A concise, stereocontrolled synthesis of spectinomycin

Article abstract of DOI:10.1016/j.tetasy.2006.03.035

A simple and concise synthesis of the antibiotic spectinomycin is described. The key step comprises a reaction cascade initiated by the β-selective 5-O-glycosylation of an N,N-blocked myo-1,3-inosadiamine 10 with a suitable actinospectosyl donor-the d-glu

Full text of DOI:10.1016/j.tetasy.2006.03.035

Tetrahedron: Asymmetry 17 (2006) 1120–1124
A concise, stereocontrolled synthesis of spectinomycinI
Eckehard Cuny and Frieder W. Lichtenthaler*
Clemens-Scho¨pf-Institut fu¨r Organische Chemie und Biochemie, Technische Universita¨t Darmstadt, D-64287 Darmstadt, Germany
Received 20 March 2006; accepted 27 March 2006
Abstract—A simple and concise synthesis of the antibiotic spectinomycin is described. The key step comprises a reaction cascade initiated
by the b-selective 5-O-glycosylation of an N,N-blocked myo-1,3-inosadiamine 10 with a suitable actinospectosyl donor—the D-glucose-
derived enol benzoate of 4,6-dideoxy-a-D-hex-3-enulosyl chloride 5—which is spontaneously followed by regio- and stereospecific cyclo-
hemiketalization and a 3-O!2-O-benzoyl group migration to directly elaborate the cis–cisoid–trans-fused pyran–dioxane–cyclohexane
framework of 1. The approach is flexible enough to be applied to other inosadiamines towards the generation of novel spectinomycin
analogues.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
to N,N-methylated 1,3-diamino-myo-inositol actinamine
3 by both a b-glycosidic and a hemiketal linkage to
The broad spectrum antibiotic spectinomycin 12 can be
perceived, in formal terms, to be a pseudodisaccharide in
which a tricarbonyl sugar, the 4,6-dideoxy-D-glycero-
hexose-2,3-diulose 2 designated actinospectose, is fused
form
a
pyran–dioxane–cyclohexane system in cis–
cisoid–trans arrangement. Whilst actinamine 3 can readily
be secured by a variety of syntheses,3 as well as by acid
hydrolysis of spectinomycin,2b the actinospectose portion
OH
OH
O
O
NHMe
MeHN
O
OH
O
O
OH
O
O
NHMe
HO
O
Me
NHMe
OH
Me
Spectinomycin 1
cis-cisoid-trans
O
NHMe
OH
OH
O
O
HO
HO
OH
O
Me
O
OH
NHMe
Me
OH
2
3
(actinospectose)
(actinamine)
q Part 39 of the series, Sugar-derived building blocks’. Part 38, see Ref. 1.
*
Corresponding author. Fax: +49 6151 166674; e-mail: lichtenthaler@chemie.tu-darmstadt.de
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.03.035

E. Cuny, F. W. Lichtenthaler / Tetrahedron: Asymmetry 17 (2006) 1120–1124
1121
does not survive the harsh acidic conditions required for
OBz
MeN
Z
cleaving the bis-glycosidic linkage (6 M HCl, 6 h reflux)
and is left as an intractable tar.
O
HO
HO
OH
4
5
+
6
Me
O
Cl
NMe
Z
A number of actinospectose derivatives have been obtained
in crystalline form by synthetic means, such as the b-di-
benzoate of its enol form 4 and the respective a-chloride
5, which can serve as an actinospectosyl donor substrate.
Under Koenigs–Knorr conditions, it not only reacts with
alcohols to smoothly give the respective b-glycosides,4
but also with 1,2-diols in a unique cascade reaction: the
essentially b-specific mono-O-glycosylation of one of the
OH groups of glycol, for example, is spontaneously
followed by cyclo-hemiketalization of the other with the
2-carbonyl, thereby generating a sterically favourable
disposition for the ensuing 3-O!2-O-benzoyl migration
(arrows in 6), which liberates the 3-carbonyl group
(Scheme 1). Of the two possible products 8 and 9, only
the cis-linked 9 is formed (TLC, 1H NMR, 71% isol.
yield4), obviously due to the favourable thermodynamics
derived from the operation of two vicinal anomeric effects:
the OH!C@O attack occurs from the lower (axial) face of
the pyran ring to product 9, in which electronic interactions
are minimized by the trans-diaxial disposition of the pyra-
noid ring oxygen and the acetalic benzoyloxy group.5
OH
10
5
a
O
O
MeN
Z
OBz
O
OH
4
5
6
Me
O
NMe
Z
OH
11
b
O
O
c
MeN
Z
OH
O
OH
4
5
6
Me
O
NMe
Z
OH
The essentially complete stereoselection in this cyclo-hemi-
ketal ‘folding’, followed by the benzoyl shift-induced liber-
ation of the enolester-blocked carbonyl, can be similarly
observed on reaction of 5 with (R,R)-1,2-cyclohexanediol6
or the steroidal diol gomphogenine, which led to the first
total synthesis of the gomphosides.6 In analogous fashion,
12
1
Scheme 2. Reagents and conditions: (a) Ag aluminosilicate (1.2 equiv),
toluene, 80 ꢁC, 3 h, 51%; (b) K2CO3, MeOH, 25 ꢁC, 10 min, 89%; (c) H2,
5% Pd/C, iPrOH, 25 ꢁC, 12 h, 90%, Z = benzyloxycarbonyl.
O
Ph
HO
OBz
O
OBz
H
O
O
O
O
HCl/AcCl
74%
HO
Ag2CO3
Me
O
OBz
Me
O
Me
O
Cl
O
6
4
5
61%
71%
X
O
O
O
OBz
OBz
OBz
O
O
BzO
Me
Me
O
O
Me
O
O
7
4 steps
55%
OBz
Me OBz
O
O
D-Glucose
O
O
O
O
O
O
Me
Scheme 1. Generation of actinospectosyl donor 5 from D-glucose and reaction with glycol.

1122
E. Cuny, F. W. Lichtenthaler / Tetrahedron: Asymmetry 17 (2006) 1120–1124
coupling of the actinospectosyl donor 5 with a suitably
blocked actinamine was surmised to entail a comparatively
simple synthetic access to spectinomycin 1, inasmuch as
previous synthetic efforts required nine steps from L-glu-
cose (overall yield 3%)7 or 23 from D-glucose8 (ꢀ1%9).
Herein we report a new, 10-step sequence of 1 from D-glu-
cose in 10.2% overall yield, which proceeds in a particularly
concise manner and proceeds along lines possibly relevant
to its biosynthesis.10
promoted with Ag2CO3. After substantial experimentation
with various insoluble silver catalysts—the more reactive
soluble promoters, such as silver triflate, were excluded as
they entail a-selectivity—silver alumosilicate13 in THF/
CH2Cl2 or in toluene was found to effect b-selective O-gly-
cosylation of 10 with donor 5 to give 11 as the major prod-
uct, isolable in 51% yield after the removal of 4-O- and 6-O-
glycosylated analogues (ca. 5% each) by preparative HPLC
(Scheme 2). The integrity of 11 to be the 20-O-benzoyl deriv-
ative of N,N-bis-(benzyloxycarbonyl)spectinomycin in its
cis–cisoid–trans-fused tricyclic framework clearly followed
from its spectral data, its ready de-O-benzoylation to the
known8,14 Z-blocked 12 as well as from its hydrogenolysis
to spectinomycin 1, which was found to be identical to the
natural product in all respects.
2. Results and discussion
After elaborating an improved procedure for the acquisi-
tion of actinospectosyl donor 5 from D-glucose—the three
steps from hydroxyglucal ester 711 were merged into one
continuous operation—its glycosidation with the readily
accessible12 N,N-bis(benzyloxycarbonyl)actinamine 10 was
probed with the reasonable7,8 expectation that the bulky
N-protecting groups would impede glycosylation at the vic-
inal hydroxyls through steric shielding. However, the accep-
tor reactivity of the 3-OH of 10 proved to be considerably
lower than that in cyclohexane-1,2-diol or in gomphoge-
nine—seemingly due to the high oxygen substitution in its
cyclohexane ring—entailing sluggish glycosylations when
Whilst the regio- and b-selectivity of the glycosylation reac-
tion is already remarkable, the stereocontrol exercised in
the hemiketal folding of the glycosidulose intermediate is
even more so as it leads from a product with two stereo-
genic centres to one with nine altogether: enjoying the
thermodynamics derived from two anomeric effects and
the propensity to form the sterically most favourable linear-
fused tricycle, the glycosidulose-C@O in 13 is attacked with
high, or exclusive, preference by the 5R-OH from the axial
O
O
Ph
Ph
O
O
NMe
NMe
Z
Z
a
H
O
H
O
5 + 10
O
O
OH
OH
6
5
4
5
4
6
Me
O
O
Me
O
O
NZ
Me
NZ
Me
OH
OH
13a
13b
X
X
X
O
NMe
Z
O
O
NMe
O
O
NMe
Z
Z
O
NMe
Z
OBz
OBz
OBz
OBz
O
OH
O
OH
O
OH
O
OH
6
5
4
5
4
5
6
5
4
6
6
4
Me
O
O
NMe
Z
Me
O
NMe Me
Z
O
NMe
Z
Me
O
O
NMe
Z
OH
OH
OH
OH
O
Z
MeN
OBz
O
OH
Z
O
BzO
Z
Me
ZN
O
NMe
Z OH
MeN
O
Me
O
O
OH
O
O
Me
Z
NMe
NMe
Me
O
O
O
O
H
NMe
Z
O
O
HO
BzO
OH
O
MeN
O
OBz
Z
OH
Me
Me
11
14
trans, transoid, trans
15
cis, transoid, trans
16
trans, cisoid, trans
cis, cisoid, trans
Scheme 3. Unique stereocontrol in the intramolecular hemiketal folding of glycosidulose intermediate 13 towards the cis,cisoid,trans-fused framework of
spectinomycin.

E. Cuny, F. W. Lichtenthaler / Tetrahedron: Asymmetry 17 (2006) 1120–1124
1123
(lower) face of the pyran ring, elaborating 11 with a pyra-
noid ring oxygen and ketalic OBz in the favourable trans-
diaxial disposition, as well as the chair conformations of
the three rings (Scheme 3). In contrast, the alternate possi-
bilities—4R-OH attack from the b-(equatorial) face
(13a!14) or of the 6S-OH in the two conceivable steric
modes (13b!15 or 16)—invariably lead to thermodynam-
ically less stable products because the central dioxane ring
is forced into a boat conformation (14 and 15), or unfa-
vourable dipolar interactions are operative (14 and 16).
O-benzoyl-2-benzoyloxy-6-deoxy-D-glucal 711 in toluene
(200 mL) that contained 3 mL of water. Upon reaching a
pale yellow solution (5–10 min), stirring was continued at
room temperature (20 min), followed by evaporation to
dryness in vacuo. The resulting amorphous residue, con-
sisting of the anomeric ulose hydrates (mainly the b-ano-
mer, Rf = 0.12–0.24 in CHCl3/CH2Cl2 5:1) and about
10% of the manno-1,2-dichloride (Rf = 0.77),4 was directly
subjected to elimination of benzoic acid by dissolution in
benzene (200 mL), addition of NaHCO3 (15 g) and water
(5 mL) and heating at reflux with stirring for 1.5 h. After
cooling, MgSO4 was stirred into the mixture (30 min) and
the inorganic salts were filtered off. Evaporation to dryness
in vacuo followed by two co-evaporations from ethanol left
a syrup, which crystallized from ethanol (30 mL) on stand-
The ease with which the glycosidulose intermediate sponta-
neously ‘folds’ into the natural cis–cisoid–trans geometry
can be taken as evidence for such a process to be operative
in non-enzyme mediated fashion in the biosynthesis of
spectinomycin from D-glucose,15 with an unprotected form
of glycosidulose 13 conceivably being the decisive
intermediate.
ing overnight in a refrigerator: 3.95 g (51%) of enolone di-
22
benzoate 4 as colourless needles; mp 82–83 ꢁC, ½aꢂD ¼ ꢁ89
(c 1, CHCl3), and 1H NMR data corresponded to literature
values.4 A second crop (0.78 g, 10%) was secured from the
mother liquor; the total yield for 7!4: 4.73 g (61%).
3. Conclusion
4.3. 3-O-Benzoyl-a-D-actinospectosyl chloride 5 [syn 3-O-
benzoyl-4,6-dideoxy-a-D-glycero-hex-3-enopyranosulosyl
chloride and (2R,6R)-2-chloro-4-benzoyloxy-6-methyl-2H-
pyran-3(6H)-one]
In conclusion, the chemistry detailed herein defines a con-
cise and general method for the construction of products
with
a cis–cisoid–trans-interconnected pyran–dioxane–
cyclohexane framework, such as the antibiotic spectino-
mycin. The key feature of the methodology is the selective,
insoluble silver salt-promoted mono-O-glycosylation of the
central OH of the 1,2,3-triol segment of the actinamine
aglycone by the glucose-derived 4,6-dideoxy-2-ketohexosyl
donor—an approach that has enough flexibility to be ap-
plied to other inosadiamine aglycones towards the genera-
tion of novel spectinomycin analogues.
Enolone dibenzoate 4 (2.40 g, 6.8 mmol) was gradually
added with stirring to 65 mL of acetyl chloride presatu-
rated with HCl gas and the mixture was allowed to stand
at room temperature for 12 h, followed by evaporation in
vacuo. The resulting syrup was co-evaporated twice from
benzene for removal of traces of acetyl chloride and subse-
quently crystallized by dissolution in ether and addition of
n-pentane to turbidity. After standing at 0–5 ꢁC for 1 day,
the colourless crystals were collected: 1.15 g (63%) of 5; mp
90–92 ꢁC, ½aꢂD ¼ þ151 (c 1, CHCl3); H NMR data corre-
sponded to the literature values.4 A second crop (600 mg,
11%) was similarly isolated from the mother liquor; total
yield: 1.75 g (74%).
22
1
4. Experimental
4.1. General
Melting points, determined with a Bock hot-stage micro-
scope, are uncorrected. Optical rotations were measured
on a Perkin–Elmer 241 polarimeter at 20 ꢁC using a cell
of 1 dm path length; concentration (c) in g/100 mL and
4.4. 20-O-Benzoyl-N,N-bis(benzyloxycarbonyl)-spectino-
mycin 11
1
solvent are given in parentheses. H and 13C NMR spectra
A slurry of N,N0-bis(benzyloxycarbonyl)-actinamine 1012
(145 mg, 0.3 mmol), silver alumosilicate catalyst13
(180 mg, 0.6 mmol) and freshly desiccated molecular sieves
were recorded on a Bruker ARX-300 spectrometer in the
solvents given. Mass spectra were acquired on Varian
MAT 311 and MAT 212 spectrometers. Microanalyses
were determined on a Perkin–Elmer 240 elemental ana-
lyzer. Analytical thin layer chromatography (TLC) was
performed on precoated Merck plastic sheets (0.2 mm silica
gel 60 F254) with detection by UV (254 nm) and/or by
spraying with H2SO4 (50%), and heating. Column and flash
chromatography were carried out on Fluka silica gel 60
(70–230 mesh) using the specified eluents.
˚
4 A (1 g) in toluene (5 mL) was stirred at ambient temper-
ature for 15 min. 3-O-Benzoyl-actinospectosyl chloride 5
(160 mg, 0.6 mmol) was then added, and the mixture re-
fluxed for 2.5 h with the exclusion of light and moisture,
whereafter 10 (Rf = 0.01, CHCl3/acetone 9:1) had been
fully consumed in favour of a major spot at Rf = 0.15
and two minor ones (ca. 5% each) at 0.20 and 0.23. Filtra-
tion through Kieselgur, washing with CH2Cl2 and evapora-
tion of the combined filtrates to dryness in vacuo left a
residue, which was subjected to HPLC (Lichrosorb RP-
18, 5 lm, MeOH/water 60:40, 1 mL/min). Removal of
4.2. 1,3-Di-O-benzoyl-b-D-actinospectose 4 [syn 1,3-di-O-
benzoyl-4,6-dideoxy-b-D-glycero-hex-3-enopyranos-2-ulose
or (2S,6R)-2,4-bis(benzoyloxy)-6-methyl-2H-pyran-3(6H)-
one]
the solvents in vacuo gave 108 mg (51%, based on 10) of
20
11 as an amorphous solid; ½aꢂD ¼ ꢁ5:3 (c 0.9, CHCl3).
1H NMR (300 MHz, CDCl3): d 1.40 (d, J5 ;6 , 3H, 6 -H3),
0
0
0
2.58 (dd, J4 ;4 14.2, J4 e;5 1.7, 1H, 40-He), 2.70 (dd, J4 ;4
0
0
0
0
0
0
Dry chlorine gas was slowly passed through a stirred and
cooled (ꢁ20 ꢁC) solution of 10.0 g (21.8 mmol) of 3,4-di-
14.2, J4 a;5 10.2, 1H, 40-Ha), 3.05, 3.07 (two 3H-s, 2
0
0

1124
E. Cuny, F. W. Lichtenthaler / Tetrahedron: Asymmetry 17 (2006) 1120–1124
NMe2), 4.26 (m, 1H, 50-H), 5.43 (s, 1H, 10-H), 7.31 (4H-s,
2 BnCH2), 7.4–8.0 (5H-m, Bz-H5). Ms (FD, 25 mA):
m/z = 705 (M++1). Anal. Calcd for C37H40N2O12
(704.74): C, 63.06; H, 5.72. Found: C, 62.90; H, 5.74.
References
1. Martin, D.; Lichtenthaler, F. W. Tetrahedron: Asymmetry
2006, 17, 756–762.
2. Isolation: (a) Mason, D. J.; Dietz, A.; Smith, R. M. Antibiot.
Chemother. 1961, 11, 118–122; Proof of structure: (b) Wiley,
P. F.; Argoudelis, A. D.; Hoeksema, H. J. Am. Chem. Soc.
1963, 85, 2652–2659; Cochran, T. G.; Abraham, D. J.;
Martin, L. L. J. Chem. Soc., Chem. Commun. 1972, 494–495;
for a pertinent review, see: Rosenbrook, W., Jr. J. Antibiot.
1979, 32, S211–S227; Clinical evaluation in gonorrhea: (c)
McCormack, W. M.; Finland, M. Ann. Int. Med. 1976, 84,
712–716; Holloway, W. J. Med. Clin. North Am. 1982, 66,
169–173.
3. (a) Nakajima, M.; Kurihara, N.; Hasegawa, A.; Kurokawa,
T. Liebigs Ann. Chem. 1965, 689, 243–247; (b) Lichtenthaler,
F. W.; Leinert, H.; Suami, T. Chem. Ber. 1967, 100, 2383–
2388; (c) Ogawa, S.; Abe, T.; Sano, H.; Kotera, K.; Suami, T.
Bull. Chem. Soc. Jpn. 1967, 40, 2405–2409; (d) Suami, T.;
Ogawa, S.; Sano, H. Bull. Chem. Soc. Jpn. 1970, 43, 1843–
1846; (e) Schubert, J.; Keller, R.; Schwesinger, R.; Prinzbach,
H. Chem. Ber. 1983, 116, 2524–2545.
4.5. N,N-Bis(benzyloxycarbonyl)-spectinomycin 12
4.5.1. A. De-O-benzoylation of 11. K2CO3 (12 mg) was
added to a methanolic solution of 23 (56 mg, 0.08 mmol,
in 5 mL) and the mixture was stirred for 30 min at room
temperature. Dilution with CH2Cl2 (50 mL), washing with
2 M HCl (20 mL) and water (2 · 20 mL), drying over
Na2SO4 and removal of CH2Cl2 in vacuo left 49 mg
20
(89%) of 12 as a colourless amorphous solid of ½aꢂD
¼
ꢁ4:3 (c 0.9, CHCl3), indistinguishable spectroscopically
1
(IR, H NMR) and chromatographically (CHCl3/MeOH
10:1, n-hexane/EtOAc 5:1, or EtOH) from a sample pre-
pared14 by carbobenzoxylation of commercial (Sigma)
spectinomycin; lit.: [a]D = ꢁ3 (CHCl3),14 ꢁ4.6 (c 0.28,
CHCl3).8
4. Lichtenthaler, F. W.; Lo¨he, A.; Cuny, E. Liebigs Ann. Chem.
1983, 1973–1985.
4.5.2. B. Glycosylation of 10 with actinospectosyl chloride 5,
followed by de-O-benzoylation. Reaction of 10 (320 mg,
1.2 mmol) with 5 (285 mg, 0.6 mmol) in toluene (15 mL)
in the presence of silver alumosilicate13 (360 mg, 1.2 mmol)
5. The alternate possibility, cyclo-hemiketalization of inter-
mediate 6 by OH!C@O attack from the upper (equatorial)
face, would lead to the trans-fused product 8 in which the
axial OBz group is in the less favourable gauche disposition to
the two vicinal ring oxygens.
˚
and molecular sieves 4 A (2 g) for 2.5 h at reflux, followed
by filtration through Kieselgur, evaporation of the filtrate
to dryness in vacuo and exposure of the syrupy residue
of crude 11 to K2CO3 in MeOH for 30 min gave, upon
workup as described under procedure A, crude 12
(Rf = 0.36 in CHCl3/MeOH 9:1) with two minor, slightly
faster spots (ca. 5% each). Purification by HPLC on
Lichrosorb RP-18 (5 lm) with MeOH/water 60:40 at
1 mL/min and removal of the solvents of the appropriate
fraction in vacuo afforded 208 mg of 12 (58%, based on
5) as a colourless solid, identical in all respects with the
product described under A.
6. Lichtenthaler, F. W.; Cuny, E.; Sakanaka, O. Angew. Chem.
2005, 117, 5024–5028; Angew. Chem., Int. Ed. 2005, 44, 4944–
4948.
7. White, D. R.; Birkenmeyer, R. D.; Thomas, R. C.; Mizsak, S.
A.; Wiley, V. H. Tetrahedron Lett. 1979, 2737–2740.
8. Hanessian, S.; Roy, R. J. Am. Chem. Soc. 1979, 101, 5839–
5841; Can. J. Chem. 1985, 63, 163–172.
9. Estimated overall yield as, for example, step 9 of the 23
involved is reported to proceed in a yield of 114%.8
10. A preliminary report on part of this work has appeared.6
11. (a) Ekborg, G.; Garegg, P. J.; Josephson, S. Carbohydr. Res.
1978, 65, 301–306; (b) Evans, M. E.; Parrish, F. W. Methods
Carbohydr. Chem. 1972, 6, 177–179, 193–196; (c) Ref. 4,
footnote 19.
4.6. Spectinomycin 1
12. Suami, T.; Nishiyama, S.; Ishikawa, H.; Okada, H.;
Kinoshita, T. Bull. Chem. Soc. Jpn. 1977, 50, 2754–
2757.
13. (a) van Boeckel, C. A. A.; Beetz, T.; Kock-van Dalen, A. C.;
van Bekkum, H. Recl. Trav. Chim. Pays-Bas 1987, 106, 596–
598; (b) Lichtenthaler, F. W.; Metz, T. Eur. J. Org. Chem.
2003, 3081–3093.
A 100 mg portion of 12 was hydrogenolyzed over 5% Pd/C
(100 mg) in 1:1 iPrOH/water (5 mL) and processed as
described8 to give, upon crystallization from aqueous
acetone, 74 mg (90%) of 1 dihydrochloride pentahydrate
20
as needles with mp 200–204 ꢁC (dec) and ½aꢂD ¼ þ14:3
20
(c 1.1, H2O) {lit. mp 210 ꢁC, ½aꢂD ¼ þ14:0 (c 5, water);16
14. Herrinton, P. M.; Klotz, K. L.; Hartley, W. M. J. Org. Chem.
1993, 58, 678–682.
mp 205–207 ꢁC (dec), [a]D = +14.8 (c 0.42, H2O)8}. IR
1
and H NMR data were identical to the natural product
15. Mitscher, L. A.; Martin, L. L.; Feller, D. R.; Martin, J. R.;
Goldstein, A. W. J. Chem. Soc., Chem. Commun. 1971, 1541–
1542; Hoeksema, H.; Knight, J. C. J. Antibiot. 1975, 28, 240–
241; Strochane, R. M.; Taniguchi, M.; Rinehart, K. L., Jr.
J. Am. Chem. Soc. 1976, 98, 3025–3027; Otsuka, H.;
Mascaretti, O. R.; Hurley, L. H.; Floss, H. G. J. Am. Chem.
Soc. 1980, 102, 6817–6820.
in all respects.
Acknowledgement
We thank the Deutsche Forschungsgemeinschaft and the
Fonds der Chemischen Industrie for support of this work.
16. Sinclair, A. C.; Winfield, A. F. Antimicrob. Agents Chemo-
ther. 1961, 503–506.