99H127N9O19
(M + Na+) 1756.
Com p ou n d 19b. To a mixture of 12a (100 mg, 0.08 mmol),
N6,N6-dibenzyl-2′,3′-O-isopropylideneadenosine 13c (48 mg,
0.10 mmol), and tetrabutylammonium iodide (60 mg, 0.16
mmol) in anhydrous DMSO (8 mL) was added powdered
potassium hydroxide (40 mg, 0.71 mmol). The mixture was
vigorously stirred at room temperature under a nitrogen
atmosphere for 4 h, quenched with cold water, and extracted
with CH2Cl2 (3 × 10 mL). The organic layer was dried
(Na2SO4) and concentrated to a syrup in vacuo to give a
residue, which was purified by column chromatography
(SiO2, 5:2 hexane/ethyl acetate) to afford the desired compound
19b as a white solid (48 mg, 36%): Rf 0.19 (hexanes:EtOAc,
Com p ou n d 22a . To a solution of 21a (801 mg, 0.46 mmol)
in dry MeOH was added potassium carbonate (200 mg, 1.44
mmol), and the resulting mixture was stirred at room tem-
perature under a nitrogen atmosphere for 6 h. The mixture
was filtered and concentrated to dryness in vacuo. The residue
was dissolved in CH2Cl2, extracted with water (10 mL) and
then with brine, dried (Na2SO4), and concentrated to give
a crude product, which was subsequently treated with tri-
fluoroacetic acid (10 mL) in a mixture of CH2Cl2 (10 mL) and
water (0.5 mL). The reaction mixture was allowed to stir at
room temperature for 1 h. The mixture was concentrated to
dryness in vacuo and purified on a column (SiO2, 20:1:0.1
CHCl3/MeOH/NH4OH) to afford compound 22a (270 mg, 53%)
as a white powder: Rf 0.25 (20:1:0.1 CHCl3/MeOH/NH4OH);
mp 75-77 °C; IR(KBr) 3323, 2926, 2861, 1641, 1601, 1072
1
5:2); mp 84-86 °C; IR (film) 2933, 1696, 1579, 1158 cm-1; H
NMR (CD3COCD3, 500 MHz) δ 8.34 (s, 1 H), 8.24 (s, 1 H),
7.44-7.09 (m, 30 H), 6.94 (d, 1 H, J ) 6.5 Hz), 6.28 (s, 1 H),
5.45 (dd, 1 H, J ) 2.5, 6.0 Hz), 5.08 (dd, 1 H, J ) 2.5, 6.5 Hz),
4.99 (br, 1 H), 4.90 (t, 2 H, J ) 15.0 Hz), 4.41 (d, 1 H, J ) 2.5
Hz), 4.77-3.11 (m, 27 H), 1.61-1.18 (m, 50 H); 13C NMR
(CD3COCD3, 125 MHz) δ 154.7, 152.3, 150.8, 141.2, 139.7,
138.3, 137.0, 128.6, 128.0, 127.8, 127.7, 127.6, 127.4, 127.1,
126.7, 126.5, 126.2, 125.9, 119.7, 113.3, 111.5, 96.6, 90.5, 85.9,
84.6, 82.0, 80.2, 80.1, 79.4, 73.5, 72.1, 71.1, 71.0, 70.7, 61.2,
58.7, 57.2, 54.4, 52.0, 47.4, 45.7, 36.3, 35.9, 34.8, 29.9, 27.8,
27.6, 27.5, 26.7, 24.7, 23.6, 22.3, 22.2; MS (FAB, NBA) for
1
cm-1; H NMR (CD3OD, 300 MHz) δ 8.36 (s, 1 H), 8.17 (s, 1
H), 7.34-7.10 (m, 25 H), 6.03 (d, 1 H, J ) 4.2 Hz), 4.98 (d, 1
H, J ) 3.6 Hz), 4.55 (d, 1 H, J ) 11.4 Hz), 4.49 (t, 1 H, J ) 4.8
Hz), 4.32 (t, 1 H, J ) 4.8 Hz), 4.17 (m, 1 H), 3.95-3.32 (m, 23
C
94H117N9O17 (M+) 1645, (M + Na+) 1668.
Alter n a tive Rou te to Com p ou n d 14a . To a mixture of
12a (100 mg, 0.08 mmol), N,6N6-di-benzoyl-2′,3′-O-isopro-
pylideneadenosine 13b (36 mg, 0.07 mmol), and tetrabutyl-
ammonium iodide (60 mg, 0.16 mmol) in anhydrous DMSO (4
mL) was added powdered potassium (20 mg, 0.34 mmol). The
mixture was vigorously stirred at room temperature under a
nitrogen atmosphere for 1 h, quenched with cold water,
extracted with CH2Cl2 (3 × 10 mL), dried, and concentrated
H), 2.63-2.34 (m, 4 H), 1.70-1.61 (m, 4 H), 1.43 (m, 2 H); 13
C
NMR (CD3OD, 75 MHz) δ 152.3, 139.3, 139.1, 138.0, 128.1-
127.8 (m), 127.3, 127.2, 126.9, 126.8, 126.7, 126.6, 118.8, 101.0,
88.5, 87.6, 83.7, 81.6, 79.8, 76.4, 74.9, 74.1, 72.8, 71.4, 70.9,
70.2, 69.6, 60.6, 56.1, 54.6, 53.2, 52.3, 50.2, 49.8, 30.0, 29.7,
29.2, 22.7; MS (FAB, NBA) for C62H77N9O10 (M+), (M + Na+)
1131.

7428 J . Org. Chem., Vol. 65, No. 22, 2000
Liu et al.
Sch em e 1
Sch em e 2
Com p ou n d 22b. The title compound was prepared as
described for 22a from compound 21b (736 mg, 0.42 mmol) to
give the desired compound 22b (261 mg, 55%) as a white
powder: Rf 0.28 (20:1:0.1 CHCl3/MeOH/NH4OH); mp 76-78
°C; IR(KBr) 3322, 2926, 2860, 1642, 1602, 1071 cm-1; 1H NMR
(CD3OD, 500 MHz) δ 8.36 (s, 1 H), 8.18 (s, 1 H), 7.35-7.12
(m, 25 H), 6.07 (d, 1 H, J ) 4.5 Hz), 5.10 (d, 1 H, J ) 3.5 Hz),
4.54 (d, 1 H, J ) 11.0 Hz), 4.53 (t, 1 H, J ) 4.5 Hz), 4.34 (t, 1
H, J ) 4.5 Hz), 4.20 (m, 1 H), 3.96-3.29 (m, 23 H), 2.65-2.36
(m, 4 H), 1.54 (m, 4 H), 1.33 (m, 4 H); 13C NMR (CD3OD, 500
MHz) δ 156.5, 153.0, 149.8, 140.6, 140.0, 139.8, 139.1, 139.0,
138.8, 129.2-128.7 (m), 128.2, 128.1, 127.9, 127.8, 127.8, 127.6,
119.7, 101.2, 89.4, 87.4, 84.6, 82.2, 80.6, 77.2, 75.9, 75.8, 75.5,
74.9, 73.6, 71.9, 71.8, 71.2, 70.5, 61.43, 57.0, 55.6, 53.8, 53.0,
50.8, 50.7, 30.9, 30.4, 30.0, 26.6, 26.5; MS (FAB, NBA) for
C
63H79N9O10 (M+) 1122, (M + Na+) 1145.

Inhibitors of Aminoglycoside 3′-Phosphotransferases
J . Org. Chem., Vol. 65, No. 22, 2000 7429
Sch em e 3
Sch em e 4
Resu lts a n d Discu ssion
We envisioned that the 5′-hydroxyl of adenosine could
be tethered to the 3′-hydroxyl of neamine, to bridge the
gap between the subsites where the two substrates bind.
The closer the length of the tether comes to the distance
of the separation between the two subsites, the better
should be the affinity for the conjoint molecule with the
given tether. If the length of the tether is either shorter
or longer than optimal, the enzyme affinity for the
compound should decrease. In principle, the binding of
one substrate (i.e., one-half of the molecule) drives the
binding of the other for entropic reasons. Therefore, when
and if the length of the given tether might match the
separation of the gap between the two subsites, there
exists the potential for strong inhibition of the enzyme,
despite the fact that most tethers cannot be expected to
be a close structural mimic of the triphosphoryl moiety
of ATP and would not have the specific interactions in
existence between APH(3′) and ATP.
We have used molecular modeling to investigate what
the proper length of the tether should be for a hypotheti-
cal phosphotransferase reaction. This analysis takes into
account the design criterion for the inhibitors in terms
of the tether length, but no assumption regarding con-
formations is made, because it is not necessary to do so.
We constructed molecular models of various lengths of
carbon chains to span such distances. We also envisioned
that the attachement of the tether to the aminoglycoside
and adenosine should be via ether linkages, which are
stable in acqueous solution. Compounds 1-4 were four
such inhibitors of our design, which were synthesized by
two routes according to the following procedures given
in Schemes 1-5.
Syn t h et ic R ou t e A. Neamine hydrochloride (5)
was prepared from methanolysis of the commercially
available neomycin sulfate, as reported earlier.17,18,19
Treatment of this compound with di-tert-butyl dicarbon-
ate afforded the tetra-N-Boc protected derivative 6.
Selective protection of C-5 and C-6 hydroxyl groups via
treatment by 1,1-dimethoxycyclohexane20 resulted in the
formation of the 5,6-cyclohexylidene derivative 7. The 3′-
hydroxyl group of 7 was selectively protected with tert-
butyldimethylsilyl (TBS) group to yield the silyl deriva-
tive 8. Several attempts to protect the C-4′-hydroxyl
group of 8 by an ether linkage failed to give any
acceptable result. However, treatment of 8 with excess
of benzyl bromide and sodium hydride produced the cyclic
carbamate derivative 9. This compound was treated with
trifluoroacetic acid to remove the cyclohexylidene and the
Boc groups to afford 10, which proved to be useful in
assignment of the sites of modifications by NMR. Re-
moval of the silyl group of 9 with tetrabutylammonium
fluoride provided the intermediate 11 having the 3′
hydroxyl group free for further structural manipulation.
The reaction of this compound with the corresponding
dibromo compounds Br(CH2)nBr (n ) 5-8), in the pres-
ence of powdered potassium hydroxide by application of
the modified William’s protocol,21 afforded in high yields
the 3′-O-bromoalkyl derivatives 12a -d , respectively
(17) Dutcher, J .; Donin, M. J . Am. Chem. Soc. 1952, 74, 3420-3422.
(18) Ford, J . H.; Bergy, M. E.; Brooks, A. A.; Garrett, E. R.; Alberti,
J .; Dryer, J . R.; Carter, H. E. J . Am. Chem. Soc. 1955, 77, 5311-5314.
(19) Grapsas, I.; Cho, Y. I.; Mobashery, S. J . Org. Chem. 1994, 59,
1918-1922.
(20) Tohma, S.; Yoneta, T.; Fukatsu, S. J . Antibiot. 1980, 33, 671-
673.

7430 J . Org. Chem., Vol. 65, No. 22, 2000
Liu et al.
F igu r e 3. Dixon plots showing competitive inhibition of APH(3′)-IIa by compound 3 against substrates ATP (panel A) and
kanamycin A (panel B). Substrate concentrations are indicated to the right for each set of determinations.
Sch em e 5
(Scheme 1). We encountered many difficulties in coupling
of compounds 12 with the adenosine derivative 13a
(prepared by literature procedures).22,23 We solved this
problem by the use of excess tetrabutylammonium iodide
as a promoter. Because of the tedious separation of the
coupling products, the subsequent basic hydrolysis with
aqueous sodium hydroxide24,25 was performed to give the
partially deprotected compounds 14a -d , which were
purified by silica gel chromatography. The acid-labile
protective groups, tert-butoxycarbonyl, cyclohexylidene,
and isopropylidene groups, were simultaneously removed
by treatment of 14 with 95% trifluoroacetic acid to
give the tetra-N-benzyl derivatives 15a -d , respectively.
Finally, hydrogenolysis of compounds 15 over palla-
(21) J ohnstone, R. A. W.; Rose M. E. Tetrahedron 1979, 35, 2169-
2173.
(22) Maguire A. R.; Meng W.-D.; Robert S. M.; Willetts A. J . Chem.
Soc., Perkin Trans. 1 1993, 15, 1795-1808.
(23) Vrudhula, V. M.; Kappler, F.; Afshar, C.; Gnell, S. L.; Lessinger,
L.; Hampton, A. J . Med. Chem. 1989, 32, 885-890.
(24) Takahashi, Y.; Tsuneda, S.; Tsuchiya, T.; Koyama, Y.; Ume-
zawa, S. Carbohydr. Res. 1992, 232, 89-105.
(25) Kobayashi, Y.; Tsuchiya, T.; Umezawa, S.; Yoneta, T.; Fukatsu,
S.; Umezawa, H. Bull. Chem. Soc. J pn. 1987, 62, 713-720.

Inhibitors of Aminoglycoside 3′-Phosphotransferases
J . Org. Chem., Vol. 65, No. 22, 2000 7431
Ta ble 1. Kin etic P a r a m eter s for In h ibition of AP H(3′)-Ia
a n d AP H(3′)-IIa by Com p ou n d s 1-4a
These findings showed that the N,N-dibenzoyl-protect-
ed adenosyl derivative 13b is the proper coupling coun-
terpart for the reaction with the neamine derivatives.
Therefore, this compound was chosen to undergo reaction
with 18a and 18b to provide the N6-benzoyl protected
adenosyl derivatives 21a and 21b in 48% and 64% yields,
respectively (Scheme 5). Subsequent benzoyl group depro-
tection of these compounds with potassium carbonate
in anhydrous methanol, followed by treatment with
trifluoroacetic acid furnished the tetra-N-benzyl-protect-
ed derivatives 22a and 22b, in good yields. Finally,
hydrogenolysis of these compounds in the presence of
the cocatalysts, palladium and palladium hydroxide on
carbon, gave the desired compounds 1 and 2 in excellent
yields (Scheme 5).
Ki (µM)
variable
inhibitor
1
substrate
APH(3′)-Ia
APH(3′)-IIa
ATP
kanamycin A
ATP
kanamycin A
ATP
kanamycin A
ATP
558 ( 65
32 ( 5
10 ( 8
3 ( 2
22 ( 14
9 ( 1
35 ( 26
224 ( 61
10 ( 4
18 ( 11
3 ( 2
2
3
4
5 ( 3
6 ( 5
17 ( 4
9 ( 5
kanamycin A
14 ( 2
a
The Km for kanamycin A and ATP for APH(3′)-Ia are 2.8 (
0.3 µM and 77 ( 4 µM, respectively. The Km for kanamycin A and
ATP for APH(3′)-IIa are 3.1 ( 0.5 µM and 69 ( 13 µM, respectively.
dium-C and palladium hydroxide-C in methanol and
acetic acid26,27 afforded the desired compounds 1-4 in
good to high yields (Scheme 2).
Compounds 1-4 were tested with APH(3′)-Ia and
APH(3′)-IIa as potential inhibitors. All four compounds
were competitive inhibitors for the enzyme. Regardless
of which substrate (kanamycin A or ATP) was varied in
the kinetics experiments, in general, the best inhibition
was afforded by compounds 2 and 3 (Table 1; Figure 3).
Furthermore, the Ki values measured for 2 and 3 were
relatively within a few folds of each other.
Syn th etic Rou te B. One of the key steps in the
synthesis of compounds 1-4 is the coupling reaction of
the appropriately protected adenosine derivative and its
aminoglycoside counterpart. During the course of syn-
thesis of these compounds we found that the N,N-
dibenzoyl-protected adenosine derivative (13b) and the
bromoalkylated neamines (18) are the best building
blocks for synthesis of these conjount molecules in good
yields. Therefore a slightly modified procedure was
employed to prepare the neamine derivative 16. To make
this compound, benzyl bromide was added to a solution
of neamine derivative 8 and NaH in one portion, and the
reaction was stopped after 2 h (in contrast to the first
synthetic route in which benzyl bromide was added
dropwise over 30 min). Products of this reaction were
compounds 9 and 16 in 34% and 48% yields, respectively
(Scheme 3). Desilylation of 16 with tetrabutylammonium
fluoride gave the key intermediate 17, which was bromo-
alkylated with dibromoalkanes (n ) 5 and 6) in the
presence of powdered potassium hydroxide to give 3-O-
bromoalkyl derivatives 18a and 18b in good yields. In
the most difficult step of the synthesis, which was the
coupling of 18 with the adenosine moiety, the major
byproduct of the reaction was elimination of hydrogen
bromide and formation of 3′-O-(4”-alkenyl) neamine
derivatives. To find the optimal condition for the coupling
reaction, three adenosine derivatives 13a -c having
various protecting groups on the amino group at the C4
position were prepared22,23,28 and used in the above
reaction (Scheme 4). Reaction of 12a with either 13a or
13b in the presence of tetrabutylammonium iodide
afforded adduct 19a , which was subsequently treated
with aqueous sodium hydroxide to give the benzoyl-
deprotected derivative 14a . The overall yield for the two-
step for these reactions were 24% from 13a and 48% from
13b, respectively. Treatment of 12a with 13c furnished
the tethered compound 19b in 36% yield. Unfortunately
19b did not undergo a facile and complete 4-N-benzyl
group deprotection to give the desired compound 1.
The tethers in compounds 1-4 are straight chain
hydrocarbons. We note that the X-ray structure for the
complex of APH(3′)-IIIa with Mg2+-ADP indicated ap-
proximately a half-dozen electrostatic interactions by the
enzyme with the magnesium-diphosphoryl moiety of ADP
(Figure 1). These interactions would obviously be absent
in a simple hydrocarbon tether in interactions of com-
pound 1-4 with the enzyme. These observations in part
explain why the affinities of the enzymes for our inhibi-
tors are only slightly better than for the substrates
themselves, despite the favorable entropic factor for
binding to molecules such as 1-4. Nonetheless, these
compounds are the best known inhibitors for APH(3′)s
to date, which occupy both subsites within the active site.
These compounds were investigated for slow binding to
APH(3′)s, both with and without preincubation of the
inhibitors. They all served as linear competitive inhibi-
tors with no evidence (data not shown) for slow binding.
Availability of these compounds allows us to attempt
to study by X-ray analysis the complexes of the APH(3′)s
with these inhibitors. This effort should generate a
structural basis for the design of the next generation of
these molecules that should enjoy more favorable inhibi-
tion of these important bacterial enzymes. These efforts
are currently under way.
Ack n ow led gm en t. This work was supported by the
National Institutes of Health.
Note Ad d ed in P r oof: While this manuscript was in
review, a report that addressed inhibition of amino-
glycoside-modifying enzymes appeared in the literature
(Sucheck, S. J .; Wang, A. L.; Koeller, K. M.; Boehr, D.
D.; Draker, K.; Sears, P.; Wright, G. D.; Wang, C. H. J .
Am. Chem. Soc. 2000, 122, 5230-5231).
(26) Pearlman, W. M. Tetrahedron. Lett. 1967, 1663-1664.
(27) Kozikowski, A. P.; Xia, Y.; Reddy, E. R.; Tu¨ckmantel, W.; Hanin,
I.; Tang, X. C. J . Org. Chem. 1991, 56, 4636-4645.
(28) (a) Kato T.; Arakawa E.; Ogawa S.; Suzumura Y. Chem. Pharm.
Bull. 1986, 34, 3635-3643. (b) Takeshi E.; Zemlicka J . J . Org. Chem.
1979, 44, 3652-3656.
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