Novel (N-ferrocenylmethyl)amines and (N-ferrocenylmethylen)imines derived from vicinal steroid amino alcohols and amines: Synthesis, molecular structure, and biological activity

Article abstract of DOI:10.1016/S0039-128X(98)00062-2

Novel steroidal (N-ferrocenylmethyl)amines with potential biologic activity and of potential interest as chiral ligands for metal complexation were synthesized. The new compounds were screened in vitro for their potential as antimicrobial agents. The synt

Full text of DOI:10.1016/S0039-128X(98)00062-2

Novel (N-Ferrocenylmethyl)amines
and (N-Ferrocenylmethylen)imines
derived from vicinal steroid amino
alcohols and amines: Synthesis,
molecular structure, and
biological activity
Reimar Krieg,* Ralf Wyrwa,† Ute Mo¨llmann,† Helmar Go¨rls,‡ and
Bruno Scho¨necker§
*Institut fu¨r Anatomie/Anatomie II, Klinikum der Universita¨t Jena; †Hans-Kno¨ll-Institut fu¨r
Naturstofforschung, Jena; ‡Institut fu¨r Anorganische und Analytische Chemie der Universita¨t
Jena, and §Institut fu¨r Organische und Makromolekulare Chemie der Universita¨t Jena, Jena,
Germany
Novel steroidal (N-ferrocenylmethyl)amines with potential biologic activity and of potential interest as chiral
ligands for metal complexation were synthesized. The new compounds were screened in vitro for their potential
as antimicrobial agents. The synthesis of the new steroidal ferrocenes, including two X-ray crystal structures and
biologic assays, are described. The 16-(ferrocenylmethyl)amino-estratrienes 4a–d, 7b, and 10b exhibited out-
standing broad antimicrobial activity particularly against mycobacteria and multi-resistant staphylococci. Thus,
they can be considered as new lead structures. In contrast, the analogous 3␣-(ferrocenylmethyl)amino-
cholestanes 12 possessed only weak activity. The reaction of the four isomeric amino alcohols 1a–d (Scheme 1)
with ferrocenecarbaldehyde was studied. 1b and 1c with 16/17-trans configuration yielded nearly quantitatively
the (E)-Schiff bases 2b and 2c (Scheme 2). In contrast to the trans-compounds, condensation of the cis-
configurated amino alcohols 1a and 1d furnished tautomeric mixtures of the Schiff bases (2a and 2d, respectively)
and their corresponding 1,3-oxazolidines (3a and 3d, respectively). The novel (N-ferrocenylmethyl)amines 4 a–d
were obtained in excellent yields by reduction of the tautomer mixtures and the uniform Schiff bases with sodium
borohydride in ethanol. Starting with the 16␤-hydroxy compound 5a, the synthesis of 16␤- and 16␣-amino-3-
methoxy-estra-1,3,5(10)-triene (6b, 9b) is described. The corresponding 16-(N-ferrocenylmethyl)amines 7b and
10b and the 3␣-(N-ferrocenylmethyl)amino-cholestanes 12 were synthesized (Scheme 3) for comparison in
biologic tests. (Steroids 63:531–541, 1998) © 1998 by Elsevier Science Inc.
Keywords: steroids; (N-ferrocenylmethyl)amines; amino alcohols; 16-amino-estra-1,3,5(10)-trienes; X-ray analysis; antimi-
crobial activity
mously. The dramatic increase in the incidence of my-
cobacterial infections accompanied by resistance of my-
cobacteria against the frontline anti-tuberculous agents is
alarming.1 About 3 million deaths annually are due to
tuberculosis. Serious problems are also caused by multi-
resistant staphylococci (MRSA)2 and glycopeptide-
resistant enterococci.3 Untreatable infections may occur
by transfer of the enterococcal vancomycin resistance to
MRSA. These problems urgently demand the develop-
ment of new drugs, preferentially those with new mech-
anisms of action.
Introduction
Since the introduction of antibiotics for the therapy of
infectious diseases, bacteria has rapidly developed mech-
anisms to inhibit drug actions. Pathogenic organisms
resistant to one or more drugs have become increasingly
prevalent over the last few decades. The difficulties in
controlling infections with antibiotics have risen enor-
Address reprint requests to Bruno Scho¨necker, Institut fu¨r Organische und
Makromolekulare Chemie der Universita¨t Jena, Humboldtstra␤e 10,
D-07743 Jena, Germany.
In continuation of our efforts to develop and investi-
gate new chelating agents from the 16,17-amino alcohols
Received April 17, 1998; accepted June 19, 1998.
Steroids 63:531–541, 1998
© 1998 by Elsevier Science Inc. All rights reserved.
655 Avenue of the Americas, New York, NY 10010
0039-128X/98/$19.00
PII S0039-128X(98)00062-2

Papers
Scheme 1 16N-16C-17C-17O torsion angles of the 16-amino-17-hydroxy-3-methoxy-estra-1,3,5(10)-trienes 1 a–d.
of 3-methoxy-1,3,5(10)-trienes, we studied the reaction
of these compounds with salicylic aldehyde,4 heteroaro-
matic ␣-aldehydes, and ferrocenecarbaldehyde followed
by the reduction of the condensation products. The de-
fined stereochemistry at the nearly rigid steroid skeleton
should be useful for the study of different reactions and
properties in dependent upon the 16N-16C-17C-17O tor-
sion angle (Scheme 1).
These compounds were screened in vitro for antimicro-
bialactivity.Thehighestactivitywasfoundfortheferrocenyl-
methylamino steroids. Therefore, we describe here the syn-
thesis and the biologic data of the ferrocenylmethylamines.
The ferrocene group is an ideal building block in the
design of new drugs due to its high stability, its low
toxicity, and its bulkiness. Moreover, the synthesis of
new ferrocene derivatives is of considerable interest with
respect to their widespread potential applications e.g., as
building blocks in molecular recognition5 and material
science.6 Chiral ferrocenes are of increasing importance
as chiral ligands in transition metal-catalyzed asymmetric
organic synthesis,7–9 in biochemistry,10 and in drug
development.11 Therefore, we were interested in a com-
bination of the ferrocenyl group with amino steroids,
some of which have antimicrobial activity. Recently,
squalamine, a natural chelating aminosteroid,12,13 its
derivatives,14 and 7-aminocholesterol15 and its deriva-
tives16 were described as having interesting antibiotic
activities.
16,17-Piperidino- and morpholino alcohols of androstane
and estra-1,3,5(10)-triene have been synthesized as com-
pounds with potential non-hormonal activities.17 Amines
with broad activity against multiresitant staphylococci, gly-
copeptide resistant enterococci, as well as mycobacteria, are
not known.
Scheme 2 Synthesis of the ferrocenes 2, 3, and 4 derived from steroid 16,17-aminoalcohols.
532 Steroids, 1998, vol. 63, October

Steroid (N-ferrocenylmethyl)amines and (N-ferrocenylmethylen)imines: Krieg et al.
Scheme 3 Synthesis of the ferrocenes 7, 10, and 12 derived from steroid 16-amines and 3-aminocholestanes.
spectra were obtained using a matrix of 3-nitrobenzyl alco-
hol (3-NBA). IR: Spectrometer Nicolet Impact 400. Melting
points are uncorrected (Boe¨tius micromelting point appara-
tus). Microanalyses: Foss-Heraeus CHN-Rapid elemental
analyzer.
Experimental
Chemistry
General methods
All reactions were carried out under argon. NMR: Bruker
AMX 400 and AC 250, TMS was the internal standard; 1H
NMR spectra were recorded at 400 or 250 MHz, 13C NMR
spectra at 100 or 62.5 MHz. Optical rotations were obtained
at 589 nm in CHCl3 or pyridine (Polamat A, Carl Zeiss
Jena). Concentrations (c) are reported in mg/mL. Mass
spectra: AMD 402, EI 70 eV ionizing voltage. FAB mass
X-ray diffraction data: The data collections were made
with a NONIUS-CAD4 diffractometer using graphite-
monochromated Mo-K␣ radiation. The crystals were
mounted in a glass capillary at 20°C for 2c and in a cold
nitrogen stream at Ϫ90°C for 4d ⅐ HCl. Data were corrected
Table 1 1H NMR Data and Ratios of Epimers for the Tautomeric Equilibria between 2 and 3 in the cis-16/17 Series a and d (CDCl3, 400
MHz, ␦ in ppm)
2a
3a
(Epimers)
Share [%]
Temperature
␦(CHAN)
␦(18-H3)
Share [%]
␦(O-CH-N)
␦(18-H3)
40°C
8.119
8.118
8.126
8.137
0.881
0.868
0.828
0.800
74
68
56
52
5.346 and 5.364
5.340 and 5.351
5.273 and 5.372a
5.236b and 5.415b
0.852 and 0.903
0.838 and 0.896
0.790 and 0.882
13.5 and 12.5
16.5 and 15.5
25 and 19
r.t.
Ϫ10°C
Ϫ30°C
29 and 19
2d
3d
(Epimers)
r.t.
8.209
8.210
0.780c
0.781c
8
9
4.887 and 5.387
4.888 and 5.398
0.731 and 0.780c
0.734 and 0.781c
59 and 33
54 and 37
r.t. (Pyridine-D5)
If not otherwise noted, all signals are singlets.
am, broad.
bd, 3J ϭ 12.4 Hz.
coverlapped.
Steroids, 1998, vol. 63, October 533

Papers
for Lorentz and polarization effects but not for absorption.18
The structures were solved by direct methods (SHELXS19)
and refined by full-matrix least squares techniques against
F2 (SHELXL-9320). For 2c, the hydrogen atoms were lo-
cated by difference Fourier synthesis and refined isotropi-
cally. For 4d ⅐ HCl, only the hydrogen atoms for the N- and
the O2-atom were located by difference Fourier synthesis
and refined isotropically. The other hydrogen atoms were
included at calculated positions with fixed thermal param-
eters; all nonhydrogen atoms were refined anisotropically.
XP was used for structure representations.21 Further details
concerning the crystal structure analysis on request from
Fachinformationszentrum Karlsruhe, Gesellschaft fu¨r
wissenschaftlich-technische Information mbH, D-76344
Eggenstein-Leopoldshafen, on quoting the depository num-
bers (CSD-(2c) 407527, CSD-(4d-HCl), 407522, the names,
and the journal citation.
calc. C 72.44, H 7.09, N 2.82; found C 72.29, H 7.22, N
2.74.
16␤-(N-ferrocenylmethylen)imino-17␣-hydroxy-3-meth-
oxy-estra-1,3,5(10)-triene (2c): Yield: 78%, bright red-
orange prisms (AcOEt/heptane). - mp 181.5–183.5°C. -
[␣]D (CHCl3) ϭ ϩ 96° (c ϭ 9.90). - IR (KBr): ␯ ϭ 3579
(OH, m, sharp), 1641 cmϪ1 (CAN, vs); 1608 (m), 1573 (w)
1
and 1496 (s; aromatic CA C). - H NMR (CDCl3): ␦ ϭ
0.977 (s, 3 H, 18-H3), 2.862 (s, broad, 2 H, 6-H2), 3.546
(pseudo-t, 3J ϭ 7.55 Hz, 1 H, 16␣-H), 3.716 (s, 1 H,
17␤-H), 3.769 (s, 3 H, 3-MeO), 4.152 (s, 5 H, cp), 4.329 (s,
2 H, cpЈ), 4.607 (s, 1 H, cpЈ), 4.641 (s, 1 H, cpЈ), 6.633 (m,
3
1 H, 4-H), 6.709 (m, 1 H, 2-H), 7.226 (d, J ϭ 7.74 Hz, 1
H, 1-H), 8.109 (s, 1 H, N ϭ CH). -13C NMR (CDCl3): ␦ ϭ
17.30 (C-18); 69.04 (5 C, cp), 158.84 (CH ϭ N). - Anal.
(C30H35NO2Fe, 497.4): calc. C 72.44, H 7.09, N 2.82; found
C 72.76, H 7.03, N 2.76.
Typical procedure for the preparation of 16-(N-ferrocenyl-
methylen)imino-17-hydroxy-3-methoxy-estra-1,3,5(10)-
trienes (2): In the case of the 16/17-cis configurations, 2a
and 2d were obtained as tautomeric mixtures with the
corresponding 1,3-oxazolidines 3a and 3d, respectively.
235.4 mg (1.1 mmol) of ferrocenecarbaldehyde was added
at room temperature to a stirred solution of 301.4 mg (1.0
mmol) 16-amino-17-hydroxy-3-methoxy-estra-1,3,5(10)-
triene22 in 15 mL of abs. methanol. The mixture was stirred
for 3 h at ambient temperature, kept at Ϫ18°C for 1 h, and
the resulting precipitate was filtered off. Solvent was re-
moved from the mother liquor to half its volume in vacuum
to give additional product. The combined red-orange solids
were recrystallized and dried or employed in the subsequent
reduction step without further purification.
Crystal data for 2c: C30H35NO2Fe, Mr ϭ 497.4 gmolϪ1
,
red prisms, size: 0.40 ϫ 0.40 ϫ 0.36 mm3, orthorhombic,
space group P212121, a ϭ 7.395,1 b ϭ 10.048,2 c ϭ 33.4694
Å, V ϭ 2486.97 Å,3 Z ϭ 4, ␳calc. ϭ 1.33 gcmϪ3, ␮ (Mo-K␣)
ϭ 6.34 cmϪ1, F(000) ϭ 1056, 4838 reflections in ϩh, ϩk,
Ϫl, measured in the range 2.30° Յ ⌰ Յ 27.41°, 3630
reflections with Fo Ͼ 4␴(Fo), 447 parameters, R ϭ 0.040,
wR2 ϭ 0.092, GOOF ϭ 1.43, Flack-parameter: 0.01,2 larg-
est difference peak: 0.34 e ÅϪ3
.
Mixture of 16␣-(N-ferrocenylmethylen)imino-17␣-
hydroxy-3-methoxy-estra-1,3,5(10)-triene (2d) and the
oxazolidines 3d: Yield: 93% (crude product), ratio of 3d
and 2d at r.t. in CDCl3: Ϸ 90: 10 (1H NMR; see Table 1 for
separated signals and integral evaluations), ochre amor-
phous powder. - A analytical sample was crystallized from
ethanol: bright ochre scaly powder, ratio of 3d and 2d at r.t.
(1H NMR, CDCl3): 92: 8. - mp 136–139°C. - IR (KBr):
1641 cmϪ1 (C ϭ N, vw); 1611 (m), 1577 (w) and 1503 (s;
16␤-(N-ferrocenylmethylen)imino-17␤-hydroxy-3-meth-
oxy-estra-1,3,5(10)-triene (2a) - Tautomeric Mixture
with 3a: Yield 80% (crude product), ratio of 2a /3a at r.t. in
CDCl3: 68: 32 (1H NMR; see Table 1 for separated signals
and integral evaluations at different temperatures); orange
crystals, mp 186–189°C. - After crystallizations from
MeOH/AcOEt, the ratio of isomers was found to be un-
changed (1H NMR control, in CDCl3); bright orange nee-
dles, mp 193.5–196°C (dec.). - [␣]D (CHCl3) ϭ ϩ 10° (c ϭ
7.03). Ϫ IR (KBr): ␯ ϭ 1634 cmϪ1 (CAN, vs); 1606 (m),
1
aromatic CAC). - H NMR (CDCl3): ␦ ϭ 2.837 (m, 2 H,
6-H2), 3.758 (s, 3 H, 3-MeO), 3.889 (m, ca. 0.9 H, 16 or
17␤-H of oxazolidine tautomer), 4.191 (s, 5 H, C5H5),
4.125–4.395 (overl., 4 H, C5H5Ј), 6.621 (m, 1 H, 4-H),
6.702 (dd, 3J ϭ 9.90 Hz, 4J ϭ 2.65 Hz, 1 H, 2-H), 7.202 (d,
3J ϭ 9.93 Hz, 1 H, 1-H). - Anal. (C30H35NO2Fe, 497.4):
calc. C 72.44, H 7.09, N 2.82; found C 72.03, H 7.08, N
2.92.
1579 (w) and 1499 (s; aromatic CAC).
- Anal.
(C30H35NO2Fe, 497.4): calc. C 72.44, H 7.09, N 2.82; found
C 72.40, H 7.17, N 2.82.
Typical procedure for the preparation of 16-(N-ferro-
cenylmethyl)amino-17-hydroxy-3-methoxy-estra-1,3,5(10)-
trienes 4 and their hydrochlorides: 497.4 mg (1 mmol) of
16-(N-ferrocenylmethylenimino)-17-hydroxy-estra-1,3,5(10)-
triene 2 or the corresponding mixture of tautomers 2/3 and
113.5 mg (3 mmol) of NaBH4 were stirred in a mixture of
10 mL of abs. ethanol and 15 mL of abs. THF at ambient
temperature. After 1 h, 1 mL of water was added. The
yellow-orange mixture was stirred further for 10 min and
then evaporated, giving a orange solid. The crude product
was poured into 30 mL of CH2Cl2 and extracted with water
(3 ϫ 50 mL). The organic layer was dried over Na2SO4,
evaporated, and the residue was purified by crystallization.
Similarly, the corresponding hydrochlorides were prepared
16␣-(N-ferrocenylmethylen)imino-17␤-hydroxy-3-meth-
oxy-estra-1,3,5(10)-triene (2b): Yield: 72%, bright ochre
needles (AcOEt/heptane). - mp 193–195°C (dec.). - [␣]D
(CHCl3) ϭ ϩ 81° (c ϭ 9.76). - IR (KBr): ␯ ϭ 1637 cmϪ1
(CAN, vs); 1610 (m), 1577(w) and 1500 (s; aromatic
1
CAC). - H NMR (CDCl3): ␦ ϭ 0.888 (s, 3 H, 18-H3),
2.836 (s, broad, 2 H, 6-H2), 3.497 (m, 1 H, 16␤-H), 3.700–
3.762 (overl., 1 H, 17␣-H) and 3.762 (s, overl., 3 H,
3-MeO), 4.168 (s, 5 H, cp), 4.344 (s, 2 H, cpЈ), 4.648 (s, 2
3
H, cpЈ), 6.621 (m, 1 H, 4-H), 6.689 (d, J ϭ 8.24 Hz, 1 H,
3
2-H), 7.203 (d, J ϭ 8.24 Hz, 1 H, 1-H), 8.085 (s, 1 H,
NACH). - 13C NMR (CDCl3): ␦ ϭ 12.39 (C-18); 69.18 (5
C, cp), 160.29 (CHAN). - Anal. (C30H35NO2Fe, 497.4):
534 Steroids, 1998, vol. 63, October

Steroid (N-ferrocenylmethyl)amines and (N-ferrocenylmethylen)imines: Krieg et al.
by treatment of the crude steroid amines in 10 mL of
methanol with 1.2 mmol of NH4Cl or HCl at r.t., evapora-
tion after 2–4 h, and further work up as described above.
13C-NMR (CDCl3): ␦ ϭ 68.91 (cp), 69.43 (cp), 69.71 (cp),
70.66 (cp), 71.23 (cp). - Anal (C30H38ClFeNO2, 536.0):
calc. C 67.22, H 6.96, N 2.61; found C 66.82, H 6.90, N
2.62.
16␤-(N-ferrocenylmethyl)amino-17␤-hydroxy-3-meth-
oxy-estra-1,3,5(10)-triene (4a): Yield: 78%, bright yellow
needles (AcOEt/hexane). - mp 166–168.5°C (dec.). - [␣]D
(CHCl3) ϭ ϩ 91° (c ϭ 10.41). - IR (KBr): ␯ ϭ 3329 (w,
sharp, OH), 3227 (m, NH); 1603 (m), 1581(w) and 1502 (s;
16␣-(N-ferrocenylmethyl)amino-17␣-hydroxy-3-methoxy-
estra1,3,5(10)-triene (4d): Yield: 84%, gold ochre solid
(methanol/water). - mp 129–133°C. - [␣]D (CHCl3) ϭ ϩ 24°
(c ϭ 6.65). - MS: m/z ϭ 499 [Mϩ] (100%). - IR (KBr): ␯ ϭ
3088 (w, sharp) and 3417 (m, broad; XH); 1610 (m), 1577(w)
and 1500 (s; aromatic CAC); no ␯C ϭ N. -1H NMR (CDCl3):
␦ ϭ 0.717 (s, 3 H, 18-H3), 2.817 (m, 2 H, 6-H2), 3.751 (s, 3 H,
3-MeO), 3.393–3.531 (overl., 4 H; 16␣-H, 17␣-H and NH-
CH2), 4.174 (overl., broad, 7 H, cp and cpЈ), 4.185 (s, 2 H, cpЈ),
6.508 (m, 1 H, 4-H), 6.685 (m, 1 H, 2-H), 7.205 (m, 1 H, 1-H).
-13C NMR (CDCl3): ␦ ϭ 16.78 (C-18), 47.97 (CH2-NH,),
68.47 (5 C, cp). - Anal. (C30H37NO2Fe, 499.5): calc. C 72.14,
H 7.47, N 2.80; found C 72.03, H 7.08, N 2.92.
1
aromatic CAC); no ␯C ϭ N. - H NMR (CDCl3, HMQC,
Cosydqf): ␦ ϭ 0.679 (s, 3 H, 18-H3), 2.822 (m, 2 H, 6-H2),
3.131 (m, 1 H, 16␣-H), 3.395 (d, 3J ϭ 8.80 Hz, 1 H,
17␣-H), 3.544 (s, broad, 2 H, NH-CH2), 3.751 (s, 3 H,
3-MeO), 4.108 (s, 5 H, cp), 4.163 (s, 2 H, cpЈ), 4.189 (s, 2
H, cpЈ), 6.602 (s, 1 H, 4-H), 6.688 (m, 1 H, 2-H), 7.103 (d,
3J ϭ 8.62 Hz, 1 H, 1-H). -13C NMR (CDCl3): ␦ ϭ 12.38
(C-18), 49.43 (CH2-NH,), 57.62 (C-16), 67.75 (cpЈ), 67.86
(cpЈ), 67.99 (cpЈ), 68.13 (cpЈ), 68.37 (5 C, cp), 78.96 (C-17).
- Anal. (C30H37NO2Fe, 499.5): calc. C 72.14, H 7.47, N
2.80; found C 71.28, H 7.19, N 2.86.
The hydrochloride 4d ⅐ HCl was prepared as described
above. - Yield: 80%, gold ochre solid, mp 203–205°C
(dec.). - MS: m/z ϭ 499 [Mϩ] (100%). - Anal.
(C30H38NO2ClFe, 536.0): calc. C 67.22, H 6.96, N 2.61;
found C 67.32, H 6.96, N 2.61.
16␣-(N-ferrocenylmethyl)amino-17␤-hydroxy-3-meth-
oxyestra-1,3,5(10)-triene (4b): Yield: 68%, bright yellow
powder (methanol/water). - mp 146–148°C. - 1H NMR
(CDCl3, HMQC): ␦ ϭ 0.869 (s, 3 H, 18-H3), 2.836 (m, 2 H,
Crystal data for 4d ⅐ HCl: C30H38NO2ClFe, Mr ϭ 536.0
gmolϪ1, red prisms, size: 0.40 ϫ 0.40 ϫ 0.30 mm3, mon-
oclinic, space group P21, a ϭ 13.278,1 b ϭ 7.415,1 c ϭ
14.8591 Å, ␤ ϭ 113.341°, V ϭ 1343.22 Å,3 Z ϭ 2, ␳calc. ϭ
1.32 gcmϪ3, ␮ (Mo-K␣) ϭ 6.88 cmϪ1, F000 ϭ 568, 3062
reflections in Ϯ h, -k, -l, measured in the range 2.30° Յ ⌰
Յ 27.41°, 2951 independent reflections Rint ϭ 0.020), 2834
reflections with Fo Ͼ 4␴(Fo), 328 parameters, 1 restraint,
R ϭ 0.032, wR2 ϭ 0.073, GOOF ϭ 1.012, Flack-parame-
3
6-H2), 2.989 (t, J ϭ 7.76 Hz, 1 H, 16␤-H), 3.551 (s, 2 H,
3
NH-CH2), 3.561 (d, overl., J ϭ 7.97 Hz, 1 H, 17␣-H),
3.757 (s, 3 H, 3-MeO), 4.090 (m, 2 H, cpЈ); 4.117 (s, overl.,
5 H, cp), 4.209 and 4.325 (each: m, 2 H, cpЈ), 6.617 (m, 1
H, 4-H), 6.699 (dd, 3J ϭ 8.56 Hz, 4J ϭ 2.79 Hz, 1 H, 2-H),
3
7.199 (d, J ϭ 8.61 Hz, 1 H, 1-H). - 13C NMR (CDCl3): ␦
ϭ 17.80 (C-18), 47.83 (CH2-NH,), 55.21 (O-Me), 68.33 (5
C, cp), 85.07 (C-17). - Anal. (C30H37NO2Fe, 499.5): calc. C
72.14, H 7.47, N 2.80; found C 71.52, H 7.22, N 2.87.
ter: -0.01,2 largest difference peak: 0.28 e ÅϪ3
.
16␣-(N-ferrocenylmethyl)amino-17␤-hydroxy-3-meth-
oxyestra-1,3,5(10)-triene-hydrochloride (4b ⅐ HCl):
Yield: 74%, bright ochre powder (methanol/water). - mp Ͼ
260°C (180–195°C dec.). - [␣]D (pyridine) ϭ ϩ 29° (c ϭ
1.74). - IR (KBr): ␯ ϭ 3429 (vs, broad, XH); 1617 (m),
16␣-Bromo-3-methoxy-estra-1,3,5(10)-triene (5b): To a
stirred suspension of 2.67 (15.0 mmol) of
g
N-bromosuccinimide and 18 mL of dry dichloromethane, a
solution of 3.93 g (15.0 mmol) of triphenylphosphine in 18
mL of dry dichlormethane was added dropwise at 10°C.
After 5 min, 1.72 g (6.0 mmol) of 16␤-hydroxy compound
5a23 were added, and the mixture was kept for 2 h at room
temperature. The reaction product 5b was isolated as white
crystals after column chromatography of the reaction mix-
ture on silica gel (60 g) with 300 mL of dichloromethane.
Yield: 1.86 g (89%) of 5b. - mp 108–111°C. - [␣]D (CHCl3)
ϭ ϩ 118° (c ϭ 10.1). - 1H NMR (CDCl3): ␦ ϭ 0.718 (s, 3
H, 18-H3), 2.831 (m, 2 H, 6-H2), 3.757 (s, 3 H, 3-MeO),
4.463 (m, 1 H, 16␤-H), 6.607 (s, 1 H, 4-H), 6.694 (m, 1 H,
2-H), 7.183 (d, 3J ϭ 8.62 Hz, 1 H, 1-H). - Anal.
(C19H25BrO, 349.3): calc. C 65.33, H 7.21, Br 22.88; found
C 65.40, H 7.41, Br 22.95.
1
1580 (w) and 1500 (s; aromatic CAC); no ␯C ϭ N. - H
NMR (CDCl3): ␦ ϭ 0.774 (s, 3 H, 18-H3), 2.819 (m, 2 H,
6-H2), 3.113 (dd, 3J ϭ 12.14 Hz, 4J ϭ 5.85 Hz,1 H, 16␤-H),
3
3.613 (d, J ϭ 13.30 Hz, 1 H, 17␣-H), 3.672–3.781 (dd,
overl., 2 H, NH-CH2) and 3.751 (s, 3 H, 3-MeO), 4.140 (s,
overl., Ϸ 5 H, cp); 4.170 (s, overl., Ϸ 2 H, cpЈ), 4.265 and
4.346 (each: s, overl., 1 H, cpЈ), 6.600 (m, 1 H, 4-H), 6.683
(dd, 3J ϭ 8.56 Hz, 4J ϭ 2.51 Hz, 1 H, 2-H), 7.163 (d, 3J ϭ
8.62 Hz, 1 H, 1-H). -13C NMR (CDCl3): ␦ ϭ 12.00 (C-18),
47.16 (CH2-NH,), 68.66 (5 C, cp).
-
Anal.
(C30H38ClNO2Fe, 536.0): calc. C 67.23, H 7.15, N 2.61;
found C 68.38, H 7.21, N 2.60.
16␤-(N-ferrocenylmethyl)amino-17␣-hydroxy-3-meth-
oxy-estra-1,3,5(10)-triene-hydrochloride (4c ⅐ HCl):
Yield: 50%, yellow crystals. - mp 203–206°C (dec.). - MS
(EI): m/e ϭ 499 [M-HCl]ϩ (100%, isotopic pattern of Fe).
-1H-NMR (CDCl3): ␦ ϭ 0.993 (s, 3 H, 18-H3), 3.127 (mc,
1 H, 16␣-H), 3.752 (s, 3 H, OCH3), 3.752–3.846 (m, overl.,
1 H, 17␤-H), 3.990 (s, 2 H, CH2-cpЈ), 4.163 (s, 5 H, cp),
4.245 (m, 2 H, cpЈ), 4.433 (m, 1 H, cpЈ), 4.541 (m, 1 H, cpЈ).
16␤-Azido-3-methoxy-estra-1,3,5(10)-triene (6a):
A
stirred solution of 1.56 g (4.5 mmol) of 16␣-bromo com-
pound 5b and 0.67 g (13.7 mmol) of lithium azide in 30 mL
of dimethyl sulfoxide was heated to 80°C for 1 h. After
cooling, water was added dropwise. The resulting precipi-
tate was filtered off, washed with water, and dried in vac-
uum. Yield: 1.33 g (96%) of 6a. - mp 80–85°C, after
crystallization from ether/methanol mp 84–87°C. - [␣]D
Steroids, 1998, vol. 63, October 535

Papers
Figure 1 X-ray structure of the azomethine 2c.
1
(CHCl3) ϭ ϩ 60° (c ϭ 10.2). - H NMR (CDCl3): ␦ ϭ
0.895 (s, 3 H, 18-H3), 2.830 (s, 2 H, 6-H2), 3.760 (s, 3 H,
3-MeO), 4.086 (m, 1 H, 16␣-H), 6.613 (s, 1 H, 4-H), 6.690
16␣-H), 8.012 (d, 3J ϭ 7.00 Hz, 1 H, 16␤-NH), 8.634 (s, 1
H, NH-CO). - Anal. (C19H27NO, 285.4): calc. C 79.95, H
9.53, N 4.90; found C 79.23, H 9.44, N 4.74.
3
(m, 1 H, 2-H), 7.181 (d, J ϭ 8.62 Hz, 1 H, 1-H). - Anal.
(C19H25N3O, 311.4): calc. C 73.28, H 8.09, N 13.49; found
C 73.18, H 7.97, N 13.41.
16␤-(N-ferrocenylmethylen)imino-3-methoxy-estra-
1,3,5(10)triene (7a): To a stirred solution of 150 mg (0.53
mmol) of 16␤-amine 6b in 6 ml of abs. methanol, 115 mg
(0.54 mmol) of ferrocenecarbaldehyde were added at room
temperature. After two hours, the precipitated crystals were
filtered off, washed with 3 ml of methanol, and dried.
Yield: 198 mg (78%) of 7a as yellow-ochre crystals. -
mp 212–216°C (dec.). - [␣]D (CHCl3) ϭ ϩ 43° (c ϭ 5.1).
- 1H NMR (CDCl3): ␦ ϭ 1.031 (s, 3 H, 18-H3), 3.798 (s, m,
overl., 4 H, 3-MeO, 16␣-H), 4.144 (s, 5 H, cp), 4.302 (m, 2
H, cpЈ), 4.608 (m, 2 H, cpЈ), 8.018 (s, 1 H, NACH). -13C
NMR (CDCl3): ␦ ϭ 19.32 (C-18), 55.21 (OCH3), 68.26
(cpЈ), 68.40 (cpЈ), 68.50 (C-16), 68.97 (cp), 70.03 (cpЈ),
81.46 (cpЈ), 157.23 (N ϭ CH), 157.38 (C-3). - Anal.
(C30H35FeNO, 481.5): calc. C 74.84, H 7.33, N 2.91; found
C 74.45, H 7.39, N 2.88.
16␤-Amino-3-methoxy-estra-1,3,5(10)-triene (6b): To a
stirred and cooled solution of 1.28 g (4.1 mmol) of the
16␤-azido compound 6a and 30 ml of dry tetrahydrofuran,
10 mL of a solution of lithium aluminium hydride in tetra-
hydrofuran (ϳ 1 M, Fluka) were added dropwise. After
stirring for 30 min at ambient temperature, ether/water was
carefully added at 0°C, the mixture was treated with sodium
tartrate solution, the aqueous phase was extracted three
times with ether, and the combined organic phases were
washed with water twice and then dried with sodium sulfate.
The solvent was removed in vacuum, and the residue was
crystallized from heptane to yield 1.05 g (89%) of the amine
6b as a white solid. - mp 90–94°C. - [␣]D (pyridine) ϭ ϩ
1
67° (c ϭ 9.6). - H NMR (CDCl3): ␦ ϭ 0.923 (s, 3 H,
18-H3), 2.850 (s, 2 H, 6-H2), 3.755 (s, 3 H, 3-MeO), 3.481
(m, 1 H, 16␣-H), 6.608 (s, 1 H, 4-H), 6.686 (m, 1 H, 2-H),
7.183 (d, 3J ϭ 8.64 Hz, 1 H, 1-H); after addition of trichlo-
roacetyl isocyanate: 0.918 (s, 1 H, 18- H3), 4.320 (m, 1 H,
16␤-(N-ferrocenylmethyl)amino-3-methoxy-estra-1,3,5(10)-
triene (7b): To a stirred solution of 50 mg (0.10 mmol) of 7a
in 15 mL of abs. methanol, 20 mg (0.53 mmol) of NaBH4 were
added at room temperature. After 1 h, a few drops of acetone
Figure 2 X-ray structure of the (N-ferrocenylmethyl)amino-hydrochloride 4d.
536 Steroids, 1998, vol. 63, October

Steroid (N-ferrocenylmethyl)amines and (N-ferrocenylmethylen)imines: Krieg et al.
Table 2 Antibacterial Activity of (N-ferrocenylmethyl)aminosteroids in the Agar Diffusion Assay (Inhibition Zones [mm])
B. subtilis
ATCC
6633
Compound
(100 ␮g/mL)
M. flavus
ATCC 10240
S. aureus E. faecalis M. smegmatis M. fortuitum M. chelonae E. coli
134/94
1528
SG 987
B
B
AS 19 G. cingulata
2b
11,5
10,5
16
15,5
14
15
15,5
15,5
13p
15
9,5
10,5
22
0
0
0
0
14,5
15,5
14
14,5
15,5
14,5
12P
14,5
0
16,5
0
0
0
16
10
0
0
0
0
0
0
0
0
0
2c
4a
4b
4b HCl
4c HCl
4d
14,5
16,5
12
15
14
15
9,5
14,5
0
22
22
20
15
15
17
21,5
19
16
17
19
16
18,5
19
12P
14,5
0
20,5
0
0
0
32
21
22,5
17
20,5
22,5
20
16
19
19
14
21,5
23,5
21
12P
19
0
10
10
14p
11
12P
13
0
19
20,5
22,5
22
4d HCl
7a
16p
20
7b
14
0
17
0
0
10a
9,5
16
10P
20,5
0
13P
21
16PP
21
10b
14
0
14p
0
12a.HCl
12b
0
0
0
0
0
0
0
0
0
12c
0
0
0
0
0
0
Ciprofloxacin
5 ␮g/mL
Rifampicin
100 ␮g/mL
INH 100 ␮g/mL
30
0
0
20p
28p
0
0
0
29
0
43
0
0
0
21
0
18p
24p
14p
30P
13
0
0
0
27
p: some colonies in the inhibition zone.
P: many colonies in the inhibition zone.
were added. The solvents were removed, and the residue was
extracted with 20 mL of dichloromethane. After concentration
to 3 mL, chromatographical purification with silica gel and
n-heptane/ethylacetate (1:1) as the solvent was carried out,
giving yellow crystals.
Yield: 35 mg (70%) of 7b: mp 133–135°C. - [␣]D
(CHCl3) ϭ ϩ 47° (c ϭ 4.1). - 1H NMR (CDCl3): ␦ ϭ 0.918
(s, 3 H, 18-H3), 3.262 (m, 1 H, 16␣-H), 3.472 (m, 2 H, cpЈ),
3.758 (s, 3 H, 3-MeO), 4.082 (m, 2 H, cpЈ), 4.172 (s, 5 H,
cp), 4.181 (m, 2 H, cpЈ). -13C NMR (CDCl3): ␦ ϭ 19.99
(C-18), 47.57 (cp), 55.20 (3-OCH3), 56.40 (C-16), 67.65
(cp), 67.68 (cp), 68.38 (cp), 87.09 (cp). - Anal.
(C30H37FeNO, 483.5): calc. C 74.53, H 7.71, N 2.89; found
C 74.91, H 8.22, N 2.91.
3-Methoxy-16␤-tosyloxy-estra-1,3,5(10)-triene (8): Four
and a half grams (15.7 mmol) of the 16␤-hydroxy com-
pound 5a,23 9.0 g (47.2 mmol) p-tosylchloride, and 45 mL
of dry pyridine were kept at 5°C for 3 days. After treating
with water, the precipitate was filtered off, washed with
water and dried. After purification with column chromatog-
raphy (75 g of silica gel, benzene), 6.6 g (95%) of 8 were
Table 3 Minimal Inhibition Concentration of Selected (N-ferrocenylmethyl)amino-steroids against Fast-growing Mycobacteria
(␮g/mL)
M. smegmatis
Compound
SG 987
M. fortuitum B.
M. chelonae B.
M. vaccae 10670
M. aurum SB 66
2b
25
25
25
25
50
50
n.d.
n.d.
6,25
n.d.
25
25
2c
4a
4b
4b HCl
4c HCl
4d
3,12
3,12
3,12
3,12
3,12
3,12
Ͼ100
3,12
12,5
3,12
Ͼ100
Ͼ100
50
3,12
3,12
3,12
3,12
1,56
3,12
Ͼ100
1,56
25
3,12
Ͼ100
Ͼ100
50
3,12
1,56
3,12
1,56
1,56
3,12
25
0,78
50
3,12
Ͼ100
Ͼ100
50
3,12
3,12
3,12
3,12
3,12
3,12
Ͼ100
3,12
12,5
3,12
Ͼ100
Ͼ100
6,25
0,78
0,05
6,25
6,25
3,12
3,12
n.d.
4d HCl
7a
7b
n.d.
10a
12,5
3,12
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Ͼ100
10b
12a.HCl
12b
Rifampicin
Isoniazid
Ciprofloxacin
6,25
0,78
Ͼ100
0,2
3,125
25
n.d. ϭ not determined.
Steroids, 1998, vol. 63, October 537

Papers
Table 4 Minimal Inhibition Concentration of 16,17-amino-alcohols and 16-amines against Fast-growing Mycobacteria (␮g/mL)
B.
subtilis
ATCC
6633
Compound
100 ␮g/mL
M. flavus
ATCC 10240
S. aureus
134/94
E. faecalis
1528
M. smegmatis
SG 987
M. chelonae
B
M. fortuitum B
1a
1b
1c
1d
6b
9b
50
50
50
50
25
50
50
50
50
100
50
50
100
100
50
12,5
12,5
12,5
12,5
12,5
12,5
25
50
50
50
25
25
25
12,5
25
50
25
12,5
25
50
25
25
50
50
12,5
12,5
100
obtained. mp 144–150°C, after crystallization with dichlo-
(C30H35FeNO, 481.5): calc. C 74.84, H 7.33, N 2.91; found
C 74.94, H 7.46, N 3.02.
romethane/methanol, mp 151–155°C. - [␣]D (CHCl3) ϭ ϩ
1
55° (c ϭ 10.3). - H NMR (CDCl3): ␦ ϭ 0.894 (s, 3 H,
18-H3), 2.435 (s, 3 H, CH3-phenyl), 3.745 (s, 3 H, 3-MeO),
4.973 (m, 1 H, 16␣-H). - Anal. (C26H32O4S, 440.6): calc. C
70.88, H 7.32, S 7.28; found C 71.05, H 7.46, S 7.31.
16␣-(N-ferrocenylmethyl)amino-3-methoxy-estra-
1,3,5(10)-triene (10b): A suspension of 200 mg (0.4 mmol)
of 10a, 10 mL of abs. methanol, and 55 mg (1.5 mmol) of
NaBH4 was reacted, as described for the synthesis of 7b.
However, the residue was extracted with ether instead of
dichloromethane. Concentration of the ether solution and
filtration of the yellow crystals gave 120 mg (60%) of 10b.
16␣-Azido-3-methoxy-estra-1,3,5(10)-triene (9a): 3.3 g
(7.5 mmol) of 8 and 0.99 g (15.7 mmol) of lithium azide in
66 mL of dimethylsulfoxide was reacted as described above
for the synthesis of 6a. Yield: 2.15 g (92%) of 9a, mp
80–89°C, after crystallization from acetone: mp 89–92°C. -
1
mp 103–107°C. - [␣]D (CHCl3) ϭ ϩ 46°(c ϭ 4.5). - H
NMR (CDCl3): ␦ ϭ 0.757 (s, 3 H, 18-H3), 3.370 (m, 1 H,
16␤-H), 3.456 (m, 2 H, CH2-N), 3.753 (s, 3 H, 3-MeO),
4.085 (m, 2 H, cpЈ), 4.104 (s, 5 H, cp), 4.139 (m, 2 H, cpЈ).
- Anal. (C30H37FeNO, 483.5): calc. C 74.53, H 7.71, N 2.89;
found C 75.18, H 8.25, N 2.89.
1
[␣]D (CHCl3) ϭ ϩ 90° (c ϭ 10.1). - H NMR (CDCl3): ␦
ϭ 0.757 (s, 3 H, 18-H3), 2.834 (s, 2 H, 6-H2), 3.759 (s, 3 H,
3-MeO), 4.052 (m, 1 H, 16␤-H), 6.614 (s, 1 H, 4-H), 6.695
3
(m, 1 H, 2-H), 7.181 (d, J ϭ 8.52 Hz, 1 H, 1-H). - Anal.
(C19H25N3O, 311.4): calc. C 73.28, H 8.09, N 13.49; found
C 73.32, H 8.30, N 13.53.
3␣-azido-5␣-cholestane (14)24: Four grams (7.37 mmol) of
3␤-tosyloxy-5␣-cholestane (13) and 0.99 g (15.23 mmol) of
NaN3 were heated in 25 mL of hexamethylphosphorous-
tris-amide (HMPT) for 3 h at 90°C. The cold mixture was
poured into 100 ml of ice water. The crude product was
filtered off and dissolved in 60 mL of ether. The organic
layer was washed twice with water and dried over Na2SO4.
The ether was evaporated, leaving a white waxy product.
The product was used without further purification. Yield:
95%, mp 49–51°C. - IR: ␯ ϭ 2103 cmϪ1 (vs, N3). - Anal.
(C27H47N3, 413.7): calc. C 78.39, H 11.45, N 10.16; found
C 78.11, H 11.40, N 9.98.
16␣-Amino-3-methoxy-estra-1,3,5(10)-triene (9b): Next,
2.62 g (8.4 mmol) of the 16␣-azido compound 9a in 80 mL
of dry tetrahydrofuran was reacted with 20 mL of a solution
of lithium aluminium hydride, as described for the the
synthesis of 6b. Crystallization from heptane yielded 2.05 g
(85%) of 9b as a white solid. mp 82–85°C. [␣]D (pyridine)
ϭ ϩ 81° (c ϭ 9.5). - 1H NMR (CDCl3): ␦ ϭ 0.746 (s, 3 H,
18-H3), 2.833 (m, 2 H, 6-H2), 3.575 (m, 1 H, 16␤-H), 3.754
(s, 3 H, 3-MeO); after addition of trichloroacetyl isocyanate:
0.789 (s, 3 H, 18-H3), 2.828 (m,2 H, 6-H2), 3.744 (s, 3 H,
3-MeO), 4.390 (m, 1 H, 16␤-H), 7.912 (d, 3J ϭ 7.23 Hz, 1
H, 16␣-NH), 8.662 (s, 1 H, NH¢CO). - Anal. (C19H27NO,
285.4): calc. C 79.95, H 9.53, N 4.90; found C 80.39, H
9.59, N 4.92.
3␣-amino-5␣-cholestane (15)24: To a solution of 2.04 g
(4.83 mmol) of 14 in 50 mL of THF, 16 mL (16 mmol) of
a 1 M solution of LiAlH4 in ether was added slowly by
syringe. After refluxing for 4 h, the reaction mixture was
cooled down to r.t. and 5 mL of a saturated solution of
NH4Cl was added. The solution was filtered, and the residue
was washed twice with ether. Then, the solution was dried
over Na2SO4. The ether was evaporated giving a white
solid. The crude amine was used without further purifica-
tion. Yield: 96%, mp 95–97°C. - Anal. (C27H49N, 387.7):
calc. C 83.65, H 12.74, N 3.61; found C 83.30, H 12.42, N
3.65.
16␣-(N-ferrocenylmethylen)amino-3-methoxy-estra-
1,3,5(10)triene (10a): Five hundred milligrams (1.75
mmol) of the 16␣-amine 9b dissolved in 25 mL of abs.
methanol and 385 mg (1.8 mmol) of ferrocenecarbaldehyde
were reacted, as described for the synthesis of 7a. After
concentration in vacuo and cooling, the separated crystals
were filtered off and dried. Yield: 680 mg (81%) of 10a as
yellow-ochre crystals. - mp 150–152°C, 108°C conversion
point. [␣]D (CHCl3) ϭ ϩ 134°(c ϭ 5.6). - 1H NMR
(CDCl3): ␦ ϭ 0.851 (s, 3 H, 18-H3), 3.762 (s, 3 H, 3-MeO),
3.854 (m, 1 H, 16␤-H), 4.145 (s, 5 H, cp), 4.318 (m, 2 H,
cpЈ), 4.623 (m, 2 H, cpЈ), 8.047 (s, 1 H, N ϭ CH). - Anal.
3␣-(N-ferrocenylmethyl)amino-5␣-cholestane-2␤-ol-
hydrochloride (12a ⅐ HCl): Next, 397 mg (0.98 mmol) of
3␣-amino-5␣-cholestane-2␤-ol25 (11) and 210 mg (0.98
538 Steroids, 1998, vol. 63, October

Steroid (N-ferrocenylmethyl)amines and (N-ferrocenylmethylen)imines: Krieg et al.
mmol) of ferrocenecarbaldehyde were dissolved in 20 mL
were cut out and separately filled with 50 ␮L of test solu-
tion. Inhibition zones were evaluated after incubation for
24 h at 37°C. M. chelonae and Glomerella were incubated
at 30°C.
of abs. methanol and 15 mL of THF. The mixture was
stirred under argon for 6 h at r.t., after which 80 mg (2.11
mmol) of NaBH4 were added. After stirring for 2 h, the
mixture was hydrolyzed with 0.5 mL of water. Subse-
quently, four drops of conc. HCl-solution were added. After
30 min, the crystals were filtered off, washed twice with 5
mL of ethanol, and dried in vacuum. Yield: 57%, yellow
needles. - mp 212–213°C (dec.). - MS (EI): m/z ϭ 601
[M-Cl]ϩ (100%, isotopic pattern of Fe). - Anal.
(C38H60ClFeNO, 638.2): calc. C 71.52, H 9.48, N 2.19, Cl
5.56; found C 71.30, H 9.72, N 2.19, Cl 5.65.
Determination of minimal inhibitory
concentrations (MIC)
For selected compounds, a broth microdilution method
according to the NCCLS-guidelines26 was used to estimate
the MICs against M. smegmatis SG 987, M. chelonae B., M.
fortuitum B., M. aurum SB66, and M. vaccae 10670. The
inoculum was 105 CFU/ml or 104 CFU per well. Plates were
read after incubation for 48 h.
3␣-(N-ferrocenylmethyl)amino-5␣-cholestane (12b):
Five-hundred twenty-three milligrams (2.44 mmol) of fer-
rocenecarbaldehyde were added to 947 mg (2.44 mmol) of
15 dissolved in 20 mL of abs. methanol and 10 ml of THF.
The mixture was stirred for 4 h at r.t. Then, 120 mg (3.17
mmol) of NaBH4 were added at 0°C. After stirring at r.t. for
2 h, the mixture was hydrolyzed with 1 mL of water. All
solvents were evaporated to dryness, leaving a yellow solid.
The residue was dissolved in 50 mL of CH2Cl2. After
extraction with water, the organic layer was dried over
Na2SO4. The solvent was evaporated, and the ochre colored
crude product was purified by recrystallization from ethyl
acetate. Yield: 75%, mp 103–105°C, MS (EI): m/z ϭ 585
Results and discussion
Chemistry
The vicinal amino alcohols 1a–d (Scheme 1) derived from
3-O-methylestrone22 were reacted with ferrocenecarbalde-
hyde in methanol at ambient temperature (Scheme 2). Both
trans amino alcohols 1b and 1c with N-C16-C17-O torsion
angles of about 90° and 150°,27 respectively, (Scheme 1)
furnished the corresponding (E)-ferrocene carbaldimines 2b
and 2c (Scheme 2) as uniform (NMR) orange colored com-
pounds in 72% and 78% yield, respectively.
The X-ray analysis of 2c (16␤, 17␣-configuration)
proved the (E)-configuration of the Schiff base (Figure 1).
The 16N-16C-17C-17O torsion angle was estimated as
135.6° and is in agreement with the value of 140° for the
corresponding Schiff base derived from salicylic alde-
hyde.28 The torsion angle 16␣H-16C-16␤NAC of 16.5°
demonstrated the preferred eclipsed to skew arrangement of
the imino bond to the 16-proton and is in agreement with
our other data on 16-salicylidenimines.28
In contrast to the trans-series, the cis-configurated amino
alcohols 1a and 1d (Scheme 1) with 16N-16C-17C-17O
torsion angles of about 30° 27,28 gave the (E)-azomethines 2
in mixtures with the oxazolidines 3. In solution, 2 was found
as an equilibrium mixture with its tautomeric oxazolidine
(3); in addition, 3 was also a mixture of the (R)- and
(S)-epimers (Scheme 2). Thus, in addition to the typical
1
[M-0.5H2O]ϩ (100%, isotopic pattern of Fe). - H-NMR
(CDCl3): ␦ ϭ 2.850 (m, 1 H, NH), 3.443 (s, 2 H, N-CH2),
4.069 (m, 2 H, cpЈ), 4.101 (s, 5 H, cp), 4.159 (m, 2 H, cpЈ).
-13C-NMR (CDCl3): ␦ ϭ 46.26 (N-CH2), 67.56 (cpЈ), 68.36
(overl., cp and cpЈ), 87.86 (s, cpЈ). - Anal. (C38H59FeN ⅐
0.5H2O, 594.7): calc. C 76.74, H 10.17, N 2.36; found. C
76.47, H 10.13, N 2.35.
3␣-(N-ferrocenylmethyl)acetamido-5␣-cholestane (12c):
Four hundred seven milligrams (0.68 mmol) of 3␣-(ferro-
cenylmethyl)amino-5␣-cholestane (12b) were dissolved in
3 mL of pyridine and 1.5 mL of acetanhydride and stirred at
room temperature for 24 h. Then, the reaction mixture was
poured into 100 mL of cold water. The solid was filtered,
washed with water, and dried in vacuum. The crude product
was purified by column chromatography on Al2O3 with
ethyl acetate as the solvent. - Yield: 75%; yellow, waxy
compound. - 1H-NMR (CDCl3): ␦ ϭ 2.146 (s, broad), 3 H,
COCH3), 4.054 (m, 2 H, cpЈ), 4.118 (s, 5 H, cp), 4.211 (m,
2 H, cpЈ). -13C-NMR (CDCl3): ␦ ϭ 67.56 (cpЈ); 67.57 (cp);
86.36 (cpЈ). - Anal. (C40H61FeNO, 627.8): calc. C 76.53, H
9.79, N 2.23; found C 76.41, H 9.87, N 2.12.
1
azomethine singlet at about 8.1–8.2 ppm (for 2), the H
NMR spectra (CDCl3, 400 MHz) exhibited two singlets in
the range of 5 ppm representing the epimeric oxazolidines 3.
Consistently, three 18-H3 singlets indicated the presence of
three isomers. By means of 1H NMR signal integration, the
isomer distribution in CDCl3 was determined (Table 1).
The ratio of tautomers and epimers was unaffected by
further recrystallization of the crude products but was, how-
ever, temperature dependent as proved by a variable-
temperature 1H NMR investigation for 2a/3a (Table 1). This
indicated a fast equilibrium at ambient temperature despite
the formal disfavored 5-endo-trig ring closure reaction
(Baldwin Rules29,30).
In the 16␤, 17␤ series, the (E)-azomethine 2a was the
dominating tautomer (e.g., at r.t. 68%). Its share increased
with increasing temperature (40°C: 74%) and diminished at
lower temperature (e.g., Ϫ30°C: 52%).
The ratio of the corresponding oxazolidine epimers 3a
exhibited a pronounced temperature dependence: tempera-
Biology
Agar diffusion assay for antimicrobial activity
The following strains were used as test-organisms:
Gram-positive bacteria: Bacillus subtilis ATCC 6633, Mi-
crococcus flavus ATCC 10240, Staphylococcus aureus
134/94 (MRSA), Enterococcus faecalis 1528 (vancomycin-
resistant), Mycobacterium chelonae B, M. fortuitum B,
Gram-negative bacterium: Escherichia coli AS19, fungus:
Glomerella cingulata. The melted and tempered agar me-
dium was inoculated with 106 CFU/ml of the test-organisms
and poured into petri dishes. Agar wells of 9-mm diameter
Steroids, 1998, vol. 63, October 539

Papers
ture decrease favoured one of both epimers (e.g., at r.t.,
16.5% and 15.5%, respectively; at Ϫ30°C, 29%, and 19%,
respectively). In contrast, in the 16␣, 17␣ series, the oxazo-
lidine isomer 3d was clearly preferred (92% at room tem-
perature, ratio of epimers 59: 33 3d). The use of pyridine-D5
as solvent shifted the equilibrium in favor of 2d only
slightly (9% 2d).
These results are supported by considerations of the
molecular geometry as well as by a simple computer aided
geometry estimation (HyperChem 228) of 2a/3a and 2d/3d,
which implicated the thermodynamical preference of the
oxazolidine tautomer 3. In the 16␤/17␤-cis series, both
epimers of 3a exhibited a steric repulsion of the oxazolidine
moiety and the 13-methyl group at the steroid skeleton. This
steric effect should contribute considerably to an equilib-
rium shift in favour of 2a.
Tables 2 and 3, all of the (16-ferrocenylmethyl)amino-
estratrienes 4a–d or their hydrochlorides exhibited out-
standing broad activity against all organisms tested includ-
ing MRSA, glycopeptide resistant enterococci, and different
strains of fast growing mycobacteria. Compounds 7b and
10b, which are 16-(ferrocenylmethyl)amino-estratrienes
without 17-O-function, showed the same broad activity to
nearly the same extent. For compounds 4b, 4d, and 7b
activity against M. tuberculosis H37 Rv was also determined
in the TAACF screening program (Tuberculosis Antimicro-
bial Acquisition & Coordinating Facility, National Institute
of Allergy and Infectious Diseases, Southern Research In-
stitute, Birmingham, Alabama, USA). Minimal inhibitory
concentrations (MICs) of 6.25 ␮g/mL for 4d and below
12.5 ␮g/mL for 4b and 7b were determined. The (E)-Schiff
bases 2a and 2b exhibited only weak activity against sen-
sitive Gram-positive bacteria.
The 3-(N-ferrocenylmethyl)amino-cholestanes 12 were
inactive. Ferrocenylmethylaminoethanol was also inactive.
Consequently, the activity of the 16-ferrocenylmethylamino
steroids must be attributed to the ferrocenyl group and the
estratriene steroid moiety. Interestingly, neither the 17-
oxygen function nor the configuration of the 16-nitrogen
function are important for high activity. Therefore, the
activities of the primary amino compounds 1a to 1d and 6b
and 9b have been determined (Table 4). From these results,
it is evident that the 16-amino group is essential for anti-
microbial activity. The ferrocenylmethyleneimino group di-
minished activity, while the more stable ferrocenylmethyl-
amino group caused a remarkable increase in antimicrobial
activity. Rifampicin, isoniazid, and ciprofloxacin, which
were used as standards, displayed another pattern of activity
and did not cover the broad range of organisms as did the
16-(ferrocenylmethyl)amino-estratrienes. Based on the
structural properties, a mode of action different from that of
known drugs should be anticipated for the ferrocenylmeth-
ylamino steroids. Investigations of other amino steroids and
their derivatives are under way.
This tendency was confirmed by the condensation reac-
tions of 1 with salicylic aldehyde. In these cases, only 1d
gave the oxazolidines4,28 in part.
31–33
The reduction of Schiff bases with NaCNBH3
or
NaBH433–35 has been shown to be effective in amine ligand
synthesis in the presence of a variety of functional groups.
We obtained the ferrocenymethylamines 4 in high yields by
reduction of the Schiff bases 2 with an excess of NaBH4. It
is remarkable that in the cis-configurated series a and d, the
reduction of the equilibrium mixtures 2/3 furnished the
amines 4a and 4d in excellent yields, even in the case of the
dominating oxazolidine 3d. This result is due to the above
mentioned fast equilibrium. Depending on the work-up pro-
cedure, compounds 4 were obtained as amines or as their
hydrochlorides.
The correct structures were determined from NMR, IR,
elemental analysis, and in part, from MS investigations. The
X-ray structure of the hydrochloride 4d is given in Figure 2
and exhibits a 16N-16C-17C-17O torsion angle of 37.4°C in
agreement with both the predicted value of 30°C27 and with
the value of the corresponding salicylidenimine (31°C).28
The synthesis of the novel (N-ferrocenylmethyl)-
aminosteroids 4 can be carried out advantageously as a one
pot procedure too without isolation of the Schiff bases.
The approaches to 7b and 10b, to the 17-unsubstituted 16␤-
and 16␣-(N-ferrocenymethyl)amines, and to the 3␣-(N-
ferrocenylmethyl)amino-cholestanes 12, are outlined in
Scheme 3. Intermediates are the hitherto unknown 16-azido
(6a, 9a) and 16-amino compounds (6b, 9b). Starting
compounds were 3-methoxy-estra-1,3,5(10)-triene-16␤-ol
(5a),23 the amino alcohol 11,25 and 3␤-tosyloxy-cholestane
(13). Synthesis of the 3␣-azidosteroid 1424 was improved
considerably by using HMPT as the solvent. The use of
dichloromethane instead of tetrahydrofuran36 as the solvent
was advantageous for the synthesis of the hitherto unknown
16␣-bromo-3-methoxy-estra-1,3,5(10)-triene (5b) from the
16␤-hydroxy compound 5a using NBS and triphenylphos-
phine.
Acknowledgment
We are grateful to Schering AG for the supply of steroids
and to the Deutsche Forschungsgemeinschaft and Freistaat
Thu¨ringen for financial support.
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