Role of the C(6)-hydroxy group in bicyclomycin: Synthesis, structure, and chemical, biochemical, and biological properties

Article abstract of DOI:10.1021/jm9708386

Bicyclomycin (1) is a commercial antibiotic whose primary site of action in Escherichia coli is the transcription termination factor rho. A recent structure-activity relationship study of 1 showed that replacing the C(6)- hydroxy group with alkoxy and thi

Full text of DOI:10.1021/jm9708386

1185
J . Med. Chem. 1998, 41, 1185-1194
Role of th e C(6)-Hyd r oxy Gr ou p in Bicyclom ycin : Syn th esis, Str u ctu r e, a n d
Ch em ica l, Bioch em ica l, a n d Biologica l P r op er ties
Alejandro Santilla´n, J r.,† Xiangdong Zhang,† J on Hardesty,† William R. Widger,‡ and Harold Kohn*,†
Departments of Chemistry and Biochemical and Biophysical Sciences, University of Houston, Houston, Texas 77204
Received December 10, 1997
Bicyclomycin (1) is a commercial antibiotic whose primary site of action in Escherichia coli is
the transcription termination factor rho. A recent structure-activity relationship study of 1
showed that replacing the C(6)-hydroxy group with alkoxy and thioalkoxy substituents led to
dramatic losses of inhibitory activity in rho biochemical assays. The origin for this structural
specificity has been explored by the synthesis and chemical, biochemical, and biological
evaluation of C(6)-amino- (13), C(6)-(hydroxylamino)-(14), and C(6)-mercaptobicyclomycin (15).
These compounds, like 1, are capable of entering into hydrogen bond donor interactions with
rho and are capable of undergoing C(6) ring opening to generate R,â-unsaturated carbonyl,
imine, or thione systems. The chemical reactivity of 13-15 was compared with that of 1. We
observed that 1, upon treatment with EtSH under moderate and basic conditions, readily
underwent C(6) hemiaminal bond cleavage followed by conjugate addition to â-methylene-R-
ketoamide 2 to give Michael addition adducts whereas 13-15 reacted by initial cleavage of
the C(1)-O(2) bond. Biochemical and biological assays of 13-15 and related analogues
demonstrated that the C(6) hydroxy group in 1 was essential for activity. We found that
replacing the C(6)-hydroxy group in 1 with weaker hydrogen bond donors led to low inhibitory
activities in the rho-dependent ATPase and transcription termination assays. None of the
bicyclomycin derivatives exhibited antibiotic activity against E. coli W3350 cells at a 32 mg/
mL concentration. The apparent specificity for the C(6)-hydroxy group in 1 suggests that an
efficient hydrogen bond donor interaction from the C(6)-hydroxy group to rho or the C(6)
hemiaminal bond cleavage to 2 or both is necessary for drug function.
Bicyclomycin (1) is a structurally unique antibiotic
possessing a diverse spectrum of biological activity.1-4
Chemical studies have demonstrated that in the pres-
ence of nucleophiles, 1 readily undergoes C(6) hemi-
aminal bond cleavage to give â-methylene-R-ketoamide
2 followed by reaction with the nucleophile to provide a
Michael addition product.5-9 The primary site of action
for 1 in Escherichia coli is the essential cellular protein
transcription termination factor rho.10 Mechanistic
studies have shown that bicyclomycin efficiently inhibits
the production of rho-dependent transcripts11 and in-
hibits rho poly(C)-stimulated ATP hydrolysis with simple
noncompetitive kinetics with respect to ATP.12
the poly(C)-stimulated ATPase15 assays and the elimi-
nation of antimicrobial activity in a filter disk assay.16
Our study included the 10 C(6)-substituted bicyclomy-
cins 3-12. Most of these compounds, unlike 1, can
neither undergo C(6)-N(10) cleavage to generate a
neutral ring-opened intermediate nor provide a C(6)
hydrogen bond to a protein residue surrounding the
bicyclomycin binding pocket in rho. Accordingly, we
have prepared and evaluated C(6)-amino- (13), C(6)-
(hydroxylamino)- (14), and C(6)-mercaptobicyclomycin
(15) to determine the importance of C(6) ring-opening
transformations and hydrogen bond donor interactions
for drug binding. In the work reported herein, we
demonstrate that replacement of the C(6)-hydroxy group
by amino, hydroxylamino, and thiol units altered the
chemical reactivity of bicyclomycin and led to significant
losses in biochemical activity.
Resu lts a n d Discu ssion
A. Ch oice of Su bstr a tes a n d Syn th esis. The
primary compounds selected for study were 13-15. For
C(6)-aminobicyclomycin (13) and C(6)-(hydroxylamino)-
bicyclomycin (14) we used methylation to progressively
remove potential hydrogen bond donors located at C(6).
Accordingly, we prepared 16, 17, and 19-21. We did
not prepare the S-methyl derivative of 15 since we
showed that C(6)-S-ethylbicyclomycin (9) did not inhibit
rho-dependent ATPase or transcription termination
activities.13 Added to our list was the C(6)-acetami-
dobicyclomycin (18). This compound was included to
In a recent structure-activity relationship (SAR)
study13 of 1 we reported that replacing the C(6)-hydroxy
group with alkoxy and thioalkoxy substituents led to
dramatic losses of bicyclomycin inhibitory activity in the
in vitro rho-dependent transcription termination14 and
* Author to whom correspondence should be addressed. Phone: 713/
743-3240. Fax: 713/743-2709. E-mail: HKohn@uh.edu.
† Department of Chemistry.
‡ Department of Biochemical and Biophysical Sciences.
S0022-2623(97)00838-8 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/06/1998

1186 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Santilla´n et al.
F igu r e 1. View of 24 showing the atom numbering scheme.
Thermal ellipsoids are 40% equiprobability envelopes, with
hydrogens as spheres of arbitrary diameter.
provide a modified C(6)-aminobicyclomycin that would
not be protonated under the assay conditions (pH 7.9).
Compounds 13-17 and 19-21 were prepared by a
method similar to that described for 3.13 Bicyclomycin
was first converted to the known bicyclomycin C(2′),C-
(3′)-acetonide (22)17,18 and then to the tentatively as-
signed mesylate 23.13 For 13, 16, and 17, compound
23 was dissolved in THF and treated with excess amine
to give acetonides 24-26. Removing the C(2′),C(3′)-
acetonide group in 24-26 with trifluoroacetic acid in
aqueous methanol yielded 13, 16, and 17, respectively.
For compounds 14 and 19-21, the mesylate 23 was
dissolved in isopropyl alcohol in the presence of an
excess amount of the hydroxylamine and then the “pH”
of the solution was adjusted to 5.5. Deprotection of the
acetonide group in 27-30 with acid gave 14 and 19-
21. We also employed isopropyl alcohol as the solvent
to prepare 15. Using thiolacetic acid in place of the
hydroxylamine, we obtained 31. Successive removal of
the acetyl and the acetonide groups in 31 with base and
acid, respectively, yielded first 32 and then 15. Syn-
thesis of 18 was accomplished first by acetylation of 24
with acetic anhydride in the presence of Proton Sponge
(Aldrich) to give 33 and then by careful deprotection of
the acetonide group with trifluoroacetic acid.
goes C(6) hemiaminal ring cleavage to give 2 followed
by conjugate addition.6-9 We have shown that at near
neutral “pH” values in THF-H2O mixtures (“pH” 7.4-
8.5) 1 reacts with EtSH to give the novel rearranged
adduct 346,9 whereas at higher “pH” values (“pH” 10-
11) the C(5a)-substituted dihydrobicyclomycin 35 pre-
dominated.9
Compounds 13-15, like 1, each has an ionizable
proton attached to the C(6) substituent. These three
compounds can potentially undergo C(6)-N(10) bond
cleavage to generate an R,â-unsaturated imine or thione
system capable of undergoing conjugate addition. In an
effort to determine the structural features that govern
bicyclomycin reactivity with nucleophiles, we treated
these compounds with EtSH. No reaction was observed
for C(6)-aminobicyclomycin (13) at “pH” 9.0, while at
“pH” 10.5, the bis-spiro adduct 36 was generated. NMR
analyses indicated the presence of a single diastereomer.
X-ray crystallographic analysis of the corresponding
p-bromobenzoate 37 identified 36 as the trans diaste-
reomer having C(1)-S and C(6)-R stereochemistry.21
Compound 37 was previously characterized by Maag
and co-workers when they determined the absolute
stereochemistry of 1.22 The EtSH-mediated reactions
of C(6)-(hydroxylamino)bicyclomycin (14) gave a differ-
ent product. We observed only 38 at both “pH” 8.8 and
10.0. Once again, NMR analyses indicated that the
reaction was stereospecific.23 Addition of EtSH to a
THF-H2O solution of C(6)-mercaptobicyclomycin (15)
maintained at “pH” 9.5 gave only 36, and NMR analyses
indicated stereospecificity.
B. Sp ectr a l Stu d ies. The 1H and 13C NMR spectral
properties for bicyclomycin acetonides 24-33 and bicy-
clomycins 13-21 agreed with the proposed struc-
tures19,20 and were consistent with those observed for
3-12.13 X-ray crystallographic analysis of 24 docu-
mented the incorporation of the amino group at C(6)
(Figure 1).21
These findings demonstrated the importance of the
C(6) substituent in bicyclomycin chemical transforma-
tions. Replacement of the C(6)-hydroxy group in 1 by
an amino, hydroxylamino, or thiol group prevented
EtSH addition reactions to the C(5)-C(5a) olefinic
group. In each case, the reaction appears to proceed
by initial cleavage of the C(1)-O(2) bond in 39 to give
40 (Scheme 1). Energy minimization studies24 indicate
C. Ch em ica l Stu d ies. A distinguishing feature of
bicyclomycin chemistry is the ease with which 1 under-

Role of the C(6)-Hydroxy Group in Bicyclomycin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1187
Sch em e 1. Proposed Pathway for Base-Mediated
Conversion of C(6)-Substituted Bicyclomycins to 36
that 40 (X ) OH) undergoes stereospecific cyclization
to 41 due in part to favorable hydrogen bonds that exist
between the C(1′)-hydroxy and C(9)-carbonyl groups and
between the C(2′)-hydroxy and N(8)-imine units. This
facial alignment of the C(1) triol group promotes ring
closure to give the C(1)-S-substituted tetrahydrofuran
41. Subsequent cyclization of 41 can then give either
the C(1)-S, C(6)-R or the C(1)-S, C(6)-S bis-spiro tet-
rahydrofuranyl adducts. Energy minimization studies
using molecular mechanics24 indicate that of these
compounds, 36 (C(1)-S, C(6)-R) has lower energy.
Information concerning the importance of the C(6)
substituent in governing bicyclomycin chemistry was
also obtained by comparing the stabilities of 1 and 13-
15 in aqueous solutions (pH 7.4, 37 °C). We found that
1, 14, and 15 had approximately the same stabilities in
buffered water solutions (t1/2 ≈ 28-31 h), but 13 was
appreciably more stable (t1/2 ≈ 155 h). HPLC analyses
of the reaction mixtures showed that 113 and 14
produced a series of unidentified, polar adducts, 13 gave
diastereomeric 42,22,25 and 15 gave 42 along with several
additional products. Identification of 42 in the reaction
mixtures of 13 and 15 was accomplished by coinjecting
(cospotting) authentic samples of 4222,25 in the HPLC
(TLC) and by running a larger scale reaction for 13 and
verifying 42 by NMR spectroscopy. Significantly, nei-
ther bicyclomycin nor its reaction products were de-
tected in the HPLC product profiles for 13-15, signi-
fying that these compounds did not hydrolyze to 1.
Comparable results were observed for 3, 5, and 9-11.13
poly(C)-stimulated ATPase activity at a concentration
of 400 µM nor did they inhibit transcription termination
at a concentration of 100 µM.13 Comparable findings
were observed for the new C(6)-substituted bicyclomy-
cins 13-21 (Table 1). In the ATPase assay15 all I50
values for 13-21 exceeded 400 µM, with the most active
bicyclomycin being 14 followed by 15 and then 13.
Similarly, the I50 values for 13-21 in the transcription
termination assay14 exceeded 100 µM.
The data in Table 1 demonstrate the exquisite de-
pendency of the C(6)-hydroxy group in bicyclomycin on
the inhibition of rho-dependent processes. Our earlier
study showed that C(6)-alkoxybicyclomycins 3-6 and
C(6)-deoxybicyclomycin (12) had little inhibitory activity
in rho functional assays.13 This result demonstrated
that steric interactions were not solely responsible for
the loss of biochemical activities when the C(6)-hydroxy
group was replaced. Our original investigation also
demonstrated that hydrogen bond acceptor interactions
from rho to the C(6)-hydroxy group in 1 did not promote
drug binding. Compounds 3-11 are all capable of
accepting a hydrogen bond, but none inhibited rho
functional processes. Two other factors were briefly
considered. First, hydrogen bond donor interactions
between the C(6)-hydroxy group in 1 and the amino acid
residue(s) in rho may be necessary for efficient drug
binding. Second, bicyclomycin may undergo ring open-
ing to â-methylene-R-ketoamide 2 upon binding to rho.
The data for C(6)-substituted bicyclomycins 3-12 did
not provide information concerning the likelihood of
these two factors. Of these compounds, only the bulky
C(6)-N-phenylbicyclomycin (11) was capable of either
donating a hydrogen bond or undergoing C(6)-N(10)
cleavage to generate a neutral ring-opened intermedi-
ate.
Our cumulative studies demonstrated that at moder-
ate “pH” values bicyclomycin underwent predominant
C(6) hemiaminal ring opening to generate â-methylene-
R-ketoamide 2. Replacing the C(6)-hydroxy group with
nitrogen and sulfur groups permitted C(1)-O(2) and
C(6)-X bond cleavage to become competitive with C(6)
hemiaminal ring opening.
D. Bioch em ica l a n d Biologica l Stu d ies. In our
initial SAR study we showed that the C(6)-substituted
bicyclomycins 3-12 neither appreciably inhibited rho
Many of the bicyclomycin C(6) substituents in this
study were capable of serving as hydrogen bond donors.
We expected that the parent compounds, 13-15, would
be the most efficient hydrogen bond donors and that
successive methylation of the C(6) heteroatom(s) would

1188 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Santilla´n et al.
Ta ble 1. Biochemical and Biological Activities of C(6)-Substituted Bicyclomycins
inhibition of ATPase activitya
TT activityd
compd no.
X
I50 (µM) (BCM)b
400 µM (%) (BCM)c
I50 (µM)e
100 µM (%)f
MICg (mg/mL)
1
13
16
17
18
14
19
20
21
15
OH
NH2
N(H)CH3
N(CH3)2
N(H)C(O)CH3
N(H)OH
N(CH3)OH
N(H)OCH3
N(CH3)OCH3
SH
60
>400 (70)
>400 (70)
>400 (70)
>400 (70)
>400 (70)
>400 (70)
>400 (70)
>400 (60)
>400 (70)
95
17 (88)
3 (88)
∼5
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
5
2
1
1
22
0
0
0
8
0.25
>32 (0.27)
>32 (0.27)
>32 (0.27)
>32 (0.31)
>32 (0.35)
>32 (0.35)
>32 (0.35)
>32 (0.35)
>32 (0.27)
1 (88)
15 (92)
45 (89)
16 (89)
13 (89)
7 (90)
28 (93)
a
b
Activity measured using the ATPase assay (ref 15). The I50 value is the average 50% inhibition concentration determined from
duplicate tests. The corresponding value obtained from bicyclomycin in a concurrently run experiment is provided in parentheses. c The
percent inhibition of ATPase activity at 400 µM. The corresponding value obtained from bicyclomycin in a concurrently run experiment
d
is provided in parentheses. Activity in the transcription termination assay was determined by the method of T. Platt and co-workers
(ref 14). e The I50 value is the average 50% inhibition concentration determined from duplicate tests. f The percentage of transcription
g
termination at 100 µM. MIC value is the average minimum inhibitory concentration of the tested compound determined from duplicate
tests using a filter disk assay (ref 16). The number in parentheses is the corresponding value obtained from bicyclomycin in a concurrently
run experiment.
Ta ble 2. Calculated ∆G°rxn and Keq Values for Equilibrium in
diminish this interaction. Of these, we noted that 13
Eq 1
is likely to exist as the protonated amine possibly
reaction series
∆G°rxn (kcal/mol)
Keq
preventing its binding to rho. Correspondingly, nitrogen
protonation should not be significant for hydroxylamine
14 (pKa alkylhydroxylamines ≈ 5-626) at the pH
employed in the ATPase and transcription termination
assays (pH 7.9). In hydroxylamine 14, the hydrogen
bond could be donated to rho from either the N-H or
O-H sites. We found that none of the compounds
capable of donating a hydrogen bond to rho from the
C(6) site (13-16, 18, 20) approached the inhibitory
activity observed for 1. However, within this series we
observed that 14 and 15 were the most effective inhibi-
tors. Partial inhibition of rho-dependent ATPase and
transcription termination were seen at 400 and 100 µM,
respectively. The steep drop in inhibitory ATPase
activity observed for 15 was reminiscent of the loss in
activity observed upon replacement of the C(3′) hydroxy
group in 1 with a thiol unit to give 43.27 These findings,
then, do not permit us to rule out the notion that the
C(6)-hydroxy group fosters antibiotic binding to rho by
hydrogen bonding to a protein acceptor residue or that
this moiety simultaneously serves as a hydrogen bond
donor and an acceptor to protein residue(s) that line the
bicyclomycin binding pocket.28
O
NH
S
-13.4
-10.1
- 8.0
6.7 × 109
2.5 × 107
7.3 × 105
ring opening) to give the â-methylene-R-ketoamide 2.
Our chemical studies demonstrated that replacing the
C(6)-hydroxy group with a C(6)-amino, -hydroxylamino,
or -mercapto substituent led to decreased reactivity with
EtSH and to the preferential scission of the C(1)-O(2)
and C(6)-XH bonds, compared with cleavage of the
C(6)-N(10) bond (Scheme 1). These results suggest
that, for bicyclomycin, â-methylene-R-ketoamide 2 may
be the preferred species that binds to rho leading to
inhibition of rho transcription termination processes.
Accordingly, we calculated the thermodynamic charac-
teristics of the model equilibrium processes shown in
eq 1, in which 45 and 47 correspond to the parent
(1)
bicyclomycin structure and the ring-opened species,
respectively. These calculations, performed at the
density functional level, and summarized in Table 2,
reveal that in the absence of protein interactions the
transformation of 45 to 47 is exothermic for X ) O, NH,
and S, with 45a possessing the greatest thermodynamic
driving force. The fact that all three reactions are
thermodynamically favorable suggests that biochemical
Another factor that may account for the observed
specificity of the C(6)-hydroxy group in bicyclomycin is
the relative ease with which the parent bicyclomycin
underwent C(6)-N(10) bond scission (C(6) hemiaminal

Role of the C(6)-Hydroxy Group in Bicyclomycin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1189
otides and RNase inhibitor were obtained from Ambion, Inc.
(Austin, TX). Polyethylenimine (PEI) thin-layer chromatog-
raphy plates used for ATPase assays were purchased from J .
T. Baker, Inc. (Phillipsburg, NJ ).
Gen er a l P r oced u r e for th e P r ep a r a tion of C(6)-Am i-
n obicyclom ycin C(2′),C(3′)-Aceton id es 24-26. To an an-
hydrous THF solution (4 mL) of 22 (1 equiv) and triethylamine
(2-3 equiv) was added methanesulfonyl chloride (2-3 equiv).
The reaction mixture was stirred at room temperature (15
min), filtered (glass wool), and treated with the desired
nucleophile (excess). The solution was then concentrated in
vacuo, and the residue was purified by PTLC (15-20%
MeOH-CHCl3) to afford the desired product.
activity in the rho-dependent assays should also be
observed for 13 and 15 if the bioactive structure is the
ring-opened â-methylene-R-ketoamide 2. However, com-
parison of equilibrium constants reveals that the equi-
librium lies 270 × further to the right when X ) O than
when X ) NH and ∼9200× further to the right than
when X ) S. Thus the hydroxy-substituted species is
expected to undergo ring opening more readily than
either the amino- or the thio-substituted compounds.
While not indicative of the absolute rate at which ring
opening occurs, these values do express the relative
thermodynamic preferences for the model reaction,
revealing a trend in reactivity that parallels our obser-
vation that bicyclomycin undergoes ring opening to the
R,â-unsaturated conjugated system 2 followed by C(5)-
C(5a) modification by nucleophiles at near neutral to
basic pH values, but the C(6)-substituted bicyclomycins
13 and 15 do not.
By use of this procedure, the following compounds were
prepared.
Syn th esis of C(6)-Am in obicyclom ycin C(2′),C(3′)-Ac-
eton id e (24). Using 22 (17 mg, 0.05 mmol), triethylamine
(15 mg, 0.15 mmol), methanesulfonyl chloride (17 mg, 0.15
mmol) and ammonia(g) (2 min, excess) gave 24 as a white solid
(10 mg, 59%): mp 183-185 °C; Rf 0.49 (10% MeOH-CHCl3);
1
IR (KBr) 3364, 3302, 1702, 1669, 1603, 1467 cm-1; H NMR
Our studies indicated that the C(6)-hydroxy group in
bicyclomycin was essential for drug function. They also
suggested that binding of the antibiotic to rho was
fostered either by a hydrogen bond donor interaction
from the C(6)-hydroxy group in 1 to the protein or by
the conversion of 1 to ring-opened â-methylene-R-
ketoamide 2 or by both. Experiments are in progress
to study these possibilities.
Bicyclomycins 13-21 were all inactive (MIC >32 mg/
mL) against W3350 E. coli in the filter disk assay16
(Table 1). This result was consistent with the lack of
significant inhibitory activities in the rho poly(C)-
stimulated ATPase and transcription termination as-
says. A similar finding was observed for 3-12.13
(CD3OD) δ 1.38 (s, 3 H, C(2′)CH3), 1.42 (s, 3 H, C(CH3)2), 1.45
(s, 3 H, C(CH3)2), 2.56-2.73 (m, 2 H, C(4)H2), 3.72 (d, J ) 8.3
Hz, 1 H, C(3′)HH′), 3.84 (ddd, J ) 1.7, 6.2, 8.9 Hz, 1 H, C(3)-
HH′), 3.96 (ddd, J ) 1.7, 6.2, 8.9 Hz, 1 H, C(3)HH′), 4.16 (s, 1
H, C(1′)H), 4.46 (d, J ) 8.3 Hz, 1 H, C(3′)HH′), 5.16 (s, 1 H,
C(5a)HH′), 5.50 (s, 1 H, C(5a)HH′); 13C NMR (CD3OD) 25.1
(C(2′)CH3), 26.8 (C(CH3)2), 28.2 (C(CH3)2), 37.1 (C(4)), 66.7
(C(3)), 72.3 (C(6)), 72.8 and 73.1 (C(1′), C(3′)), 86.5 (C(2′)), 89.2
(C(1)), 111.6 (C(CH3)2), 116.6 (C(5a)), 151.6 (C(5)), 168.8 and
172.4 (C(7), C(9)) ppm; MS (+CI) 342 [M + 1]+; Mr (+CI)
342.166 20 [M + 1]+ (calcd for C15H24N3O6 342.166 51).
Syn th esis of C(6)-(N-Meth yla m in o)bicyclom ycin C(2′),
C(3′)-Aceton id e (25). Using 22 (32 mg, 0.09 mmol), triethyl-
amine (19 mg, 0.19 mmol), methanesulfonyl chloride (21 mg,
0.19 mmol), and methylamine (29 mg, 0.94 mmol) gave 22 (10
mg, 31% recovery) and 25 as a white solid (14 mg, 42%): mp
158-162 °C; Rf 0.57 (10% MeOH-CHCl3); IR (KBr) 3446,
3311, 1686, 1457 cm-1; 1H NMR (CD3OD) δ 1.37 (s, 3 H, C(2′)-
CH3), 1.43 (s, 3 H, C(CH3)2), 1.45 (s, 3 H, C(CH3)2), 2.56 (dd, J
) 6.9, 15.3 Hz, 1 H, C(4)HH′), 2.63-2.70 (m, 1 H, C(4)HH′),
3.72 (d, J ) 8.4 Hz, 1 H, C(3′)HH′), 3.83-3.97 (m, 2 H, C(3)-
H2), 4.15 (s, 1 H, C(1′)H), 4.45 (d, J ) 8.4 Hz, 1 H, C(3′)HH′),
5.13 (s, 1 H, C(5a)HH′), 5.50 (s, 1 H, C(5a)HH′); 13C NMR (CD3-
OD) 25.0 (C(2′)CH3), 26.8 (C(CH3)2), 28.2 (C(CH3)2), 29.9
(NCH3), 37.4 (C(4)), 67.0 (C(3)), 73.0 and 73.2 (C(1′), C(3′)),
77.1 (C(6)), 86.4 (C(2′)), 89.0 (C(1)), 111.7 (C(CH3)2), 116.5
(C(5a)), 150.6 (C(5)), 169.8 and 171.5 (C(7), C(9)) ppm; MS
(+CI) 356 [M + 1]+; Mr (+CI) 356.181 99 [M + 1]+ (calcd for
Con clu sion s
This report highlights the essential role of the C(6)-
hydroxy group in bicyclomycin. Simple replacement of
this unit with an amino, hydroxylamino, or mercapto
moiety altered the chemical reactivity of the drug
analogue leading to the near total elimination of bicy-
clomycin inhibitory activities in rho functional assays
and resulting in the loss of antimicrobial activity against
W3350 E. coli.
C
16H26N3O6 356.182 16).
Exp er im en ta l Section
Syn th esis of C(6)-(N,N-Dim eth yla m in o)bicyclom ycin
Gen er a l Meth od s. Proton (1H NMR) and carbon (13C
NMR) nuclear magnetic resonance spectra were recorded on
300 and 75 MHz spectrometers, respectively. Low-resolution
and high-resolution (CI) mass spectral investigations were run
at the University of Texas at Austin by Dr. M. Moini. “pH”
measurements of mixed organic-water solutions were deter-
mined using electrodes calibrated against aqueous pH 4.0 and
7.0 buffer solutions. The solvents and reactants were of the
best commercial grade available and were used without further
purification unless noted. Tetrahydrofuran was distilled from
Na metal and benzophenone. Thin-layer chromatography was
run on general purpose silica gel plates (20 × 20 cm; Aldrich
No. Z12272-6).
Rho protein was isolated from E. coli AR120 containing the
overexpressing plasmid p39-AS29 following previously pub-
lished protocols.29 Rho purity was determined by SDS-PAGE,
and concentrations were determined by Lowry protein deter-
mination.30 Procedures identical to those previously de-
scribed27 were used in the evaluation of the C(6)-substituted
bicyclomycins in the poly(C)-dependent ATPase,15 rho-depend-
ent transcription termination,14 and antimicrobial assays.16
[γ-32P]ATP and [R-32P]UTP (3000 Ci/mmol) were purchased
from Dupont-New England Nuclear (Doraville, GA); nucle-
C(2′),C(3′)-Aceton id e (26). Using 22 (30 mg, 0.09 mmol),
triethylamine (18 mg, 0.18 mmol), methanesulfonyl chloride
(20 mg, 0.18 mmol), and dimethylamine (40 mg, 0.88 mmol)
gave 22 (12 mg, 40% recovery) and 26 as a white solid (11 mg,
34%): mp 98-104 °C; Rf 0.55 (10% MeOH-CHCl3); IR (KBr)
3440, 3326, 1687, 1458 cm-1; 1H NMR (CD3OD) δ 1.38 (s, 3 H,
C(2′)CH3), 1.40 (s, 3 H, C(CH3)2), 1.44 (s, 3 H, C(CH3)2), 2.37
(s, 6 H, N(CH3)2), 2.50-2.63 (m, 2 H, C(4)H2), 3.60-3.70 (m, 1
H, C(3)HH′), 3.73 (d, J ) 8.4 Hz, 1 H, C(3′)HH′), 4.05-4.11
(m, 1 H, C(3)HH′), 4.15 (s, 1 H, C(1′)H), 4.42 (d, J ) 8.4 Hz, 1
H, C(3′)HH′), 5.36 (s, 1 H, C(5a)HH′), 5.53 (s, 1 H, C(5a)HH′);
13C NMR (CD3OD) 25.1 (C(2′)CH3), 26.8 (C(CH3)2), 28.0
(C(CH3)2), 37.0 (C(4)), 39.0 (N(CH3)2), 68.2 (C(3)), 72.8 and 73.2
(C(1′), C(3′)), 82.4 (C(6)), 86.4 (C(2′)), 88.1 (C(1)), 111.5
(C(CH3)2), 119.9 (C(5a)), 149.6 (C(5)), 170.9 and 171.1 (C(7),
C(9)) ppm; the spectral assignments were in agreement with
the HMQC experiment; MS (+CI) 370 [M + 1]+; Mr (+CI)
370.197 91 [M + 1]+ (calcd for C17H28N3O6 370.197 81).
Gen er a l P r oced u r e for th e P r ep a r a tion of C(6)-(N-
Su bst it u t ed Hyd r oxyla m in o)b icyclom ycin C(2′),C(3′)-
Aceton id es 27-30. To an anhydrous THF solution (4 mL) of
22 (1 equiv) and triethylamine (3 equiv) was added methane-
sulfonyl chloride (3 equiv). The reaction mixture was stirred

1190 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Santilla´n et al.
at room temperature (15 min), filtered (glass wool), and
concentrated in vacuo. The residue was dissolved in isopropyl
alcohol (4 mL) and treated with the desired nucleophile (10
equiv). The “pH” of the reaction mixture was adjusted to
approximately 5.5 with dilute aqueous NaOH and stirred at
room temperature (20 min). The “pH” of the reaction mixture
was adjusted to 7.0 with dilute aqueous NaOH and concen-
trated in vacuo. The residue was suspended in H2O (8 mL)
and extracted with ethyl acetate (4 × 8 mL). The organic
layers were combined, dried (Na2SO4), filtered, and concen-
trated in vacuo. The residue was purified by PTLC (10%
MeOH-CHCl3) to afford the desired product.
1H NMR (CD3OD) δ 1.38 (s, 3 H, C(2′)CH3), 1.39 (s, 3 H,
C(CH3)2), 1.43 (s, 3 H, C(CH3)2), 2.54-2.61 (m, 2 H, C(4)H2),
2.69 (s, 3 H, NCH3), 3.53 (s, 3 H, OCH3), 3.61-3.68 (m, 1 H,
C(3)HH′), 3.73 (d, J ) 8.4 Hz, 1 H, C(3′)HH′), 4.08-4.14 (m, 1
H, C(3)HH′), 4.16 (s, 1 H, C(1′)H), 4.43 (d, J ) 8.4 Hz, 1 H,
C(3′)HH′), 5.31 (s, 1 H, C(5a)HH′), 5.65 (s, 1 H, C(5a)HH′);
13C NMR (CD3OD) 25.1 (C(2′)CH3), 26.8 (C(CH3)2), 28.0
(C(CH3)2), 37.0 (C(4)), 37.2 (NCH3), 60.3 (OCH3), 67.8 (C(3)),
72.7 and 73.2 (C(1′), C(3′)), 83.2 (C(6)), 86.4 (C(2′)), 88.0 (C(1)),
111.5 (C(CH3)2), 118.5 (C(5a)), 149.5 (C(5)), 169.3 and 170.5
(C(7), C(9)) ppm; MS (+CI) 386 [M + 1]+; Mr (+CI) 386.192 73
[M + 1]+ (calcd for C17H28N3O7 386.192 73).
By use of this procedure, the following compounds were
prepared.
Syn t h esis of C(6)-S-Acet ylb icyclom ycin C(2′),C(3′)-
Aceton id e (31). To an anhydrous THF solution (4 mL) of 22
(56 mg, 0.16 mmol) and triethylamine (33 mg, 0.33 mmol) was
added methanesulfonyl chloride (38 mg, 0.33 mmol). The
reaction mixture was stirred at room temperature (15 min),
filtered (glass wool), and concentrated in vacuo. The residue
was dissolved in isopropyl alcohol (4 mL) and treated with
thiolacetic acid (125 mg, 1.64 mmol). The “pH” of the reaction
mixture was adjusted to approximately 5.5 with dilute aqueous
NaOH and stirred at room temperature (20 min). The “pH”
of the reaction mixture was adjusted to 7.0 with dilute aqueous
NaOH and concentrated in vacuo. The residue was suspended
in H2O (8 mL) and extracted with ethyl acetate (4 × 8 mL).
The organic layers were combined, dried (Na2SO4), filtered,
and concentrated in vacuo. The residue was purified by PTLC
(10% MeOH-CHCl3) to provide 22 (18 mg, 32% recovery) and
31 as a white solid (23 mg, 35%): mp 157-160 °C; Rf 0.67
Syn th esis of C(6)-(Hyd r oxyla m in o)bicyclom ycin C(2′),
C(3′)-Aceton id e (27). Using 22 (37 mg, 0.11 mmol), triethyl-
amine (22 mg, 0.22 mmol), methanesulfonyl chloride (25 mg,
0.22 mmol), and NH2OH‚HCl (74 mg, 1.1 mmol) gave 22 (5
mg, 14% recovery) and 27 as a white solid (15 mg, 39%): mp
119-122 °C; Rf 0.47 (10% MeOH-CHCl3); IR (KBr) 3426,
1
3271, 1690 cm-1; H NMR (CD3OD) δ 1.37 (s, 3 H, C(2′)CH3),
1.43 (s, 3 H, C(CH3)2), 1.44 (s, 3 H, C(CH3)2), 2.49-2.68 (m, 2
H, C(4)H2), 3.73 (d, J ) 8.4 Hz, 1 H, C(3′)HH′), 3.86-3.98 (m,
2 H, C(3)HH′), 4.13 (s, 1 H, C(1′)H), 4.43 (d, J ) 8.4 Hz, 1 H,
C(3′)HH′), 5.11 (s, 1 H, C(5a)HH′), 5.32 (s, 1 H, C(5a)HH′);
13C NMR (CD3OD) 24.7 (C(2′)CH3), 26.8 (C(CH3)2), 28.2
(C(CH3)2), 35.5 (C(4)), 67.1 (C(3)), 73.4 (C(3′)), 73.8 (C(1′)), 78.4
(C(6)), 86.2 (C(2′)), 88.8 (C(1)), 111.7 (C(CH3)2), 116.2 (C(5a)),
147.3 (C(5)), 168.7 and 171.0 (C(7), C(9)) ppm; MS (+CI) 358
[M + 1]+; Mr (+CI) 358.161 87 [M + 1]+ (calcd for C15H24N3O7
358.161 43).
(10% MeOH-CHCl3); IR (KBr) 3440, 3325, 1699, 1458 cm-1
;
1H NMR (CD3OD) δ 1.41 (s, 3 H, C(2′)CH3), 1.44 (s, 6 H,
C(CH3)2), 2.34 (s, 3 H, SC(O)CH3), 2.61 (dd, J ) 6.6, 15.9 Hz,
1 H, C(4)HH′), 2.75 (dd, J ) 9.0, 15.9 Hz, 1 H, C(4)HH′), 3.71-
3.81 (m, 1 H, C(3)HH′), 3.74 (d, J ) 8.4 Hz, 1 H, C(3′)HH′),
4.04 (dd, J ) 6.6, 13.2 Hz, 1 H, C(3)HH′), 4.09 (s, 1 H, C(1′)H),
4.38 (d, J ) 8.4 Hz, 1 H, C(3′)HH′), 5.23 (s, 1 H, C(5a)HH′),
5.38 (s, 1 H, C(5a)HH′); 13C NMR (CD3OD) 24.1 (C(2′)CH3),
26.8 (C(CH3)2), 27.9 (C(CH3)2), 30.2 (SC(O)CH3), 37.3 (C(4)),
66.6 (C(3)), 73.4 (C(6)), 73.6 (C(3′)), 75.0 (C(1′)), 86.1 (C(2′)),
87.3 (C(1)), 111.6 (C(CH3)2), 118.4 (C(5a)), 148.8 (C(5)), 168.8
and 168.9 (C(7), C(9)), 194.0 (SC(O)CH3) ppm; MS (+CI) 401
[M + 1]+; Mr (+CI) 401.137 11 [M + 1]+ (calcd for C17H25N2O7S
401.138 25).
Syn th esis of C(6)-Mer ca p tobicyclom ycin C(2′),C(3′)-
Aceton id e (32). To a solution of 31 (25 mg, 0.06 mmol) in
degassed MeOH (3 mL) was added aqueous 0.1 M NaOH (3
mg, 0.06 mmol). The solution was stirred (0 °C, 1 h),
neutralized (aqueous dilute HCl), and concentrated in vacuo.
The residue was suspended in H2O (4 mL) and extracted with
ethyl acetate (4 × 8 mL). The organic layers were combined,
dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by PTLC (2 × 5% MeOH-CHCl3) to afford
32 as a white solid (15 mg, 67%): mp 192-196 °C; Rf 0.51
(10% MeOH-CHCl3); IR (KBr) 3446, 3289, 1673, 1650, 1458
cm-1; 1H NMR (CD3OD) δ 1.38 (s, 3 H, C(2′)CH3), 1.41 (s, 3 H,
C(CH3)2), 1.44 (s, 3 H, C(CH3)2), 2.62 (dd, J ) 7.5, 16.1 Hz, 1
H, C(4)HH′), 2.76 (dd, J ) 8.4, 16.1 Hz, 1 H, C(4)HH′), 3.72
(d, J ) 8.3 Hz, 1 H, C(3′)HH′), 3.76-3.84 (m, 1 H, C(3)HH′),
3.92-3.98 (m, 1 H, C(3)HH′), 4.16 (s, 1 H, C(1′)H), 4.45 (d, J
) 8.3 Hz, 1 H, C(3′)HH′), 5.24 (s, 1 H, C(5a)HH′), 6.18 (br s, 1
H, C(5a)HH′); 13C NMR (CD3OD) 25.0 (C(2′)CH3), 26.8 (C(CH3)2),
28.2 (C(CH3)2), 37.0 (C(4)), 67.4 (C(3)), 71.7 (C(6)), 73.1 (C(1′)),
73.3 (C(3′)), 86.5 (C(2′)), 89.5 (C(1)), 111.7 (C(CH3)2), 121.7
(C(5a)), 152.6 (C(5)), 166.9 and 167.0 (C(7), C(9)) ppm, the
structural assignments were in agreement with the HMBC
and HMQC experiments; MS (+CI) 359 [M + 1]+; Mr (+CI)
359.128 60 [M + 1]+ (calcd for C15H23N2O6S 359.127 68).
Syn th esis of C(6)-(N-Meth ylh yd r oxyla m in o)bicylom y-
cin C(2′),C(3′)-Aceton id e (28). Using 22 (41 mg, 0.12 mmol),
triethylamine (24 mg, 0.24 mmol), methanesulfonyl chloride
(28 mg, 0.24 mmol), and N-methylhydroxylamine hydrochlo-
ride (102 mg, 1.2 mmol) gave 22 (8 mg, 20% recovery) and 28
as a white solid (14 mg, 31%): mp 109-113 °C; Rf 0.53 (10%
MeOH-CHCl3); IR (KBr) 3291, 1688 cm-1; 1H NMR (CD3OD)
δ 1.38 (s, 3 H, C(2′)CH3), 1.39 (s, 3 H, C(CH3)2), 1.43 (s, 3 H,
C(CH3)2), 2.54-2.60 (m, 2 H, C(4)H2), 2.68 (s, 3 H, NCH3),
3.64-3.70 (m, 1 H, C(3)HH′), 3.73 (d, J ) 8.6 Hz, 1 H, C(3′)-
HH′), 4.05-4.11 (m, 1 H, C(3)HH′), 4.17 (s, 1 H, C(1′)H), 4.42
(d, J ) 8.6 Hz, 1 H, C(3′)HH′), 5.30 (s, 1 H, C(5a)HH′), 5.68 (s,
1 H, C(5a)HH′); 13C NMR (CD3OD) 25.1 (C(2′)CH3), 26.8
(C(CH3)2), 28.0 (C(CH3)2), 37.0 (C(4)), 41.8 (NCH3), 67.4 (C(3)),
72.7 and 73.2 (C(1′), C(3′)), 83.0 (C(6)), 86.4 (C(2′)), 88.1 (C(1)),
111.5 (C(CH3)2), 119.0 (C(5a)), 148.7 (C(5)), 170.0 and 170.6
(C(7), C(9)) ppm; MS (+CI) 372 [M + 1]+; Mr (+CI) 372.176 51
[M + 1]+ (calcd for C16H26N3O7 372.177 08).
Syn th esis of C(6)-(Meth oxyam in o)bicyclom ycin C(2′),C-
(3′)-Aceton id e (29). Using 22 (35 mg, 0.10 mmol), triethyl-
amine (21 mg, 0.21 mmol), methanesulfonyl chloride (23 mg,
0.21 mmol), and methoxyamine hydrochloride (95 mg, 1.0
mmol) gave 22 (6 mg, 17% recovery) and 29 as a white solid
(13 mg, 34%): mp 92-96 °C; Rf 0.60 (10% MeOH-CHCl3); IR
1
(KBr) 3417, 3296, 1692 cm-1; H NMR (CD3OD) δ 1.36 (s, 3
H, C(2′)CH3), 1.43 (s, 3 H, C(CH3)2), 1.44 (s, 3 H, C(CH3)2),
2.51 (dd, J ) 7.2, 16.1 Hz, 1 H, C(4)HH′), 2.63 (dd, J ) 8.4,
16.1 Hz, 1 H, C(4)HH′), 3.59 (s, 3 H, OCH3), 3.72 (d, J ) 8.6
Hz, 1 H, C(3′)HH′), 3.84-3.99 (m, 2 H, C(3)H2), 4.12 (s, 1 H,
C(1′)H), 4.43 (d, J ) 8.6 Hz, 1 H, C(3′)HH′), 5.10 (s, 1 H, C(5a)-
HH′), 5.31 (s, 1 H, C(5a)HH′); 13C NMR (CD3OD) 24.6 (C(2′)-
CH3), 26.8 (C(CH3)2), 28.2 (C(CH3)2), 37.7 (C(4)), 63.3 (OCH3),
67.1 (C(3)), 73.4 (C(3′)), 73.8 (C(1′)), 77.9 (C(6)), 86.3 (C(2′)),
88.8 (C(1)), 111.7 (C(CH3)2), 116.1 (C(5a)), 147.2 (C(5)), 168.6
and 170.7 (C(7), C(9)) ppm; MS (+CI) 372 [M + 1]+; Mr (+CI)
372.175 70 [M + 1]+ (calcd for C16H26N3O7 372.177 08).
Syn th esis of C(6)-Aceta m id obicyclom ycin C(2′),C(3′)-
Aceton id e (33). To an anhydrous THF (2 mL) solution of 24
(12 mg, 0.04 mmol) and acetic anhydride (18 mg, 0.18 mmol)
was added Proton Sponge (11 mg, 0.05 mmol). The solution
was stirred at room temperature (24 h) and concentrated in
vacuo. The residue was purified by PTLC (10% MeOH-
CHCl3) to provide 24 (5 mg, 42% recovery) and 33 (8 mg, 58%)
Syn th esis of C(6)-(N,O-Dim eth ylh yd r oxyla m in o)bicy-
clom ycin C(2′),C(3′)-Aceton id e (30). Using 22 (40 mg, 0.12
mmol), triethylamine (24 mg, 0.23 mmol), methanesulfonyl
chloride (27 mg, 0.23 mmol), and N,O-dimethylhydroxylamine
hydrochloride (116 mg, 1.2 mmol) gave 22 (8 mg, 20% recovery)
and 30 as a white solid (14 mg, 31%): mp 76-80 °C; Rf 0.58
(10% MeOH-CHCl3); IR (KBr) 3446, 3314, 1693, 1457 cm-1
;

Role of the C(6)-Hydroxy Group in Bicyclomycin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1191
as a white solid: mp 156-160 °C; Rf 0.52 (10% MeOH-CHCl3);
3414, 1686, 1456 cm-1; 1H NMR (CD3OD) δ 1.34 (s, 3 H, C(2′)-
CH3), 2.31 (s, 3 H, NCH3), 2.56 (dd, J ) 6.9, 16.1 Hz, 1 H,
C(4)HH′), 2.66 (dd, J ) 8.5, 16.1 Hz, 1 H, C(4)HH′), 3.51 (d, J
) 11.4 Hz, 1 H, C(3′)HH′), 3.67 (d, J ) 11.4 Hz, 1 H, C(3′)-
HH′), 3.81 (dd, J ) 8.5, 12.9 Hz, 1 H, C(3)HH′), 3.93 (dd, J )
6.9, 12.9 Hz, 1 H, C(3)HH′), 4.08 (s, 1 H, C(1′)H), 5.13 (s, 1 H,
C(5a)HH′), 5.47 (s, 1 H, C(5a)HH′); 13C NMR (CD3OD) 24.2
(C(2′)CH3), 29.4 (NCH3), 37.6 (C(4)), 66.1 (C(3)), 68.5 (C(3′)),
72.1 (C(1′)), 77.2 (C(6)), 78.2 (C(2′)), 89.5 (C(1)), 116.5 (C(5a)),
150.9 (C(5)), 170.1 and 171.7 (C(7), C(9)) ppm; MS (+CI) 316
[M + 1]+; Mr (+CI) 316.151 15 [M + 1]+ (calcd for C13H22N3O6
316.150 86).
1
IR (KBr) 3420, 1686, 1541 cm-1; H NMR (CD3OD) δ 1.40 (s,
6 H, C(2′)CH3, C(CH3)2), 1.44 (s, 3 H, C(CH3)2), 2.06 (s, 3 H,
C(O)CH3), 2.58-2.75 (m, 2 H, C(4)H2), 3.66-3.75 (m, 1 H, C(3)-
HH′), 3.73 (d, J ) 8.6 Hz, 1 H, C(3′)HH′), 4.04-4.09 (m, 1 H,
C(3)HH′), 4.11 (s, 1 H, C(1′)H), 4.39 (d, J ) 8.6 Hz, 1 H, C(3′)-
HH′), 5.24 (s, 1 H, C(5a)HH′), 5.37 (s, 1 H, C(5a)HH′); 1H NMR
(THF-d8) δ 1.34 (s, 3 H, C(2′)CH3), 1.35 (s, 3 H, C(CH3)2), 1.40
(s, 3 H, C(CH3)2), 2.01 (s, 3 H, C(O)CH3), 2.69 (dd, J ) 9.5,
16.1 Hz, 1 H, C(4)HH′), 3.66 (d, J ) 8.3 Hz, 1 H, C(3′)HH′),
3.66-3.73 (m, 1 H, C(3)HH′), 4.01 (dd, J ) 6.3, 12.5 Hz, 1 H,
C(3)HH′), 4.12 (d, J ) 8.4 Hz, 1 H, C(1′)H), 4.37 (d, J ) 8.3
Hz, 1 H, C(3′)HH′), 5.04 (s, 1 H, C(5a)HH′), 5.18 (s, 1 H, C(5a)-
HH′), 5.28 (d, J ) 8.4 Hz, 1 H, C(1′)OH), 8.07, 8.11, 8.15 (s, 3
H, C(6)NH, N(8)H, N(10)H), the C(4)HH′ was not detected and
is believed to be beneath the impurity peak at 2.56 ppm; 13C
NMR (CD3OD) 22.8 (C(O)CH3), 24.5 (C(2′)CH3), 26.8 (C(CH3)2),
27.9 (C(CH3)2), 36.7 (C(4)), 66.9 (C(3)), 72.5 (C(6)), 73.4 (C(3′)),
74.3 (C(1′)), 86.2 (C(2′)), 87.4 (C(1)), 111.6 (C(CH3)2), 117.0
(C(5a)), 148.7 (C(5)), 168.7, 169.0 and 173.6 (C(7), C(9), C(O)-
CH3)) ppm; MS (+CI) 384 [M + 1]+; Mr (+CI) 384.175 82 [M
+ 1]+ (calcd for C17H26N3O7 384.177 08).
Syn t h esis of C(6)-N,N-Dim et h yla m in ob icyclom ycin
(17). Using 26 (10 mg, 0.03 mmol) gave 17 as a white solid (4
mg, 45%): mp 98-104 °C; Rf 0.54 (20% MeOH-CHCl3); IR
1
(KBr) 3414, 1686, 1460 cm-1; H NMR (CD3OD) δ 1.33 (s, 3
H, C(2′)CH3), 2.36 (s, 6 H, N(CH3)2), 2.48-2.63 (m, 2 H, C(4)-
H2), 3.48 (d, J ) 11.4 Hz, 1 H, C(3′)HH′), 3.62 (d, J ) 11.4 Hz,
1 H, C(3′)HH′), 3.62-3.79 (m, 1 H, C(3)HH′), 4.00-4.06 (m, 1
H, C(3)HH′), 4.07 (s, 1 H, C(1′)H), 5.35 (s, 1 H, C(5a)HH′), 5.52
(s, 1 H, C(5a)HH′); 13C NMR (CD3OD) 24.1 (C(2′)CH3), 37.2
(C(4)), 39.0 (N(CH3)2), 67.0 (C(3)), 68.5 (C(3′)), 71.9 (C(1′)), 78.2
(C(2′)), 82.5 (C(6)), 88.5 (C(1)), 119.7 (C(5a)), 149.6 (C(5)), 170.8
and 171.3 (C(7), C(9)) ppm; MS (+CI) 330 [M + 1]+; Mr (+CI)
330.166 48 [M + 1]+ (calcd for C14H24N3O6 330.166 51).
Gen er a l P r oced u r e for t h e P r ep a r a t ion of C(6)-
Su bstitu ted Bicyclom ycin s 13-21. To a 50% aqueous
methanol solution (3 mL) of the C(6)-substituted acetonides
was added trifluoroacetic acid (3 drops). The reaction solution
was stirred at room temperature (2 h) and concentrated in
vacuo. The residue was purified by PTLC (20% MeOH-
CHCl3) to provide the desired product.
Syn th esis of C(6) Aceta m id obicyclom ycin (18). Using
33 (16 mg, 0.04 mmol) gave an unidentified adduct plus 18 as
a white solid (5 mg, 35%): mp 185-187 °C; Rf 0.25 (20%
MeOH-CHCl3); IR (KBr) 3406, 3239, 1686, 1520 cm-1 1H
;
By use of this procedure, the following compounds were
prepared.
NMR (CD3OD) δ 1.34 (s, 3 H, C(2′)CH3), 2.06 (s, 3 H, C(O)-
CH3), 2.58-2.74 (m, 2 H, C(4)H2), 3.48 (d, J ) 11.4 Hz, 1 H,
C(3′)HH′), 3.62 (d, J ) 11.4 Hz, 1 H, C(3′)HH′), 3.70 (dd, J )
8.9, 13.3 Hz, 1 H, C(3)HH′), 4.00-4.08 (m, 1 H, C(3)HH′), 4.06
(s, 1 H, C(1′)H), 5.21 (s, 1 H, C(5a)HH′), 5.32 (s, 1 H, C(5a)-
HH′); 13C NMR (CD3OD) 22.9 (C(O)CH3), 24.1 (C(2′)CH3), 36.8
(C(4)), 65.9 (C(3)), 68.5 (C(3′)), 72.1 (C(1′)), 72.7 (C(6)), 78.2
(C(2′)), 88.7 (C(1)), 116.8 (C(5a)), 149.0 (C(5)), 168.9, 169.0 and
173.5 (C(7), C(9), C(O)CH3) ppm; MS (+CI) 344 [M + 1]+; Mr
(+CI) 344.146 20 [M + 1]+ (calcd for C14H22N3O7 344.145 78).
Syn th esis of C(6)-Am in obicyclom ycin (13). Using 24
(7 mg, 0.02 mmol) gave 13 as a white solid (3 mg, 62%): mp
108-111 °C; Rf 0.23 (20% MeOH-CHCl3); IR (KBr) 3420,
1
3306, 1686 cm-1; H NMR (CD3OD) δ 1.34 (s, 3 H, C(2′)CH3),
2.61-2.67 (m, 2 H, C(4)H2), 3.50 (d, J ) 11.3 Hz, 1 H, C(3′)-
HH′), 3.66 (d, J ) 11.3 Hz, 1 H, C(3′)HH′), 3.80 (ddd, J ) 2.0,
6.3, 8.0 Hz, 1 H, C(3)HH′), 3.95 (ddd, J ) 2.0, 6.3, 8.0 Hz, 1 H,
C(3)HH′), 4.07 (s, 1 H, C(1′)H), 5.16 (s, 1 H, C(5a)HH′), 5.46
(s, 1 H, C(5a)HH′); 13C NMR (CD3OD) 24.2 (C(2′)CH3), 37.3
(C(4)), 65.7 (C(3)), 68.5 (C(3′)), 72.1 (C(1′)), 72.4 (C(6)), 78.2
(C(2′)), 89.6 (C(1)), 116.7 (C(5a)), 151.9 (C(5)), 169.1 and 172.6
(C(7), C(9)) ppm; MS (+FAB) 302 [M + 1]+; Mr (+FAB)
302.134 32 [M + 1]+ (calcd for C12H20N3O6 302.135 21).
Syn th esis of C(6)-(N-Meth ylh yd r oxyla m in o)bicyclo-
m ycin (19). Using 28 (14 mg, 0.04 mmol) gave 19 as a white
solid (6 mg, 48%): mp 119-123 °C; Rf 0.38 (20% MeOH-
CHCl3); IR (KBr) 3408, 3258, 1686 cm-1; 1H NMR (CD3OD) δ
1.33 (s, 3 H, C(2′)CH3), 2.48-2.63 (m, 2 H, C(4)H2), 2.67 (s, 3
H, NCH3), 3.47 (d, J ) 11.3 Hz, 1 H, C(3′)HH′), 3.62 (d, J )
11.3 Hz, 1 H, C(3′)HH′), 3.65-3.72 (m, 1 H, C(3)HH′), 4.01-
4.07 (m, 1 H, C(3)HH′), 4.09 (s, 1 H, C(1′)H), 5.28 (s, 1 H, C(5a)-
HH′), 5.67 (s, 1 H, C(5a)HH′); 13C NMR (CD3OD) 24.1
(C(2′)CH3), 37.2 (C(4)), 41.8 (NCH3), 66.3 (C(3)), 68.5 (C(3′)),
71.8 (C(1′)), 78.2 (C(2′)), 83.0 (C(6)), 88.6 (C(1)), 118.9 (C(5a)),
148.7 (C(5)), 170.2 (C(7), C(9) overlap) ppm; MS (+CI) 332 [M
+ 1]+; Mr (+CI) 332.145 74 [M + 1]+ (calcd for C13H22N3O7
332.145 78).
Syn th esis of C(6)-(Meth oxya m in o)bicyclom ycin (20).
Using 29 (12 mg, 0.03 mmol) gave 20 as a white solid (7 mg,
61%): mp 115-121 °C; Rf 0.40 (20% MeOH-CHCl3); IR (KBr)
3427, 3244, 1686, 1461 cm-1; 1H NMR (CD3OD) δ 1.33 (s, 3 H,
C(2′)CH3), 2.48-2.59 (m, 1 H, C(4)HH′), 2.60-2.68 (m, 1 H,
C(4)HH′), 3.52 (d, J ) 11.3 Hz, 1 H, C(3′)HH′), 3.57 (s, 3 H,
OCH3), 3.68 (d, J ) 11.3 Hz, 1 H, C(3′)HH′), 3.86-3.93 (m, 2
H, C(3)H2), 4.08 (s, 1 H, C(1′)H), 5.10 (s, 1 H, C(5a)HH′), 5.29
(s, 1 H, C(5a)HH′); 13C NMR (CD3OD) 24.2 (C(2′)CH3), 37.8
(C(4)), 63.2 (OCH3), 66.2 (C(3)), 68.5 (C(3′)), 72.2 (C(1′)), 78.1
(C(6)), 78.2 (C(2′)), 89.7 (C(1)), 116.1 (C(5a)), 147.5 (C(5)), 168.9
and 170.9 (C(7), C(9)) ppm; MS (+CI) 332 [M + 1]+; Mr (+CI)
332.144 59 [M + 1]+ (calcd for C13H22N3O7 332.145 78).
Syn th esis of C(6)-(N-Hydr oxylam in o)bicyclom ycin (14).
Using 27 (14 mg, 0.04 mmol) gave 14 as a white solid (8 mg,
61%): mp 120-123 °C; Rf 0.20 (20% MeOH-CHCl3); IR (KBr)
3405, 3226, 1686 cm-1; 1H NMR (CD3OD) δ 1.34 (s, 3 H, C(2′)-
CH3), 2.54-2.58 (m, 1 H, C(4)HH′), 2.61-2.65 (m, 1 H, C(4)-
HH′), 3.52 (d, J ) 11.4 Hz, 1 H, C(3′)HH′), 3.67 (d, J ) 11.4
Hz, 1 H, C(3′)HH′), 3.86-3.92 (m, 2 H, C(3)H2), 4.08 (s, 1 H,
C(1′)H), 5.11 (s, 1 H, C(5a)HH′), 5.31 (s, 1 H, C(5a)HH′); 13C
NMR (CD3OD) 24.1 (C(2′)CH3), 37.9 (C(4)), 66.2 (C(3)), 68.5
(C(3′)), 72.2 (C(1′)), 78.2 (C(2′)), 78.5 (C(6)), 89.6 (C(1)), 116.3
(C(5a)), 147.5 (C(5)), 169.9 and 171.2 (C(7), C(9)) ppm; MS
(-CI) 317 [M]-; Mr (-CI) 317.121 09 [M]- (calcd for C12H19N3O7
317.122 30).
Syn th esis of C(6)-Th iolbicyclom ycin (15). Using 32 (20
mg, 0.03 mmol) gave 15 as a white solid (11 mg, 62%): mp
143-147 °C; Rf 0.27 (30% MeOH-CHCl3); IR (KBr) 3390, 1686
1
cm-1; H NMR (CD3OD) δ 1.33 (s, 3 H, C(2′)CH3), 2.63 (dd, J
) 7.0, 16.1 Hz, 1 H, C(4)HH′), 2.76 (dd, J ) 8.5, 16.1 Hz, 1 H,
C(4)HH′), 3.53 (d, J ) 11.3 Hz, 1 H, C(3′)HH′), 3.68 (d, J )
11.3 Hz, 1 H, C(3′)HH′), 3.81 (dd, J ) 8.5, 12.8 Hz, 1 H, C(3)-
HH′), 3.92 (dd, J ) 7.0, 12.8 Hz, 1 H, C(3)HH′), 4.09 (s, 1 H,
C(1′)H), 5.23 (s, 1 H, C(5a)HH′), 6.20 (s, 1 H, C(5a)HH′); 13C
NMR (CD3OD) 24.1 (C(2′)CH3), 37.1 (C(4)), 66.6 (C(3)), 68.4
(C(3′)), 71.8 (C(6)), 72.2 (C(1′)), 78.3 (C(2′)), 90.1 (C(1)), 121.9
(C(5a)), 152.8 (C(5)), 177.3 and 177.8 (C(7), C(9)) ppm; MS
(+CI) 319 [M + 1]+; Mr (+CI) 319.097 44 [M + 1]+ (calcd for
Syn th esis of C(6)-(N,O-Dim eth ylh yd r oxyla m in o)bicy-
clom ycin (21). Using 30 (12 mg, 0.03 mmol) gave 21 as a
white solid (5 mg, 46%): mp 124-127 °C; Rf 0.61 (20% MeOH-
CHCl3); IR (KBr) 3421, 3269, 1687, 1456 cm-1; 1H NMR (CD3-
OD) δ 1.33 (s, 3 H, C(2′)CH3), 2.53-2.63 (m, 2 H, C(4)H2), 2.68
(s, 3 H, NCH3), 3.46 (d, J ) 11.4 Hz, 1 H, C(3′)HH′), 3.53 (s, 3
H, OCH3), 3.61 (d, J ) 11.4 Hz, 1 H, C(3′)HH′), 3.62-3.69 (m,
1 H, C(3)HH′), 4.03-4.08 (m, 1 H, C(3)HH′), 4.08 (s, 1 H,
C
12H19N2O6S 319.096 38).
Syn th esis of C(6)-(N-Meth yla m in o)bicyclom ycin (16).
Using 25 (12 mg, 0.03 mmol) gave 16 as a white solid (5 mg,
47%): mp 105-108 °C; Rf 0.40 (20% MeOH-CHCl3); IR (KBr)

1192 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Santilla´n et al.
C(1′)H), 5.30 (s, 1 H, C(5a)HH′), 5.65 (s, 1 H, C(5a)HH′); 13C
NMR (CD3OD) 24.1 (C(2′)CH3), 37.2 (C(4)), 37.3 (NCH3), 60.2
(OCH3), 66.7 (C(3)), 68.5 (C(3′)), 71.8 (C(1′)), 78.2 (C(2′)), 83.2
(C(6)), 88.5 (C(1)), 118.2 (C(5a)), 149.5 (C(5)), 169.6 and 170.3
(C(7), C(9)) ppm; MS (+CI) 346 [M + 1]+; Mr (+CI) 346.162 23
[M + 1]+ (calcd for C14H24N3O7 346.161 43).
Gen er a l P r oced u r e for th e Rea ction of Bicyclom ycin s
1 a n d 13-15 w ith EtSH. To a degassed (Ar, 5 min) THF-
H2O solution (3:1, 4 mL) containing the bicyclomycin (1 equiv)
was added EtSH (∼16 equiv). The “pH” of the reaction
mixture was adjusted with aqueous dilute NaOH, and the
solution was maintained at room temperature under an Ar
atmosphere. The solvent was removed in vacuo, and the
residue was purified by PTLC (20% MeOH-CHCl3) to afford
the product(s).
C(5a)HH′), 5.46 (s, 1 H, C(5a)HH′); 13C NMR (CD3OD) 21.3
(C(2′)CH3), 35.3 (C(4)), 62.0 (C(3)), 77.6, 78.2 and 78.5 (C(1′),
C(2′), C(3′)), 80.0 (C(6)), 90.1 (C(1)), 116.8 (C(5a)), 143.7 (C(5)),
168.6 and 169.3 (C(7), C(9)) ppm; MS (+CI) 318 [M + 1]+; Mr
(+CI) 318.132 29 [M + 1]+ (calcd for C12H20N3O7 318.130 13).
Rea ction of 14 w ith EtSH a t “p H” 10.0. Using 14 (14.0
mg, 0.04 mmol) and EtSH (44 mg, 0.64 mmol) resulted in a
drop of the solution “pH” to 9.6 during the reaction (17 h) and
provided 38 (3 mg, 21%) as a semisolid: Rf 0.25 (20% MeOH-
CHCl3); 1H NMR (CD3OD) δ 1.33 (s, 3 H, C(2′)CH3), 2.35-
2.47 (m, 2 H, C(4)H2), 3.67 (t, J ) 6.9 Hz, 2 H, C(3)H2), 3.77
(d, J ) 9.5 Hz, 1 H, C(3′)HH′), 3.90 (d, J ) 9.5 Hz, 1 H, C(3′)-
HH′), 4.48 (s, 1 H, C(1′)H), 5.19 (s, 1 H, C(5a)HH′), 5.46 (s, 1
H, C(5a)HH′).
Rea ction of 15 w ith EtSH a t “p H” 9.5. Using 15 (8 mg,
0.02 mmol) and EtSH (21 mg, 0.34 mmol) resulted in a drop
of “pH” to 9.0 during the reaction time (18 h) and provided
3613 (3 mg, 42%) as a white solid: Rf 0.70 (20% MeOH-CHCl3);
1H NMR (CD3OD) δ 1.33 (s, 3 H, C(2′)CH3), 2.77-2.87 (m, 2
H, C(4)H2), 3.82 (d, J ) 9.5 Hz, 1 H, C(3′)HH′), 3.96 (d, J )
9.5 Hz, 1 H, C(3′)HH′), 4.09-4.20 (m, 2 H, C(3)H2), 4.42 (s, 1
H, C(1′)H), 5.34 (br s, 1 H, C(5a)HH′), 5.40 (br s, 1 H, C(5a)-
HH′).
By use of this procedure, the following compounds were
prepared.
Rea ction of 1 w ith EtSH a t “p H” 9. Using 1 (10 mg, 0.03
mmol) and EtSH (33 mg, 0.53 mmol) resulted in a drop of “pH”
to 8.3 during the reaction time (16 h) and provided 346,9 as a
white solid (4 mg, 33%): Rf 0.65 (20% MeOH-CHCl3); 1H NMR
(CD3OD) δ 1.15 (s, 3 H, C(2′)CH3), 1.24 (t, J ) 7.5 Hz, 3 H,
SCH2CH3), 1.81-1.93 (m, 1 H, C(4)HH′), 2.30 (dt, J ) 6.5, 14.1
Hz, 1 H, C(4)HH′), 2.58 (q, J ) 7.5 Hz, 2 H, SCH2CH3), 2.90
(d, J ) 14.1 Hz, 1 H, C(5a)HH′), 3.00 (d, J ) 14.1 Hz, 1 H,
C(5a)HH′), 3.64 (d, J ) 12.0 Hz, 1 H, C(3′)HH′), 3.68-3.78
(m, 1 H, C(3)HH′), 3.90 (s, 1 H, C(1′)H), 4.03 (d, J ) 12.0 Hz,
1 H, C(3′)HH′), 3.92-4.06 (m, 1 H, C(3)HH′).
Syn th esis of 37. To an anhydrous dioxane solution (2 mL)
of 36 (6 mg, 0.02 mmol), p-bromobenzyl chloride (14 mg, 0.06
mmol), and diisopropylethylamine (8 mg, 0.06 mmol) was
added a catalytic amount of DMAP. The solution was stirred
at room temperature (4 h) and concentrated in vacuo. The
residue was purified by PTLC (2 × 20% ethyl acetate-
benzene) to provide 36 (3 mg, 50% recovery) and 3722 (2 mg,
33%) as a white solid: mp 215-218 °C, Rf 0.60 (10% MeOH-
CHCl3); IR (KBr) 3447, 3206, 3113, 1735, 1688, 1590, 1485
Rea ction of 1 w ith EtSH a t “p H” 10.5. Using 1 (21 mg,
0.07 mmol) and EtSH (69 mg, 1.11 mmol) resulted in a drop
of “pH” to 9.9 during the reaction time (14 h) and provided
the following compounds.
1
cm-1; H NMR (CD3OD) δ 1.41 (s, 3 H, C(2′)CH3), 2.70-2.79
Compound 35:9 yield, 8 mg (33%); Rf 0.45 (20% MeOH-
CHCl3); 1H NMR (CD3OD) δ 1.23 (t, J ) 7.2 Hz, 3 H,
SCH2CH3), 1.32 (s, 3 H, C(2′)CH3), 2.05-2.26 (m, 4 H, C(4)H2,
C(5)H, C(5a)HH′), 2.42-2.55 (m, 2 H, SCH2CH3), 3.15 (d, J )
11.7 Hz, 1 H, C(5a)HH′), 3.51 (d, J ) 11.4 Hz, 1 H, C(3′)HH′),
3.67 (d, J ) 11.4 Hz, 1 H, C(3′)HH′), 3.73-3.84 (m, 1 H, C(3)-
HH′), 3.90-4.00 (m, 1 H, C(3)HH′), 4.03 (s, 1 H, C(1′)H).
Compound 34:6,9 yield, 10 mg (40%); Rf 0.65 (20% MeOH-
CHCl3); 1H NMR (CD3OD) δ 1.15 (s, 3 H, C(2′)CH3), 1.24 (t, J
) 7.3 Hz, 3 H, SCH2CH3), 1.80-1.94 (m, 1 H, C(4)HH′), 2.32
(dt, J ) 6.6, 14.1 Hz, 1 H, C(4)HH′), 2.58 (q, J ) 7.3 Hz, 2 H,
SCH2CH3), 2.89 (d, J ) 14.0 Hz, 1 H, C(5a)HH′), 3.00 (d, J )
14.0 Hz, 1 H, C(5a)HH′), 3.64 (d, J ) 12.2 Hz, 1 H, C(3′)HH′),
3.68-3.78 (m, 1 H, C(3)HH′), 3.90 (s, 1 H, C(1′)H), 4.03 (d, J
) 12.2 Hz, 1 H, C(3′)HH′), 3.97-4.03 (m, 1 H, C(3)HH′).
Rea ction of 13 w ith EtSH a t “p H” 9.5. Using 13 (6 mg,
0.02 mmol) and EtSH (20 mg, 0.32 mmol) resulted in a drop
of “pH” to 9.0 during the reaction time (2 d). TLC analysis
indicated no reaction. PTLC of the reaction solution led to
the recovery of 13 (3 mg, 50% recovery) as a white solid: Rf
(m, 2 H, C(4)H2), 3.84 (dd, J ) 6.8, 13.7 Hz, 2 H, C(3)H2), 3.98
(d, J ) 9.5 Hz, 1 H, C(3′)HH′), 4.08 (d, J ) 9.5 Hz, 1 H, C(3′)-
HH′), 5.29 (br s, 1 H, C(5a)HH′), 5.32 (br s, 1 H, C(5a)HH′),
5.56 (s, 1 H, C(1′)H), 7.66 (d, J ) 8.4 Hz, 2 H, ArH), 8.01 (d, J
) 8.4 Hz, 2 H, ArH); 13C NMR (CD3OD) 21.0 (C(2′)CH3), 32.7
(C(4)), 68.4 (C(3)), 77.8, 78.7 and 80.2 (C(1′), C(2′), C(3′)), 89.0,
91.0 (C(1), C(6)), 111.4 (C(5a)), 129.5 (Ar, C(4′′)), 132.8 (2 Ar,
C(2′′), 132.9 (2 Ar, C(3′′)), 133.1 (Ar, C(1′′)), 150.5 (C(5)), 166.2,
168.4 and 169.4 (C(7), C(9), C(O)Ar) ppm; MS (+CI) 467 (41),
469 (59) [M + 1]+; Mr (+CI) 467.045 85 [M + 1]+ (calcd for
C
19H2079BrN2O7 467.045 39).
X-r a y Cr ysta llogr a p h ic Stu d y of 37.21 Crystals of 37
belong to the space group P1 (triclinic) with a ) 6.077(1) Å, b
) 10.039(2) Å, c ) 11.403(2) Å, R ) 114.62(1)°; â ) 97.05(1)°,
γ ) 102.22(1)°; V ) 600 Å3, Dcalcd ) 1.62 g cm-3, and Z ) 1.
Data were collected at -50 °C, and the structure was refined
to Rf ) 0.034, Rw ) 0.025 for 2178 reflections with I > 3σ(I).
X-r a y Cr ysta llogr a p h ic Stu d y of 24.21 Crystals of 24
belong to the space group P212121 (orthorhombic) with a )
7.155(1) Å, b ) 9.104(3) Å, c ) 25.169(7) Å; V ) 1639 Å3, Dcalcd
) 1.38 g cm-3, and Z ) 4. Data were collected at -50 °C, and
the structure was refined to Rf ) 0 034, Rw ) 0.028 for 1359
reflections with I > 3σ(I).
Gen er a l P r oced u r e for th e Deter m in a tion of th e
Sta bility of Bicyclom ycin Der iva tives. The stability of
bicyclomycins (1, 13-15) in aqueous buffered solution (40 mM
Tris, pH ) 7.4) was determined by HPLC. A 2 mg/mL solution
of the bicyclomycin derivative was maintained at 37 ( 1 °C.
The pH of the reaction mixture was determined before and
after the reaction. The pH at the end of the reaction was 7.4
( 0.1. Linear plots of ln A0/A (λ ) 214 nM) versus time were
obtained for an average of 10 points per bicyclomycin deriva-
tives over the course of the reaction. Most of the reactions
were monitored for more than two half lives and the identified
products were verified by co-injection of authentic samples
with the reactions and further verified by TLC analysis. Using
a least squared program the half life of the derivative was
calculated.
1
0.22 (20% MeOH-CHCl3); H NMR (CD3OD) δ 1.33 (s, 3 H,
C(2′)CH3), 2.61-2.67 (m, 2 H, C(4)H2), 3.50 (d, J ) 11.3 Hz, 1
H, C(3′)HH′), 3.66 (d, J ) 11.3 Hz, 1 H, C(3′)HH′), 3.80-3.84
(m, 1 H, C(3)HH′), 3.92-3.96 (m, 1 H, C(3)HH′), 4.07 (s, 1 H,
C(1′)H), 5.16 (s, 1 H, C(5a)HH′), 5.46 (s, 1 H, C(5a)HH′).
Rea ction of 13 w ith EtSH a t “p H” 10.0. Using 13 (6 mg,
0.02 mmol) and EtSH (20 mg, 0.32 mmol) resulted in a drop
of “pH” to 9.5 during the reaction time (22 h) and provided
3613 (2 mg, 36%) as a white solid: Rf 0.68 (20% MeOH-CHCl3);
1H NMR (CD3OD) δ 1.33 (s, 3 H, C(2′)CH3), 2.74-2.82 (m, 2
H, C(4)H2), 3.82 (d, J ) 9.6 Hz, 1 H, C(3′)HH′), 3.96 (d, J )
9.6 Hz, 1 H, C(3′)HH′), 4.07-4.18 (m, 2 H, C(3)H2), 4.41 (s, 1
H, C(1′)H), 5.34 (br s, 1 H, C(5a)HH′), 5.40 (br s, 1 H, C(5a)-
HH′).
Rea ction of 14 w ith EtSH a t “p H” 8.8. Using 14 (5.0
mg, 0.02 mmol) and EtSH (16 mg, 0.32 mmol) resulted in a
drop of the solution “pH” to 8.2 during the reaction (46 h) and
provided 38 (2 mg, 40%) as a semisolid: Rf 0.25 (20% MeOH-
1
CHCl3); IR (KBr) 3434, 1681 cm-1; H NMR (CD3OD) δ 1.33
(s, 3 H, C(2′)CH3), 2.37-2.44 (m, 2 H, C(4)H2), 3.67 (t, J ) 6.9
Hz, 2 H, C(3)H2), 3.77 (d, J ) 9.5 Hz, 1 H, C(3′)HH′), 3.90 (d,
J ) 9.5 Hz, 1 H, C(3′)HH′), 4.48 (s, 1 H, C(1′)H), 5.19 (s, 1 H,
Sta bility of C(6)-Am in obicyclom ycin (13). An aqueous
buffer solution (2 mL, 40 mM Tris, pH ) 7.4) of 13 (5 mg, 0.02
mmol) was maintained at 37 ( 1 °C (125 h). During the course

Role of the C(6)-Hydroxy Group in Bicyclomycin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1193
tibiot. 1972, 25, 610-612. (b) Miyamura, S.; Ogasawara, N.;
Otsuka, H.; Niwayama, S.; Tanaka, H.; Take, T.; Uchiyama, T.;
Ochiai, H. Antibiotic 5879 produced by Streptomyces Aizunesis,
identical with bicyclomycin J . Antibiot. 1973, 26, 479-484.
(4) Williams, R. M.; Durham, C. A. Bicyclomycin: Synthetic,
mechanistic, and biological studies. Chem. Rev. 1988, 88, 511-
540 and references cited therein.
(5) Someya, S.; Iseki, M.; Tanaka, N. Active groups of bicyclomycin
and the reaction with thiols. J . Antibiot. 1979, 32, 402-407.
(6) Abuzar, S.; Kohn, H. Observations on the activation of bicyclo-
mycin. J . Am. Chem. Soc. 1988, 110, 4089-4090.
(7) Abuzar, S.; Kohn, H. Studies on the reactivity of bicyclomycin
with nucleophilic amino acid derivatives. J . Org. Chem. 1989,
54, 4000-4003.
(8) Abuzar, S.; Kohn, H. Studies on the reactivity of bicyclomycin
with amines. J . Am. Chem. Soc. 1989, 111, 4895-4903.
(9) Abuzar, S.; Kohn, H. Studies on the reactivity of bicyclomycin
with thiols. J . Am. Chem. Soc. 1990, 112, 3114-3121.
(10) Zweifka, A.; Kohn, H.; Widger, W. R. Transcription termination
factor rho: The site of bicyclomycin inhibition in Escherichia
coli. Biochemistry 1993, 32, 3564-3570.
(11) Magyar, A.; Zhang, X.; Kohn, H.; Widger, W. R. The antibiotic
bicyclomycin affects the secondary RNA binding site of Escheri-
chia coli transcription termination factor Rho. J . Biol. Chem.
1996, 271, 25369-25374.
(12) Park, H.-g.; Zhang, X.; Moon, H.-s.; Zwiefka, A.; Cox, K.; Gaskell,
S. J .; Widger, W. R.; Kohn, H. Bicyclomycin and dihydrobicy-
clomycin inhibition kinetics of Escherichia coli rho-dependent
transcription termination factor ATPase activity. Arch. Biochem.
Biophys. 1995, 323, 447-454.
(13) Santilla´n, J r., A.; Park, H.-g.; Zhang, X.; Lee, O.-S.; Widger, W.
R.; Kohn, H. Role of the [4.2.2] bicyclic unit in bicyclomycin:
Synthesis, structure, chemical, biochemical, and biological prop-
erties. J . Org. Chem. 1996, 61, 7756-7763.
(14) Wu, A. M.; Christie, G. E.; Platt, T. Tandem termination sites
in the tryptophan operon of Escherichia coli. Proc. Natl. Acad.
Sci. U.S.A. 1981, 78, 2913-2917.
of the reaction a change of pH was recorded from 7.4 to 7.7.
The reaction was then concentrated in vacuo, and the residue
was purified by PTLC (2 × 15% MeOH-CHCl3) to provide
4222,25 (6 mg, 63%) as a white solid: Rf 0.56 (20% MeOH-
CHCl3); 1H NMR (CD3OD) δ 1.33 (s, 3 H, C(2′)CH3), 2.72-
2.84 (m, 2 H, C(4)H2), 3.80-3.84 (m, 1 H, C(3′)HH′), 3.95-
3.97 (m, 1 H, C(3′)HH′), 4.05-4.22 (m, 2 H, C(3)H2), 4.41-
4.42 (m, 1 H, C(1′)H), 5.25-5.41 (m, 2 H, C(5a)H2).
Molecu la r Or bita l Ca lcu la tion s. Density functional
calculations were performed with the Gaussian92/DFT31 pro-
gram package using the internally stored 6-31G** 32 basis set
at the B3LYP33 level. Geometry optimizations of all species
in eq 1 were performed under their respective point groups
(D2h for ethylene (44), Cs for ethylamine (46), and C1 for 45a -c
and 47a -c). The only additional constraints were imposed
upon 47a -c in which the newly formed R,â-unsaturated
ketone, imine, or thione was held in a cisoid conformation
maintaining a 45° dihedral angle between the CdC and CdX
bonds, mimicking the lowest energy conformation of â-meth-
ylene-R-ketoamide 2 as determined by molecular modelling
using PC MODEL.24 Single-point calculations in which this
dihedral was held at 0° and the dihedral angle between the
CdO and CdX bonds was widened to 45° resulted in consistent
energy changes smaller than 0.5 kcal/mol lending confidence
in the energy values obtained from this model compound. All
converged geometries were confirmed as minima by perform-
ing vibrational frequency calculations. Free energies of all
compounds were calculated at STP by applying thermal and
entropy corrections obtained from the frequency calculations
to the electronic energies. The thermal corrections account
for zero-point vibrational energy as well as contributions from
the population of various translational, rotational, and vibra-
tional energy levels assuming an ideal gas in the canonical
ensemble.
(15) Sharp, J . A.; Galloway, J . L.; Platt, T. A kinetic mechanism for
the poly(C)-dependent ATPase of the Escherichia coli transcrip-
tion termination protein, rho. J . Biol. Chem. 1983, 258, 3482-
3486.
Ack n ow led gm en t. We thank the National Insti-
tutes of Health (Grant GM37934) and the Robert A.
Welch Foundation (Grants E-607, H.K., E-1381, W.R.W.)
for their support of this research. We thank Dr. M.
Kawamura and the Fujisawa Pharmaceutical Co., Ltd.,
J apan, for the gift of bicyclomycin, Dr. T. Platt (Uni-
versity of Rochester) for the overproducing strain of rho,
Dr. A. Magyar (University of Houston) for his help in
the transcription termination assays, and Dr. J ames
Korp (University of Houston) for conducting the X-ray
crystallographic analyses of 24 and 37. We thank Dr.
Thomas Albright (University of Houston) for his con-
tributions to the computational studies.
(16) Ericsson, H. M.; Sherris, J . C. Antibiotic sensitivity testing. Acta
Pathol. Microbiol. Scand. 1971, Suppl. 217, 1-90.
(17) Kamiya, T.; Maeno, S.; Kitaura, Y. Belgium patent 847 475.
(18) (a) Zhang, Z.; Kohn, H. The role of the C-1 triol group in
bicyclomycin. J . Chem. Soc., Chem. Commun. 1994, 1343-1344.
(b) Zhang, Z.; Kohn, H. Chemical, biochemical, and biological
studies on select C(1) triol modified bicyclomycins. J . Am. Chem.
Soc. 1994, 116, 9815-9826.
(19) J ackman, L. M.; Sternhall, S. Applications of Nuclear Magnetic
Resonance Spectroscopy in Organic Chemistry, 2nd ed.; Perga-
mon Press: Oxford, 1969.
(20) Stothers, J . B. Carbon-13 NMR Spectroscopy; Academic Press:
New York, 1972.
(21) The authors have deposited X-ray crystallographic data, a
description of the structure determination, and tables of atomic
coordinates and isotropic thermal parameters, bond lengths and
angles, anisotropic thermal parameters, and refined and calcu-
lated hydrogen atom coordinates with the Cambridge Crystal-
lographic Data Centre. The data can be obtained, on request,
from the Director, Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge CB2 lEZ, U.K.
(22) Maag, H.; Blount, J . F.; Coffen, D. L.; Steppe, T. V.; Wong, F.
Structure, absolute configuration, and total synthesis of an acid-
catalyzed rearrangement product of bicyclomycin. J . Am. Chem.
Soc. 1978, 100, 6786-6788.
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C NMR
spectra of all new compounds prepared in this study and X-ray
crystallographic information (data collection, final atomic
positional parameters, atomic thermal parameters (anisotropic
displacement parameters, H atom coordinates and isotropic
displacement parameters), bond distances and angles) for 24
and 37 (60 pages). Ordering information is given on any
current masthead page.
(23) Comparable findings were observed for C(6)-O-methylhydroxy-
laminobicyclomycin (20); see: Santilla´n, A., J r. Ph.D. Thesis,
University of Houston, 1997.
(24) Molecular mechanics calculations were performed using the
program PC MODEL available from Serena Software, Bloom-
ington, IN.
(25) Kohn, H.; Abuzar, S. Studies on the chemical reactivity of
bicyclomycin: Acid hydrolysis. J . Org. Chem. 1988, 53, 2769-
2773.
(26) Smith, P. A. S. The Chemistry of Open-Chain Organic Nitrogen
Compounds; W. A. Benjamin, Inc.: New York, 1966; Chapter 8,
pp 1-118.
(27) Park, H.-g.; Zhang, X.; Widger, W. R.; Kohn, H. Role of the C(1)
trial group in bicyclomycin: Synthesis and biochemical and
biological properties. J . Org Chem. 1996, 61, 7750-7755.
(28) For representative examples, see: (a) Wells, T. N. C.; Fersht,
A. R. Hydrogen bonding in enzymatic catalysis analysed by
protein engineering. Nature 1985, 316, 656-657. (b) Sams, C.
F.; Vyas, N. K.; Quiocho, F. A.; Matthews, K. S. Predicted
structure of the sugar-binding site of the lac repressor. Nature
1984, 310, 429-430.
Refer en ces
(1) Tanaka, N. Antibiotics (N.Y.) 1979, 5, 18-25.
(2) (a) Miyoshi, T.; Miyari, N.; Aoki, H.; Kohsaka, M.; Sakai, H.;
Imanaka, H. Bicyclomycin,
a new antibiotic I. Taxonomy,
isolation and characterization. J . Antibiot. 1972, 25, 569-575.
(b) Kamiya, T.; Maeno, S.; Hashimoto, M.; Mine, Y. Bicyclomycin,
a new antibiotic. II. Structural elucidation and acyl derivatives.
J . Antibiot. 1972, 25, 576-581. (c) Nishida, M.; Mine, Y.;
Matsubara, T. Bicyclomycin, a new antibiotic. III. In vitro and
in vivo antimicrobial activity. J . Antibiot. 1972, 25, 582-593.
(d) Nishida, M.; Mine, Y.; Matsubara, T.; Goto, S.; Kuwahara,
S. Bicyclomycin, a new antibiotic. IV. Absorption, excretion and
tissue distribution. J . Antibiot. 1972, 25, 594-601.
(3) (a) Miyamura, S.; Ogasawara, N.; Otsuka, H.; Niwayama, S.;
Takana, H.; Take, T.; Uchiyama, T.; Ochiai, H.; Abe, K.; Koizumi,
K.; Asao, K.; Matuski, K.; Hoshino, T. Antibiotic No. 5879, a new
water-soluble antibiotic against gram-negative bacteria. J . An-

1194 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Santilla´n et al.
(29) Mott, J . E.; Grant, R. A.; Ho, Y.-S.; Platt, T. Maximizing gene
expression from plasmid vectors containing the λ PL promoter:
strategies for overproducing transcription termination factor rho.
Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 88-92.
type basis sets for use in molecular orbital studies of organic
molecules. J . Chem. Phys. 1972, 56, 2257-2261. (b) Francl, M.
M.; Pietro, W. J .; Hehre, W. J .; Binkley, J . S.; Gordon, M. S.;
Defrees, D. J .; Pople, J . A. Self-consistent molecular orbital
methods. XXIII. A polarization-type basis set for second-row
elements. J . Chem. Phys. 1982, 77, 3654-3665.
(30) Lowry, O. H.; Rosebrough, N. J .; Farr, A. L.; Randall, R. J .
Protein measurement with the Folin phenol reagent. J . Biol.
Chem. 1951, 193, 265-275.
(33) (a) Becke, A. D. Density-functional exchange-energy approxima-
tion with correct asymptotic behavior. Phys. Rev. A 1988, 38,
3098-3100. (b) Lee, C.; Yang, W.; Parr, R. G. Development of
the Colle-Salvetti correlation-energy formula into a functional
of the electron density. Phys. Rev. B. 1988, 37, 785-789. (c)
Becke, A. D. Density-functional thermochemistry. III. The role
of exact exchange. J . Chem. Phys. 1993, 98, 5648-5652.
(31) Frisch, M. J .; Trucks, G. W.; Schlegel, H. B.; Gill, P. M. W.;
J ohnson, B. G.; Wong, M. W.; Foresman, J . B.; Robb, M. A.;
Head-Gordon, M.; Replogle, E. S.; Gomperts, R.; Andres, K. L.;
Raghavachari, K.; Binkley, J . S.; Gonzalez, C.; Martin, R. L.;
Fox, D. J .; Defrees, D. J .; Baker, J .; Stewart, J . J . P.; Pople, J .
A. Gaussian 92/ DFT, Revision G.4, Gaussian, Inc., Pittsburgh,
PA, 1993.
(32) (a) Hehre, W. J .; Ditchfield, R.; Pople, J . A. Self-consistent
molecular orbital methods. XII. Further extensions of Gaussian-
J M9708386