Approaches towards the stabilization of hemiaminal function at ornithine unit of mulundocandin

Article abstract of DOI:10.1016/j.bmcl.2003.12.084

Semisynthetic modifications at position-12 (ornithine-5-position, hemiaminal function) of mulundocandin were carried out to improve its chemical stability. New carbon-carbon (C-C) and carbon-hydrogen (C-H) linkage at hemiaminal function -12 has been achieved. Lewis acid catalyzed introduction of electron rich aryl group at position-12 of mulundocandin is developed. Synthesized mulundocandin analogues were evaluated for their chemical stability and antifungal activity against C. albicans and A. fumigatus.

Full text of DOI:10.1016/j.bmcl.2003.12.084

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1123–1128
Approaches towards the stabilization of hemiaminal function at
ornithine unit of mulundocandin§
Bansi Lal*,y and Vitthal Genbhau Gundy
Quest Institute of LifeScience, Nicholas Piramal India Limited, Mulund, Mumbai-400080, India
Received 24 October 2003; revised 19 December 2003; accepted 20 December 2003
Abstract—Semisynthetic modifications at position-12 (ornithine-5-position, hemiaminal function) of mulundocandin were carried
out to improve its chemical stability. New carbon–carbon (C–C) and carbon–hydrogen (C–H) linkage at hemiaminal function -12
has been achieved. Lewis acid catalyzed introduction of electron rich aryl group at position-12 of mulundocandin is developed.
Synthesized mulundocandin analogues were evaluated for their chemical stability and antifungal activity against C. albicans and A.
fumigatus.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
esis of b-(1,3)-d-glucan, an integral component of the
cell wall of certain fungi.4 The most of the echino-
candins are unstable and have limited water solubility,
hence their development as drug candidates has been
hampered. Two new echinocandin antifungals, caspo-
fungin5 and micafungin6 have been introduced in the
market for the treatment of the invasive Aspergillus
infection.6a A few more echinocandines as antifungals
are under development with an aim to provide
improved therapy for fungal infections.7
The incidence of virulent fungal infection, especially
systemic mycoses in immuno deficient host are on the
increase during the last decades.1 The majority of life
threatening fungal infections is caused by opportunistic
pathogens such as Candida spp., Aspergillus spp., Pneu-
mocystis carinii and Cryptococcus neoformans.2 The
increase is attributed to indiscriminate use of broad
spectrumantibiotics in organ transplantation. Polyenes
and azoles are two classes of compounds used for the
treatment of fungal infections. Amphotercin B is used
alone or in combination with 5-flucytosine. Polyene anti-
fungals are broad spectrumantifungals, however their
utility is limited due to nephorotoxicity. Azoles are fungi-
static agents, they have tendency to develop drug resis-
tance.3 Both Polyene and azoles target fungal cell wall
membrane, a structure shared by both mammalian and
fungal cells, hence these drugs have possibility of toxicity.
Mulundocandin (MCN or 1),8 belongs to an echino-
candins class of antifungal lipopeptides. They inhibit the
synthesis of b-(1,3)-d-glucan, an essential component of
the fungal cell wall that is absent in mammalian cell.
Thus, the inhibition of b-(1,3)-d-glucan synthesis repre-
sents a fungal specific, potentially non-toxic target.
Naturally occurring echinocandins have very reactive
hemiaminal function at ornithine unit, which undergoes
ring opening and rearrangement, leading to biologically
less active linear peptides in acidic or basic medium.9
Various approaches are reported in the literature for
modifications at ornithine-5-position of echinocandins
to obtain chemically stable and water-soluble analo-
gues.10 We have previously described semisynthetic
modifications at hemiaminal function of the fungicidal
lipopeptide, mulundocandin (1) that resulted in
improved chemical stability without significant loss of
anti-Candida activity.11 In this work, we wish to report
the formation of C–C and C–H linkage at position-12
(=ornithine-5) position of mulundocandin with an aim
Therefore, there is a considerable need for the develop-
ment of new antifungal agents with improved proper-
ties. Echinocandin class of natural products is potent
lipopeptide antifungal agents, which inhibit the synth-
Keywords: Mulundocandin; Hemiaminal stabilization; Antifungal.
§Supplementary data associated with this article can be found at doi:
10.1016/j.bmcl.2003.12.084
* Corresponding author. Tel.: +91-022-25641286; fax: +91-022-
25641953; e-mail: bansilal@nicholaspiramal.co.in
y
This work was done at Hoechst Research Center, Mulund, Mumbai-
400080, Maharashtra, India.
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.084

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B. Lal, V. G. Gund / Bioorg. Med. Chem. Lett. 14 (2004) 1123–1128
to improve further stability and modify the biological
activity.
assigned to the methylene protons (–CH2NH2). Com-
pound 4 displayed increased chemical stability as well as
improved water solubility on being converting into a
hydrochloride salt.
2. Chemistry
Another approach to synthesize carbon derivatives at
Orn-5-position of muludocandin was through introduc-
tion of an aryl group. The crucial step during this
approach involved the utilization of the very reactive N-
acyliminium ion derived intermediate from hemiaminal
at position-12. The intermolecular and intramolecular
amidoalkylations with p-nucleophiles have been reported
in the literature.13 The Orn-5-methoxymulundocandin
(5) was chosen as a starting material for this reaction,
this in turn was prepared by treating mulundocandin
with excess methanol and catalytic amount of PTSA in
dry dioxane. We were aware of the fact that it will be
very hard to stabilize the N-acyliniumion of mulund-
candin, due to inherited unstable nature of this center,
yet we decided to go ahead with an aimto introduce an
aryl group. We had to select an aryl component, which
will be electron rich so that it can capture acyliniumion
fast. We chose 3,4-dimethoxy benzene as the electron
rich component. (This type of reaction has not been
attempted on any of the echinocandins so far.) Com-
pound 5 was treated with catalytic amount of titanium
tetrachloride in 1,2-dimethoxyethane at À70 ꢀC under
nitrogen atmosphere for 5 min and then reacted with
3,4-dimethoxy benzene (veratrole ) at À78 ꢀC to À50 ꢀC
for 1.5 h. LC-MS analysis of the reaction mixture
showed the presence of acid degraded product as the
major component and the required compound 6 with
The Orn-5-sulfone mululndocandin (2) was used as
starting material for this reaction. The sulfone 2 was
synthesized by reacting 1 with methylthioglycolate in
presence of catalytic amount of p-toulene sulfonic acid,
followed by oxidation of the corresponding thioether
with OXONE.12 Sulfone-2 on reaction with sodium
cyanide in anhydrous DMF at ambient temperature
gave Orn-5-cyanomulundocandin (3) in 36% yield as
shown in Scheme 1. H NMR spectrumof 3 had all the
peaks of mulundocandin, except that B7-H appeared as
a broad singlet at d 5.17. A band at 2320 cmÀ1 (CN) in
IR spectrumand peak at 119.69 ppmin
1
13C NMR
spectrumconfirmed the presence of cyano group in the
molecule.
Compound 3 on reduction with cobalt chloride hex-
ahydrate and sodium borohydride mixture in methanol
gave the required amino compound in low yield. Cata-
lytic hydrogenation using Pd–C or Raney-Ni in metha-
nol solvent failed. However, hydrogenation using Raney
Ni catalyst in methanolic ammonia gave the desired
aminomethyl analogue 4 in 53% yield (Scheme 1). The
compound was purified on RP18 column using 50–90%
acetonitrile/water, the compound displayed (M+Na)+
peak at 1043.5 (mw: 1021.21) in ESI-MS spectrum. In
1H NMR spectrumhad an additional multiplet at d 2.1,
Scheme 1. Formation of C–C linkage at Orn-5 position of mulundocandin.

B. Lal, V. G. Gund / Bioorg. Med. Chem. Lett. 14 (2004) 1123–1128
1125
some unidentified minor products. Purification of the
reaction mixture on a RP-18 column using acetonitrile
in water as the eluent gave Orn-5-(3,4-methoxyphenyl)-
mulundocandin (6)17 in 8% yield (Scheme 2). Attempts
to improve the yield of 6 by changing the reaction con-
ditions like reaction solvent and Lewis acid catalyst was
less successful.
either of the deoxymulundocandins. The major product
isolated was identified as degraded mulundocandin,
presumably, as more time was required for completion
of the reaction in these solvents. When we used TFA as
a solvent, reaction got completed within 10 min giving
desired, Orn-5-deoxymulundocandin (7, 38%), HTyr-4-
deoxymulundocandin (8, 19%) and dideoxymulundo-
candin (9, 14%), along with a small amount of acid
degraded MCN (Scheme 3). When reaction time and
temperature was increased, more of dideoxymulundo-
candin was formed.
1
In H NMR spectrumof 6, B7-H disappeared fromits
characteristic low field position, two doublets for D7-H,
0
0
D7 -H and D8-H, D8 -H appeared at d 7.18 and 6.78,
respectively. The three aromatic protons from 3,4-
dimethoxybenzene appeared as doublet at d 7.38,
J=1.47 Hz, doublets of doublet at d 7.10, J=8.55 and
1.47 Hz, and another doublet at d 6.9, J=8.55 Hz. The
two methoxy group of veratrol appeared as two singlets
at d 3.4–3.6 respectively. Its structure was also con-
firmed by (M+Na) peak at 1150.6 (mw=1128.31) in its
ESI-MS spectrum.
1
Comparison of H NMR spectrumof 7 with mulundo-
candin showed two methylene protons of iminol carbon
(–NHCH2). The two doublets of homotyrosine aro-
matic ring protons appeared at d 7.06 and 6.74. In mass
spectra the required (M+Na)+ peak appeared at
1014.5 in ESI-MS spectrum(mw: 992.177). The struc-
ture of 8 was confirmed by comparing with authentic
compound.14 Dideoxymulundocandin (9) showed two
gemcoupled doublet of doublets at d 2.95–3.05 for
two benzylic protons and multiplet at d 2.55–2.7 for iminol
Several reports have appeared in the literature regarding
synthesis and SAR studies of deoxy-echinocandins and
pneumocandins.13 In the present study, selective
removal of Orn-5-hydroxyl and/or HTyr-4 hydroxyl
of mulundocandin was attempted. Initial experiments of
reduction studies with varied concentration of TFA and
NaBH4 or NaCNBH4 as reducing agent in dry dioxane
as well as in acetic acid as solvent gave low yield of
1
protons (NHCH2) in H NMR spectrum. In ESI-MS
+
spectrum(M+Na)
and 976.6 as a molecular ion peak.
appeared at 998.7 as a base peak
Protection of Orn-5-hydroxyl with benzyl ether
increased chemical stability with retention of in vivo
Scheme 2. Introduction of aryl group at Orn-5-position of mulundocandin.
Scheme 3. Reduction studies at mulundocandin.

1126
B. Lal, V. G. Gund / Bioorg. Med. Chem. Lett. 14 (2004) 1123–1128
Scheme 4. Reduction studies on Orn-5-benzyloxy mulundocandin.
anti-Candida activity. Benzylic deoxymulundocandin is
reported to have the same in vitro and in vivo activities
against most Candida species; and in fact, it shows bet-
ter in vivo activity against some candida species.15 Thus,
it was hoped that selective removal of HTyr-4-hydroxyl
group of the Orn-5-benzyloxy mulundocandin (10) will
give analogue, which will be more stable than mulun-
docandin due to absence of hemiaminal function, and
with deoxy at benzylic position of homotyrosine unit
may improve activity. Sodium cyanoborohydride or
sodiumborohydride in acidic mediumare known to
cleave benzyl ether linkages and also to remove benzylic
hydroxyls giving corresponding deoxygenated com-
pounds. Thus, the reduction of 10, in a manner similar
to described for mulundocandin, with sodium borohy-
dride in TFA at 0 ꢀC gave Orn-5-benzyloxy-HTyr-4-
deoxymulundocandin (11) in 14%, Orn-5-deoxymulun-
docandin (7) in 11% and dideoxymulundocandin (9) in
15% yield (Scheme 4). In ESI MS spectrum, the com-
pound 11 (mw: 1082.293) showed (M+Na)+ peak at
1104.7, which indicates the loss of only one oxygen
Fromthe Table 1 it is apparent that, the analogues 3–11
in vitro activity is comparable to that of mulundo-
candin, but with much improved chemical stability in
both acidic and basic conditions. The in vitro activities
of analogues 4 and 6 are comparable to that of mulun-
docandin. Compound 4 has the added advantage of
improved stability and aqueous solubility in its salt
form.
Reduction of mulundocandin generated compounds 7–
9, of which 7 retains the in vitro activity (25 mm and
23hd mm zone size against C. albicans and A. fumigatus,
Table 1. In vitro activities of mulundocandin analogues against C.
albicans (IV) and A. fumigatus (AF-1)
Compd
Concentration
(mg/mL) in MeOH
Zone size (mm)
C. albicans
A. fumigatus
2
3
4
5
6
7
1
2
5
1
2
5
1
2
5
1
2
5
1
2
5
1
2
5
1
2
1
2
5
2
5
2
5
0.1
1.1
2.0
20
22
26
19
22
25
22
22
25
25
26h
27
24
25
29
25
26
29
30
33
24h
33
38
12
16
15
17
24
29
32
20hd
24vh
29hd
18hd
20hd
27hd
9/30hd
23hd
25hd
25hd
24h/37h
35vhd
30hd
30hd
32hd
23hd
23hd
25hd
29hd
30hd
25h
1
functionality than in the starting compound. H NMR
showed the presence of benzyloxy protons suggesting
that the hemiaminal position is protected in the form of
benzyl ether. Proton NMR was consistent with the
structure.
We had expected that this compound, with stabilized
hemiaminal function and reduced at HTyr-4 position,
may show better activity, but the result we obtained was
not promising. All these Orn-5 modified analogues were
qualitatively studied for their chemical stability by
keeping in aqueous methanolic solution at pH 5 and 9
for 24 hrs at roomtemp, followed by TLC and HPLC
analysis and were found to be more stable than MCN.
8
9
30hd
35hd
24h
2.1. Biological studies of mulundocandin analogues
10
11
2.1.1. In vitro. The synthesized mulundocandin analo-
gues were screened for their antifungal activity in vitro
by standard agar well method against C. albicans (IV)
and A. fumigatus (AF-1), in Sabouraud agar bioassay
plates.16 The zones produced by the mulundocandin
analogues were compared with the zone of mulundo-
candin and the results of the assay are given in Table 1.
26h
15hd
18hd
24hd
29hd
35vh
1
Mulundocandin
h=hazy, vh=very hazy, hd=hazy diffused, vhd=very hazy diffused.

B. Lal, V. G. Gund / Bioorg. Med. Chem. Lett. 14 (2004) 1123–1128
1127
Table 2. Anti-Candida activity of mulundocandin derivatives by
intraperitoneal injection in Swiss mice
generating novel series of echinocandin analogues for
SAR studies. The stability study using TLC and HPLC
analysis has established that all the synthesized deriva-
tives are more stable than muludocandin.
Approx.CFU count
(per gmkidney)/Dose
(ip, mg/kgꢁ6; B.i.d)
3.1. Nomenclature of mulunddocandin
Activity of analogue 8
1.8ꢁ108 (20 mg)
1.5ꢁ108 (20 mg)
3.8ꢁ105 (20 mg)
2.5ꢁ108
Activity of mulundocandin
Activity of fluconozole
Control (no treatment is given)
N1- [(6S, 9S, 14aS, 15S, 16S, 20S, 23S, 25aS, 2R, 11R,
12R)-23-((1S,2S)-1,2-dihydroxy-2-(4-hydroxyphenyl)
ethyl)-2,11,12,15-tetrahydroxy-6-((1R)-1-hydroxyethyl)-
20-hydroxymeth-yl-16-methyl-5,8,14,19,22,25-hexaoxo-
perhydrodiazolo [2,1-c:2,1-/][1,4,7,10,13,16] hexaazacyclo-
henicosin-9-yl]-12-methyltetradecanamide (Fig. 1).
Activity of analogue 9
7.7ꢁ106 (20 mg)
1.25ꢁ107 (20 mg)
1.0ꢁ104 (20 mg)
6.7ꢁ107
Activity of mulundocandin
Activity of fluconozole
Control (No treatment is given)
Acknowledgements
respectively) of MCN with much improved aqueous
stability in both acidic and basic solutions. The benzylic
deoxy mulundocandin 8 shows better in vitro activity
than mulundocandin, in accordance with literature
report.15
The authors wish to thank Antiinfective Research
Department, Hoechst AG, Germany for the constant
supply of Mulundocandin and Microbiology Dept of
Hoechst Research Center for testing our compounds.
Prof. Susheel Durani of IIT Mumbai for helpful sug-
gestions. We also thank Dr. Ramakrishna, N. V. S. and
his group for providing spectra.
2.2. In vivo testing of MCN analogues against C. albicans
using Swiss mice model16
Antifungal activity of analogues 8 and 9 was evaluated
in vivo in C. albicans infected Swiss mice after intraper-
itoneal administration. The colony formation unit
(CFU) in kidney homogenate after administrating
MCN derivatives were determined and compared with
MCN, a standard antifungal in clinical use (Flucona-
zole) and an untreated control group. The results are
summarized in Table 2. The compound 9 with both
Orn-5- and HTyr-4-hydroxyls removed shows one log
reduction in CFU by intraperitoneal injection.
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The hemiaminal function of mulundocandin has been
stabilized by formation of C–C and C–H derivatives at
Orn-5 position without significant loss of activity. We
have developed synthetic route for introduction of aryl
group at Orn-5 position of mulundocandin (MCN) and
activity is retained, which can further be exploited for
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Figure 1. Mulundocandin (1).

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17. 12-(3,4-Dimethoxyphenyl)-mulundocandin (6). To the
three-neck 100 mL RB flask fitted with a nitrogen inlet,
calciumchloride guard tube and rubber septumwas
added Orn-5-methoxymulundocandin 5 (0.11 g, 0.108
mmol) in 1,2-dimethoxyethane (3 mL) and mixture was
stirred at À70 ꢀC under nitrogen atmosphere. Titanium
tetrachloride (25 mL) was added to the above solution and
stirred for 5 min. 3,4-Dimethoxybenzene (0.04 mL, 3.13
mmol) was added to the above solution and stirred at À50
to À70 ꢀC for 1.5 h. TLC analysis showed no starting
compound but mixture of nonpolar compounds was
formed. Reaction mixture was quenched with aq
NaHCO3, evaporated to smaller volume, poured into
water (20 mL) and extracted with n-BuOH. The organic
extract was evaporated to dryness and the crude residue
was purified through HPLC. (Semipreparative RP-18
column, 10 m, 16ꢁ 250 mm, l=220 and 270 nm, flow
rate=8 mL/min., isocratic system, 70% CH3CN/H2O).
Lyophilization of the appropriate fractions gave 6 (0.01 g,
8.2%). Partial 1H NMR: d 7.38 (d, 1H, J=1.47 Hz, ArH),
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7.18 (d, 2H, J=8.50 Hz, D7-H & D7 -H), 7.10 (dd, 1H,
J=8.55 Hz and 1.47 Hz, ArH), 6.9 (d, 1H, J=8.55 Hz,
0
ArH), 6.78 (d, 1H, J=8.50, D8-H & D8 -H), 5.1 (s, 1H,
B7), 3.4–3.6 (2ꢁs, 6H, 2ꢁArOCH3). IR (KBr): nmax 3300–
3400, 2915, 1650, 1615, 1520, 1445, 1240, 1080 cmÀ1. ESI
MS (ES+): for C56H85N7O17; Calculated: 1128.317;
Found: (M+Na)+=1150.6. UV (MeOH) lmax (e MÀ1
cmÀ1): 206 nm(22924).