Discovery, SAR, synthesis, pharmacokinetic and biochemical characterization of A-192411: A novel fungicidal lipopeptide-(I)

Article abstract of DOI:10.1016/S0960-894X(02)00978-2

The echinocandin class of cyclic lipopepetides has been simplified to discover potent antifungal compounds. Namely A-192411 shows good in vitro activity against common pathogenic yeasts and has an acceptable safety window in vivo. Discovery, limited SAR, synthesis, biochemical and pharmaco-dynamic profiles of A-192411 are presented.

Full text of DOI:10.1016/S0960-894X(02)00978-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 489–493
Discovery, SAR, Synthesis, Pharmacokinetic and Biochemical
Characterization of A-192411:A Novel Fungicidal Lipopeptide-(I)
Weibo Wang,* Qun Li, Lisa Hasvold, Beth Steiner, Daniel A. Dickman, Hong Ding,
Akyio Clairborne, Hui-Ju Chen, David Frost, Robert C. Goldman, Kennan Marsh,
Yu-Hua Hui, Brian Cox, Angela Nilius, Darlene Balli, Paul Lartey,
Jacob J. Plattner and Youssef L. Bennani*,y
Infectious Diseases Research, Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064, USA
Received 4 December 2001; accepted 8 October 2002
Abstract—The echinocandin class of cyclic lipopepetides has been simplified to discover potent antifungal compounds. Namely
A-192411 shows good in vitro activity against common pathogenic yeasts and has an acceptable safety window in vivo. Discovery,
limited SAR, synthesis, biochemical and pharmaco-dynamic profiles of A-192411 are presented.
# 2002 Elsevier Science Ltd. All rights reserved.
Human fungal infections have come to prominence
during the past two decades due to an increase in
patients with HIV-infection, chemotherapy-induced
neutropenia; organ transplantation, hemodialysis or
extensive use of broad-spectrum antibiotics and gluco-
corticosteroids.1,2 The echinocandin family of natural
hexacyclic-lipopeptides has been of interest in anti-
fungal research over the past three decades due to their
fungicidal profiles. Increased interest became apparent
during the early-mid eighties after the delineation of
their mode of action as b-1,3-glucan synthetase inhibi-
tors. This enzyme is part of a membrane bound protein
complex that utilizes UDP-glucose as a substrate to
produce oligomeric glucose, which is further processed
by a variety of enzymatic steps to mesh the fungal cell
wall. Inhibition of such a process induces cell wall break-
age, leads to cellular lysis and death.3 We, and others,
have recently reported structure–activity relationship
studies, syntheses, in vitro and in vivo antifungal eval-
uation of a number of cyclic hexapeptides with simplified
structures, superior stability and water solubility as com-
pared to the natural product Echinocandin B.4 As the
search of such agents continues, we present in the next
two papers, the discovery path to, and synthesis of
A-192411 (1, Table 1), a structurally simplified cyclic
hexapeptide with acceptable in vitro5 and in vivo pro-
files (following paper).
Our previous lead compound: A-199930 (Fig. 1) showed
in vitro fungicidal activity on several Candida strains in
the range of 0.2–2 mg/mL and in vivo efficacy in a mouse
chronic candidiasis model with ED50 <1.25 mg/kg
(100% cure and 90% survival rates) and ED50 <0.6
mg/kg in a mouse acute infection setting. However,
further safety evaluations of A-199930 in rats showed
NOEL <0.1 mg/kg. The main side effect was a ꢀ50%
drop in mean arterial pressure at low infusion con-
centrations. While such an event was not observed in
the anesthetized dog during CV safety study, A-199930
proved to be unsafe at low doses in cyn. Monkey. The
cardiovascular deficiency of A-199930 could be blocked
in vivo by the presence of mepyramine, an H1-antago-
nist. The increased levels of histamine release upon drug
treatment were traced to a mast cell degranulation
effect, which, was dose-dependently blocked by both
benzylalkonium chloride and pertussis toxin. A hista-
mine-radioligand-immunoassay was established and
several hundred cyclic hexapeptides were screened for
their ability to induce histamine release from mast cells.
Limited SAR could be established from such an assay:
compounds with high proximal positive charge density
(i.e., 4-guanidino, lysine; e.g., A-199930 and several
related derivatives) showed high histamine-releasing
*Corresponding author. Fax:+1-216-361-9495; e-mail: ybennani@
athersys.com
yPresent address: Department of Medicinal Chemistry, Athersys Inc.,
3201 Carnegie Avenue, Cleveland, OH 44115-2634, USA.
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00978-2

490
W. Wang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 489–493
Table 1.
Organism
Compd
MIC (mg/mL)
1
2
3
4
5
6
7
8
9
10
11
Candida albicans ATCC 10231
Candida albicans 579A
Candida albicans CCH 442
Candida albicans ATCC 38247
Candida albicans ATCC 62376
Candida zeylanoides NRRL Y-112
Candida kefyr ATCC 28838
Torulopsis glabrata ATCC 15545
Cryptococcus albidus ATCC 34140
Candida albicans CAF-2
0.2
0.2
0.2
0.2
0.39
0.2
0.2
0.2
0.1
0.78
0.39
0.78
0.78
0.39
1.56
0.78
1.56
6.25
0.39
6.25
0.78
0.39
0.78
0.39
0.78
3.12
0.78
0.78
6.25
0.1
0.78
0.78
0.78
0.39
0.39
1.56
1.56
1.56
0.39
0.2
0.2
0.39
0.2
0.39
1.56
0.39
1.56
0.78
0.78
0.78
0.78
0.78
0.78
0.78
0.2
0.2
0.1
0.2
0.2
0.2
1.56
1.56
0.39
0.39
0.39
0.78
0.39
0.2
1.56
1.56
25
0.1
25
0.1
0.1
0.1
0.2
0.1
0.1
1.56
0.2
0.01
0.01
0.2
0.1
0.1
0.78
0.2
3.2
0.1
0.2
0.2
0.39
1.56
1.56
12.5
0.2
0.39
1.56
1.56
12.5
0.2
12.5
0.1
12.5
25
0.1
25
12.5
0.1
25
12.5
0.1
3.12
6.25
0.1
1.56
Aspergillus niger ATCC 16404
6.25
6.25
0.2
6.25
Figure 1.

W. Wang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 489–493
491
potency. Overall, compounds showing the best in vitro
and in vivo antifungal profiles also turned out to be the
most potent histamine-releasing agents. From these
studies, we found A-192411 (compound 1) to possess
acceptable in vitro, in vivo and CV safety profiles (Fig.
1). MIC data, generated using NCCLS method, are
presented on a small subset of derivatives around com-
pound 1, Table 1.
NaOH to give the corresponding pentyl ether in 90%
yield. The latter underwent palladium mediated Suzuki-
coupling with 4-borono-benzoic diacid (Pd(PPh3)4,
DMF-water, 100 ꢁC) to give the desired side chain C
(Fig. 1) in 82% yield.7
Final couplings
Tripeptides A (17) and B (20) were first coupled at the
hydroxyproline and homotyrosine sites to give the cor-
responding linear hexapeptide in 57% yield (i) Tripeptide
B:diethyl amine (DIEA), CH3CN; (ii) Tripeptide A, CMC-
HOBT; (iii) DIEA, CH3CN; (iv) LiOH, THF–water; and
(v) DPPA-NaHCO3-DMF]. The latter underwent suc-
cessive N-Fmoc deprotection (ornithine), ester hydro-
lysis (azidoproline) and ring-cyclization to the
corresponding peptide 21 in 60% overall yield. Azide
reduction and guanidylation proceeded to give the cor-
responding bis-Cbz-guanidine in 85% overall yield,
Scheme 3. N-Boc removal, under TFA conditions, fol-
lowed by N-acylation with the terphenyl side chain C and
hydrogenation to remove all three N-Cbz groups, in a
near quantitative yield, gave A-192411 as its acetic acid
salt after HPLC purification. This convergent synthesis
(27 steps) was used to prepare multi-gram quantities of
the antifungal agent A-192411 suitable for in vitro and in
vivo preclinical evaluation and characterization.
While most cyclic peptides maintained relatively inter-
esting potencies as anti-microbial agents (Table 1, 3, 9,
and 11), their assessment in mast cell-histamine release
directed us towards compound 1 as a preferred entity
for further in vivo characterization. A-192411 contains
two novel amino acids and a lipophilic side chain
attached at the l-ornithine a-amino position. This com-
pound was synthesized through a convergent approach
consisting of couplings between two tripeptides A and B
and the 400-O-pentyl terphenyl carboxylate side chain C;
(Fig. 1). Previous syntheses of Echinocandin B were
reported by the Ohfune and Evans groups.6
Tripeptide A contains two unnatural amino acids, an
aryl-N-glycine homotyrosine and the 4-(S)-azido-pro-
line (Fig. 1). The aryl-N-glycine homotyrosine moiety
14 was prepared from the reaction of 2-chloroanisole 12
with (l)-N-methylcarbamoyl aspartic anhydride, under
Friedel–Craft’s conditions, to give the corresponding
amino acid in 88% yield. Ketone-hydrogenation affor-
ded the desired homotyrosine in 90% yield (not shown).
The resulting N-methyl carbamate-amino acid (AA) was
further treated with HBr and reprotected as the
N-Fmoc-AA 13, obtained in 90% yield. The latter
underwent ortho-nitration (70% yield) followed by
hydrogenation and N-acylation with Cbz-protected gly-
cine afforded precursor 14 in 68% yield, (Scheme 1).
A-192411 showed solubility >3 mg/mL is water, and
0.77 mg/mL phosphate buffered saline. The measured
octanol:water partition coefficient (logD) is 2.45 and
2.47 at pH 6.5 and 7.4, respectively. The high aqueous
solubility allowed ready formulation for iv delivery, as
was used in the preclinical animal studies. The in vitro
rat, dog and human plasma protein binding at 5, 10,
and 50 mg/mL of A-192411 acetate showed no apparent
gender-related differences observed for all species.
Plasma protein binding did not appear to be concen-
tration-dependent (range 5–50 mg/mL, in rats and in
humans) except in dogs, the mean plasma protein bind-
ing of A-192411 was lower (91.0%) at 5 mg/mL when
compared to the 10 or 50 mg/mL (98.9 and 99.6%,
respectively). The pharmacokinetic evaluation of
A-192411 was conducted in mouse, rat, dog and mon-
key at 1 mg/kg and is summarized in Table 2. In mon-
keys the plasma concentration profile was fit to a two
compartmental model, characterized by a terminal
elimination half-life of 3.3 h (range 2.2–5.9 h) after a 1
mg/kg intravenous dose. The terminal phase volume of
distribution of A-192411 was 0.9 L/kg (range 0.6–1.3 L/
kg). A-192411 had a plasma clearance of 0.17 L/h kg
(range 0.11–0.26 L/h kg). In dogs, it was characterized by
a long terminal elimination half-life of 30.4 h (range 23.1–
36.9 h), with a terminal phase volume of the distribution
Separately, 4-(R)-hydroxyproline methyl ester 15,
which, was used in the preparation of both tripeptides A
and B, was coupled to (l)-N-Boc-threonine to give
dipeptide 16 in 98% yield. The latter underwent hydro-
lysis, N-Boc deprotection (84% yield) and coupling to
(L)-N-a-Boc-e-Fmoc-Ornithine to give tripeptide B (17)
in 50% yield, (Scheme 2). Similarly, ester 15 was N-Boc-
protected and treated, in a one-pot reaction, with
methanesulfonyl chloride followed by sodium azide, to
give the desired derivative 18 in 89% yield. The latter was
N-deprotected in TFA and coupled to N-Boc-threonine to
give the corresponding dipeptide 19 in 90% yield. Sub-
sequent TFA deprotection and coupling to ortho-N-glycyl
homotyrosine moiety 14 (Scheme 1), afforded tripeptide A
(20) in an overall yield of 72–82%. The terphenyl side
chain was prepared as follows: 4-(40-bromophenyl)-phenol
was O-alkylated with iodopentane in the presence of
Scheme 1. (a) AlCl3, (L)-N-methylcarbamoyl Asp. Anhydride; (b) H2, Pd/C; (c) (i) HBr/AcOH; (ii) FmocOSu; (d) NaNO2, AcOH; (e) (i). H2, Pd/C;
(ii) Z-GlyOSu, THF.

492
W. Wang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 489–493
Scheme 2. (a) Boc-Thr-OH, EDCI-HOBT; (b) (i) 50% TFA–DCM; (ii) LiOH–THF–water: (iii) Boc-Orn(Fmoc)-Osu; (c) (i) (Boc)2O; (ii) MsCl,
Et3N; (iii) NaN3; (d) (i) 50% TFA–DCM; (ii) Boc-Thr-OH, EDC-HOBT; (e) (i) 50% TFA-DCM; (ii) 14, EDC-HOBT.
Scheme 3. (a) (i) PPh3, THF–water; (ii) ZNHC(=NHZ)-SMe, HgCl2, Et3N, DMF; (iii) 50% TFA-DCM; (b) Side chain C or (16); DIPEA, DMF;
(c) H2, Pd(OH)2/C, AcOH, EtOH.
Table 2. Pharmacokinetic evaluation after a 1 mg/kg iv dose in
mouse, rat, dog and monkey
substantially different in different animals species (Table
2). The plasma elimination half lives following a 1 mg/
kg intravenous dose were 3.3, 7.4, 11.5, and 30.4 h for
monkey, rat, mouse, and dog, respectively. Bioavail-
ability from a single ip-dose of A-192411, in mouse, was
constant over the 1.25–20 mg/kg dose range. There was
no apparent rapid increase in plasma histamine levels in
monkeys following an iv dose of A-192411, with the
levels remaining low throughout the sampling interval.
Species
t1/2
(h)
Vv
(L/kg)
Vab
(L/kg)
AUC0-1
Clp
(L/hꢂ kg)
(mgꢂ h/mL)
Mouse
Rat*
Dog
11.5
7.4
30.4
3.3
0.4
0.6
0.6
0.2
1.0
2.2
1.3
0.9
16.84
11.66
36.39
6.55
0.06
0.14
0.03
0.17
Monkey
of 1.3 L/kg (range 0.9–1.7 L/kg) and a plasma clearance
of 0.03 L/hꢂ kg. Similarly, it was characterized by a
relative long terminal elimination half-life of 11.5 h after
intravenous dosing. The terminal phase volume of the
distribution was 1.0 L/kg with a plasma clearance of
0.06 L/hꢂ kg. Elimination half-lives, after ip or iv doses,
were similar with values of 10.5, 13.6, and 13.2 h for
1.25, 5, and 20 mg/kg ip doses, respectively. Both Cmax
and AUC increased proportionally with the increase in
dose over the range of 1.25–20 mg/kg. In conclusion,
the pharmacokinetic characteristics of A-192411 were
A-192411 inhibits the synthesis of fungal b-1,3-glucan, a
major polymer of the fungal cell wall both in vitro and
in vivo. The IC50 for inhibition of glucan synthesis in
vitro is 10–20 mg/mL, depending on the assay prepara-
tion conditions (Fig. 2). Glucan synthesis is pre-
ferentially inhibited over other macromolecular
pathways when whole cells are treated with A-192411.
Formal lysis of cells occurred following drug addition,
as evidenced by the release of high molecular weight
RNA from C. albicans (Fig. 2). The ‘kill-kinetics’ and
extent of cellular lysis parallel loss of viability (Fig. 3).

W. Wang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 489–493
493
and amino groups) potently induced histamine release
from mast cells and lead to drop in mean arterial pres-
sure. We have selected 1 for further in-vivo character-
ization (see following manuscript) based on its in-vitro
anti-fungal activity and its safety profile in both rodents
and monkeys.
Acknowledgements
We would like to thank Dr. Larry Klein and his team
for their support.
References and Notes
1. (a) Alexander, B. D.; Perfect, J. R. Drugs 1997, 54, 657. (b)
Klepsner, M. E.; Ernst, E. J.; Pfaller, M. A. Trends Microbiol.
1997, 5, 372.
Figure 2. Inhibition of glucan synthesis.
2. (a) Fostel, J. M.; Lartey, P. A. Drug Dev. Today 2000, 5, 25.
(b) Bossche, H. V. Expert Opin. Ther. Patents 2002, 12, 151.
3. Turner, W. W.; Rodriguez, M. J. Curr. Pharm. Des. 1996, 2,
209.
4. (a) Abruzzo, G. K.; Flattery, A. M.; Gill, C. J.; Kong, L.;
Smith, J. G.; Pikounis, V. B.; Balkovec, J. M.; Boufard, A. F.;
Dropinski, J. F.; Rosen, H.; Kropp, H.; Bartizal, K. Anti-
microbial Agents Chemother. 1997, 2333. (b) Jamison, J. A.;
LaGrandeur, L. M.; Rodriguez, M. J.; Turner, W. W.; Zeck-
ner, D. J. J. Antibiot. 1998, 51, 239. (c) Klein, L. L.; Li, L.;
Chen, H.-J.; Curty, C. B.; DeGoey, D. A.; Grampovnik, D. J.;
Leone, C. L.; Thomas, S. A.; Yueng, C. M.; Funk, K. W.;
Kishmore, V.; Lundell, E. O.; Wodka, D.; Meulbroek, J. A.;
Alder, J. D.; Nilius, A. M.; Lartey, P. A.; Plattner, J. J. Biorg.
Med. Chem. 2000, 8, 1677. (d) Groll, A. H.; Walsh, T. J.
Expert Opin. Investig. Drugs 2001, 1545. (e) Klein, L. L.; Li, L.
Curr. Pharm. Des. 1999, 5, 57.
5. Nilius, A. M.; Raney, P. M.; Hensey-Rudolff, D. M.;
Wang, W.; Li, Q.; Flamm, K. R. Antimicrob. Agents Che-
mother. 2000, 44, 1242.
Figure 3. Lysis of Cand. Alb. By 3H-RNA release.
6. (a) Kurokawa, N.; Ohfune, Y. J. Am. Chem. Soc. 1986,
108, 6043. (b) Evans, D. A. A.; Weber, A. E. J. Am. Chem.
Soc. 1987, 109, 7151.
7. Debono, M.; Turner, W. W.; LaGrandeur, L.; Bur-
khardt, F. J.; Nissen, J. S.; Nichols, K. K.; Rodriguez,
M. J.; Zweifel, M. J.; Zeckner, D. J.; R. S. Gordee,
R. S.; Tang, J.; Parr, T. R., Jr. J. Med. Chem. 1995, 38,
3271.
In conclusion, we have described the path to select
A-192411 from SAR studies on simplified echinocandin-
like motifs. This process was guided by unexpected species-
specific toxicological findings revolving primarily around
cardiovascular deficiencies. We have found that hex-
acyclic-lipopeptides with clustered basic sites (guanidine