8-amido-bearing pseudomycin B (PSB) analogue: Novel antifungal agents

Article abstract of DOI:10.1016/S0960-894X(00)00606-5

During the course of a structure-activity relationship (SAR) study on novel depsinonapeptide pseudomycin B, we synthesized a total of 12 8-amidopseudomycin analogues via standard two-step sequence from either ZPSB 2 or AllocPSB 3. A number of these amides exhibited good in vitro antifungal activities.

Full text of DOI:10.1016/S0960-894X(00)00606-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 123±126
8-Amido-Bearing Pseudomycin B (PSB) Analogue:
Novel Antifungal Agents
Yan-Zhi Zhang, Xicheng Sun, Douglas J. Zeckner, Roberta K. Sachs, William L. Current
and Shu-Hui Chen*
Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
Received 20 September 2000; accepted 23 October 2000
AbstractÐDuring the course of a structure±activity relationship (SAR) study on novel depsinonapeptide pseudomycin B, we syn-
thesized a total of 12 8-amidopseudomycin analogues via standard two-step sequence from either ZPSB 2 or AllocPSB 3. A number
of these amides exhibited good in vitro antifungal activities. # 2001 Published by Elsevier Science Ltd.
Introduction
site.4 To circumvent this irritation potential found with
PSB, we carried out rather extensive structure±activity
relationship (SAR) investigations on both the pseudo-
mycin side chain5 and the cyclic lipopeptide core structure6
in the hope of improving the toxicity pro®le of PSB by
means of modi®cation of the acid functions on residues 3
and 8. In a recent disclosure from this laboratory, we
reported a 3-imido and an intramolecular lactone bearing
(linking 8a-hydroxyl group and the acid function at resi-
due 3) derivatives of PSB that were devoid of in vitro
antifungal activity as well as tail vein toxicity.6 To con-
tinue the core modi®cation, we prepared a series of 8-
amido containing pseudomycin B analogues. These new
PSB analogues were designed based on the recent ®nd-
ings from Bruzzese et al., who reported that some
amido-derivatives of amphotericin B exhibited reduced
toxicity relative to the parent drug.7 In this commu-
nication, we wish to report the synthesis and pre-
liminary evaluation of these 8-amides 4±15 shown in
Figure 1.
The increasing incidence of systemic fungal infections in
hospitalized patients, coupled with the shortage of
e€ective and safe antifungal agents have stimulated
renewed research interests in search for broad spectrum
antifungal agents. Clinically, Candida albicans, Crypo-
tococcus neoformans and Aspergillus fumigatus are the
three major pathogens responsible for the life-threaten-
ing systemic fungal infections. Despite the severe side
e€ects or narrow spectrum of activity, amphotericin B
(AMB) and several triazole based drugs (e.g., ¯ucon-
azole) still remain as the limited available drugs for the
treatment of systemic fungal infections.1 Clearly, the
discovery and development of novel broad spectrum
and safe antifungal agents constitutes a key step
towards curing these fungal infections. Towards this
end, we were inspired by several reports by Strobel et
al.2 detailing the preliminary biological pro®les of pseu-
domycins, a novel class of depsinonapeptides produced
by Pseudomonas syringea in plants. Within the pseudo-
mycin family, pseudomycin B (PSB 1) was identi®ed as
the most potent member, which showed excellent activ-
ity against all strains of Candida as well as Crypto-
coccus.3,4 It also demonstrated weak activity against
Aspergillus fumigatus.3,4 In spite of its promising anti-
fungal activity, the development of natural product
pseudomycin B (PSB) as a therapeutic agent was
complicated by its irritation potential at the injection
Chemical Synthesis
Attempted regioselective synthesis of 8-amides from
either ZPSB 2 or AllocPSB 3 using HOBt/EDCI proto-
col was unsuccessful. In this case, almost equal amounts
of 3-amides, 3,8-diamides, along with 8-amides were
produced. Separation of the desired 8-amides from their
respective 3-amides proved to be nontrivial. After care-
ful survey of the peptide coupling reagents, we dis-
covered that replacement of HOBt/EDCI with PyBOP
*Corresponding author. Tel.: +1-317-276-2076; fax: +1-317-276-
5431; e-mail: chen_shu-hui@lilly.com
0960-894X/01/$ - see front matter # 2001 Published by Elsevier Science Ltd.
PII: S0960-894X(00)00606-5

124
Y.-Z. Zhang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 123±126
Figure 1. Structures of pseudomycin analogues.
Table 1. Yields and molecular weight of the 8-amidopseudomycin analogues 4±15
Product
Method of preparation
Yields (%)
Deprotection
Molecular formula
C51H88ClN13O18
Molecular weighta,b,c
Coupling
4
5
6
7
8
9
10
11
12
13
14
15
A
C
C
B
C
B
C
C
C
C
B
B
10
91
78
34
49
28
44
71
95
39
40
12
68
94
75
91
62
86
58
46
74
73
83
61
1207
1221
1235
1249
1247
1263
1261
1251
1278
1292
1278
1369
C
52H90ClN13O18
C53H92ClN13O18
54H94ClN13O18
C54H92ClN13O18
55H96ClN13O18
C
C
C55H94ClN13O18
C53H92ClN13O19
C
55H97ClN14O18
C56H99ClN14O18
54H92ClN13O20
C61H98ClN13O20
C
aMethod A: 2/PyBOP/Rink resin; TFA; H2/10%Pd/C in 1%HOAc/MeOH.
bMethod B: 2/PyBOP/DMF; H2/10%Pd/C in 1%HOAc/MeOH.
cMethod C: 3/PyBOP/DMF; Bu3SnH/PdCl2(PPh3)2/HOAc/THF.
Table 2. Proton NMR assignments for 1a and 5b
led to the expected regioselective 8-amidation. Follow-
ing this new route, a total of 12 8-amides 4±15 were
prepared from either 2 or 3. In all 12 examples listed in
Table 1, the desired 8-amides were isolated as the major
products along with their corresponding 3-amides.8 The
regioselectivity for 8-amidation over 3-amidation was
at least greater than 5:1. As indicated in Table 1, seven
8-amides (5, 6, 8, and 10±13) were prepared from
AllocPSB 3, whereas the remaining ®ve 8-amides (4, 7,
9, 14, and 15) were synthesized from ZPSB 2. It should
be mentioned that compound 4 was prepared using
Rink resin via solid-phase chemistry. In most cases, the
yields for coupling reactions ranged from 40 to 95%.
The low coupling yields obtained with 4 and 15 were
probably due to the bulky amines used in both cases.
The ®nal deprotection step was carried out using either
hydrogenation (for Cbz-protected analogues)9 or
Bu3SnH/Pd(0) mediated deallylation (for Alloc pro-
tected ones).10 As also shown in Table 1, satisfactory
mass spectra were obtained for all 8-amides prepared.
Along with PSB, the detailed proton assignments for
8-methylamide 5 were listed in Table 2. The structural
assignments of 5, 8, 12, and 14 were secured based on
the interactions between the 8-NHR protons with their
corresponding H8b protons observed in the ROSEY
experiments (see Fig. 1 for structures). The structures
for the remaining 8-amido PSB derivatives were
assigned in a similar fashion.
Positions
PSB 1
5
Positions
PSB 1
5
Residue 1
a
b1
b2
Residue 2
Residue 5
a
4.59
4.38
4.53
4.54
4.40
4.51
4.29
1.99
2.14
2.89
2.91
4.24
1.99
2.13
2.91
2.91
b1
b2
g1
a
b1
b2
g1
4.13
2.01
2.07
2.90
2.97
4.16
2.00
2.07
2.91
2.97
g2
Residue 6
a
b
4.27
3.92
1.18
4.32
3.91
1.16
g2
Residue 3
g
Residue 7
a
b1
4.55
2.82
2.87
4.63
2.85
2.85
b
6.53
1.70
6.36
1.70
g
Residue 8
NHC(O)Me
a
b
Residue 9
b2
Residue 4
a
b1
b2
g1
g2
d1
d2
e
Ð
4.96
4.75
2.66
4.86
4.60
4.13
1.75
1.78
1.26
1.32
1.53
1.56
2.84
4.20
1.72
1.78
1.30
1.35
1.55
1.56
2.85
a
b
g1
g2
4.87
4.31
3.44
3.50
4.86
4.32
3.45
3.52
Side chain
Side chain
20a
20b
30
2.25
2.36
3.86
2.26
2.40
3.90
40
1.38
ꢀ1.22
0.83
1.40
ꢀ1.23
0.83
50-130
140
a 1H NMR of 1 was recorded in acetonitrile-d3/D2O/CF3CO2D.
b 1H NMR of 5 was recorded in acetonitrile-d3/D2O.

Y.-Z. Zhang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 123±126
125
Table 3. In vitro and in vivo antifungal activity of novel 8-amidopseudomycin derivatives
MICa (mg/mL)
ED50 (ip) (mg/kg  4)
Tail vein (iv) assay
b
Compd.
R
C. albicans
C. neoform.
A. fumigatus
4
5
6
7
8
9
10
11
12
13
14
15
PSB
H
Me
Et
n-Pr
c-Pr
n-Bu
c-Bu
0.312
1.25
10
20
5.0
20
5.0
5.0
5.0
20
2.5
10
<1
0.156
1.25
5.0
0.312
1.25
1.25
0.625
1.25
1.25
0.08
1.25
0.078
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
Ð
Clean
Clean
Ð
Clean
Clean
Clean
Clean
Clean
Clean
Ð
14
>20
>20
>20
Ð
>20
Ð
>20
>20
2.4±7.2
CH2CH2OH
CH2CH2NMe2
(CH2)3NMe2
GlyOMe
PheOMe
Ð
Clean
Clean
Positive
0.625
aMIC: Lowest drug concentration required to inhibit 90±100% of visible fungal growth compared to controls.
bED50: Drug concentration required to cure 50% of fungal infection compared with untreated animals.
Biological evaluation
tail vein irritation while retaining excellent in vitro
antifungal activity.
In vitro assays
All 12 8-amides were tested against C. albicans (A26), C.
neoformans (M1 106), and A. fumigatus (WM1), three
major fungi responsible for systemic fungal infections.
As shown in Table 3, all 8-amides showed excellent
activity towards C. neoformans, with MIC values in the
range of <0.01 to 1.25 mg/mL. However, none of these
analogues exhibited better activity against this pathogen
than that achieved by PSB. Based on the results shown
in Table 3, it is also evident that all 12 8-amides exhib-
ited only weak activity against A. fumigatus. When these
newly synthesized 8-amides were assayed against C.
albicans, several trends can be gleaned from the data
accumulated so far: (1) The in vitro potency decreases
according to the following order: 4 (R=H) >5 (R=Me)
> 6 (R=Et) > 7 (R=n-Pr)=9 (R=n-Bu); 12 > 13; 14
(GlyOMe) > 15 (PheOMe). Evidently, better activities
were found with those bearing smaller alkyl groups. In
fact, compound 4 (R=H) was identi®ed as the most
potent analogue within this series, being 2-fold more
potent than PSB 1. (2) c-Alkyl amides 8 and 10 dis-
played better activity than their corresponding n-alkyl
bearing counterparts 7 and 9, respectively. (3) Polar
termini bearing analogues 11±13 were found to be at
least 8-fold less active than the parent.
In vivo ecacy study
Most of the newly synthesized 8-amides were evaluated
for their in vivo ecacy against murine systemic Candi-
diasis. Experimentally, mice were ®rst infected by an
intravenous injection in the lateral tail vein. The testing
compounds (formulated in 4% hydroxypropyl cyclo-
dextrin, sodium acetate pH 7 bu€er and 1.75% dextrose)
were given to mice (six per group) four times at 0, 4, 24,
and 48 h post-infection. Infected sham-treated mice (10
animals) were dosed with the vehicle alone. Untreated
controls were moribund within 3±4 days post-infection.
The 50% e€ective dose (ED50) was determined using the
method of Reed and Muench.11 Judging from the data
listed in Table 3, it is rather disappointing to see that
none of the 8-amides tested showed comparable in vivo
ecacy to that achieved by pseudomycin B 1. The rea-
son for the lack of correlation between in vitro potency
and in vivo ecacy is currently unclear.
In summary, we completed the synthesis of a novel ser-
ies of 8-amido-bearing pseudomycin B analogues. These
analogues displayed very di€erent in vitro activity, ran-
ging from 2-fold more potent to 30-fold less potent than
the parent compound. It is encouraging to note that the
8-NH2 containing analogue 4, the bioisostere of PSB 1,
was found to be more potent than the parent yet devoid
of tail vein irritation. On the basis of our testing results,
it is also evident that the formation of a new amide
bond at the hydroxylated aspartic acid (residue 8) was
bene®cial for improving toxicity pro®le, yet detrimental
to in vivo ecacy.
Tail vein irritation
Selective members of the novel 8-amides were evaluated
in the tail vein toxicity assay in vivo. In this experiment,
the testing compounds (formulated in 5% dextrose and
sterile water) were given to mice (two per compound)
with a single intravenous injection at 75, 50, and 25
mg/kg. Following dosing, mice were observed closely
for clinical signs of histamine induced pathology. As
brie¯y outlined in Table 3, in sharp contrast to the
parent, all 10 8-amides evaluated (4, 5, 7, 8, 9, 10,
11, 12, 14, and 15) failed to induce tail vein irrita-
tion. It is particularly interesting to note that com-
pound 4, the bioisostere of the parent, was devoid of
Acknowledgements
We would like to thank R. Boyer and Dr. J. Paschal for
NMR support. We are also indebted to Drs. M.
Rodriguez, J. Munroe and B. Laguzza for their support
and encouragement.

126
Y.-Z. Zhang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 123±126
5. Jamison, J.; Levy, S.; Sun, X.; Zeckner, D.; Current, W.;
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