Identification of a fossil sterane biomarker in crude oil - An androstane with a modified carbon skeleton

Article abstract of DOI:10.1002/ejoc.201300788

Three constitutional isomers of androstane were prepared for comparison with three unknown fossil C₁₉H₃₂ organic biomarkers ("19A", "19B", and "19C" in elution order in geological samples from Oman. 3β-Methyl-A-nor-androstane was pre

Full text of DOI:10.1002/ejoc.201300788

FULL PAPER
DOI: 10.1002/ejoc.201300788
Identification of a Fossil Sterane Biomarker in Crude Oil – an Androstane
with a Modified Carbon Skeleton
Matthias Bender,[a,b] Marc Schmidtmann,[a] Jürgen Rullkötter,[b] Roger E. Summons,[c] and
Jens Christoffers*[a]
Dedicated to Professor Dr. Karl-Heinz Dötz on the occasion of his 70th birthday
Keywords: Natural products / Steroids / Total synthesis / Structure elucidation / Fossil biomarkers / Crude oil
Three constitutional isomers of androstane were prepared for
comparison with three unknown fossil C19H32 organic bio-
markers (“19A”, “19B”, and “19C” in elution order) in geolo-
gical samples from Oman. 3β-Methyl-A-nor-androstane was
prepared in six steps (8% overall yield) from testosterone.
The key steps of this sequence were an Eschenmoser frag-
mentation and recyclization of the A ring. A mixture of four
stereoisomers of 3-methyl-A-nor-androstane (4% overall
yield) was prepared by ionic hydrogenation of the olefin after
A-ring recyclization in three steps. 17-Methyl-18-nor-andros-
tane was synthesized in four steps as a mixture of isomers
(58% overall yield) from dihydrotestosterone with a Wagner–
Meerwein shift of the 13β-methyl group to C-17 as the key
step. Pure 17β- and 17α-methyl-18-nor-13α-androstane (4
and 2% overall yield, respectively) were obtained in three
additional steps after α-oxidation of the Wagner–Meerwein
rearrangement product and subsequent reduction. The syn-
thesis of 18-nor-D-homo-13β-androstane (12% yield over
seven steps from dihydrotestosterone) involved oxidative
cleavage of the C–C double bond in the Wagner–Meerwein
rearrangement product from the previous synthesis and sub-
sequent recyclization of the D ring followed by reduction.
The relative configurations of all final products were con-
firmed by X-ray crystallography. A comparison of the syn-
thetic standards with the saturated hydrocarbon fraction of
an Oman crude oil by gas chromatography (GC–MS) coinjec-
tion on three different GC columns and comparison of mass
spectra revealed that unknown compound 19C is 18-nor-D-
homo-13β,14α-androstane, whereas the isomeric 3-methyl-
A-nor-androstanes and 17-methyl-18-nor-androstanes elute
close to 19A and 19B, respectively, but do not match the un-
knowns.
from Eastern Siberia, India, and Australia contain the same
hydrocarbons.[1c] As there is empirical evidence that the oc-
currence and isomer distribution of unknown biomarkers
correlate with geological age and the salinity of the water
during sediment accumulation, there is considerable interest
in revealing the precise structures of the unknowns to trace
them back to potential biogenic ancestors in specific pre-
cursor organisms. The concentrations of the unknown bi-
omarkers in the Oman samples are too low for them to be
readily isolated as pure compounds for rigorous spectro-
scopic structural identification.
Introduction
Based on extended organic geochemical investigations,
mass spectral information indicated that samples of approx-
imately 550-million-year-old crude oils and sedimentary
rocks from the Oman Salt Basin contains (at least) three
gas chromatographically separated isomers of putative C19
steranes (C19H32, m/z = 260; abbreviated as 19A, 19B, and
19C in order of elution) with modified androstane carbon
skeletons and unknown constitutions.[1] Coeval sediments
In our previous synthetic studies, we prepared steranes
1–3 (Figure 1), all of which have m/z = 260 [M]+.[2] Their
mass spectra and gas chromatographic retention times were
compared with those of the putative unknown C19 steranes.
Neither the spectral data nor the GC data matched closely
enough to infer a structural identity of the natural hydro-
carbons as any one of the synthetic standards. In continua-
tion of these efforts, we selected structures 4–6 as additional
reference compounds. Compound 4 is a regioisomer of 2α-
methyl-A-nor-androstane (3), in which we translocated the
[a] Institut für Chemie, Carl von Ossietzky Universität Oldenburg,
26111 Oldenburg, Germany
E-mail: jens.christoffers@uni-oldenburg.de
Homepage: www.christoffers.chemie.uni-oldenburg.de
[b] Institut für Chemie und Biologie des Meeres (ICBM), Carl von
Ossietzky Universität Oldenburg,
26111 Oldenburg, Germany
[c] Massachusetts Institute of Technology, Department of Earth,
Atmospheric and Planetary Sciences,
77 Massachusetts Avenue, E25-633, Cambridge, MA 02139,
USA
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300788
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Identification of a Fossil Sterane Biomarker in Crude Oil
Figure 2. ORTEP representation of the molecular structure of
ketone 12a.
Figure 1. Target compounds of our previous (1–3) and present syn-
thetic studies (4–6).
methyl group to the 3-position. 17-Methyl-18-nor-andros- tions in Figures 2–4). Finally, the 18-methyl group was in-
tane (5) represents the shift of the 18-methyl group to the corporated into the D ring to afford D-homo-18-nor-andro-
17-position (for atom numbering, see ORTEP representa- stane (6), another synthetic target.
Figure 3. ORTEP representation of the molecular structure of ketone 18a.
Figure 4. ORTEP representation of the molecular structure of hydrazone 19.
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and the A/B rings fused trans. As this scenario provokes a
1,3-strain of two pseudoaxial methyl groups on the A ring,
the hydrogenation may have been accompanied by palla-
dium-catalyzed epimerization at C-5. Alternatively, prior to
hydrogenation, the tetrasubstituted C-3(5) double bond in
Results and Discussion
The synthesis of 3-methyl-A-nor-androstane (4) started
with testosterone (7), which was submitted to a Weitz–
Scheffer[3] oxidation to give epoxide 8[4] (Scheme 1) as a
mixture of two diastereomers [55% yield, diastereomeric
ratio, dr (α/β)[5] = 30:70]. The latter was submitted to
Eschenmoser fragmentation[6] with tosylhydrazine to give
4,5-seco-ynone 10[7] in 60% yield, which was subsequently
reductively cyclized to 3-methyl-A-nor-androst-3(5)-en-17-
ol (9, 63%).[7] Catalytic hydrogenation of the C–C double
bond furnished the so far unknown alcohol 11a as a single
diastereomer. Its configuration was established after pyrid-
inium chlorochromate (PCC) oxidation[8] to ketone 12a
(99%), which is a solid material that yielded single crystals
suitable for X-ray structure determination (Figure 2).[9] Sur-
prisingly, the A and B rings of 12a are cis fused with 3β-
Me and 5β-H, which implies a formal anti dihydrogenation
of olefin 9. We actually presumed a syn hydrogenation from
the α face to put the 3-Me group into the β configuration
Scheme 2. Synthesis of four diastereomers of hydrocarbon 4 by
ionic hydrogenation of olefin 9. Reagents and conditions: (a) 1.
40 equiv. TFA, 20 equiv. Et3SiH, CH2Cl2, –20 to 23 °C, 16 h; 2.
KOH, MeOH, H2O, 23 °C, 0.5 h; (b) PCC, CH2Cl2, 23 °C, 16 h;
(c) excess. H2NNH2·H2O, KOH, DEG, 200 °C, 16 h.
Scheme 1. Synthesis of 3β-methyl-A-nor-5β-androstane (4a). Rea-
gents and conditions: (a) H2O2, NaOH, H2O, MeOH, 4 °C, 24 h;
(b) pTosNHNH2, CH2Cl2, AcOH, 0 to 23 °C, 2 h; (c) Li, NH3(l),
THF, –60 °C, 3 h, then excess NH4Cl; (d) H2 (3 bar), cat. Pd/C,
EtOAc, 60 °C, 16 h; (e) PCC, CH2Cl2, 23 °C, 16 h; (f) excess
H2NNH2·H2O, KOH, DEG, 200 °C, 16 h.
Scheme 3. Synthesis of five diastereomers of hydrocarbon 5 from
dihydrotestosterone (13). Reagents and conditions: (a) excess
H2NNH2·H2O, KOH, DEG, 200 °C, 16 h; (b) TosCl, cat. 4-dimeth-
ylaminopyridine (DMAP), pyridine, 55 °C, 5 d; (c) 5 equiv.
EtMgBr, toluene, 110 °C, 4 h; (d) H2 (1 bar), cat. Pd/C, EtOAc,
23 °C, 6 d.
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Identification of a Fossil Sterane Biomarker in Crude Oil
9 may have migrated (under Pd catalysis) to C-2–C-3 or as 11. Upon coinjection with the saturated hydrocarbon
C-3–C-4. A different diastereomer of 12a (with 5α-H) was fraction of an Oman crude oil, product 4a as well as its
actually reported previously.[10] Finally, ketone 12a was sub- stereoisomers eluted close to 19A but did not match the
mitted to the Huang-Minlon version[11] of the Wolff– naturally occurring unknown C19 compound. The mass
Kishner reduction in diethylene glycol (DEG) to give hy- spectra of the synthesis products slightly differed from that
drocarbon 4a (42%), again as a single diastereomer.
To access the diastereomers of 4a, we decided to perform
of 19A to different degrees.
The synthetic plan for 17-methyl-18-nor-androstane (5)
dihydrogenation of olefin 9 under ionic conditions via a envisaged a shift of the 13-methyl group to the 17-position
carbenium ion at either C-3 or C-5 (Scheme 2). An appro- by a Wagner–Meerwein rearrangement, as proposed by
priate protocol utilizes a mixture of trifluoroacetic acid Miescher und Kägi.[13] Therefore, we started with commer-
(TFA) and Et3SiH.[12] Indeed, the reaction basically pro- cially available dihydrotestosterone (13) and first removed
ceeded without any stereoselectivity, and four diastereomers the carbonyl group by
a Huang-Minlon reduction
of alcohol 11 were obtained [ratio (11a/b/c/d) 27:8:53:12 by (Scheme 3). Alcohol 14[14] was activated by sulfonylation,
retention time order and relative intensity, GC]. PCC oxi- and the tosylate 16[15] was treated with an excess of ethyl
dation and Huang-Minlon reduction gave hydrocarbon 4, Grignard reagent.[16] The methyl group shifted from the 13-
again as a mixture of four diastereomers in the same ratio position to the 17-position. A mixture of four tetrasubsti-
tuted olefins (ratio 4:12:80:4 by retention time order and
relative intensity, GC) was obtained in 91% yield; com-
pound 15[14] is assumed to be the major component (ca.
80%) of this mixture. This rearrangement is presumed to
Scheme 4. Synthesis of single diastereomers 5a and 5b (17β- and
17α-methyl-18-nor-13α-androstane) of hydrocarbon 5. Reagents
and conditions: (a) 20 equiv. CrO3, 40 equiv. pyridine, CH2Cl2,
23 °C, 16 h; (b) H2 (4 bar), cat. Pd/C, EtOAc, 40 °C, 3 d; (c) 2,4- Scheme 5. Synthesis of hydrocarbons 6a and 6b. Reagents and con-
(O2N)2C6H3NHNH2, H2SO4, EtOH, H2O, 23 °C, 10 min; (d) ex- ditions: (a) KMnO4, Me3NBnCl, CH2Cl2, 40 °C, 20 h; (b) cat.
cess H2NNH2·H2O, KOH, DEG, 200 °C, 16 h; (e) excess
H2NNH2·H2O, KOH, toluene, DEG, 110 °C, 5 h; then 200 °C,
16 h.
KOH, H2O, MeOH, 40 °C, 7 h; (c) H2 (4 bar), cat. Pd/C, pyridine,
23 °C, 1 d; (d) Li, NH3(l), THF, –60 °C, 3 h; (e) excess
H2NNH2·H2O, KOH, DEG, 200 °C, 16 h.
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proceed via a secondary carbenium ion, and the role of the hydrogenation of 17 occurred from the α face, which seemed
Grignard reagent is proposed to be that of a Lewis acid, to be less shielded by the axial 10β methyl group. To our
which is definitely unusual for such a reagent. Therefore, we surprise, we found hydrazone 19 also with cis annulation
wondered whether the rearrangement could also be induced of the C and D rings and the 17-methyl group in the α
by anhydrous MgBr2·OEt2. Indeed, a mixture of tetrasub- configuration (Figure 4); thus, compound 18b would be the
stituted olefins was obtained (77% yield), but only 66% of product of an anti dihydrogenation. Therefore, we first
this mixture was 15. However, catalytic hydrogenation of 15 checked whether product 19 has the same configuration as
and its isomers gave hydrocarbon 5 as a mixture of five starting material 18b, because epimerization could have oc-
stereoisomers (ratio 53:6:7:28:6 by retention time order and curred during hydrazone formation. Reductive cleavage of
intensity, GC). Upon investigation by GC–MS, the second hydrazone 19 with SnCl2 in tetrahydrofuran (THF)[19] gave
isomer of this mixture exhibited a GC retention time and ketone 18, which was identical to diastereomer 18b by
mass spectrum very similar to those of unknown Oman NMR and according to GC retention time. Thus, epimeriz-
biomarker 19B.[1a]
ation at C-17 must have occurred after syn dihydrogenation
To prepare a single stereoisomer of 5 with known config- of 17 from the α face. Such epimerizations were observed
uration, the mixture of olefin 15 (ca. 80%) and its regioiso- previously under Pd/C catalysis[20] in an apparently formal
mers was submitted to Collins oxidation[17] in the α position anti dihydrogenation.
to the C–C double bond (Scheme 4). Upon this transforma-
Ketones 18a and 18b were submitted to Huang-Minlon
tion, the minor isomers of 15 appear to be completely re- reduction, and hydrocarbons 5a (48%) and 5b (32%) were
moved, and enone 17 was obtained as a single isomer (34%; obtained. Actually, when a mixture of 18a and 18b was sub-
its constitution was established by 2D NMR experiments). mitted to this reduction, isomer 18a was converted signifi-
Catalytic hydrogenation gave ketone 18[18] as a mixture of cantly faster than 18b; thus, the latter was recovered to-
two stereoisomers 18a and 18b (ratio ca. 1:1). This mixture gether with product 5a. Compound 5a perfectly coelutes
was difficult to separate, but pure isomers 18a (35%) and with Oman crude oil component 19B[1a] on capillary col-
18b (33%) were obtained by repeated column chromatog- umns coated with DB-1MS and DBXLB, but has a slightly
raphy. The relative configurations of both materials were shorter retention time on a DB-5MS column and, thus, the
elucidated by X-ray crystallography. Although we directly two compounds are not structurally identical. Hydrocarbon
obtained single crystals of 18a, we failed to obtain single 5b has a GC retention time very similar to that of 5a; it
crystals of 18b. Therefore, we prepared hydrazone 19, which actually elutes as a trailing shoulder of 5a and was initially
fortuitously showed good crystallinity. ORTEP representa- not recognized as the sixth component in the original iso-
tions of both structures are depicted in Figures 3 and 4. mer mixture of 5. Nevertheless, upon coinjection with the
Rings C and D are cis annulated in 18a (Figure 3) and the Oman petroleum hydrocarbons on the DB-5MS column,
17-methyl group is in the β configuration; therefore, syn di- compound 5b elutes slightly earlier than 19B and, thus, also
Figure 5. GC–MS coinjection experiments of 6b and 19C.[1a] Chromatogram C is the m/z = 260Ǟ203 transition for the saturated
hydrocarbon fraction of an Oman crude oil (OMO 021), chromatogram B is compound 6b, and chromatogram A is the admixture of the
crude oil hydrocarbons containing 19C plus 6b.The box at the top of the GC peak shows a perfect match on a DB-5MS column.
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Identification of a Fossil Sterane Biomarker in Crude Oil
does not match the unknown. The mass spectra of 19B, 5a, retention times of synthetic androstane derivatives 4, 5, and
and 5b are virtually identical. The use of GC columns with 6 were compared with those of three putative unknown
even higher polarity was considered but discarded because C19H32 steranes (named 19A, 19B and 19C) in sediments
the dominant factor for the GC retention time of an apolar and crude oils from the Oman Salt Basin. 3-Methyl-A-nor-
saturated hydrocarbon is its boiling point.
androstane (4, four diastereomers were investigated) and
18-nor-D-homo-Androstane (6) is another androstane 17-methyl-18-nor-androstane (5, six diastereomers) had GC
isomer that formally involves use of the 13-methyl group retention times close to those of 19A and 19B, respectively,
for D-ring extension. Starting with a mixture of olefin 15 but did not match those of the unknowns closely enough
and its regioisomers, we oxidatively cleaved the C–C double to infer the structural identity of one of these two natural
bond[21] to obtain diketone 20, which was subsequently re- hydrocarbons. On the other hand, one of the diastereoiso-
cyclized to give enone 21 (Scheme 5). Catalytic hydrogen- mers of 6, namely, 18-nor-D-homo-13β,14α-androstane (6b),
ation in pyridine as solvent gave saturated ketone 22 (73% is identical to the hitherto unknown compound 19C, which
yield) as a mixture of cis and trans isomers (22a/22b 77:23). was (apart from an identical mass spectrum) proved by co-
Huang-Minlon reduction furnished the target hydrocarbon injection of the natural material on three different GC col-
6
[22] also as a mixture of cis and trans isomers (6a/6b 3.3:1). umns. Compounds 4, 5, and 6 were synthesized in several
A comparison of the analytical data revealed a match of steps from testosterone (7) or dihydrotestosterone (13), and
trans compound 6b with Oman crude oil constituent 19C[1a] the constitutions and relative configurations of dia-
on three different gas chromatographic columns (example stereomerically pure final products were established by X-
in Figure 5) and identical mass spectra.
ray crystallography. Further investigation will focus on the
We elucidated the relative configurations of 22a/22b and elucidation of constituents 19A and 19B by conceptualiza-
6a/6b as follows: The reduction of enone 21 under Birch tion of alternative target compounds for synthesis and the
conditions[23] gave a single ketone 22b, which was identical preparation of all-isomer mixtures of the products to ac-
to the minor isomer of 22 obtained by hydrogenation. This count for eventual geochemical epimerization reactions
compound 22b gave single crystals suitable for X-ray struc- during diagenesis (low-temperature geochemical transfor-
ture determination. Figure 6 shows an ORTEP representa- mation, usually Ͻ50 °C) and catagenesis (higher tempera-
tion of its molecular structure in the solid state; trans fusion tures, “oil window”) in the course of the geological history.
of the C and D rings is clearly visible. Consequently, hydro-
carbon 6b was obtained after Huang-Minlon reduction and
we were also able to grow single crystals of this compound
(Figure 7).
Experimental Section
General: Preparative column chromatography was performed with
Merck SiO2 (35–70 μm, type 60 A) with hexane, tert-butyl methyl
ether (MTBE), and ethyl acetate (EtOAc) as eluents. TLC was per-
formed with Merck aluminium plates coated with SiO2 F254.
1H
and 13C NMR spectra were recorded with a Bruker Avance DRX
500 instrument. The multiplicities of carbon signals were deter-
mined with DEPT experiments. MS and HRMS spectra of synthe-
sis products were obtained with a Finnigan MAT 95 (EI and CI)
and a Waters Q-TOF Premier (ESI) spectrometer. IR spectra were
recorded with a Bruker Tensor 27 spectrometer equipped with a
“GoldenGate” diamond attenuated total reflectance (ATR) unit.
GC–MS retention time experiments on petroleum hydrocarbons,
both alone and in admixtures with synthetic standards, were con-
ducted in multiple reaction monitoring (MRM) mode, and the
compound-specific 260 Da molecular ion to 203 Da fragment tran-
sition was targeted. We used a Micromass AutoSpec Ultima mass
spectrometer interfaced to an Agilent 6890 N gas chromatograph,
and the GC was fitted with a fused silica capillary column (60 m;
0.25 mm i.d.; 0.25 μm film thickness; J&W Scientific) with He as
carrier gas. The phases tested were DB-5MS, DB-1MS, and DB-
XLB (30 m for this column), and identity was assured by co-elution
on all three. The GC temperature program was 60 (2 min) to 150 °C
at 10 K/min and to 330 °C (held 19 min) at 3 K/min. The AutoSpec
source was operated in electron ionization (EI, 70 eV) mode at
250 °C with 8 kV accelerating voltage. Additional full scan analyses
were conducted over a range of m/z = 50–600. The data were ac-
quired and processed by using MassLynx 4.0 (Micromass Ltd.).
Figure 6. ORTEP representation of the molecular structure of
ketone 22b.
Figure 7. ORTEP representation of the molecular structure of hy-
drocarbon 6b.
17β-Hydroxy-4ξ,5ξ-epoxyandrostan-3-one (8): A solution of testos-
terone (7, 2.50 g, 8.67 mmol) in MeOH (100 mL) was treated at
0 °C successively with H2O2 (17 mL, 30% in H2O) and NaOH solu-
Conclusions
We identified a new steroidal hydrocarbon as a constitu-
ent of crude oil. The mass spectra and gas chromatographic tion (10 mL, 2% in H2O). The resulting mixture was stirred for
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J. Christoffers et al.
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24 h at 0 °C, and the solvent was evaporated under vacuum. The
crude material was treated with H2O (40 mL) and extracted with
EtOAc (3ϫ 50 mL). The combined organic layers were dried
(MgSO4), filtered, and concentrated under vacuum. After
chromatography (SiO2, MTBE, Rf = 0.44), epoxytestosterone 8
(1.45 g, 4.76 mmol, 55%) was obtained as a mixture of α- and β-
temperature overnight, and the NH3 evaporated. Water (5 mL) was
added, and the solution was extracted with MTBE (3ϫ 30 mL).
The combined organic solvents were dried (MgSO4) and filtered,
and the solvent was evaporated. The residue was chromatographed
(SiO2, hexane/MTBE 1:1, Rf = 0.37) to furnish olefin 9 (90 mg,
0.32 mmol, 63%) as colorless solid, m.p. 117–119 °C. [α]2D0 = 61.7
1
epoxides (α/β 0.3:0.7).[5] 1H NMR (500 MHz, CDCl3): δ = 0.75 (s, (CH2Cl2, 1 g/l). H NMR (500 MHz, CDCl3): δ = 0.69–0.80 (m, 2
0.7ϫ3 H, β isomer), 0.77 (s, 0.3ϫ3 H, α isomer), 0.95–1.12 (m, 4
H), 1.06 (s, 0.3ϫ3 H, α isomer), 1.15 (s, 0.7ϫ3 H, β isomer), 1.18–
1.33 (m, 2.3 H), 1.35–1.48 (m, 2.9 H), 1.52–1.65 (m, 3.9 H), 1.68–
H), 0.76 (s, 3 H), 0.88–0.94 (m, 1 H), 0.90 (s, 3 H), 1.02–1.08 (m,
1 H), 1.27 (qd, J = 12.5, 6.2 Hz, 1 H), 1.36–1.51 (m, 6 H), 1.57 (s,
3 H), 1.58–1.73 (m, 3 H), 1.78–1.84 (m, 2 H), 2.00–2.11 (m, 2 H),
1.73 (m, 0.3 H), 1.80–1.86 (m, 2.6 H), 2.02–2.25 (m, 3 H), 2.26– 2.26–2.36 (m, 2 H), 3.62 (t, J = 7.8 Hz, 1 H) ppm. 13C{1H} NMR
2.41 (m, 1 H), 2.96 (s, 0.7 H, β isomer), 3.02 (s, 0.3 H, α isomer),
3.63 (t, J = 8.6 Hz, 0.7 H, β isomer), 3.66 (t, J = 8.6 Hz, 0.3 H, α
(125 MHz, CDCl3): δ = 11.07 (CH3), 13.56 (CH3), 18.02 (CH3),
22.26 (CH2), 22.56 (CH2), 23.54 (CH2), 30.37 (CH2), 31.57 (CH2),
isomer) ppm. 13C{1H}NMR (125 MHz, CDCl3): β isomer: δ = 35.41 (CH2), 36.07 (CH), 36.67 (CH2), 38.14 (CH2), 43.17 (C),
11.04 (CH3), 18.93 (19-CH3), 21.11 (CH2), 23.31 (CH2), 26.08 49.72 (C), 50.59 (CH), 55.22 (CH), 81.85 (CH), 125.99 (C), 141.44
(CH2), 29.70 (CH2), 29.87 (CH2), 30.31 (CH2), 32.49 (CH2), 35.43 (C) ppm. IR (ATR): ν = 3282 (br m), 2956 (m), 2921 (s), 2871 (m),
˜
(CH), 36.18 (CH2), 37.23 (C), 43.00 (C), 46.57 (CH), 50.40 (CH),
62.62 (CH), 70.26 (C), 81.51 (CH), 206.83 (C) ppm; α isomer: δ =
2840 (m), 1469 (w), 1448 (m), 1436 (m), 1378 (w), 1366 (w), 1349
(m), 1333 (w), 1318 (w), 1278 (w), 1261 (w), 1205 (w), 1183 (w),
11.04 (CH3), 16.49 (19-CH3), 20.97 (CH2), 23.31 (CH2), 28.45 1160 (w), 1133 (m), 1114 (m), 1075 (m), 1054 (s), 1034 (m), 1018
(CH2), 29.07 (CH2), 29.57 (CH2), 30.36 (CH2), 33.05 (CH2), 35.43 (m), 987 (w), 963 (w), 937 (w), 923 (w), 910 (w), 892 (w), 875 (w),
(CH), 36.42 (CH2), 36.74 (C), 42.87 (C), 50.19 (CH), 50.75 (CH),
830 (w) cm–1. MS (EI, 70 eV): m/z (%) = 274 (21) [M]+, 259 (100),
62.82 (CH), 70.10 (C), 81.64 (CH), 206.99 (C) ppm. IR (ATR): ν 241 (7), 164 (6), 147 (12), 122 (10), 109 (14), 93 (14). HRMS (EI):
˜
= 3401 (br m), 2938 (s), 2871 (m), 2851 (m), 1709 (s), 1468 (m),
1448 (m), 1379 (m), 1341 (m), 1319 (w), 1307 (w), 1266 (m), 1248
(m), 1205 (m), 1182 (m), 1138 (m), 1114 (m), 1079 (m), 1055 (s),
1020 (m), 984 (w), 953 (m), 937 (w), 911 (w), 884 (m), 861 (m)
cm–1. MS (ESI+): m/z = 327 [M + Na]+.
calcd. for C19H30O [M]+ 274.2297; found 274.2299.
17β-Hydroxy-3-methyl-A-nor-5β-androstane (11a): A suspension of
olefin 9 (90 mg, 0.32 mmol) and Pd/C (35 mg, 33 μmol, 10% w/w
Pd) in EtOAc (8 mL) was hydrogenated with H2 (3 bar) for 16 h at
60 °C. The mixture was filtered through SiO2 (hexane/MTBE 1:1,
Rf = 0.41), and the solvent was evaporated to yield 11a (83 mg,
0.30 mmol, 94%) as a colorless solid, m.p. 69 °C. [α]2D0 = 45.2
(CH2Cl2, 1 g/l). 1H NMR (500 MHz, CDCl3): δ = 0.73 (s, 3 H),
0.83–0.98 (m, 3 H), 0.90 (d, J = 6.6 Hz, 3 H), 0.94 (s, 3 H), 1.00–
1.06 (m, 2 H), 1.09–1.16 (m, 1 H), 1.20–1.34 (m, 4 H),1.36–1.43
(m, 2 H), 1.45–1.50 (m, 2 H), 1.52–1.60 (m, 3 H), 1.65–1.70 (m, 1
H), 1.76–1.86 (m, 2 H), 1.95–2.07 (m, 2 H), 3.61 (t, J = 8.5 Hz, 1
H) ppm. 13C{1H} NMR (125 MHz, CDCl3): δ = 11.18 (CH3),
20.27 (CH3), 21.83 (CH2), 21.87 (CH3), 21.88 (CH2), 23.40 (CH2),
26.13 (CH2), 30.60 (CH2), 30.76 (CH2), 33.62 (CH), 36.25 (CH),
36.91 (CH2), 37.09 (CH2), 43.19 (C), 43.84 (C), 45.50 (CH), 51.10
17β-Hydroxy-5-oxo-4,5-seco-androst-3-yne (10):[7] A solution of ep-
oxy ketone 8 (2.6 g, 8.5 mmol) in a mixture of CH2Cl2 and AcOH
(1:1, 45 mL) was added dropwise over a period of 1 h to a cooled
(ice/water bath) solution of p-tosylhydrazine (1.7 g, 8.9 mmol) in
CH2Cl2/AcOH (1:1, 45 mL). The solution was warmed to ambient
temperature and stirred for a further 2 h. After removal of the sol-
vent under vacuum, the mixture was redissolved in CH2Cl2
(100 mL) and successively washed with saturated NaHCO3 solu-
tion (100 mL) and water (100 mL). The solution was dried
(MgSO4) and filtered. After evaporation, the residue was chro-
matographed (SiO2, hexane/MTBE 1:1, Rf = 0.23) to furnish 10
(1.47 g, 5.10 mmol, 60%) as a yellow oil. [α]2D0 = 31.6 (CH2Cl2,
(CH), 55.68 (CH), 81.98 (CH) ppm. IR (ATR): ν = 3328 (br w),
˜
1 gdm–3). 1H NMR (500 MHz, CDCl3): δ = 0.79 (s, 3 H), 0.98 (td, 2946 (m), 2927 (m), 2866 (m), 1449 (m), 1373 (m), 1345 (w), 1321
J = 11.5, 7.3 Hz, 1 H), 1.05–1.10 (m, 1 H), 1.08 (s, 3 H), 1.12–1.19
(w), 1310 (w), 1246 (w), 1205 (w), 1121 (m), 1051 (s), 1036 (m), 994
(m, 1 H), 1.22–1.36 (m, 2 H), 1.42–1.54 (m, 4 H), 1.59–1.65 (m, 1
(w), 963 (w), 927 (w), 906 (w), 863 (w), 831 (w) cm–1. MS (EI,
H), 1.73 (dq, J = 11.0, 3.3 Hz, 2 H), 1.83 (dt, J = 12.6, 3.3 Hz, 1 70 eV): m/z (%) = 276 (63) [M]+, 261 (25), 244 (7), 234 (14), 218
H), 1.90–1.95 (m, 2 H), 2.00–2.17 (m, 4 H), 2.24–2.28 (m, 1 H),
(16), 217 (100), 203 (7), 201 (6), 163 (8), 149 (46), 122 (16), 109
2.51 (td, J = 14.5, 6.3 Hz, 1 H), 3.64 (t, J = 8.5 Hz, 1 H) ppm. (37), 81 (26), 67 (18), 55 (21). HRMS (EI): calcd. for C19H32O
13C{1H} NMR (125 MHz, CDCl3): δ = 11.07 (CH3), 13.68 (CH2), [M]+ 276.2453; found 276.2448.
20.58 (CH3), 21.13 (CH2), 23.34 (CH2), 30.25 (CH2), 30.57 (CH2),
3-Methyl-A-nor-5β-androstan-17-one
(12a):
PCC
(83 mg,
33.54 (CH2), 34.84 (CH), 36.02 (CH2), 37.95 (CH2), 42.91 (C),
47.41 (CH), 50.36 (CH), 50.73 (C), 68.07 (CH), 81.42 (CH), 84.85
0.38 mmol) was added to a solution of alcohol 11a (43 mg,
0.16 mmol) in CH2Cl2 (5 mL). The solution was stirred for 16 h at
ambient temperature, then treated with MTBE (10 mL) and filtered
through a short pad of SiO2 (washed with 100 mL MTBE). The
solvent was removed under vacuum to give ketone 12a (44 mg,
0.16 mmol, 99%) as a crystalline solid (m.p. 96 °C), which was used
without further purification. [α]2D0 = 108.3 (CH2Cl2, 1 g/l). 1H NMR
(500 MHz, CDCl3): δ = 0.85 (s, 3 H), 3.02 (d, J = 7.1 Hz, 3 H),
0.95 (s, 3 H), 1.00–1.17 (m, 3 H), 1.19–1.45 (m, 6 H), 1.46–1.64 (m,
5 H), 1.65–1.70 (m, 1 H), 1.76–1.86 (m, 2 H), 1.89–1.94 (m, 1 H),
1.96–2.08 (m, 2 H), 2.42 (dd, J = 19.3, 9.0 Hz, 1 H) ppm. 13C{1H}
NMR (125 MHz, CDCl3): δ = 13.80 (CH3), 20.22 (CH3), 21.49
(CH2), 21.74 (CH3), 21.76 (CH2), 21.77 (CH2), 25.45 (CH2), 30.68
(CH2), 31.89 (CH2), 33.59 (CH), 35.71 (CH), 35.91 (CH2), 36.85
(CH2), 43.86 (C), 45.52 (CH), 47.99 (C), 51.54 (CH), 55.58 (CH),
(CH), 214.46 (C) ppm. IR (ATR): ν = 3405 (br w), 3306 (w), 2939
˜
(m), 2872 (w), 2855 (w), 2118 (w), 1700 (s), 1454 (w), 1429 (w),
1378 (w), 1348 (w), 1319 (w), 1278 (w), 1249 (w), 1208 (w), 1160
(w), 1132 (w), 1070 (m), 1054 (m), 1023 (w), 952 (w), 921 (w), 852
(w), 831 (w), 737 (w), 704 (w), 627 (m) cm–1. MS (CI, isobutane):
m/z (%) = 289 (23) [M + H]+, 271 (14), 236 (100). HRMS (ESI+):
calcd. for C19H28NaO2 [M + Na]+ 311.1987; found 311.1981.
17β-Hydroxy-3-methyl-A-nor-androst-3(5)-ene (9): Absolute THF
(1 mL) was added to a solution of Li (36 mg, 5.20 mmol) in liquid
NH3 (ca. 5 mL) at –78 °C. The mixture was stirred for 30 min, and
a solution of 4,5-seco-ynone 10 (150 mg, 0.52 mmol) in absolute
THF (4 mL) was added dropwise. The reaction mixture was stirred
for 3 h at –78 to –50 °C. Solid NH4Cl (556 mg, 10.4 mmol) was
added in small portions, and the solution was warmed to ambient
221.42 (C) ppm. IR (ATR): ν = 2950 (m), 2931 (m), 2899 (m), 2862
˜
5940
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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 5934–5945

Identification of a Fossil Sterane Biomarker in Crude Oil
(m), 2821 (w), 1736 (vs), 1455 (m), 1408 (w), 1380 (m), 1339 (w), (17) [M]+, 261 (19), 217 (100), 203 (7), 175 (7), 166 (11), 149 (55),
1304 (w), 1291 (w), 1269 (w), 1212 (w), 1170 (w), 1150 (w), 1116
147 (22), 133 (12), 121 (19), 109 (45), 95 (45), 81 (54), 67 (46), 55
(w), 1063 (m), 1053 (m), 1015 (m), 966 (w), 916 (w), 899 (w), 863 (51); (b): m/z (%) = 276 (18) [M]+, 261 (22), 243 (7), 217 (100), 201
(w), 833 (w) cm–1. MS (EI, 70 eV): m/z (%) = 274 (100) [M]+, 259
(20), 242 (10), 230 (39), 203 (41), 164 (6), 149 (15), 121 (11), 109
(37), 108 (43), 95 (26), 81 (21). HRMS (EI): calcd. for C19H30O
[M]+ 274.2297; found 274.2295.
(29), 175 (13), 166 (15), 149 (43), 147 (21), 133 (14), 121 (27), 109
(41), 107 (43), 95 (44), 81 (71), 79 (59), 67 (48), 55 (73).
Four Isomers of 3-Methyl-A-nor-5β-androstan-17-one (12): PCC
(142 mg, 0.66 mmol) was added to a solution of alcohol 11 (73 mg,
0.26 mmol) in CH2Cl2 (10 mL). The solution was stirred for 16 h
at ambient temperature, treated with MTBE (20 mL), and filtered
through a short pad of SiO2 (washed with 100 mL MTBE). The
solvent was removed under vacuum to obtain ketone 12 (70 mg,
0.25 mmol, 99%) as a mixture of four diastereomers [ratio 12a/b/
c/d 27:8:53:12]. GC–MS (EI, 70 eV): 12a: m/z (%) = 274 (100)
[M]+, 259 (20), 242 (10), 230 (39), 203 (41), 164 (6), 149 (15), 121
(11), 109 (37), 108 (43), 95 (26), 81 (21); (b): m/z (%) = 274 (28)
[M]+, 259 (18), 241 (22), 230 (16), 217 (21), 203 (55), 199 (9), 189
(5), 175 (9), 161 (21), 149 (45), 147 (33), 133 (24), 122 (35), 108
(84), 95 (66), 93 (72), 81 (80), 79 (82), 67 (100); (c): m/z (%) = 274
(36) [M]+, 259 (11), 241 (19), 230 (40), 217 (31), 203 (43), 199 (26),
189 (11), 175 (12), 161 (30), 149 (33), 147 (30), 133 (22), 121 (47),
108 (91), 95 (87), 93 (86), 81 (76), 79 (85), 67 (100); (d): m/z (%) =
274 (30) [M]+, 259 (13), 241 (15), 230 (17), 217 (17), 203 (45), 199
(7), 189 (9), 175 (13), 161 (19), 149 (35), 147 (41), 133 (24), 122
(39), 108 (100), 109 (98), 95 (61), 93 (75), 81 (67), 79 (86), 67 (970).
3β-Methyl-A-nor-5β-androstane (4a): A solution of ketone 11a
(30 mg, 0.11 mmol), H2NN2H·H2O (0.2 mL, 1.1 mmol, 65% hy-
drazine), and toluene (1 mL) was heated to 100 °C for 2 h. DEG
(4 mL) was added, and the toluene was removed by distillation.
The reaction mixture was treated with KOH (15 mg, 0.27 mmol)
and then stirred at 200 °C for 16 h. After cooling to ambient tem-
perature, the solution was diluted with brine (10 mL) and extracted
with hexane (2ϫ 10 mL). The combined organic layers were dried
(MgSO4) and filtered, and the solvent was removed under reduced
pressure. After chromatography (SiO2, hexane, Rf = 0.76), the hy-
drocarbon 4a (12 mg, 46 μmol, 41%) was obtained as a colorless
oil. [α]2D0 = 60 (CH2Cl2, 1 g/l). 1H NMR (500 MHz, CDCl3): δ =
0.69 (s, 3 H), 0.88–0.93 (m, 1 H), 0.91 (d, J = 6.1 Hz, 3 H), 0.95
(s, 3 H), 0.98–1.06 (m, 2 H), 1.07–1.16 (m, 5 H), 1.18–1.25 (m, 2
H), 1.27–1.36 (m, 2 H), 1.39–1.46 (m, 3 H), 1.53–1.57 (m, 2 H),
1.61–1.72 (m, 4 H), 1.80–1.88 (m, 1 H), 1.97–2.07 (m, 1 H) ppm.
13C{1H} NMR (125 MHz, CDCl3): δ = 17.57 (CH3), 20.33 (CH3),
20.61 (CH2), 21.95 (CH3), 22.08 (CH2), 22.30 (CH2), 25.53 (CH2),
26.98 (CH2), 30.83 (CH2), 33.65 (CH), 36.56 (CH), 36.97 (CH2),
39.27 (CH2), 40.50 (CH2), 41.09 (C), 43.94 (C), 45.63 (CH), 54.68
Four Isomers of 3-Methyl-A-nor-5β-androstane (4): A solution of
ketone 12 [62 mg, 0.23 mmol; four isomers, ratio 12a/b/c/d
27:8:53:12], KOH (32 mg, 0.58 mmol), and H2NNH2·H2O (111 mg,
2.23 mmol) in DEG (5 mL) was heated for 16 h to 200–220 °C.
After cooling to ambient temperature, the mixture was diluted with
brine (15 mL) and extracted with MTBE (3ϫ 20 mL). The com-
bined organic layers were dried (MgSO4) and concentrated after
filtration. The residue was purified by chromatography (SiO2, hex-
ane/MTBE 1:1, Rf = 0.62) to yield 4 (12 mg, 46 μmol, 20%; four
isomers, ratio 4a/b/c/d 27:8:53:12) as a colorless oil. MS (EI, 70 eV):
4a: m/z (%) = 260 (50) [M]+, 245 (46), 232 (7), 217 (40), 203 (100),
189 (22), 175 (59), 161 (10), 149 (34), 135 (38), 121 (22), 109 (24),
107 (25), 95 (58), 81 (34), 67 (20); isomer b: m/z (%) = 260 (26)
[M]+, 245 (74), 232 (5), 217 (26), 203 (100), 189 (23), 175 (34), 163
(10), 149 (55), 135 (30), 121 (17), 107 (20), 95 (35), 81 (24), 67 (18),
55 (13); isomer c: m/z (%) = 260 (10) [M]+, 245 (12), 232 (10), 217
(13), 203 (100), 189 (9), 175 (9), 161 (6), 149 (12), 135 (13), 121
(12), 107 (17), 95 (22), 81 (16), 67 (10); isomer d: m/z (%) = 260
(29) [M]+, 245 (22), 232 (3), 217 (10), 203 (100), 189 (10), 175 (40),
162 (10),149 (33), 135 (22), 121 (11), 107 (13), 95 (25), 81 (17), 67
(13), 55 (10).
(CH), 55.71 (CH) ppm. IR (ATR): ν = 2947 (m), 2925 (m), 2858
˜
(m), 1451 (m), 1376 (m), 1297 (w), 1260 (w), 1215 (w), 1191 (w),
1162 (w), 1088 (w), 1063 (w), 1015 (w), 968 (w), 946 (w), 928 (w),
911 (w), 902 (w), 799 (w) cm–1. MS (EI, 70 eV): m/z (%) = 260 (50)
[M]+, 245 (46), 232 (7), 217 (40), 203 (100), 189 (22), 175 (59), 161
(10), 149 (34), 135 (38), 121 (22), 109 (24), 107 (25), 95 (58), 81
(34), 67 (20). HRMS (EI): calcd. for C19H32 [M]+ 260.2504; found
260.2501.
Four Isomers of 3-Methyl-A-nor-androstan-17β-ol (11): TFA (2.47 g,
21.7 mmol) was added to
a solution of olefin 9 (149 mg,
0.54 mmol) in CH2Cl2 (20 mL) at –20 °C. The solution was stirred
for 10 min, treated with Et3SiH (1.26 g, 10.9 mmol) at –20 °C, and
then stirred for 16 h at ambient temperature. The reaction mixture
was diluted with H2O (20 mL) and extracted with CH2Cl2 (3ϫ
20 mL). The solvent was removed under vacuum to give the trifluo-
roacetates of the title compounds (186 mg, 0.49 mmol) as a mixture
of four diastereomers (ratio a/b/c/d 27:8:53:12), which were ana-
lyzed by GC–MS (EI, 70 eV). (a): m/z (%) = 372 (6) [M]+, 357 (7),
315 (15), 287 (6), 258 (10), 243 (20), 217 (18), 201 (25), 173 (13),
149 (32), 133 (22), 109 (40), 95 (100), 81 (91), 69 (69), 55 (91); (b):
5α-Androstan-17β-ol (14):[14] A solution of dihydrotestosterone (13,
m/z (%) = 372 (4) [M]+, 357 (28), 315 (23), 287 (7), 258 (13), 243 2.00 g, 6.89 mmol), KOH (965 mg, 17.2 mmol), and H2NNH2·H2O
(72), 217 (25), 201 (25), 173 (14), 149 (74), 133 (22), 109 (42), 95
(3.45 g, 68.9 mmol) in DEG (35 mL) was heated for 16 h to 200–
220 °C. After cooling to ambient temperature, the mixture was di-
(67), 81 (100), 81 (90), 69 (87), 55 (95); (c): m/z (%) = 372 (5)
[M]+, 357 (5), 344 (9), 315 (47), 287 (3), 258 (5), 243 (14), 217 (14), luted with hydrochloric acid (ca. 15 mL, 2 mol/L) and extracted
201 (47), 173 (7), 149 (21), 133 (20), 109 (29), 107 (46), 95 (67), 81
with MTBE (3ϫ 100 mL). The combined organic layers were dried
(95), 69 (78), 55 (100); (d): m/z (%) = 372 (8) [M]+, 357 (5), 330 (4), (MgSO4) and concentrated after filtration. The residue was purified
315 (25), 287 (15), 258 (5), 243 (25), 217 (10), 201 (22), 173 (21),
149 (26), 133 (19), 109 (29), 107 (32), 95 (62), 81 (82), 69 (86), 55
(100). The trifluoroacetates were then treated with a solution of
KOH (10 mL, 10% w/w in MeOH/H2O, 9:1), and the mixture was
stirred for 30 min at ambient temperature. The reaction mixture
by chromatography (SiO2, hexane/MTBE 2:1, Rf = 0.38) to yield
14 (1.63 g, 5.90 mmol, 86%) as a colorless solid, m.p. 160 °C.
1
[α]2D0 = 11.7 (CH2Cl2, 1 gdm–3). H NMR (500 MHz, CDCl3): δ =
0.66 (td, J = 12.3, 4.1 Hz, 1 H), 0.73 (s, 3 H), 0.79 (s, 3 H), 0.84–
0.98 (m, 3 H), 1.01–1.06 (m, 2 H), 1.15–1.30 (m, 8 H), 1.35–1.45
was diluted with H2O (20 mL) and extracted with MTBE (3ϫ (m, 3 H), 1.48–1.68 (m, 6 H), 1.78 (dt, J = 12.2, 3.1 Hz, 1 H),
50 mL). The combined organic layers were dried (MgSO4) and fil-
tered, and the solvent was removed under vacuum to give alcohol
11 (135 mg, 0.49 mmol, 90%) as a colorless oil. Two peaks [a/b
23:77] were detected by GC–MS (EI, 70 eV). (a): m/z (%) = 276
2.01–2.08 (m, 1 H), 3.63 (t, J = 8.0 Hz, 1 H) ppm. 13C{1H} NMR
(125 MHz, CDCl3): δ = 11.12 (CH3), 12.23 (CH3), 20.36 (CH2),
22.15 (CH2), 23.35 (CH2), 26.78 (CH2), 28.92 (CH2), 29.02 (CH2),
30.50 (CH2), 31.71 (CH2), 35.55 (CH), 36.32 (C), 36.80 (CH2),
Eur. J. Org. Chem. 2013, 5934–5945
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5941

J. Christoffers et al.
38.70 (CH2), 42.96 (C), 47.09 (CH), 51.12 (CH), 54.87 (CH), 82.01 (100), 189 (12), 175 (13), 164 (9), 163 (11), 162 (8), 161 (18), 149
FULL PAPER
(CH, C-17) ppm. IR (ATR): ν = 3276 (br m), 2975 (w), 2918 (s),
(32), 148 (28), 135 (51), 121 (21), 109 (31), 107 (37), 95 (52), 81
˜
2868 (m), 2845 (m), 1468 (w), 1447 (w), 1375 (w), 1353 (w), 1336 (49), 67 (32), 55 (17); (b, 5a): m/z (%) = 260 (49) [M]+, 245 (39),
(w), 1323 (w), 1246 (w), 1136 (w), 1116 (w), 1087 (w), 1053 (s), 231 (5), 218 (2), 217 (2), 204 (41), 203 (100), 189 (9), 175 (10), 164
1010 (m) cm–1. MS (EI, 70 eV): m/z (%) = 276 (74) [M]+, 261 (36),
219 (18), 217 (100), 203 (9), 166 (11), 150 (23), 149 (72), 109 (48),
81 (36), 55 (33), 41 (16). HRMS (EI): calcd. for C19H32O [M]+
276.2453; found 276.2460.
(22), 163 (12), 162 (4), 161 (16), 149 (29), 148 (23), 135 (80), 121
(28), 107 (41), 95 (57), 81 (58), 67 (40), 55 (21); (c): m/z (%) = 260
(59) [M]+, 245 (100), 232 (6), 218 (13), 217 (37), 204 (40), 203 (72),
190 (15), 189 (30), 175 (16), 164 (9), 163 (15), 162 (7), 161 (18), 149
(50), 148 (25), 135 (50), 121 (36), 109 (44), 107 (41), 95 (80), 81
(54), 67 (43), 55 (25); (d): m/z (%) = 260 (53) [M]+, 245 (41), 218
(4), 217 (3), 204 (51), 203 (100), 189 (13), 175 (9), 164 (9), 163 (11),
162 (6), 161 (17), 149 (27), 148 (24), 135 (57), 121 (22), 109 (31),
107 (34), 95 (52), 81 (47), 67 (31), 55 (18); (e): m/z (%) = 260 (56)
[M]+, 245 (35), 231 (7), 218 (9), 217 (4), 204 (37), 203 (95), 189
(11), 175 (15), 164 (20), 163 (11), 162 (7), 161 (21), 149 (44), 148
(30), 135 (100), 121 (40), 109 (36), 107 (48), 95 (73), 81 (66), 67
(45), 55 (29).
5α-Androstan-17β-yl-4-toluenesulfonate (16):[15]
A solution of
alcohol 14 (3.50 g, 12.7 mmol), p-TosCl (9.68 g, 50.8 mmol), and 4-
dimethylaminopyridine (DMAP, 155 mg, 1.27 mmol) in absolute
pyridine (40 mL) was stirred for 5 d at 55 °C. The solution was
diluted with MTBE (100 mL) and washed successively with hydro-
chloric acid (1 mol/L, 100 mL), H2O (100 mL), saturated NaHCO3
solution (100 mL), and water (100 mL). The organic phase was
dried (MgSO4) and filtered. After evaporation of the solvent, the
crude solid was recrystallized from MeOH (ca. 200 mL) to give
tosylate 16 (4.61 g, 10.7 mmol, 84%) as a colorless solid, m.p.
137 °C. [α]2D0 = –8.3 (MTBE, 1 gdm–3). 1H NMR (500 MHz,
CDCl3): δ = 0.56–0.61 (m, 1 H), 0.75 (s, 3 H), 0.77 (s, 3 H), 0.80–
0.91 (m, 4 H), 0.96–1.02 (m, 1 H), 1.11–1.30 (m, 6 H), 1.31–1.43
(m, 2 H), 1.47–1.68 (m, 9 H), 1.87–1.95 (m, 1 H), 2.44 (s, 3 H),
4.24 (t, J = 8.4 Hz, 1 H), 7.31 (d, J = 8.2 Hz, 2 H), 7.77 (d, J =
8.2 Hz, 2 H) ppm. 13C{1H} NMR (125 MHz, CDCl3): δ = 11.77
(CH3), 12.15 (CH3), 20.03 (CH2), 21.61 (CH3), 22.07 (CH2), 23.23
(CH2), 26.70 (CH2), 27.65 (CH2), 28.74 (CH2), 28.92 (CH2), 31.47
(CH2), 35.18 (C), 36.17 (CH), 36.24 (CH2), 38.62 (CH2), 43.00 (C),
46.95 (CH), 50.05 (CH), 54.53 (CH), 90.17 (CH), 127.78 (2ϫCH),
17-Methyl-5α-androst-13-en-16-one (17): Anhydrous CrO3 (774 mg,
7.74 mmol) was added to a solution of absolute pyridine (1.22 g,
15.5 mmol) in absolute CH2Cl2 (4 mL) at 0 °C. The mixture was
stirred for 10 min at 0 °C and then for 40 min at ambient tempera-
ture. After the addition of a solution of olefin 15 (100 mg,
0.39 mmol) in absolute CH2Cl2 (0.5 mL), the reaction mixture was
stirred for 16 h at ambient temperature. After decantation from the
brown precipitate, the solution was treated with MTBE (10 mL).
The mixture was filtered through a short pad of SiO2 (washed with
100 mL MTBE), and the resulting filtrate was concentrated under
vacuum. The residue was chromatographed (SiO2, hexane/MTBE
1:1, Rf = 0.26) to give enone 17 (36 mg, 0.13 mmol, 34%) as a
colorless solid, m.p. 121 °C. [α]2D0 = 30 (CH2Cl2, 1 g/l). 1H NMR
(500 MHz, CDCl3): δ = 0.70 (s, 3 H), 0.95 (td, J = 13.1, 4.1 Hz, 1
H), 1.04–1.16 (m, 5 H), 1.18–1.29 (m, 5 H), 1.36–1.45 (m, 1 H),
1.48–1.56 (m, 1 H), 1.65 (s, 3 H), 1.68–1.75 (m, 2 H), 1.79–1.85 (m,
1 H), 1.96–2.13 (m, 3 H), 2.16–2.22 (m, 1 H), 2.49 (dd, J = 18.5,
6.5 Hz, 1 H), 2.83–2.85 (m, 1 H) ppm. 13C{1H} NMR (125 MHz,
CDCl3): δ = 7.55 (CH3), 12.06 (CH3), 22.00 (CH2), 25.61 (CH2),
26.66 (CH2), 28.38 (CH2), 28.58 (CH2), 28.78 (CH2), 33.10 (CH2),
36.50 (C), 38.70 (CH2), 39.94 (CH2), 44.72 (CH), 46.55 (CH), 46.94
(CH), 52.58 (CH), 132.16 (C), 175.45 (C), 209.27 (C) ppm. IR
129.60 (2ϫCH), 134.29 (C), 144.29 (C) ppm. IR (ATR): ν = 2971
˜
(w), 2921 (m), 2904 (w), 2879 (w), 2846 (w), 1598 (w), 1444 (w),
1371 (m), 1358 (s), 1292 (w), 1188 (m), 1171 (s), 1097 (m), 993 (m),
966 (s), 889 (m), 868 (s), 844 (m), 827 (m), 814 (s), 667 (s), 575 (s),
551 (s) cm–1. MS (EI, 70 eV): m/z (%) = 430 (10) [M]+, 415 (11),
258 (100), 243 (61), 217 (27), 175 (9), 161 (17), 149 (56), 148 (59),
121 (17), 109 (33). HRMS (EI): calcd. for [M]+ C26H38O3S
430.2542; found 430.2537.
17-Methyl-18-nor-5α-androst-13(17)-ene
(15):[14]
EtMgBr
(19.5 mmol, 6.50 mL, 3 m solution in Et2O) was added to a stirred
solution of tosylate 16 (1.68 g, 3.90 mmol) in absolute toluene
(20 mL) at 100 °C. After the Et2O was removed by distillation, the
suspension was stirred for 4 h at 110 °C. The suspension was cooled
to 0 °C, treated with cold water (10 mL), and extracted with MTBE
(2ϫ 20 mL). The combined organic layers were dried (MgSO4) and
concentrated after filtration under high vacuum to give a mixture
of olefins (all with m/z = 258, GC–MS; 919 mg, 3.56 mmol, 91%)
containing compound 15 in 80% purity. 13C{1H} NMR (125 MHz,
CDCl3): δ = 12.21 (CH3), 13.44 (CH3), 22.25 (CH2), 25.30 (CH2),
25.77 (CH2), 26.84 (CH2), 28.18 (CH2), 28.86 (CH2), 29.06 (CH2),
32.52 (CH2), 36.39 (C), 37.16 (CH2), 38.87 (CH2), 45.46 (CH),
46.61 (CH), 52.78 (CH), 54.01 (CH), 127.34 (C), 136.82 (C) ppm.
MS (EI, 70 eV): m/z (%) = 258 (44) [M]+, 243 (25), 201 (7), 188 (7),
175 (6), 162 (10), 161 (12), 149 (59), 148 (64), 133 (21), 119 (20),
107 (40), 94 (100), 79 (88), 67 (45), 55 (51).
(ATR): ν = 2975 (w), 2920 (m), 2857 (m), 1693 (s), 1633 (s), 1451
˜
(m), 1441 (m), 1398 (w), 1385 (w), 1357 (w), 1341 (w), 1327 (w),
1311 (w), 1295 (w), 1285 (w), 1267 (w), 1251 (w), 1238 (w), 1200
(w), 1733 (w), 1153 (w), 1095 (w), 1064 (w), 1027 (w), 985 (w), 968
(w), 909 (w), 891 (w), 845 (w) cm–1. MS (EI, 70 eV): m/z (%) = 272
(100) [M]+, 244 (13), 175 (6), 162 (16), 149 (42), 135 (9), 110 (50),
79 (10), 67 (9), 55 (8). HRMS (EI, 70 eV): calcd. for C19H28O
[M]+ 272.2140; found 272.2143.
17-Methyl-5α-androstan-16-one (18a and 18b): A suspension of en-
one 17 (200 mg, 0.73 mmol) and Pd/C (78 mg, 73 μmol, 10% w/w
Pd) in EtOAc (10 mL) was hydrogenated (4 bar H2) for 3 d at
40 °C. The mixture was filtered through SiO2 (washed with 100 mL
MTBE). The solvent was removed under vacuum, and the crude
material was chromatographed (SiO2, hexane/MTBE 5:1) to give
ketone 18b (40 mg, 15 mmol, 20%) in the first fraction (Rf = 0.53)
as a colorless solid. The second fraction (Rf = 0.42–0.53) was a
mixture of ketones 18a and 18b [120 mg, 0.44 mmol, 60%, 18a/18b
70:30 (by 13C NMR)]. The mixed fraction was chromatographed
three times to give pure ketone 18a (68 mg, 0.25 mmol, 35%) as a
colorless solid and another portion of ketone 18b (28 mg,
0.10 mmol, 13%). Isomer 18a: M.p. 76 °C. [α]2D0 = 35 (CH2Cl2, 1 g/
Five Isomers of 17-Methyl-18-nor-androstane (5): A suspension of
15 (50 mg, 0.19 mmol; 80% purity) and Pd/C (10 mg, 10 μmol,
10% w/w Pd) in EtOAc (1 mL) was hydrogenated with H2 (1 atm)
at ambient temperature for 6 d. The mixture was filtered through
SiO2 (washed with 50 mL MTBE), and the solvent was removed
under vacuum to give a mixture of five isomers of 5 [44 mg,
0.17 mmol, 89%; ratio a/b (5a)/c/d/e 53:6:7:18:9] as a colorless oil,
which was analyzed by GC–MS. GC–MS (EI, 70 eV): (a): m/z (%)
= 260 (62) [M]+, 245 (40), 231 (5), 218 (4), 217 (3), 204 (55), 203
1
l). H NMR (500 MHz, CDCl3): δ = 0.68 (s, 3 H), 0.82–0.90 (m, 3
H), 0.99–1.06 (m, 2 H), 1.07 (d, J = 7.6 Hz, 3 H), 1.12–1.25 (m, 6
H), 1.32–1.41 (m, 1 H), 1.47–1.49 (m, 1 H), 1.57–1.70 (m, 5 H),
5942
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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 5934–5945

Identification of a Fossil Sterane Biomarker in Crude Oil
1.81–1.86 (m, 2 H), 2.17 (dd, J = 18.4, 3.2 Hz, 1 H), 2.32 (pent, J
acid (10 mL, 2 mol/L) and extracted with MTBE (2ϫ 50 mL). The
= 8.0 Hz, 1 H), 2.41–2.46 (m, 2 H) ppm. 13C{1H} NMR (125 MHz, combined organic layers were dried (MgSO4) and filtered, and the
CDCl3): δ = 12.05 (CH3), 13.06 (CH3), 21.66 (CH2), 21.92 (CH2), solvent was removed under reduced pressure. The crude product
23.39 (CH2), 26.64 (CH2), 28.57 (CH2), 28.89 (CH2), 33.38 (CH2), was chromatographed (SiO2, hexane, Rf = 0.77) to give 5a (15 mg,
36.47 (C), 37.42 (CH), 38.14 (CH), 38.54 (CH2), 42.68 (CH), 43.64 60 μmol, Rf = 0.80, 48%) in the first fraction as a colorless oil. In
(CH2), 45.46 (CH), 46.45 (CH), 49.90 (CH), 223.04 (C) ppm. IR the second fraction, ketone 18b (11 mg, 40 μmol, Rf = 0.47, 33%)
1
(ATR): ν = 2971 (w), 2934 (m), 2919 (m), 2848 (m), 1732 (vs), 1455 was recovered. [α]2D0 = –31.7 (CH2Cl2, 1 g/l). H NMR (500 MHz,
˜
(w), 1444 (w), 1408 (w), 1380 (w), 1370 (w), 1353 (w), 1309 (w), CDCl3): δ = 0.66 (td, J = 11.1, 2.6 Hz, 1 H), 0.70 (s, 3 H), 0.73–
1281 (w), 1158 (w), 1145 (w), 1131 (w), 1039 (w), 1024 (w), 1000
0.88 (m, 3 H), 0.91 (d, J = 6.5 Hz, 3 H), 0.97–1.05 (m, 3 H), 1.08–
(w), 977 (w), 957 (w), 918 (w), 898 (w), 852 (w), 795 (w) cm–1. MS 1.16 (m, 2 H), 1.18–1.26 (m, 4 H), 1.31–1.35 (m, 1 H), 1.38–1.51
(EI, 70 eV): m/z (%) = 274 (100) [M]+, 259 (21), 245 (5), 217 (42),
(m, 5 H), 1.62–1.80 (m, 5 H), 1.81–1.90 (m, 2 H) ppm. 13C{1H}
203 (11), 189 (10), 175 (6), 163 (40), 149 (15), 135 (7), 123 (8), 121
NMR (125 MHz, CDCl3): δ = 12.16 (CH3), 19.21 (CH3), 19.89
(11), 110 (19), 95 (20), 86 (30), 84 (48), 67 (23), 55 (20), 49 (51). (CH2), 22.15 (CH2), 25.39 (CH2), 26.84 (CH2), 28.19 (CH2), 29.09
HRMS (EI): calcd. for C19H30O [M]+ 274.2291; found 274.2283. (CH2), 29.19 (CH2), 32.72 (CH2), 32.83 (CH2), 33.72 (CH), 36.42
Isomer 18b: M.p. 64 °C. [α]2D0 = 58 (CH2Cl2, 1 g/l). 1H NMR
(500 MHz, CDCl3): δ = 0.69 (s, 3 H), 0.78 (td, J = 11.0, 2.0 Hz, 1
(C), 37.78 (CH), 38.62 (CH2), 46.77 (CH), 47.29 (CH), 47.60 (CH),
53.17 (CH) ppm. IR (ATR): ν = 2920 (s), 2853 (s), 1445 (m), 1378
˜
H), 0.84–0.91 (m, 2 H), 1.01 (d, J = 7.0 Hz, 3 H), 1.02–1.26 (m, 8 (w), 1305 (w), 1259 (m), 1192 (w), 1162 (w), 1094 (m), 1030 (m),
H), 1.34–1.44 (m, 1 H), 1.49–1.52 (m, 1 H), 1.58–1.67 (m, 3 H),
1.72–1.86 (m, 5 H), 2.07–2.14 (m, 1 H), 2.19–2.29 (m, 2 H) ppm.
974 (w), 958 (w), 930 (w), 922 (w), 840 (w) 801 (m), 705 (w), 686
(w) cm–1. MS (EI, 70 eV): m/z (%) = 260 (70) [M]+, 245 (39), 231
13C{1H} NMR (125 MHz, CDCl3): δ = 12.13 (CH3), 12.70 (CH3), (5), 204 (40), 203 (100), 189 (8), 175 (12), 164 (18), 161 (16), 149
18.85 (CH2), 22.03 (CH2), 25.65 (CH2), 26.67 (CH2), 28.77 (CH2), (22), 148 (22), 135 (60), 121 (20), 109 (18), 107 (28), 95 (34), 81
28.89 (CH2), 32.52 (CH2), 36.34 (C), 36.88 (CH), 38.50 (CH2), (38), 67 (28), 55 (18). HRMS (EI): calcd. for C19H32 [M]+ 260.2499;
41.74 (CH), 42.96 (CH), 43.60 (CH2), 43.75 (CH), 46.59 (CH), found 260.2491.
52.74 (CH), 222.05 (C) ppm. IR (ATR): ν = 2979 (w), 2918 (m),
˜
17α-Methyl-18-nor-13α-androstane (5b): A solution of ketone 18b
(48 mg, 0.17 mmol), H2NNH2·H2O (85 mg, 1.7 mmol, 65% hy-
drazine), KOH (24 mg, 0.43 mmol), and toluene (2 mL) in DEG
(8 mL) was heated to reflux for 5 h. The water/toluene azeotrope
was removed by distillation, and the remaining reaction mixture
was heated to 200 °C for 16 h. After cooling to ambient tempera-
ture, the solution was diluted with hydrochloric acid (5 mL, 2 m)
and extracted with MTBE (3ϫ 50 mL). The combined organic lay-
ers were dried (MgSO4) and filtered, and the solvent was removed
under reduced pressure. The crude product was chromatographed
(SiO2, hexane, Rf = 0.77) to give 5b (15 mg, 54 μmol, 32%) as a
colorless oil. [α]2D0 = –45 (CH2Cl2, 3 g/l). 1H NMR (500 MHz,
2874 (m), 2853 (w), 1730 (vs), 1442 (m), 1402 (w), 1374 (w), 1175
(w), 1154 (w), 1133 (w), 1061 (w), 1061 (w), 1038 (w), 1011 (w),
978 (w), 959 (w), 902 (w), 869 (w), 844 (w) cm–1. MS (EI, 70 eV):
m/z (%) = 274 (44) [M]+, 259 (10), 217 (29), 203 (9), 189 (9), 175
(5), 163 (58), 149 (21), 147 (16), 133 (11), 123 (19), 110 (48), 95
(69), 81 (96), 67 (100), 55 (85). HRMS (EI): calcd. for C19H30O
[M]+ 274.2297; found 274.2288.
17α-Methyl-5α-androstane-16-(2,4-dinitrophenyl)hydrazone (19): A
solution of ketone 18b (20 mg, 7.3 μmol) in EtOH (2.5 mL) was
added to
a solution of 2,4-dinitrophenylhydrazine (100 mg,
0.50 mmol) and H2SO4 (0.5 mL) in H2O (0.75 mL). The reaction
mixture was stirred for 10 min at 23 °C. The orange precipitate was CDCl3): δ = 0.65 (td, J = 11.4, 2.3 Hz, 1 H), 0.69 (s, 3 H), 0.72–
collected by filtration, washed with EtOH/H2O (5 mL, 1:1), and
dried in high vacuum to give phenylhydrazone 19b (20 mg,
4.4 μmol, 60%) as an orange solid, m.p. 154 °C. Single crystals
were obtained from CH2Cl2/hexane. 1H NMR (500 MHz, CDCl3):
δ = 0.70 (s, 3 H), 0.76–0.80 (m, 1 H), 0.86–0.96 (m, 2 H), 1.01–1.16
0.88 (m, 2 H), 0.90 (d, J = 6.5 Hz, 3 H), 0.94–1.04 (m, 3 H), 1.07–
1.15 (m, 2 H), 1.18–1.25 (m, 4 H), 1.30–1.35 (m, 1 H), 1.37–1.54
(m, 6 H), 1.61–1.80 (m, 5 H), 1.81–1.89 (m, 2 H) ppm. 13C{1H}
NMR (125 MHz, CDCl3): δ = 12.16 (CH3), 19.22 (CH3), 19.85
(CH2), 22.13 (CH2), 25.35 (CH2), 26.82 (CH2), 28.13 (CH2), 29.06
(m, 4 H), 1.19 (d, J = 6.5 Hz, 3 H), 1.22–1.30 (m, 4 H), 1.34–1.43 (CH2), 29.16 (CH2), 32.67 (CH2), 32.80 (CH2), 33.64 (CH), 36.38
(m, 1 H), 1.50–1.53 (m, 1 H), 1.57–1.68 (m, 3 H), 1.73–1.75 (m, 2
H), 1.81–1.91 (m, 3 H), 2.44–2.53 (m, 2 H), 2.62–2.70 (m, 1 H),
7.97 (d, J = 9.9 Hz, 1 H), 8.28 (dd, J = 9.9, 2.1 Hz, 1 H), 9.12 (d,
(C), 37.73 (CH), 38.57 (CH2), 46.71 (CH), 47.24 (CH), 47.55 (CH),
53.13 (CH) ppm. IR (ATR): ν = 2919 (s), 2853 (s), 1445 (m), 1378
˜
(m), 1351 (w), 1306 (w), 1264 (w), 1247 (w), 1192 (w), 1107 (w),
J = 2.1 Hz, 1 H), 10.86 (s, 1 H) ppm. 13C{1H} NMR (125 MHz, 1086 (w), 1055 (w), 1029 (w), 975 (w), 958 (w), 930 (w), 922 (w),
CDCl3): δ = 12.15 (CH3), 15.10 (CH3), 18.90 (CH2), 22.02 (CH2), 893 (w), 844 (w), 794 (w) cm–1. MS (EI, 70 eV): m/z (%) = 260 (76)
24.84 (CH2), 26.63 (CH2), 28.70 (CH2), 28.85 (CH2), 32.56 (CH2), [M]+, 245 (40), 231 (8), 218 (3), 217 (3), 204 (43), 203 (100), 189
32.96 (CH2), 36.35 (CH), 36.88 (C), 38.43 (CH2), 38.78 (CH), 43.77
(CH), 45.36 (CH), 46.51 (CH), 52.77 (CH), 116.29 (CH), 123.59
(9), 175 (11), 164 (17), 161 (14), 149 (22), 148 (22), 135 (61), 121
(20), 109 (18), 107 (28), 95 (39), 81 (45), 67 (35), 55 (26). HRMS
(CH), 128.72 (C), 129.91 (CH), 137.43 (C), 145.14 (C), 170.58 (C) (EI): calcd. for C19H32 [M]+ 260.2499; found 260.2504.
ppm. IR (ATR): ν = 3314 (w), 3107 (w), 2920 (m), 2855 (m), 1623
˜
13,17-seco-18-nor-17-homo-Androsta-13,17-dione (20): A solution
of the mixture of olefins containing 15 (470 mg, 1.82 mmol: 80%
purity, 1.45 mmol of 15) in absolute CH2Cl2 (5 mL) was added to
a solution of KMnO4 (711 mg, 4.50 mmol) and dry benzyltrimeth-
ylammonium chloride (835 mg, 4.50 mmol) in absolute CH2Cl2
(45 mL) at 23 °C. The solution was heated for 20 h to 40 °C. After
cooling to ambient temperature, the solution was diluted with
aqueous AcOH (50%, 50 mL). The layers were separated, and the
aqueous layer was extracted with CH2Cl2 (3ϫ 50 mL). The com-
bined organic layers were dried (MgSO4) and filtered, and the sol-
vents were evaporated. The residue was chromatographed (SiO2,
hexane/MTBE 1:1, Rf = 0.37) to furnish diketone 20 (288 mg,
(s), 1590 (s), 1519 (m), 1501 (m), 1426 (m), 1412 (m), 1366 (w),
1335 (s), 1333 (s), 1271 (s), 1269 (m), 1220 (m), 1134 (m), 1070 (m),
1050 (m), 1022 (m), 923 (m), 831 (m), 743 (m), 702 (m), 686 (m)
cm–1. MS (ESI+): m/z = 493 [M + K]+, 477 [M + Na]+, 455 [M +
H]+. HRMS (ESI): calcd. for C25H34N4NaO4 [M + Na]+ 477.2478;
found 477.2489.
17β-Methyl-18-nor-13α-androstane (5a): A suspension of ketones
18a and 18b [34 mg, 0.12 mmol; 18a/18b 70:30], H2NNH2·H2O
(240 mg, 4.8 mmol, 65% hydrazine), and KOH (97 mg, 1.73 mmol)
in DEG (1.5 mL) was heated for 16 h to 200 °C. After cooling to
ambient temperature, the solution was diluted with hydrochloric
Eur. J. Org. Chem. 2013, 5934–5945
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5943

J. Christoffers et al.
FULL PAPER
0.99 mmol, 68%) as a yellow oil. [α]2D0 = 6.7 (MTBE, 1 g/l). 1H
were dried (MgSO4) and filtered, and the solvent was removed un-
NMR (500 MHz, CDCl3): δ = 0.70 (s, 3 H), 0.95 (td, J = 12.9, der reduced pressure. After chromatography (SiO2, hexane, Rf =
3.9 Hz, 1 H), 1.05–1.43 (m, 11 H), 1.45–1.52 (m, 2 H), 1.63–1.73
(m, 3 H), 1.82–1.88 (m, 1 H), 1.96–1.99 (m, 1 H), 2.04–2.08 (m, 1
0.77), the alkanes 6a and 6b (ratio 3.3:1, 20 mg, 80 μmol, 65%)
were obtained as a colorless oil. MS (EI, 70 eV), 6a: m/z (%) = 260
H), 2.10 (s, 3 H), 2.65 (td, J = 13.3, 5.5 Hz, 1 H), 2.34–2.40 (m, 2 (100) [M]+, 245 (48), 231 (9), 218 (4), 204 (47), 203 (92), 189 (9),
H), 2.46–2.53 (m, 1 H) ppm. 13C{1H} NMR (125 MHz, CDCl3): δ
175 (15), 164 (27), 149 (22), 148 (35), 135 (53), 121 (30), 109 (17),
= 11.91 (CH3), 19.38 (CH2), 21.95 (CH2), 26.39 (CH2), 26.59
107 (22), 95 (34), 81 (46), 67 (32), 55 (18); data for isomer 6b vide
(CH2), 28.52 (CH2), 28.63 (CH2), 29.83 (CH3), 32.30 (CH2), 36.62 infra.
(C), 38.67 (CH2), 41.02 (CH2), 41.99 (CH2), 42.60 (CH), 46.11
D-homo-13β,14α-Androstan-17-one (22b): Absolute THF (1 mL)
(CH), 52.68 (CH), 54.84 (CH), 209.12 (C), 212.73 (C) ppm. IR
was added to a solution of Li (18 mg, 2.6 mmol) in NH3 (4 mL) at
–78 °C, and the resulting solution was stirred for 30 min. A solu-
tion of enone 21 (140 mg, 0.51 mmol) in absolute THF (3 mL) was
then added dropwise. After 2.5 h of stirring at –78 to –50 °C, the
reaction mixture was treated with solid NH4Cl (275 mg,
5.14 mmol) in small portions. The solution was warmed to ambient
temperature overnight, and the NH3 evaporated. The slurry was
dissolved in H2O (5 mL) and extracted with MTBE (3ϫ 50 mL).
The combined organic layers were dried (MgSO4) and filtered, and
the solvents were evaporated. The crude material was chromato-
graphed (SiO2, hexane/MTBE 4:1, Rf = 0.39) to give 22b (107 mg,
0.39 mmol, 76%) as a colorless solid, m.p. 106 °C. [α]2D0 = –38.9
(CH2Cl2, 1 g/l). 1H NMR (500 MHz, CDCl3): δ = 0.72 (s, 3 H),
0.78–1.30 (m, 14 H), 1.32–1.43 (m, 2 H), 1.49–1.52 (m, 1 H), 1.66–
1.72 (m, 3 H), 1.78 (dq, J = 12.7, 3.2 Hz, 1 H), 1.98 (dq, J = 12.7,
3.2 Hz, 1 H), 2.02–2.07 (m, 1 H), 2.23–2.31 (m, 3 H), 2.35–2.40 (m,
1 H) ppm. 13C{1H} NMR (125 MHz, CDCl3): δ = 12.09 (CH3),
22.07 (CH2), 24.67 (CH2), 26.66 (CH2), 28.77 (CH2), 28.87 (CH2),
30.35 (CH2), 31.15 (CH2), 34.43 (CH2), 36.43 (C), 38.53 (CH2),
40.90 (CH), 41.33 (CH2), 43.30 (CH), 46.31 (CH), 47.50 (CH),
(ATR): ν = 2920 (m), 2854 (w), 1708 (vs), 1445 (w), 1354 (w), 1284
˜
(w), 1213 (w), 1196 (w), 1162 (w), 1109 (w), 1089 (w), 960 (w), 925
(w) cm–1. MS (EI, 70 eV): m/z (%) = 290 (100) [M]+, 272 (33), 257
(14), 233 (33), 220 (50), 219 (53), 149 (22), 109 (56). HRMS (EI):
calcd. for C19H30O2 [M]+ 290.2246; found 290.2240.
D-homo-Androst-13(17a)-en-17-one (21): Aqueous NaOH solution
(10%, 0.04 mL) was added to a solution of diketone 20 (300 mg,
1.03 mmol) in MeOH/H2O (10:1, 11 mL), and the resulting mixture
was subsequently stirred for 7 h at 40 °C. Water (20 mL) was then
added, and the mixture was allowed to stand at ambient tempera-
ture for 16 h. The precipitate was collected by filtration and washed
with H2O (5 mL). The crude solid was purified by chromatography
(SiO2, hexane/MTBE 2:1; Rf = 0.43) to give compound 21 (196 mg,
0.71 mmol, 69%) as a colorless solid, m.p. 143 °C. [α]2D0 = –43.3
(CH2Cl2, 1 g/l). 1H NMR (500 MHz, CDCl3): δ = 0.69 (s, 3 H),
0.90 (td, J = 12.9, 3.9 Hz, 1 H), 0.95–1.31 (m, 10 H), 1.34–1.42 (m,
1 H), 1.46–1.57 (m, 2 H), 1.65–1.72 (m, 2 H), 1.90–2.01 (m, 3 H),
2.12–2.27 (m, 3 H), 2.23–2.45 (m, 2 H), 5.77 (br s, 1 H) ppm.
13C{1H} NMR (125 MHz, CDCl3): δ = 11.92 (CH3), 21.90 (CH2),
25.61 (CH2), 26.23 (CH2), 26.56 (CH2), 28.57 (CH2), 28.63 (CH2),
31.51 (CH2), 35.95 (CH2), 36.42 (C), 36.43 (CH2), 38.55 (CH2),
43.25 (CH), 43.87 (CH), 46.00 (CH), 52.63 (CH), 123.79 (CH),
48.67 (CH ), 53.40 (CH), 212.12 (C) ppm. IR (ATR): ν = 2950 (m),
˜
2
2916 (s), 2851 (s), 1705 (vs), 1444 (m), 1419 (w), 1379 (w), 1326
(w), 1274 (w), 1263 (w), 1246 (w), 1205 (w), 1186 (w), 1103 (w),
1089 (w), 1024 (w) cm–1. MS (EI, 70 eV): m/z (%) = 274 (75)
[M]+, 259 (38), 256 (12), 241 (31), 230 (32), 218 (45), 217 (100), 201
(13), 199 (34), 189 (16), 175 (12), 171 (11), 162 (21), 149 (66), 147
(32), 131 (26), 119 (25), 110 (29), 109 (34), 108 (24), 107 (37), 105
(32), 95 (49), 93 (41), 91 (58), 81 (75), 79 (60), 67 (58), 55 (40).
HRMS (EI): calcd. for C19H30O [M]+ 274.2297; found 274.2290.
167.03 (C), 199.99 (C) ppm. IR (ATR): ν = 2947 (m), 2918 (m),
˜
2855 (m), 2811 (w), 1663 (vs), 1620 (m), 1466 (w), 1445 (w), 1429
(w), 1380 (w), 1361 (w), 1261 (m), 1209 (m), 1178 (w), 1122 (w),
970 (w), 957 (w), 902 (w), 880 (m) cm–1. MS (EI, 70 eV): m/z (%)
= 272 (48) [M]+, 230 (16), 163 (50), 164 (22), 149 (21), 135 (14),
110 (100). HRMS (EI): calcd. for C19H28O [M]+ 272.2140; found
272.2137.
D-homo-13β,14α-Androstane (6b): A mixture of ketone 22b (40 mg,
0.15 mmol), H2NNH2·H2O (0.07 mL, 1.5 mmol, 65% hydrazine),
and KOH (21 mg, 0.38 mmol) in DEG (2 mL) was heated for 16 h
to 200 °C. After cooling to ambient temperature, the solution was
diluted with hydrochloric acid (10 mL, 2 mol/L) and extracted with
hexane (2ϫ 10 mL). The combined organic layers were dried
(MgSO4) and filtered, and the solvent was removed under reduced
pressure. After chromatography (SiO2, hexane, Rf = 0.77), sterane
6b (20 mg, 80 μmol, 53%) was obtained as a colorless solid, m.p.
D-homo-Androstan-17-ones (22a and 22b): A suspension of enone
21 (40 mg, 0.15 mmol) and Pd/C (8 mg, 7 μmol, 10% w/w Pd) in
pyridine (3 mL) was hydrogenated for 24 h with H2 (4 bar) at ambi-
ent temperature. The mixture was filtered through SiO2 (washed
with 100 mL MTBE). After evaporation of the solvent, the crude
product was chromatographed (hexane/MTBE 4:1, Rf = 0.42–0.39)
to give the ketones 22a and 22b (ratio 3.3:1, 27 mg, 0.10 mmol,
67%) as a colorless oil. Isomer 22a: 13C{1H} NMR (125 MHz,
CDCl3): δ = 12.78 (CH3), 19.54 (CH2), 22.51 (CH2), 27.08 (CH2),
27.85 (CH2), 29.22 (CH2), 29.37 (CH2), 31.33 (CH2), 31.42 (CH2),
31.95 (CH), 38.81 (C), 37.00 (CH2), 38.53 (CH), 38.93 (CH2), 42.00
(CH), 43.34 (CH2), 46.73 (CH), 54.18 (CH), 213.17 (C) ppm. MS
(EI, 70 eV): m/z (%) = 274 (60) [M]+, 259 (15), 256 (30), 241 (32),
227 (4), 218 (33), 217 (49), 204 (23), 199 (27), 189 (11), 171 (9), 159
(13), 149 (100), 147 (30), 131 (21), 119 (17), 110 (22), 109 (24), 108
(25), 107 (26), 95 (35), 93 (29), 91 (35), 81 (50), 79 (40), 67 (40), 55
(25).
1
53 °C. [α]2D0 = –21.7 (CH2Cl2, 1 g/l). H NMR (500 MHz, CDCl3):
δ = 0.54–0.61 (m, 1 H), 0.66–0.70 (m, 1 H), 0.71 (s, 3 H), 0.72–1.03
(m, 8 H), 1.10–1.25 (m, 8 H), 1.34–1.42 (m, 1 H), 1.47–1.51 (m, 1
H), 1.55–1.59 (m, 2 H), 1.63–1.73 (m, 5 H), 1.91–1.97 (m, 2 H)
ppm. 13C{1H} NMR (125 MHz, CDCl3): δ = 12.17 (CH3), 22.17
(CH2), 24.92 (CH2), 26.45 (CH2), 26.80 (CH2), 26.91 (CH2), 29.01
(CH2), 29.03 (CH2), 30.01 (CH2), 30.79 (CH2), 34.52 (CH2), 34.54
(CH2), 36.38 (C), 38.56 (CH2), 41.43 (CH), 42.80 (CH), 46.45
(CH), 49.14 (CH), 53.84 (CH) ppm. IR (ATR): ν = 2946 (m), 2915
˜
(s), 2846 (s), 1464 (w), 1442 (w), 1376 (w), 1346 (w), 1304 (w), 1289
(w), 1263 (w), 1238 (w), 1190 (w), 1172 (w), 1134 (w), 1103 (w),
1082 (w), 1053 (w), 1042 (w), 1016 (w), 986 (w), 972 (w), 964 (w),
945 (w), 931 (w), 906 (w), 883 (w), 869 (w), 847 (w), 838 (w), 808
(w), 794 (w) cm–1. MS (EI, 70 eV): m/z (%) = 260 (100) [M]+, 246
(6), 245 (35), 218 (5), 204 (36), 203 (78), 189 (5), 176 (6), 175 (7),
164 (13), 149 (13), 135 (26), 121 (15), 109 (20), 95 (36), 81 (40), 67
D-homo-Androstanes (6a and 6b): A suspension of ketones 22a and
22b (34 mg, 0.12 mmol), H2NNH2·H2O (0.06 mL, 1.2 mmol, 65%
hydrazine), and KOH (17 mg, 0.31 mmol) in DEG (1 mL) was
heated to 200 °C for 16 h. After cooling to ambient temperature,
the solution was diluted with hydrochloric acid (5 mL, 2 m) and
extracted with hexane (3ϫ 10 mL). The combined organic layers
5944
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Eur. J. Org. Chem. 2013, 5934–5945

Identification of a Fossil Sterane Biomarker in Crude Oil
(27), 55 (18), 41 (10). HRMS (EI): calcd. for C19H32 [M]+ 260.2504;
found 260.2508.
[9]
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-934100 (for 19), and -934101 (for 22b) contain the supplemen-
tary crystallographic data for this paper. These data can be
obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
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Acknowledgments
The authors gratefully acknowledge Wolfgang Saak for X-ray crys-
tallography. Bernd Kopke is thanked for his support in comparative
GC–MS analysis of synthetic and fossil steranes. The authors are
furthermore grateful to Dr. Amy E. Kelly, Shell, Houston, for sup-
plying saturated hydrocarbon fractions of crude oils for coinjec-
tions. The work at MIT was supported by grants from the National
Aeronautics and Space Administration Astrobiology Institute to
R. E. S. MTBE was obtained as a generous gift from Evonik Indus-
tries, Marl, Germany.
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Received: May 28, 2013
Published Online: July 26, 2013
Eur. J. Org. Chem. 2013, 5934–5945
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5945