Synthesis of symmetrical bis-steroid pyrazines connected via D-rings

Article abstract of DOI:10.1135/cccc20001597

Bis-steroid pyrazines fused through rings D of the steroid skeleton were synthesized. Methods for the preparation of the corresponding rings A fused derivatives (cephalostatin type) were checked on simple androstanes and then a symmetrical D fused analogue, 5α-androstano[16″,17″-5′,6′]pyrazino[2′, 3′-16,17]-5α-androstane-3β,3″β-diol, was prepared. Partially substituted bis-steroid pyrazines in both series were prepared and their use for the preparation of higher fused compounds was discussed. No significant cytostatic activity was found on parent bis-steroid pyrazines both A and D fused.

Full text of DOI:10.1135/cccc20001597

On Steroids
1597
SYNTHESIS OF SYMMETRICAL BIS-STEROID PYRAZINES
CONNECTED via D-RINGS+
1,
2
3
4
Ivan ČERNÝ *, Vladimír POUZAR , Miloš BUDĚŠÍNSKÝ and Pavel DRAŠAR
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
1
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; e-mail: cerny@uochb.cas.cz,
2
3
4
pouzar@uochb.cas.cz, budesinsky@uochb.cas.cz, drasar@uochb.cas.cz
Received May 22, 2000
Accepted August 25, 2000
Bis-steroid pyrazin es fused th rough rin gs D of th e steroid skeleton were syn th esized.
Meth ods for th e preparation of th e correspon din g rin gs A fused derivatives (ceph alostatin
type) were ch ecked on sim ple an drostan es an d th en a sym m etrical D fused an alogue,
5α-an drostan o[16′′,17′′-5′,6′]pyrazin o[2′,3′-16,17]-5α-an drostan e-3β,3′′β-diol, was prepared.
Partially substituted bis-steroid pyrazin es in both series were prepared an d th eir use for th e
preparation of h igh er fused com poun ds was discussed. No sign ifican t cytostatic activity was
foun d on paren t bis-steroid pyrazin es both A an d D fused.
Key w ord s: Steroids; Ceph alostatin an alogues; Sym m etrical bis-steroid pyrazin es; NMR spec-
troscopy; Cytostatic activity.
Th e size an d rigidity of a steroid skeleton ren der th is un it a poten tial build-
in g block for con structin g larger m olecular structures with in terestin g bio-
logical properties (m em bran e m odification s, ion h ostin g an d tran sport,
self-assem bly, etc.) Man y exam ples of dim eric an d oligom eric steroids, both
syn th etic an d n atural, are kn own 2. From th is class of com poun ds, th ose
h avin g two steroid skeleton s fused togeth er th rough pyrazin e rin g were
very in ten sively studied in con n ection with ceph alostatin 3 an d its an a-
logues. Several syn th etic approach es m ade available both sym m etrical an d
un sym m etrical bis-steroid pyrazin es4–9. Most of th e syn th eses of sym m etri-
cal pyrazin es used vicin al am in o keton es, available from oth er n itrogen de-
rivatives, such as oxim es4, en am in es5 or azides6 (Sch em e 1). Th e aim of our
work was first to ch eck selected m eth ods for con den sation th rough both
rin g A an d rin g D on sim ple an drostan e derivatives. Secon dly, we in ten ded
to prepare som e partially substituted derivatives suitable for an addition al
con den sation cycle, wh ich m ay deliver h igh ly fused system s.
+ Part CDIX in th e series On Steroids; Part CDVIII see ref.1
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

1598
Černý, Pouzar, Buděšínský, Drašar:
Th e pyrazin e derivative 3 in rin g A fused series is kn own : a th orough de-
scription of experim en ts is given in ref.4, wh ereas ref.10 m en tion ed th e syn -
th esis on ly briefly. An un successful attem pt at th e syn th esis of rin g D fused
pyrazin e 12 was m en tion ed4.
tert-BuOK,
isopentyl nitrite
R1
R2
O
R1
R2
O
N
H2, Pd/C, EtOH
HCl
1. NaOH
A
B
C
OH
tert-BuOH
2. O2, TsOH, EtOH
NH3 Cl
R1
R2
O
R1
R2
O
R1
O
R1
R2
N
R2
R1
NaN3, AcOH
DMF
PhMe3NBr3
THF
1. Ph3P, H2O, THF
2. O2, TsOH, EtOH
R2
Br
N3
N
R1
(H-R2)
O
NaN3, NaI
DMF
H2, Pd/C, AcOH
EtOAc, MeOH
NH2
SCHEME 1
RESULTS AND DISCUSSION
In th e rin g A fused series, we tested m eth ods startin g from brom oketon e 1.
Prelim in ary experim en ts were based on its reaction with sodium azide in
dim eth ylform am ide in th e presen ce of sodium iodide at elevated tem pera-
ture (Sch em e 1: m eth od C, ref.5). Th e foam y en am in e product on subse-
quen t h ydrogen ation on palladium on carbon in a m ixture of eth yl acetate
an d acetic acid gave pyrazin e 3, but on ly in a low yield. Better results were
ach ieved by usin g n ucleoph ilic substitution of brom in e in brom oketon e 1
with sodium azide in dim eth ylform am ide in th e presen ce of acetic acid11
suppressin g th e resultin g azide destruction (Sch em e 1: m eth od B). In agree-
m en t with th e fin din g on ch olestan e derivatives11
, azide 2 with
2α-con figuration was formed. The reduction of azide 2 was performed by hy-
drogen ation on palladium on carbon in eth an ol with th e addition of
h ydroch loric acid. Th e crude in term ediate (am in e h ydroch loride) was tran s-
form ed in to pyrazin e 3 by alkalin ization with a con com itan t con den sation
to a dih ydropyrazin e m ixture an d subsequen t arom atization by air oxida-
tion in eth an ol with 4-toluen esulfon ic acid (Sch em e 2).
Wh en com pared with th e publish ed procedures leadin g to 3, th e use of
azide in stead of oxim e circum ven ted problem s with oxim ation selectivity
(cf. ref.4) an d th e overall yield was in our case better; for an oth er ap-
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

On Steroids
1599
proach 10, very close to ours, on ly syn th etic sch em e with out experim en tal
data was given .
OH
OH
N3
O
Br
(i)
O
2
1
H
H
OH
(ii)
H
N
N
H
3
HO
(i) NaN3, AcOH, DMF; (ii) 1. H2, Pd/C, EtOH, HCl; 2. K2CO3, H2O; 3. O2, TsOH, EtOH
SCHEME 2
Th e pyrazin e 3 was partially acetylated with acetic an h ydride in pyridin e:
in a m ixture of diacetate 4, m on oacetate 5 an d un reacted startin g pyrazin e
diol 3, th e m on oacetate 5 prevailed (Sch em e 3) an d could be separated by
colum n ch rom atograph y.
Oxidation of th e free h ydroxy groups on bis-steroid pyrazin es 3 an d 4
with ch rom ium (IV) oxide in sulfuric acid (Jon es reagen t) did n ot affect th e
pyrazin e rin g; we prepared both diketon e 6 an d keton e 7. Deacetylation of
com poun d 7 gave bis-steroid pyrazin e 8 with h ydroxy an d keto groups on
rem ote sides of th e m olecule.
At th is stage, we plan n ed to repeat th e above syn th etic route in order to
obtain a m olecule con tain in g four steroid un its. We failed, h owever, in th e
preparation of th e correspon din g brom oketon e from 7 or 8 both by th e re-
action with trim eth ylph en ylam m on ium brom ide perbrom ide in tetra-
h ydrofuran an d with copper(II) brom ide in a m eth an ol–ben zen e m ixture. It
is n ecessary to n ote th at all bis-steroid pyrazin es un der study h ave lim ited
solubility in com m on organ ic solven ts an d th is com plicates th e treatm en t
an d n arrows th e reaction selection . Th e oxim e approach was a little m ore
prom isin g: th e reaction of 8 with potassium tert-butoxide an d isopen tyl n i-
trite in tert-butyl alcoh ol or in a tert-butyl alcoh ol–pyridin e m ixture gave
oxim e 9 (see NMR in Experim en tal) but on ly in very low yield (about 10%,
m ost of th e un reacted startin g com poun d was recovered). Even an attem pt
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

1600
Černý, Pouzar, Buděšínský, Drašar:
OAc
H
N
N
4
(i)
3
H
OH
+
AcO
H
N
N
H
(ii)
AcO
5
(ii)
O
H
N
N
6
O
H
N
N
H
O
H
RO
O
7, R = Ac
8, R = H
(iv)
(iii)
OH
N
9
(i) Ac2O, pyridine; (ii) CrO3, H2SO4, CH2Cl2, Me2CO; (iii) KOH, MeOH, THF;
(iv) 1. tert-BuOK, tert-BuOH, DMF; 2. isopentyl nitrite
SCHEME 3
at in creasin g solubility by a tert-butyldim eth ylsilyl protection of th e
h ydroxy group failed; th e resultin g protected derivative was in soluble as
well (derivative of 8, R = TBDMS). Despite th e preparation problem s, we
tried to h ydrogen ate oxim e 9 over palladium on carbon in acetic acid. Th e
experim en t was don e on th e scale of several m illigram s, so th e results are
on ly ten tative, but n o fused product was iden tified after alkali addition .
Th e on ly separated product seem s to be th e 17-h ydroxy-16-oxo derivative
of th e startin g bis-steroid pyrazin e (MS FAB: 587 [M + 1], sim ilar derivatives
of sim ple steroids were foun d in trace am oun ts in th e m ixture after th e re-
duction of azide 2 by th e triph en ylph osph in e m eth od).
Th e above results posed a question about th e stability of bis-steroid
pyrazin es fused th rough rin gs D. To address th is issue, we prepared azide 11
from keton e 10 via th e sam e route as in th e case of com poun ds 1 an d 2 in
th e rin g A fused series (Sch em e 4). Hydrogen ation an d subsequen t treat-
m en t as in th e case of 3 did n ot give an y crystallin e product directly from
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

On Steroids
1601
th e reaction m ixture. After purification by colum n ch rom atograph y, we ob-
tain ed a crystallin e m aterial, but it was a n on separable m ixture, in wh ich ,
in addition to th e bis-steroid pyrazin e 12, oth er com poun ds were presen t,
m ost probably dih ydropyrazin es with a sim ilar ch rom atograph ic m obility.
After som e un successful experim en ts, we ch an ged th e m eth od of azide
reduction an d usin g a procedure6 based on th e triph en ylph osph in e
m eth od, we were able to prepare th e bis-steroid pyrazin e 12, h owever, in
a yield lower th an in rin g A fused series. To prom ote th e oxidation /
deh ydrogen ation step, we varied th e con dition s, but n eith er prolon ged stir-
rin g, elevated tem perature, ch an ge of th e solven t, n or addition of cop-
per(II) acetate h ad an y m arked effect on th e yield.
O
O
(i)
Br
N3
HO
HO
10
11
H
H
H
OR2
(ii)
N
N
12, R1 = R2 = H
R1O
13, R1 = R2 = Ac
14, R1 = Ac, R2 = H
(iii)
H
(iv)
H
O
N
N
RO
15, R = Ac
16, R = H
(v)
H
(i) NaN3, AcOH, DMF; (ii) 1. Ph3P, THF; 2. H2O; 3. O2, TsOH, EtOH; (iii) Ac2O, pyridine;
(iv) CrO3, H2SO4, CH2Cl2, Me2CO; (v) KOH, MeOH, THF
SCHEME 4
Ch em ical properties of rin g D fused bis-steroid pyrazin es are sim ilar to
th eir rin g A fused coun terparts; after acetylation , it was possible to separate
diacetate 13 an d m on oacetate 14. Oxidation of m on oacetate 14 gave
keton e 15 an d its deacetylation h ydroxyketon e 16. Wh en com pared with
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Černý, Pouzar, Buděšínský, Drašar:
rin g A fused an alogues, th e rin g D fused bis-steroid pyrazin es are little m ore
soluble. However, prelim in ary experim en ts with brom in ation gave th e
sam e results as in th e rin g A fused series an d n itrosation of 15 afforded on ly
a 2,4-bis oxim e derivative with out an y practical use for furth er con den sa-
tion .
In sum m ary, we accom plish ed sim ple syn th eses of m odel bis-steroid
pyrazin es an d prepared rin g D fused derivatives. Th e exten sion of th e
pyrazin e couplin g of steroids to m ore th an two steroid un its will be a diffi-
cult task due to an extrem ely low solubility of bis-steroid pyrazin es an d to a
relatively h igh ch em ical reactivity of th e pyrazin e n ucleus.
Cytostatic activity of th e paren t bis-steroid pyrazin es 3 an d 12 was tested
on th eir in vitro in h ibition of th e cell growth usin g th e m eth odology de-
scribed in ref.12. No sign ifican t in h ibition on L1210, L929, HeLa S3 an d
CCRF-CEM cell cultures were foun d at th e con cen tration of 10 µm ol l–1
(precipitation in culture m edium was observed).
EXPERIMENTAL
Meltin g poin ts were determ in ed on a Boetius m icro m eltin g poin t apparatus (Germ an y). Op-
tical rotation s were m easured on a Perkin –Elm er 141 MC polarim eter; [α]D values are given
in 10–1 deg cm 2 g–1. IR spectra (waven um bers in cm –1) were recorded on a Bruker IFS 88
spectrom eter. 1H NMR spectra were taken on a Varian UNITY-200 (200 MHz) or on Varian
UNITY-500 (1H at 500 MHz, 13C at 125.7 MHz) in strum en ts at 23 °C, in deuterioch loroform ,
with tetram eth ylsilan e as in tern al stan dard. Ch em ical sh ifts are given in ppm (δ-scale), cou-
plin g con stan ts (J) an d width of m ultiplets (W) in Hz. Hom on uclear 2D-COSY spectra were
used for structural assign m en t of proton sign als in com poun ds 3, 6, 9 an d 13. Partial assign -
m en t in 1D-spectra of oth er com poun ds was don e on th e basis of ch em ical sh ift values an d
th e com parison with data on structurally sim ilar com poun ds. Carbon sign als in com poun ds
3, 6, 9, 12, 13 an d 15 were assign ed usin g: (i) distin guish in g of CH3-, CH2-, CH- an d C-type
carbon atom s in APT spectra, (ii) literature data on structuraly sim ilar com poun ds an d (iii)
1H–13C correlation in an HMQC spectrum of com poun d 13. Th e sign als in NMR spectra of
sym m etrical bis-steroid pyrazin es are n um bered with on ly on e set of n um bers. Mass spectra
(FAB) were recorded on a VG An alytical ZAB-EQ spectrom eter. Th in -layer ch rom atograph y
(TLC) was perform ed on silica gel G (ICN Biom edicals, detection by sprayin g with con cen -
trated sulfuric acid followed by h eatin g); preparative TLC was don e on 200 × 200 m m plates
(layer th ickn ess 0.4 m m ). For colum n ch rom atograph y, n eutral silica gel 60–120 µm was
used. Prior to evaporation on a rotary evaporator in vacuo (bath tem perature 50 °C), solu-
tion s in organ ic solven ts were dried over an h ydrous MgSO4.
2α-Azido-17β-h ydroxy-5α-an drostan -3-on e (2)
To a solution of brom oketon e 1 (ref.13; 1.5 g, 4.06 m m ol) in DMF (25 m l) acetic acid (0.25 m l,
4.37 m m ol) an d sodium azide (1.0 g, 15.38 m m ol) were added. Th e m ixture was stirred at
room tem perature for 20 m in an d th en poured on ice. Crystallin e m aterial was collected by
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

On Steroids
1603
filtration , wash ed with water an d dried. Yield of 2 was 1.03 g (77%), an alytical sam ple was
purified by colum n ch rom atograph y in a m ixture of ben zen e–aceton e (20 : 1) an d crystal-
lized from h ot aceton e, m .p. 170–173 °C (dec.), [α ]2D5 –83 (c 0.95, ch loroform ). IR: 3 614
(O–H); 2 107 (–N3); 1 723 (C=O); 1 054 (C–O). 1H NMR (200 MHz): 3.99 dd, 1 H, J = 6.3,
13.0 (H-2β); 3.65 t, 1 H, J = 8.2 (H-17α); 1.10 s, 3 H (3 × H-19); 0.76 s, 3 H (3 × H-18). For
C
19H29N3O2 (331.5) calculated: 68.85% C, 8.82% H, 12.68% N; foun d: 68.98% C, 8.90% H,
12.42% N.
5′′α-An drostan o[2′′,3′′-5′,6′]pyrazin o[2′,3′-2,3]-5α-an drostan e-17β,17′′β-diol (3)
A solution of azide 2 (1.0 g, 3.02 m m ol) in eth an ol (100 m l) an d 6 M h ydroch loric acid (1 m l)
was h ydrogen ated over 10% palladium on carbon (100 m g) at atm osph eric pressure an d at
room tem perature for 2 h . Th e catalyst was filtered off on celite, wh ich was wash ed with
eth an ol, an d th e com bin ed solution s were evaporated. Water (5 m l) an d aqueous saturated
KHCO3 were added, th e yellow solid was filtered off, dissolved in eth an ol an d evaporated.
Th e residue was twice coevaporated with a m ixture of ben zen e an d eth an ol (1 : 1, 20 m l)
an d th en eth an ol (20 m l) an d 4-toluen esulfon ic acid m on oh ydrate (50 m g) were added. Th e
m ixture was sh ortly boiled to dissolve all solids an d th en in ten sively stirred in an open flask
at room tem perature for 24 h . Durin g th is tim e a solid was precipitatin g. Th e product was
filtered off an d wash ed with aqueous eth an ol to give 480 m g (55%) of 3, m .p. 340 °C (dec.).
IR (KBr): 3 394 (O–H); 1 398 (pyrazin e rin g); 1 059, 1 046 (C–O). 1H NMR (500 MHz): 3.66 t,
2 H, J = 8.7 (H-17); 2.92 d, 2 H, J = 16.5 (H-1β); 2.78 dd, 2 H, J = 17.5, 5.1 (H-4α); 2.59 bdd,
2 H, J = 17.5, 12.5 (H-4β); 2.52 bd, 2 H, J = 16.5 (H-1α); 2.07 ddt, 2 H, J = 13.5, 5.8, 9.2
(H-16β); 1.86 ddd, 2 H, J = 12.5, 3.9, 2.8 (H-12β); 1.75 dq, 2 H, J = 12.8, 3.5 (H-6α); 1.70 m ,
2 H (H-11α); 1.66 m , 2 H (H-5); 1.64 m , 2 H (H-7β); 1.62 m , 2 H (H-15α); 1.46 m , 2 H
(H-16α); 1.45 m , 2 H (H-11β); 1.40 m , 2 H (H-8); 1.35 m , 2 H (H-7α); 1.28 dq, 2 H, J = 5.9,
12.2 (H-15β); 1.12 dt, 2 H J = 4.1, 12.8 (H-12α); 0.99 ddd, 2 H, J = 12.2, 11.0, 7.2 (H-14);
0.94 m , 2 H (H-6β); 0.88 ddd, 2 H, J = 12.2, 10.6, 4.0 (H-9); 0.813 s, 6 H (6 × H-19); 0.766 s,
6 H (6 × H-18). 13C NMR: 148.86 (C-2), 148.47 (C-3), 81.92 (C-17), 53.81 (C-9), 50.90 (C-14),
45.99 (C-1), 42.89 (C-13), 41.84 (C-5), 36.69 (C-12), 35.70 (C-10), 35.50 (C-4), 35.40 (C-8),
31.18 (C-7), 30.50 (C-16), 28.30 (C-6), 23.40 (C-15), 20.82 (C-11), 12.01 (C-19), 11.06 (C-18).
MS, m/z: 573 (M + 1). For C38H56N2O2 (572.9) calculated: 79.67% C, 9.85% H, 4.89% N;
foun d: 79.73% C, 9.90% H, 4.71% N.
5′′α-An drostan o[2,3′′-5′,6′]pyrazin o[2′,3′-2,3]-5α-an drostan e-17β,17′′β-diol Diacetate (4)
an d 5′′α-An drostan o[2′′,3′′-5′,6′]pyrazin o[2′,3′-2,3]-5α-an drostan e-17β,17′′β-diol
17-Acetate (5)
To a stirred solution of diol 3 (1 g, 1.75 m m ol) in pyridin e (25 m l), acetic an h ydride (50%
solution in pyridin e, 0.6 m l, 3.18 m m ol) was added dropwise an d stirrin g was con tin ued at
room tem perature for 48 h . Th e m ixture was poured on ice, th e solid was filtered off,
wash ed with water an d dried. Ch rom atograph y on silica gel (100 m l) in a m ixture of ch loro-
form –eth yl acetate (100 : 1 to 50 : 1) gave successively diacetate 4 (50 m g, 4%), m on oacetate
5 (460 m g, 43%) an d recovered pyrazin e 3 (372 m g, 37%).
Diacetate 4: m .p. 330 °C (dec., eth an ol), [α ]2D5 +61 (c 0.5, ch loroform ). IR: 1 724 (C=O);
1 399 (pyrazin e rin g); 1 258, 1 032. 1H NMR (200 MHz): 4.62 t, 2 H, J = 8.3 (H-17); 2.91 d,
2 H, J = 16.4 (H-1β); 2.79 dd, 2 H, J = 18.0, 5.8 (H-4α); 2.57 dd, 2 H, J = 18.0, 11.9 (H-4β);
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

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Černý, Pouzar, Buděšínský, Drašar:
2.52 bd, 2 H, J = 16.4 (H-1α); 2.04 s, 6 H (CH3CO); 0.81 s, 12 H (6 × H-18, 6 x H-19). MS,
m/z: 657 (M + 1). For C42H60N2O4 (657.0) calculated: 76.97% C, 9.21% H, 4.26% N; foun d:
77.12% C, 9.29% H, 4.10% N.
Monoacetate 5: m .p. 345 °C (dec., eth an ol). IR: 3 450 (O–H); 1 738 (C=Oacetate); 1 397
(pyrazin e rin g); 1 247, 1 239, 1 030 (C–O); 1 045 (C–OH). 1H NMR (200 MHz): 4.62 t, 1 H,
J = 8.3 (H-17α); 3.66 bt, 1 H, J = 8.3 (H-17′′α); 2.95 d, 2 H, J = 17.4 (H-1β); 2.82 dd, 2 H, J =
18.2, 4.3 (H-4α); 2.60 dd, 2 H, J = 18.2, 12.2 (H-4β); 2.53 d, 2 H, J = 17.4 (H-1α); 2.04 s, 3 H
(CH3CO); 0.81 s, 9 H (6 × H-19, 3 × H-18); 0.77 s, 3 H (3 × H-18′′). MS, m/z: 615 (M + 1). For
C
40H58N2O3 (614.9) calculated: 78.13% C, 9.51% H, 4.56% N; foun d: 78.27% C, 9.61% H,
4.29% N.
5′′α-An drostan o[2′′,3′′-5′,6′]pyrazin o[2′,3′-2,3]-5α-an drostan e-17β,17′′β-dion e (6)
To diol 3 (120 m g, 0.21 m m ol) stirred in a m ixture of dich lorom eth an e (5 m l) an d aceton e
(5 m l) th e Jon es reagen t (0.3 m l, 0.54 m m ol) was added dropwise. After 10 m in , th e excess
reagen t was destroyed by propan -2-ol (0.3 m l), aqueous saturated KHCO3 (about 10 m l) was
added an d organ ic solven ts were rem oved on a rotary evaporator. Th e aqueous suspen sion
was extracted with ch loroform (3 × 5 m l), th e com bin ed extracts were wash ed with aqueous
saturated KHCO3 an d water, dried an d th e solven t was evaporated. Boilin g with aceton e left
93 m g (78%) of keton e 6. An alytical sam ple was recrystallized from a ch loroform –eth er m ix-
ture, m .p. 320 °C (dec.). IR (KBr): 1 743 (C=O); 1 399 (pyrazin e rin g). 1H NMR (500 MHz):
2.93 d, 2 H, J = 16.7 (H-1β); 2.81 dd, 2 H, J = 18.0, 5.0 (H-4α); 2.61 bdd, 2 H, J = 18.0, 12.0
(H-4β); 2.54 bd, 2 H, J = 16.7 (H-1α); 2.46 ddd, 2 H, J = 19.2, 9.0, 0.8 (H-16β); 2.09 dt, 2 H,
J = 19.2, 9.0 (H-16α); 1.98 dddd, 2 H, J = 12.2, 9.0, 5.8, 0.8 (H-15α); 1.89 m , 2 H (H-7β);
1.78 ddd, 2 H, J = 13.0, 4.0, 3.0 (H-11α); 1.70 m , 2 H (H-5); 1.68 m , 2 H (H-6α); 1.57 dq,
2 H, J = 3.7, 11.0 (H-8); 1.54 tt, 2 H, J = 12.4, 9.0 (H-15β); 1.49 dq, 2 H, J = 4.0, 13.0 (H-11β);
1.39 dq, 2 H, J = 3.8, 13.3 (H-6β); 1.31 ddd, 2 H, J = 12.6, 11.0, 5.8 (H-14); 1.31 dt, 2 H, J =
4.0, 13.0 (H-12α); 1.06 ddt, 2 H, J = 12.0, 3.6, 13.0 (H-7α); 0.95 ddd, 2 H, J = 12.5, 10.5, 4.0
(H-9); 0.895 s, 6 H (6 × H-18); 0.832 s, 6 H (6 × H-19). 13C NMR: 220.94 (C-17), 148.72
(C-2), 148.41 (C-3), 53.78 (C-5), 51.33 (C-14), 47.64 (C-13), 45.89 (C-1), 41.76 (C-9), 35.81
(C-16), 35.76 (C-4), 35.41 (C-10), 34.90 (C-8), 31.54 (C-12), 30.45 (C-6), 28.11 (C-7), 21.78
(C-15), 20.50 (C-11), 13.71 (C-18), 11.98 (C-19). MS, m/z: 569 (M + 1). For C38H52N2O2
(568.9) calculated: 80.24% C, 9.21% H, 4.82% N; foun d: 79.97% C, 9.30% H, 4.75% N.
17′′-Oxo-5′′α-an drostan o[2′′,3′′-5′,6′]pyrazin o[2′,3′-2,3]-5α-an drostan e-17β-yl Acetate (7)
To m on oacetate 5 (400 m g, 0.65 m m ol) stirred in a m ixture of dich lorom eth an e (10 m l) an d
aceton e (10 m l) th e Jon es reagen t (0.4 m l, 0.72 m m ol) was added dropwise. After 20 m in ,
th e excess reagen t was destroyed by propan -2-ol (0.4 m l), aqueous saturated KHCO3 (about
50 m l) was added an d organ ic solven ts were rem oved on a rotary evaporator. Th e aqueous
suspen sion was extracted by ch loroform (3 × 15 m l), th e com bin ed extracts were wash ed by
aqueous saturated KHCO3 an d water, dried an d th e solven t was evaporated. Th e product was
purified on preparative TLC plates in a m ixture of ch loroform –m eth an ol (50 : 1) yieldin g
310 m g (78%) of keton e 7, m .p. 330 °C (dec., m eth an ol), [α ]2D5 +117 (c 0.5, ch loroform ). IR
(KBr): 1 740 (C=O); 1 398 (pyrazin e rin g); 1 246, 1 032 (C–O). 1H NMR (200 MHz): 4.62 t,
1 H, J = 8.3 (H-17α); 2.04 s, 3 H (CH3CO); 0.89 s, 3 H (3 × H-18′′); 0.83 s, 3 H (3 × H-19′′);
0.81 s, 6 H (3 × H-19, 3 × H-18). MS, m/z: 613 (M + 1). For C40H56N2O3 (612.9) calculated:
78.39% C, 9.21% H, 4.57% N; foun d: 78.46% C, 9.28% H, 4.41% N.
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17′′β-Hydroxy-5′′α-an drostan o[2′′,3′′-5′,6′]pyrazin o[2′,3′-2,3]-5α-an drostan -17-on e (8)
Acetate 7 (200 m g, 0.32 m m ol) in a m ixture of ben zen e (10 m l) an d m eth an ol (10 m l) was
deacetylated with sodium m eth oxide (100 m g Na in 5 m l of m eth an ol; 0.6 m l, 0.52 m m ol)
at room tem perature for 24 h . Solid CO2 (about 0.5 cm 3) was added an d th e solven ts were
evaporated. Th e residue was extracted with ch loroform (3 × 10 m l), th e com bin ed extracts
were con cen trated an d th e product was purified on th e preparative TLC plates in a ch loro-
form –m eth an ol (50 : 1) m ixture. Boilin g with a m ixture of m eth an ol–water (100 : 1) gave
136 m g (73%) of keton e 8, m .p. 335 °C (dec.), [α ]2D5 +134 (c 0.5, ch loroform ). IR (KBr): 3 461
(O–H); 1 742, 1 729 (C=O); 1 397 (pyrazin e rin g); 1 048 (C–O). 1H NMR (200 MHz): 3.65 t, 1 H,
J ≈ 8 (H-17′′); 0.90 s, 3 H (3 × H-18); 0.83 s, 3 H (3 × H-19); 0.82 s, 3 H (3 × H-19′′); 0.77 s, 3 H
(3 × H-18′′). MS, m/z: 571 (M + 1). For C38H54N2O2 (570.9) calculated: 79.95% C, 9.53% H,
4.91% N; foun d: 79.92% C, 9.37% H, 4.73% N.
17′′β-Hydroxy-5′′α-an drostan o[2′′,3′′-5′,6′]pyrazin o[2′,3′-2,3]-5α-an drostan -16,17-dion e
16-Oxim e (9)
1H NMR (500 MHz): 10.05 bs (oxim e H); 3.66 t, 1 H, J ≈ 9.3 (H-17α); 2.97 dd, 1 H, J = 17.5,
6.6 (H-15′′α); 2.94 d, 1 H, J = 17.0 (H-1′′β); 2.92 d, 1 H, J = 17.0 (H-1β); 2.83 bdd, 1 H, J =
17.5, 5.2 (H-4′′α); 2.79 bdd, 1 H, J = 17.5, 5.2 (H-4α); 2.62 bdd, 1 H, J = 17.5, 11.5 (H-4′′β);
2.60 bdd, 2 H, J = 17.5, 11.5 (H-4β); 2.55 bd, 1 H, J = 17.0 (H-1′′α); 2.53 bd, 1 H, J = 17.0
(H-1α); 2.16 dd, 1 H, J = 17.5, 13.2 (H-15′′β); 2.07 dddd, 1 H, J = 13.6, 9.3, 9.0, 6.0 (H-16β);
≈1.68 m , 2 H (H-5α, H-5′′α); ≈1.62 m , 1 H (H-15α); ≈1.46 m , 1 H (H-16α); ≈1.45 m , 1 H
(H-14′′α); ≈1.27 m , 1 H (H-15β); 0.988 s, 3 H (3 × H-18′′); 0.852 s, 3 H (3 × H-19′′); 0.814 s,
3 H (3 × H-19); 0.768 s, 3 H (3 × H-18′′). 13C NMR: 205.09 (C-17′′), 156.55 (C-16′′), 149.16
(C-2′′), 148.73 (C-3), 148.49 (C-2), 148.26 (C-3′′), 81.90 (C-17), 53.78 (C-9), 53.58 (C-9′′),
50.89 (C-14), 48.76 (C-13′′), 46.39 (C-14′′), 45.86 (C-1), 45.56 (C-1′′), 42.87 (C-13), 41.77
(C-5), 41.68 (C-5′′), 36.67 (C-12), 35.79 (C-10′′), 35.66 (C-10), 35.38 (C-8), 35.34 (C-4), 35.22
(C-4′′), 34.30 (C-8′′), 31.14 2 C (C-7, C-15′′), 30.46 2 C (C-7′′, C-16), 28.25 (C-6), 27.98
(C-6′′), 25.50 (C-12′′), 23.38 (C-15), 20.80 (C-11), 20.34 (C-11′′), 14.08 (C-18′′), 12.00 (C-19),
11.97 (C-19′′), 11.03 (C-18).
16β-Azido-3β-h ydroxy-5α-an drostan -17-on e (11)
To a solution of brom oketon e 10 (ref.14; 3.0 g, 8.12 m m ol) in DMF (60 m l) acetic acid (0.5 m l,
8.74 m m ol) an d sodium azide (1.8 g, 27.69 m m ol) were added. Th e m ixture was stirred at
room tem perature for 20 m in an d th en poured on ice. Crystallin e m aterial was collected by
filtration , wash ed with water an d dried. Yield of 11 was 2.65 g (98%), an alytical sam ple was
crystallized from h ot aceton e, m .p. 184–185 °C (dec., ref.15 gives m .p. 113–116 °C), [α ]D25 –28
(c 1.0, ch loroform ). IR: 3 610 (O–H); 2 102 (–N3); 1 749 (C=O); 1 034 (C–O). 1H NMR: 3.71 t,
1 H, J = 8 (H-16α); 3.58 m , 1 H, W = 32 (H-3α); 2.32 ddd, 1 H, J = 11.6, 8.4, 4.6 (H-15α);
0.92 s, 3 H (3 × H-19); 0.83 s, 1 H (3 × H-18). For C19H29N3O2 (331.5) calculated: 68.85% C,
8.82% H, 12.68% N; foun d: 69.03% C, 8.85% H, 12.57% N.
5′′α-An drostan o[16′′,17′′-5′,6′]pyrazin o[2′,3′-16,17]-5α-an drostan e-3β,3′′β-diol (12)
A dry m ixture of azide 11 (1.0 g, 3.02 m m ol) an d triph en ylph osph in e (2.0 g, 7.62 m m ol)
was stirred un der argon for 10 m in an d th en tetrah ydrofuran (20 m l) was added th rough
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Černý, Pouzar, Buděšínský, Drašar:
septum . Th e m ixture was stirred for addition al 1 h ; durin g th is period a gas was evolvin g
from th e solution . Water (1.0 m l, 55.56 m m ol) was added an d th e m ixture was stirred for
24 h . Th e solution was tran sferred to a 250 m l roun d flask, th e reaction vessel was wash ed
with tetrah ydrofuran an d th e com bin ed solution s were con cen trated. Th e product was
coevaporated twice with toluen e (20 m l), th en eth an ol (10 m l) an d 4-toluen esulfon ic acid
m on oh ydrate (50 m g) were added. Th e m ixture was sh ortly boiled to dissolve all solids an d
in ten sively stirred in an open flask at room tem perature for 24 h . Th e solven ts were evapo-
rated, th e residue was coevaporated twice with toluen e (20 m l) an d ch rom atograph ed on sil-
ica gel (50 m l) in a m ixture of ch loroform –m eth an ol (100 : 1). Th e glassy m ain fraction was
boiled with eth an ol to give 160 m g (18%) of crystallin e solid 12, m .p. >340 °C (from 320 °C
subl.), [α ]2D5 +53 (c 0.5, ch loroform ). IR (KBr): 3 415 (O–H); 1 041 (C–O). 1H NMR (200 MHz):
3.61 m , 2 H, W = 32 (H-3); 2.78 dd, 2 H, J = 15.8, 5.6 (H-15α); 2.59 dd, 2 H, J = 15.8, 10.2
(H-15β); 2.21 m , 2 H, W = 12 (H-12β); 1.01 s, 6 H (6 × H-19); 0.89 s, 6 H (6 × H-18). 13C
NMR: 163.84 (C-17), 155.51 (C-16), 71.21 (C-3), 54.83 (C-9), 54.72 (C-14), 44.99 (C-5), 44.11
(C-13), 38.13 (C-4), 36.72 (C-1), 35.80 (C-10), 34.12 (C-8), 33.56 (C-12), 31.86 (C-15), 31.58
(C-2), 31.45 (C-7), 28.45 (C-6), 20.77 (C-11), 17.56 (C-18), 12.32 (C-19). MS, m/z: 573 (M + 1).
For C38H56N2O2 (572.9) calculated: 79.67% C, 9.85% H, 4.89% N; foun d: 79.76% C, 9.91% H,
4.68% N.
5′′α-An drostan o[16′′,17′′-5′,6′]pyrazin o[2′,3′-16,17]-5α-an drostan e-3β,3′β-diol Diacetate (13)
an d 5′′α-An drostan o[16′′,17′′-5′,6′]pyrazin o[2′,3′-16,17]-5α-an drostan e-3β,3′′β-diol
3-Acetate (14)
To a stirred solution of diol 12 (1 g, 1.75 m m ol) in pyridin e (25 m l), acetic an h ydride (50%
solution in pyridin e; 0.6 m l, 3.18 m m ol) was added dropwise. Stirrin g was con tin ued at
room tem perature for 48 h . Th e m ixture was poured on ice, th e solid was filtered off,
wash ed with water an d dried. Ch rom atograph y on silica gel (100 m l) in a m ixture of ch loro-
form –eth yl acetate (50 : 1 to 30 : 1) gave successively diacetate 13 (70 m g, 6%), m on oacetate
14 (527 m g, 49%) an d recovered diol 12 (278 m g, 27%).
Diacetate 13: m .p. 342–343 °C (eth an ol), [α ]2D5 +42 (c 0.5, ch loroform ). IR: 1 724 (C=O);
1 261, 1 254, 1 025. 1H NMR (500 MHz): 4.77 tt, 2 H, J = 11.4, 5.0 (H-3); 2.77 dd, 2 H, J =
15.0, 6.4 (H-15α); 2.59 m , 2 H, W = 30 (H-15β); 2.21 m , 2 H (H-12β); 2.03 s, 6 H (CH3CO);
1.85 m , 2 H (H-2α); 1.78 m , 2 H (H-7α); 1.75 m , 2 H (H-1β); 1.73 m , 2 H (H-11α); 1.70 m ,
2 H (H-8); 1.67 m , 2 H (H-14); 1.62 m , 2 H (H-4α); 1.52 m , 4 H (H-11β, H-12α); 1.50 m , 2 H
(H-2β); 1.37 m , 2 H (H-4β); 1.35 m , 4 H (H-6α, H-6β); 1.23 m , 2 H (H-5); 1.05 m , 2 H (H-1α);
1.03 m , 2 H (H-7β); 1.005 s, 6 H (6 × H-18); 0.903 s, 6 H (6 × H-19); 0.85 m , 2 H (H-9).
13C NMR: 170.66 (C=O), 163.77 (C-17), 155.45 (C-16), 73.54 (C-3), 54.66 (C-9), 54.59
(C-14), 44.79 (C-5), 44.09 (C-13), 36.47 (C-1), 35.81 (C-10), 34.11 (C-8), 33.97 (C-4), 33.51
(C-12), 31.85 (C-15), 31.49 (C-7), 28.34 (C-6), 27.41 (C-2), 21.44 (CH3CO), 20.72 (C-11),
17.56 (C-18), 12.22 (C-19). MS, m/z: 657 (M + 1). For C42H60N2O4 (657.0) calculated:
76.97% C, 9.21% H, 4.26% N; foun d: 77.03% C, 9.35% H, 4.17% N.
Monoacetate 14: m .p. 312–313 °C (eth an ol), [α ]2D5 +47 (c 0.1, ch loroform ). IR: 3 437 (O–H);
1 738 (C=Oacetate); 1 247, 1 030 (C–O). 1H NMR (500 MHz): 4.70 m , 1 H, W = 32 (H-3); 3.61 m ,
1 H, W = 32 (H-3′′); 2.77 dd, 2 H, J = 6.5, 15.0 (H-15α, H-15′′α); 2.60 m , 2 H (H-15β,
H-15′′β); 2.21 m , 2 H (H-12β, H-12′′β); 2.03 s, 3 H (CH3CO); 1.007 s, 6 H (3 × H-18, 3 ×
H-18′′); 0.904 s, 3 H (3 × H-19); 0.888 s, 3 H (3 × H-19′′). MS, m/z: 615 (M + 1). For
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

On Steroids
1607
C40H58N2O3 (614.9) calculated: 78.13% C, 9.51% H, 4.56% N; foun d: 78.31% C, 9.65% H,
4.38% N.
3′′-Oxo-5′′α-an drostan o[16′′,17′′-5′,6′]pyrazin o[2′,3′-16,17]-5α-an drostan -3β-yl Acetate (15)
To m on oacetate 14 (80 m g, 0.13 m m ol) stirred in a m ixture of dich lorom eth an e (3 m l) an d
aceton e (3 m l) th e Jon es reagen t (0.2 m l, 0.36 m m ol) was added dropwise. After 10 m in th e
excess reagen t was destroyed with propan -2-ol (0.2 m l), aqueous saturated KHCO3 was
added an d organ ic solven ts were rem oved on a rotary evaporator. Th e aqueous suspen sion
was extracted with ch loroform (3 × 2 m l), th e com bin ed extracts were wash ed by aqueous
saturated KHCO3 an d water, dried an d th e solven t was evaporated. Boilin g with eth an ol left
55 m g (69%) of keton e 15, m .p. 330–331 °C, [α ]2D5 +55 (c 0.5, ch loroform ). IR (KBr): 1 738
(C=Oacetate); 1 713 (C=Oketon e); 1 250, 1 027 (C–O). 1H NMR (500 MHz): 4.70 m , 1 H, W = 32
(H-3); 2.78 m , 2 H (H-15α, H-15′′α); 2.60 m , 2 H (H-15β, H-15′′β); 2.02 s, 3 H (CH3CO);
1.092 s, 3 H (3 × H-19′′); 1.035 s, 3 H (3 × H-18′′); 1.008 s, 3 H (3 × H-18); 0.905 s, 3 H (3 ×
H-19). 13C NMR: 211.70 (C-3′′); 170.70 (C=O); 163.95, 163.61 (C-17, C-17′′); 155.61, 155.33
(C-16, C-16′′); 73.55 (C-3); 54.68, 54.61, 54.48, 54.26 (C-9, C-9′′, C-14, C-14′′); 46.75 (C-5′);
44.80 (C-5); 44.63 (C-4′′); 44.12, 44.08 (C-13, C-13′′); 38.21 (C-1′′); 38.08 (C-2′′); 36.49 (C-1);
35.98, 35.81 (C-10, C-10′′); 34.12, 34.04 (C-8, C-8′′); 33.98 (C-4); 33.52, 33.50 (C-12, C-12′′);
31.85, 2 C (C-15, C-15′′); 31.50 (C-7); 31.24 (C-7′′); 28.69 (C-6′′); 28.34 (C-6); 27.41 (C-2);
21.42 (CH3CO); 21.00, 20.72 (C-11,C-11′′); 17.56, 2 C (C-18, C-18′′); 12.22 (C-19); 11.47
(C-19). MS, m/z: 613 (M + 1). For C40H56N2O3 (612.9) calculated: 78.39% C, 9.21% H,
4.57% N; foun d: 78.46% C, 9.28% H, 4.41% N.
3′′β-Hydroxy-5′′α-an drostan o[16′′,17′′-5′,6′]pyrazin o[2′,3′-16,17]-5α-an drostan -3-on e (16)
Acetate 15 (100 m g, 0.16 m m ol) in a m ixture of ben zen e (10 m l) an d m eth an ol (10 m l) was
deacetylated with sodium m eth oxide (100 m g Na in 5 m l of m eth an ol; 0.3 m l, 0.26 m m ol)
at room tem perature for 24 h . Solid CO2 (about 0.5 cm 3) was added an d th e solven ts were
evaporated. Th e residue was extracted with ch loroform (3 × 2 m l), th e com bin ed extracts
were con cen trated an d th e product was purified on preparative TLC plates in a ch loro-
form –m eth an ol (10 : 1) m ixture. Boilin g with a m ixture of m eth an ol–water (100 : 1) gave
50 m g (54%) of keton e 16, m .p. 320 °C (dec.), [α ]2D5 +65 (c 0.5, ch loroform ). IR (KBr): 3 446
(O–H); 1 713 (C=O); 1 045 (C–O). 1H NMR (200 MHz): 3.60 m , 1 H, W = 32 (H-3′′); 2.78 m ,
2 H (H-15′′α, H-15α); 2.60 m , 2 H (H-15′′β, H-15β); 1.09 s, 3 H (3 × H-19); 1.03 s, 3 H (3 ×
H-18); 1.01 s, 3 H (3 × H-18′′); 0.88 s, 3 H (3 × H-19′′). MS, m/z: 571 (M + 1). For
C
38H54N2O2 (570.9) calculated: 79.95% C, 9.53% H, 4.91% N; foun d: 79.85% C, 9.38% H,
4.62% N.
We thank Ms L. Kloučková for excellent technical assistance. We are grateful to Dr I. Votruba and
his co-workers for cytostatic activity evaluation. We are indebted to Ms K. Matoušková for taking and
Dr P. Fiedler for interpretation of IR spectra. Our thanks are due to Ms M. Snopková for measurements
of 200 MHz 1H NMR spectra. Mass spectra were measured by the staff of the Laboratory of Mass
Spectrometry (Dr K. Ubik, Head). The elemental analyses were carried out in the Analytical Laboratory
(Dr L. Válková, Head). This work was supported by the Grant Agency of the Czech Republic (grant
No. 203/97/0695), in part by Ministry of Education, Youth and Sports of the Czech Republic (project
LB98233) and from COST OC D12.20.
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