Convenient synthesis of new pregnenolone oximinyl oxalate dimers

Article abstract of DOI:10.1007/s10600-008-9051-x

Three new symmetrical pregnenolone oxyminyl oxalate dimers (8-10) were synthesized from the corresponding pregnenolone oximes (3, 5, and 7) at room temperature. All dimers were characterised by spectroscopic means, notably HRFABMS and comprehensive NMR spectroscopic data analyses.

Full text of DOI:10.1007/s10600-008-9051-x

Chemistry of Natural Compounds, Vol. 44, No. 3, 2008
CONVENIENT SYNTHESIS OF NEW PREGNENOLONE
OXIMINYL OXALATE DIMERS
L. Nahar,1* S. D. Sarker,2 and A. B. Turner1
UDC 547.918
8 10
Three new symmetrical pregnenolone oxyminyl oxalate dimers ( - ) were synthesized from the
3 5
7
corresponding pregnenolone oximes ( , , and ) at room temperature. All dimers were characterised by
spectroscopic means, notably HRFABMS and comprehensive NMR spectroscopic data analyses.
Key words: pregnenolone, oxime, esterification, oxalate dimers, NMR.
In continuation of the search for biologically active steroid-based dimers [1], we have used pregnenolone (1) as the
starting substance. Pregnenolone, a well-known vertebrate hormone, is a primary steroid on the pathway from cholesterol to
the androgens, estrogens, progesterone, and the corticoids, and also the starting material for several other pharmacologically
active steroids [2, 3]. As part of our initiative for synthesizing new steroid dimers towards the generation of a libraryof steroidal
dimers for relevant pharmacological screening, we now report on the synthesis of three new pregnenolone oximinyl oxalate
dimers (8-10), which contain an oxalate ester linkage between the 20-one oxime positions of two molecules.
Oximes, as they contain a hydroxyl group linked to nitrogen, behave like alcohols, and are considered to be important
reaction intermediates for the synthesis of various nitrogen-containing compounds. To synthesize the target dimer bis(3β-
acetoxypregn-5-en)-20-on-oximinyl oxalate (8), it was necessaryto prepare the steroid monomer 3β-acetoxypregn-5-en-20-one
oxime (3). In order to functionalize C-20 of pregnenolone (1), its 3β-hydroxy group was protected by acetylation [4-6]. The
acetylated pregnenolone 2 was treated with hydroxylamine hydrochloride to obtain 20-one oxime 3 [4]. An increase of 15 mass
units in its molecular mass (compared to that of 2) obtained from the ESIMS spectrum ([M+H]+ and [M+Na]+ at m/z 374 and
396, respectively) confirmed the presence of a C=N-OH instead of a C=O group. The 1H NMR showed a broad 1H singlet at
δ 8.88 for hydroxyl proton, and an olefinic quaternary carbon at δ 158.7 observed in its 13C NMR spectrum was characteristic
O
O
for C=N-OH group.
R
1
R
2
Ac O
2
HONH ⋅ HCl
2
Py, 18h
Et N, Py, 6h
3
HO
AcO
R
1
2, 4, 6
N OH
R
1
R
2
O
N
(COCl)
Steroid
O
N
2
O
Steroid
Py, 18h, r.t.
O
AcO
R
3, 5, 7
8, 9, 10
2, 3: R = 5-dehydro, R1 = R2 = H; 4, 5: R = 5-dehydro, R1 = H, R2 = 16-dehydro
6, 7: 5α-H, R1 = 11α-OAc, R2 = 16-dehydro
1) Japp Laboratory, Department of Chemistry, Universityof Aberdeen, Meston Walk, Old Aberdeen, Aberdeen AB24
3UE, Scotland, UK; 2) School of Biomedical Sciences, Universityof Ulster at Coleraine, Cromore Road, Coleraine BT52 1SA,
Co. Londonderry, Northern Ireland, UK, e-mail: lnahar2003@yahoo.co.uk. Published in Khimiya Prirodnykh Soedinenii,
No. 3, pp. 250-252, May-June, 2008. Original article submitted December 15, 2005.
0009-3130/08/4403-0315 ©2008 Springer Science+Business Media, Inc.
315

TABLE 1. 13C NMR (CDCl3, 100 MHz) Data of Dimers 8-10
Chemical shifts (δ, ppm)
C atom
8
9
10
1, 1′
2, 2′
3, 3′
4, 4′
5, 5′
6, 6′
7, 7′
8, 8′
9, 9′
10, 10′
11, 11′
12, 12′
13, 13′
14, 14′
15, 15′
16, 16′
17, 17′
36.8
27.8
73.8
38.1
139.7
122.2
31.7
32.0
50.0
36.6
21.0
38.4
44.0
56.2
24.1
23.4
56.6
13.2
19.3
167.5
17.1
170.5
21.4
170.5
-
36.9
27.7
73.8
34.6
140.2
122.0
32.1
30.2
50.2
36.7
20.9
38.1
46.5
56.7
31.4
139.5
149.7
13.4
15.4
159.5
19.2
170.5
21.4
170.5
-
37.1
27.7
73.1
34.4
44.6
28.8
33.2
33.5
55.5
36.7
70.5
42.2
46.3
56.9
31.9
139.2
149.0
12.7
13.4
159.0
16.7
170.5
21.4
170.5
21.9
170.7
18, 18′
19, 19′
20, 20′
21, 21′
2×CO (oxalate)
2×Me (3β-OAc)
2×CO (3β-OAc)
2×Me (11α-OAc)
2×CO (11α-OAc)
-
-
Similarly, twoother oximes, 3β-acetoxypregna-5,16-dien-20-oneoxime(5)and3β,11α-diacetoxy-5α-pregn-16-en-20-
one oxime (7) were synthesised, respectively, from acetylated ketonic derivatives 4 and 6 as the precursors for corresponding
oximinyl oxalate dimers (9 and 10). The identity of 5 was confirmed by comparison of its mp, IR, MS, and 1H NMR data with
published data [7]. The ESIMS spectrum of 5 revealed the [M+H]+ and [M+Na]+ ions, respectively, at m/z 372 and 394, which
confirmed the molecular formula, C23H33NO3, for this compound. An olefinic quaternary carbon at δ 154.5 in its 13C NMR
spectrum was characteristic for the C=N-OH group.
The oxime 7 was synthesized previously as a reaction intermediate and identified by forming 3β,11α-diacetoxy-5α-
androstan-17-one [8], but no physical or spectroscopic data for this compound could be found in the literature. The HRFABMS
spectrum of 7 revealed the [M+H]+ ion at m/z 432.274978 which confirmed the molecular formula, C25H37NO5, for this
compound. This fact was confirmed further from a broad 1H singlet at δ 7.51 (for the hydroxyl proton) in its 1H NMR and a
signal for an olefinic quaternary carbon at δ 153.3 for C=N-OH in its 13C NMR spectrum.
Bis(3β-acetoxypregn-5-en)-20-on-oximinyl oxalate (8) was synthesized from the reaction between 3β-acetoxypregn-
5-en-20-one oxime (3) and oxalyl chloride. The HRFABMS spectrum of 8 exhibited the [M+H]+ ion at m/z 801.50536
corresponding toC48H69N2O8 and thus confirmed the formation of the dimer. In the13C NMRspectrum (Table 1), the chemical
shifts of four carbonyl carbons, two of the oxalates and two of the acetate moieties, appeared at δ 170.5. The oxalate carbonyls
contributed to the downfield shift of C-20 carbon that appeared at δ 167.5 as opposed to δ 158.7 for the parent oxime.
Conjugated oximes show enhanced reactivity in biological environment compared to free oximes [8]. The steroid
monomer, 3β-acetoxypregna-5,16-dien-20-one oxime (5), is an important keyintermediate for the synthesis ofantitumor drugs
[9, 10]. It has recently been found that 3β-acetoxypregna-5,16-dien-20-one oxime (5) is a potent inhibitor of human P45017α
[7]. Therefore, synthesis of the dimer of 5 via oxalic acid spacer appeared to be an interesting prospect as the dimer may have
the potential of exerting enhanced antitumor properties compared to its monomer. Bis (3β-acetoxypregna-5,16-dien)-20-on-
oximinyl oxalate (9) was synthesized from 3β-acetoxypregna-5,16-dien-20-one oxime (5) using oxalyl chloride. The molecular
316

mass of 9 was confirmed from its HRFABMS spectrum where the [M+H]+ was observed at m/z 797.47406 corresponding to
C48H65N2O8.
Anotherconjugated16-dehydrooximedimer wassynthesizedin asimilar manner. Bis(3β,11α-diacetoxy-5α-pregn-16-
en)-20-on-oximinyl oxalate (10) was synthesised from 3β,11α-diacetoxy-5α-pregn-16-en-20-one oxime (7) using oxalyl
chloride. In its HRFABMS spectrum, the [M+H]+ ion at m/z 917.51633 corresponding to C52H73N2O12 confirmed the identity
of this dimer. The 13C NMR spectral behavior of 9 and 10 (Table 1) was similar to that observed for the dimer 8, especially in
relation to the chemical shifts of the carbonyl carbons and C-20 olefinic quaternary carbon. In both cases, all four carbonyl
carbons gave a single resonance at δ 170.5, and the C-20 carbon signal was deshielded compared to those of their parent
monomers.
EXPERIMENTAL
General Procedures[1]. Pregnenolone(1), 3β-hydroxypregna-5,16-dien-20-one,3β,11α-dihydroxy-5α-pregn-16-en-
20-one oxalyl chloride, and dry pyridine were purchased from Aldrich and used as received.
3β-Acetoxypregn-5-en-20-one (2). A stirred solution of pregnenolone (1, 1.0 g, 3.16 mmol) in dry pyridine (5 mL)
was added to Ac2O (3 mL) and stirred for 18 h at room temperature. Then the reaction mixture was quenched with ice-H2O,
and a white precipitate was formed. The precipitate was collected, taken into ether, dried over MgSO4, and evaporated to afford
1
the title compound 2 (990 mg, 87%) as a white solid, mp: 136-137°C (lit. mp: 135-137°C [4], IR [5], MS [5], H NMR and
13C NMR [6]). A similar synthetic procedure was followed for the synthesis of compounds 4 and 6.
3β-Acetoxypregn-5-en-20-one Oxime (3). A stirred solution of (2, 930 mg, 2.59 mmol) in dry pyridine (5 mL) was
treated with hydroxylamine hydrochloride (360 mg, 5.19 mmol) and Et3N (5 mL). The mixture was refluxed for 6 h. Finally,
the reaction mixture was quenched with ice-H2O, and a white precipitate was formed. The precipitate was collected and taken
into EtOAc, dried over MgSO4, and evaporated to afford the title compound 3 (851 mg, 88%) as a white solid, mp: 183-185°C
−
1
(lit. mp: 187-189°C [4]). IR (CHCl3, νmax cm ): 3313br (O-H), 2937s (C-H), 2854s (C-H), 1732vs (ester C=O), 1654m (C=C),
1439m, 1369m, 1249s (ester C-O), 1041m and 668m. 1HNMR(400 MHz, CDCl3, δ): 0.63 (s, 3H, 18-Me), 1.00 (s, 3H, 19-Me),
2.01 (s, 3H, 21-Me), 1.99 (s, 3H, 3β-OCO-Me), 4.57 (m, 1H, 3α-OCH), 5.36 (br m, 1H, 6-CH), 8.88 (br s, 1H, =N-OH).
13C NMR (100 MHz, CDCl3, δ): 37.0 (C′-1), 27.8 (C-2), 73.9 (C-3), 38.1 (C-4), 139.7 (C-5), 122.4 (C-6), 31.8 (C-7), 32.0
(C-8), 50.1 (C-9), 36.6 (C-10), 21.0 (C-11), 38.6 (C-12), 43.8 (C-13), 56.8 (C-14), 24.2 (C-15), 23.1 (C-16), 56.1 (C-17), 13.1
(C-18), 15.2 (C-19), 158.7 (C-20), 19.3 (C-21), 170.6 (3E-OCO-Me), 21.5 (3E-OCO-Me). ESIMS: m/z 374 [M+H]+, 396
[M+Na]+.
3β-Acetoxypregna-5,16-dien-20-one Oxime (5). The title compound 5 (510 mg, 52%), mp 182-183°C (lit. mp 180-
185°C [7], IR and 1H NMR [7]) was synthesized from 3β-acetoxypregna-5,16-dien-20-one (4, 950 mg, 2.66 mmol) using
the procedure described for compound 3. 13C NMR (100 MHz, CDCl3, δ): 36.9 (C-1), 27.7 (C-2), 74.0 (C-3), 38.2 (C-4),
140.1 (C-5), 122.3 (C-6), 31.6 (C-7), 30.3 (C-8), 50.4 (C-9), 36.8 (C-10), 20.9 (C-11), 35.6 (C-12), 46.6 (C-13), 57.1 (C-14),
31.6 (C-15), 133.0 (C-16), 151.5 (C-17), 11.7 (C-18), 15.9 (C-19), 154.5 (C-20), 19.3 (C-21), 170.7 (3β-OCO-Me), 21.5
(3β-OCO-Me). ESIMS: m/z 372 [M+H]+, 394 [M+Na]+.
3β,11α-Diacetoxy-5α-pregn-16-en-20-one Oxime (7). The title compound 7 (500 mg, 48%), mp 120-122°C was
synthesised from 3β,11α-diacetoxy-5α-pregn-16-en-20-one (6, 1.0 g, 2.40 mmol) using the procedure described for compound
−
1
3. IR (CHCl3, νmax cm ): 3378 br (O-H), 2928s (C-H), 2857s (C-H), 1731s (ester C=O), 1590w (C=C), 1443m, 1372m, 1247s
(ester C-O), 1021m, 962m and 753s. 1H NMR (400 MHz, CDCl3, δ): 0.89 (s, 3H, 18-Me), 0.91 (s, 3H, 19-Me), 1.99 (s, 3H,
21-Me), 1.91 (s, 3H, 11α-OCO-Me), 1.91 (s, 3H, 3β-OCO-Me), 4.61 (m, 1H, 3α-OCH), 5.17 (m, 1H, 11β-OCH), 5.96 (t,
J = 2.1, 1H, 16-CH=C), 7.51 (br s, 1H, =N-OH). 13C NMR (100 MHz, CDCl3, δ): 36.9 (C-1), 27.7 (C -2), 73.1 (C-3), 34.4
(C-4), 44.7 (C-5), 31.4 (C-6), 31.9 (C-7), 33.4 (C-8), 55.4 (C-9), 37.2 (C-10), 71.5 (C-11), 42.7 (C-12), 46.6 (C-13), 56.9
(C-14), 28.9 (C-15), 132.1 (C-16), 150.8 (C-17), 11.0 (C-18), 12.8 (C-19), 153.3 (C-20), 17.0 (C-21), 170.7 (11α-OCO-Me),
22.0 (11α-OCO-Me), 170.5 (3β-OCO-Me), 21.4 (3β-OCO-Me). FABMS: m/z 432 [M+H]+, 454 [M+Na]+. HRFABMS calc.
for C25H38NO5: 432.274978; found: 432.274978.
Bis(3β-acetoxypregn-5-en)-20-on-oximinyl Oxalate (8). A stirred solution of 20-one oxime 3 (200 mg, 0.54 mmol)
in dry pyridine (5 mL) was treated with oxalyl chloride (34 mg, 0.27 mmol) in DCM. After 18 h, water (10 mL) was added
slowlytothe resulting mixture, precipitation occurred, and the precipitate was filtered off. It was then washed several times with
317

water to remove pyridine hydrochloride. Finally, the solid was dissolved in ether (10 mL) and evaporated to dryness at 35°C.
Recrystallization from a mixture of (2:1) CHCl3 and EtOAc yielded a white solid as the title compound 8 (125 mg, 58%),
−
1
mp: 204-205°C. IR (CHCl3, νmax cm ): 2943s (C-H), 2853s (C-H), 1764s (oxime oxalate C=O), 1732s (oxime oxalate and
acetate C=O), 1638m (C=C), 1438m, 1374m, 1247s (acetate C-O), 1134s (C-O), 1033m and 761m. 1H NMR (400 MHz,
CDCl3, δ): 0.64 (s, 6H, 18-Me and 18′-Me), 1.00 (s, 6H, 19-Me and 19′-Me), 2.02 (s, 6H, 21-Me and 21′-Me), 1.99 (s, 6H,
3-OCO-Me and 3′-OCO-Me), 4.57 (br m, 2H, 3-CH-O and 3′-CH-O), 5.35 (br m, 2H, 6-CH and 6′-CH) and 13C NMR
(Table 1). FABMS: m/z 801 [M+H]+, 823 [M+Na]+. HRFABMS calc. for C48H69N2O8: 801.50536; found: 801.50536. Similar
synthetic procedure was followed for the synthesis of dimers 9 and 10.
Bis(3β-acetoxypregna-5,16-dien)-20-on-oximinyl Oxalate (9). Thetitlecompound9(150mg, 47%, mp: 165-167°C)
was synthesized using 20-one oxime 5 (300 mg, 0.81 mmol in dry pyridine (5 mL) and oxalyl chloride (51 mg, 0.40 mmol).
−
1
IR (CHCl3, νmax, cm ): 3313 br (O-H) 2937s (C-H), 2854s (C-H), 1764s (oxime oxalate C=O), 1732s (oxime oxalate and
acetate C=O), 1654m(C=C), 1439m, 1369m, 1249s (acetate C-O), 1132s (C-O), 1041m and 668m. 1HNMR(400 MHz, CDCl3,
δ): 0.83 (s, 6H, 18-Me and 18′-Me), 0.98 (s, 6H, 19-Me and 19′-Me), 2.09 (s, 6H, 21-Me and 21′-Me), 1.99 (s, 6H, 3-OCO-Me
and 3′-OCO-Me), 4.56 (m, 2H, 3-CH-O and 3′-CH-O), 5.33 (br m, 2H, 6-CH and 6′-CH), 6.33 (br m, 2H, 16-CH and 16′-CH)
and 13C NMR (Table 1). FABMS: m/z 797 [M+H]+, 819 [M+Na]+. HRFABMS calc. for C48H65N2O8: 797.47406; found:
797.47406.
Bis (3β,11α-diacetoxy-5α-pregn-16-en)-20-on-oximinyl Oxalate (10). The title compound 10 (424 mg, 38%, mp
229-230°C) was obtained from 20-one oxime 7 (1.0 g, 2.42 mmol) in dry pyridine (15 mL) and oxalyl chloride (154 mg,
−
1
1.21 mmol). IR (CHCl3, νmax, cm ): 3364 br (O-H) 2930s (C-H), 2860s (C-H), 1765s (oxime oxalate C=O), 1731s (oxime
oxalate and acetate C=O), 1594w (C=C), 1449m, 1373m, 1258s (acetate C-O), 1131s (C-O), 1027m, 916m and 733s. 1H NMR
(400 MHz, CDCl3, δ): 0.91 (s, 6H, 18-Me and 18′-Me), 0.94 (s, 6H, 19-Me and 19′-Me), 1.89 (s, 6H, 11-OCO-Me and
11′-OCO-Me), 1.94 (s, 6H, 21-Me and 21′-Me), 1.91 (s, 6H, 3-OCO-Me and 3′-OCO-Me), 4.60 (m, 2H, 3-CH-O and 3′-CH-O),
5.18 (m, 2H, 11-CH-O and 11′-CH-O), 5.97 (t, J = 2.1, 2H, 16-CH and 16′-CH) and 13C NMR (Table 1). FABMS: m/z 917
[M+H]+, 939 [M+Na]+. HRFABMS calc. for C52H73N2O12: 917.51631; found: 917.51633.
ACKNOWLEDGMENT
The authors thank the funding body, the University of Aberdeen Development Trust, for a PhD studentship (LN) and
EPSRC National Mass Spectrometry Service Centre (Department of Chemistry, University of Wales Swansea, Swansea, UK)
for MS and HRMS analysis.
REFERENCES
1.
2.
L. Nahar, S. D. Sarker, and A. B. Turner, Chem. Nat. Comp., 42, 549 (2006).
J. B. Dence, Steroids and Peptides, John Wiley and Sons: New York, 1980.
3.
4.
5.
6.
P. M. Dewick, Medicinal Natural products – A Biosynthetic Approach, John Wiley and Sons: New York, 1997.
B. U. Khodzhaev, R. Shakirov, and S. Y. Yunusov, Chem. Nat. Compd., 16, 278 (1980).
M. P. S. Ishar, N. K. Girdhar, K. Kumar, and R. Kaur, Indian J. Chem. Sect. B., 38, 1253 (1999).
Z. Szendi, P. Forgo, and F. Sweet, Steroids, 60, 442 (1995).
7.
8.
9.
10.
R. W. Hartmann, M. Hector, S. Haidar, P. B. Ehmer, W. Reichert, and J. Jose, J. Med. Chem., 43, 4266 (2000).
V. Meystre, J. Kalvoda, G. Anner, and A. Wettstein, Helv. Chim. Acta., 46, 2844 (1963).
C. I. Hong, Antitumour Steroids, by Blickenstaff, RT. Academic Press: New York, 1992.
G. H. Rasmusson, G. F. Reynolds, N. G. Steinberg, E. Walton, G. F. Patel, T. Liang, M. A. Cascieri,
A. H. Cheung, J. R. Brooks, and C. Bermann, J. Med. Chem., 29, 2298 (1986).
318