Synthesis and biological activity of novel macrocyclic antifungals: Modification of the tyrosine moiety of the lipopeptidolactone FR901469

Article abstract of DOI:10.1016/S0960-894X(01)00317-1

A series of tyrosine-modified derivatives of the macrocyclic lipopeptidolactone FR901469 have been prepared and evaluated for in vitro and in vivo antifungal activity and for hemolytic activity towards red blood cells. Compound 14 displayed significantly reduced hemolytic potential at 1 mg/mL and a comparable protective effect to FR901469 in a mouse candidiasis model.

Full text of DOI:10.1016/S0960-894X(01)00317-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1843–1849
Synthesis and Biological Activity of Novel Macrocyclic
Antifungals: Modification of the Tyrosine Moiety of the
Lipopeptidolactone FR901469
David Barrett,a,* Akira Tanaka,a Keiko Harada,a Etsuko Watabe,b Katsuyuki Makib
and Fumiaki Ikedab
aMedicinal Chemistry Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 2-1-6 Kashima, Yodogawa-ku,
Osaka 532-8514, Japan
bMedicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 2-1-6 Kashima, Yodogawa-ku,
Osaka 532-8514, Japan
Received 15March 2001; accepted 28 April 2001
Abstract—A series of tyrosine-modified derivatives of the macrocyclic lipopeptidolactone FR901469 have been prepared and eval-
uated for in vitro and in vivo antifungal activity and for hemolytic activity towards red blood cells. Compound 14 displayed sig-
nificantly reduced hemolytic potential at 1 mg/mL and a comparable protective effect to FR901469 in a mouse candidiasis model.
# 2001 Elsevier Science Ltd. All rights reserved.
Introduction
critical in order to improve the overall profile of this
natural product, since work on echinocandin B sug-
gested a relationship between hemolysis and toxicity.8,9
We have already reported some of our efforts on the
site-specific chemical modification of FR901469. In
particular, we have described selective direct modifica-
tion of the ornithine residue,10 replacement of the lac-
tone by an amide group,11 and complete replacement of
ornithine by a novel substituted glutamic acid residue.12
In the course of these studies, it became apparent that
the hemolysis associated with the natural product could
be reduced significantly by chemical modification whilst
maintaining the potent in vitro and, especially, in vivo
antifungal activity. Examination of the structure of 1
reveals a number of amino acid residues that are
As a part of our efforts to discover novel water-soluble
antifungal agents suitable for parenteral administration,
we recently described the isolation and antifungal
activity of the novel macrocyclic lipopeptidolactone
FR901469 (1) (Fig. 1).1,2 This compound belongs to a
new family of natural product fungal 1,3-b-glucan syn-
thase inhibitors,3 traditionally represented by the echi-
nocandins and papulacandins, but more recently
expanded by the discovery of arbocandins4 and enfu-
mafungin-type steroid derivatives.5 Scientists at Nippon
Roche recently described the discovery of aerothricins, a
series of lipopeptides closely related to FR901469.6,7
The validity of 1,3-b-glucan synthase as a clinically sig-
nificant antifungal target has recently been clearly
demonstrated and three compounds (caspofungin,
micafungin, anidulafungin) are in various late-stage
clinical trials.3 Indeed, caspofungin very recently
received FDA approval as a therapy for refractory
invasive aspergillosis.
As described earlier,1,2 FR901469 possesses potent
antifungal activity, but also displays a tendency to cause
lysis of red blood cells (100% at a concentration of
1 mg/mL). Reduction of this hemolysis was considered
*Corresponding author. Tel.: +81-6-6390-1285; fax: +81-6-6304-
5435; e-mail: david_barrett@po.fujisawa.co.jp
Figure 1.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00317-1

1844
D. Barrett et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1843–1849
potentially amenable to site-specific chemical modifica-
tion. A particularly attractive candidate for selective
and specific chemical modification is the tyrosine moi-
ety, owing to the presence of the phenol moiety, leading
to activation towards electrophilic aromatic substitution
reactions. Furthermore, the relatively high acidity asso-
ciated with the tyrosine hydroxyl group renders it
amenable to selective alkylation reactions with a variety
of alkylating reagents.
the carboxylic acid moiety with 1-tert-butoxycarbonyl-
ethylenediamine and removal of the BOC group affor-
ded 12. Compounds 11 and 13 were prepared from Z-
protected FR901469 (21) by alkylation, tert-butyl ester
deprotection, amidation and finally by hydrogenation to
remove the carbobenzyloxy protecting group, as out-
lined in Scheme 2. Compound 14 was prepared by
alkylation of nitrophenol 22 (an intermediate in the
preparation of 8), reduction, acylation and finally by
single step removal of all protecting groups.
Earlier work in the pneumocandin series of compounds
revealed the critical importance of the homotyrosine
phenolic hydroxyl group for 1,3-b-glucan synthase
inhibitory activity.13,14 In particular, removal of this
hydroxyl group by reduction or blocking as a phosphate
ester completely abolished enzyme activity. In the work
described in this paper, we aimed to examine whether
the presence of the tyrosine phenol group in FR901469
is required for antifungal activity and to investigate
whether tyrosine-specific modification can lead to novel
compounds with an improved hemolytic profile com-
pared to 1. We herein report the synthesis and evaluation
of a series of tyrosine-modified derivatives of FR901469.
All reactions and purifications were monitored by
reverse phase HPLC. Purifications were performed by
reverse phase ODS column chromatography and pro-
ducts obtained by pooling of the appropriate fractions,
adjusting to pH 3 with 1 N HCl, passage through a short
column of Amberlyst A-26 (chloride form) and freeze-
drying. All compounds were characterized by 1H NMR,
FAB-MS, IR and elemental analysis. Purity was assessed
by HPLC.
Biological Methods
In vitro antifungal activity
Chemistry
The minimum inhibitory concentration (MIC) values
shown in Table 2 were determined by the agar dilution
method using Sabouraud Dextrose agar, as described in
our earlier paper.10 MIC was read as the lowest con-
centration required to inhibit visible growth of the
organism. MICs in Table 3 were determined by the
broth microdilution method, according to NCCLS
M27-A guidelines.15
The novel tyrosine-modified derivatives prepared in this
work were obtained by the methods summarized in
Schemes 1 and 2. Chlorination at the ortho-position
adjacent to the tyrosine hydroxyl group was achieved
with sodium hypochlorite in aqueous acetic acid (46%).
Nitro derivative 3 was obtained by direct nitration with
sodium nitrite in aqueous acetic acid (75%).14 Reduc-
tion of 3 with sodium borohydride in the presence of
10% palladium on carbon in MeOH–AcOH–H2O
afforded amine 4 (39%). Conversion of acetate 5 to the
b-alanine derivative 7 was achieved by nitration, reduc-
tion under hydrogenolysis conditions, acylation with b-
alanine and deprotection of the tert-BOC group. Rela-
ted analogues 9 and 15–20 were prepared via the com-
mon intermediate 8, readily prepared from 1 via tert-
butoxycarbonyl-protected derivative 6. Nitration (78%)
and hydrogenation (77%) of 6 afforded 8 in good yield.
Acylation of 8 with various acids and acid derivatives
afforded the respective amide derivatives in moderate
yield (Table 1). These acylation reactions proceeded in
modest yields due to formation of varying amounts of
the corresponding bis-acylated species resulting from
acylation of the phenol hydroxyl group in addition to
the amino group. The desired mono-acylated inter-
mediates could be readily obtained by reverse phase
ODS column chromatography. Deprotection of pro-
tecting groups by stirring in neat TFA at room tem-
perature afforded the final antifungal agents as
amorphous solids.
In vivo antifungal activity
Disseminated candidiasis was induced in ICR mice by
intravenous inoculation of 0.2 mL of a Candida albicans
FP633 cell suspension via the lateral tail vein. A single
dose of each compound was administered sub-
cutaneously 60 min after challenge. ED50 was estimated
on the basis of survival at day 14 after challenge.
Hemolytic activity
A microtiter red blood cell (RBC) hemolysis assay was
used to determine the potential of compound to hemo-
lyze ICR mouse RBCs at 1 mg/mL, as described earlier.10
Results and Discussion
Introduction of a chloro or nitro group adjacent to the
tyrosine phenol group of 1 resulted in no significant
change in antifungal activity (2 and 3) (Tables 2 and 4).
In particular, nitro derivative 3 displayed comparable in
vivo efficacy in the candidiasis model. This result sug-
gests that the tyrosine phenol in 1 and the homotyrosine
phenol in the echinocandins may not have the same role
in the inhibition mechanism of glucan synthase. This
follows from results reported by Merck researchers, who
established that introduction of nitro to pneumocandins
The tyrosine hydroxyl-modified analogues 10–13 were
obtained as shown in Scheme 2. Compound 10 was
prepared in two steps from 6 by O-alkylation with tert-
butyl bromoacetate in the presence of K2CO3 (84%),
followed by deprotection with TFA (100%). Reprotec-
tion of the ornithine amino group as BOC, coupling of

Scheme 1. Synthesis of tyrosine-modified FR901469 derivatives (1).

Scheme 2. Synthesis of tyrosine-modified FR901469 derivatives (2).

D. Barrett et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1843–1849
Table 1. Acylation and deprotection reactions of amine 8
1847
Final compound
Reagent
Methoda
Acylation yield (%)
31
Deprotection yield (%)
81
9
A
15
16
B
—
34
20b
66
A
17
18
19
20
C
A
C
A
36
37
29
60
97
100
33
5
5
aA: Acid–HOBT–WSCD.HCl–DMF–rt; B: succinic anhydride–DMAP(cat)–DMF–rt; C: DCHA salt–EtNiPr2–HOBT–WSCD.HCl–DMF–rt
(DCHA, dicyclohexylamine).
bOverall yield from 8.
Table 2. In vitro antifungal and hemolytic activity of tyrosine-modified FR901469 derivatives (1)
MIC (mg/mL)
C. a.b
C. g.
C. t.
C. p.
Compounda
FP633
FP579
FP587
8001
8002
15001
Hemolytic activity (%)
FR901469
NH2
NH2
NH2
NH2
H
Cl
NO2
NH2
0.39
0.39
0.39
0.78
0.39
0.78
0.78
1.56
0.39
0.78
0.78
0.78
0.39
0.78
0.78
1.56
0.39
0.78
0.78
0.78
0.39
0.78
0.78
1.56
100
97
100
100
2
3
4
9
7
NH2
0.78
1.56
1.56
1.56
0.78
1.56
1.56
1.56
1.56
1.56
0.78
1.56
49
23
NHCOCH3
510
NHCOCH3
H
0.39
0.78
0.78
1.56
1.56
1.56
5
aAll compounds are hydrochloride salts, except 5.
bC. a., Candida albicans; C. g., Candida guilliermondi; C. t., Candida tropicalis; C. p., Candida parapsilosis.

1848
D. Barrett et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1843–1849
Table 3. In vitro antifungal and hemolytic activity of tyrosine-modified FR901469 derivatives (2)
MIC (mg/mL)
C. a.b
FP633
A. f.
FP13058004
100
Compounda
R1
R2
H
13004
Hemolytic activity (%)
FR901469
OH
0.250.250.25 0.5
0.50.50.1215
10
11
12
13
14
H
H
H
H
76
0.51
1
0.251
85
1
2
0.51
1
45
1
2
5
3
0.50.50.1205.5
0.51
23
15
16
OH
OH
0.250.5
79
1
2
2
2
1
0.52
84
17
OH
2
2
65
18
19
20
OH
OH
OH
1
0.51
46
0.51
0.250.5
75
0.50.50.5 1
60
aAll compounds are hydrochloride salts.
bC. a., Candida albicans; A. f., Aspergillus fumigatus.

D. Barrett et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1843–1849
1849
Table 4. In vivo antifungal activity: efficacy in disseminated murine
candidiasis
blocking of this OH group in the echinocandins
removes enzyme activity. An alternative explanation for
the potent activity of tyrosine-modified analogues might
be that FR901469 can also express antifungal activity
by another mechanism not involving glucan synthesis,
for example, membrane destabilization. With regards
this possibility, researchers at Nippon Roche have
reported that the tyrosine O-methyl derivative of 1 pos-
sessed good glucan synthase inhibitory activity.7 Fur-
ther studies on the glucan synthase inhibitory potency
and membrane effects of these derivatives should help to
clarify this point in more detail.
Compound
ED50 (mg/kg)
0.44–0.88a
FR901469
3
14
18
19
20
Amphotericin B
Fluconazole
b
0.45(0.5)
0.72 (1.5)
0.97 (2)
0.8 (1.6)
1.02 (2.1)
0.132
>20
aRange for FR901469 over a number of experiments.
bRatio of ED50(drug)/ED50(FR901469) for the same experiment.
References and Notes
led to a significant reduction in glucan synthase inhibi-
tion and MFC.14 Amino derivative 4 had slightly
weaker in vitro antifungal activity. However, these
modifications had no effect at all on hemolysis at 1 mg/
mL, indicating the need for further modification.
Increasing polarity by introduction of a b-alanine resi-
due (9) reduced hemolysis significantly (49% vs 100% at
1 mg/mL). Interestingly, compound 7 with the ornithine
group blocked as acetamide and a b-alanyl residue on
the tyrosine, had even lower hemolysis (23%), although
not as low as acetamide 5.10
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15. National Committee for Clinical Laboratory Standards.
Reference Method for Broth Dilution Antifungal Suscept-
ibility Testing of Yeasts; Approved Standard. NCCLS docu-
ment M27-A. National Committee for Clinical Laboratory
Standards: Wayne, PA, USA, 1997.
A number of other acylated derivatives, replacing the b-
alanine by various polar residues, were prepared and are
summarized in Table 3. It is clear from the data pre-
sented for these derivatives (15–20) that addition of
amino groups and/or carboxylic acid groups results in a
better hemolytic profile, but antifungal activity is also
reduced. Furthermore, the in vivo efficacies of com-
pounds 18, 19, and 20 were approximately half that of
FR901469, indicating no advantage over 1.
Introduction of substituents to the tyrosine phenol
group by alkylation was readily achieved. The car-
boxylic acid derivative 10 displayed strong in vitro
antifungal activity, but did not have a significant effect
on hemolysis (76% at 1 mg/mL). Amino derivative 12
had the lowest hemolytic activity amongst these simple
derivatives, however MIC was considered too weak to
warrant further consideration. The optimum result was
obtained with compound 14. Introduction of a carboxy-
methyl group to the phenol OH and a b-alanylamide to
the ortho-position resulted in a compound with the
lowest hemolysis (23%) and good in vivo activity in the
candidiasis model.
In summary, a series of tyrosine-modified analogues of
the unique macrocyclic lactone FR901469 has been
prepared. Several derivatives with good in vivo anti-
fungal efficacy and reduced hemolytic potential were
identified. Compound 14 in particular displayed good in
vivo efficacy in a candidiasis model and significantly
reduced hemolysis at 1 mg/mL. The results described
herein support the notion that the tyrosine phenol OH
in 1 and the homotyrosine phenol OH in the echino-
candins may not have the same role in the inhibition
mechanism of 1,3-b-glucan synthase, since removal or