Semisynthetic derivatives of purpuromycin as potential topical agents for vaginal infections

Article abstract of DOI:10.1021/jm960672t

Purpuromycin (1) is an antibiotic with a broad spectrum of antimicrobial activity, encompassing bacteria, fungi, and protozoa, particularly those involved in vaginal infections. With the aim of enhancing the solubility and reducing the serum binding, a ch

Full text of DOI:10.1021/jm960672t

J . Med. Chem. 1997, 40, 967-971
967
Sem isyn th etic Der iva tives of P u r p u r om ycin a s P oten tia l Top ica l Agen ts for
Va gin a l In fection s
Aldo Trani,* Clelia Dallanoce, Gianbattista Panzone, Franca Ripamonti, Beth P. Goldstein,† and Romeo Ciabatti
Biosearch Italia S.p.A., via R. Lepetit 34, 21040 Gerenzano (VA), Italy
Received September 26, 1996X
Purpuromycin (1) is an antibiotic with a broad spectrum of antimicrobial activity, encompassing
bacteria, fungi, and protozoa, particularly those involved in vaginal infections. With the aim
of enhancing the solubility and reducing the serum binding, a chemical program of modifications
was undertaken on the natural compound, and a new interesting series of derivatives at the
naphthoquinone system was synthesized and evaluated as potential topical agents for vaginal
infections. In particular three semisynthetic derivatives, 7′-amino (8a ), 7′-methylamino (8b),
7′-ethylamino (8c), of 7′-demethoxypurpuromycin seemed to be the most promising. They were
tested for in vitro activity against three of the most important vaginal pathogens and showed
activity similar to that of purpuromycin against Candida isolates while they were significantly
more active against Trichomonas vaginalis and Gardnerella vaginalis, which are cultured in
media containing blood or serum. This is probably due to the fact that the activity of the
derivatives is less antagonized by these supplements than that of purpuromycin.
In tr od u ction
A variety of different microorganism coexist in the
normal flora of the vagina, and several other organisms
(including bacteria, fungi, and protozoa) can also colo-
nize the vagina, causing pathologies known as vaginitis
and vaginosis. Vaginosis is generally due to infection
with Gardnerella vaginalis, probably acting synergisti-
cally with obligate anaerobes such as Bacteroides and
Mobiluncus spp. Organisms commonly causing vagini-
tis include the yeast Candida albicans and the proto-
zoan Trichomonas vaginalis. Topical and/or systemic
antifungal agents may be prescribed to treat these
Candida infections, while systemic or topical metron-
idazole and topical disinfectants are used against Tri-
chomonas and anaerobic bacteria.1-3
F igu r e 1. Structure of purpuromycin (1).
ture of purpuromycin. The derivatives obtained by
modification of the ester group in position 7 and the
hydroxyl group in position 4 did not show interesting
activity.7,8 This paper deals with the synthesis and the
antimicrobial activities of the most promising series of
derivatives, obtained by introducing an amine residue
on the naphthoquinone system.
Purpuromycin (1), an antibiotic produced by filamen-
tous bacterium Actinoplanes ianthinogenes,4 contains
naphthazarin and isocoumarin moieties linked through
a spiroketal system. It has a broad spectrum of
antimicrobial activity, including fungi, bacteria, and
protozoa.5 However, in systemic animal model infec-
tions, purpuromycin showed poor activity, attributable
to low solubility and little absorption, and perhaps also
to a strong affinity for protein, as revealed by an
antagonism of its in vitro activity by serum. Because
topical therapy, including with nonspecific disinfectants,
is still widely used as an adjunct to more targeted
therapy of vaginal infections, we have been evaluating
the potentiality of purpuromycin and semisynthetic
derivatives of this antibiotic for such an indication,
while seeking to improve its bioavailability through
chemical modification.
Ch em istr y
The common reactions which usually occur on quino-
9-11
ne rings
turned out not to be feasible for purpuro-
micin (1) (Figure 1). This is probably due to the rapid
tautomerism of the naphthazarin system resulting in
the simultaneous existence of benzenoid and quinoid
properties in both rings.12 For this reason the synthesis
of 7′-amino-7′-demethoxy derivatives of purpuromycin
was accomplished by employing a route (Scheme 1) in
which 1 is first protected at the 4-hydroxyl group with
a tetrahydropyranyl ether, and this species 2 is con-
verted into the corresponding 4′,9′,10-triacetyl deriva-
tive 3 with acetic anhydride. Treatment of 3 with
pyridinium bromide perbromide yielded the 6′-bromo
derivative 4, which was deprotected at position 4 with
a catalytic amount of p-toluenesulfonic acid in methanol
to provide compound 5. 4′,9′,10-Triacetyl-6′-bromopur-
puromycin (5) was used as starting material for the
preparation of the series of amine derivatives 8a -m
(Scheme 1). The nucleophilic substitution of the 7′-
methoxy group with amines is possible due to the
presence in the ortho position of the bromine, which is
then removed by catalytic hydrogenation. This nucleo-
philic displacement works only with primary amines.
When the key intermediate 5 was submitted to reaction
with amines in wet THF or DMF at room temperature,
In general, reduction in lipophilicity and an increase
in positive charge reduce the interaction of compounds
with serum proteins.6 With the aim of increasing
solubility and reducing serum binding, a chemical
program of derivatization was undertaken on the struc-
* Corresponding author.
† Current address: Applied Microbiology Inc., 771 Old Saw Mill
River Rd, Tarrytown, NY 10591.
X Abstract published in Advance ACS Abstracts, February 1, 1997.
S0022-2623(96)00672-3 CCC: $14.00 © 1997 American Chemical Society

968 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Trani, et al.
Sch em e 1a
a
Reagents: (a) DHP/H+, THF; (b) (CH3CO)2O, py; (c) PyBr2HBr, CH2Cl2; (d) MeOH/H+; (e) R4-NH2, THF or DMF; (f) H2-5% Pd/C,
CH3COONa, THF-MeOH.
Ta ble 1. In vitro Antimicrobial Activity of Purpuromycin Derivatives (MIC, µg/mL)
strains
1
8a
8b
8c
8d
8e
8f
8g
8h
0.5
1
32
0.13
0.13
1
8i
8l
8m
Staphylococcus aureus L165
0.03
0.06
8
0.03
0.06
1
0.03
0.06
0.03
0.016 0.06
0.06
0.25
0.008 0.06
0.016 0.06
0.03
0.03
0.03
2
0.016 0.13
0.03
2
0.06
0.13
1
0.06
0.13
0.03
0.13 0.13
0.13 0.13
0.25
0.13 0.06
0.5 0.06
0.13 0.13
0.06 0.06
0.13 0.13
1
Staphylococcus aureus L165 106cfu/ml
Staphylococcus aureus L165 30% bovine serum
Staphylococcus epidermidis ATCC12228
Staphylococcus haemolyticus L602
Streptococcus pyogenes L49
0.06
1
0.13
2
1
8
1
1
2
1
2
2
0.03
0.03
0.25
0.06
0.06
0.03
0.25
0.06
0.03
8
0.016 0.016 0.13
0.06
0.13
0.03
0.06
nda
0.03
0.06
0.13
0.016 0.13
0.06
0.13
0.06
0.03
0.13
0.03
0.13
Streptococcus pneumoniae L44
Enterococcus faecalis ATCC7080
Propionibacterium acnes ATCC6919
Bacteroides fragilis ATCC25285
Neisseria gonorrhoeae L997
Haemophilus influenzae ATCC19418
Escherichia coli L47
Pseudomonas aeruginosa ATCC10145
Proteus vulgaris ATCC881
0.016 0.016 0.06
0.06 0.03 0.06
0.008 0.008 0.06
0.06
0.03
0.06
0.016 0.06
0.06
4
0.008 0.016 0.004 nda
0.06 0.016 0.5
0.03
0.06
0.13
0.06
0.06
0.06
0.06
0.06
0.06
0.5
0.06
0.13
0.13
0.03
0.06
0.5
0.25
2
0.5
0.13 0.13
0.13
0.5
>128
64
32
1
>128 >128 >128 >128 >128 >128 g128 >128 16
>128 >128
>128 >128 >128 >128 >128 >128 >128 g128 >128 128 >128 >128
32
1
>128 >128 >128 >128 >128 >128 g128 >128 128 >128 >128
32
32
Candida albicans L145
Trichophyton mentagrophytes L634
2
2
>128
8
>128 g128 >128 64
>128 >128
0.25
e0.13 e0.13 64
>128 128
g128 g128 128 >128 >128
a
nd not determined.
we observed an immediate color change of the reaction
mixture from yellow to violet due to basic hydrolysis of
the acetyl groups with formation of 6′-bromopurpuro-
mycin (6). Subsequently the nucleophilic displacement
of the 7′-methoxy group with the amine residue occurred
to yield the 6′-bromo-7′amine derivatives 7a -l. By
using 36% ammonia in THF a mixture of 6 with 7a was
recovered after 48 h at room temperature. By contrast,
when more nucleophilic amines, e.g. methylamine, were
used, the reaction was very fast, lasting minutes, and
in this case we preferred to stop the reaction, even if 6
was still present, to avoid the subsequent opening of
the lactone ring. Finally dehalogenation of 7a -l was
accomplished by hydrogenolysis with 5% Pd/C at atmo-
spheric pressure to produce the amine derivatives
8a -m .
An tim icr obia l Activity
The antimicrobial activity of purpuromycin deriva-
tives 8a -m were determined in comparison with the
parent compound 1 (Table 1). The derivatives as well
as purpuromycin had excellent activity against Gram-
positive bacteria and fastidious Gram-negative species
(Bacteroides, Neisseria, Haemophilus). Compounds
8a -m lost the activity that 1 had against Enterobac-
teriaceae (Escherichia coli and Platanus vulgaris).
None of the compounds were active against Pseudomo-
nas aeruginosa. The derivatives, with the exception of
8h and 8m , had somewhat better activity than 1 against
Streptomyces aureus in the presence of 30% bovine
serum. Only three derivatives, 8a-c were active against
C. albicans and Trichophyton mentagrophytes. These
three compounds were evaluated, in comparison with

Semisynthetic Derivatives of Purpuromycin
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 969
Ta ble 2. In vitro anti-Candida Activity of Selected Compounds
(MIC, µg/mL)
Ta ble 4. Activity of Selected Compounds against Trichomonas
vaginalis
species (n. isolates)
1
8a
8b
8c
compd
MIC, µg/mL
compd
MIC, µg/mL
C. tropicalis (2)
C. glabrata (1) 1
C. albicans (24)
MIC50
128
128
64
128
0.25-32
2
2
64-128
1
8a
8b
8-16
0.5
1
8c
metronidazole
4-8
0.25-1
32
32
0.25-2
1
2
2-128a
64
0.25-8
2
4
MIC90
128
bated in 5% CO2 in air; Propionibacterium acnes and Bacteroi-
des fragilis in nitrogen-carbon dioxide-hydrogen (80:10:10);
other organisms in air. Incubation times were 48 h for N.
gonorrhoeae, H. influenzae, P. acnes, and B. fragilis; 72 h for
T. mentagrophytes; 20-24 h for other organisms. The growth
media were as follows: Oxoid Iso-Sensitest broth for staphy-
lococci, E. fecalis, E. coli, P. vulgaris, and P. aeruginosa; Difco
Todd Hewitt broth for streptococci; Difco GC Base broth + 1%
(v/v) BBL IsoVitaleX for N. gonorrhoeae; Difco Brain Heart
Infusion broth + 1% (v/v) Difco supplement C for H. influenzae;
Wilkins-Chalgren broth (Difco) for P. acnes and B. fragilis;
phosphate-buffered Yeast Nitrogen Base broth (Difco) supple-
mented with glucose (1% w/v) and L-asparagine (0.15% w/v)
for Candida sp.; Difco YM broth for T. mentagrophytes. MICs
for G. vaginalis, shown in Table 3, were determined by an agar
dilution method in Difco Casman medium supplemented with
5% (v/v) whole rabbit blood and 0.15% (v/v) lysed rabbit blood.
Inocula were approximately 105 colony-forming units. Incuba-
tion was for 48 h at 37 °C in N2-CO2-H2 (80:10:10). MICs
for T. vaginalis (Table 4) were determined by a broth microdi-
lution method14 in 0.2 mL of Trichomonas Culture Medium
Base (Merck) + 10% horse serum in flat-bottomed microtiter
wells. The inoculum was approximately 105 cells/mL. After
48 h of incubation at 37 °C in N2-CO2-H2, the wells were
viewed directly with a microscope to detect the presence of
viable (motile) protozoa.
a
Partial inhibition of growth at lower concentrations.
Ta ble 3. Activity of Selected Compounds against 12 Strains of
Gardnerella vaginalis
compd
MIC range
MIC50
MIC90
1
8-16
8
0.06
1
2
8
0.06
2
2
8a
8b
8
0.06-13
1-2
0.5-2
1, for their in vitro activity against clinical isolates of
Candida (Table 2), against G. vaginalis (Table 3), and
against T. vaginalis (Table 4), three of the most impor-
tant vaginal pathogens. Compound 1 had excellent
activity against clinical isolates of C. albicans and was
also active against a single isolate of Candida glabrata
but not against isolates of Candida tropicalis. Among
the derivatives, 8b and 8c had anti-Candida activities
similar to that of 1, although the MICs of the derivatives
were higher for some isolates. MICs of 8a covered a
wider range, and the growth of most of the isolates was
not completely inhibited even at high concentrations of
this compound.
Against G. vaginalis, all three derivatives had activity
significantly better than that of 1. Two of the com-
pounds (8a and 8b) also had activity significantly better
than that of 1 against T. vaginalis; their MICs were
similar to that of the standard anti-Trichomonas drug,
metronidazole.
Ch em istr y: Ma ter ia ls a n d Meth od s. Evaporation of the
solvents was carried out with a rotary evaporator at 50 °C
under vacuum. HPLC analysis was used to monitor the
progress of reactions, homogeneity of chromatographic frac-
tions, and purity of the compounds using a Hewlett-Packard
1090L instrument equipped with a UV detector at 254 nm and
a Hibar LiChroCart 125-4 column, packed with LiChrospher
100 RP-18 (5 µm) injection volume, 10 µL; flow rate, 1 mL/
min; mobile phases, (A) 0.02 M NaH2PO4 (pH 4.7); (B) CH3-
CN].
8b and purpuromycin were tested for activity against
a large number of clinical isolates of anaerobic bacteria,
many of them associated with bacterial vaginosis, as
reported by Goldstein et al.13 8b was generally more
gradient 1
gradient 2
time (min)
%B in A
time (min)
%B in A
0
40
0
2
40
2
25
60
20
50
35
40
25
50
36
stop
30
1
active than purpuromycin, with MICs often
/
those
16
31
stop
of 1, whose MICs ranged from 1 to 8 µg/mL for most
species. Both 8b (MICs e 0.03 µg/mL) and purpuro-
mycin (MICs 0.25-0.5 µg/mL) were particularly active
against 20 isolates of Mobiluncus.
35
35
35
The reported tR values are relative to the tR value of
purpuromycin obtained in the same analysis. Thin layer
chromatography (TLC) was performed on precoated plates of
0.25 mm thickness and preparative plates of 1 mm thickness
(silica gel 60 F254, Merck). Column chromatography was
performed using silica gel 60 (70-230 mesh) (Merck) buffered
with KH2PO4.14 Melting points were determined on a Buchi
apparatus and are uncorrected. The compounds were ana-
lyzed for C, H, N, and halogens using samples previously dried
at 140 °C under N2. Weight loss was determined by thermo-
gravimetric analysis (TGA) at 140 °C, and inorganic residue
was determined after the samples were heated at 900 °C in
O2. The results were (0.4% of the theoretical values. LC/
MS negative and positive ion spectra were obtained on an HP
5985 B instrument; (source 250 °C, 0.9 Torr). HPLC Hewlett-
Packard 1090L equipped with Hewlett-Packard ODS Hypersil
100 × 4.6 mm, 5 µm column; eluent CH3CN/THF/H2O (70:10:
30 v/v). FAB/MS positive ion spectra were obtained on a
Kratos MS-50 instrument fitted with a standard FAB source
and a high-field magnet; the sample (10 µmol) was dispersed
in a few microliters of 2-thioglycerol-diglycerol (1:1) matrix
and bombarded with a 6-9 keV beam of Xe atoms. IR spectra
were recorded as a Nujol mull with a Perkin-Elmer 580
spectrophotometer. 1H NMR spectra were obtained with a
Bruker AM 500 instrument equipped with an Aspect 3000
The nitrogen-containing compounds 8-c were the
most active among all of the derivatives previously
obtained by chemical modification of 1 in positions 4 and
7.7,8 8-8c were insoluble in water but showed a
reduced serum antagonism (Table 1), satisfying one of
our aims, and have potentially useful activity against
pathogens associated with bacterial vaginosis, trichomo-
niasis, and candidiasis. These compounds have been
selected for further study as potential topical agents in
vaginal infections.
Exp er im en ta l Section
Micr obiologica l Ma ter ia ls a n d Meth od s. For the or-
ganisms listed in Tables 1 and 2, the minimal inhibitory
concentrations (MIC) were determined by broth microdilution
methodology.15 Inocula were approximately 104 colony-forming
units per milliliter (cfu/mL) for bacteria and Candida sp.
except for anaerobes (5 × 105 cfu/mL) and 1% (v/v) of a
suspension of mycelia and conidia for T. mentagrophytes.
Incubation was at 37 °C, except for T. mentagrophytes (30 °C).
Neisseria gonorrhoeae and Haemophils influenzae were incu-

970 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Trani, et al.
Ta ble 5. Reaction Conditions and Physical-Chemical Data for Compounds 7a -l
molar ratio:
amine/5
compd
amine
solvent
time (h)
yield (%)
anal. (C, H, Br, N)
HPLC (tR rel)a
MS, m/ za
7a
7b
7c
7d
7e
7f
7g
7h
7i
NH4OH, 36% aq
CH3NH2, 35% aq
C2H5NH2, 70% aq
C3H7NH2
CF3CH2NH2
CNCH2NH2
HOC2H4NH2
PhCH2NH2
(CH3)2NC2H4NH2
BOCNHC2H4NH2
50
1.5
1.9
7.4
14.8
5
4
5.5
2
THF
THF
THF
THF
DMF
DMF
DMF
THF
THF
DMF
48
2
37
33
84
96
70
96
98
87
92
78
C25H16BrNO12
C26H18BrNO12
C27H20BrNO12
C28H22BrNO12
C27H17BrF3NO12
C27H17BrN2O12
C27H20BrNO13
C32H22BrNO12
C29H25BrN2O12
C32H29BrN2O14
1.16b
1.12b
1.34b
2.54c
1.52b
0.91b
0.56b
2.89c
0.73c
1.58b
601, [M]- d
615, [M]- d
629, [M]- d
643, [M]- d
684, [MH]+ e
641, [MH]+ e
646, [MH]+ e
691, [M]- d
672, [M]- d
689, [MH]+ e
15
20
96
18
6
2
2
1
7l
5
a
b
d
See the Experimental Section for HPLC and MS conditions. Gradient 2. c Gradient 1. LC/MS. e FAB/MS.
Ta ble 6. Workup Conditions and Physical-Chemical Data for Compounds 8a -l
reaction
time, h
yield
(%)
anal.
(C, H, Br, N)
HPLC
compd
workup
C. c.a (CHCl3/AcOH, 99:1, v/v)
(tR rel)a
MS, m/ za
8a
8b
8c
8d
8e
8f
8g
8h
8i
2
3
4
2
1.5
3
4
1.5
2
48
50
40
34
20
35
23
62
45
C25H17NO12
C26H19NO12
C27H21NO12
C28H23NO12
C27H18F3NO12
C27H18N2O12
C27H21NO13
C32H23NO12
C29H26N2O12
0.74b
0.80b
0.92b
1.72c
1.61b
0.68b
0.44b
2.15c
0.55c
523, [M]- d
538, [MH]+ e
551, [M]- d
565, [M]- d
606, [MH]+ e
563, [MH]+ e
568, [MH]+ e
613, [M]- d
594, [M]- d
C. c. (CHCl3/MeOH, 99.5:0.5, v/v)
C. c. (CHCl3/MeOH/AcOH, 98:2:0.1, v/v)
C. c. (CHCl3/MeOH/AcOH, 98:2:0 to 96:4:0.1, v/v)
C. c. (CHCl3/MeOH/AcOH, 98:2:0 to 96:4:0.1, v/v)
C. c. (CHCl3/MeOH/AcOH, 100:0.5:0.05 to 80:20:0.1, v/v)
C. c. (CH2Cl2/MeOH, 99:1, v/v)
C. c. (CHCl3/MeOH/AcOH, 100:0:0 to 95:5:0.1, v/v)
the residue is taken up in absolute EtOH, filtered,
and washed with Et2O
8l
3
the same as 8i
74
C32H30N2O14
1.29b
667, [MH]+ e
a
b
d
See Experimental Section for C. c. (column chromatography), HPLC, and MS conditions. Gradient 2. c Gradient 1. LC/MS. e FAB/
MS.
Ta ble 7. 1H NMR (δ, ppm) Data for Purpuromycin and Compounds 7a -l and 8a -l
3-H
3′-H
(d), (d)
7,7′OCH3 4-H 6′-H
(s), (s) (m) (s)
5,6-H
(s), (s)
10-H 9′-H 4′-H
(s) (s) (s)
compd (m), (m)
substituents
1
2.52, 2.79 3.43, 3.62 3.87, 3.89 4.95 6.39 7.50, 7.75 10.66 11,87 13.10
7a
7b
7c
7d
7e
7f
7g
7h
7i
2.51, 2.78 3.43, 3.63 3.88
2.51, 2.76 3.42, 3.62 3.86
2.50, 2.77 3.42, 3.62 3.87
2.50, 2.77 3.41, 3.61 3.87
2.51, 2.77 3.43, 3.63 3.88
2.52, 2.75 3.42, 3.64 3.86
2.49, 2.78 3.42, 3.59 3.85
2.46, 2.73 3.32, 3.53 3.87
2.52, 2.78 3.44, 3.63 3.86
4.95
4.94
4.95
4.95
4.96
4.95
4.94
4.94
4.96
7.51, 7.76 10.66 11.50 13.50 7.95 (2H, s)
7.49, 7.74 10.62 11.40 13.61 3.13 (3H, d); 7.70 (1H, q)
7.50, 7.74 10.54 11.38 13.60 1.22 (3H, t); 3.81 (2H, q); 7.61 (1H, t)
7.50, 7.74 nd
12.70 13.63 0.89 (3H, t); 1.62 (2H, q); 3.75 (2H, q); 7.60 (1H, t)
7.50, 7.75 8.30 10.65 13.22 4.65 (2H, m); 5.85 (1H, t)
7.51, 7.77 10.66 11.41 13.21 4.65 (2H, d); 7.73 (1H, t)
7.50, 7.75 nd
7.49, 7.73 nd
7.50, 7.74 nd
nd
nd
nd
13.55 3.61 (2H, m); 3.65 (2H, m); 7.33 (1H, t)
nd
nd
5.05 (2H, s); 7.24-7.41 (5H, m); 7.87 (1H, t)
2.84 (3H, s); 2.86 (3H, s); 3.36 (2H, m);
4.10 (2H, m); 7.58 (1H, t)
7l
2.50, 2.79 3.42, 3.65 3.87
4.96
7.51, 7.78 10.68 11.47 13.55 1.34 (9H, s); 3.22 (2H, m); 3.87 (2H, m);
7.00 (1H, t); 7.55 (1H, t)
8a
8b
8c
8d
8e
8f
8g
8h a
8i
2.49, 2.77 3.39, 3.60 3.87
2.50, 2.78 3.41, 3.61 3.88
2.49, 2.77 3.40, 3.60 3.87
2.48, 2.76 3.40, 3.60 3.87
2.50, 2.77 3.41, 3.61 3.86
2.50, 2.76 3.42, 3.62 3.88
2.49, 2.76 3.40, 3.60 3.87
2.50, 2.77 3.39, 3.59 3.86
2.50, 2.78 3.42, 3.61 3.87
4.96 5.73 7.50, 7.74 10.70 11.57 14.04 7.45 (2H, broad)
4.97 5.53 7.51, 7.76 10.68 11.55 14.19 2.82 (3H, d); 7.93 (1H, q)
4.96 5.59 7.50, 7.74 nd
nd
14.15 1.16 (3H, t); 3.32 (2H, q); 7.79 (1H, t)
4.96 5.60 7.50, 7.74 10.61 11.55 14.13 0.90 (3H, t); 1.59 (2H, m); 3.16 (2H, q); 7.82 (1H, t)
4.95 6.02 7.50, 7.75 10.64 11.53 13.77 4.14 (2H, m); 7.93 (1H, t)
4.96 5.90 7.50, 7.75 10.65 nd
4.95 5.66 7.50, 7.75 10.65 11.50 14.14 3.28 (2H, m); 3.58 (2H, m); 7.61 (1H, t)
4.95 5.50 7.50, 7.74 nd nd nd 4.47 (2H, d); 8.39 (1H, t)
nd
4.47 (2H, d); nd (1H, t)
4.97 5.82 7.51, 7.75 10.55 11.50 13.93 2.80 (6H, s); 3.31 (2H, m); 3.63 (2H, m);
7.86 (1H, t)
4.97 5.69 7.51, 7.77 10.65 11.55 14.12 1.36 (9H, s); 3.14 (2H, m); 3.23 (2H, m);
7.01 (1H, t);7.83 (1H, t)
8l
2.49, 2.78 3.43, 3.60 3.87
a
CD3SOCD3 + CF3COOD.
console at 500 MHz. The spectra were recorded at 40 °C in
DMSO-d6 or CDCl3 solution (internal standard TMS, δ 0.00
ppm).
product (8.2 g, 70% yield): TLC Rf 0.35 (chloroform-methanol,
98:2, v/v); HPLC tR rel 2.7 (gradient 2); IR 3600-3100, 1735,
1
1690, 1605, 1250 cm-1; H NMR (DMSO-d6) δ 1.45-1.75 (m,
4-O-(Tetr a h yd r o-2H-p yr a n -2-yl)p u r p u r om ycin (2). Di-
hydropyran (50 mL, 0.548 mol), camphorsulfonic acid (500 mg,
2.15 mmol), and molecular sieve 4Å (50 g) were added to a
stirred solution of purpuromycin (1) (10 g, 0.0186 mol) in
anhydrous CH2Cl2, and the mixture was heated at reflux for
2 h. After the reaction mixture was cooled to 0 °C, it was
washed with 0.5% chilled aqueous NaHCO3 (1 L) and water
(1 L) and dried (Na2SO4). The solvent was concentrated under
reduced pressure, and the residue was suspended in ether,
filtered, and washed with the same solvent to afford the
6H), 2.70 and 2.82 (2m, 2H), 3.41 and 3.61 (2d, 2H), 3.59-
3.63 (m, 2H), 3.87 and 3.89 (2s, 6H), 4.95-5.05 (m, 1H), 5.01
(m, 1H), 6.39 (s, 1H), 7.50 and 7.80 (2s, 2H), 10.68 (s, 1H),
11.88 (s, 1H), 13.12 (s, 1H) anomeric mixture; MS (LC) m/ z
622 [M]+. Anal. (C31H26O14) C, H.
4′,9′,10-Tr ia cet yl-4-O-(t et r a h yd r o-2H -p yr a n -2-yl)p u r -
p u r om ycin (3). A mixture of the tetrahydropyranyl ether 2
(2.07 g, 3.33 mmol), anhydrous pyridine (3 mL), and acetic
anhydride (50 mL) was stirred overnight at room temperature
under an atmosphere of argon. The reaction mixture was

Semisynthetic Derivatives of Purpuromycin
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 971
poured into ice water (300 mL) with stirring and extracted
with ethyl acetate (3 × 200 mL). The ethyl acetate extract
was washed with 0.5% aqueous NaHCO3 (1 × 200 mL) and
water (3 × 200 mL) and dried over anhydrous Na2SO4. After
removal of solvent, the solid residue was washed with ether,
filtered, and dried in vacuo to give pure 3 (2.1 g, 85% yield):
mp 160 °C dec; HPLC tR rel 1.9 (gradient 2); 1H NMR (DMSO-
d6) δ 1.45-1.75 (m, 6H), 2.18 (s, 3H), 2.26 (s, 3H), 2.35 (s, 3H),
2.75 and 2.88 (2m, 2H), 3.50-3.60 (m, 2H), 3.51 and 3.72 (2d,
2H), 3.79 and 3.86 (2s, 6H), 4.95-5.12 (m, 1H), 5.03 (m, 1H),
6.12 (s, 1H), 7.76 and 7.94 (2s, 2H) anomeric mixture; MS (LC)
m/ z 748 [M]+. Anal. (C37H32O17) C, H.
ladium on activate carbon (300 mg). This reaction mixture
was hydrogenated at room temperature and atmospheric
pressure until the starting material disappeared and then was
filtered on Celite (filter aid) and acidified with 0.1 N HCl. After
removal of solvent, the residue was worked up to afford
compounds 8a -l (Table 6, workup conditions and physical-
1
chemical data; Table 7, H NMR data).
7′-[(2-Am in oet h yl)a m in o]-7′-d em et h oxyp u r p u r om y-
cin Tr iflu or oa ceta te (8m ). A solution of 8l (175 mg, 0.262
mmol) and trifluoroacetic acid (1.75 mL) in dichloromethane
(35 mL) was stirred at room temperature for 3 h with
progressive precipitation of the product. The solvent was
evaporated, and the residue was taken up in ether, filtered,
and vacuum dried to afford 8m (170 mg, 95% yield): HPLC tR
rel 0.62 (gradient 1); IR 3500-3100, 1710 (shoulder), 1682,
4′,9′,10-Tr ia cetyl-6′-br om o-4-O-(tetr a h yd r o-2H-p yr a n -
2-yl)p u r p u r om ycin (4). To compound 3 (4.15 g, 5.54 mmol)
in methylene chloride (500 mL) were added pyridinium
bromide perbromide (3.5 g, 0.011 mmol) and pyridine (2.5 mL).
The reaction mixture was stirred overnight at 30 °C, washed
with aqueous Na2S2O5 (2 × 200 mL) and water (2 × 200 mL),
and dried over anhydrous Na2SO4. The solvent was evapo-
rated by adding toluene (50 mL) to eliminate pyridine. To the
solid residue was added ether, and the precipitate was filtered,
rinsed with ether, and vacuum dried to afford 4 (4.3 g, 94%
1589, 1522, 1331, 1229, 1204, 1144, 1003, 943, 798, 721 cm-1
;
1H NMR (DMSO-d6) δ 2.50 and 2.77 (2m, 2H), 3.02 (m, 2H),
3.41-3.49 (m, 2H), 3.42 and 3.62 (2d, 2H), 3.87 (s, 3H), 4.97
(m, 1H), 5.79 (s, 1H), 7.51 and 7.76 (2s, 2H), 7.78 (broad, 4H),
phenolic protons not determined; MS (FAB) m/ z 567 [MH]+.
Anal. (C27H22N2O12‚CF3CO2H) H; C: calcd, 51.18; found, 51.58;
N: calcd, 4.12; found, 4.05.
1
yield): HPLC tR rel 2.73 (gradient 2); H NMR (DMSO-d6) δ
Ack n ow led gm en t. The authors thank Dr. Pietro
Ferrari, Dr. Marina Galimberti, and Dr. Marino Nebu-
loni, of the Analytical Sciences Department, for deter-
mination of 1H NMR, mass spectra, and elemental
analysis, respectively.
1.45-1.80 (m, 6H), 2.19 (s, 3H), 2.26 (s, 3H), 2.38 (s, 3H), 2.65
and 2.79 (2m, 2H), 3.42 and 3.59 (2d, 2H), 3.50-3.60 (m, 2H),
3.87 and 4.09 (2s, 6H), 4.96 (m, 1H), 5.04 (m, 1H), 7.75 and
7.94 (2s, 2H) anomeric mixture; MS (LC) m/ z 825 [M]-. Anal.
(C37H31BrO17) C, H, Br.
4′,9′,10-Tr ia cetyl-6′-br om op u r p u r om ycin (5). A mixture
of 4 (30 g, 36 mmol) and catalytic p-toluenesulfonic acid (300
mg) in methanol (3 L) was heated to reflux for 5 h. The
reaction solution was concentrated to a small volume, and the
resulting precipitate was filtered, washed with ether, and
vacuum dried to give 5 (24.2 g, 90% yield): 2.19 (s, 3H), 2.26
Refer en ces
(1) Lossick, J . G.; Kent, H. L. Trichomoniasis: Trends in Diagnosis
and Management. Am. J . Obstet. Gynecol. 1991, 165, 1217-
1222.
(2) Adamson, G. D. Three-day Treatment of Vulvovaginal Candidi-
asis. Am. J . Obstet. Gynecol. 1988, 158, 1002-1005.
(3) Tobin, J . M.; Loo, P.; Granger, S. E. Treatment of Vaginal
Candidosis: A Comparative Study of the Efficacy and Accept-
ability of Itraconazole and Clotrimazole. Genitourin. Med. 1992,
68, 36-38.
1
(s, 3H), 2.38 (s, 3H), 1.15 (gradient 2); H NMR (DMSO-d6) δ
2.15 (s, 3H), 2.32 (s, 3H), 2.35 (s, 3H), 2.78 and 2.81 (2m, 2H),
3.47 and 3.71 (2d, 2H), 3.88 and 4.10 (2s, 6H), 5.01 (m, 1H),
7.78 and 7.94 (2s, 2H). Anal. (C32H23BrO16) C, H, Br.
6′-Br om opu r pu r om ycin (6) an d 6′-Br om o-7′-dem eth oxy-
7′-a m in op u r p u r om ycin (7a ). To a stirred solution of 5 (300
mg, 0.403 mmol) in tetrahydrofuran (100 mL) was added 36%
NH4OH (1 mL), and the mixture was stirred at room temper-
ature for 48 h. The reaction mixture was then neutralized
with 1 N HCl and the solvent evaporated in vacuo. The crude
material obtained was purified by flash column chromatogra-
phy eluted with chloroform-methanol (99:1 v/v) to afford pure
6 (100 mg, 40% yield) and 7a (90 mg, 37% yield). 6: HPLC tR
rel 1.83 (gradient 2); 1H NMR (DMSO-d6) δ 2.51 and 2.79 (2m,
2H), 3.45 and 3.64 (2d, 2H), 3.87 and 4.17 (2s, 6H), 4.95 (m,
1H), 7.50 and 7.75 (2s, 2H), phenolic protons not determined;
MS (LC) m/ z 616 [M]-. Anal. (C26H17BrO13) C, H, Br. 7a :
HPLC tR rel 1.16 (gradient 2); 1H NMR (DMSO-d6) δ 2.51 and
2.78 (2m, 2H), 3.43 and 3.63 (2d, 2H), 3.88 (s, 3H), 4.95 (m,
1H), 7.51 and 7.76 (2s, 2H), 7.95 (s, 2H), 10.66 (s, 1H), 11.50
(4) Coronelli, C.; Pagani, H.; Bardone, M. R.; Lancini, G. C.
Purpuromycin, a New Antibiotic Isolated from Actinoplanes
ianthinogenes n. sp. J . Antibiot. 1974, 27, 161-168.
(5) Parenti, F.; Goldstein, B.; Simioni, L. (Gruppo Lepetit S.p.A) Use
of Purpuromycin in the Treatment of Vaginal Infections and
Pharmaceutical Composition Containing Purpuromycin. Euro-
pean Patent 389924.
(6) Pitkin, D. H.; Mico, B. A.; Sitrin, R. D.; Nisbet, L. J . Charge
and Lipophilicity Govern the Pharmacokinetics of Glycopeptide
Antibiotics. Antimicrob. Agents Chemother. 1986, 29, 440-444.
(7) Trani, A.; Kettenring, J .; Ripamonti, F.; Goldstein, B.; Ciabatti,
R. Chemical Modifications of the Antibiotic Purpuromycin. Il
Farmaco 1993, 48 (5), 637-651.
(8) Trani, A.; Dallanoce, C.; Ferrari, P.; Goldstein, B.; Ripamonti,
F.; Ciabatti, R. Syntheses and Antimicrobial Activities of 4-pur-
puromycin Derivatives. Accepted for publication in Il Farmaco.
(9) Fieser, L. F.; Turner, R. B. The Addition of Sulfhydryl Deriva-
tives to 2-Methyl-1,4-naphthoquinone. J . Am. Chem. Soc. 1947,
69, 2335-2338.
(s, 1H), 13.50 (s, 1H); MS (LC) m/ z 601 [M]-. Anal. (C25H16
-
(10) The Chemistry of the Quinonoid Compounds. Part II. Patai 1974,
1079.
BrNO12) C, H, N, Br.
Gen er a l Meth od A for Der iva tives 7b-l. To a stirred
solution of 5 (743 mg, 1 mmol) in the solvent (100 mL) was
added the amine, and the mixture was stirred at room
temperature until the reaction was complete. The mixture was
treated with 1 N HCl up to color change from violet to red,
and then water was added under stirring until complete
precipitation of the product. The suspension was centrifuged
(4500 rpm, 10 °C, 10 min), and the precipitate was separated
from the supernatant and dissolved in acetone. The solvent
was evaporated, and the residue was taken up in ethanol
absolute, filtered, washed with ether, and vacuum dried to
afford compounds 7a -l (Table 5, reaction conditions and
(11) Huisman, H. O. Investigations on Quinones and Quinone-
derivatives. Recl. Trav. Chim. Pays-Bas 1950, 69, 1133-1156.
(12) Moore, R. E.; Scheuer, P. J . Nuclear Magnetic Resonance Spectra
of Substituted Naphthoquinones. Influence of Substituents on
Tautomerism, Anisotropy, and Stereochemistry in the Napht-
hazarin System. J . Org. Chem. 1966, 31, 3272-3283.
(13) Goldstein, B. P.; King, A.; Ripamonti, F.; Trani, A.; Phillips, I.
In-vitro Activity of Purpuromycin and MDL 63,604 against
Microorganisms that Cause Vaginitis and Vaginosis. J . Anti-
microb. Chemother. 1995, 36, 1061-1065.
(14) Bardone, M. R.; Martinelli, E.; Zerilli, L.; Coronelli, C. Structure
Determination of Purpuromycin, a new Antibiotic. Tetrahedron
1974, 30, 2747-2754.
(15) National Committeee for Clinical Laboratory Standards. Ap-
proved standart M7-A2. Methods for dilution antimicrobial
susceptibility tests for bacteria that grow aerobically, 2nd ed.;
National Committee for Clinical Laboratory Standards: Vill-
anova, (PA), Italy, 1990.
1
physical-chemical data; Table 7, H NMR data).
Gen er a l Meth od B for Der iva tives 8a -l. To a stirred
solution of compound 7 (1 mmol) in a mixture of tetrahydro-
furan (100 mL) and methanol (25 mL) were added under an
argon atmosphere sodium acetate (0.5 mmol) and 5% pal-
J M960672T