Semisynthetic modifications of hemiaminal function at ornithine unit of mulundocandin, towards chemical stability and antifungal activity

Article abstract of DOI:10.1016/j.bmc.2003.08.003

Mulundocandin (1), is an echinocandin class of lipopeptide. It has wide spectrum of antifungal activity against Candida and Aspergillus species. Semisynthetic modification at Ornithine-5-hydroxyl (hemiaminal function) of 1 was carried out to improve solut

Full text of DOI:10.1016/j.bmc.2003.08.003

Bioorganic & Medicinal Chemistry 11 (2003) 5189–5198
Semisynthetic Modifications of Hemiaminal Function at Ornithine
Unit of Mulundocandin, Towards Chemical Stability and
Antifungal Activity§
Bansi Lal,a,* Vitthal Genbhau Gund,a,c Ashok Kumar Gangopadhyay,a S. R. Nadkarni,b
Vidula Dikshit,b D. K. Chatterjeeb and R. Shirvaikarb
aDepartment of Medicinal Chemistry, Quest Institute of LifeSciences, Nicholas Piramal India Limited, Mulund (w)
Mumbai 4000 80, India
bDepartment of Natural Products, Quest Institute of LifeSciences, Nicholas Piramal India Limited, Mulund (w)
Mumbai 4000 80, India
cDepartment of Chemistry, University of Sherbrooke, 2500 Boul. University, Sherbrooke-J1K 2R1, Quebec, Canada
Received 1 May 2003; revised 7 May 2003; accepted 11 August 2003
Abstract—Mulundocandin (1), is an echinocandin class of lipopeptide. It has wide spectrum of antifungal activity against Candida
and Aspergillus species. Semisynthetic modification at Ornithine-5-hydroxyl (hemiaminal function) of 1 was carried out to improve
solution stability and hence in vivo activity. Synthesis of ether (C-OR), thioether (C-SR) and C–N linkage at hemiaminal function
have been described. All synthetic analogues were evaluated for their stability in aqueous solution and found to be more stable than
mulundocandin. Antifungal activity of Orn-5 analogues was evaluated both in vitro against Candida albicans and Aspergillus
fumigatus by agar well method and in vivo (oral and intraperitoneal) in C. albicans infected Swiss mice. Results of in vivo assays of
analogues 2–9 by the oral route suggests that the introduction of either oxygen nucleophiles (-OR) or sulphur nucleophiles (-SR), at
either Orn-5 or at both Orn-5 and HTyr-4 positions, results in retaining the activity of the parent compound with improved aqu-
eous stability in most cases. Compound 9 has shown improved antifungal activity in comparison to mulundocandin by oral appli-
cation in Swiss mice.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
fungal that has broad-spectrum activity, but its utility is
limited due to nephrotoxicity.3 The other available
agents are highly effective and widely used azoles.4
However, since azoles are fungistatic agents, the con-
cern of developing resistance to these drugs are quite
high.5 The need for a new antifungal agent is due to the
alarming rise in the number of AIDS cases and the
subsequent suppression of the immune system in these
patients. Other reasons that have spurred the develop-
ment of new systemic antifungal agents include the
increase in the frequent use of antineoplastic agents and
long term use of antibiotics. The polyenes and azoles,
target the fungal cell membrane, a structure shared by
both mammalian and fungal cells, and thus these drugs
have inherent toxicity.
The prevalence of systemic fungal infections, especially
disseminated systemic mycoses in immunodeficient
hosts has increased significantly during the last two
decades.1 This increase is due to greater use of broad-
spectrum antibiotics, immunosuppressive agents,
hyperalimentation products and central venous cathe-
ters, intensive care of low birth weight infants, organ
transplantation and the acquired immunodeficiency
syndrome (AIDS) epidemic.2 Prior to the development
of echnocandins, the option for the treatment of fungal
infections are limited to two class of compounds namely
the polyenes and the azoles. Amphotericin B, used alone
or in combination with 5-flucytosine, is a polyene anti-
A newer class of cell wall active agents that have been
developed to the point of seeing clinical candidates is
the cyclic lipopeptides belonging to echinocandin
family.6 This group has the potential of providing a
§This work was completed at Research Centre, Hoechst Marion
Roussel Limited, Mulund (w) Mumbai 400 080, India.
*Corresponding author. Tel.: +91-22-592-2631; fax: +91-22-564-
1953; e-mail: bansilal@nicholaspiramal.co.in
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.08.003

5190
B. Lal et al. / Bioorg. Med. Chem. 11 (2003) 5189–5198
broad-spectrum fungicide with much lower toxicity than
the current antifungal agents, through the inhibition of
the biosynthesis of (1,3)-b-d-glucan, an essential com-
ponent of the fungal cell wall.7 This novel mode of
action and potent antifungal activity has led to the
development of several interesting drug candidates
during the past several years. Caspofungin and mica-
fungin are new echinocandin antifungals that have
recently been introduced in the market for the treatment
of invasive Aspergillus infections in patients unresponsive
to or unable to receive amphotericin B.8 Other echino-
candins as antifungals are under development in attempts
to provide improved therapy for fungal infections.9
acyliminium ions are almost always generated insitu, in
view of their limited stability and high reactivity. The
mechanism of amidoalkylation reaction is shown in
Scheme 1.
Formation of ether linkage at Orn-5-position of mu-
lundocandin (Scheme 2)
Under acidic conditions, Orn-5-hydroxyl of mulundo-
candin is protonated, leading to the formation of N-
acyliminium ion, which either reacts with an internal
nucleophile to furnish a rearranged compound,12 or
with an external nucleophile to furnish an ether deriva-
tive or combination. The formation of the rearranged
product could be suppressed by performing the reaction
in the presence of a large excess of an external nucleo-
phile. Thus, treatment of mulundocandin with excess of
benzyl alcohol in the presence of catalytic amounts of p-
toluene sulphonic acid at room temperature furnished
two nonpolar products, monobenzylated 2 (67%) at
Orn-5 position and dibenzylated 3 (13%) at Orn-5- and
HTyr-4 positions, as shown in Scheme 2. The ratio of
products 2 and 3 depends upon the amount of the acid
used, the reaction temperature and duration. Com-
pound 2 and 3 are purified by flash column chromato-
graphy over silica gel in >95% purity and are well
characterized by using 1H NMR, 13C NMR, DEPT-135
and ESI MS spectra (see Experimental). Subsequent
attempt was made to introduce smaller group like
methyl ether so as to minimize the steric impact on the
molecule, thereby retaining its original activity but pos-
sibly with increased stability. Thus, Orn-5-methoxy-
mulundocandin (4) and Orn-5- and HTyr-4-dimethoxy-
mulundocandin (5) were prepared in a manner similar
to that used for compound 2 using excess methanol.
Compound 4 was obtained, as the major product while
compound 5 was the minor product formed in this
reaction in 70 and 5% yields, respectively (Scheme 2).
1H NMR of 5 showed two doublets at d 7.25 and 7.15
Mulundocandin (1), an echinocandin class of antifungal
lipopeptide, was isolated from the culture broth of a
strain of Aspergillus sydowi.10 It exhibited excellent in
vitro activity against Candida species, especially against
C. albicans and C. glabrata isolates.11
Naturally occurring echinocandins have reactive
hemiaminal function due to Orn-5-hydroxyl group
and is responsible for its unstability. It undergoes
ring opening and rearrangement leading to biologically
inactive compounds in acidic, basic and even neutral
solutions.12 Various approaches are reported in the lit-
erature for modifications at ornithine-5-position of
echinocandins to obtain stable and water-soluble
analogues.13
Mulundocandin also bear a hemiaminal function at C-5
of Orn residue and suffers from similar stability prob-
lem. We observed that water: acetonitrile (1:1) solution
of mulundocandin in acidic (pH=3) as well as in base
(pH=8.5) undergo rapid decomposition. Particularly in
basic pH the T1/2 was found to be less than 1 h. Even in
neutral condition substantial decomposition was
observed (T1/2=4 days in phosphate buffer of pH 7.2
and T1/4=5 days in sodium acetate buffer). In this work
we describe the approach for the stabilization of
mulundocandin in order to improve its stability and hence
the in vivo antifungal activity, through formation of ether
(C-OR), thioether (C-SR) and C–N linkage.14 The crucial
step during the present study involved the utilization of the
very reactive N-acyliminium ion intermediate.
0
for D7-H and D7 -H and two more doublets merged at d
0
6.82 for D8-H and D8 -H. The effect due to stereo-
isomerisms at HTyr-4 position was apparent in the
chemical shift of aromatic protons and the diaster-
eomers are formed in the ratio of 1:1. In case of 3 also,
two stereoisomers at HTyr-4 position could be detected
based on TLC and HPLC analysis, but the aromatic
protons of H-Tyr unit did not reveal any NMR differ-
entiation, possibly because the benzyloxy group is away
from the aromatic p cloud. We have not made any
attempt to separate diastereomers arising out of the
reactions unless the biological activity of the resulting
compounds are significantly higher than mulundocandin
and comparable to marketed product.
Chemistry
It is observed from literature that most of the reactions
of N-acyliminium ions are of intermolecular type.15 The
presence of strong electron withdrawing carbonyl group
makes N-acyliminium ions more electrophilic. The N-
Scheme 1. Mechanism of amidoalkylation reaction.

B. Lal et al. / Bioorg. Med. Chem. 11 (2003) 5189–5198
5191
Scheme 2. Formation of ether/thioether linkage at Orn-5 position of mulundocandin.
The strategy adopted for the introduction of C–C bond
or C–N bond at Orn-5-position, was through introduc-
tion of thio-ether, which could be activated to sulfone (a
good leaving group) by mild oxidation. An initial
experiment utilizing ethyl mercaptan and PTSA combi-
nation led to the formation of Orn-5- and HTyr-4-di-
thioether derivative as the major product (diastereo-
meric ratio=2:3) while the desired mono-thioether
derivative at Orn-5 position was obtained in very small
yield. Presumably, the high reactivity of sulphur
nucleophile is responsible for the observed low selectiv-
ity. Literature report13f indicated that the use of methyl
thioglycolate leads to the formation of Orn-5-
SCH2COOCH3 as the major product in pneumocandin
Bo. Thus, on treatment with methyl thioglycolate in
presence of catalytic amounts of p-toluene sulphonic
acid, mulundocandin gave compounds 6 and 7 in 61 and
16% yields, respectively.
of catalytic amount of p-toluene sulfonic acid to give 8
in 50% yield and 9 in 18% yield.
The analogues 2–5 are useful starting materials for fur-
ther modifications to improve the aqueous solubility
and antifungal activity of mulundocandin.14
Formation of C–N linkage at Orn-5-position of mu-
lundocandin (Scheme 3)
Another approach for the stabilization of hemiaminal
function with desired antifungal activity of mulundo-
candin with additional advantage of water solubility
could be achieved by formation of C–N linkage at Orn-
5 position. Balkovec et al.,13,16 reported nucleophilic
displacement of activated sulfone by nitrogen nucleo-
phile to get N-substituted ornithine analogues of echi-
nocandins. Using similar approach compound 6 was
subjected to oxidation13,16 with OXONE1. The resul-
tant reactive sulfone intermediate 10 was reacted with
the potential nitrogen neuclophiles shown in Scheme 3.
Orn-5- and HTyr-4-di-SCH2CO2CH3 mulundocandin
(7) also displayed stereoisomerisms at HTyr-4 position,
which can be visualized by two doublets appearing at d
1
In the H NMR spectrum of sulfone 10, B7-H appeared
0
7.25, 7.12 for D7-H and D7 -H with coupling constants
downfield at d 5.6, in comparison to d 5.39 in the
thioether 6, due to the deshielding nature of the sulfone
(–SO2CH2CO2CH3) group. Subsequent treatment of
sulfone intermediate 10 with 4-(2-aminoethyl) morpho-
line in anhydrous dioxan at 25–60 ꢀC, gave the desired
compound 12 in 70% yield. Compound 13 was synthe-
sized similarly in 64% yield using imidazole as the
nucleophile. The products could be purified by HPLC
8.55 Hz and two additional doublets at d 6.8 for D8-H
1
0
and D8 -H in the H NMR spectrum and the diastereo-
mers were in the ratio of 1:3. The research group at
Merck reported13e that SPh group at Orn-5 position
could be replaced directly by primary or secondary
amines, leading to N-substitution. Taking the lead
Mulundocandin was treated with thiophenol in presence

5192
B. Lal et al. / Bioorg. Med. Chem. 11 (2003) 5189–5198
Scheme 3. Formation of C–N linkage at Orn-5 position of mulundocandin.
on a C-18 column using 50–90% acetonitrile/water as
the eluent. The compound (13) was characterized by an
(M+Na)+ peak at 1080 (calcd M+=1057) and a base
peak at 1011, corresponding to the loss of 68 units due
Hz) due to the deshielding nature of azide. Due to
racemization at HTyr-4 position, the aromatic protons
of H-Tyr unit appeared as two doublets at d 7.28 and
7.14 with coupling constants 8.88 Hz and were assigned
1
0
to D7-H, D7 -H and another pair of doublets centred at
to the elimination of imidazole moiety. The H NMR
0
spectrum of the compound was similar to that of
mulundocandin but with singlets at d 7.8 and 7.65, that
were assigned to the three-imidazole protons.
d 6.83 were assigned to D8-H, D8 -H. Attempts to
reduce the diazide 14, to corresponding di-amino com-
pound, using a variety of reported procedures, did not
succeed. Disulfone 11 on treatment with 4-(2-amino-
ethyl) morpholine in dioxan gave 15 in 44% yield.
Sulfone 11 was obtained from thioether 7, following the
same procedure as described above by treatment with
OXONE1 in acetonitrile–water mixture. This was then
subjected to displacement reaction nitrogen nucleo-
philes as shown in Scheme 3. The diazide derivative 14
was thus prepared in 68% yield by the treatment of the
sulfone 11 with sodium azide in anhydrous dioxan.
Compound 14 displayed a characteristic IR band for
azide group at 2100 cmꢁ1 and the (M+Na)+ peak at
1080 (calcd M+=1057) in ESI-MS, with the base peak
at 1037 resulting from the loss of the azide unit. The B7
proton appeared downfield at d 5.39 (d, 1H, J=1.86
Some of the compounds as representative example were
studied for their stability in water–acetonitrile (1:1)
solution at neutral, acidic (pH=3), and alkaline
(pH=8.5) conditions followed by HPLC analysis. We
observed that compounds 2 and 4 were stable in both
neutral and basic pH however in acidic pH T1/2 was
found to be ꢂ24 h. On the other hand, compounds 6
and 9 were found to be stable in neutral and acidic pH
while in basic pH T1/2 was ꢂ24 h. The extent of
decomposition of compounds 12 and 13 were found to

B. Lal et al. / Bioorg. Med. Chem. 11 (2003) 5189–5198
5193
less than 10% over a period of 5 days. Thus modifi-
cation of aminal function of mulundocandin in the pre-
sent study resulted in more stable mulundocandin
analogues that can be exploited in future.
ideal position for modification. Amongst the ether deri-
vatives at Orn-5 position, analogue 4 show better in
vitro activity against C. albicans and A. fumigatus. The
activity of N-substituted compounds 12–15 was found
to be much lower in comparison to O-ether and S-ether
analogues. Compound 13 showed 22 and 20hd mm zone
size against C. albicans and A. fumigatus, respectively, at
1 mg/mL of concentration and showed improved aqu-
eous stability than mulundocandin.
Biological studies of Mulundocandin analogues
In vitro. Mulundocandin analogues were screened in
vitro by standard agar well method against C. albicans
(IV) and A. fumigatus (AF-1), in Sabouraud agar bioas-
say plates.17 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.
In vivo testing of mulundocandin analogues against C. al-
bicans using swiss mice model18
Antifungal activity of mulundocandin analogues was
evaluated in vivo in C. albicans infected Swiss mice after
oral and intraperitoneal administration. The colony
formation unit (CFU) in kidney homogenate after
administrating mulundocandin derivatives were deter-
mined and compared with mulundocandin, and fluco-
nazole. The experimental details of activity evaluation
are given below and the results are summarized in
Tables 2 and 3.
From the Table 1, it is apparent that, the analogues
modified at Orn-5 position (2–15) have retained in vitro
activity comparable to that of mulundocandin (1).
Replacement of Orn-5-hydroxyl by either benzyloxy (2)
or methoxy (4) or methylthioglycolate (6), results in
slight reduction of in vitro activity compared with
mulundocandin. Compound 4 shows better in vitro
activity than 2, possibly because comparatively smaller
methoxy group replaces Orn-5-hydroxyl. Compounds 3,
5, 7 and 9, di-alkylated at Orn-5- and HTyr-4 positions
showed lower in vitro activity than 2, 4, 6 and 8, possi-
bly, suggesting that HTyr-4 position may not be the
Swiss mice, 4–6 weeks old and 18–22 g body weights
were used for in vivo studies. The inoculum was pre-
pared by culturing C. albicans on Sabouraud agar and
cell density adjusted to 106 cells/mouse for non-lethal
model. Mice were infected by injecting the conidia of C.
albicans through the lateral tail vein, with 0.25 mL of
the inoculum on D0. Treatment was commenced at zero
hr after infection and continued from D0 to D+4. Con-
trol groups (untreated) consisted of four mice infected
but not treated as the negative control and mice infected
and treated with the standard antifungal fluconazole as
the positive control. A test sample of mulundocandin,
mulundocandin analogues and fluconazole were pre-
pared in tween 80 and distilled water and tested at 100,
80 and 50 mg/kg, respectively, administered by the oral
route and at 20 mg/kg each by the intraperitoneal route.
Dosing protocol consisted of drug administrating on
D0, D+1, D+2, D+3 and D+4 by either of the routes
mentioned above. After the treatment was over, the
animals were sacrificed after two days. Paired kidneys
were removed aseptically and cultured for the presence
of C. albicans in sterile saline. The kidneys of each
group were homogenized and 10-fold serial dilutions
were prepared. A 10-mL aliquot from each dilution was
plated on Sabouraud agar plate, incubated at 30 ꢀC for
24–48 h and the CFU’s were determined. The number
of CFU per gram of paired kidneys was calculated.
Activity was interpreted by the difference shown in the
load of organisms per gram kidney weights of treated
group and untreated control group.
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
2
5
2
5
1
2
5
2
5
1
2
5
2
5
2
5
2
5
1
2
5
2
5
1
2
5
1
2
5
2
5
12
16
11
14
25
26h
27
18
20
20
22
26
10h
19
16
18h
15
18
21
23
24
13
16
22
24
24
15
21
25
17h
19h
24
29
32
24h
26h
18hd
20hd
25hd
24h/37h
35vhd
30h
5
6
30vhd
20hd
24vh
29hd
20hd
26hd
17hd
21hd
15hd
17vhd
20hd
21vh
27hd
20hd
24hd
20hd
24hd
30hd
17hd
19hd
8/25hd
13vh
16vhd
24hd
29hd
35vh
7
8
9
10
12
13
14
15
The results of in vivo assays of compounds 2–9, after
oral administration, suggests that both ether (-OR) and
thioether (-SR) at either Orn-5 or at both Orn-5 and
HTyr-4 positions are well tolerated in mulundocandin
1
Mulundocandin
0.1
1.0
2.0
in its biological potency. Compound
9 showed
improved activity corresponding to two-log reduction in
CFU as compared with 1 by the oral route at 80 mg/kg
and are comparable with fluconazole, the standard
antifungal compound.
h, hazy; vh, very hazy; hd, hazy diffused; vhd, very hazy diffused. (50
mL of solution used for testing).

5194
B. Lal et al. / Bioorg. Med. Chem. 11 (2003) 5189–5198
Table 2. Anti-Candida activity of mulundocandin (MCN) derivatives by oral application in Swiss mice
Compd
Activity of MCN derivatives
Activity of MCN
Activity of Fluconazole
Control
(no treatment is given)
(approx CFU count, per g kidney)/dose (mg/kgꢃ5 per os)
2
1.1ꢃ106
6.8ꢃ107
NTa
NT
2.0ꢃ108
2.0ꢃ108
4.0ꢃ108
4.0ꢃ108
2.6ꢃ107
2.6ꢃ107
2.6ꢃ107
2.6ꢃ107
(80 mg)
(100 mg)
3
20.0ꢃ106
6.8ꢃ107
(80 mg)
(100 mg)
4
8.4ꢃ107
8.8ꢃ107
1.5ꢃ103
(80 mg)
(100 mg)
(50 mg)
5
6.4ꢃ107
8.8ꢃ107
1.5ꢃ103
(80 mg)
(100 mg)
(50 mg)
6
2.0ꢃ106
2.4ꢃ106
1.5ꢃ104
(80 mg)
(100 mg)
(50 mg)
7
3.4ꢃ106
2.4ꢃx106
1.5ꢃ104
(80 mg)
(100 mg)
(50 mg)
8
2.5ꢃ107
2.4ꢃ106
1.5ꢃ104
(80 mg)
(100 mg)
(50 mg)
9
2.8ꢃ104
2.4ꢃ106
1.5ꢃ104
(80 mg)
(100 mg)
(50 mg)
aNT, not tested.
Table 3. Anti-Candida activity of mulundocandin (MCN) derivatives by intraperitoneal injection in Swiss mice
Compd
Activity of MCN derivatives
Activity of MCN
Activity of Fluconazole
Control
(no treatment is given)
(approx CFU count, per g kidney)/dose (mg/kgꢃ5 per os)
3
1.2ꢃ107
4.8ꢃ106
NTa
NT
NT
6.6ꢃ106
5.9ꢃ107
5.9ꢃ107
6.7ꢃ107
(20 mg)
(20 mg)
4
1.6ꢃ107
7.2ꢃ107
(20 mg)
(20 mg)
5
1.1ꢃ108
7.2ꢃ107
(20 mg)
(20 mg)
7
1.2ꢃ108
1.25ꢃ107
1.0ꢃ104
(20 mg)
(20 mg)
(20 mg)
aNT, not tested.
the blocking of Tyr-4 hydroxy group believed to be
required for antifungal activity discussed earlier. Results
of in vivo study of 2–9 through oral route also suggest
the maintenance of activity. Compound 9 has shown
improved activity (two log difference in CFU in compar-
ison to mulundocandin), which is close to fluconazole.
We cannot explain at this stage why 9 showed higher
in vivo activity in comparison to ether or C-N com-
pounds. One possibility could be the better stability of 9
in acidic pH in comparison to 2 and 4 surviving initial
transport phase followed by oxidative hydrolysis at Orn-5
position releasing active substance. Further studies
will be required to carry out to ascertain the exact
reason. However the analogues 2 and 4 are useful starting
material for further modification towards increased
water solubility. This part of the work will be published
elsewhere.
Conclusion
We have achieved our initial objective of stabilizing
mulundocandin through ether, thioether or C–N linkage
at Orn-5 position that is quite obvious from the compar-
ison of the result of stability studies. Mulundocandin
undergoes rapid degradation in acidic and basic pH parti-
cularly at pH 8.5 wherein T1/2 was found to be <1 h
while mulundocandin analogues showed much higher
stability. Most significantly in neutral solution T1/2 of
mulundocandin was found to be 4 days while synthetic
analogues shows very little sign of decomposition. The
in vitro activity of these compounds suggest that intro-
duction of ether or thioether at Orn-5 position main-
tained the antifungal activity of mulundocandin to a
great extent while improving stability. However, the
disubstituted analogues showed lower activity due to

B. Lal et al. / Bioorg. Med. Chem. 11 (2003) 5189–5198
5195
Experimental
Nomenclature of mulundocandin
General Remarks
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 - hexaox-
operhydrodiazolo[2,1 - c:2,1 - /][1,4,7,10,13,16] hex-
aazacyclohenicosin-9-yl]-12-methyltetradecanamide.
All reagents and solvents were purchased from com-
mercial supplier and were used as received unless
otherwise stated. Yields reported are isolated yields of
the materials. All mulundocandin analogues synthesized
during this study were >95% pure based on HPLC
analysis. Dioxan was freshly distilled over sodium prior
to use. Melting points (mp) were recorded on a Kofler
hot-plate apparatus and were uncorrected. 1H NMR
spectra and 13C NMR spectra were recorded on 300 and
75 MHz, respectively, on a Brucker ACP-300 spectro-
meter, using CD3ODas solvent, unless otherwise men-
tioned. Chemical shifts values are expressed in d scale
(parts per million) using TMS as internal standard.
Coupling constant (J) values were reported in Hz.
Alphabets B7, D7, D7 , D8, and D8 on structures of
compounds 2–15 are used to identify the protons posi-
tion (Fig. 1). ESI MS were recorded on a Fisons VG
QUATTRO II instrument. IR spectra were recorded as
KBr wafers on a Perkin-Elmer 782: Infrared spectro-
photometer. UV spectra were recorded on Chemito
2500: UV–vis Recording spectrometer. TLC was per-
formed on precoated silica gel aluminium plates con-
taining a fluorescent indicator (1.05554, Silica gel 60
F254, Merck). Flash chromatography was performed
using Acme’s silica gel (230–400 mesh) and MeOH–
CHCl3 mixtures as eluent. Visualization of the compo-
nents on TLC plates was achieved either by exposure to
iodine vapour or UV light or by spraying 50% H2SO4
followed by charring. Perkin-Elmer HPLC (Diode
Array Detector 235, Binary LC pump 250) was used for
purification using Knauer Eurosphere 100, RP C-18
column (size 250 mmꢃ16 mm and particle size 10 mm)
and CH3CN/H2O gradient as an eluent with a flow rate
of 5–8 mL/min and was monitored at l=220 and 270 nm.
The purity of compounds was checked on YMC-Pack,
AQ-313 S-5 120A ODS, RP C-18 column (size, 250 mmꢃ6
mm; particle size 5 mm; mobile phase, 70% CH3CN/H2O;
flow rate 1 mL/min; monitored at l=220 and 270 nm).
Experimental procedures
N1-[(6S,9S,14aS,15S,16S,20S,23S,25aS,2R,11R)-12-ben-
zyloxy-23-((1S,2S)-1,2-dihydroxy-2-(4-hydroxyphenyl)-
ethyl)-2,11,15-trihydroxy-6-((1R)-1-hydroxyethyl)-20-h
droxymeth-yl-16-methyl-5,8,14,19,22,25-hexaoxoperhydro-
diazolo[2,1 - c:2,1 -/][1,4,7,10,13,16]hexa-azacyclohenico-
0
0
sin-9-yl]-12-methyltetradecanamide
(2)
and
N1-
[(6S,9S,14aS,15S,16S, 20S,23S,25aS,2R,11R)-12-benzy-
loxy-23-((1S)-2-benzyloxy-1-hydroxy-2-(4-hydroxyphen-
yl)ethyl)-2,11,15-trihydroxy-6-((1R)-1-hydroxyethyl)-20-
hydroxymethyl-16-methyl-5, 8,14, 19,22,25-hexaoxoper-
hydrodiazolo[2,1 -c:2,1-/] [1,4,7,10,13,16] hexaazacyclo-
henic-osin-9-yl]-12-methyltetradecanamide (3). To
a
stirred solution of mulundocandin (5.2 g, 5.15 mmol) in
anhydrous dioxan (150 mL), under nitrogen atmo-
sphere, anhydrous benzyl alcohol (10.45 g, 96.6 mmol),
and p-toluene sulfonic acid (0.32 g, 1.66 mmol) were
added and the reaction mixture was stirred at ambient
temperature for 1 h. After 1 h TLC (20% MeOH/
CHCl3) showed disappearance of starting compound.
The reaction was quenched by the addition of saturated
aqueous NaHCO3 at 5–10 ꢀC and evaporated to a
smaller volume (25 mL). The crude mixture was diluted
with water (250 mL), extracted with n-BuOH (3ꢃ150
mL), washed with water (200 mL) followed by brine
(200 mL). Combined organic extract was dried over
anhydrous Na2SO4. Evaporation of solvent under
vacuum gave crude gummy product. Which was pur-
ified by flash column chromatography on silica gel with
0–15% MeOH/CHCl3 as 5% step gradient eluent. Eva-
poration of the appropriate fractions gave white com-
pound 2 (3.8 g, 67.ꢀ1%; mp: 196–99 ꢀC) and 3 (0.82 g,
13.3%; mp: 182–83 C). Compound 2: 1H NMR: d 7.28–
7.41 (m, 5H, –OCH2Ph). 7.17 (d, 2H, J=8.41 Hz, D7
0
0
and D7 ), 6.78 (d, 2H, J=8.37 Hz, D8 and D8 ), 5.32 (d,
1H, J=1.60 Hz, B7 or iminol proton), 4.68 (s, 2H,
–OCH2Ph), 13C NMR (DMSO-d6): 172.07, 171.51,
170.46, 170.27, 169.59, 168.14, 156.57, 138.78, 132.47,
128.19, 127.94, 127.35, 127.08, 114.65, 79.01, 75.19,
74.24, 73.19, 69.23, 68.99, 68.66, 68.04, 66.10, 62.27,
60.82, 56.29, 55.67, 53.49, 51.84, 51.28, 49.23, 37.26,
36.99, 35.99, 35.13, 34.72, 33.73, 29.36, 29.03, 28.90,
28.52, 26.45, 25.42, 19.38, 19.06, 11.19, 10.81. IR (KBr):
nmax 3350–3450, 2930, 1650, 1615, 1520, 1450, 1385,
1220, 1070 cmꢁ1. ESI-MS (ES+): for C55H83N7O16;
Calculated: 1097; found: (M+Na)+=1120 (base peak).
UV (MeOH) lmax (e Mꢁ1 cmꢁ1): 225, 277 nm (14016,
1595). Compound 3: 1H NMR: d 7.24–7.31 (m, 10H,
0
2ꢃOCH2Ph) 7.12 (d, 2H, J=8.55 Hz, D7 and D7 ), 6.74
0
(d, 2H, J=8.54 Hz, D8 and D8 ), 5.17 (d, 1H, J=3.66
Figure 1. Mulundocandin (1).

5196
B. Lal et al. / Bioorg. Med. Chem. 11 (2003) 5189–5198
Hz, B7), 4.4–4.53 (2ꢃs, 4H, –OCH2Ph). IR (KBr): nmax
3350–3450, 2930, 1650, 1615, 1520, 1450, 1385, 1220,
1070 cmꢁ1. ESI-MS (ES+): for C62H89N7O16; calcd
1187; found: (M+Na)+=1210 (base peak). UV
(MeOH): lmax (e Mꢁ1 cmꢁ1): 228, 275 nm (14113, 1767).
the addition of aqueous NaHCO3 at 5–10 ꢀC and eva-
porated to smaller volume (25 mL). The mixture was
diluted with water (250 mL). The semisolid material was
extracted with n-BuOH (3ꢃ150 mL), washed with water
(200 mL), followed by brine (200 mL). Combined
organic extract was dried over anhydrous Na2SO4. The
solvent was removed under reduced pressure to give
crude gummy product. The crude material was purified
by flash chromatography over silica gel with 0–15%
MeOH/CHCl3 as 5% step gradient eluent. Evaporation
of the appropriate fractions gave white compound 6
(3.171 g, 60.ꢀ7%; mp: 183–85 ꢀC) and 7 (0.885 g, 15.7%;
N1 - [(6S,9S,14aS,15S,16S,20S,23S,25aS,2R,11R) - 23 -
((1S,2S) - 1,2 - dihydroxy - 2 - (4 - hydrox - yphenyl)e-
thyl)2,11,15-trihydroxy-6-((1R)-1-hydroxyethyl)-20-hy-
droxymethyl - 12 - methoxy - 16 - methyl - 5,8,14,19,22,25 -
hexaoxoperhydrodiazolo[2,1 - c:2,1 - /][1,4,7,10,13,16]hex-
aaz-acyclohenicosin-9-yl]-12-methyltetradecanamide (4)
and N1-[(6S,9S,14aS,15S,16S, 20S,23S,25aS,2R,11R)-
23-((1S)-1-hydroxy-2-(4-hydroxyphenyl)-2-methoxyethyl)
-2,11,15-trihydroxy-6-((1R)-1-hydroxyethyl)-20-hydroxy-
methyl-12-methoxy-16-methyl-5,8,14,19, 22,25-hexaoxoper-
hydrodiazolo[2,1-c:2,1-/][1,4,7,10,13,16]hexaazaclohenicosin-
9-yl]-12-methyltetradecanamide (5). To a stirred solu-
tion of mulundocandin (2.2 g, 2.18 mmol) in anhydrous
dioxan (50 mL), under nitrogen atmosphere, anhydrous
MeOH (6.0 mL, 147.9 mmol) and p-toluene sulfonic
acid (0.12 g, 0.624 mmol) were added and the reaction
mixture was stirred at ambient temperature for 0.5 h.
Reaction progress was monitored by TLC (20%
MeOH/CHCl3). The reaction workup and purification
process is similar to the described for compound 2 and
3. Evaporation of the appropriate fractions gave white
compound 4 (1.55 g, 69.5%; mp: 188 ꢀC dec.) and 5
1
mp: 115–17 C). Compound 6: H NMR: d 7.2 (d, 2H,
0
J=8.54 Hz, D7 & D7 ), 6.8 (d, 2H, J=8.54 Hz, D8 &
0
D8 ), 5.39 (br, 1H, B7), 3.75 (s, 3H,-SCH2COOCH3),
3.45, 3.65 (2ꢃd, 2H, J=15.78 Hz, –SCH2COOCH3). IR
(KBr): nmax 3350, 2920, 1730, 1660–1620, 1520, 1440,
1385 cmꢁ1. ESI MS (ES+): for C51H81N7O17S; calcd:
1095; found: (M+Na)+=1118 (base peak). UV
(MeOH) lmax (e Mꢁ1 cmꢁ1): 225, 277 nm (5769, 9428).
1
Compound 7: H NMR: d 7.25, 7.12 (2ꢃd, 2H, J=8.55
0
0
Hz, D7 & D7 ), 6.8 (2ꢃd, 2H, J=8.55 Hz, D8 and D8 ),
5.41 (br, 1H, B7), 3.75 (s, 3H, –SCH2COOCH3), 3.65,
3.8 (2ꢃs, 3H, –SCH2COOCH3), 3.45, 3.64 (2ꢃd, 2H,-
SCH2COOCH3), 3.21–2.85 (m, 2H, –SCH2COOCH3).
IR (KBr): nmax 3300–3400, 2930, 1740, 1680–1610, 1520,
1435, 1380, 1260, 1070 cmꢁ1. ESI–MS (ES+): for
C54H85N7O18S2; calcd: 1183; found: (M+Na)+=1206
(base peak). UV (MeOH) lmax (e Mꢁ1 cmꢁ1): 227 nm
(2421).
ꢀ
1
(0.109 g, 4.8%; mp 172–75 C). Compound 4: H NMR:
0
d 7.19 (d, 2H, J=8.55 Hz, D7 & D7 ), 6.89 (d, 2H,
0
J=8.55 Hz, D8 and D8 ), 5.12 (d, 1H, J=1.65 Hz, B7),
3.38 (s, 3H, –OCH3). IR (KBr): nmax 3300–3400, 2920,
1660, 1625, 1515, 1440, 1385 cmꢁ1. ESI MS (ES+): for
C49H79N7O16; calcd 1021; found: (M+Na)+=1044
(base peak); UV (MeOH) lmax (e Mꢁ1 cmꢁ1): 223, 277
N1 - [(6S,9S,14aS,15S,16S,20S,23S,25aS,2R,11R) - 23 -
((1S,2S)-1,2-dihydroxy-2-(4-hydroxyphenyl)ethyl)-2,11,
15-trihydroxy-6-((1R)-1-hydroxyethyl)-20-hydroxymethyl-
16-methyl- -5,8,14,19,22,25-hexaoxo-12-phenylsulfanyl-
perhydrodiazo - lo[2,1 - c:2,1 - /][1,4,7,10,13,16]hexaazacy-
clohenicosin-9-yl]-12-methyltetradecanamide (8) and N1-
[(6S,9S,14aS,15S,16S,20S,23S,25aS,2R,11R) - 2,11,15 -
trihydroxy-6-((1R)-1-hydroxyethyl)-23-((1S)-1-hydroxy-
2-(4-hydroxyphenyl)-2-phenylsulfanylethyl)-20-hydroxy-
methyl-16-methyl-5,8, 14,19,22,25-hexaoxo-12-phenyl-
sulfanylperhydrodiazolo[2,1-c:2,1-/][1,4,7,10,13,16]hexa -
1
nm (8085, 557). Compound 5: H NMR: d 7.25, 7.15
0
(2ꢃd, 2H, J=8.37 Hz, D7 and D7 ), 6.82 (2ꢃd (merged),
0
2H, J=8.37 Hz, D8 & D8 ), 5.12 (br, 1H, B7), 3.42 (2ꢃs,
6H, 2 ꢃ OCH3). IR (KBr): nmax 3300–3400, 2915, 1650,
1630, 1520, 1445, 1390, 1240, 1080 cmꢁ1. ESI MS
(ES+): for C50H81N7O16; calcd 1035; found:
(M+Na)+=1058 (base peak). UV (MeOH) lmax (e
M
ꢁ1 cmꢁ1): 223, 275 nm (5526, 506).
azacyclohenicosin-9-yl]-12-methyltetradecanamide
(9).
To a stirred solution of mulundocandin (2.3 g, 2.28
mmol) in anhydrous dioxan (100 mL), under nitrogen
atmosphere were added anhydrous thiophenol (4.29 g,
38.95 mmol), and p-toluene sulfonic acid (0.23 g, 1.196
mmol) and the reaction mixture was stirred at ambient
temperature for 3 h. The workup and purification pro-
cess were similar to that described for compounds 6 and
7. Yield of the white solids 8 (1.241 g, 49.4%; mp: 189–
90 ꢀC dec.) and 9 (0.478 g, 17.5%; mp: 175 ꢀC dec.).
Methyl-2-[(6S,9S,14aS,15S,16S,20S,23S,25aS,2R,11R)-
23-((1S,2S)-1,2-dihydroxy-2-(4-hydroxyphenyl)ethyl)-
2,11,15-trihydroxy-6-((1R)-1-hydroxyethyl)-20-hydroxy-
methyl - 16 - methyl - 9 - (11 - methyltridecylcarboxamido) -
5,8,14,19,22,25-hexaoxoperhydrodiazolo[2,1-c:1/][1,4,7,-
10,13,16]hexaazacyloenicosin - 12 - ylsulfanyl]acetate (6)
and Methyl - 2 - [(6S, 9S,14aS,15S,16S,20S,23S,25aS,2-
R,11R)-2,11,15-trihydroxy-6-((1R)-1-hydroxyethyl)-23-
((1S)-1-hydroxy-2-(4-hydroxyphenyl)-2-methyloxycarbo-
nylmethylsulfanylethyl)-20-hydroxymethyl-16-methyl-9-
(11-methyltridecycarboxamido)-5,8,14,19,22,25-hexaoxo-
perhyd-rodiazolo[2,1-c:1-/][1,4,7,10,13,16]hexaazacyclo-
henicosin-12-ylsulfanyl]acetate (7). To a stirred solution
of mulundocandin (4.8 g, 5.15 mmol) in anhydrous
dioxan (150 mL), under nitrogen atmosphere, anhy-
drous methylthioglycolate (11.87 g, 111.83 mmol) and
p-toluene sulfonic acid (0.338 g, 1.758 mmol) were
added and the reaction mixture was stirred at ambient
temperature for 1.5 h. The reaction was quenched by
1
Compound 8: H NMR: d 7.58 (m, 2H,-SPh), 7.33 (t,
3H, J=2.63 Hz,-SPh), 7.2 (d, 2H, J=8.39 Hz, D7 &
0
0
D7 ), 6.8 (d, 2H, J=8.39 Hz, D8 and D8 ), 5.69 (br, 1H,
B7). IR (KBr): nmax 3400–3300, 2940, 1670, 1630, 1525,
1460, 1390, 1250, 1075 cmꢁ1. ESI MS (ES+): for
C54H81N7O15S; calcd: 1099; found: (M+Na)+=1122
(base peak). UV (MeOH) lmax (e Mꢁ1 cmꢁ1): 228, 265
1
nm (22,336, 4703). Compound 9: H NMR: d 7.58 (m,
2H, –SPh), 7.30 (t, 3H, J=3.3 Hz,-SPh), 7.18–7.25 (m,
0
5H, –SPh), 6.91 (d, 2H, J=8.4 Hz, D7 and D7 ), 6.61 (d,
0
2H, J=8.4 Hz, D8 and D8 ), 5.69 (br, 1H, B7). IR

B. Lal et al. / Bioorg. Med. Chem. 11 (2003) 5189–5198
5197
(KBr): nmax 3400–3300, 2940, 1680–1620, 1520, 1450,
of organic solvent from appropriate fractions under
vacuum followed by lyophilization provided product 12
(0.07 g, 70.5%). Yield is calculated from nearly 90%
pure starting compound. 1H NMR: d 7.2 (d, 2H,
1380, 1240, 1075 cmꢁ1
.
ESI MS (ES+): for
C60H85N7O14S2; calcd 1191; found: (M+Na)+=1214
(base peak). UV (MeOH) lmax (e Mꢁ1 cmꢁ1): 255 nm
(4892).
0
J=8.55 Hz, D7 and D7 ), 6.8 (d, 2H, J=8.55 Hz, D8
0
and D8 ), 5.04 (br, 1H, B7), 3.7–3.8 (m, 4H, 2 ꢃ OCH2),
Methyl-2-[(6S,9S,14aS,15S,16S,20S,23S,25aS,2R,11R)-
23-((1S,2S)-1,2-dihydroxy-2-(4-hydroxyphenyl)ethyl)-
2,11,15-trihydroxy-6-((1R)-1-hydroxyethyl)-20-hydroxy-
methyl - 16 - methyl - 9 - (11 - methyltridecylcarboxamido) -
5,8,14,19,22,25-hexaoxoperhydrodiazolo[2,1-c:1-/][1,4,7,10,
13,16]hexaazacyclohenicosin-12-ylsulfonyl]acetate (10).
To a stirred solution of thioether 6 (0.515 g, 0.47
mmol) in acetonitrile (35 mL) and water (35 mL) at
ambient temperature, OXONE1 (0.577 g, 0.94 mmol)
was added. After a period of 1 h TLC in 20% MeOH/
CHCl3 showed complete conversion of 6 to a more
polar product. Reaction mixture was evaporated under
reduced pressure to smaller volume (25 mL). White
solid precipitated out was filtered off, washed with water
(25 mL) dried under high vacuum to yield sulfone 10
(0.45 g, 84.9%; purity 90% by HPLC). This was used as
such without further purification. mp: 63–65 ꢀC; 1H
2.35–2.2 (m, 8H, 4 ꢃ NCH2). IR (KBr): nmax 3300–
3400, 2930, 1680–1620, 1520, 1435, 1380, 1260, 1070
cmꢁ1. ESI MS (ES+): for C54H89N9O16; calcd 1119;
found: (M+Na)+=1142 (base peak).
N1 - [(6S,9S,14aS,15S,16S,20S,23S,25aS,2R,11R) - 12 -
(1H-1,3-diazolo-1-yl)-23-((1S,2S)-1,2-dihydroxy-2-(4-hy-
droxyphenyl)ethyl)-2,11,15-trihydroxy-6-((1R)-1-hydro-
xyethyl)-20-hydroxymethyl-16-methyl-5,8,14,19,22,25-
hexaoxoperhydrodiazolo[2,1-c:2,1-/][1,4,7,10, 13,16]hex-
aazacyclohenicosin-9-yl]-12-methyltetradecanamide (13).
To a stirred solution of Orn-5-sulfone 10 (0.1 g, 0.089
mmol) in anhydrous dioxan (10 mL) under nitrogen
atmosphere, imidazole (0.024 g, 0.356 mmol) was added
and the reaction mixture was stirred at 55–60 ꢀC for 1 h.
Reaction progress was monitored by TLC (20%
MeOH/CHCl3). After 1 h the reaction mixture was
diluted with water (100 mL), extracted with n-BuOH
(3ꢃ50 mL). It was washed with water (100 mL) fol-
lowed by brine (100 mL). Combined organic extract was
dried over anhydrous Na2SO4 and was concentrated
under vacuum to give a crude product. The crude pro-
duct was purified in a similar manner as shown for
compound 12, to furnish compound 13 (0.06 g, 64%)
Yield is calculated from nearly 90% pure starting com-
pound. 1H NMR: d 7.8 (s, 1H, -Imidazole-H), 7.65 (br s,
2H, -Imidazole-H), 7.18, (d, 2H, J=8.55 Hz, D7 and
0
NMR: d 7.18 (d, 2H, J=8.58 Hz, D7 & D7 ), 6.8 (d, 2H,
0
J=8.58 Hz, D8 and D8 ), 5.6 (br, 1H, B7), 3.92–4.08 (m,
2H, –SO2CH2CO2CH3), 3.85 (s, 3H, –SO2CH2-
CO2CH3). IR (KBr): nmax 3500–3400, 2920, 2890, 1680-
1625, 1525, 1445, 1225, 1080 cmꢁ1. ESI–MS (ES+): for
C51H81N7O19S; calcd 1127; found: (M+Na)+=1150
(base peak). UV (MeOH) lmax (e Mꢁ1 cmꢁ1): 223, 276
nm (31366, 3587).
Methyl-2-[(6S,9S,14aS,15S,16S,20S,23S,25aS,2R,11R)-
2,11,15-trihydroxy-6-((1R)-1-hydroxyethyl)-23-((1S)-1-
hydroxy-2-(4-hydroxyphenyl)-2-methyloxycarbonylme-
thylsul-fonylethyl)-20-hydroxymethyl-16-methyl-9-(11-
methyltridecylcarboxamido)-5,8,14,19, 22,25-hexaoxo-
perhydrodiazolo[2,1 - c:1 - /][1,4,7,10,13,16]hexaazacyclo-
henicosin-12-ylsulfonyl]acetate (11). Compound 11
(Orn-5- and HTyr-4-disulfone mulundocandin) was
prepared from di-thioether 7, using the process outlined
for preparation of Orn-5-sulfone 10 and was used
immediately for further reactions without purification.
0
0
D7 ), 6.8 (d, 2H, J=8.55 Hz, D8 and D8 ), 5.30 (br s, 1H,
B7). IR (KBr): nmax 3350–3400, 2931, 1650, 1620, 1520,
1455, 1390, 1225, 1065 cmꢁ1. ESI MS (ES+): for
C51H79N9O15; calcd 1057; found: (M+Na)+=1080,
1012 (base peak).
N1-[(6S,9S,14aS,15S,16S,20S,23S,25aS,2R,11R)-12-azido
-23-((1R)-2-azido-1-hydroxy-2-(4-hydroxyphenyl)ethyl)-
2,11,15-trihydroxy-6-((1R)-1-hydroxyethyl)-20-hydroxy-
meth-yl-16-methyl-5,8,14,19,22,25-hexaoxoperhydrodia-
zolo[2,1-c:2,1-/][1,4,7,10,13,16] hexaazacyclohenicosin-9-
yl]-12-methyltetradecanamide (14). Using the process
outlined for the preparation of 12, a solution of Orn-5
and HTyr-4-disulfone mulundocandin (11) (0.2 g, 0.16
mmol), anhydrous sodium azide (0.104 g, 1.6 mmol) in
anhydrous dioxan (10 mL), was stirred at 45–50 ꢀC for 2
h. After usual workup, the crude product was purified
by HPLC using Semi-preparative RP-18 column (size,
250ꢃ16 mm; particle size 10 m; mobile phase 70% ace-
tonitrile/water; flow rate 8 mL/min; monitoring wave-
length l=220 and 270 nm). Removal of organic solvent
from appropriate fractions under vacuum followed by
lyophilization provided compound 14 (0.115 g, 67.8%;
mp: 175 ꢀC). Yield is calculated from nearly 90% pure
N1 - [(6S,9S,14aS,15S,16S,20S,23S,25aS,2R,11R) - 23 -
((1S,2S) - 1,2 - dihydroxy - 2 - (4 - hydroxyphenyl)ethyl) -
2,11,15-trihydroxy-6-((1R)-1-hydroxyethyl)-20-hydroxy-
methyl - 16 - methyl - 12 - (2 - morpholinoethylamino) -
5,8,14,19,22,25-hexaoxoperhydrodiazolo[2,1-c:2,1-/][1,4,
7,10,13,16]hexaazacyclohenicosin-9-yl]-12-methyltetrade-
canamide (12). To a stirred solution of Orn-5-sulfone 10
(0.1 g, 0.089 mmol) in anhydrous dioxan (10 mL) under
nitrogen atmosphere, 4-(2-aminoethyl) morpholine
(0.495 g, 3.8 mmol) was added and the reaction mixture
was stirred at 55–60 ꢀC for 1 h. The reaction mixture
was diluted with water (150 mL), extracted with n-
BuOH (3ꢃ100 mL). It was washed with water (150 mL)
followed by brine (150 mL). Combined organic extract
was dried over anhydrous Na2SO4, filtered and was
concentrated under vacuum to give crude product. The
crude product was purified by flash column chromato-
graphy over reverse-phase (RP, C-18) by using 50–90%
acetonitrile/water as 10% step gradient eluent. Removal
1
starting compound. H NMR: d 7.28, 7.14 (2ꢃd, 2H,
0
J=8.88 Hz, D7 and D7 ), 6.83 (dd merged, 2H, J=8.88
0
Hz, D8 and D8 ), 5.39 (d, 1H, J=1.86 Hz, B7). IR
(KBr): nmax 3300–3400, 2930, 2100, 1650, 1620, 1515,
1440, 1240, 1070 cmꢁ1
C48H75N13O14;
. ESI MS (ES+): for
Calculated: 1057; found:

5198
B. Lal et al. / Bioorg. Med. Chem. 11 (2003) 5189–5198
(M+Na)+=1080, 1037 (base peak). UV (MeOH) lmax
(e Mꢁ1 cmꢁ1): 221, 275 nm (8266, 1985).
6. (a) Debono, M.; Turner, W. W.; LaGrandeur, L.; Bur-
khardt, F. J.; Nissen, J. S.; Nichols, K. K.; Rodriguez, M. J.;
Zweifel, M. J.; Zekner, D. J.; Gordee, R. S.; Tang, J.; Parr,
T. R., Jr J. Med. Chem. 1995, 38, 3271. (b) Masurekar, P. S.;
Fountoulakis, J. M.; Hallada, T. C.; Sosa, M. S.; Kaplan, L.
J. Antibiot. 1992, 45, 1867. (c) Iwamoto, T.; Fujie, A.; Nitta,
K.; Hashimoto, S.; Okuhara, M.; Kohsaka, M. J. Antibiot.
1994, 47, 1092.
7. Debono, M.; Gordee, R. S. Ann. Rev. Microbiol. 1994, 48,
471.
8. Pound, M. W.; Drew, R. H.; Perfect, J. R. Curr. Opin.
Infect. Dis. 2002, 15, 183.
9. (a) Ablordeppey, S. Y.; Fan, P.; Ablordeppey, J. H.;
Mardenborough, L. Curr. Med. Chem. 1999, 6, 1151 (A
review with 537 refs). (b) Green, L. J.; Marder, P.; Mann,
L. L.; Chio, L.-C.; Current, W. L. Antimicrob. Agents Che-
mother. 1999, 43, 830. (c) Tomishima, M.; Ohki, H.; Yanada,
A.; Takasugi, H.; Maki, K.; Tawara, S.; Tanaka, H. J. Anti-
biot. 1999, 52, 674.
10. Roy, K.; Mukhopadhyay, T.; Reddy, G. C. S.; Desikan,
K. R.; Ganguli, B. N. J. Antibiot. 1987, 40, 275.
11. Hawser, S.; Borgonovi, M.; Markus, A.; Isert, D. J. Anti-
biot. 1999, 52, 305.
N1-[(6S,9S,14aS,15S,16S,20S,23S,25aS,2R,11R)-2,11,15
-trihydroxy-6-((1R)-1-hydroxy-ethyl)-23-((1R)-1-hydroxy
-2-(4-hydroxyphenyl)-2-(2-morpholinoethylamino)ethyl)-
20-hydroxymethyl-16-methyl-12-(2-morpholinoethyla-
mino)-5,8,14,19,22,25-hexaoxoper-hydrodiazolo[2,1-c:2,1-
/][1,4,7,10,13,16]hexaazacyclohenicosin - 9-yl]-12-methyl-
tetrade-canamide (15). Using the process outlined for
the preparation of 12, a solution of Orn-5- and HTyr-4-
disulfone mulundocandin (0.2 g; 0.16 mmol), 4-(2-ami-
noethyl)-morpholine (0.208 g, 1.6 mmol) in anhydrous
dioxan (10 mL), was stirred at 45–50 ꢀC for 2 h. The
crude product was purified as described for compound
14. Removal of organic solvent from appropriate frac-
tions under vacuum followed by lyophilization provided
compound 15 (0.093 g, 43.8%). Yield is calculated from
1
nearly 90% pure starting compound. H NMR: d 7.26
0
(t, 2H, J=8.55 Hz, D7 and D7 ), 6.8 (d, 2H, J=8.55 Hz,
0
D8 and D8 ), 5.04 (br, 1H, B7), 3.7–3.8 (m, 8H,
12. Bouffard, F. A.; Hammond, M. L.; Arison, B. H. Tetra-
hedron Lett. 1995, 36, 1405.
4ꢃOCH2), 2.4–2.27 (m, 16H, 8ꢃNCH2). IR (KBr): nmax
3300–3400, 2930, 1680–1620, 1520, 1435, 1380, 1260,
1070 cmꢁ1. ESI–MS (ES+): for C60H101N11O16; calcd
1231; found: (M+Na)+=1254 (base peak).
13. (a) Balkovec, J. M.; Bouffard, F. A.; Dropinski, J. F.
(Merck and Co. Inc., USA). PCT. Int. Appl. WO 9,613,272, 9
May 1996. (b) Balkovec, J. M.; Christian, R. M. (Merck and
Co., Inc.). Eur. Pat. Appl. EP 459,564, 4 Dec. 1991. (c) Bouf-
fard, F. A. (Merck and Co., Inc., USA). Eur. Pat. Appl. EP
538,002, 21 Apr. 1993. (d) Bouffard, F. A.; Dropinski J. F.
(Merck and Co., Inc. USA). Eur. Pat. Appl. EP 539,088, 28
Apr. 1993. (e) Belyk, K. M.; Bender, D. R.; Black, R. M.;
Hughes, D. L.; Leonard, W. (Merck and Co., Inc.). US
5,552,521, 03 Sep. 1996. (f) Boffard, F. A. (Merck and Co.,
Inc.). PCT, WO 96/22784, 01 Aug. 1996.
Acknowledgements
The authors wish to thank Anti-infective Research
Department, Hoechst AG, Germany, for the constant sup-
ply of Mulundocandin. We also thank Dr. Ramakrishna,
N.V.S. and his group for providing analytical support.
14. (a) Lal, B: Gund, V. G.; Gangopadhyay, A. K. (Aventis
Pharma Deutschland G.m.b.H., Germany). PCT Int. Appl.,
WO 0107468 A2 200110201, 2001. (b) Gund, V. G. Semisyn-
thetic Studies On Mulundocandin: An Antifungal Lipopeptide.
PhDThesis, Nov 2001, Indian Institute Of Technology, Bom-
bay, India.
15. (a) Zaugg, H. E. Synthesis 1970, 2, 49. (b) Zaugg, H. E.
Synthesis 1984, 85. (c) Zaugg, H. E. Synthesis 1984, 181. (d)
Speckamp, W. N.; Hiemstra, H. Tetrahedron 1985, 41, 4367.
16. Bouffard, F. A.; Zambias, R. A.; Dropinski, J. F.; Balk-
ovec, J. M.; Hammond, M. L.; Abruzzo, G. K.; Bartizal, K. F.;
Marrinan, J. A.; Powels, M. A.; Schmatz, D. M. J. Med.
Chem. 1994, 37, 222.
17. (a) Barry, A. L. Procedure and Therotical Considerations
for Testing Antimicrobial Agents in Agar Media. In Anti-
biotics in Laboratory Medicine, 3rd ed., Lorian V. ed., Wil-
liams and Wilkins: Baltimore, MD, 1991; p 16. (b) Denning,
D. W.; Radford, S. A.; Oakley, K. L.; Hall, L.; Johnson,
E. M.; Warnock, D. W. J. Antimicrob. Chemother. 1997, 40,
401.
References and Notes
1. (a) Walsh, T. J.; Jarrosinski, P. F.; Fromtling, R. A. Diagn.
Microbiol. Infect. Dis 1990, 13, 37. (b) Samonis, G.; Bafalou-
kos, D. In vivo 1992, 6, 183. (c) Pizzo, P. A.; Young, L. S. Am.
J. Med. 1984, 76, 101. (d) Walsh, T. J.; Gonzalez, C.; Lyman,
C. A.; Chanock, S. J.; Pizzo, P. A. Adv. Pediatr. Infect. Dis.
1996, 11, 187.
2. (a) Walsh, T. J. Hematol. Oncol. Clin. North Am. 1993, 7,
1003. (b) Warnock, D. W. J. Antimicrob. Chemother. 1995,
36 (Suppl B), 73. (c) Buchner, T.; Roos, N. Ann. Hematol
1992, 65, 153. (d) Dean, D. A.; Burchard, K. W. Am. J. Surg.
1996, 171, 374. (e) Khan, Z. K.; Gyanchandani, A. PINSA-B:
Proc. Indian Natl. Sci. Acad., Part B 1998, 64, 1. (f) Powderly,
W. G. J. Phys. Assoc. AIDS Care 1994, 32.
3. Gallis, H. A.; Drew, R. H.; Pickard, W. W. Rev. Inf. Dis.
1990, 12, 308.
4. Bailey, E. M.; Drakovsky, D. J.; Ryback, M. J. Pharmaco-
therapy 1990, 10, 146.
18. (a) Walsh, T. J.; Lee, J. W.; Kelly, P.; Bacher, J.; Lec-
ciones, J.; Thomas, V.; Lyman, C.; Coleman, D.; Gordee, R.;
Pizzo, P. A. Antimicrob. Agents Chemother. 1991, 35, 1321. (b)
Nawada, R.; Amitani, R.; Tanaka, E.; Niimi, A.; Suzuki, K.;
Murayama, T.; Kuze, F. J. Clin. Microb. 1996, 34, 1433. (c)
Valentin, A.; Guennec, R. L.; Rodriguez, E.; Reynes, J.; Mal-
lie, M.; Bastide, J.-M. Antimicrob. Agents Chemother. 1996,
40, 1342. (d) Bartizal, K.; Abruzzo, G.; Trainor, C.; Krupa,
D.; Nollstadt, K.; Schmatz, D.; Hammond, M.; Balkovec, J.;
Van Middlesworth, F. Antimicrob. Agents Chemother. 1992,
36, 1648.
5. (a) Fox, R.; Neal, K. R.; Leen, C. L. S.; Ellis, M. E.;
Mandal, B. K. J. Infect. 1991, 22, 201. (b) Smith, D.; Boag, F.;
Midgley, J.; Gazzard, B. J. Infect. 1991, 23, 345. (c) Hitchcock,
C. A.; Pye, G. W.; Trok, F. J.; Johnson, E. M.; Warnock,
D . W.Antimicrob. Agents Chemother. 1993, 37, 1962. (d)
Sanglard, D.; Kuchler, K.; Ischer, F.; Pagani, J.-L.; Monod,
M.; Bille, J. Antimicrob. Agents Chemother. 1995, 39, 2378. (e)
Marybeth, F.; Barrett, J. F. Expert Opin. Invest. Drugs 1998,
7, 175 (a review with 221 refs).