Conformationally restricted analogs of deoxynegamycin

Article abstract of DOI:10.1016/j.bmcl.2004.04.036

Deoxynegamycin (1b) is a protein synthesis inhibitor with activity against Gram-negative (GN) bacteria. A series of conformationally restricted analogs were synthesized to probe its bioactive conformation. Indeed, some of the constrained analogs were found to be equal or better than deoxynegamycin in protein synthesis assay (1b, IC₅₀=8.2μM; 44, IC ₅₀=6.6μM; 35e₂, IC₅₀=1μM). However, deoxynegamycin had the best in vitro whole cell antibacterial activity (Escherichia coli, MIC=4-16μg/mL; Klebsiella pneumoniae, MIC=8μg/mL) suggesting that other factors such as permeation may also be contributing to the overall whole cell activity. A new finding is that deoxynegamycin is efficacious in an E. coli murine septicemia model (ED₅₀=4.8mg/kg), providing further evidence of the favorable in vivo properties of this class of molecules.

Full text of DOI:10.1016/j.bmcl.2004.04.036

Bioorganic & MedicinalChemistry Letters 14 (2004) 3103–3107
Conformationally restricted analogs of deoxynegamycinq
B. Raju,* Sampathkumar Anandan, Shihai Gu, Prudencio Herradura,
Hardwin O’Dowd, Bum Kim, Marcela Gomez, Corinne Hackbarth, Charlotte Wu,
Wen Wang, Zhengyu Yuan, Richard White, Joaquim Trias and Dinesh V. Patel
Vicuron Pharmaceuticals, Inc., Department of Chemistry, 34790 Ardentech Court, Fremont, CA 94555, USA
Received 8 August 2003; accepted 10 April2004
Abstract—Deoxynegamycin (1b) is a protein synthesis inhibitor with activity against Gram-negative (GN) bacteria. A series of
conformationally restricted analogs were synthesized to probe its bioactive conformation. Indeed, some of the constrained analogs
were found to be equalor better than deoxynegamycin in protein synthesis assay ( 1b, IC50 ¼ 8.2 lM; 44, IC50 ¼ 6.6 lM; 35e2,
IC50 ¼ 1 lM). However, deoxynegamycin had the best in vitro whole cell antibacterial activity (Escherichia coli, MIC ¼ 4–16 lg/mL;
Klebsiella pneumoniae, MIC ¼ 8 lg/mL) suggesting that other factors such as permeation may also be contributing to the overall
whole cell activity. A new finding is that deoxynegamycin is efficacious in an E. coli murine septicemia model(ED ¼ 4.8 mg/kg),
50
providing further evidence of the favorable in vivo properties of this class of molecules.
ꢀ 2004 Published by Elsevier Ltd.
R
NH
O
O
1. Introduction
1
2
NH
O
O
2
H N
2
N
H N
2
N
N
H
OH
N
H
OH
A surge in resistance1 to the existing armamentarium of
clinically used antibacterial agents has stimulated the
search for novelantibiotics with different mechanisms of
action2 that are not subject to cross-resistance. Antibio-
tics, which are naturalproducts, have palyed a pre-
dominant role in antibacterial therapy. Chemistry has in
many cases improved on nature and semi-synthetic
variants of the originalantibiotic are the marketed
products. Total synthesis of the typically complex anti-
biotic structures has been mostly an academic challenge
rather than a practicalalternative for their manufacture.
In a limited number of cases the structure of an antibio-
tic has been sufficiently simple to allow reasonable
synthetic access, for example, chloramphenicol is manu-
1a.
1b.
R
= OH (Negamycin)
= H (Deoxynegamycin)
2
1
1
R
Boc
NH
Boc
O
NH
O
N
3
/ BocHN
HO
OH
or
OR
R CHO
4
3
O
3
6
5a. R = t-Bu
3
3
+
5b.
R = Me
O
4a.
4b.
R
= t-Bu
= Bz
2
N
R
2
R
2
H N
2
O
Figure 1. Design and retrosynthetic analysis.
thetically accessible. We report here on the synthesis and
activity of several analogs that are conformationally
restricted at the N-terminalregion.
3
factured by totalsynthesis. Negamycin is an antibiotic
with activity against Gram-negative bacteria and has a
mechanism involving inhibition of protein synthesis. As
a starting point for making improved negamycin ana-
logs, we chose to work with deoxynegamycin,4 a closely
related molecule that is less complex and more syn-
The desired b-amino acids of type 3 can be obtained
from appropriately protected aspartic acid 5 (Fig. 1)
through functionalization of a-carboxylgroup. Alter-
natively, aldehyde 6 can serve as a starting materialto
synthesize the b-amino acids of type 3 through multi-
component condensation reaction.
5
2. Chemistry
q Part of this work was presented at 42nd ICAAC. Annualmeeting of
the American Society for Microbiology. September 27–30, 2002, San
Diego, CA.
Homologation of D-ornithine derivative 7 (Scheme 1)
under Arndt–Eistert reaction6 conditions followed by
ester hydrolysis produced the desired b-amino acid 9.
* Corresponding author. Fax: +1-510-739-3003; e-mail: braju@
vicuron.com
0960-894X/$ - see front matter ꢀ 2004 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2004.04.036

3104
B. Raju et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3103–3107
Boc
NH
Boc
NH
Boc
HN
Boc
NH
Boc
HO C
a - c
O
2
O
NHBoc
CO t-Bu
HN
16
NHBoc
CO H
a - c
d
O
d, e
CbzHN
CbzHN
7
CbzHN
2
( )
OMe
( )
CO H
2
2
n
n
( )
OH
9. n = 3
n
OH
14
15
8
Boc
HN
NHBoc
NHBoc
CO H
NHBoc
HO C
2
f, b, c
d, e
Scheme 1. Synthesis of 3,6-diaminohexanoic acid. Reagents and con-
ditions: (a) i-butylcholroformate, NMM, THF )15 ꢁC, 45 min then
CH2N2 in ether; (b) C6H5CO2Ag, Et3N, MeOH (65% for two steps);
(c) LiOH, dioxane, water (85%).
CO t-Bu
2
H
CO t-Bu
2
2
17
18
O
19
g - i
Boc
NHBoc
NHBoc
NHBoc
N
j, e
CO t-Bu
PMB
2
CO H
2
21
20
The synthesis of b-amino acids 13a, 13c, and 13d relied
on L-aspartic acid derivative 10 as a chiralsource
Scheme 3. Synthesis of cyclopropane-based b-amino acids. Reagents
and conditions: (a) DIC, DMAP, t-BuOH; (b) N2CHCO2Et,
Rh2(OAc)4, DCM, rt (27–31%); (c) LiOH, H2O, EtOH (79–80%); (d)
DPPA, Et3 N, t-BuOH, 110 ꢁC (40–66%); (e) 2 M KOH in MeOH (60–
97%); (f) PPh3MeBr, KHMDS, THF, )78 ꢁC to rt (80%); (g) p-
methoxybenzylamine, HATU, DIEA, DMF, 0 ꢁC to rt (82%); (h)
BH3, THF, 0 ꢁC to rt to 45 ꢁC (94%); (i) (Boc)2O, K2CO3, THF, rt
(75%); (j) CAN, CH3CN–H2O (2:1), rt (65%).
(Scheme 2). Hence, aspartic acid 10 was reduced to
corresponding alcohol and oxidized to aldehyde under
Swern7 oxidation conditions. Condensation of aldehyde
8
with triethyl2-fluoro-2-phosphonoacetate or with tri-
ethyl2-phosphonoacetate gave oelfin derivatives 11a–d,
cis/trans isomers were separated by silica gel chroma-
tography. Hydrolysis of the ethyl ester in 11a, 11c, and
11d followed by reduction9 of the acid produced alco-
hols 12a, 12c, and 12d, respectively. These alcohols were
converted to the corresponding mesylates and treated
with sodium azide to furnish the azides. Removalof the
t-butylester under basic conditions provided azido acids
13a, 13c, and 13d.
azide, obtained from acid 18, upon Curtius rearrange-
ment11 using t-butanolas a sovl ent and further removal
of the t-butylester group under basic conditions gave
the desired b-amino acid 19 as a mixture of diastereoi-
somers. Acid 18 was coupled with p-methoxybenzyl
amine and the resultant amide was reduced to yield
secondary amine, which was capped with t-butoxycar-
bonyl(Boc) group. Removalof p-methoxybenzylgroup
under oxidative conditions12 followed by base hydrolysis
of the t-butylester furnished the desired b-amino acid 21
as a mixture of diastereoisomers.
Aspartic acid 10 and homoallylglycine 14 served as
chiralsource to access cycolpropane-based
b-amino
acids 16, 19, and 21 as a mixture of diastereomers
(Scheme 3). Homoallylglycine 14 was converted to the
corresponding t-butylester and subjected to rhodium
catalyzed carbene insertion reaction.10 The cyclopro-
pane derivative was selectively hydrolyzed to furnish
cyclopropane carboxylic acid 15. Curtius rearrange-
ment11 of acylazide derived from acid 15 in the presence
of t-butanol as a solvent followed by removal of the t-
butylester by base hydroyl sis gave b-amino acid 16 as a
mixture of diastereomers. Aldehyde 17, obtained from
aspartic acid as depicted in Scheme 2, was converted to
an alkene derivative. Alkene was subjected to the rho-
dium catalyzed carbene insertion10 reaction followed by
selective removal of the ethyl ester under basic condi-
tions to get acid 18 as a mixture of diastereomers. Acyl
The synthesis of cyclopentane-based b-amino acid is
depicted in Scheme 4. c-Amino acid 22 was reduced9 to
alcohol and then oxidized to aldehyde. Condensation of
aldehyde with t-butyl diethylphosphonoacetate followed
by 1,4-addition of p-methoxybenzylamine gave b-amino
ester 23 as a mixture of diastereomers. Protection of
secondary amine in 23 with Boc group, removal p-
12
methoxybenzylgroup under oxidative conditions, and
hydrolysis of the t-butylester furnished the desired b-
amino acid 24.
The synthesis of thiazole-based b-amino acids was
accomplished as described in Scheme 5 starting from
aspartic acid 25 as a chiralsynthon. Diazoketone, pre-
pared from aspartic acid 25, was reacted with hydro-
bromic acid to give the bromoketone13 26 (Scheme 5).
On the other hand, diazoketone was subjected to Wolff
Boc
NH
Boc
NH
11a.
11b.
R
R
= H, R =CO Et
2 2
1
1
R
1
O
O
= CO Et,
R = H
2
2
a - c
HO
11a : 11b (10:1 ratio)
OtBu
R
OtBu
2
11c.
11d.
R
R
= F, R = CO Et
2 2
1
1
O
10
= CO Et,
R = F
2
2
11c : 11d (1:10 ratio)
d, a
Boc
NH
Boc
NH
R
1
O
R
1
O
e - g
PMB
R
OtBu
2
Boc
HN
Boc
NH
R
OH
2
NH O
Boc
HN
Boc
HN
O
e - g
a - d
O
OtBu
OH
13a.
13c.
13d.
R
R
R
= H,
R
= CH N
12a.
12c.
12d.
R
R
R
= H, R = CH OH
2 2
1
1
1
2
2
2
3
3
1
1
1
= F,
R
= CH
N
= F,
= CH OH, R = F
2
R = CH OH
2
= F
2 2
OH
= CH N ,
R
22
23
24
2
3
2
2
Scheme 2. Synthesis of alkene-based b-amino acids. Reagents and
conditions: (a) i-butylchloroformate, NMM, THF )15 ꢁC, 45 min then
NaBH4, H2O, 0 ꢁC to rt, 2 h (75%); (b) oxalyl chloride, DMSO, Et3N,
)60 to )40 ꢁC, 1 h (95%); (c) n-BuLi, triethyl2-fluoro-2-phospho-
noacetate, THF, )78 ꢁC or NaH, triethyl2-phosphonoacetate, 0 ꢁC to
rt (65%); (d) LiOH, THF, H2O, rt, 16 h (100%); (e) CH3SO2Cl, pyr-
idine DCM, 0 ꢁC, 2 h (100%); (f) NaN3, DMF, 80 ꢁC, 16 h (80%); (g)
2 M KOH in MeOH, rt, 16 h (100%).
Scheme 4. Synthesis of cyclopentane-based b-amino acid. Reagents
and conditions: (a) i-butylchloroformate, NMM, THF )15 ꢁC, 45 min
and then NaBH4, H2O, 0 ꢁC to rt, 2 h (75%); (b) DMSO, oxalyl
chloride, TEA, )60 to )40 ꢁC, 1 h (75%); (c) t-butyldieth-yl
phosphonoacetate, NaH, THF, 0 ꢁC to rt (63%); (d) p-methoxybenzyl
amine, 110 ꢁC, 60 h (42%); (e) (Boc)2O, Pd–C (10%), EtOAc (65%); (f)
CAN, CH3CN–H2O (2:1), rt, 16 h (89%); (g) 2 M NaOH in MeOH, rt,
16 h (95%).

B. Raju et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3103–3107
3105
Br NHBoc
CO Me
NHBoc
O
O
NHBoc
CO Me
O
O
O
RHN
( )
a, b
c, e
N
CO H
b, d or c, d
or d
N
HO
2
2
a
N
P
1
2
R
N
H
OH
n
2
R
1
N
H
O
R
OH
1
O
O
S
26
25
a, e
34
27a. n = 0, R = H
27b. n = 1, R = Boc
27c. n = 2, R = Boc
35
Boc
NH
NH
2
9
CbzHN
H N
2
1b
a
S
NHBoc
CO H
O
NHBoc
RHN
( )
NHBoc
CO Me
Boc
NH
a, b
c, e
2
n
2
H
F
N
H
NH
NH
2
N
3
13a
H N
2
Br
CO Me
2
CO H
2
30a. n = 0, R = H
30b. n = 1, R = Boc
30c. n = 2, R = Boc
28
29
Boc
NH
F
2
2
N
3
13c
13d
H N
2
b
c
Scheme 5. Synthesis of thiazole-based b-amino acids. Reagents and
conditions: (a) i-butylchloroformate, NMM, THF )15 ꢁC, 45 min then
CH2N2 in ether; (b) HBr, AcOH, ether; (c) thiourea or Boc-
NH(CH2)nC(@S)NH2 (n ¼ 1, 2), EtOH; (d) LiOH, H2O, THF;
(e) C6H5CO2Ag, H2O, THF.
Boc
NH
N
H N
2
3
NH
F
F
Boc
HN
Boc
H N
2
HN
NH
2
16
19
d
Boc
HN
rearrangement in the presence of water to provide acid
28. Bromoketone 29 was prepared from acid 28 as de-
scribed above. Condensation of bromoketones 26 and
29 with thiourea followed by base hydrolysis of methyl
ester afforded b-amino acid 27a and 30a, respectively.
These acids were isolated as lithium salt upon lyophil-
lization. Further, the condensation of bromoketones 26
and 29 with thioamides derived from t-butyloxygylci-
namide and 3-(N-t-butoxycarboylamino)propionamide
followed by esters hydrolysis gave acids 27b, 27c, 30b,
and 30c.
NHBoc
NH
H N
2
2
e1
e2
NH
H N
2
2
NHBoc
NHBoc
NH
2
H N
2
f
21
24
Boc
NH
NH
2
Boc
HN
H N
2
g
NH
2
NHBoc
H N
2
RHN
N
N
( )
n
( )
n
Synthesis of b-amino acid 33 is outlined in Scheme 6.
The protection of amine 31 with Boc group followed by
oxidation of secondary alcohol gave the corresponding
ketone. This ketone upon Wittig olefination followed by
protection of the carbamate nitrogen with Boc group
gave the cyclohexene 32. The [2+2] cycloaddition14
reaction between cyclohexene 32 and isocyanate gave
b-lactam, which was treated with di-tert-butyldicar-
bonate in the presence of DMAP followed by base
hydrolysis to furnish b-amino acid 33.
S
S
h. n = 0, R = H
i. n = 1, R = Boc
j. n = 2, R = Boc
27a. n = 0, R = H
27b. n = 1, R = Boc
27c. n = 2, R = Boc
S
S
NH
NHBoc
2
RHN
( )
H N
2
( )
n
n
N
N
30a. n = 0, R = H
30b. n = 1, R = Boc
30c. n = 2, R = Boc
k. n = 0, R = H
l. n = 1, R = Boc
m. n = 2, R = Boc
Boc
NH
NH
2
n
33
NH
NHBoc
2
The structurally diverse set of b-amino acids synthesized
(Schemes 1–6) in the present study were incorporated in
deoxynegamycin template as summarized in Scheme 7.
b-Amino acid 9 was coupled with hydrazine 4a and the
resultant product was subjected to catalytic hydroge-
nation followed by acidic treatment to afford 1b. Azido
b-amino acids 13a, 13c, and 13d were coupled with
hydrazine 4a, the azide was reduced to amine and the
resultant products were treated with hydrochloric acid
in dioxane to furnish 35a–c, respectively. b-Amino acid
19 was coupled with hydrazine 4a and the diastereomers
were separated by silica gel chromatography.15 Global
Scheme 7. Conformationally restricted deoxynegamycin analogs.
Reagents and conditions: (a) HATU, DIEA, DMF, 4a, 3 h, 0 ꢁC to rt
(70–90%); (b) PPh3, THF, H2O (65–80%); (c) H2, Pd–C, EtOAc; (d)
4 M HClin dioxane (quantitative).
removalof protecting groups under acid conditions
furnished the desired analogs 35e1 and 35e2. b-Amino
acids 16, 21, 24, 27a–c, 30a–c, and 33 were coupled with
hydrazine 4a and the resultant products were treated
with hydrochloric acid in dioxane to give 35d and 35f–n,
respectively.
In the synthesis of cyclohexyl-based deoxynegamycin
analog, reduction of aryl ring in the penultimate step is
the key reaction (Scheme 8). Thus, a three component
condensation of aldehyde (36a or 36b), malonic acid,
and ammonium acetate gave b-amino acids 38a and 38b
along with cinnamic acids 37a and 37b, respectively. The
reduction of nitro group in 38a under catalytic hydro-
genation conditions furnished diamine 39a. Acetyl
group in 38b was removed under acidic conditions to get
diamine 39b. Diamines 39a and 39b were protected with
Boc group then the resultant diBoc derivatives were
OH
Boc
NH
O
e - g
a - d
OH
NHBoc
33
N(Boc)
32
NH
31
2
2
Scheme 6. Synthesis of cyclohexane-based b-amino acid. Reagents and
conditions: (a) Boc2O, Et3N, THF (95%); (b) (ClCO)2, DMSO,
CH2Cl2, Et3N (94%); (c) H3C–PPh3ÆBr, KHMDS, THF (91%); (d)
LiHMDS, Boc2O, THF (99%); (e) ClSO2NCO, Et2O (5%); (f) Boc2O,
DMAP, MeCN (93%); (g) LiOH, THF/H2O (65%).

3106
B. Raju et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3103–3107
NH
phosphonoacetate under Wittig–Horner conditions to
2
B
A
CHO
COOH
B
B
A
a
give a,b-unsaturated ester. 1,4-Addition of a,b-unsatu-
rated ester with chiralilthium amide, derived from ( R)-
(+)-N-benzyl-a-methylbenzylamine and n-butyllithium,
gave the enatiomerically pure b-amino ester. Benzyl
protecting groups from b-amino ester were removed
under catalytic transfer hydrogenation conditions. The
resultant primary amine was protected with Boc group
and base hydrolysis of t-butylester gave b-amino acid
43. b-Amino acid 43 was coupled with hydrazine 4a, the
protecting groups removed under acidic conditions, and
the aromatic ring reduced under catalytic hydrogenation
conditions to produce the desired compound 44.
+
CO
H
2
A
37a
37b
38a. A = H, B = NO
2
36a. A = H, B = NO
36b. A = NHAc, B = H
b (38a)
or
c (38b)
2
38b. A = NHAc, B = H
Boc
O
O
NH
NH
2
B
A
N
B
A
N
H
OtBu
d, e
CO
H
2
40a. A = H, B = NHBoc
40b. A = NHBoc, B = H
39a. A = H, B = NH
39b. A = NH , B = H
2
f
2
O
O
NH
NH
2
2
B
A
N
B
A
N
CO H
2
g
N
H
N
H
CO H
2
41a. A = H, B = NH
41b. A = NH , B = H
42a. A = H, B = NH
2
2
42b. A = NH , B = H
2
2
Scheme 8. Synthesis of cyclohexane-based b-amino acids. Reagents
and conditions: (a) malonic acid, NH4OAc, EtOH, reflux, 24 h (70%);
(b) H2, Pd–C, MeOH, rt 50 psi, 16 h (100%); (c) 6 M HClreflux, 20 h
(99%); (d) (Boc)2O, Na2CO3, H2O, rt (5%); (e) hydrazine, HATU,
DIEA, DMF, 0 ꢁC to rt, 12 h (60%); (f) 4 M HClin dioxane, H 2O, rt
4 h (100%); (g) H2, PtO2, AcOH, H2O, rt, 50 psi (50%).
3. Results and discussion
Methods were developed to synthesize a number of
conformationally restricted b-amino acids. Incorpora-
tion of these b-amino acids into deoxynegamycin tem-
plate has resulted in structurally novel and
conformationally restricted analogs. These analogs were
evaluated in whole cell assays17 and as inhibitors of cell-
free protein synthesis.18 Deoxynegamycin showed good
antibacterialactivity ( E. coli, MIC ¼ 4–16 lg/mL; K.
pneumoniae, MIC ¼ 8 lg/mL) in the whole cell assay and
also good protein synthesis inhibition in cell-free protein
synthesis assay (IC50 ¼ 8.2 lM). However, to the best of
our knowledge there is no in vivo data reported for
deoxynegamycin in the literature. Therefore, we evalu-
ated deoxynegamycin in an E. coli murine septicemia
model19 and the efficacy was comparable to that of
negamycin (Deoxynegamycin: ED50 ¼ 4.8 mg/kg; Nega-
mycin ED50 ¼ 5.1 mg/kg). Thus, the combined in vitro
and in vivo antibacterialprofiel of deoxynegamycin
validates it as a good starting point for the synthesis of a
structurally novel analogs with improved properties.
coupled with hydrazine 4a using HATU as coupling
reagent to give 40a and 40b, respectively. Removal of
the protecting groups from 40a and 40b under acidic
conditions gave 41a and 41b, respectively. Reduction of
the aromatic ring of 41a and 41b gave the corresponding
cyclohexane derivatives 42a and 42b, respectively.
Reduction of pyridine ring in the penultimate step is the
key step in the synthesis of piperidine based deoxynega-
mycin analog.16 Hence, pyridine-3-carboxaldehyde
(Scheme 9) was condensed with the t-butyldiethy-l
Boc
NH
O
NH
O
O
2
N
CHO
N
OH
HN
N
OH
N
a - e
f - h
H
42
43
44
The conformationalconstraint introduced through a
double bond, as in analogs 35a, 35b, and 35c, in the N-
terminalregion of deoxynegamycin has resulted in either
complete loss or significant decrease in antibacterial
activity (Table 1). Although the activity of the alkene
analog 35c in cell-free protein synthesis is very similar to
that of deoxynegamycin, it has no antibacterialactivity
Scheme 9. Synthesis of piperidine-based b-amino acid. Reagents and
conditions: (a) (EtO)2P(O)CH2CO2tBu, NaH, DMF (99%); (b) (R)-
(+)-N-benzyl-a-methylbenzylamine n-BuLi, THF (88%); (c) 1,4-cyclo-
hexadiene, Pd–C (10%), AcOH (93%); (d) (Boc)2O, aq Na2CO3, THF
(84%); (e) 2 M NaOH in MeOH, rt (86%); (f) hydrazine, HATU,
DIEA, DMF, 0 ꢁC to rt (54%); (g) 4 M HClin dioxane, rt (96%); (h)
H2ÆPtO2, AcOH, 55 psi (71%).
Table 1. MICs17 and protein synthesis inhibition data (TC/TL)18
MIC (lg/mL)
Organisms/Compounds
1b
35a
35b
35c
35d
35e1
35e2
35f
35g
42a
256
42b
44
E. coli ATCC 25922
E. coli MG1655
16
8
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
64
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
128
64
128
64
>128
>128
>128
>128
>128
128
64
E. coli MG1655 tolC
K. pneumoniae ATCC13882
Enterobacter cloacae
ATCC35030
4
32
32
32
32
8
64
128
64
256
4
128
128
Pseudomonas aeruginosa PA01
Staphylococcus aureus
ATCC25293
64
64
>256
>256
>256
>256
>256
>256
256
256
>128
>128
>128
>128
>128
>128
256
128
>256
256
256
128
>128
>128
Streptococcus pneumoniae 1005
TC/TL IC50 (lM)
64
256
>80
256
>80
256
11
128
11
ND
>82
ND
1
ND
>82
128
>82
128
128
>128
8.2
10.7
13.2
6.6
Analogs 35h–n are inactive (MIC>256 lg/mL and IC50>80 lM).

B. Raju et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3103–3107
3107
Hori, M. J. Antibiot. 1972, 25, 685; (e) Uehara, Y.; Hori,
U.; Umezawa, H. Biochim. Biophys. Acta 1974, 374, 82; (f)
Uehara, Y.; Hori, U.; Umezawa, H. Biochem. Biophys.
Acta 1976, 447, 406; (g) Uehara, Y.; Hori, U.; Umezawa,
H. Biochem. Biophys. Acta 1976, 442, 251; (h) Kondo, S.;
Yamamoto, H.; Maeda, K.; Umezawa, H. J. Antibiot.
1971, 24, 732; (i) Kondo, S.; Iinuma, K.; Yoshida, K.;
Yokose, K.; Ikeda, Y.; Shimazaki, M.; Umezawa, H. J.
Antibiot. 1976, 29, 208; (j) Uehara, Y.; Hori, M.; Kondo,
S.; Hamada, M.; Umezawa, X. J. Antibiot. 1976, 29, 937;
(k) Kondo, S.; Yoshida, K.; Ikeda, T.; Iinuma, K.;
Honma, Y.; Hamada, M.; Umezawa, H. J. Antibiot.
1977, 30, 1137; (l) Streicher, W.; Reinshagen, H. Chem.
Ber. 1975, 108, 813; (m) Streicher, W.; Reinshagen, H.;
Turnowsky, F. J. Antibiot. 1978, 31, 725.
in the whole cell assay. Introduction of a cyclopropyl
ring adjacent to the N-terminalamine, as in anaol g 35d,
resulted in significant loss in whole cell antibacterial
activity although the activity of this analog in cell-free
protein assay is very similar to that of deoxynegamycin
(1b). On the other hand, introduction of cyclopropyl
ring adjacent to the internalamine ( 35e1, 35e2, and 35f)
has resulted in complete loss of antibacterial activity.
However, the cyclopropyl analog 33e2 is 8-fold more
active than deoxynegamycin in the cell-free protein
synthesis assay. The cyclohexyl analog 35n was inactive.
Insertion of five and six member ring in the N-terminal
region of deoxynegamycin resulted in analogs (35g, 42a,
and 42b), which are less active than deoxynegamycin
(1b). Piperidine-based analog 44 has no appreciable
antibacterialactivity in the whoel celal ssay but its
activity in cell-free protein synthesis assay is comparable
to that of deoxynegamycin. The conformationalcon-
straint introduced by insertion of arylor heteroaryl
rings (analogs 27a–c, 30a–c, 41a, and 41b) has resulted
in complete loss of antibacterial activity.
4. Curran, W. V.; Boothe, J. H. J. Antibiot. 1978, 31, 914.
5. Cole, D. C. Tetrahedron 1994, 50, 9517.
6. Muller, A.; Vogt, C.; Sewald, N. Synthesis 1998, 837, and
references cited therein.
7. Mancuso, A. J.; Swern, D. Synthesis 1981, 165.
8. Burton, D. J. Chem. Rev. 1996, 96, 1641.
9. Rodriguez, M.; Llinaries, M.; Doulut, S.; Heitz, A.;
Martinez, J. Tetrahedron Lett. 1991, 32, 923.
10. Donaldson, W. A. Tetrahedron 2001, 57, 8589.
11. Shioiri, T.; Ninomiya, K.; Yamada, S. J. Am. Chem. Soc.
1972, 94, 6203.
12. Yoshimura, J.; Yamaura, M.; Suzuki, T.; Hashimoto, H.
Chem. Lett. 1983, 1001.
13. Ye, T.; McKervey, M. A. Chem. Rev. 1994, 94, 1091.
14. Palomo, C.; Oiarbide, M.; Bindi, S. J. Org. Chem. 1998,
63, 2469.
15. Acid 19 is an inseparable mixture of diastereomers. Upon
coupling with the hydrazine derivative, the diastereomers
were separated by silica gel column chromatography.
Removalof protecting groups from higher Rf diastereo-
mer under acidic conditions afforded 35e1. Similar trans-
formation on lower Rf diastereomer resulted in 35e2. The
stereochemistry of these analogs at cyclopropane juncture
cannot be assigned by NMR due to the presence of
rotomers.
16. (a) Davies, S. G.; Ichihara, O. Tetrahedron: Asymmetry
1991, 2, 183; (b) Rico, J. G.; Lindmark, R. J.; Rogers, T.
E.; Bovey, P. R. J. Org. Chem. 1993, 58, 7948.
17. Microdilution MICs were determined in Mueller–Hinton
broth supplemented with 50% human serum and an
inoculum size of 5 · 105 cfu/mL.
Our earlier studies20 were indicative of an active-trans-
port mediated event for permeability of highly charged
molecules such as negamycin and related analogs across
the outer membrane of Gram-negative bacteria. In the
present study we were able to walk through the con-
formationalspace by the insertion of a set of confor-
mationally restricted b-amino acids in the N-terminal
region of deoxynegamycin. Among these analogs, 35e2
is 8-fold more active than deoxynegamycin (1b) in the
cell-free protein synthesis assay (Table 1). However, this
analog has no antibacterial activity in the whole cell
assay, suggesting that it may not be getting transported
efficiently into the bacterial cell. In conclusion, the
transport structure–activity relationship (SAR) may not
be in parallel with the target SAR for this class of
molecules. These observations may play a critical role in
the future discovery of new deoxynegamycin based
antibacterialagents.
18. Inhibition of protein synthesis was measured with a cell-
free transcription/translation coupled assay (E. coli S30
Extract System, Promega, Madison, WI) using pGEMbgal
as a DNA template. Compounds were pre-incubated with
the reaction mixture for 10 min prior to the addition of
template. After 1 h incubation, ONPG was added and
b-galactosidase activity was monitored at 420 nm.
19. In vivo efficacy: Mice were infected ip with approximately
1 · 105 cfu of E. coli ATCC25922. The compound was
administered iv at 1 and 5 h after infection. Survivalwas
monitored for 7 days and the ED50 calculated by nonlinear
regression. Ampicillin was used as a standard in this study
(ED50 ¼ 2.3 mg/kg).
References and notes
1. (a) Swartz, M. N. Proc. Natl. Acad. Sci. 1994, 91, 2420; (b)
Gold, H. S.; Moellering, R. C. N. Engl. J. Med. 1996, 335,
1445.
2. (a) Miesel, L.; Greene, J.; Blak, T. A. Nat. Rev. Genet.
2003, 4, 442; (b) Walsh, C. In Antibiotics: Actions, Origins,
Resistance; Walsh, C., Ed.; ASM: Washington DC, 2003,
and references cited therein.
3. (a) Hamada, M.; Takeuchi, T.; Kondo, S.; Ikeda, Y.;
Naganawa, H.; Maeda, K.; Okami, Y.; Umezawa, H. J.
Antibiot. 1970, 23, 170; (b) Mizuno, S.; Nitta, K.;
Umezawa, H. J. Antibiot. 1970, 23, 581; (c) Mizuno, S.;
Nitta, K.; Umezawa, H. J. Antibiot. 1970, 23, 589; (d)
Uehara, Y.; Kondo, S.; Umezawa, H.; Suzukake, K.;
20. Raju, B.; Mortell, K.; Anandan, S.; O’Dowd, H.; Gao, H.;
Gomez, M.; Hackbarth, C.; Wu, C.; Wang, W.; Yuan, Z.;
White, R.; Trias, J.; Patel, D. V. Bioorg. Med. Chem. Lett.
2003, 13, 2413.