Synthesis and evaluation of novel pseudomycin side-chain analogues. Part 3

Article abstract of DOI:10.1016/S0960-894X(01)00627-8

To increase the therapeutic utility of C-18 side-chain bearing pseudomycin analogue 2, we prepared additional analogues and prodrugs of 2 containing further modifications at various positions within its core structure. Each of the newly synthesized deriva

Full text of DOI:10.1016/S0960-894X(01)00627-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 3055–3059
Synthesis and Evaluation of Novel Pseudomycin Side-Chain
Analogues. Part 3
Xicheng Sun,y Yan-Zhi Zhang,{ Doug Zeckner, William Current and Shu-Hui Chen*
Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
Received 15 August 2001; accepted 10 September 2001
Abstract—To increase the therapeutic utility of C-18 side-chain bearing pseudomycin analogue 2, we prepared additional analogues
and prodrugsof 2 containing further modifications at various positions within its core structure. Each of the newly synthesized
derivatives( 10–15) exhibited reduced tail vein toxicity relative to the parent compound. Some of the new pseudomycin derivatives
(e.g., 14) also showed improved in vivo antifungal activity relative to its corresponding parent compound. # 2001 Elsevier Science
Ltd. All rightsreserved.
Introduction
carried out rather extensive SAR modifications at both
the hydrophilic core structure4a and the lipophilic side-
chain portion.4b,4c Towardsthe latter goal, we ysnthe-
sized and evaluated many types of side-chain analogues
of pseudomycin B.4b,4c Based on the in vitro antifungal
activity data obtained with these newly prepared PSB
side-chain analogues, two C-18 aliphatic side-chain
bearing analogues( 2 and 3) exhibited improved activity
(up to 8-fold) against A. fumigatus relative to PSB 1 (see
The rising incidence of serious fungal infections (e.g.,
systemic fungal infection), especially in the immuno-
compromised patients, attests to the need for more
effective therapies.1 All current agents have some ser-
ious liabilities: inadequate spectrum of activity (e.g.,
fluconazole), limited dosage forms, narrow therapeutic
window (e.g., amphotericin B), and rapid emergence of
fungal resistance. Thus the search for new agents, par-
ticularly those with a novel mode of action, continues to
attract more and more attention from the medical com-
munity. These efforts led to the discovery of several new
triazole based antifungal agents (e.g., voriconazole,2a
posaconazole,2b BMS-207147,2c etc.) aswell asesveral
cyclic peptide based novel agents such as aur-
eobasidins,2d echinocandin,2e FR901469,2f and recently
disclosed pseudomycins.3 Compared with the com-
monly used antifungal drug amphotericin B, pseudo-
Fig. 1). Furthermore, compound
2 also showed
impressive MIC towards C. albicans (MIC=0.625 mg/
mL) and C. neoformans (MIC=0.01 ug/mL) without
tail vein toxicity being observed at the dose of ꢀ10 mg/
kg. Although slight discoloration and swelling in tail
vein irritation assay were observed with 2 at the top
dose (20 mg/kg), the level of tail vein irritation detected
with 2 wasbetter than that found with the parent PSB 1.
Despite its encouraging in vitro activity, compound 2
exhibited only marginal in vivo activity in the mouse
disseminated Candidiasis model (@5 mg/kg dose). All
animalsreceiving 2 at higher dose (10 or 20 mg/kg) died
shortly after dosing. It is evident that in order to
translate its promising in vitro activity into in vivo
efficacy, the inherent irritation potential of 2 must be
circumvented.
mycin
B
1
(the most potent member within
pseudomycin family) demonstrated improved in vitro
and in vivo activity against Candida albicans and
Cryptococcus neoformans.3c
To further expand the therapeutic utility of pseudomy-
cin B (e.g., activity against Aspergillus fumigatus), we
Two main approacheswe used to improve the toxicity
profile of 2 utilized both analoging and prodruging
strategies. The rationales behind these approaches were
based upon our recent success achieved through core
modificationssuch as N-acylated prodrugs(e.g., 4–6)5,6
and 3-acid derivatizations(e.g., etser 7 and amides 8
*Corresponding author. Tel.: +1-317-276-2076; fax: +1-317-276-
1177; e-mail: chen_shu-hui@lilly.com
yCurrent address: Array Biopharma Inc., 1885 33rd St., Boulder, CO
80301, USA.
{Current address: Cubist Pharmaceticals, Inc., 24 Emily St., Cam-
bridge, MA 02139, USA.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00627-8

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X. Sun et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3055–3059
Figure 1. Structures of pseudomycin derivatives.
and 9).7,8 All six PSB prodrugs and analogues high-
lighted herein (4–9) exhibited impressive in vivo efficacy
in the murine Candidiasis model without adverse tail
vein irritation being detected (see Fig. 1). Thus, to take
advantage of these exciting findings, we decided to pre-
pare additional C-18 side-chain bearing derivatives (10–
15) in hopes of discovering novel pseudomycin deriva-
tives possessing improved overall biological and tox-
icology profilesin compaorins to the parent
pseudomycin B. Herein, we wish to report the synthesis
and preliminary evaluation of these new pseudomycin
derivatives( 10–15) (Fig. 2).
As shown in Scheme 3, two 3-amido pseudomycin ana-
logues 14 and 15 were synthesized from Cbz-protected
intermediate 16 according to the previously established
regioselective method.8 Thus, with the aid of 5 equiv of
EtPr2N, TBTU mediated coupling reaction between 16
and N,N-dimethylethyldiamine provided the desired 3-
amido intermediate 24 (60%). Compound 24 wasthen
converted, upon removal of Cbz protecting groups, to
the final product 15 in 87% yield. Following the same
two-step sequence, the requisite 3-cyclopropyl derivative
14 wasprepared, albeit in low overall yield. The low
coupling yield obtained with 23 wasin part due to the
formation of the 3- and 8-bisamide side product. With
the intention to prepare large amountsof 14 for in vivo
evaluation, we devised a more efficient synthesis as also
shown in Scheme 3. In this case, ZPSN 19 wasfirts
transformed into N-Boc protected derivative 25, which
wasnext converted to the 3-cyclopropyl amido nucleus
27 via N-Boc protected intermediate 26 in ꢁ28% over-
all yield. It should be mentioned that the formation of
unwanted bisamide was no longer an issue in this case.
The 3-monoamide 27 wasthen converted ms oothly to
the desired product 14 via previously published side-
chain N-acylation protocol.9
Chemical Synthesis
The preparation of three C-18 side-chain bearing pseu-
domycin prodrugs 10–12 isoutlined in Scheme 1. In this
event, direct N-acylation of 24 with the previously
reported prodrug linkers5,6 17a, 17b, and 18 afforded
the desired prodrugs 10 (45%), 11 (43%), and 12 (26%),
respectively. All three products isolated via preparative
HPLC showed proton NMR and mass spectra
consistent with the structures reported.
Scheme 2 shows the synthetic sequence employed
towards 3- and 8-bisester analogue 13. Treatment of a
methnol solution ZPSN 19 with catalytic amountsof
HCl gas(disolved in THF) 7 afforded the bis-OMe core
20 in high yield. Coupling of 20 with pre-activated
HOBt C-18 side-chain ester 21 provided the
corresponding adduct 22 (32%), which wasfurther
converted to the desired product 13 (52%) via standard
hydrogenation.
Biological and Tail Vein Toxicity Evaluation
All six newly synthesized C-18 side-chain bearing pseu-
domycin derivatives 10–15 were evaluated in the fol-
lowing three assays: (i) in vitro MIC against C. albicans,
C. neoformans and A. fumigatus, the three major fungi
responsible for systemic fungal infections; (ii) in vivo
against murine disseminated Candidiasis mouse model
Figure 2. Novel core analoguesand prodrugsof compound 2.

X. Sun et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3055–3059
3057
Scheme 1. Synthesis of N-acylated prodrugs 10–12.
Scheme 2. Synthesis of ester derivative 13.
Scheme 3. Synthesis of 3-amido derivatives 14–15. Conditions: (A) Boc2O/TEA/DMF, 65%; (B) TBTU/c-PrNH2/i-Pr2EtN/DMF, 28%; (C) TFA/
CH2Cl2, 95%; (D) 21/DMF+THF, 35%; (E) H2/Pd/C/1% HOAc/MeOH, 70%.

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X. Sun et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3055–3059
Table 1. In vitro and in vivo evaluation of pseudomycin derivatives 1–15
Entry
Compd
MIC (mg/mL)
In Vivo (mg/Kgꢂ4)
Tail vein toxicity (20 mg/kg)
C. albicans
C. neoformans
A. fumigatus
ED50 (anal.)/ED50 (PSB)
A
B
C
D
E
F
G
H
I
1
2
3
4
10
5
11
6
12
7
13
8
14
0.625
0.625
1.25
0.039
0.01
0.02
20
2.5-5.0
5.0
2.0–7.0
>20a
>20
11.4 (7.0)
>14
6.4 (2.8)
>20
7.8 (3.8)
>19
7.8 (2.8)
>20
Positive
Positive
Positive
>20
>20
>20
>20
>20
>20
2.5
>20
>20
>20
>20
>20
>20
0.01
1.25
<0.01
0.039
>20
>20
>20
>20
>20
>20
10-20
20
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
J
K
L
M
5.0
0.156
2.5-5.0
>20
5.0
<5.0 (6.6)
67% survival extension
@ 10 mg/kg doseb
<5.0 (4.5)
>20
N
O
9
15
0.156
5.0
<0.01
0.156
5.0
20
Negative
Negative
MIC: Lowest drug concentration required to inhibit 90–100% of visible fungal growth compared to controls. ED50: Drug concentration required to
achieve 50% survival of fungal infection compared with untreated animals.
aExtension of survival was recorded at low dose [5 (ꢁ20%) mg/kg].
bExtension of survival was observed at all doses [5 (37%), 10 (67%) and 20 (22%) mg/kg].
(ip); and (iii) in vivo tail vein toxicity assay in healthy
mice. The detailed testing protocols for these assays are
described in refs 6 and 8. The antifungal activity and tail
vein toxicity profilesof the reference compounds( 1–9)
are included in Table 1 for comparison. The ED50
valuesreported were calculated uisng the method of
Reed and Muench.10
but also a clean result in tail vein toxicity assay. When
evaluated against murine disseminated Candidiasis
model, 67 and 37% extension of survival was obtained
with mice receiving 14 at the dose of 10 and 5 mg/kg,
respectively. This level of in vivo activity observed with
14 isin fact better than that obtained with itsparent
analogue 2. Careful comparison of animal death pattern
observed with 2 and 14 revealed that 3-amido analogue
14 waslestoxic than 2. On the other hand, 3-dimethyl-
aminoethyl amide 15 possessed weaker in vitro potency
in comparison to 2. When tested in vivo, compound 15
wasdevoid of both tail vein irritation aswell as
antifungal efficacy.
Asdocumented in Table 1, compound 2 showed similar
MIC against C. albicans and C. neoformans to that
found with PSB 1. In addition, compound 2 exhibited
improved in vitro activity against A. fumigatus relative
to its parent compound. As also shown in Table 1, three
N-acylated pseudomycin B prodrugs 4–6 displayed
slightly reduced in vivo efficacy relative to the parent
PSB. However, unlike PSB 1, prodrugs 4–6 were found
to be free of tail vein irritation. Asexpected, all three C-
18 side-chain bearing prodrugs 10, 11, and 12 exhibited
reduced in vitro activity relative to analogue 2. In con-
trast to their corresponding parent 2, compounds 10–12
did not induce tail vein irritation. Disappointingly
however, none of the newly synthesized prodrugs 10–12
demonstrated significant in vivo efficacy.
In summary, we have synthesized six C-18 side-chain
bearing pseudomycin derivatives. These include four
prodrugs( 10–13) and two analogues( 14 and 15). Unlike
parent compound 2, all six newly synthesized derivatives
were free of tail vein irritation (at 20 mg/kg dose). In
contrast to the pseudomycin B prodrugs (4–6),5,6 three
N-acylated prodrugsof 2 (10–13) failed to demonstrate
significant antifungal activity in vivo. Consistent with
our previously finding with PSB,8 3-amidation on com-
pound 2 led to the 3-cyclopropyl amido analogue 14
with improved in vivo efficacy aswell asa reduction in
tail vein toxicity in comparison to its parent 2.
As listed in entries J and K, both 3,8-bisester derivatives
7 and 13 displayed excellent safety profile in the tail vein
irritation assay. When evaluated in the in vivo, unlike its
C-14 side-chain bearing counterpart 7 (ED50<5.0 mg/kg),
the newly prepared analogue 13 failed to show
significant in vivo efficacy.
Acknowledgements
The authorsare indebted to Drs. J. Munroe, B. Laguzza,
and J. McDonald for their support. We would also like
to thank Dr. M. Rodriguez for helpful discussion.
Asshown in entriesL and N, two 3-amido PSB analo-
gues 8 and 9 exhibited comparable in vitro and in vivo
activity to that observed with PSB, without adverse tail
vein irritation. More interestingly, 3-dimethylami-
noethyl-amdo bearing analogue 9 displayed improved in
vitro activity against Aspergillus than the parent. The 3-
cyclopropylamido analogues 14 showed not only
impressive balanced in vitro activity against all three
major fungi responsible for systemic fungal infections
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