Syntheses and antifungal activities of novel 3-amido bearing pseudomycin analogues

Article abstract of DOI:10.1016/S0960-894X(01)00101-9

As a result of our core SAR effort, we discovered a large number of 3-amido pseudomycin B (PSB) analogues (e.g., 4e LY448212 and 5b LY448731) that retain good in vitro and in vivo (IP) activities against Candida and Cryptococcus without inherent tail vein irritation. Several dimethylamino termini bearing 3-amides (e.g., 5b) also exhibited improved potency against Aspergillus in vitro. When evaluated in a two-week rat toxicology study, it was found that all animals receiving 4e (up to 75 mg/kg) were found to be normal. On the basis of these observations, we are convinced that it is possible to broaden the antifungal spectrum and improve the safety profile of pseudomycin analogues at the same time.

Full text of DOI:10.1016/S0960-894X(01)00101-9

Bioorganic & Medicinal Chemistry Letters 11 (2001) 903–907
Syntheses and Antifungal Activities of Novel 3-Amido
Bearing Pseudomycin Analogues
Yan-Zhi Zhang, Xicheng Sun, Douglas J. Zeckner, Roberta K. Sachs, William L. Current,
Jaswant Gidda, Michael Rodriguez and Shu-Hui Chen*
Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
Received 27 December 2000; accepted 5 February 2001
Abstract—As a result of our core SAR effort, we discovered a large number of 3-amido pseudomycin B (PSB) analogues (e.g., 4e
LY448212 and 5b LY448731) that retain good in vitro and in vivo (IP) activities against Candida and Cryptococcus without inher-
ent tail vein irritation. Several dimethylamino termini bearing 3-amides (e.g., 5b) also exhibited improved potency against Asper-
gillus in vitro. When evaluated in a two-week rat toxicology study, it was found that all animals receiving 4e (up to 75 mg/kg) were
found to be normal. On the basis of these observations, we are convinced that it is possible to broaden the antifungal spectrum and
improve the safety profile of pseudomycin analogues at the same time. # 2001 Elsevier Science Ltd. All rights reserved.
Due to the growing population of immunocompromised
patients, fungal infections are becoming a major medi-
cal concern.1 In particular, those systemic fungal infec-
tions (SFIs), defined as infections involving deep viscera
such as lung and liver, can cause serious life-threatening
diseases even in healthy humans. Statistically, Candida
albicans, Cryptococcus neoformans and Aspergillus
fumigatus account for more than 90% of SFIs. The
emergence of fungal pathogens resistant to current
therapies further compounds the dearth of antifungal
agents.2 Currently available antifungal drugs for treat-
ment of SFIs are limited to amphotericin B (AMB) and
a few azole based compounds (e.g., fluconazole). Unfor-
tunately, the utility of these drugs is further restricted
either by severe host toxicity or by lack of broad spec-
trum of activity against all three major fungi mentioned
above. In light of the urgent need for the development
of safe and effective drugs for the treatment of SFIs, we
became interested in the SAR modifications of pseudo-
mycins, a novel class of lipodepsipeptides reported
recently.3 In a series of recent publications from this
institution, we have disclosed biological and toxicity
profiles of pseudomycin B.4 In comparison with AMB,
PSB 1 exhibited better activity against C. albicans and
C. neoformans both in vitro and in vivo.3c In addition,
we have also reported on preliminary findings of pseu-
domycin side-chain5 and core SAR efforts including N-
acylated pseudomycin prodrugs6,7 and 8-amido pseu-
domycin analogues 3.8 Our results showed that several
PSB prodrugs indeed exhibited good in vivo efficacy
without inherent tail vein toxicity and long term end
organ toxicity. Encouraged by these promising results,
we decided to expand our core SAR effort by synthe-
sizing novel 3-amido PSB analogues 4, 5, and 6 as
shown in Figure 1. Our interest in preparing analogue 5
stemmed from the observations that certain vancomycin
related glycopeptides bearing basic dialkylamino ter-
mini exhibited improved antibacterial activity.9 In this
communication, we wish to report the synthesis, anti-
fungal activity and safety data of these 3-amido pseu-
domycin analogues (4–6).
The syntheses of 3-amido PSB analogues (4–6) were
accomplished using our recently developed chemoselec-
tive reactions. After many unsuccessful attempts, we
discovered that treatment of a DMF solution of ZPSB 2
(1 equiv) with 1.3 equiv of TBTU (O-benzotriazol-1-yl-
N,N,N0,N0-tetramethyluronium tetrafluoroborate) in the
presence of 6 equiv of EtPr2N afforded mainly the
desired Cbz protected 3-amide intermediates, which
were converted next, upon standard hydrogenation, to
the final 3-amido bearing PSB analogues. Following this
three-step sequence, we synthesized a total of 13 straight
alkyl chain bearing amides 4a–m, 10 basic termini and
five amino acid containing analogues (5 and 6). The
yields of the TBTU mediated 3-amidation reaction and
*Corresponding author. Tel.: +1-317-276; fax: +1-317-276-5431;
e-mail: chen_shu-hui@lilly.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00101-9

904
Y.-Z. Zhang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 903–907
Figure 1. Structures of pseudomycin B analogues.
subsequent deprotection are listed in Tables 1 and 2. It
should be mentioned that all of the 3-amides synthe-
sized showed satisfactory mass spectra (see data below).
0.15 ppm) of the 3b1 and 3b2 protons for 5b relative to
the parent compound. The detailed proton assignments
for 5b (LY448731) and PSB are listed in Table 3. The
structures of other 3-amido PSB analogues were
assigned in a similar fashion.
The structures of several newly synthesized 3-amides (4–
6) were confirmed by detailed NMR analyses. All NMR
spectra were obtained on a Bruker AMX550 spectro-
meter. The samples were dissolved in a 3:1 mixture of
acetonitrile-d3/deuterium oxide plus one drop of TFA-
d1. Careful inspection of the proton NMR spectra of the
3-amides (e.g., 5b) indicated an upfield shift (ꢀ0.10–
All 3-amido pseudomycin B analogues were evaluated
first in the following three assays: in vitro assay for MIC
determinations; in vivo efficacy against murine systemic
Candidiasis (ip) and in vivo tail vein irritation assay (iv).
Analogues with good in vivo efficacy and clean results
Table 1. Yields and molecular weights of 3-amido pseudomycin B analogues 4a–m
Compd
R
Coupling yield (%)
Deprotection yield (%)
Molecular formula
Molecular weight (salt free)
4a
4b
4c
4d
4e
4f
4g
4h
4I
4j
H
Me
Et
n-Pr
c-Pr
n-Bu
5a
37
36
52
90
83
77
44
C51H88ClN13O18
C52H90ClN13O18
C53H92ClN13O18
C54H94ClN13O18
C54H92ClN13O18
C55H96ClN13O18
C56H98ClN13O18
C56H98ClN13O18
C57H100ClN13O18
C58H102ClN13O18
C59H104ClN13O18
C60H106ClN13O18
C61H108ClN13O18
1207
1221
1235
1249
1247
1263
1277
1277
1291
1305
1319
1333
1347
41
b
99
b
n-Amyl
i-Amyl
n-Hexyl
n-Heptyl
n-Octyl
n-Nonanyl
n-Decyl
50
22
36
35
32
36
35
100
98
73
92
42
84
84
4k
4l
4m
aCompound 4a was synthesized using Rink amide resin (purchased from Advanced ChemTech).
bCompound 4f was provided initially by our external collaborators.
Table 2. Yields and molecular weights of 3-amido PSB analogues 5a–j and 6a–e
Compd
R
Coupling yield (%)
Deprotection yield (%)
Molecular formula
Molecular weight
5a
5b
5c
5d
5e
5f
5g
5h
5I
5j
6a
6b
6c
6d
6e
NH(CH2)2NH2
NH(CH2)2NMe2
NH(CH2)2Net2
NH(CH2)3NMe2
NH(CH2)3Net2
NH(CH2)4NMe2
NH(CH2)6NMe2
NH(CH2)7NMe2
NMe2
47
48
22
24
41
47
57
49
27
63
—
37
25
32
30
27
78
72
95
93
61
94
83
77
85
—
52
80
90
88
C53H93ClN14O18
C55H97ClN14O18
C57H101ClN14O18
C56H99ClN14O18
C58H103ClN14O18
C57H101ClN14O18
C59H105ClN14O18
C60H107ClN14O18
C53H92ClN13O18
C56H99ClN14O18
C54H92ClN13O20
C61H98ClN13O20
C58H96ClN15O20
C58H96ClN15O20
C58H101ClN16O20
1250
1278
1306
1292
1320
1306
1334
1348
1235
1292
1277
1369
1359
1350
1378
NMe(CH2)2NMe2
GlyOMe
PheOMe
HisOMe
LysOMe
ArgOMe

Y.-Z. Zhang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 903–907
905
from the tail vein irritation assay were selected for
further evaluation in the following testing: in vivo effi-
cacy against murine systemic Cryptococosis (ip) and
Aspergillosis (ip); dose elevation test in mice and two
week long term toxicity in rats (iv).
The MIC values reported were defined as the lowest
drug concentration required to inhibit 90–100% visible
growth compared to controls.
Protocol for in vivo efficacy against murine Candidiasis:
3-Amides of interest along with PSB and amphotericin
B (both used as positive control) were dosed four times
at 0, 4, 24, and 48 post-infection with testing analogue
given at 20, 10, and 5 mg/kg. The 50% effective doses
(ED50) were determined using the method of Reed and
Muench.10
Protocol for in vitro assay: all 3-amido analogues (4, 5,
and 6) and PSB (positive control) were evaluated
against the following three major fungi responsible for
systemic fungal infections. These are Candida albicans,
Cryptococcus neoformans, and Aspergillus fumigatus.
Protocol for tail vein irritation test: each testing com-
pound was dosed four times to mice (Outbred, male
ICR mice, ꢀ18–20 g weight) at 0, 24, 48, and 72 h. Mice
were observed closely for signs of irritation including
erythema, swelling, discoloration, necrosis and tail loss.
Table 3. Proton NMR assignments for PSB and 5b (in CD3CN/D2O/
TFA-d3)
Positions
PSB 1
448731
Positions
PSB 1
448731
Residue 1
a
b1
b2
Residue 2
a
b1
b2
g1
Residue 5
a
4.59
4.38
4.53
4.59
4.42
4.50
4.29
1.99
2.14
2.89
2.91
4.29
2.00
2.15
2.91
2.91
Subsequently, a few selected 3-amides were also eval-
uated against murine Cryptococosis (analogue dosed
with BID ꢁ4 at the doses of 5.0, 1.25, 0.312, and
0.078 mg/kg) and Aspergillosis (analogue dosed with
BID ꢁ3 at 50, 10, and 1 mg/kg) in vivo.
b1
b2
g1
4.13
2.01
2.07
2.90
2.97
4.16
2.01
2.07
2.91
2.98
g2
Residue 6
a
b
4.27
3.92
1.18
4.27
3.92
1.18
Protocol for dose elevation study: mice were treated
with a single injection (iv) of the testing compounds
(dissolved in 5% dextrose and sterile water) at the dose
of 50, 75, 100, and 125 mg/kg. Outbred, male ICR mice
(ꢀ18–20 g, Harlan Sprague–Dawley, Indianapolis, IN)
were used in this experiment. Two mice were included in
each group. Following dose, mice were observed closely
(for seven days) for clinical signs of histamine induced
pathology, including dyspnea, agitation, convulsions
and death.
g2
Residue 3
g
Residue 7
a
b1
4.55
2.82
2.87
—
—
—
4.56
2.66
2.77
3.42/3.52
3.16
2.80/2.81
b
g
6.53
1.70
6.53
1.71
b2
10a, 10b
20
40,50-Me
Residue 4
Residue 8
a
b
Residue 9
a
b1
b2
g1
g2
d1
d2
e
4.13
1.75
1.78
1.26
1.32
1.53
1.56
2.84
4.14
1.73
1.77
1.27
1.33
1.56
1.56
2.85
4.96
4.75
4.96
4.75
a
b
g1
g2
4.87
4.31
3.44
3.50
4.87
4.32
3.46
3.51
After careful reviewing of the data shown in Tables 4
and 5, we made the following observations: In vitro
Activity: (1) All straight alkyl chain bearing 3-amides
4a–m exhibited excellent activity against Cryptococcus
with MIC values ranging from <0.01–5.0 mg/mL. They
also displayed weak activities against Aspergillus with
MIC values around 20 mg/mL. Their activity towards
Side chain
Side chain
20a
20b
30
2.25
2.36
3.86
2.25
2.38
3.87
40
1.38
ꢀ1.22
0.83
1.39
ꢀ1.23
0.83
50-130
140
Table 4. In vitro, in vivo and tail vein toxicity of 3-amides 4a–m
Compd
R
MICa (mg/mL)
Tail vein toxicity
ED50 (ED50PSB)b (mg/kgꢁ4, IP)
C. albicans
C. neoformans
A. fumigatus
PSB
4a
4b
4c
4d
4e
4f
4g
4h
4i
—
H
Me
Et
n-Pr
c-Pr
n-Bu
0.625
2.5
5.0
0.625
1.25
0.156
5.0
0.312
0.312
5.0
20
20
0.01
<0.01
0.312
0.156
0.078
<0.01
<0.01
<0.01
<0.01
0.156
0.312
0.625
5.0
>20
10
>20
20
>20
20
20
>20
>20
>20
>20
>20
>20
>20
Yes
Yes
3.2–8.4
<.8 (4.5)
7.4 (4.5)
<.0 (4.2)
6.0 (8.4)
<5.0 (7.2)
5.9 (3.8)
8.5 (6.6)
10.9 (8.4)
>12 (3.2)
Toxic
Pat. yesc
Not tested
No
No
No
No
Yes
No
No
No
No
n-Amyl
i-Amyl
n-Hexyl
n-Heptyl
n-Octyl
n-Nonanyl
n-Decyl
4j
4k
4l
>20 (7.1)
>20
>20 (7.1)
20
>20
4m
1.25
No
aMIC values were defined as the lowest drug concn required to inhibit 90–100% visible growth compared to controls.
bED50: drug concentration required to achieve 50% survival of fungal infection compared with untreated animals.
cPartial normal/partial yes meaning: one normal, one abnormal.

906
Y.-Z. Zhang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 903–907
Candida varies according to the nature of alkyl groups.
Generally speaking, better activities against Candida
were obtained with analogues bearing smaller alkyl
groups. (2) All of the N0,N0-diMe(Et)alkyl-3-amides 5
listed in Table 5 showed excellent activity against Cryp-
tococcus. The activity of 5a–j for Candida ranged from
0.16 to 20 mg/mL. Importantly, several such 3-amides
(e.g., 5a, 5b, and 5f) demonstrated improved potencies
(ꢀ4-fold) against Aspergillus in comparison to the parent
(see Table 5). (3) Five amino acid containing 3-amides
6a–e showed modest to excellent activities against Can-
dida and Crypotococcus, respectively. When tested
against Aspergillus, these analogues exhibited similar
potencies to that observed with the parent (see Table 5).
bearing amide 6b displayed poor activity. Comparison
of the efficacy data obtained from 6b and 6d or 6e
seemed to indicate that better in vivo activities were
associated with those analogues bearing basic termini.
Tail vein irritation: (1) With the exception of 4a, 4b,
and 4h, most of the straight chain 3-amides prepared
were found to be clean in this assay (Table 4). (2) As
shown in Table 5, only two analogues within the
dialkylamino-termini amide series, 5a and 5f, were
capable of inducing tail vein irritation. The remaining
eight analogues passed this test. (3) It is encouraging
to note that three out of five amino acids containing
3-amides (6a, 6d, and 6e) were proved to be clean in
this experiment. The remaining two analogues (6b
and 6c) showed some improvement over the parent
PSB with respect to tail vein irritation potential (see
Table 5).
In vivo activity against Candidiasis: (1) Seven straight
chain alkyl bearing 3-amides listed in Table 4 (4b–h)
exhibited comparable in vivo activity against murine
Candidiasis. It is also evident that further extension of
alkyl chain beyond six carbons (e.g., 4j and 4k) was
detrimental to in vivo efficacy. (2) In contrast to the
trends observed with 4, all nine basic termini bearing 3-
amides (5a–h and 5j), regardless of the length of the
alkyl linker, demonstrated similar impressive in vivo
efficacy against Candidiasis (ED50 values <5.0 mg/kg).
(3) Within the amino acid series, four out of five analo-
gues (6a and 6c–e) showed good efficacy against Candi-
diasis (ED50 values <5.0 mg/kg). The bulky PheOMe
To summarize the data discussed so far, it is important
to point out that no clear correlation existed between in
vitro potency and in vivo efficacy. As shown in Table 5,
compound 5b (with excellent MIC against Candida)
possessed equivalent in vivo efficacy to 5f (with MIC
value >20 mg/mL for Candida). It is also evident that
many newly prepared 3-amides passed the primary in
vivo efficacy screening without inherent tail vein toxicity.
These include 4c–g, 5b–d, 5f–j, 6a, and 6d–f.
Table 5. In vitro, in vivo (Candidiasis) and tail vein toxicity of 3-amides 5a–j and 6a–e
Compd
R
MICa (mg/mL)
Tail vein toxicity
ED50 (ED50PSB)b (mg/kgꢁ4, IP)
C. albicans
C. neoformans
A. fumigatus
PSB
5a
5b
5c
5d
5e
5f
5g
5h
5i
—
(CH2)2NH2
(CH2)2NMe2
(CH2)2NEt2
(CH2)3NMe2
(CH2)3NEt2
(CH2)4NMe2
(CH2)6NMe2
(CH2)7NMe2
NMe2
0.625
10
0.156
10
10
20
20
5.0
10
10
5.0
1.0
1.25
0.312
1.25
5.0
0.01
>20
5.0
5.0
10
Yes
Yes
No
No
No
Yes
No
No
3.2–9.0
<5.0 (7.1)
<5.0 (4.5)
4.9 (3.5)
<5.0 (3.9)
<4.0
<5.0 (7.1)
<5.0 (3.5)
<4.0
<4.5 (5.4)
4.0 (3.2)
<5.0 3.8)
>15 (9.0)
<5.0 (3.2)
<5.0 (8.4)
<5.0 (5.4)
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.01
10
>20
5.0
20
>20
10
20
20
>20
20
No
No
No
5j
NMe(CH2)2NMe2
GlyOMe
PheOMe
6a
6b
6c
6d
6e
No
1.25
Pat. yesc
Pat. yesc
No
HisOMe
LysOMe
ArgOMe
<0.01
<0.01
0.01
>20
>20
No
aMIC values were defined as the lowest drug concd required to inhibit 90–100% visible growth compared to controls.
bED50: drug concentration required to achieve 50% survival of fungal infection compared with untreated animals.
cPartial normal/partial yes meaning: one normal, one abnormal.
Table 6. Additional efficacy and toxicity profiles of LY448212 (4e) and 448731(5b)
Compd
Cryptococosis
ED50 (mg/kg)a
Aspergillosis survival extension
Dose elevation
1 mg/kg
20 mg/kg
50 mg/kg
75 mg/kg
100 mg/kg
125 mg/kg
4e
5b
4d
6d
2.9
2.5
—
—
1.8
29%
23%
—
—
Not tested
Not tested
—
—
46%
Normal
Normal
Normal
Normal
Death
Pat. Normb
Death
Normal
Normal
Death
Death
Death
Death
Death
Death
Death
Death
Death
Death
Death
PSB 1
Not tested
aED50: drug concentration required to achieve 50% survival of fungal infection compared with untreated animals.
bPartial normal/partial yes meaning: one normal, one abnormal.

Y.-Z. Zhang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 903–907
907
Consequently, 4e and 5b were further evaluated in vivo
against Cryptococosis and Aspergillosis. As can be seen
in Table 6, both analogues tested demonstrated good in
vivo efficacy against Cryptococosis with ED50 values
ranging from 2.5 to 2.9 mg/kg. Although no significant
efficacy towards Aspergillosis was obtained with 4e and
5b, both analogues were found to be capable of extend-
ing survival time in animals infected with this fungus at
doses as low as 1 mg/kg.
and 6d: >15) than that observed with the parent. Fur-
thermore, 4e and 5b also demonstrated good activity in
the murine systemic Cryptococosis model. More impor-
tantly, 3-cyclopropylamide 4e passed our long term
toxicity test in rats (detailed results not shown). To take
all of the findings presented herewith as a whole, it is
evident that the desirable antifungal activity of pseudo-
mycin B analogues can be preserved without inherent
tail vein irritation and long term organ toxicity.
To further assess the safety profiles of 3-amides, a total
of 3-amides (4d, 4e, 5b, and 6d) were selected, on the
basis of tail vein irritation testing, for additional eva-
luation in the dose elevation study (see Table 6). The
positive control, PSB, was found to be safe only at the
dose of 25 mg/kg (data not shown). When PSB was
dosed at 50 mg/kg and higher, severe toxicity resulted.
In comparison, 3-amides 4e and 5b were found to be
safe at the dose of 50 mg/kg. More impressively, the
other two amides 4d and 6d passed this test at the dose
of 75 mg/kg.
Acknowledgements
The authors would like to thank M. Zweifel for the
scale-up synthesis of compound 4f. We are also
indebted to Drs. J. Munroe, B. Laguzza and J. McDo-
nald for their support and encouragement.
References
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as pradimicins.
In light of its good in vivo and tail vein toxicity profile,
4e was selected for two-week toxicity study in rats.
Towards this end, amide 4e was given to rats at doses
of 50 and 75 mg/kg for 14 days. To our satisfaction, all
drug treated animals (at highest dose) were found to
be normal at the end of this experiment. No clinical
observations were recorded. Careful analysis of blood
samples collected from those drug treated rats indicated
that blood chemistry was found to be normal also.
In conclusion, we succeeded in the preparation of three
kinds of 3-amido bearing pseudomycin analogues (4–6)
via a three-step sequence in a regioselective fashion. In
general, all of the newly synthesized 3-amides exhibited
excellent in vitro activity against Cryptococcus. Their
potencies towards Candida varied according to the nat-
ure of the amide linkages, with MIC values ranging
from 0.156 to 20 mg/mL. Several members of dialkyl-
amino termini bearing amides exhibited improved
potencies against Aspergillus, with MIC values at ꢀ5 mg/
mL. In addition to in vitro testing, we also evaluated all
of the 3-amides synthesized in the tail vein toxicity assay
and in vivo efficacy assay against Candidiasis. On the
basis of these testing results, we identified a number of
3-amides with good in vivo efficacy yet without tail vein
toxicity, such as 4d, 4e, 5b, and 6d. When tested in the
dose elevation study, all four 3-amides (4d, 4e, 5b, and
6d) demonstrated improved safety profiles (Therapeutic
Index value calculated for 4d: 12.5; 4e: >10; 5b: >10;
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