Probing functional diversity in pactamycin toward antibiotic, antitumor, and antiprotozoal activity

Article abstract of DOI:10.1016/j.bmc.2013.01.037

A total of eight new analogs of pactamycin were prepared and tested alongside pactamycin and three of its natural congeners for antibacterial, anticancer, and antiprotozoal activities. The present study highlights the effects of changing the urea and anil

Full text of DOI:10.1016/j.bmc.2013.01.037

Bioorganic & Medicinal Chemistry 21 (2013) 1775–1786
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Probing functional diversity in pactamycin toward antibiotic,
antitumor, and antiprotozoal activity
a
a
Stephen Hanessian a, , Ramkrishna Reddy Vakiti , Amit Kumar Chattopadhyay ,
⇑
Stéphane Dorich a, Christian Lavallée b
a Department of Chemistry, Université de Montréal, Station Centre Ville, C.P. 6128, Montréal, Qc, Canada H3C 3J7
b Hôpital Maisonneuve-Rosemont, 5415 boul l’Assomption, Montréal, Qc, Canada H1T 2M4
a r t i c l e i n f o
a b s t r a c t
Article history:
A total of eight new analogs of pactamycin were prepared and tested alongside pactamycin and three of
its natural congeners for antibacterial, anticancer, and antiprotozoal activities. The present study high-
lights the effects of changing the urea and aniline groups especially with regard to anticancer and anti-
protozoal activities.
Received 20 December 2012
Revised 15 January 2013
Accepted 21 January 2013
Available online 31 January 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Pactamycin
Analogs
Urea
Aniline
Antibacterial
Anticancer
Antiprotozoal
1. Introduction
co-workers10 on the biosynthesis of pactamycin have traced its
components to small molecule precursors by isotopic labelling.
Pactamycin represents a structurally unique natural product
belonging to the aminocyclopentitol family1 (Fig. 1). Its isolation
in 1961 from a fermentation broth of Streptomyces pactum by the
former Upjohn Company scientists,2 was followed by its structure
elucidation by chemical and spectroscopic methods,3 and eventu-
ally as a derivative by X-ray crystallography.4 Early studies by
the Upjohn group have shown that pactamycin exhibited in vitro
activity against a limited panel of Gram-positive and Gram-nega-
tive bacteria as well as cytotoxicity against some cancer cell lines.5
However, further interest in pactamycin was curtailed because of
its toxicity.
The highly functionalized and unique structure of pactamycin
has generated interest in recent years on several fronts.6 Pioneer-
ing X-ray crystallographic studies of a pactamycin-RNA complex
from Thermus thermophilus by Ramakrishnan and co-workers7
showed a unique mode of binding at the 30S site. Early studies
on the biosynthesis of pactamycin were reported by Rinehart and
co-workers.8 More recently, Kudo and co-workers9 cloned the bio-
synthetic gene cluster involved in the formation of the cyclopen-
tane ring of pactamycin. Elegant studies by Mahmud and
Furthermore, they have identified the biosynthetic gene cluster
that produces pactamycin (1), de-6-methylsalicylyl pactamycin
(2), pactamycate (3), de-6-methylsalicylyl pactamycate (4), and
7-deoxypactamycin (5a) (Fig. 1).
Synthetic approaches toward the synthesis of the cyclopentane
core of pactamycin were sparse except for preliminary reports
from the Isobe11 and Knapp12 groups in 2005 and 2007, respec-
tively. A total synthesis of pactamycin and pactamycate was re-
ported in 2011 by our group.13 More recently, conceptually
different approaches to the substituted core structure of pactamy-
cin were independently divulged by Johnson,14 Looper,15 and
Nishikawa.16
In spite of the sustained interest in the mode of action of pacta-
mycin as an inhibitor of protein biosynthesis in prokaryotes,6 and
the intriguing interactions with RNA’s,7 little was known regarding
its activity beyond the limited testing done at the former Upjohn
Company.5 Recently, interest in pactamycin and its relatively few
congeners available from biosynthetic studies in small amounts
has been highlighted by the discovery of its antiprotozoal activity.
Thus, Õmura and co-workers17 reported that 7-deoxypactamycin
(5a) exhibited activity against Trypanosoma brucei and Plasmodium
falciparum at levels that were eightfold higher in potency
compared to pactamycin. In a more recent report, Õmura and
⇑
Corresponding author. Fax: +1 514 434 5728.
E-mail address: stephen.hanessian@umontreal.ca (S. Hanessian).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.037

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S. Hanessian et al. / Bioorg. Med. Chem. 21 (2013) 1775–1786
O
Me
Me
NH2
OH
O
O
O
NH
OH
OR1
O
N
H
Me
Me
Me
Me
R2
NH2
NH2
7
HO
O
1
2
NH
OH
OR1
NH
OH
OR1
5
O
R1 = MSA, pactamycate (3)
R1 = H, de-6-MSA-pactamycate (4)
3
4
N
H
Me
N
H
Me
Me2N
Me2N
OH
OH
6
R1 = MSA, pactamycin(1)
R1 = H, de-6-MSA-pactamycin (2)
R1 = MSA, R2 = Me, 7-deoxypactamycin (5a)
R1 = H, R2 = Me, jogyamicin (5b)
R1 = H, R2 = H, TM-025 (5c)
OH
O
Me
MSA (6-methylsalicylyl)
Figure 1. Structures of pactamycin, pactamycate, their de-6-methylsalicylyl analogs, jogyamicin, 7-deoxypactamycin, and TM-025.
co-workers18 showed that jogyamycin (5b), the de-6-meth-
ylsalicylyl 7-deoxypactamycin congener, was also a potent antitry-
panosomal agent and considerably better than pactamycin. Finally,
superior activity of a new metabolite of pactamycin (5c, TM-025)
against malaria parasites was recently divulged by Mahmud and
co-workers.19
cleavage of the epoxide group in 1213 in the presence of four differ-
ent anilines gave the aniline analogs 13a–13d as single diastereo-
mers.20 Acid-catalyzed cleavage of the oxazoline moiety afforded
the aminoalcohols 14a–14d, which were converted to the isocya-
nates 15a–15d. Treatment with dimethylamine led to the
N,N-dimethylurea derivatives 16a–16d, which were eventually
converted to 19a–19d as described in Scheme 1.
2. Results and discussion
2.1. Chemistry
2.2. Biological activity studies
Our synthesis plan toward pactamycin was conceived so as to
allow the preparation of functionally modified analogs.13 We
were intrigued by the role that the unusual 6-methyl salicylyl
ester (6-MSA) moiety in pactamycin could play in the in vitro
activity as an antibiotic when our work began some years ago.
Only within the past three years has it been demonstrated that
de-6-methylsalicylyl pactamycin (2) and its 7-modified congen-
ers were endowed with equal if not better antiprotozoal activity,
but diminished antibacterial activity.19 With this knowledge, we
focussed on the synthesis of de-6-methylsalicylyl pactamycin
analogs in which the aniline and urea moieties were modified,
starting from appropriate advanced intermediates13 (Fig. 2).
Maintaining the original aniline moiety with the m-acetyl
group, we prepared a series of N,N-dialkyl ureas (11a–11d) varying
the bulk of the substituents by treating the isocyanate 613 with a
series of amines. The resulting substituted ureas (7a–7d), were
converted to the 7-hydroxy analog by treatment with DIBAL-H to
give 8a–8d. Subsequent oxidative transformation to the ketones
9a–9d, cleavage of the acetal to 10a–10d, and Zn-mediated reduc-
tion of the azide group led to the N,N-disubstituted urea analogs of
de-6-methylsalicylyl pactamycin 11a–11d (Scheme 1).
The antibacterial activities of these and related derivatives
against a panel of six representative microorganisms are shown
in Table 1. Pactamycin and de-6-MSA pactamycin remained the
most active against Escherichia coli and Staphylococcus aureus,
closely followed by the m-fluoro, and m-trifluoromethyl aniline
analogs. Modification of the urea group led to diminution or loss
of activity showing its paramount importance.
The cytotoxicity of the same analogs against a panel of four
cancer cell lines is shown in Table 2. Excellent activity was exhib-
ited against a colorectal HCT116 cell lines by pactamycin (1) and
de-6-MSA pactamycin (2). Among the modified urea analogs, the
pyrrolidine urea 11b appeared to be the best. Unfortunately, all
other analogs were either inactive or weakly active against the
other cell lines.
The most interesting results were obtained against Plasmodium
falciparum (Table 3). A clear demarcation in the tolerance of N,N-
dialkylurea groups was observed as the size increased. Thus, the
threshold of activity was maintained up to the pyrrolidine urea
(11b) (IC50 = 9 nM), but rapidly fell for the piperidine and morpho-
line analogs (11c, 11d). Among the anilines, excellent activity was
observed in the case of the m-fluoro and m-trifluoromethyl aniline
analogs (19b and 19d) against the D6 strain. In addition the same
analogs were also highly active against chloroquine-resistant Dd2
and 7G8 strains.
Next, keeping the N,N-dimethylurea group, we substituted the
original m-acetyl-1-aniline moiety at C2 by aniline and m-substi-
tuted anilines (19a–19d) (Scheme 2). Thus Yb(OTf)3 mediated
R1
R1 = H, F, OMe, CF3, COCH3
Me
Me
NH2
7
HO
N
N
R2 =
O
1
2
3
NH
OH
Me
5
HN
4
NMe2
R2
Me
N
N
O
OH
6
OH
Figure 2. Two series of modifications toward the pactamycin analogs.

S. Hanessian et al. / Bioorg. Med. Chem. 21 (2013) 1775–1786
1777
diethylamine;
pyrrolidine;
piperidine;
Me
Me
Me
Me
N3
N3
PMBzO
O
NH
O
O
NH
O
O
PMBzO
DIBAL-H, CH2Cl2
-78 °C to rt
morpholine
N
H
Me
N
Me
C
O
0 °C to rt
R
OH
OH
6
7a-d
7a (91%); 7b (88%)
7c
7d
(89%);
(83%)
O
Me
Me
Me
N3
1. OsO4 (cat.), NMO,
THF: acetone: H2O
(5:5:1), rt
Me N3
HO
O
TFA: MeCN: H2O
(5:1:1)
HO
O
NH
O
O
NH
O
N
H
Me
N
H
2. NaIO4, THF: H2O
(1:1), rt
R
0 °C to rt
R
O
OH
Me
OH
8a-d
9a-d
9a (85%); 9b (78%)
8a (87%); 8b (85%)
9c
9d (74%)
(74%);
8c
8d
(85%)
(86%);
O
Me
O
Me
Me
N
N
a
b
=
=
Me
Me
N3
Me
NH2
HO
O
HO
O
NH
OH
OH
Zn, NH4Cl
NH
OH
N
H
Me
N
H
EtOH: H2O (3:1),
rt
R
c
R
=
N
N
OH
Me
OH
OH
10a-d
10b
(87%)
11a-d
d
O
=
11a
11b (
86%)
(88%);
10a
(85%);
10c (88%); 10d (83%)
11c (88%); 11d (84%)
Scheme 1. Synthesis of disubstituted urea analogs of de-6-methylsalicylyl pactamycin.
Visualization of the developed chromatogram was performed by
UV absorbance, aqueous cerium ammonium molybdate, iodine,
or aqueous potassium permanganate. Flash chromatography was
performed on 230–400 mesh silica gel with the indicated solvent
systems. Melting points are uncorrected. Infrared spectra were re-
corded on a FT-IR spectrometer and are reported in reciprocal cen-
timeters (cmꢀ1). Routine nuclear magnetic resonance spectra were
recorded either on AMX-300, AV-300, AV-400, or AV-700 spec-
trometer. Chemical shifts for 1H NMR spectra are recorded in parts
per million from tetramethylsilane with the solvent resonance as
the internal standard (CHCl3, d 7.27 ppm). Data are reported as fol-
lows: chemical shift, integration, multiplicity (s = singlet, d = dou-
blet, t = triplet, q = quartet, qn = quintet, m = multiplet and
br = broad) and coupling constant in Hz. Chemical shifts for 13C
NMR spectra are recorded in parts per million from tetramethylsil-
ane using the central peak of the solvent resonance as the internal
standard (CDCl3, d 77.00 ppm). All spectra were obtained with
complete proton decoupling. Optical rotations were determined
at 589 nm at ambient temperature. Data are reported as follows:
3. Conclusion
In conclusion, we have prepared a series of modified urea and
aniline analogs of de-6-MSA pactamycin and studied the influence
of systematic modifications on the biological activities compared
to pactamycin and de-6-MSA pactamycin. Antibacterial activity
against Escherichia coli and Staphylococcus aureus was maintained
only in the m-fluoro and m-trifluoromethyl aniline analogs. There
appears to be a limit to steric tolerance in the antitumor activity
of urea analogs against colorectal cancer cell line with the pyrrol-
idine analog of de-6MSA pactamycin being the most active. Varia-
tion in the m-position of the aniline resulted in excellent activity
against Plasmodium falciparum indicating better tolerance
compared to changes in the urea moiety. Moreover, equally high
activity was observed against chloroquine-resistant strains by the
m-fluoro and m-trifluoromethyl anline analogs. Further studies
toward a better understanding of structure–activity relationships
toward an extended panel of tumor cell lines and protozoal organ-
isms will be reported in due course.
[a] concentration (c in g/100 mL), and solvent. High-resolution
D
mass spectra were performed by the Centre régional de spectrosco-
pie de masse de l’Université de Montréal using fast atom bombard-
ment (FAB) or electrospray ionization (ESI) techniques.
Low-resolution mass spectra were obtained using electrospray
ionization (ESI).
4. Experimental
4.1. General
All non-aqueous reactions were run in flame-dried glassware
under a positive pressure of argon with exclusion of moisture from
reagents and glassware using standard techniques for manipulat-
ing air-sensitive compounds. Anhydrous solvents were obtained
using standard drying techniques. Unless stated otherwise, com-
mercial grade reagents were used without further purification.
Reactions were monitored by analytical thin-layer chromatogra-
phy (TLC) performed on pre-coated, glass-backed silica gel plates.
4.1.1. General procedure for synthesis of compounds 7a–7d
To isocyanate 6 (49 mg, 0.085 mmol) was added 0.1 mL of sec-
ondary amines (neat) at 0 °C and the reaction mixture was left
warming to room temperature. It was directly subjected to flash
column chromatography using [40–50]% ethyl acetate in hexanes
to afford 7a–7d as colorless oils.

1778
S. Hanessian et al. / Bioorg. Med. Chem. 21 (2013) 1775–1786
R
Me
Me
N3
N3
R
NH2
Yb(OTf)3
O
O
NH
OH
OTBDPS
2N HCl, THF
0 °C to rt
N
O
N
Me
toluene, 80 °C
Me
OTBDPS
OH
12
OH
MeO
MeO
13a-d
13a R=H, 74%; 13b R=F, 83%
13c
13d
R=OMe, 86%;
R=CF3, 52%
R
R
1. TASF, DMF, 0 °C to rt
2. 2,2-DMP, CSA,
CH2Cl2, 0 °C to rt
Me
Me
N3
N3
NH
OH
OTBDPS
PMBzO
H2N
Me
NH
O
PMBzO
Me2NH
N
Me
C
O
-46 °C to rt
3. activated charcoal,
TEA, diphosgene,
CH2Cl2, -46 °C
O
OH
OH
14a-d
15a-d
14a R=H, 53%; 14b R=F, 53%
15a R=H, 86%; 15b R=F, 81%
14c
14d
R=OMe, 45%;
R=CF3, 53%
15c
15d
R=OMe, 47%; R=CF3, 82%
R
R
Me
N3
Me N3
TFA: MeCN: H2O
(5:1:1)
HO
O
PMBzO
DIBAL-H, CH2Cl2
-78 °C to rt
NH
O
NH
O
O
N
H
Me
N
H
0 °C to rt or 33 °C
Me2N
Me2N
O
O
Me
OH
OH
16a-d
17a-d
16a R=H, 88%; 16b R=F, 93%
17a R=H, 91%; 17b R=F, 98%
16c
16d
17c
17d
R=CF3, 91%
R=OMe, 86%;
R=CF3, 93%
R=OMe, 75%;
R
R
Me N3
HO
Me NH2
HO
O
NH
OH
NH
OH
Zn, NH4Cl
O
N
N
H
H
EtOH: H2O (3:1),
rt
Me2N
Me2N
OH
OH
Me
Me
OH
OH
18a-d
19a-d
18a R=H, 76%; 18b R=F, 85%
19a
19b
R=H, 80%;
R=F, 75%
18c 18d
R=OMe, 70%;
R=CF3, 82%
19c R=OMe, 80%; 19d R=CF3, 75%
Scheme 2. Synthesis of de-6-methylsalicylyl aniline analogs of pactamycin.
4.1.1.1. (S)-1-((5S,6R,7R,8S,9S)-8-Azido-7-(3,3-diethylureido)-6-
hydroxy-2,2,6-trimethyl-9-((3-(prop-1-en-2-yl)phenyl)amino)-
1,3-dioxaspiro[4.4]nonan-7-yl)ethyl
(7a).
4-methoxybenzoate
Table 1
Yield: 91% (50 mg from 49 mg); ½a D20
ꢁ
+90.9 (c 1.00,
Antibacterial activities (minimum inhibitory concentration) for compounds 1–4, 11a–
d and 19a–d
CHCl3); IR (neat): mmax 3403, 2977, 2107, 1708, 1644, 1605,
1582, 1511, 1257, 1168, 1032, 849, 769 cmꢀ1
;
1H NMR
Compound E. coli S. aureus K.
A.
P.
E. faecalis
(400 MHz, CDCl3): 7.98 (2H, d, J = 8.9 Hz), 7.17 (1H, t,
d
(mg/L) (mg/L) pneumoniae baumanii aeruginosa (mg/L)
J = 8.0 Hz), 6.94 (2H, d, J = 8.9 Hz), 6.89–6.85 (2H, m), 6.69 (1H,
dd, J = 8.0, 1.8 Hz), 6.57 (1H, q, J = 6.6 Hz), 5.66 (1H, s), 5.33 (1H,
s), 5.06 (1H, t, J = 1.4 Hz), 4.42 (1H, d, J = 9.8 Hz), 4.25–4.20 (3H,
m), 3.97 (2H, s), 3.88 (3H, s), 3.34–3.20 (4H, m), 2.13 (3H, s),
1.58 (1H, d, J = 6.6 Hz), 1.57 (3H, s), 1.43 (3H, s), 1.35 (3H, s),
1.15 (6H, t, J = 7.1 Hz); 13C NMR (100 MHz, CDCl3): d 164.5,
163.5, 156.7, 147.2, 143.5, 142.6, 131.6, 129.2, 122.3, 116.0,
113.8, 112.5, 112.2, 111.1, 109.9, 90.2, 83.0, 71.9, 70.6, 68.1,
65.9, 65.2, 55.4, 41.7, 26.0, 25.7, 22.4, 21.8, 17.2, 13.9; HRMS-ESI
(m/z): calcd for C34H47N6O7 [M+H]+ 651.35007, found 651.35183.
(mg/L)
(mg/L)
(mg/L)
1
16
4
8
>128
>128
>128
>128
64
0.12
2
8
>128
>128
>128
4
32
8
16
16
128
128
128
64
2
128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
2a
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
3
>128
>128
>128
>128
128
128
>128
>128
64
4
4a
11a
11b
11ba
11c
11d
19a
19b
19c
19d
19da
8
8
128
128
64
>128
>128
32
8
64
>128
>128
64
4
16
2
>128
>128
>128
128
>128
64
4.1.1.2.
(S)-1-((5S,6R,7R,8S,9S)-8-Azido-6-hydroxy-2,2,6-tri-
32
>128
128
128
128
64
64
methyl-9-((3-(prop-1-en-2-yl)phenyl)amino)-7-(pyrrolidine-1-
carboxamido)-1,3-dioxaspiro[4.4]nonan-7-yl)ethyl 4-methoxy-
16
32
2
128
benzoate (7b).
Yield: 88% (66 mg from 67 mg); ½a D20
ꢁ
+88.8 (c
a
Compounds tested after 24 h storage at 0 °C.
1.00, CHCl3); IR (neat): mmax 3388, 2979, 2107, 1709, 1649, 1605,

S. Hanessian et al. / Bioorg. Med. Chem. 21 (2013) 1775–1786
1779
Table 2
4.1.1.4.
(S)-1-((5S,6R,7R,8S,9S)-8-Azido-6-hydroxy-2,2,6-tri-
Cytotoxicity values for compounds 1–4, 11a–d and 19a–d
methyl-7-(morpholine-4-carboxamido)-9-((3-(prop-1-en-2-yl)
phenyl)amino)-1,3-dioxaspiro[4.4]nonan-7-yl)ethyl 4-meth-
oxybenzoate (7d).
Compound HCT116
(colorectal)
PC3
(prostate)
IC50
WI-38
(lung)
MDA-231
(breast)
IC50 (lM)
Yield: 83% (59 mg from 62 mg); ½a D20
ꢁ
IC50
(l
M)
(lM)
IC50
(l
M)
+81.9 (c 1.00, CHCl3); IR (neat): mmax 3406, 2984, 2109, 1708,
1605, 1581, 1511, 1382, 1257, 1168, 1116, 1030, 849, 768 cmꢀ1
;
1
2
3
4
11a
11b
11c
11d
19a
19b
19c
19d
0.07
0.07
>1
0.24
0.31
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
0.5
0.26
>1
1H NMR (400 MHz, CDCl3): d 8.00 (2H, d, J = 8.9 Hz), 7.15 (1H, t,
J = 7.8 Hz), 6.95 (2H, d, J = 8.9 Hz), 6.88 (1H, d, J = 7.8 Hz), 6.84
(1H, d, J = 1.9 Hz), 6.69 (1H, dd, J = 7.9, 1.9 Hz), 6.44 (1H, q,
J = 6.6 Hz), 5.77 (1H, s), 5.33 (1H, s), 5.06 (1H, t, J = 1.4 Hz), 4.40
(1H, d, J = 9.9 Hz), 4.28 (1H, d, J = 9.2 Hz), 4.22 (1H, d, J = 9.9 Hz),
4.11 (1H, s), 4.00–3.94 (2H, m), 3.88 (3H, s), 3.71–3.60 (4H, m),
3.42–3.28 (4H, m), 2.13 (3H, s), 1.57 (3H, d, J = 6.6 Hz), 1.55 (3H,
s), 1.42 (3H, s), 1.35 (3H, s); 13C NMR (100 MHz, CDCl3): d 164.7,
163.7, 157.2, 147.0, 143.5, 142.6, 131.6, 129.2, 122.1, 116.1,
113.9, 112.5, 112.3, 111.1, 109.9, 90.2, 82.8, 71.6, 70.6, 68.2,
66.4, 66.1, 65.2, 55.4, 44.3, 25.9, 25.7, 22.2, 21.8, 17.2; HRMS-
>1
>1
>1
0.23
0.09
>1
0.40
0.15
>1
>1
>1
>1 (35%)
>1
0.75
0.44
0.12
>1
>1
>1
>1
>1
0.38
>1
0.81
0.19
0.39
0.10
>1 (34%)
>1
>1 (42%)
ESI (m/z): calcd for
665.33075.
C
34H45N6O8 [M+H]+ 665.32934, found
Table 3
Typical antimalarial activity for compounds 1–4, 11a–d and 19a–d21
Compound
D6
IC50 (nM)
Dd2
IC50 (nM)
7G8
IC50 (nM)
4.1.2. General procedure for synthesis of 8a–8d
To a stirred solution of 7a–7d (49 mg, 0.075 mmol) in dry
CH2Cl2 (3 mL), DIBAL-H (0.26 mL, 1.5 M in toluene, 0.385 mmol)
was added slowly at ꢀ78 °C under argon and the mixture was stir-
red for 1.5 h. The reaction mixture was then quenched by slow
addition of methanol and warmed to room temperature. A satu-
rated aqueous potassium sodium tartrate solution was added, the
reaction mixture stirred for 1 h, then extracted with ethyl acetate.
The combined organic layers were washed with brine, dried over
anhydrous Na2SO4 and concentrated under reduced pressure. Flash
column chromatography using [20–25]% ethyl acetate in hexanes
gave compounds 8a–8d as colorless liquids.
1
2
3
4
11a
11b
11c
11d
19a
19b
19c
19d
<2.5
<2.5
>2500
>2500
29
<2.5
<2.5
>2500
>2500
40
2.5
2.5
>2500
>2500
42
9
9
10
>2500
>2500
14.6
6.5
13.7
6.7
2003
>2500
13.9
7.4
11.5
3.5
1596
>2500
18.5
<2.5
19.8
<2.5
4.1.2.1. 3-((5S,6R,7R,8S,9S)-8-Azido-6-hydroxy-7-((S)-1-hydroxy-
ethyl)-2,2,6-trimethyl-9-((3-(prop-1-en-2-yl)phenyl)amino)-
1511, 1382, 1257, 1168, 849, 757 cmꢀ1
;
1H NMR (400 MHz,
CDCl3): d 7.99 (2H, d, J = 8.9 Hz), 7.14 (1H, t, J = 7.9 Hz), 6.95 (2H,
d, J = 8.9 Hz), 6.87 (1H, d, J = 14.1 Hz), 6.86 (1H, d, J = 1.9 Hz),
6.69 (1H, dd, J = 7.9, 1.9 Hz), 6.47 (1H, q, J = 6.6 Hz), 5.56 (1H, s),
5.33 (1H, s), 5.06 (1H, s), 4.42 (1H, d, J = 9.8 Hz), 4.29–4.19 (3H,
m), 4.01–3.93 (2H, m), 3.88 (3H, s), 3.41–3.21 (4H, m), 2.13 (3H,
s), 1.91 (1H, br s), 1.93–1.87 (4H, m), 1.43 (3H, s); 13C NMR
(100 MHz, CDCl3): d 164.6, 163.5, 156.4, 147.2, 143.6, 142.6,
131.6, 129.2, 122.3, 116.0, 113.8, 112.5, 112.2, 111.1, 109.8, 90.2,
82.8, 71.9, 70.6, 68.3, 65.9, 65.2, 55.4, 45.7, 26.0, 25.7, 25.6, 22.4,
1,3-dioxaspiro[4.4]nonan-7-yl)-1,1-diethylurea (8a).
Yield:
87% (34 mg from 49 mg); ½a D20
ꢁ
+19.3 (c 1.00, CHCl3); IR (neat): mmax
3424, 2983, 2935, 2104, 1635, 1602, 1580, 1528, 1489, 1455, 1407,
1374, 1287, 1259, 1216, 1118, 1057, 890, 855, 784 cmꢀ1 1H NMR
;
(400 MHz, CDCl3): d 7.67 (1H, d, J = 11.2 Hz), 7.23 (1H, t, J = 7.9 Hz),
6.92 (1H, d, J = 7.7 Hz), 6.73 (1H, s), 6.57 (1H, dd, J = 8.0, 2.0 Hz),
5.39 (1H, s), 5.38 (1H, s), 5.29 (1H, d, J = 11.2 Hz), 5.10 (1H, s),
4.85 (1H, s), 4.30 (1H, d, J = 9.9 Hz), 4.26 (1H, d, J = 9.9 Hz), 3.98–
3.88 (2H, m), 3.69 (1H, s), 3.35 (2H, q, J = 7.1 Hz), 2.16 (3H, s),
1.47 (3H, s), 1.44 (3H, s), 1.43 (3H, s), 1.24 (6H, t, J = 7.1 Hz), 1.20
(3H, d, J = 6.5 Hz); 13C NMR (75 MHz, CDCl3): d 157.3, 145.8,
143.4, 143.0, 129.6, 115.8, 112.6, 112.1, 110.6, 110.5, 91.4, 88.7,
73.9, 73.5, 72.8, 66.3, 65.5, 42.3, 26.3, 25.9, 21.9, 21.0, 17.8, 13.8;
HRMS-ESI (m/z): calcd for C26H41FN6O5 [M+H]+ 517.31329, found
517.31392.
21.8, 17.2; HRMS-ESI (m/z): calcd for
649.33442, found 649.33597.
C
34H45N6O7 [M+H]+
4.1.1.3.
(S)-1-((5S,6R,7R,8S,9S)-8-Azido-6-hydroxy-2,2,6-tri-
methyl-7-(piperidine-1-carboxamido)-9-((3-(prop-1-en-2-yl)
phenyl)amino)-1,3-dioxaspiro[4.4]nonan-7-yl)ethyl 4-meth-
oxybenzoate (7c).
+85.7 (c 1.00, CHCl3); IR (neat): mmax 3406, 2937, 2108, 1708,
Yield: 89% (72 mg from 70 mg); ½a D20
ꢁ
4.1.2.2. N-((5S,6R,7R,8S,9S)-8-Azido-6-hydroxy-7-((S)-1-hydroxy-
ethyl)-2,2,6-trimethyl-9-((3-(prop-1-en-2-yl)phenyl)amino)-
1,3-dioxaspiro[4.4]nonan-7-yl)pyrrolidine-1-carboxamide
1643, 1605, 1581, 1511, 1382, 1326, 1256, 1168, 1101, 1031,
850, 769 cmꢀ1
;
1H NMR (400 MHz, CDCl3): d 7.99 (2H, d,
(8b).
Yield: 85% (52 mg from 65 mg); ½a D20
ꢁ
+30.0 (c 1.00,
J = 8.9 Hz), 7.17 (1H, t, J = 7.9 Hz), 6.95 (2H, d, J = 8.9 Hz), 6.87
(1H, d, J = 7.7 Hz), 6.86 (1H, d, J = 2.0 Hz), 6.69 (1H, dd, J = 7.8,
1.9 Hz), 6.51 (1H, q, J = 6.6 Hz), 5.80 (1H, s), 5.33 (1H, s), 5.06
(1H, t, J = 1.4 Hz), 4.41 (1H, d, J = 9.8 Hz), 4.26–4.20 (3H, m),
3.99–3.97 (2H, m), 3.88 (3H, s), 3.42–3.38 (2H, m), 3.33–3.28
(2H, m), 2.13 (3H, s), 1.62–1.51 (12H, m), 1.43 (3H, s), 1.28
(3H, s); 13C NMR (100 MHz, CDCl3): d 164.6, 163.6, 157.0,
147.1, 143.5, 142.6, 131.6, 129.2, 122.3, 116.0, 114.0, 113.8,
112.5, 112.2, 111.1, 109.8, 90.2, 82.9, 71.9, 70.7, 68.1, 66.0,
65.2, 60.4, 55.4, 45.5, 25.9, 25.7, 25.5, 24.5, 22.3, 21.8, 17.2;
HRMS-ESI (m/z): calcd for C35H47N6O7 [M+H]+ 663.35007, found
663.3517.
CHCl3); IR (neat): mmax 3408, 2984, 2104, 1634, 1602, 1581,
1531, 1487, 1396, 1325, 1258, 1212, 1146, 1060, 856, 784,
730 cmꢀ1
; d 7.54 (1H, d,
1H NMR (400 MHz, CDCl3):
J = 11.4 Hz), 7.23 (1H, t, J = 7.9 Hz), 6.92 (1H, d, J = 7.9 Hz),
6.73 (1H, s), 6.57 (1H, dd, J = 8.0, 2.0 Hz), 5.38 (1H, s), 5.24
(1H, s), 5.23 (1H, d, J = 11.0 Hz), 5.11 (1H, t, J = 1.3 Hz), 4.67
(1H, s), 4.27 (2H, s), 4.01–3.91 (2H, m), 3.69 (1H, s), 3.49–
3.37 (4H, m), 2.16 (3H, s), 2.02–1.98 (4H, m), 1.46 (6H, s),
1.43 (3H, s), 1.24 (3H, d, J = 6.5 Hz); 13C NMR (100 MHz,
CDCl3): d 156.8, 145.8, 143.4, 142.9, 129.6, 115.8, 112.6,
112.1, 110.6, 110.5, 91.4, 88.4, 73.8, 73.1, 72.9, 66.6, 65.4,

1780
S. Hanessian et al. / Bioorg. Med. Chem. 21 (2013) 1775–1786
45.9, 26.3, 25.6, 21.9, 20.8, 17.8; HRMS-ESI (m/z): calcd for
26H39FN6O5 [M+H]+ 515.29764, found 515.29897.
1.24 (6H, t, J = 7.2 Hz), 1.19 (3H, d, J = 6.5 Hz); 13C NMR
(100 MHz, CDCl3): d 198.2, 157.2, 146.2, 138.6, 129.9, 118.5,
117.9, 111.8, 110.8, 91.3, 88.6, 74.2, 73.4, 72.8, 66.4, 65.5, 42.3,
26.7, 26.3, 26.0, 20.9, 17.7, 13.8; HRMS-ESI (m/z): calcd for
C
4.1.2.3. N-((5S,6R,7R,8S,9S)-8-Azido-6-hydroxy-7-((S)-1-hydroxy-
ethyl)-2,2,6-trimethyl-9-((3-(prop-1-en-2-yl)phenyl)amino)-1,3-di-
oxaspiro[4.4]nonan-7-yl)piperidine-1-carboxamide (8c). Yield:
C
25H39N6O6 [M+H]+ 519.29256, found 519.29356.
86% (48 mg from 70 mg); ½a D20
ꢁ
+29.6 (c 1.00, CHCl3); IR (neat): mmax
3374, 2986, 2935, 2855, 2104, 1631, 1602, 1579, 1528, 1374, 1258,
4.1.3.2. N-((5S,6R,7R,8S,9S)-9-((3-Acetylphenyl)amino)-8-azido-
6-hydroxy-7-((S)-1-hydroxyethyl)-2,2,6-trimethyl-1,3-dioxaspi-
ro[4.4]nonan-7-yl)pyrrolidine-1-carboxamide (9b). Yield: 78%
1209, 1120, 1059, 890, 855, 784, 730 cmꢀ1 1H NMR (400 MHz,
;
CDCl3): d 7.52 (1H, d, J = 11.3 Hz), 7.23 (1H, t, J = 7.9 Hz), 6.92 (1H, d,
J = 7.6 Hz), 6.73 (1H, s), 6.57 (1H, dd, J = 8.1, 2.0 Hz), 5.51 (1H, s), 5.38
(1H, s), 5.25 (1H, d, J = 11.3 Hz), 5.10 (1H, s), 4.67 (1H, s), 4.28 (2H, s),
4.39–3.91 (2H, m), 3.69 (1H, s), 3.51–3.47 (2H, m), 3.40–3.35 (2H, m),
2.16 (3H, s), 1.70–1.60 (6H, m), 1.59 (3H, s), 1.46 (3H, s), 1.45 (3H, s),
1.43 (3H, s), 1.21(3H, d, J = 6.5 Hz); 13C NMR (100 MHz, CDCl3): d
157.4, 145.8, 143.4, 143.0, 129.6, 115.9, 112.6, 112.1, 110.6, 91.3,
88.5, 74.1, 73.2, 72.6, 66.7, 65.5, 45.7, 29.7, 26.4, 25.9, 25.6, 24.4, 21.9,
20.8, 17.8; HRMS-ESI(m/z): calcdfor C27H41N6O5 [M+H]+ 529.31329,
found 529.31370.
in two steps (39 mg from 50 mg); ½a D20
ꢁ
+37.4 (c 1.00, CHCl3); IR
(neat): mmax 3184, 2984, 2104, 1681, 1637, 1602, 1534, 1487, 1398,
1357, 1324, 1263, 1146, 1060, 914, 855, 781, 731, 688 cmꢀ1 1H
;
NMR (400 MHz, CDCl3): d 7.52 (1H, d, J = 11.4 Hz), 7.37–7.32 (2H,
m), 7.22 (1H, s), 6.83 (1H, m), 5.37 (1H, d, J = 11.1 Hz), 5.22 (1H, s),
4.66 (1H, s), 4.24 (2H, ABq, J = 9.9 Hz), 3.95 (1H, m), 3.93 (1H, d,
J = 11.1 Hz), 3.63 (1H, s), 3.50–3.38 (4H, m), 2.60 (3H, s), 2.07–1.97
(4H, m), 1.46 (3H, s), 1.45 (3H, s), 1.45 (3H, s), 1.23 (3H, d,
J = 6.5 Hz); 13C NMR (100 MHz, CDCl3): d 198.2, 156.7, 146.2, 138.6,
129.9, 118.5, 117.9, 111.9, 110.7, 91.3, 88.4, 74.0, 73.0, 72.9, 66.7,
65.4, 45.9, 26.7, 26.3, 25.7, 25.6, 20.7, 17.8; HRMS-ESI (m/z): calcd
for C25H37N6O6 [M+H]+ 517.27691, found 517.27822.
4.1.2.4.
N-((5S,6R,7R,8S,9S)-8-Azido-6-hydroxy-7-((S)-1-hydroxy-
ethyl)-2,2,6-trimethyl-9-((3-(prop-1-en-2-yl)phenyl)amino)-1,3-di-
oxaspiro[4.4]nonan-7-yl)morpholine-4-carboxamide (8d). Yield:
4.1.3.3. N-((5S,6R,7R,8S,9S)-9-((3-Acetylphenyl)amino)-8-azido-
6-hydroxy-7-((S)-1-hydroxyethyl)-2,2,6-trimethyl-1,3-dioxaspi-
ro[4.4]nonan-7-yl)piperidine-1-carboxamide (9c). Yield: 74%
85% (39 mg from 58 mg); ½a D20
ꢁ
+39.7 (c 1.00, CHCl3); IR (neat): mmax
3377, 2986, 2106, 1640, 1602, 1580, 1538, 1373, 1263, 1210, 1119,
1059,855,730 cmꢀ1;1HNMR(400 MHz,CDCl3):d7.25–7.21(2H,m),
6.92 (1H, d, J = 7.8 Hz), 6.73 (1H, s), 6.57 (1H, dd, J = 8.0, 1.9 Hz), 5.51
(1H, s), 5.38 (1H, s), 5.21 (1H, d, J = 11.4 Hz), 5.10 (1H, s), 4.57 (1H, s),
4.28 (2H, ABq, J = 9.9 Hz), 4.02–3.92 (2H, m), 3.77–3.74 (4H, m), 3.70
(1H,s),3.54–3.50(2H,m),3.41–3.37(2H,m),2.16(3H,s),1.46(3H,s),
1.45 (3H, s), 1.42 (3H, s), 1.21 (3H, d, J = 6.5 Hz); 13C NMR (100 MHz,
CDCl3): d 157.2, 145.4, 143.0, 142.7, 129.3, 115.6, 112.3, 111.8, 110.3,
110.2, 91.0, 88.1, 73.7, 72.8, 72.2, 66.5, 66.2, 65.1, 44.1, 26.0, 25.2,
21.5, 20.4, 17.5; HRMS-ESI (m/z): calcd for C26H39FN6O6 [M+H]+
531.29256, found 531.29410.
in two steps (34 mg from 46 mg); ½a D20
ꢁ
+38.8 (c 1.00, CHCl3); IR
(neat): mmax 3371, 2988, 2937, 2857, 2106, 1682, 1603, 1532, 1487,
1438, 1373, 1357, 1323, 1270, 1233, 1209, 1125, 1059, 1023, 913,
854, 781, 732, 688 cmꢀ1; 1H NMR (400 MHz, CDCl3): d 7.49 (1H, d,
J = 11.3 Hz), 7.38–7.35 (2H, m), 7.31 (1H, s), 6.83 (1H, m), 5.49 (1H,
s), 5.65 (1H, d, J = 11.2 Hz), 4.65 (1H, s), 4.25 (2H, ABq, J = 9.9 Hz),
3.98–3.90 (2H, m), 3.64 (1H, s), 3.52–3.46 (2H, m), 3.40–3.34 (2H,
m), 2.60 (3H, s), 2.19 (1H, s), 1.68–1.60 (6H, m), 1.47 (3H, s), 1.45
(3H, s), 1.43 (3H, s), 1.19 (3H, d, J = 6.5 Hz); 13C NMR (100 MHz,
CDCl3): d 198.2, 157.4, 146.2, 138.6, 129.9, 118.6, 117.9, 111.8,
110.7, 91.3, 88.5, 74.3, 73.2, 72.6, 66.7, 65.4, 45.7, 26.7, 26.3, 25.7,
24.4, 20.7, 17.8; HRMS-ESI (m/z): calcd for C26H39N6O6 [M+H]+
531.29256, found 531.29356.
4.1.3. General procedure for synthesis of 9a–9d
To a stirred solution of 8a–8d (34 mg, 0.066 mmol) in THF
(1 mL), acetone (1 mL) and H2O (0.2 mL) were added NMO
(54 mg, 0.46 mmol) and a catalytic amount of OsO4 (0.1 mL, 4%
wt in H2O) at 0 °C. After stirring for 2 h at rt, a saturated aqueous
sodium bisulfite solution was added, the reaction mixture stirred
for 30 min, then extracted with ethyl acetate. The combined organ-
ic layers were washed with water, brine, dried over anhydrous
Na2SO4 and concentrated under reduced pressure. Flash column
chromatography using 80% ethyl acetate in hexanes gave the tetrol
as clear oil, which was directly used for the next reaction without
characterisation. To the stirred solution of above tetrol in THF
(1 mL) and H2O (1 mL) was added NaIO4 (28 mg, 0.13 mmol) at rt
and stirred for 3 h. The reaction mixture was extracted with ethyl
acetate and the combined organic layers were washed with brine,
dried over anhydrous Na2SO4 and concentrated under reduced
pressure. Flash column chromatography using [40–50]% ethyl ace-
tate in hexanes gave compounds 9a–9d as colorless liquids.
4.1.3.4. N-((5S,6R,7R,8S,9S)-9-((3-Acetylphenyl)amino)-8-azido-
6-hydroxy-7-((S)-1-hydroxyethyl)-2,2,6-trimethyl-1,3-dioxa-
spiro[4.4]nonan-7-yl)morpholine-4-carboxamide (9d). Yield:
74% in two steps (29 mg from 39 mg); ½a D20
ꢁ
+43.6 (c 1.00, CHCl3);
IR (neat): mmax 3378, 2987, 2935, 2858, 2107, 1682, 1637, 1602,
1537, 1437, 1374, 1304, 1265, 1119, 1060, 1021, 914, 854, 781,
732 cmꢀ1 1H NMR (400 MHz, CDCl3): d 7.36–7.34 (2H, m), 7.28–
;
7.19 (2H, m), 6.83 (1H, m), 5.49 (1H, s), 5.34 (1H, d, J = 11.2 Hz),
4.56 (1H, s), 4.25 (1H, ABq, J = 10.0 Hz), 3.96 (1H, m), 3.93 (1H, d,
J = 11.2 Hz), 3.80–3.70 (4H, m), 3.65 (1H, s), 3.55–3.49 (2H, m),
3.41–3.36 (2H, m), 2.60 (3H, s), 2.19 (3H, s), 1.46 (3H, s), 1.44 (3H,
s), 1.42 (3H, s), 1.20 (3H, d, J = 6.5 Hz); 13C NMR (100 MHz, CDCl3):
d 198.2, 157.5, 146.1, 138.6, 129.9, 118.7, 117.9, 111.8, 110.7, 91.3,
88.4, 74.4, 73.1, 72.6, 66.9, 66.7, 65.4, 44.5, 26.7, 26.3, 25.6, 20.6,
17.8; HRMS-ESI (m/z): calcd for C25H37N6O7 [M+H]+ 533.27182,
found 533.27306.
4.1.3.1. 3-((5S,6R,7R,8S,9S)-9-((3-Acetylphenyl)amino)-8-azido-6-
hydroxy-7-((S)-1-hydroxyethyl)-2,2,6-trimethyl-1,3-dioxaspi-
4.1.4. General procedure for synthesis of 10a–10d
ro[4.4]nonan-7-yl)-1,1-diethylurea (9a).
Yield: 85% in two
To the stirred solution of 9a–9d (29 mg, 0.056 mmol) in aceto-
nitrile (0.2 mL) and H2O (0.2 mL), TFA (1 mL) was added at 0 °C and
stirred for 45 min at rt, then the reaction mixture was cooled to
0 °C and quenched very slowly with a saturated aqueous NaHCO3
solution and extracted with ethyl acetate. The combined organic
layers were washed with brine, dried over anhydrous Na2SO4 and
concentrated under reduced pressure. Flash column chromatogra-
phy using [83–95]% ethyl acetate in hexanes gave compounds 10a–
10d as colorless liquids.
steps (29 mg from 34 mg); ½a D20
ꢁ
+30.5 (c 1.00, CHCl3); IR (neat):
mmax 3373, 2985, 2935, 2105, 1682, 1634, 1603, 1531, 1436,
1374, 1358, 1288, 1119, 1057, 855, 783, 688 cmꢀ1 1H NMR
;
(400 MHz, CDCl3): d 7.66 (1H, d, J = 11.2 Hz), 7.39–7.31 (2H,
m), 7.23 (1H, s), 6.83 (1H, d, J = 7.6 Hz), 5.42 (1H, d,
J = 11.1 Hz), 5.37 (1H, s), 4.82 (1H, s), 4.25 (2H, ABq,
J = 11.0 Hz), 3.96 (1H, d, J = 11.1 Hz), 3.90 (1H, m), 3.64 (1H, s),
3.34 (4H, q, J = 7.2 Hz), 2.61 (3H, s), 1.47 (3H, s), 1.43 (6H, s),

S. Hanessian et al. / Bioorg. Med. Chem. 21 (2013) 1775–1786
1781
4.1.4.1. 3-((1R,2R,3S,4S,5S)-4-((3-Acetylphenyl)amino)-5-azido-
2,3-dihydroxy-1-((S)-1-hydroxyethyl)-3-(hydroxymethyl)-2-
0.62 mmol), zinc powder (20 mg, 0.31 mmol) at rt and stirred for
6 h. The reaction mixture was quenched with aqueous ammonia
(2 mL) and extracted with CH2Cl2. The combined organic layers
were washed with brine, dried over anhydrous Na2SO4 and con-
centrated under reduced pressure. The residue was purified by
flash column chromatography using [5–8]% methanol in chloro-
form to give compounds 11a–11d.
methylcyclopentyl)-1,1-diethylurea
(10a). Yield:
87%
(23 mg from 29 mg); ½a D20
ꢁ
+31.6 (c 1.00, CHCl3); IR (neat): mmax
3418, 2977, 2934, 2103, 1681, 1633, 1603, 1531, 1439, 1358, 1302,
1113, 1056, 917, 783, 757, 688 cmꢀ1; 1H NMR (400 MHz, CDCl3): d
7.64 (1H, d, J = 11.2 Hz), 7.38–7.30 (2H, m), 7.25 (1H, s), 6.87 (1H, d,
J = 7.2 Hz), 5.47 (1H, s), 5.38 (1H, d, J = 11.2 Hz), 4.70 (1H, s), 4.17
(1H, d, J = 11.6 Hz), 3.97 (1H, m), 3.91 (1H, d, J = 11.2 Hz), 3.66 (1H,
d, J = 11.6 Hz), 3.63 (1H, s), 3.41–3.25 (4H, m), 3.22 (1H, s), 2.57
(3H, s), 2.23 (1H, br s), 1.44 (3H, s), 1.25–1.20 (9H, m); 13C NMR
(100 MHz, CDCl3): d 198.8, 157.9, 146.6, 138.9, 130.3, 119.3, 119.0,
112.7, 89.0, 84.8, 74.7, 74.2, 73.7, 67.0, 61.6, 42.7, 27.1, 21.1, 18.2,
14.2; HRMS-ESI (m/z): calcd for C22H35N6O6 [M+H]+ 479.26126,
found 479.26241.
4.1.5.1. 3-((1R,2R,3S,4S,5S)-4-((3-Acetylphenyl)amino)-5-amino-
2,3-dihydroxy-1-((S)-1-hydroxyethyl)-3-(hydroxymethyl)-2-meth-
ylcyclopentyl)-1,1-diethylurea (11a). Pale yellow oil, yield: 88%
(8.4 mg from 10 mg); ½a D20
ꢁ
+41.2 (c 0.33, CHCl3); IR (neat): mmax
3381, 2976, 2931, 1679, 1603, 1515, 1359, 1298, 1081, 1056, 913,
; d 8.05 (1H, d,
784, 732 cmꢀ1 1H NMR (400 MHz, CDCl3):
J = 10.0 Hz), 7.28–7.21 (2H, m), 7.05 (1H, s), 6.84 (1H, m), 5.68
(1H, s), 5.50 (1H, d, J = 10.4 Hz), 4.13 (1H, d, J = 11.6 Hz), 3.92 (1H,
m), 3.76 (1H, d, J = 11.6 Hz), 3.42–3.29 (4H, m), 2.96 (1H, s), 2.57
(3H, s), 2.10–1.82 (4H, m), 1.48 (3H, s), 1.37–1.13 (6H, m), 1.05 (3H,
d, J = 6.0 Hz); 13C NMR (100 MHz, CDCl3): d 198.6, 158.2, 146.9,
138.3, 129.6, 118.9, 118.4, 110.9, 88.7, 84.7, 74.2, 71.5, 68.2, 62.6,
61.8, 42.2, 26.7, 21.5, 18.2, 13.8; HRMS-ESI (m/z): calcd for
4.1.4.2.
N-((1R,2R,3S,4S,5S)-4-((3-Acetylphenyl)amino)-5-azido-
2,3-dihydroxy-1-((S)-1-hydroxyethyl)-3-(hydroxymethyl)-2-meth-
ylcyclopentyl)pyrrolidine-1-carboxamide
(10b). Yield:
87%
(26 mg from 32 mg); ½a D20
ꢁ
+22.2 (c 1.00, CHCl3); IR (neat): mmax
3396 (br), 2978, 2937, 2937, 2875, 2103, 1681, 1633, 1603, 1531,
1487, 1402, 1326, 1234, 1136, 755, 689 cmꢀ1
;
1H NMR (400 MHz,
C
22H37N4O6 [M+H]+ 453.27076, found 453.26901.
CDCl3): d 7.56 (1H, d, J = 11.2 Hz), 7.34–7.23 (2H, m), 6.86 (1H, d,
J = 7.2 Hz), 5.35 (1H, s), 5.34 (1H, d, J = 11.6 Hz), 4.59 (1H, s), 4.15 (1H,
d, J = 11.2 Hz), 3.98 (1H, m), 3.94 (1H, d, J = 11.2 Hz), 3.66 (1H, d,
J = 11.6 Hz), 3.62 (1H, s), 3.42 (4H, br s), 3.33 (1H, s), 1.99 (4H, br s),
1.45 (3H, s), 1.24 (3H, d, J = 6.4 Hz); 13C NMR (100 MHz, CDCl3): d
198.4, 157.0, 146.2, 138.4, 129.9, 118.8, 118.6, 112.3, 88.4, 84.4, 73.9,
73.0, 66.6, 61.2, 46.0, 26.6, 25.6, 20.6, 17.8; HRMS-ESI (m/z): calcd for
4.1.5.2. N-((1R,2R,3S,4S,5S)-4-((3-Acetylphenyl)amino)-5-amino-
2,3-dihydroxy-1-((S)-1-hydroxyethyl)-3-(hydroxymethyl)-2-meth-
ylcyclopentyl)pyrrolidine-1-carboxamide (11b). Pale yellow
solid, yield: 86% (9.8 mg from 12 mg); ½a D20
ꢁ
+29.5 (c 1.00, CHCl3);
IR (neat): mmax 3381, 2977, 2918, 2875, 1679, 1603, 1519, 1486,
1398, 1357, 1337, 1235, 1136, 1072, 911, 781, 731, 689 cmꢀ1 1H
;
C
22H33N6O6 [M+H]+ 477.24561, found 477.24459.
NMR (400 MHz, CDCl3): d 7.89 (1H, d, J = 10.8 Hz), 7.28–7.24 (2H,
m), 7.19 (1H, s), 6.84 (1H, d, J = 3.2 Hz), 6.74 (1H, s), 5.49 (1H, s)
5.46 (1H, d, J = 10.8 Hz), 4.13 (1H, d, J = 11.6 Hz), 3.94 (1H, m),
3.77–3.72 (2H, m), 3.43 (4H, br s), 2.96 (1H, s), 2.55 (3H, s), 1.97
(4H, br s), 1.49 (3H, s), 1.08 (3H, d, J = 6.4 Hz); 13C NMR (100 MHz,
CDCl3): d 198.7, 157.8, 146.9, 138.2, 129.6, 119.0, 118.4, 110.8,
88.6, 84.8, 73.9, 71.7, 68.0, 62.6, 61.8, 46.0, 29.7, 26.7, 25.6, 21.5,
18.2; HRMS-ESI (m/z): calcd for C22H35N4O6 [M+H]+ 451.25511,
found 451.25380.
4.1.4.3. N-((1R,2R,3S,4S,5S)-4-((3-Acetylphenyl)amino)-5-azido-
2,3-dihydroxy-1-((S)-1-hydroxyethyl)-3-(hydroxymethyl)-2-meth-
ylcyclopentyl)piperidine-1-carboxamide (10c). Yield: 88% (21 mg
from 26 mg); ½a 2D0
ꢁ
+26.8 (c 1.00, CHCl3); IR (neat): mmax 3381, 2937,
2856, 2103, 1681, 1627, 1603, 1531, 1487, 1442, 1325, 1270, 1114,
1060, 912, 732 cmꢀ1 1H NMR (400 MHz, CDCl3): d 7.55 (1H, d,
;
J = 11.2 Hz), 7.37–7.31 (2H, m), 7.26 (1H, s), 6.87 (1H, d, J = 7.2 Hz),
5.60 (1H, s), 5.35 (1H, d, J = 11.2 Hz), 4.62 (1H, s), 4.16 (1H, d,
J = 11.6 Hz), 3.99 (1H, m), 3.91 (1H, d, J = 11.2 Hz), 3.67 (1H, d,
J = 11.6 Hz), 3.63 (1H, s), 3.49–3.36 (4H, m), 3.20 (1H, s), 2.58 (3H, s),
1.66–1.60 (6H, m), 1.46 (3H, s), 1.22 (3H, d, J = 6.8 Hz); 13C NMR
(100 MHz, CDCl3): d 198.3, 157.6, 146.2, 138.5, 129.9, 118.9, 118.5,
112.4, 88.4, 84.4, 74.3, 73.7, 73.2, 66.7, 61.2, 45.9, 26.7, 25.8, 24.4,
20.6, 17.8; HRMS-ESI (m/z): calcd for C23H35N6O6 [M+H]+ 491.26126,
found 491.26460.
4.1.5.3. N-((1R,2R,3S,4S,5S)-4-((3-Acetylphenyl)amino)-5-amino-
2,3-dihydroxy-1-((S)-1-hydroxyethyl)-3-(hydroxymethyl)-2-meth-
ylcyclopentyl)piperidine-1-carboxamide (11c). Pale yellow solid,
yield: 88% (7.5 mg from 9 mg); ½a D
mmax 3379, 2935, 2850, 1678, 1602, 1515, 1442, 1273, 1060, 910,
731, 688 cmꢀ1 1H NMR (400 MHz, CDCl3):
7.93 (1H, d,
ꢁ
20 +24.2 (c 0.45, CHCl3); IR (neat):
;
d
J = 10.4 Hz), 7.28–7.20 (3H, m), 6.85 (1H, d, J = 6.8 Hz), 5.63 (1H,
s), 5.47 (1H, d, J = 10.0 Hz), 4.11 (1H, d, J = 11.6 Hz), 3.94 (1H, m),
3.78–3.73 (2H, m), 3.49–3.39 (4H, m), 3.08 (1H, s), 2.58 (3H, s),
1.66–1.60 (6H, m), 1.49 (3H, s), 1.06 (3H, d, J = 6.0 Hz); 13C NMR
(100 MHz, CDCl3): d 198.6, 158.6, 146.9, 138.3, 129.7, 118.9, 118.4,
111.0, 88.6, 84.7, 74.1, 71.3, 68.3, 62.7, 61.8, 45.9, 26.7, 25.8, 24.5,
4.1.4.4.
N-((1R,2R,3S,4S,5S)-4-((3-Acetylphenyl)amino)-5-azido-
2,3-dihydroxy-1-((S)-1-hydroxyethyl)-3-(hydroxymethyl)-2-meth-
ylcyclopentyl)morpholine-4-carboxamide (10d). Yield: 83%
(22 mg from 28 mg); ½a D20
ꢁ
+35.6 (c 1.00, CHCl3); IR (neat): mmax
3378, 2977, 2921, 2859, 2104, 1675, 1627, 1603, 1537, 1434, 1395,
21.5, 18.2; HRMS-ESI (m/z): calcd for C
23H37N4O6 [M+H]+
465.27076, found 465.27025.
1359, 1333, 1304, 1260, 1118, 1077, 1003, 916, 786, 730, 686 cmꢀ1
;
1H NMR (400 MHz, CDCl3): d 7.38–7.32 (2H, m), 7.26–7.23 (2H, m),
6.87 (1H, d, J = 7.2 Hz), 5.61 (1H, s), 5.34 (1H, d, J = 11.6 Hz), 4.52 (1H,
s), 4.17 (1H, d, J = 11.6 Hz), 4.01 (1H, m), 3.92 (1H, d, J = 11.6 Hz),
3.76–3.73 (4H, m), 3.65 (1H, d, J = 11.6 Hz), 3.63 (1H, s), 3.51–3.46
(2H, m), 3.43–3.38 (2H, m), 3.23 (1H, s), 2.58 (3H, s), 2.12 (1H, s), 1.45
(3H, s), 1.22 (3H, d, J = 6.4 Hz); 13C NMR (100 MHz, CDCl3): d 198.3,
157.8, 146.1, 138.5, 130.0, 119.1, 118.6, 112.3, 88.4, 84.5, 74.3, 73.7,
73.1, 66.8, 66.5, 61.1, 44.6, 26.7, 20.5, 17.8; HRMS-ESI (m/z): calcd for
4.1.5.4. N-((1R,2R,3S,4S,5S)-4-((3-Acetylphenyl)amino)-5-amino-
2,3-dihydroxy-1-((S)-1-hydroxyethyl)-3-(hydroxymethyl)-2-meth-
ylcyclopentyl)morpholine-4-carboxamide (11d). Pale yellow
solid, yield: 84% (8.8 mg from 11 mg); ½a D20
ꢁ
+29.3 (c 0.75, CHCl3);
IR (neat): mmax 3381, 2917, 2850, 1678, 1603, 1515, 1440, 1359,
1335, 1303, 1269, 1118, 1072, 909, 732 cmꢀ1 1H NMR (400 MHz,
;
CDCl3): d 7.65 (1H, d, J = 10.4 Hz), 7.35–7.28 (2H, m), 7.20 (1H, s),
6.84 (1H, d, J = 3.2 Hz), 5.55 (1H, s), 5.50 (1H, d, J = 10.4 Hz), 4.13
(1H, d, J = 11.6 Hz), 3.94 (1H, m), 3.78 (5H, br s), 3.53–3.40 (4H, m),
2.95 (1H, s), 2.57 (3H, s), 1.47 (3H, s), 1.05 (3H, d, J = 6.4 Hz); 13C
NMR (100 MHz, CDCl3): d 198.7, 158.8, 146.8, 138.3, 129.7, 118.9,
118.5, 110.8, 88.6, 84.8, 74.0, 71.4, 68.4, 66.6, 62.7, 61.6, 44.7, 26.7,
C
22H33N6O7 [M+H]+ 493.24052, found 493.24255.
4.1.5. General procedure for synthesis of 11a–11d
To a stirred solution of 10a–10d (10 mg, 0.021 mmol) in EtOH/
H2O (3:1, 2 mL) were added ammonium chloride (33 mg,

1782
S. Hanessian et al. / Bioorg. Med. Chem. 21 (2013) 1775–1786
21.3, 18.2; HRMS-ESI (m/z): calcd for C22H35N4O7 [M+H]+
467.25003, found 467.24941.
(1H, d, J = 6. Hz), 3.82 (3H, s), 3.80 (1H, d, J = 11.0 Hz), 3.71 (3H, s),
1.58 (3H, d, J = 6.6 Hz), 1.16 (3H, s), 1.08 (9H, s); 13C NMR
(75 MHz, CDCl3): d 164.0, 162.7, 160.9, 148.4, 135.7, 135.5, 131.1,
130.4, 130.2, 130.1, 127.9, 127.8, 118.9, 113.7, 105.9, 103.7, 98.7,
84.5, 80.0, 78.6, 70.9, 68.1, 66.4, 55.3, 54.9, 26.9, 18.9, 17.3, 17.0;
HRMS (ESIMS): calcd for C40H48N5O6Si [M+H]+ 722.3368, found
722.3369.
4.1.6. General procedure for synthesis of 13a–13d
To a stirred solution of 12 (130 mg, 0.217 mmol) in toluene
(4 mL), the anilines (0.2 mL, 2.173 mmol) and Yb(OTf)3 (67.5 mg,
0.108 mmol) were added at rt and heated at 80 °C and stirred for
9–26 h at the same temperature. The reaction mixture was cooled
to rt, then quenched with water and extracted with ethyl acetate.
The combined organic layers were washed with 0.5 N HCl, satu-
rated aqueous NaHCO3 solution, dried over anhydrous Na2SO4
and concentrated under reduced pressure. The residue was puri-
fied by flash column chromatography using 10% ethyl acetate in
hexanes to afford compounds 13a–13d as pale yellow viscous liq-
uids. Further elution at 12% EtOAc in hexanes recovered the unre-
acted epoxide.
4.1.6.4.
(4S,5R,6R,7S,8S,9S)-9-Azido-7-(((tert-butyldiphenylsi-
lyl)oxy)methyl)-2-(4-methoxyphenyl)-4,6-dimethyl-8-((3-(trifluo-
romethyl)phenyl)amino)-3-oxa-1-azaspiro[4.4]non-1-ene-6,7-diol
(13d). Yield: 52% (122 mg from 200 mg); 20 equiv of m-trifluoro-
methyl aniline was required; ½a D20
ꢁ
+2.4 (c 1.00, CHCl3); IR (neat): mmax
3415, 2934, 2098, 1639, 1611, 1514, 1427, 1344, 1258, 1167, 1115,
1068, 743, 700 cmꢀ1 1H NMR (400 MHz, CDCl3): d 7.90 (2H, d,
;
J = 8.9 Hz), 7.62 (2H, d, J = 6.8 Hz), 7.47–7.34 (6H, m), 7.28–7.22 (4H,
m), 6.98 (1H, d, J = 7.7 Hz), 6.94 (2H, d, J = 8.9 Hz), 6.85–6.80 (2H, m),
5.61 (1H, s), 4.98 (1H, q, J = 6.6 Hz), 4.78 (1H, s), 4.60 (1H, d,
J = 9.6 Hz), 4.33 (1H, dd, J = 6.8, 9.6 Hz), 4.07 (1H, d, J = 11.2 Hz), 3.94
(1H, d, J = 6.8 Hz), 3.85–3.82 (4H, m), 1.61 (3H, d, J = 6.6 Hz), 1.21 (3H,
s), 1.10 (9H, s); 13C NMR (100 MHz, CDCl3): d 164.2, 162.8, 147.2,
135.7, 135.4, 131.0, 130.9, 130.5, 130.3, 130.1, 129.9, 128.0, 127.9,
118.9, 115.4, 114.2, 114.1, 113.8, 109.8, 109.6, 84.5, 84.4, 80.1, 78.6,
70.9, 68.1, 66.4, 55.4, 26.9, 19.0, 17.4, 17.0; HRMS (ESIMS): calcd for
4.1.6.1.
(4S,5R,6R,7S,8S,9S)-9-Azido-7-(((tert-butyldiphenylsi-
lyl)oxy)methyl)-2-(4-methoxyphenyl)-4,6-dimethyl-8-(phenylami-
no)-3-oxa-1-azaspiro[4.4]non-1-ene-6,7-diol (13a). Yield: 74%
(110 mg from 130 mg); ½a D20
ꢁ
+6.56 (c 1.00, CHCl3); IR (neat): mmax
3409, 2938, 3864, 2102, 1642, 1606, 1516, 1466, 1430, 1366, 1349,
1312, 1259, 1173, 1153, 1108, 1059, 1033, 842, 824, 746, 703,
616 cmꢀ1; 1H NMR (300 MHz, CDCl3): d 7.87 (2H, d, J = 9.0 Hz), 7.64
(1H, d, J = 1.4 Hz), 7.62 (1H, d, J = 1.6 Hz), 7.49–7.37 (6H, m), 7.27–
7.13 (4H, m), 6.91 (2H, d, J = 9.0 Hz), 6.75 (1H, tt, J = 1.0, 7.3 Hz),
6.69 (2H, dd, J = 1, 7.6 Hz), 5.58 (1H, s), 4.95 (1H, q, J = 6.6 Hz), 4.85
(1H, s), 4.38–4.30 (2H, m), 4.13 (1H, d, J = 11.1 Hz), 3.97–3.89 (1H,
m), 3.83 (3H, s), 3.80 (1H, d, J = 11.1 Hz), 1.59 (3H, d, J = 6.6 Hz),
1.17 (3H, s), 1.08 (9H, s); 13C NMR (75 MHz, CDCl3): d 164.0, 162.7,
147.0, 135.8, 135.5, 131.1, 131.0, 130.4, 130.2, 129.4, 127.8, 118.9,
117.7, 113.1, 84.5, 84.4, 80.1, 78.6, 71.0, 68.1, 66.4, 55.3, 26.9, 18.9,
C
40H45F3N5O5Si [M+H]+ 760.31366, found 760.31417.
4.1.7. General procedure for synthesis of 14a–14d
To a stirred solution of 13a–13d (56 mg, 0.081 mmol) in THF
(1.4 mL) was added 2.0 N HCl (0.6 mL) at 0 °C and the solution
was allowed to warm up to room temperature. After being stirred
for 18 h at rt, the reaction mixture was cooled to 0 °C, quenched
with a saturated aqueous NaHCO3 solution and extracted with
ethyl acetate. The combined organic layers were washed with
brine, dried over anhydrous Na2SO4 and concentrated under re-
duced pressure. Purification by column chromatography using
10% EtOAc in hexanes gave the starting oxazoline (33–39%). Elu-
tion with [10–25]% EtOAc in hexanes afforded compounds 14a–
14d as pale yellow viscous liquids.
17.3, 17.0; HRMS (ESIMS): calcd for
692.3263, found 692.3268.
C
39H46N5O5Si [M+H]+
4.1.6.2.
(4S,5R,6R,7S,8S,9S)-9-Azido-7-(((tert-butyldiphenylsi-
lyl)oxy)methyl)-8-((3-fluorophenyl)amino)-2-(4-methoxyphenyl)-
4,6-dimethyl-3-oxa-1-azaspiro[4.4]non-1-ene-6,7-diol (13b). Yield:
83% (98 mg from 110 mg); ½a D20
ꢁ
+2.3 (c 1.00, CHCl3); IR (neat): mmax
3392, 2933, 2107, 1704, 1606, 1512, 1259, 1168, 1104, 1073, 822,
4.1.7.1. (S)-1-((1R,2R,3S,4S,5S)-1-Amino-5-azido-3-(((tert-butyl-
diphenylsilyl)oxy)methyl)-2,3-dihydroxy-2-methyl-4-(phenyla-
mino)cyclopentyl)ethyl 4-methoxybenzoate (14a). Yield: 53%
770, 738, 702 cmꢀ1 1H NMR (400 MHz, CDCl3): d 7.80 (2H, d,
;
J = 8.9 Hz), 7.55 (2H, dd, J = 1.2, 7.9 Hz), 7.40 (2H, dd, J = 1.2,
7.9 Hz), 7.40–7.26 (4H, m), 7.21–7.17 (2H, m), 7.03–6.96 (1H, m),
6.83 (2H, d, J = 8.9 Hz), 6.39–6.26 (4H, m), 5.48 (1H, s), 4.88 (1H, q,
J = 6.6 Hz), 4.71 (1H, s), 4.73 (1H, d, J = 9.7 Hz), 4.19 (1H, dd, J = 6.8,
9.7 Hz), 4.00 (1H, d, J = 11.1 Hz), 3.85 (1H,d, J = 6.8 Hz), 3.76 (3H, s),
3.72 (1H, d, J = 11.1 Hz), 1.51 (3H, d, J = 6.6 Hz), 1.11 (3H, s), 1.01
(9H, s); 13C NMR (75 MHz, CDCl3): d 165.3, 164.1, 162.9, 162.7,
148.9, 148.8, 135.8, 135.5, 131.1, 130.9, 130.5, 130.4, 130.4, 130.3,
130.1, 129.7, 127.9, 127.8, 127.7, 118.8, 113.7, 108.7, 104.3, 104.1,
100.0, 99.7, 84.5, 84.4, 84.3, 80.1, 78.6, 70.8, 68.2, 66.3, 55.3, 26.9,
18.9, 17.4, 17.0; HRMS (ESIMS): calcd for C39H45FN5O5Si [M+H]+
710.31685, found 710.31712.
(30 mg from 56 mg); ½a D20
ꢁ
+96.8 (c 1.00, CHCl3); IR (neat): mmax
3391, 2934, 2170, 1704, 1604, 1511, 1463, 1382, 1316, 1259, 1168,
1104, 1072, 1029, 846, 821, 747, 701, 504 cmꢀ1
;
1H NMR
(400 MHz, CDCl3): 8.02 (2H, d, J = 8.9 Hz), 7.59 (2H, d,
d
J = 6.8 Hz), 7.48 (2H, d, J = 6.8 Hz), 7.44–7.26 (4H, m), 7.26–7.17
(4H, m), 6.95 (2H, d, J = 8.9 Hz), 6.76 (1H, t, J = 7.3 Hz), 6.66 (2H, d,
J = 7.8 Hz), 5.76 (1H, q, J = 6.5 Hz), 4.86 (1H, s), 4.47 (1H, d,
J = 11.2 Hz), 4.23 (1H, dd, J = 4.6, 11.1 Hz), 4.04 (1H, d, J = 11.0 Hz),
3.92 (1H, d, J = 4.6 Hz), 3.88 (3H, s), 3.73 (1H, d, J = 11.0 Hz), 1.34
(3H, d, J = 6.5 Hz), 1.26 (3H, s), 1.04 (9H, s); 13C NMR (100 MHz,
CDCl3): d 165.0, 163.6, 146.5, 135.7, 135.6, 131.6, 131.3, 131.3,
130.0, 129.8, 129.5, 127.7, 122.3, 117.8, 113.8, 113.3, 83.7, 80.8,
74.0, 70.8, 70.5, 66.8, 65.4, 55.4, 26.9, 18.9, 18.1, 15.1; HRMS
4.1.6.3.
(4S,5R,6R,7S,8S,9S)-9-Azido-7-(((tert-butyldiphenylsi-
lyl)oxy)methyl)-2-(4-methoxyphenyl)-8-((3-methoxyphenyl)
amino)-4,6-dimethyl-3-oxa-1-azaspiro[4.4]non-1-ene-6,7-diol
(ESIMS): calcd for
710.3371.
C
39H48N5O6Si [M+H]+ 710.3368, found
(13c). Yield: 86% (140 mg from 150 mg); ½a D20
ꢁ
+5.14 (c 1.00,
CHCl3); IR (neat): mmax 3418, 2934, 2099, 1641, 1610, 1514, 1496,
4.1.7.2. (S)-1-((1R,2R,3S,4S,5S)-1-Amino-5-azido-3-(((tert-butyl-
diphenylsilyl)oxy)methyl)-4-((3-fluorophenyl)amino)-2,3-dihy-
droxy-2-methylcyclopentyl)ethyl 4-methoxybenzoate (14b). Yield:
1463, 1455, 1427, 1364, 1345, 1305, 1258, 1213, 1170, 1113,
1055, 1037, 908, 840, 822, 794, 739, 702 cmꢀ1
;
1H NMR
(300 MHz, CDCl3): d 7.87 (2H, d, J = 8.9 Hz), 7.64 (2H, dd, J = 1.4,
8.0 Hz), 7.48 (2H, dd, J = 1.4, 8.0 Hz), 7.44–7.30 (4H, m), 7.29–7.21
(2H, m), 7.06 (1H, t, J = 8.0 Hz), 6.90 (2H, d, J = 8.9 Hz), 6.35–6.24
(3H, m), 5.56 (1H, br s), 4.94 (1H, q, J = 6.6 Hz), 4.81 (1H, s), 4.46–
4.36 (1H, m), 4.35–4.26 (1H, m), 4.13 (1H, d, J = 11.0 Hz), 3.91
53% (30 mg from 55 mg); ½a D20
ꢁ
+105.6 (c 1.00, CHCl3); IR (neat):
mmax 3418, 2935, 2100, 1640, 1621, 1614, 1515, 1495,1427, 1365,
1346, 1258, 1171, 1152, 1114, 1057, 1033, 840, 823, 740,
702 cmꢀ1 1H NMR (400 MHz, CDCl3): d 7.92 (2H, d, J = 8.9 Hz),
;
7.49 (2H, d, J = 6.7 Hz), 7.41 (2H, d, J = 6.7 Hz), 7.30 (2H, q,

S. Hanessian et al. / Bioorg. Med. Chem. 21 (2013) 1775–1786
1783
J = 7.3 Hz), 7.27–7.15 (4H, m), 7.02 (1H, m), 6.89 (2H, d, J = 8.9 Hz),
6.34 (1H, dt, J = 1.8, 8.4 Hz), 6.28 (1H, dd, J = 1.8, 8.1 Hz), 6.22 (2H,
td, J = 2.2, 11.5 Hz), 5.67 (1H, q, J = 6.5 Hz), 4.73 (1H, s), 4.51 (1H, d,
J = 11.0 Hz), 4.07 (1H, dd, J = 4.6, 11.0 Hz), 3.88–3.80 (5H, m), 3.68
(1H, d, J = 11.0 Hz), 1.28 (3H, d, J = 6.5 Hz), 1.17 (3H, s), 0.96 (9H, s);
13C NMR (100 MHz, CDCl3): d 165.1, 163.7, 148.3, 135.7, 135.5,
131.6, 131.3, 131.3, 131.1, 130.6, 130.5, 130.1, 129.9, 127.7, 127.8,
122.3, 113.8, 109.0, 104.4, 100.1, 99.9, 83.7, 80.8, 74.1, 70.8, 70.6,
66.8, 65.4, 55.5, 26.9, 18.9, 18.1, 15.2; HRMS (ESIMS): calcd for
To a stirred solution of crude acetonide in dry THF (2 mL), acti-
vated charcoal (10 mg), Et3N (0.037 mL, 0.266 mmol) and diphos-
gene (0.024 mL, 0.199 mmol) were added slowly at ꢀ46 °C under
argon and the reaction mixture was stirred for 60 min. The reaction
was quenched with saturated aqueous NaHCO3 solution and ex-
tracted with ethyl acetate. The combined organic layers were
washed with brine, dried over anhydrous Na2SO4 and concentrated
under reduced pressure. Flash column chromatography using [22–
26]% ethyl acetate in hexanes gave compounds 15a–15d as color-
less liquids.
C
39H47FN5O6Si [M+H]+ 728.3274, found 728.3286.
4.1.8.1. (S)-1-((1R,2R,3S,4S,5S)-1-Amino-5-azido-2,3-dihydroxy-
4.1.7.3. (S)-1-((1R,2R,3S,4S,5S)-1-Amino-5-azido-3-(((tert-butyldi-
phenylsilyl)oxy)methyl)-2,3-dihydroxy-4-((3-methoxyphenyl)
3-(hydroxymethyl)-2-methyl-4-(phenylamino)cyclopentyl)ethyl
4-methoxybenzoate (15a). Yield: 86% (63 mg from 94 mg); ½a D20
ꢁ
amino)-2-methylcyclopentyl)ethyl
4-methoxybenzoate
(14c). Yield: 45% (65 mg from 140 mg); ½a D20
ꢁ
+99.2 (c 1.00,
+14.27 (c 1.00, CHCl3); IR (neat): mmax 3390, 2989, 2934, 2271,
2105, 1688, 1604, 1511, 1455, 1382, 1317, 1260, 1169, 1065, 1029,
CHCl3); IR (neat): mmax 3395, 3072, 2934, 2858, 2107, 1704, 1605,
848, 771, 750, 694 cmꢀ1 1H NMR (400 MHz, CDCl3): d 8.02 (2H, d,
;
1512, 1496, 1463, 1428, 1259, 1212, 1168, 1104, 1073, 1030, 822,
770, 737, 702 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.00 (2H, d,
;
J = 8.9 Hz), 7.24 (2H, d, J = 7.4 Hz), 6.97 (2H, d, J = 8.9 Hz), 6.78 (1H,
t, J = 7.4 Hz), 6.69 (2H, d, J = 7.7 Hz), 5.48 (1H, q, J = 6.4 Hz), 4.52
(1H, br s), 4.35 (1H, br s), 4.31 (1H, d, J = 10.1 Hz), 4.21 (1H, d,
J = 10.1 Hz), 4.12 (1H, br s), 3.89 (3H, s), 3.46 (1H, d, J = 2.6 Hz),
1.45–1.40 (9H, m), 1.24 (3H, s); 13C NMR (75 MHz, CDCl3): d 167.9,
164.0, 132.3, 129.7, 124.0, 121.1, 118.5, 114.0, 113.5, 111.3, 90.8,
85.6, 75.5, 73.8, 72.4, 67.0, 65.9, 55.5, 26.0, 25.1, 18.0, 16.1; HRMS
(ESIMS): calcd for C27H32N5O7 [M+H]+ 538.2296, found 538.2308.
J = 9.0 Hz), 7.58 (2H, dd, J = 1.4, 8.0 Hz), 7.50 (2H, d, J = 1.4, 8.0 Hz),
7.43–7.20 (7H, m), 7.08 (1H, d, J = 8.0 Hz), 6.96 (2H, d, J = 9.0 Hz),
6.33 (1H, dd, J = 2.2, 8.1 Hz), 6.24 (1H, dd, J = 2.0, 8.0 Hz), 6.20 (1H,
t, J = 2.2 Hz), 5.74 (1H, q, J = 6.5 Hz), 4.82 (1H, s), 4.52 (1H, d,
J = 11.2 Hz), 4.18 (1H, dd, J = 2.7, 11.2 Hz), 3.98 (1H, d, J = 10.9 Hz),
3.90 (1H, d, J = 4.5 Hz), 3.88 (3H, s), 3.74 (1H, d, J = 10.9 Hz), 3.72
(3H, s), 1.36 (3H, d, J = 6.5 Hz), 1.24 (3H, s), 1.04 (9H, s); 13C NMR
(100 MHz, CDCl3): d 165.1, 163.6, 160.9, 147.9, 135.8, 135.6, 131.6,
131.4, 131.3, 130.2, 130.1, 129.8, 127.8, 127.7, 122.4, 113.8, 106.1,
103.8, 99.0, 83.8, 80.8, 74.1, 70.9, 70.5, 66.9, 65.4, 55.0, 26.9, 18.9,
4.1.8.2.
(S)-1-((1R,2R,3S,4S,5S)-1-Amino-5-azido-4-((3-fluoro-
phenyl)amino)-2,3-dihydroxy-3-(hydroxymethyl)-2-methylcycl-
18.1, 15.1; HRMS (ESIMS): calcd for
740.3474, found 740.34816.
C
40H50N5O7Si [M+H]+
opentyl)ethyl 4-methoxybenzoate (15b). Yield: 81% (45 mg
from 70 mg); ½a 2D0
ꢁ
+11.79 (c 1.00, CHCl3); IR (neat): mmax 3398,
2988, 2939, 2272, 2104, 1687, 1606, 1512, 1496, 1382, 1261, 1169,
1070, 1029, 851, 771 cmꢀ1 1H NMR (400 MHz, CDCl3): d 8.05 (2H,
;
4.1.7.4. (S)-1-((1R,2R,3S,4S,5S)-1-Amino-5-azido-3-(((tert-butyldi-
phenylsilyl)oxy)methyl)-2,3-dihydroxy-2-methyl-4-((3-(trifluo-
romethyl)phenyl)amino)cyclopentyl)ethyl 4-methoxybenzoate
d, J = 8.9 Hz), 7.17 (1H, ABq, J = 8.1 Hz), 6.97 (2H, d, J = 8.9 Hz),
6.50–6.36 (3H, m), 5.48 (1H, q, J = 6.3 Hz), 4.67 (1H, d, J = 11.5 Hz),
4.42 (1H, s), 4.27 (1H, d, J = 10.1 Hz), 4.18 (1H, d, J = 10.1 Hz), 4.06
(1H, dd, J = 2.6, 11.5 Hz), 3.89 (3H, s), 3.43 (1H, d, J = 2.6 Hz), 1.44–
1.41 (9H, m), 1.24 (3H, s); 13C NMR (100 MHz, CDCl3): d 167.8,
165.3, 164.4, 162.9, 147.9, 147.8, 132.3, 130.9, 130.8, 124.0, 121.0,
114.0, 111.5, 109.0, 109.0, 105.1, 104.9, 100.5, 100.2, 90.6, 85.5,
75.5, 73.7, 72.3, 67.0, 65.8, 55.5, 26.0, 25.2, 17.9, 16.1; HRMS
(ESIMS): calcd for C27H31FN5O7 [M+H]+ 556.2202, found 556.2220.
(14d). Yield: 53% (124 mg from 230 mg); ½a D20
ꢁ
+102.4 (c 1.00,
CHCl3); IR (neat): mmax 3392, 2934, 2107, 1698, 1606, 1512, 1423,
1344, 1259, 1167, 1114, 1069, 1030, 847, 821, 770, 699 cmꢀ1 1H
;
NMR (400 MHz, CDCl3): d 8.01 (2H, d, J =8.7 Hz), 7.55 (2H, d,
J = 7.4 Hz), 7.47 (2H, d, J = 7.4 Hz), 7.39 (2H, q, J = 7.7 Hz), 7.31–
7.21 (6H, m), 6.97 (3H, d, J = 8.7 Hz), 6.81 (1H, s), 6.75 (1H, d,
J = 8.2 Hz), 5.80–5.73 (1H, m), 4.79 (1H, s), 4.73 (1H, d, J = 10.9 Hz),
4.20 (1H, dd, J = 4.5, 10.9 Hz), 3.93 (1H, d, J = 4.5 Hz), 3.92–3.88
(4H, m), 3.79 (1H, d, J = 11.0 Hz), 1.35 (3H, d, J = 6.5 Hz), 1.27 (3H,
s), 1.04 (9H, s); 13C NMR (100 MHz, CDCl3): d 165.1, 163.7, 146.7,
135.7, 135.6, 131.6, 131.3, 130.1, 129.9, 129.9, 127.8, 127.7, 122.3,
115.7, 114.1, 114.1, 113.8, 109.5, 109.5, 83.7, 80.8, 74.1, 70.8, 70.3,
66.9, 65.3, 55.4, 26.8, 18.9, 18.0, 15.1; HRMS (ESIMS): calcd for
4.1.8.3. (S)-1-((1R,2R,3S,4S,5S)-1-Amino-5-azido-2,3-dihydroxy-3-
(hydroxymethyl)-4-((3-methoxyphenyl)amino)-2-methylcycl-
opentyl)ethyl 4-methoxybenzoate (15c). Yield: 47% (39 mg
from 110 mg); ½a 2D0
ꢁ
+11.2 (c 1.00, CHCl3); IR (neat): mmax 3389,
2990, 2938, 2271, 2104, 1688, 1606, 1512, 1496, 1383, 1318,
1261, 1212, 1169, 1103, 1064, 1030, 909, 854, 733, 689 cmꢀ1 1H
;
C
40H47F3N5O6Si [M+H]+ 778.32422, found 778.32531.
NMR (400 MHz, CDCl3): d 8.05 (2H, d, J = 8.9 Hz), 7.14 (1H, t,
J = 8.1 Hz), 6.97 (2H, d, J = 8.9 Hz), 6.34 (1H, dd, J = 2.2, 7.7 Hz),
6.30 (1H, dd, J = 1.7, 8.0 Hz), 6.24 (1H, t, J = 2.2 Hz), 5.48 (1H, q,
J = 6.3 Hz), 4.53 (1H, d, J = 11.6 Hz), 4.38 (1H, s), 4.28 (1H, d,
J = 10.1 Hz), 4.20 (1H, d, J = 10.1 Hz), 4.10 (1H, dd, J = 2.6,
11.6 Hz), 3.88 (3H, s), 3.79 (3H, s), 3.47 (1H, d, J = 2.6 Hz), 1.45
(3H, s), 1.43 (3H, d, J = 6.3 Hz), 1.42 (3H, s), 1.25 (3H, s); 13C NMR
(75 MHz, CDCl3): d 167.8, 164.3, 161.0, 147.5, 132.3, 130.4, 124.0,
121.1, 114.0, 111.3, 106.2, 103.9, 99.4, 90.7, 85.5, 75.5, 73.7, 72.4,
66.9, 65.9, 55.5, 55.1, 26.0, 25.1, 18.0, 16.1; HRMS (ESIMS): calcd
for C28H34N5O8 [M+H]+ 568.24019, found 568.24056.
4.1.8. General procedure for synthesis 15a–15d
To a stirred solution of 14a–14d (94 mg, 0.134 mmol) in dry
DMF (3 mL) was added TAS-F (110 mg, 0.401 mmol) at 0 °C under
argon and allowed to room temperature. After being stirred for 1 h
at rt, the reaction mixture was cooled to 0 °C, quenched with a pH
7 phosphate buffer solution and extracted with ethyl acetate. The
combined organic layers were washed with water, brine, dried
over anhydrous Na2SO4 and concentrated under reduced pressure.
To a stirred solution of crude amino triol in dry DCM (2 mL), CSA
(37.3 mg, 0.16 mmol) and 2,2-dimethoxypropane (0.4 mL) were
added sequentially at 0 °C under argon and warm up to room tem-
perature. After being stirred for 2 h at rt, the reaction mixture was
cooled to 0 °C, quenched with saturated aqueous NaHCO3 solution
and extracted with DCM. The combined organic layers were
washed with brine, dried over anhydrous Na2SO4 and concentrated
under reduced pressure.
4.1.8.4. (S)-1-((1R,2R,3S,4S,5S)-1-Amino-5-azido-2,3-dihydroxy-
3-(hydroxymethyl)-2-methyl-4-((3-(trifluoromethyl)phenyl)
amino)cyclopentyl)ethyl 4-methoxybenzoate (15d). Yield: 82%
(54 mg from 80 mg); ½a D20
ꢁ
+19.0 (c 1.00, CHCl3); IR (neat): mmax
3382, 2969, 2104, 1687, 1605, 1513, 1342, 1317, 1261, 1168, 1124,
1069, 852, 771, 697 cmꢀ1; 1H NMR (400 MHz, CDCl3): d 8.05 (2H, d,

1784
S. Hanessian et al. / Bioorg. Med. Chem. 21 (2013) 1775–1786
J = 8.9 Hz), 7.33 (1H, t, J = 7.8 Hz), 7.02 (1H, d, J = 7.8 Hz), 6.97 (2H,
d, J = 8.9 Hz), 6.78 (1H, t, J = 7.4 Hz), 6.89 (1H, s), 6.85 (1H, d,
J = 8.1 Hz), 5.49 (1H, q, J = 6.3 Hz), 4.75 (1H, d, J = 11.5 Hz), 4.47
(1H, s), 4.35 (1H, br s), 4.24 (2H, ABq, J = 10.1 Hz), 4.15–4.09 (1H,
m), 3.88 (3H, s), 3.47 (1H, d, J = 2.7 Hz), 2.04 (1H, s), 1.45–1.43 (9H,
m), 1.26 (3H, s); 13C NMR (100 MHz, CDCl3): d 167.9, 164.4, 146.3,
132.3, 130.1, 125.4, 124.1, 122.7, 121.0, 115.8, 114.9, 114.0, 111.5,
110.0, 110.0, 90.6, 85.6, 75.4, 73.8, 72.3, 66.9, 66.9, 65.8, 55.5, 25.9,
25.2, 17.9, 16.1; HRMS (ESIMS): calcd for C28H31F3N5O7 [M+H]+
606.21701, found 606.21775.
4.1.9.4. (S)-1-((5S,6R,7R,8S,9S)-8-Azido-7-(3,3-dimethylureido)-
6-hydroxy-2,2,6-trimethyl-9-((3-(trifluoromethyl)phenyl)amino)-
1,3-dioxaspiro[4.4]nonan-7-yl)ethyl
4-methoxybenzoate
(16d). Yield: 93% (54 mg from 54 mg); ½a D20
ꢁ
+101.07 (c 1.00,
CHCl3); IR (neat): mmax 3403, 2938, 2109, 1708, 1651, 1606, 1512,
1452, 1343, 1258, 1167, 1122, 1068, 1031, 849, 766, 697 cmꢀ1 1H
;
NMR (400 MHz, CDCl3): d 7.97 (2H, d, J = 8.9 Hz), 7.31–7.24 (2H,
m), 7.00–6.85 (5H, m), 6.47 (1H, q, J = 6.5 Hz), 5.70 (1H, s), 4.48–
4.44 (1H, m), 4.39 (1H, d, J = 9.9 Hz), 4.23 (1H, s), 4.16 (1H, d,
J = 9.9 Hz), 3.97–3.93 (2H, m), 3.85 (3H, s), 2.89 (6H, s), 1.56–1.54
(6H, m), 1.39 (3H, s), 1.30 (3H, s); 13C NMR (75 MHz, CDCl3): d
164.5, 163.6, 157.9, 147.4, 131.5, 128.2, 125.5, 122.8, 12212, 116.3,
114.7, 114.7, 113.8, 109.9, 109.7, 109.6, 90.0, 82.7, 71.7, 7036, 68.0,
65.7, 65.1, 55.4, 36.5, 36.5, 25.9, 25.6, 22.2, 17.1; HRMS (ESIMS):
calcd for C30H38F3N6O7 [M+H]+ 651.27486, found 651.27541.
4.1.9. General procedure for synthesis of 16a–16d
To 15a–15d (82 mg, 0.152 mmol) was added neat 0.5 mL
dimethylamine (upon condensing the gas at ꢀ46 °C) and the reac-
tion mixture was left warming to room temperature. It was di-
rectly subjected to flash column chromatography using [32–40]%
ethyl acetate in hexanes to afford compounds 16a–16d as colorless
liquids.
4.1.10. General procedure for synthesis of 17a–17d
To a stirred solution of 16a–16d (74 mg, 0.127 mmol) in dry
DCM (3.5 mL), DIBAL-H (0.34 mL, 1.5 M in toluene, 0.51 mmol)
was added slowly at ꢀ78 °C under argon and stirred for 1.5 h.
The reaction mixture was then quenched with slow addition of
methanol and warmed to room temperature. A saturated aqueous
potassium sodium tartrate solution was added to the reaction mix-
ture and stirred for 1 h, extracted with ethyl acetate, the combined
organic layers were washed with brine, dried over anhydrous
Na2SO4 and concentrated under reduced pressure. Flash column
chromatography using [24–28]% ethyl acetate in hexanes gave
compounds 17a–17d as colorless liquids.
4.1.9.1. (S)-1-((5S,6R,7R,8S,9S)-8-Azido-7-(3,3-dimethylureido)-6-
hydroxy-2,2,6-trimethyl-9-(phenylamino)-1,3-dioxaspiro[4.4]
nonan-7-yl)ethyl 4-methoxybenzoate (16a). Yield: 88%
(78 mg from 82 mg); ½a D20
ꢁ
+96.8 (c 1.00, CHCl3); IR (neat): mmax
3401, 2987, 2937, 2109, 1711, 1652, 1605, 1512, 1372, 1317,
1258, 1168, 1101, 1057, 1030, 848, 732, 694 cmꢀ1
;
1H NMR
(300 MHz, CDCl3): 7.98 (2H, d, J = 9.0 Hz), 7.18 (2H, t,
d
J = 7.6 Hz), 6.94 (2H, d, J = 9.0 Hz), 6.79–6.69 (3H, m), 6.44 (1H,
q, J = 6.5 Hz), 5.70 (1H, s), 4.39 (1H, d, J = 9.8 Hz), 4.15 (1H, d,
J = 9.8 Hz), 4.15–4.09 (1H, m), 3.99–3.89 (2H, m), 3.86 (3H, s),
2.90 (6H, s), 1.54–1.52 (6H, m), 1.39 (3H, s), 1.30 (3H, s); 13C NMR
(75 MHz, CDCl3): d 164.5, 163.3, 157.9, 147.2, 131.6, 129.4, 122.2,
118.4, 113.8, 113.5, 109.8, 90.1, 82.8, 72.0, 70.6, 68.0, 65.9, 65.1,
55.4, 36.5, 29.6, 25.6, 22.4, 17.0; HRMS (ESIMS): calcd for
4.1.10.1. 3-((5S,6R,7R,8S,9S)-8-Azido-6-hydroxy-7-((S)-1-hydroxy-
ethyl)-2,2,6-trimethyl-9-(phenylamino)-1,3-dioxaspiro[4.4]nonan-
7-yl)-1,1-dimethylurea (17a). Yield: 91% (52 mg from 74 mg); ½a D20
ꢁ
+40.9 (c 1.00, CHCl3); IR (neat): mmax 3419, 2988, 2935, 2106, 1644,
1602, 1538, 1505, 1372, 1311, 1260, 1139, 1117, 1061, 1047, 860, 750,
693, 532 cmꢀ1; 1H NMR (300 MHz, CDCl3): d 7.44 (1H, d, J = 11.4 Hz),
7.24 (1H, t, J = 8.6 Hz), 6.77 (1H, t, J = 7.3 Hz), 6.61 (2H, d, J = 8.6 Hz),
5.41 (1H, s), 5.18 (1H, d, J = 11.2 Hz), 4.60 (1H, s), 4.24 (2H, s), 4.00–
3.84 (2H, m), 3.65 (1H, s), 3.00 (6H, s), 1.44 (3H, s), 1.43 (3H, s), 1.39
(3H, s), 1.17 (3H, d, J = 6.5 Hz); 13C NMR (75 MHz, CDCl3): d 158.3,
145.8, 129.7, 118.2, 113.1, 110.5, 97.3, 83.3, 74.0, 73.1, 72.7, 66.7, 65.4,
36.7, 26.3, 25.6, 20.7, 17.7; HRMS (ESIMS): calcd for C21H33N6O5
[M+H]+ 449.25069, found 449.25239.
C
29H39N6O7 [M+H]+ 583.28747, found 583.28885.
4.1.9.2. (S)-1-((5S,6R,7R,8S,9S)-8-Azido-7-(3,3-dimethylureido)-9-
((3-fluorophenyl)amino)-6-hydroxy-2,2,6-trimethyl-1,3-dioxa-
spiro[4.4]nonan-7-yl)ethyl 4-methoxybenzoate (16b). Yield:
93% (12 mg from 12 mg); ½a D20
ꢁ
+78.0 (c 1.00, CHCl3); IR (neat):
mmax 3405, 2932, 2109, 1709, 1650, 1607, 1513, 1495, 1372, 1317,
1258, 1168, 1102, 1058, 1031, 848, 768, 733 cmꢀ1 1H NMR
;
(400 MHz, CDCl3): d 7.97 (2H, d, J = 8.9 Hz), 7.14–7.06 (1H, m),
6.94 (2H, d, J = 8.9 Hz), 6.53–6.38 (4H, m), 5.67 (1H,s), 4.39 (1H, d,
J = 9.8 Hz), 4.29 (1H, d, J = 8.4 Hz), 4.10 (2H, d, J = 9.8 Hz), 3.93–
3.86 (5H, m), 2.89 (6H, s), 1.54 (3H, m), 1.52 (3H, d, J = 6.6 Hz),
1.39 (3H, s), 1.31 (3H, s); 13C NMR (75 MHz, CDCl3): d 165.7, 164.5,
163.6, 162.4, 157.9, 149.1, 149.0, 131.6, 130.6, 130.4, 122.1, 113.8,
109.8, 109.3, 105.0, 104.8, 100.4, 100.0, 89.8, 82.6, 72.0, 70.1, 67.8,
65.8, 65.1, 55.4, 36.5, 25.6, 22.4, 17.0; HRMS (ESIMS): calcd for
4.1.10.2. 3-((5S,6R,7R,8S,9S)-8-Azido-9-((3-fluorophenyl)amino)-
6-hydroxy-7-((S)-1-hydroxyethyl)-2,2,6-trimethyl-1,3-dioxaspiro
[4.4]nonan-7-yl)-1,1-dimethylurea (17b). Yield: 98% (43 mg
from 56 mg); ½a 2D0
ꢁ
+41.8 (c 1.00, CHCl3); IR (neat): mmax 3380,
2995, 2933, 2108, 1737, 1622, 1593, 1536, 1515, 1498, 1375, 1339,
1260, 1214, 1181, 1156, 1062, 1049, 967, 941, 857, 757, 685 cmꢀ1
;
C
29H38FN6O7 [M+H]+ 601.2781, found 601.2804.
1H NMR (300 MHz, CDCl3): d 7.45 (1H, d, J = 11.4 Hz), 7.24–7.21 (1H,
m), 6.54 (1H, ddt, J = 0.8, 2.3, 8.5 Hz), 6.37 (1H, ddd, J = 0.8, 2.3,
8.1 Hz), 6.30 (1H, td, J = 2.3, 11.4 Hz), 5.39 (1H, s), 5.31 (1H, d,
J = 11.2 Hz), 4.62 (1H, s), 4.21 (2H, ABq, J = 9.9 Hz), 3.98–3.85 (1H,
m), 3.80 (1H, d, J = 11.2 Hz), 3.62 (1H, s), 2.99 (6H, s), 1.43 (6H, s),
1.38 (3H, s), 1.21 (3H, d, J = 6.5 Hz); 13C NMR (75 MHz, CDCl3): d
165.4, 163.0, 158.2, 147.8, 147.7, 130.9, 130.8, 110.7, 108.9, 108.9,
104.8, 104.6, 100.0, 99.8, 91.2, 88.3, 74.2, 73.0, 72.8, 66.7, 65.3, 36.7,
26.3, 25.6, 20.6, 17.7; HRMS (ESIMS): calcd for C21H31FN6NaO5
[M+Na]+ 489.2232, found 489.2254.
4.1.9.3. (S)-1-((5S,6R,7R,8S,9S)-8-Azido-7-(3,3-dimethylureido)-6-
hydroxy-9-((3-methoxyphenyl)amino)-2,2,6-trimethyl-1,3-dioxa-
spiro[4.4]nonan-7-yl)ethyl 4-methoxybenzoate (16c). Yield:
86% (36 mg from 39 mg); ½a D20
ꢁ
+90.9 (c 1.00, CHCl3); IR (neat): mmax
3397, 2988, 2937, 2109, 1710, 1658, 1606, 1512, 1496, 1463, 1372,
1258, 1213, 1167, 1102, 1054, 1031, 849, 732 cmꢀ1 1H NMR
;
(300 MHz, CDCl3): d 7.97 (2H, d, J = 9.0 Hz), 7.08 (1H, t, J = 8.7 Hz),
6.94 (2H, d, J = 9.0 Hz), 6.44 (1H, q, J = 6.5 Hz), 6.38–6.27 (3H, m),
5.70 (1H, s), 4.38 (1H, d, J = 9.8 Hz), 4.24–4.09 (3H, m), 3.95–3.89
(2H, m), 3.86 (3H, s), 3.75 (3H, s), 2.89 (6H, s), 1.53 (3H, s), 1.53 (3H,
d, J = 6.5 Hz), 1.39 (3H, s), 1.33 (3H, s); 13C NMR (75 MHz, CDCl3): d
164.6, 163.5, 160.8, 157.9, 148.6, 131.6, 130.1, 122.2, 113.8, 109.8,
106.5, 103.4, 99.6, 90.0, 82.8, 72.0, 70.6, 68.0, 65.9, 65.1, 55.4, 55.0,
36.5, 26.0, 25.7, 22.3, 17.0; HRMS (ESIMS): calcd for C30H41N6O8
[M+H]+ 613.29804, found 613.29907.
4.1.10.3. 3-((5S,6R,7R,8S,9S)-8-Azido-6-hydroxy-7-((S)-1-hydroxy-
ethyl)-9-((3-methoxyphenyl)amino)-2,2,6-trimethyl-1,3-dioxaspiro
[4.4]nonan-7-yl)-1,1-dimethylurea (17c). Yield: 75% (36 mg from
62 mg); ½a 2D0
ꢁ
+47.5 (c 1.00, CHCl3); IR (neat): mmax 3418, 2988, 2936,
2106, 1643, 1614, 1538, 1495, 1373, 1308, 1259, 1212, 1164, 1139,
1061, 1046, 856, 761, 733, 689 cmꢀ1 1H NMR (400 MHz, CDCl3): d
;

S. Hanessian et al. / Bioorg. Med. Chem. 21 (2013) 1775–1786
1785
7.47 (1H, d, J = 11.3 Hz), 7.13 (1H, t, J = 8.1 Hz), 6.34 (1H, dd, J = 2.2,
8.1 Hz), 6.22 (1H, dd, J = 1.7, 8.1 Hz), 6.16 (1H, t, J = 2.2 Hz), 5.41 (1H, s),
5.22 (1H, d, J = 11.3 Hz), 4.63 (1H, s), 4.23 (2H, s), 3.93 (1H, sept,
J = 6.5 Hz), 3.84 (1H, d, J = 11.3 Hz), 3.78 (3H, s), 3.65 (1H, s), 2.99 (6H,
s), 1.43 (3H, s), 1.42 (3H, s), 1.38 (3H, s), 1.18 (3H, d, J = 6.5 Hz); 13C
NMR (75 MHz, CDCl3): d 161.1, 158.3, 147.2, 130.5, 110.5, 106.0, 103.6,
99.0, 91.3, 83.3, 74.0, 73.1, 72.7, 66.6, 65.3, 55.1, 36.7, 26.3, 25.5, 20.7,
17.7; HRMS (ESIMS): calcd for C22H35N6O6 [M+H]+ 479.26126, found
479.26244.
130.8, 109.4, 109.4, 105.3, 105.0, 100.7, 100.4, 88.3, 84.4, 74.0, 73.8,
73.0, 66.7, 61.2, 36.7, 36.6, 20.4, 17.7; HRMS (ESIMS): calcd for
C
18H28FN6O5 [M+H]+ 427.20997, found 427.21009.
4.1.11.3.
3-((1R,2R,3S,4S,5S)-5-Azido-2,3-dihydroxy-1-((S)-1-
hydroxyethyl)-3-(hydroxymethyl)-4-((3-methoxyphenyl)amino)-
2-methylcyclopentyl)-1,1-dimethylurea (18c). Yield: 70% (9 mg
from 14 mg); ½a 2D0
ꢁ
+16.9 (c 1.00, CHCl3); IR (neat): mmax 3413, 2929,
2104, 1615, 1537, 1514, 1374, 1305, 1261, 1212, 1164, 1110, 1038,
913, 762, 733 cmꢀ1 1H NMR (400 MHz, CDCl3): d 7.45 (1H, d,
;
4.1.10.4. 3-((5S,6R,7R,8S,9S)-8-Azido-6-hydroxy-7-((S)-1-hydroxy-
ethyl)-2,2,6-trimethyl-9-((3-(trifluoromethyl)phenyl)amino)-1,3-
dioxaspiro[4.4]nonan-7-yl)-1,1-dimethylurea (17d). Yield: 91%
J = 11.3 Hz), 7.14 (1H, t, J = 8.1 Hz), 6.37 (1H, dd, J = 1.7, 8.1 Hz), 6.26
(1H, dd, J = 1.2, 8.1 Hz), 6.22 (1H, t, J = 1.7 Hz), 5.50 (1H, s), 5.14 (1H,
d, J = 11.7 Hz), 4.54 (1H, s), 4.17 (1H, d, J = 11.7 Hz), 4.02–3.91 (1H,
m), 3.84–3.76 (4H, m), 3.65 (1H, s), 3.59 (1H, d, J = 11.7 Hz), 2.99 (6H,
s), 1.81 (1H, br s), 1.61 (2H, br s), 1.41 (3H, s), 1.20 (3H, d, J = 6.5 Hz);
13C NMR (100 MHz, CDCl3): d 161.1, 158.5, 147.1, 130.6, 106.5, 104.2,
99.7, 88.2, 84.6, 73.9, 73.8, 73.0, 66.8, 61.4, 55.1, 36.7, 20.5, 17.7;
HRMS (ESIMS): calcd for C19H30N6O6 [M+H]+ 439.22996, found
439.23098.
(110 mg from 130 mg); ½a D20
ꢁ
+44.0 (c 1.00, CHCl3); IR (neat): mmax
3377, 2988, 2936, 2105, 1641, 1535, 1148, 1373, 1343, 1281, 1163,
1122, 1062, 855, 785, 693 cmꢀ1 1H NMR (400 MHz, CDCl3): d 7.46
;
(1H, d, J = 11.3 Hz), 7.31 (1H, t, J = 7.9 Hz), 7.00 (1H, d, J = 7.6 Hz),
6.82 (1H, s), 6.77 (1H, d, J = 8.2 Hz), 5.42 (2H, d, J = 11.3 Hz), 4.67
(1H, s), 4.22 (2H, ABq, J = 9.9 Hz), 3.96–3.87 (1H, s), 3.85 (1H, d,
J = 11.3 Hz), 3.6 (1H, s), 2.99 (6H, s), 1.43 (3H, s), 1.43 (6H, s), 1.39
(3H, s), 1.21 (3H, d, J = 6.5 Hz); 13C NMR (100 MHz, CDCl3): d 158.2,
146.1, 130.1, 125.4, 122.7, 116.0, 114.6, 114.5, 110.7, 109.3, 109.2,
91.2, 88.2, 74.9, 72.8, 66.6, 65.3, 41.9, 36.6, 26.2, 25.6, 20.5, 17.7;
HRMS (ESIMS): calcd for C22H32F3N6O5 [M+H]+ 517.23808, found
517.23916.
4.1.11.4. 3-((1R,2R,3S,4S,5S)-5-Azido-2,3-dihydroxy-1-((S)-1-hydro-
xyethyl)-3-(hydroxymethyl)-2-methyl-4-((3-(trifluoromethyl)
phenyl)amino)cyclopentyl)-1,1-dimethylurea (18d). Yield:
82% (14 mg from 18 mg); ½a D20
ꢁ
+26.9 (c 1.00, CHCl3); IR (neat):
mmax 3408, 2934, 2104, 1633, 1538, 1488, 1375, 1342, 1284,
1164, 1122, 1068, 908, 787, 735, 699 cmꢀ1 1H NMR (400 MHz,
;
4.1.11. General procedure for synthesis of 18a–18d
CDCl3: d 7.48 (1H, d, J = 11.3 Hz), 7.33 (1H, t, J = 7.8 Hz), 7.03 (1H,
d, J = 7.8 Hz), 6.87 (1H, s), 6.81 (1H, d, J = 8.2 Hz), 5.50 (1H, s),
5.40 (1H, d, J = 11.3 Hz), 4.58 (1H, s), 4.13 (1H, d, J = 11.6 Hz),
4.02–3.92 (1H, m), 3.84 (1H, d, J = 11.1 Hz), 3.63 (1H, d,
J = 11.6 Hz), 3.60 (1H, s), 3.15 (1H, s), 2.99 (6H, s), 2.05 (1H, br
s), 1.69 (1H, br s), 1.42 (3H, s), 1.23 (3H, d, J = 6.5 Hz); 13C NMR
(100 MHz, CDCl3): d 158.4, 146.1, 132.2, 131.9, 130.2, 116.6,
115.0, 114.9, 109.8, 109.8, 88.3, 84.1, 74.1, 73.8, 73.8, 73.0, 66.5,
61.1, 36.7, 20.4, 17.7; HRMS (ESIMS): calcd for C19H28F3N6O5
[M+H]+ 477.20678, found 477.20767.
To the stirred solution of 17a–17d (13 mg, 0.029 mmol) in ace-
tonitrile (0.1 mL) and H2O (0.1 mL); TFA (0.5 mL) was added at 0 °C
and the solution was stirred at 33 °C for variable times (90 min for
17a, 75 min for 17b and 60 min for 17c, rt for 90 min in the case of
17d), cooled to 0 °C, quenched very slowly with saturated aqueous
NaHCO3 solution and extracted with ethyl acetate. The combined
organic layers were washed with brine, dried over anhydrous
Na2SO4 and concentrated under reduced pressure. Flash column
chromatography using [70–80]% ethyl acetate in hexanes gave
compounds 18a–18d as colorless liquids.
4.1.12. General procedure for synthesis of 19a–19d
4.1.11.1. 3-((1R,2R,3S,4S,5S)-5-Azido-2,3-dihydroxy-1-((S)-1-hydro-
xyethyl)-3-(hydroxymethyl)-2-methyl-4-(phenylamino)cyclo-
pentyl)-1,1-dimethylurea (18a). Yield: 76% (9 mg from
To a stirred solution of 18a–18d (9 mg, 0.022 mmol) in EtOH/
H2O (3:1, 1.4 mL) were added ammonium chloride (35 mg,
0.662 mmol), zinc powder (22 mg, 0.331 mmol) at rt and the mix-
ture was stirred for 8 h. The reaction mixture was quenched with
aqueous ammonia (2 mL) and extracted with CH2Cl2, the combined
organic layers were washed with brine, dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The residue
was purified by flash column chromatography using [4–5]% meth-
anol in chloroform to give 19a–19d as a pale yellow oils.
13 mg); ½a 2D0
ꢁ
+13.5 (c 1.00, CHCl3); IR (neat): mmax 3412, 2928,
2104, 1634, 1602, 1537, 1506, 1374, 1307, 1249, 1110, 1036,
912, 752, 734, 694 cmꢀ1 1H NMR (400 MHz, CDCl3): d 7.44 (1H,
;
d, J = 11.4 Hz), 7.30–7.21 (2H, m), 6.80 (1H, t, J = 7.3 Hz), 6.67
(2H, d, J = 7.8 Hz), 5.51 (1H, s), 5.13 (1H, d, J = 10.7 Hz), 4.53 (1H,
s), 4.17 (2H, d, J = 11.8 Hz), 4.03–3.91 (1H, m), 3.81 (1H, d,
J = 8.9 Hz), 3.64 (1H, s), 3.62 (1H, d, J = 11.8 Hz), 3.00 (6H, s),
2.15 (1H, br s), 1.74 (1H, br s), 1.41 (3H, s), 1.21 (3H, d,
J = 6.5 Hz); 13C NMR (100 MHz, CDCl3): d 158.5, 145.7, 129.7,
118.7, 113.9, 88.2, 84.6, 73.9, 73.8, 72.9, 66.8, 61.4, 36.7, 20.4,
17.7; HRMS (ESIMS): calcd for C18H29N6O5 [M+H]+ 409.21939,
found 409.22051.
4.1.12.1.
3-((1R,2R,3S,4S,5S)-5-Amino-2,3-dihydroxy-1-((S)-1-
hydroxyethyl)-3-(hydroxymethyl)-2-methyl-4-(phenylamino)
cyclopentyl)-1,1-dimethylurea (19a). Yield: 80% (7 mg from
9 mg); ½a 2D0
ꢁ
+29.3 (c 1.00, CHCl3); IR (neat): mmax 3382, 2927,
2724, 1603, 1505, 1373, 1297, 1089, 1041, 912, 750 cmꢀ1
;
1H
NMR (400 MHz, CDCl3): d 7.85 (1H, d, J = 10.5 Hz), 7.20 (2H, t,
J = 7.8 Hz), 7.05 (1H, s), 6.73 (1H, t, J = 7.3 Hz), 6.64 (2H, d,
J = 7.8 Hz), 5.56 (1H, s), 5.20 (1H, d, J = 10.9 Hz), 4.11 (1H, d,
J = 11.7 Hz), 3.99–3.87 (1H, m), 3.72 (1H, d, J = 11.7 Hz), 3.67 (1H,
d, J = 10.5 Hz), 2.99 (6H, s), 1.79 (5H, br s), 1.45 (3H, s), 1.05 (3H,
d, J = 6.4 Hz); 13C NMR (100 MHz, CDCl3): d 159.4, 146.6, 129.6,
117.9, 113.4, 88.4, 84.8, 74.0, 71.3, 68.6, 62.4, 61.9, 36.8, 21.5,
18.2; HRMS (ESIMS): calcd for C18H31N4O5 [M+H]+ 383.2289,
found 383.22804.
4.1.11.2. 3-((1R,2R,3S,4S,5S)-5-Azido-4-((3-fluorophenyl)amino)-
2,3-dihydroxy-1-((S)-1-hydroxyethyl)-3-(hydroxymethyl)-2-meth-
ylcyclopentyl)-1,1-dimethylurea (18b). Yield: 85% (10 mg from
12.5 mg); ½a D
1622, 1590, 1538, 1374, 1334, 1248, 1180, 1152, 1110, 1037, 763,
733 cmꢀ1 1H NMR (400 MHz, CDCl3): d 7.51 (1H, d, J = 11.3 Hz),
ꢁ
20 +18.9 (c 1.00, CHCl3); IR (neat): mmax 3408, 2931, 2104,
;
7.20–7.13 (1H, m), 6.47 (1H, dt, J = 2.0, 8.4 Hz), 6.41 (1H, dd, J = 1.8,
8.2 Hz), 6.35 (1H, td, J = 2.2, 11.3 Hz), 5.50 (1H, s), 5.29 (1H, d,
J = 11.3 Hz), 4.56 (1H, s), 4.10 (1H, d, J = 11.6 Hz), 4.00–3.91 (1H, m),
3.77 (1H, d, J = 11.3 Hz), 3.65–3.60 (2H, m), 3.17 (1H, s), 2.99 (6H, s),
4.1.12.2. 3-((1R,2R,3S,4S,5S)-5-Amino-4-((3-fluorophenyl)amino)-
2,3-dihydroxy-1-((S)-1-hydroxyethyl)-3-(hydroxymethyl)-2-meth-
ylcyclopentyl)-1,1-dimethylurea (19b). Yield: 75% (7.6 mg from
2.07 (1H, br s), 1.73 (1H, br s), 1.40 (3H, s), 1.22 (3H, d, J = 6.5 Hz); 13
C
NMR (100 MHz, CDCl3): d 165.4, 163.0, 158.5, 147.7, 147.6, 130.9,

1786
S. Hanessian et al. / Bioorg. Med. Chem. 21 (2013) 1775–1786
10 mg); ½a 2D0
ꢁ
+33.2 (c 1.00, CHCl3); IR (neat): mmax 3382, 2927, 1719,
1621, 1515, 1455, 1374, 1337, 1180, 1150, 1088, 1041, 918,
4.1.14. In vitro antibacterial activity
Minimum inhibitory concentrations (MIC) were measured by a
standard broth microdilution method following the Clinical Labo-
ratory Standard Institute recommendations.23 Isolates tested were
Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923,
Klebsiella pneumoniae ATCC 700603, a clinical isolate of Acinetobac-
ter baumanii, Pseudomonas aeruginosa ATCC 27853 and Enterococ-
cus faecalis ATCC 29212. Plates were read after an incubation
period of 20 h.
759 cmꢀ1 1H NMR (400 MHz, CDCl3): d 7.86 (1H, d, J = 10.8 Hz),
;
7.17–7.03 (2H, m), 6.46–6.28 (3H, m), 5.62 (1H, s), 5.39 (1H, d,
J = 10.5 Hz), 4.06 (1H, d, J = 11.6 Hz), 3.97–3.84 (1H, m), 3.73 (1H, d,
J = 11.6 Hz), 3.60 (1H, dd, J = 1.4, 10.5 Hz), 2.99 (6H, s), 2.95 (1H, s),
1.77 (1H, br s), 1.45 (3H, s), 1.04 (3H, d, J = 6.5 Hz); 13C NMR
(100 MHz, CDCl3): d 165.4, 162.9, 159.3, 148.3, 130.6, 109.5, 109.4,
104.3, 104.1, 99.9, 99.6, 88.4, 84.6, 73.9, 71.3, 68.4, 62.4, 61.7, 36.8,
21.4, 18.2; HRMS (ESIMS): calcd for C18H30FN4O5 [M+H]+ 401.21947,
found 401.2182.
Acknowledgements
4.1.12.3.
3-((1R,2R,3S,4S,5S)-5-Amino-2,3-dihydroxy-1-((S)-1-
We thank NSERC and FQRNT for financial support. We express
special thanks to Professor Taifo Mahmud (Oregon State Univer-
sity) and Jane X. Kelly (Portland VA Medical Center) for the antima-
larial tests. We thank Pierre Melançon and Dr. Jean-François
Lavallée at IRIC (Montreal) for the antitumor assays.
hydroxyethyl)-3-(hydroxymethyl)-4-((3-methoxyphenyl)amino)-
2-methylcyclopentyl)-1,1-dimethylurea (19c). Yield: 80% (7 mg
from 9 mg); ½a 2D0
ꢁ
+36.3 (c 1.00, CHCl3); IR (neat): mmax 3385, 2934,
1614, 1515, 1455, 1374, 1212, 1162, 1090, 1041, 912, 823, 762, 732,
690 cmꢀ1 1H NMR (400 MHz, CDCl3): d 7.86 (1H, d, J = 10.7 Hz),
;
7.15–7.02 (2H, m), 6.28 (1H, d, J = 8.2 Hz), 6.25 (1H, d, J = 8.2 Hz),
6.17 (1H, s), 5.56 (1H, s) 5.25 (1H, d, J = 10.7 Hz), 4.08 (1H, d,
J = 11.7 Hz), 3.98–3.87 (1H, m), 3.77 (3H,s), 3.69 (1H, d, J = 11.7 Hz),
3.60 (1H, d, J = 10.7 Hz), 3.05–2.91 (7H, s), 1.81 (2H, s), 1.44 (3H, s),
1.05 (3H, d, J = 6.4 Hz); 13C NMR (100 MHz, CDCl3): d 161.0, 159.4,
148.0, 130.0, 106.4, 102.8, 99.4, 88.4, 84.7, 71.3, 68.6, 62.4, 61.9, 55.1,
36.8, 21.5, 18.2; HRMS (ESIMS): calcd for C19H33N4O6 [M+H]+
413.23946, found 413.23835.
References and notes
1. For a review on aminocyclopentitols, see: (a) Berecibar, A.; Grandjean, C.;
Siriwardena, A. Chem. Rev. 1999, 99, 779; (b) Kurteva, V. B.; Afonso, C. A. M.
Chem. Rev. 2009, 109, 6809.
2. Argoudelis, A. D.; Jahnke, H. F.; Fox, J. A. Antimicrob. Agents Chemother. 1962,
191.
3. (a) Wiley, P. F.; Jahnke, H. K.; MacKellar, F. A.; Kelly, R. B.; Argoudelis, A. D. J.
Org. Chem. 1970, 35, 1420; For the 1H NMR spectrum of pactamycin, see: (b)
Dobashi, K.; Isshiki, K.; Sawa, T.; Obata, T.; Hamada, M.; Naganawa, H.; Takita,
T.; Takeuchi, T.; Umezawa, H. J. Antibiot. 1986, 39, 1779; For the 13C NMR
spectrum of the corrected structure of pactamycin, see: (c) Weller, D. D.; Haber,
A., ; Rinehart, K. L., Jr.; Wiley, P. F. J. Antibiot. 1978, 31, 997.
4. Duchamp, D. J. Abstract J. Am. Crystal. Assoc. Winter Meeting. Albuquerque,
1972, April, p. 23.
5. (a) Bhuyan, B. K.; Dietz, A.; Smith, C. G. Antimicrob. Agents Chemother. 1962,
184; (b) Bhuyan, B. K. Biochem. Pharmacol. 1967, 16, 1411.
6. (a) Summers, D. F.; Maizel, J. V., Jr. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 2852; (b)
Taber, R.; Rekosh, D.; Baltimore, D. J. Virol. 1971, 8, 395; (c) Kappen, L. S.;
Suzuki, H.; Goldberg, I. H. Proc. Natl. Acad. Sci. 1973, 70, 22; (d) Mankin, A. S. J.
Mol. Biol. 1997, 274, 8; (e) Ballesta, J. P.; Cundliffe, E. J. Bacteriol. 1991, 173,
7213; (f) Dinos, G.; Wilson, D. N.; Teraoko, Y.; Szaflarski, W.; Fucini, P.;
Kalpaxis, ; Nierhaus, K. H. Mol. Cell 2004, 13, 113; (g) Maroney, P. A.; Yu, Y.;
Fisher, J.; Nilsen, T. W. Nat. Struct. Biol. 2006, 13, 1102.
4.1.12.4.
3-((1R,2R,3S,4S,5S)-5-Amino-2,3-dihydroxy-1-((S)-1-
hydroxyethyl)-3-(hydroxymethyl)-2-methyl-4-((3-(trifluoromethyl)
phenyl)amino)cyclopentyl)-1,1-dimethylurea (19d). Yield: 75%
(10 mg from 14 mg); ½a D20
ꢁ
+32.7 (c 1.00, CHCl3); IR (neat): mmax
3381, 2933, 1614, 1520, 1449, 1374, 1346, 1283, 1164, 1122, 1068,
909, 787, 733, 698 cmꢀ1 1H NMR (400 MHz, CDCl3): d 7.88 (1H, d,
;
J = 10.7 Hz), 7.32–7.20 (1H, m), 7.06 (1H, s), 6.96 (1H, d, J = 7.6 Hz),
6.81 (1H, s), 6.77 (1H, d, J = 8.0 Hz), 5.62 (1H, s), 5.48 (1H, d,
J = 10.3 Hz), 4.06 (1H, d, J = 11.5 Hz), 3.97–3.84 (1H, m), 3.73 (1H, d,
J = 11.5 Hz), 3.67 (1H, d, J = 10.3 Hz), 2.99 (6H, s), 2.93 (1H, s), 1.86 (3H,
br s), 1.45 (3H, s), 1.04 (3H, d, J = 6.4 Hz); 13C NMR (100 MHz, CDCl3): d
159.3, 146.8, 131.9, 131.6, 129.9, 125.5, 122.8, 116.4, 114.1, 114.1,
109.1, 109.1, 88.4, 84.5, 74.0, 73.9, 71.3, 68.6, 62.4, 61.5, 36.8, 21.4,
18.2; HRMS (ESIMS): calcd. for C19H30F3N4O5 [M+H]+ 451.21628, found
451.21758.
7. Brodersen, D. E.; Clemons, W. M., Jr.; Carter, A. P.; Morgan-Warren, R. J.;
Wimberly, B. T.; Ramakrishnan, V. Cell 2000, 103, 1143.
8. (a) Weller, D. D.; Rinehart, K. L., Jr. J. Am. Chem. Soc. 1978, 100, 6757; (b)
Rinehart, K. L., Jr.; Potgieter, M.; Delaware, D. L. J. Am. Chem. Soc. 1981, 103,
2099; Adams, E. S.; Rinehart, K. L., Jr. J. Antibiot. 1994, 47, 1456.
9. Kudo, F.; Kasama, Y.; Hirayama, T.; Eguchi, T. J. Antibiot. 2007, 60, 492.
10. Ito, T.; Roongsawang, N.; Shirasaka, N.; Lu, W.; Flatt, P. M.; Kasanah, N.;
Miranda, C.; Mahmud, T. ChemBioChem 2009, 10, 2253.
11. Tsujimoto, T.; Nishikawa, T.; Urabe, D.; Isobe, M. Synlett 2005, 433.
12. Knapp, S.; Yu, Y. Org. Lett. 2007, 9, 1359.
13. Hanessian, S.; Vakiti, R. R.; Dorich, S.; Banerjee, S.; Lecomte, F.; DelValle, J. R.;
Zhang, J.; Deschenes-Simard, B. Angew. Chem., Int. Ed. 2011, 50, 3497.
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15. Looper, R. E.; Haussener, T. J. Org. Lett. 2012, 14, 3632.
4.1.13. In vitro antimalarial activity
In vitro antimalarial activity was determined by the malaria
SYBR Green-based fluorescence (MSF) assay described previously22
with slight modification.21 Stock solutions of each test drug were
prepared in sterile distilled water at a concentration of 10 mM.
The drug solutions were serially diluted with culture medium
and distributed to asynchronous parasite cultures on 96-well
16. Matsumoto, N.; Tjujimoto, T.; Nakazaki, A.; Isobe, M.; Nishikawa, T. RSC Adv.
2012, 2, 9448.
17. Otoguro, K.; Iwatsuki, M.; Ishiyama, A.; Namatame, M.; Nishihara-Tukashima,
A.; Shibahara, S.; Kondo, S.; Yamada, H.; Õmura, S. J. Antibiot. 2010, 63, 1.
18. Iwatsuki, M.; Nishihara-Tsukashima, A.; Ishiyama, A.; Namatame, M.;
Watanabe, Y.; Handasah, S.; Pranamuda, H.; Marwoto, A.; Matsumoto, A.;
Takahashi, Y.; Otogura, K.; Õmura, S. J. Antibiot. 2012, 65, 169.
19. Lu, W.; Roongsawang, N.; Mahmud, T. Chem. Biol. 2011, 18, 425.
20. See also: Serrano, P.; Llebaria, A.; Delgado, A. J. Org. Chem. 2002, 67, 7165.
21. Kelly, J. X.; Smilkstein, M. J.; Cooper, R. A.; Lane, K. D.; Johnson, R. A.; Janowsky,
A.; Dodean, R. A.; Hinrichs, D. J.; Winter, R.; Riscoe, M. Antimicrob. Agents
Chemother. 2007, 4133.
22. Smilkstein, M.; Sriwilaijaroen, N.; Kelly, J. X.; Wilairat, P.; Riscoe, M. Antimicrob.
Agents Chemother. 2004, 1803.
23. CLSI Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow
Aerobically; Approved Standard, 8th ed.; Clinical and Laboratory Standards
Institute: Wayne, PA, 2009. CLSI document M07-A8.
plates in quadruplicate in a total volume of 100
0.2% parasitemia with a 2% hematocrit in a total volume of
100 L. Automated pipetting and dilution were carried out with a
programmable Precision 2000 robotic station (Bio-Tek, Winooski,
VT). The Plates were then incubated for 72 h at 37 °C. After incuba-
lL to achieve
l
tion, 100 lL of lysis buffer with 0.2 lL/ml SYBR Green I (54, 66)
was added to each well. The plates were incubated at room temper-
ature for an hour in the dark and then placed in a 96-well fluores-
cence plate reader (Spectramax Gemini-EM; Molecular Diagnostics)
with excitation and emission wavelengths at 497 nm and 520 nm,
respectively, for measurement of fluorescence. The 50% inhibitory
concentration (IC50) was determined by nonlinear regression analy-
sis of logistic dose-response curves (GraphPad Prism software).