STUDIES in the CHEMISTRY of ERGOSTEROL a Thesis Submitted

STUDIES IN THE CHEMISTRY OF ERGOSTEROL

A thesis submitted by

ANTHONY GERARD MARTIN BARRETT

in partial fulfilment of the requirements

for the degree of Doctor of Philosophy of London University.

Department of Chemistry, August 1975

Imperial College, London SW7 2AY. •ir CON=NTS

Introduction 1.

Synthesis of Cholecalciferol Metabolites 9.

Structural Elucidation of the Toxisterols 45. s

An Investigation of Steroid Organoiron Chemistry 84.

Experimental 3.08.

References 186.

Publications 204.

•

• I am indebted to Professor Sir Derek H. R. Barton

for his help, encouragement and 2atience dnring. this work.

I also thank Dr. David A. Widdowson for his cosupervision

and friendship. I am grateful to Dr. Richard A. Russell

for investiating the role of solvent in ergosterol

photolysis and isolation of toxisterols B and B ; 2 21 22 13 Dr. Lawrence Phillips and Martyn H. Pendlebury for C n.m.r. • measurements and assignments; Dr. Peter F. Lindley and

Professor C. H. Carlisle for the X-ray crystallographic

study of toxisterol2 B21 3,5- dinitrobenzoate.

I thank my colleagues in the Hofmann Laboratory for

helpful discussion and a drink or two.

• •

ABSTRACT

Photochemical and thermal pathways in the preparation

of ergocalciferol (1) from ergosterol (4) are outlined.

The isolation and syntheses of hydroxylated analogues of

calciferol, important for their biological activity, is

reviewed. •

Photofragmentation and toxisterol formation during the 2 photolysis of ergosterol is described. A series of new

compounds have been isolated and their structures elucidated.

A novel dimerisation reaction of allylic bromides,

benzoates and trimethylsilylethers on reaction with iron

carbonyls is described. Both ergocalciferol iron

tricarbonyl (210) and preergocalciferol iron tricarbonyl

(208a) have been prepared photochemically.

• •

INTRODUCTION

Ergocalciferol or vitamin D2 (1) is important in man

in the prevention of rickets. It is unique amongst

vitamins in being toxic in excess causing calcification of

soft tissue. Recent results have shown the active metabolite,

formed by hydroxylation in the liver and kidneys, is the

, 25 - dihydroxy-derivative (2)

2t3

21 22 2.4 27 1y13.--'2° 12 23 215 17 26 a

HD- Ha'

1 2

•

Cholecalciferol or vitamin D3 (3) is also an antirachitic

compound, differing from ergocalciferol (1) in 7Jossessing

the cholesterol side chain.

1 •

•

Early work in the elucidation of photochemical and

thermal pathways in the conversion of ergosterol (4) into 1 ergocalciferol (1) has been reviewed. However the structures

of the known photoProducts of ergosterol (4) (or where data

is lacking of 7-dehydrocholesterol (5) ) are here summarised. • 6) results in • Irradiationofergostero""orlunlisterol,( the ring B diene opening in the symmetry allowed conrotatory ** fashion 7ivin:- Preergccalciferol (7).

Footnote:

* The prefix ergo- or s•ffix -2 denotes compounds in the the ergocalciferol (1) series and analogues in the cholecalciferol (5) series by chole- or -3.

** 1 ere atDrlicable the preferred conformation in solution is illustrated. s

HO i

5(C81-117 side chain) •

h))

Preergocalciferol (7) is the central molecule in subsequent

transformations. The thermally allo-,:ed 1, 7-antarafacial

hydrogen shift gives ergocalciferol (1) in an equilibrium

process. Thotochemical conrotatory cyclisation of

preergocalciferol (7) rives both ergosterol (4) and

11.1misterol (6); tliermal ) disrotatory cyclisation

gives pyroergocalciferol (?) and isoT)yrocrgocalciferol (9). Both

9,10 syn ring 3 dienes ( ) and (9) undergo photochemical

- 3 - • disrotatory cyclisation giving photopyroergocalciferol (10)

and photoisopyroer,7;ocalciferol (11) reol)ectively.

•

8 10

9 11

The formation of the cyclobutenes (10) and (11) is thermally

reversible presumably in a non concerted fashion. • Irradiation of cholocalciferol (3) gives suprasterol -I 3 (12) and supnenol,-II (13) as the major products formed 2 by 4115 2iii; cycloadditions.

•

12 13

•

Also two vinylallenes (14)) (15) an: two vinylcyclobutenes (16)) (17) have been isolated. The vinylcyclobutenes (16),(17) on

heating open in the conrotatory fashion giving 5, 6-trans-

cholecalciferol (18).

OH • OH

14 15

HO• R3

R3 - C H - 8H17

- 6 - • Genertion of the triplet state during photolysis

rives rise to cis/tr:Ins isomerisation or dirnerisation.

Preergocalciferol (7) gives reversibly the oxygen labile

tachysterol7 (19), so called because of its rapid reaction

with dienophiles. Ergocalciferol (1) reaches equilibrium

with 5, 6-trans-ergocalciferol (12). three 7-7 dimers

(20), (21) and (22) are formed on the triplet sensitised

• irradiation of 7-dehydrocholesterol (5).

HO OH

21 22

C9 H 17

- 7-

• Lewis acid treatment of ergocalciferol (1),

preergocalciferol (7), 5,(.-trans-7 ei7ocalciferol

or tachysterol2 (19) gives isoergocalciferol (23) and

finally isotachysterol2 (24). Isotachysterol reaches 2 4 equilibrium with cis-isotachysterol2 (25) on irradiation

presumably via the triplet state.

23 v 24

•

SYNMESIS Cif CHOL:',CALCIFEROL NTETA3OLI?ES

There is a time lapse after ingestion of cholecalciferol

(3) prior to its influence on calcium metabolism. This

results from hydroxylation in the liver and kidneys to give

104, , 25-dihydroxycholecalciferol (26)5 the hormone controlling

intestinal calcium transport and bone calcium mobilisation.

Although catabolism of cholecalciferol is unknown, pathways

in metabolite production have been elucidated and are

summarised in Scheme I. In addition both 25-hydroxyergocalciferol

(32) and 1v_ , 25-dihydroxyergocalciferol (2)5 have been

isolated. The biochemical, Physiological and clinical

properties of cholecalciferol (3) and its metabolites have 9, 10 been extensively reviewed. •

a./

•

Ha" - HO- OH

32 2

- 9 - •

SCHEME 1

• LONG CHAIN FA T T Y ACID EST ERS7

•

• •

6 27 2 65 30

_ 10_ a

Synthesis of hydroxylated derivatives of cholecalciferol

was ncessary to confirm structural assignments of isolated

metabolites and for the study of structure/activity

relationships and clinical application.

140( -7:ydroxylation

The 1t,e,l_ -hydroxyl substituent has been introduced by 1 11 oxidation of A precusors. Barton et al prepared

cholest-5-ene-1 , 35-diol (34) from cholesta-1,4,6-

trien-3-one (35) by alkaline hydrogen peroxide epoxidation

and subsequent reduction with excess lithium and ammonium

chloride in THY/ammonia. The mechanism outlined was suggested

by the authors.

HO - 45%

33 35

T. H. F. INH 3 HO Diacetylation, allylic bromination with N, N-

dibromoAimethylL.ydantoin follo-Jed by dehydrobromination

with trimethylphosphite in xylene gave cholesta-5,7-diene-

1 Q( , diacetate (37). Photolysis, thermal

equilibration of the Irevitamin (33) and saponification

gave 19( -hydroxy-cholecalciferol (27) (m.p. 132-133°,

/1(...71)=. + 26° (Et20) ). The product (27) has been 12 successfully applied in biolocical experiments.

(Me0)1P xylene AcO -Br AcO 37

AcO

88 27

D.D.H.

4 * Footnote Formation of the 0 '6 compound always A 5,7 accompanies LA in nalogenation/dehydrohalogenation.

- 12 - •

Israeli chemists ' have reduced the epoxydienone (36)

in THE/ammonia by repeated reaction with aliquots of

lithium and ammonium chloride to give cholesterol (33),

cholest-5-ene-10,C. , 31p-diol (34) and cholest-6-ene-10c

*-diol (39). Diacetylation)bromination and dehydrobromination

of (39) have 4'6-diacetate (40) and 5'7-diacetate (37).

The authors claimed preparation of but did not characterise

lc< -hydroxycholecalciferol (27) from (37).

Ac0

1 Ac 0, pyri dine 2 HMPA, Et3 NMe 2 Br 2 (Me()) PO- HO Ac0 'Br 2

•

Ac0 Ac0

37 4 0

- 13 -

•

The lc< -hydroxyl has also been introduced by hydroboration/

peroxide oxidation. However tlais h s the disadvanta::e in

giving both lc< -and 2c( - hydroxy substitution. Japanese 14 workers have prepared but failed to fully characterise

1o(.. -hydroxycholecalciferol (27) from cholest-1,4-dien-3-one ('1). Cholest-1,4-lien-3-one (41) was deconjugated with potasium t-butoxide in miso to give cholest-1,5-dien-3-one

(42 ). Sodium or calcium borohydride reduction followed by

hydroboration/oxidation gave cholest-5-ene-loc, 3p-diol (34)

and cholest-5-ene-2ci, , (43). Diacetylation, allylic

bromination, dehydrobromination and lithium aluminium hydride

reduction of (34) gave cholesta-5, 7-diene-104 , 3p-diol (44).

Subsequent -chotolysis and thermal equilibration gave

lc< -hydroxycholecalciferol (27).

o 1 KOBti! DMSO NaBH4 , Me OH, 0

2 H2O or Ca(BkI .Et014,-10o 2 42

41 42

HO HO 1 B2 i-/6' TH F

2 1-I .H0- HO 2 02 HO

43 34

1 Ac20, pyridine

2 D.D.N. 27 3 (Me0) P, xylene 3 HO 4 HAIN 0 4' Et2 44 - 1 - • By the same route coolest-5-ne-20( , 3p-diol (43) was

converted into 2o( -hydroxycholecalciferol (45). The

'oroduct (45) obtained as an oil was consistent with n.m.r.

and u.v. spectra only; no other data being reported. 14

•

• HO" -

Deconjugation of cholesta-1,,G-trien-3-one (35) and 1 5,7 borohydride reduction gave the -3p-alcohol which

was trapped with 4--ohenyl-1,2,4-triazoline-3,5-dione. S a Peracid eDoxidation and subsequent lithiUm aluminium

hydride reduction gave cholesta-5,7-diene-10( ,3p-diol (44).15

-15 - NaNN: KOBut. DM SO 2 1420 3 Ca(BH4)2 HO

1 3-C1C 144 CO3 N 2 LiA1H4,THF HO HO

N 44 Ph

16 nirst et a1 described the preparation of

cholest-5-ene-1 aC , 313 -diol (34) from cholest-1-en-

6 - 01-3-one (46) usin the 6p alcohol as a masked La 5

system. Epo::idation and subsequent borohydride reduction

of (46) gave diol (47) -eyich was selectively acetylated

(the 6p -alcohol being hindered by 19-methyl 1,3-diaxial congestion) giving (V). Dehydration followed by lithium

aluminium hydride reduction gave cholest-5-ene-lc< , 3(3 -diol (34).

- 16 -

Transformation into 1*.hydroxycholecalciferol (27) was carried out as described before (N-bromosuccinimide being used for bromination and 2,4,6-trimethylpyridine/ xylene for dehydrobromination),Fiirst obtained (27) as a crystalline solid (m.p. 138-139.50 , EtKjD +290 ( Et20 )).

1 H202, NO-

2 NaBH4

46 47

6 steps 34 27

Japanese workers17 have prepared cholest-5-ene-].o(, 3(A-diol (34) from cholest-l-en-6e-o1-3-one acetate (46, acetate) by a similar route. Lythgoe and coworkers have prepared lck-hydroxycholecalciferol (27) by total synthesis18. Lactone (49) on ik-epoxidation and subsequent base cleavage gave diol (50) which was reduced to aldehyde (51). Wittig reaction with

- 17 - 9H 1 Ac20, pyridine

1 3-CI CAC 0314 2 (COCI)2,benzene t)3 2 Na0Me, Me0H 3 LiA1140Bu , 02 H

49 50

OAc QH

1 CICH=PPh , Et 0 3 2 2 NaNH NH 2 ' NH3 CHO

51 52

chloromethylenetriphenylhosrhorane and subsequent

dehydrochlorination gave acetylene (52). Saponification,

trimethylsilylation and treatment with n -butyllithium gave lithium acetylide (53)

1' - •

C8H17 IP\

H2 Lindlar's catalyst

quinoline

AcO 55 56

17 QAc

1 L 27 2 Zemplen methanolysis AcO

Condensation with chloroketone (54) gave enyne (55).

After TMS ether hydrolysis and acetylation chromous

perchlorate reduction gave dienyne (7,6). Hydrogenation

of (56) gave ID:, -hydroxyprecholecalciferol diacetate (57)

which on thermal equilibration and Zemplen methanolysis gave

10( -hydroxycholecalciferol (27). The authors reported 250 (27) as a white solid (m.p. 15b-6°, 7D25C = + 2.?.° ( Et 2D) )

19 - 19 K. odicek and 72e1c have claimed to have prepared

cholest-5-ene-10( , 3(3 -diol (34) from epoxydiol (47)

via monomethanesulphonate (5C) but their reported data 11 for (34) has been shown to be incorrect. That the

enoxide ring of (5) was acid stable must be open to

question.

Pyridine Q, 1 A c20' 2 MeS02C1, pyridine

3 KOH,MeOH HO

47 58

LiAl H4

34 HO

Cholest-5-ene-1 o< , 3 13 -di ol (34) has also been

• prepared by photocyclisation of 5,10-secosteroid (59).20

59a 59 b - 20 -

hi)

dioxan Ac0

1 9

34 25-Hydroxylation •

The synthesis of cholest-5-ene-3 V , 25-diol (60) from

s iHmasterol (61) protecting the A 5 system by 21 i -steroid (62) formation has been described.- Czonolysis

of (62) and subsequent reduction gave 22-alcohol (63).

The derived 22-toluene-41-sulbhonate (6 was very

susce:Aible to nucleophilic attacl: and gave

- 21 -

• coolest-5-9ne-3 p ,25-diol (60) on reaction with

li thio-2-methylbut-3-yn-2-ol followed by hydrof7enation

and acid hydrolysis.

4-MeC 1 6 H45SO CI, 03,-78°, pyridine, Fi pyridine cH2c12

2 Me0H , pyridine 2 NaAIH2(OCH2CH2OMe) OMe benzene 61 62

OH OTs 4-Me C6H4S02C1,.., •petrol , dioxan ,HMI=1 • ..." pyridine Li-CCCM e2OTHP

63 64

• • 60

Cholest-5-ene-3(1 ,25-diol (60) has also been prepared

from 3 p -hydroxypregn-5-en-20-one (65) protecting the -

5 system by i'-steroid formation.22 The side chain

was assembled by Grignard reaction followed by Carroll

rearrangement.

- 22 - =\higc

Et2 0 1-10 65 2 Me0H, pyridine OMe H

Off—rd eca 1 i n Et H2 ' 0 Pt02 ' 0H s-collidine 121

6 0 (25%overai I:)

Oxidation of desmosterol acetate

3 -ol acetate) with 3-chloroperbenzoic (cholesta-5,24-dien-3 1 23 acid and subsequent lithium aluminium hydride reduction 24 or hydroxymercuration/borohydrie demer6uration have

been used to 'repare cholest-5-ene-3p ,25-diol (60).

- 23 - • )25,29 (67)2,26 Grignard reaction on Ictones (66

and esters (68)27 and (69)27 have been used to introduce

the 25-hydroxyl group.

AcO 66 67

1 4-MeC 6H4SO2CI ' O2Me

pyridine PhH, petrol

2 PhH ,ET, CH 2 (M e 0)3P xy lene AcO 3 AgO2CPh,Et3N Me0 H CO2Me

• MeMgBr

E t20 AcO 68

1 tn.)

2 6, AcO Photolysis of cholestanol acetate (71) in 69

peracetic acid gave the 50(, 25-diol derivative.

AcO

- 214 - 25,26 DeLuca et al have converted choThsta-5,

7-diene-3p ,25-diol into 25-hydroxycholecaloiferol (2P) but only cited a mass ion measurement and qualitative ultraviolet spectrum in evidence. Campbell, Squires and 27 Babcock isolated 25-hydroxycholecalciferol (28)as a crystalline hydrate (m.p. 81-3°) and satisfactorily characterised it. S. J. Haltes and N. F. Van Vliet29 reported 25-hydroxycholecalciferol (28) as a white solid

(m.p. 95-100°).

The synthesis of 1 0( , 25-dihydroxycholec lciferol (26) from cholest-5-ene-3p ,25-diol (60) introducin: the lo4. - 1,4'6 hydroxyl by epoxidation of the derived A - 3-one has been described.30

HO H 1 D.D.Q. 1 Ac20 .pyridine 2 H202, HO- 2 D.D.H. A 3 Li , NH3, N H4C1 3(Me0)3P, xylene HO HO 60

HO 26

-25 - The authors reported lc< , 25-dihydroxycholecalciferol (26)

as a white crystalline solid (m.p. 84-Pe, /-coe, 7 + 0 ( Et 1 29 20) ). DeLuca and coworkers' claimed the synthesis of lc< , 25-dihydroxycholecalciferol (26).

At best their synthetic methodology is tedious and unoriginal chemistry: the intermediates and final product were not characterised. The 25-hydroxyl was introduced by Grignard reaction on a bis nor precusor and the 5,6-double bond was protected via 6 -ethylene ketal formation.

OMe

1 Ac20, pyridine 2 HNO3 1 MeMgBr

3 Zn HOAc , H20 2 Cr03 pyridine AcO CH OH 4 HOCH2 2

OH 1 Br2,CCI4 2 CaCO3 , DM F 3 H 202'HO- HO" 4 LiA11-14,E9J

- 26 -

9H H9

NaBH4

iso-propanol H

o 1 Ac 0 pyridine,90 2 2 H30+

3 N a BH4 , iso-propano I AcO 4 POCI3 , pyridine

1 D.D.H.

2 (Me0)3P xylene

3 hi) 4

HOB/ 5 KOH , Me0H 26

DeLuca et al claimed the synthesis of 10< -hydroxycholecalciferol 32 (28) by a similar route. 4

Japanese workers 3 have 17repared cholest-5-ene-

1c< , 3(a , 25-triol_ (73) from desmosterol acetate (74)

prepared by degradation of fucosterol acetate (75) (a sterol

from brown algae). The 25-hydroxyl was introduced by

hydroxymercuration/borohydride demercuration and the 11 1 0Z, -hydroxyl by Barton's -nroce',ure.

- 27-

1 03 , CH2C12, -78° 2 NaBH4, Me0H 3 P2O5, PhH

75 74

1 Hg (0A02 T H F H2O 2 NaBH4 , NaOH H2O

1 DD Q 2 H202 . 1-10-

3 Li , NH4C1 , NH3 ,T HF HO 73

The conversion of cholest-5-ene-3 , 25-diol (60) into cholest-5-ene-1 of , 3 ( , 25-triol (73) has been described by Japanese3/4 and American35 chemists. Both groups introduced the 1 a( -hydroxyl protecting the 5 system as a 6 i3 -alcohol, selective oxidation of the 3 r) -alcohol

-bromination/aehydrobromination and epoxidation.

THP 1 B2H6

2 H202 ,H0-

3 Cr03 , pyridine THP0 THP

NaBH4 , Me0H

pyridine 1 Bcz 1-10Ac HBr, 2 Ac20 , IH 3 HC1 Me0H 2 CaCO3 DMF

4 Cr03 , pyri dine , dichloromethane OAc

H Ac OAc

pyridine

1 H2 02 , HO

2 Na B1-14 , Me OH , Et20 Ac20 , pyridine

3 Na OH , Me0H

1 POC13,PYridineA c0

2 LiA1H4 1 Hg(0Ac)2,THF.. 73 3 Ac20 2 NaB1-14,Na01-(,? AcO AcO Me0H

- 29 - Uskokovic and coworkers'obtained triol (73) in

overall yield from cholest-5-ene-3 , 25-diol (60).

1 Br2 dioxan

1 N aBH4,methanol Li Br Li2CO3,211v1F.

2H301- 3 H202,E10-

4 MeS02CI pyridine

Al/ Hg ethanol , water, 73 NaHCO3, -15°

• 7 6 oso2me 7 7 a SO2Me The intermediate epoxide (76) .,Tas cleaved by reduction

:pith- aluminium amalsam sivin;7 triol mono-methane.sulphonate (77 ).

Other Hydroxy-Derivatives

DeLuca36 reported the synthesis of cholest-5-ene-

3p , 24, 25-triol (78) from ketone ( :SG) and its conversion

to 24, 25-Jihydroxycholecrilciferol (29).

- 30 - •

OAc

2 Na I benzene,ethanol AcO 3 AcOH' Me CO KH CO3 A 2 66

• fAc

MeMg I , Et2.0

PhH 78

6 steps OH

HO' 29 Kodicek37 also claimed to have synthesised but failed to

characterise 24, 25-dihydroxycholecalciferol (29) using

desmosterol acetate (74)as starting material.

- 31 • 38 Recently the configuration at C-24 in

cholest-5-one-3 19. , 24, 25-triol (78) has been assigned

by application of the modified Horeau procedure.39

Both 24R - (294 and 24S - (29b) 24, 25-dihydroxycholecalciferol

have been prepared by standard routes.38

78 29 (a) R = OH, 131 = H ( b ) R = H, = OH

Cholest-5-ene-104; , 3(3 , 24R, 25-tetraol (79a) and its 40 11 24R-epimer (796) were prepared using Barton's procedure

and converted into the corresponding cholecalciferols (30a)

and (30b). The products ho.;:ever were not fully characterised.

- 32 - HO

78 •

6 steps

• HO-

Both 243- (20a) and 243- (Mb) 24-hydroxy,:holec:_Jciferol 41 have been prepared from cholest-5-ene-3

•

(a) R = OH, (b)R= H, RIjOH 6 steps

- 33 - a The products were not fully cin.racterised.

Cholest-':.-ene-3p , 25, 26-triol C1) and 43 25,26-dihydroxycholecalciferol (31) have been prepared

introducin: the sire chain dial by osmium tetroxide

oxidation of cholest-5, 25-dien-3 -ol'acetate (`'2).

1 Hg(01102. THE

2 NaBH4' HO- 3 POC/3 AcO

74 82

THP

Os04. Et2o,

6 steps a Pelc has claimed the ':Dreparation of 4c4. -hydroxycholecalciferol

('3) from cholest-5-ene-3 r , 11-c< -diol diacetate (54)

but was unable to effect dehydrobromination of the allyl

bromide (35) and thus prepare 4 p -hydroxycholecalciferol

(86). The product (83) was characterised by qualitative

ultraviolet sectrum and mass ion measurement only.

Ac0

•

-35- • Although 22S-hydroxycholecalciferol ("7a) has not

been pre-nared 22C-hydroxyer7ocalciferol (87b) is available

from epoxidation of ergosterol adduct (88).

87b , a =cholesterol side chain

In order to study the bioninctionof the 3 -hydroxyl

it was necessary to 1)repare 3-deoxy- analogues of the 46,47 metabolites. Both 14( -hydroxy-3-deoxycholecalciferol ("9a) 48 and 10 ,25-dihydroxy-3-deoxycholecalciferol (89b) have been

pre'oared. Deoxygenation at C-3 was carried out by selecti7e

toluene-4-sul7honylation and lithium aluminium hydride 46 Y: reduction ' or by reduction of 3-ketone (90).'

- 36- •

R HO H9

4-MeC6H4S4 pyridine

2 LiA I H4 , Et20

•

6 steps (a) R H (b) R = OH

1 N2.1-14. H2.0).

2 H2!Pdi C a

1 H30+

2 Ac2.0 , pyridine

3 NaBH4

4 POCI3 , pyridine

- 37 - 49 h Okamura and coworkers ave prepared 3-deoxy-A-homocholecalcifcrol

(91) ring exp:Anding cholest-6-en-3-one (92) with diazomethane.

H 1 CH2N2 1 D.D.H.

h 2 NH2NH2 2 04e0)3P, 3 KOH xylene H H

-'he 19-substituted cholecalciferols (93) and (94) ha-re

been prepared by Olotolysis of cholesta-5,7-diene-3 ,

19-diol diacetate (95)50 and reaction of cholecalciferol (3)

with singlet oxygen in methanol' respectively.

Ac0 1 1 Cr - tyridine Lill, PhH, H CH2C12 Ac0 2 H2NI'NTS02 Ac0 NNHSO

•

95 93

11111!!" Na0C1, H24q, 01,1e Me0H, 0-56P" ONe 11111 OMe

HO 116' 3 94 In order to study biological activity synthesis of

isomers of 25-hydroxycholecalciferol (r.) was undertaken.

25-Hydroxyisotachysterol3 (96)52 and 5,6-trans- 25-hydroxycholecalciferol (97)" were -oreDared from 25-hydroxycholecalciferol (2S).

-39- •

96 28 97

25-Hydroxytachysterol (9?:) was obtained from photolysis

of cholesta-5,7-diene-3 ,25-diol (99). Subsequent

hydrogenation gave 25-hydroxydihydrotachysterol (100).54 3

•

- 40 -

DeLuca et al 55 claimed (on the basis of qualitative

u.v. data only) the synthesis of calciferols (101a),

('101b), (101c) and 5,6-trans-calciferols (102a), (102b),

(102c) and have studied their biological activity.

(1) D.D.H. H

(2) P(OMe) xylene ,Et 0 3' 2 2 LiA1H Et 0 petrol (3) 4' 2 (4) by

101 • (a) R' = COMe (5)A

(b) R' = Me--CH(CH ) COMe (c) RI = Me--CH(CH ) CO Me 2 3 2 3 2 R

• 102

(a) R = CH(OH)Me

(b) R = Me--CH(CH )CH(OH)Me 23 (c) R = Me--CH(CH ) OH 2 4

- 41 -

Japanese workers have pre:ared 56 and fully

characterised androst-5-ene-1 0< , 3 -dio1-17-one (103), androst-5-ene-2c4. , 36 -diol-17-one (104), pregn-5-ene-

10k , 3 0, -diol-20-one (105) and preg.-5-ene-20()3i1 -diol- 20-one (106) but have not yet converted these into the calciferol analogues.

•

11 DNSO H (2)H2 0, 0°

(3) Ca(BH ) 4 2 (4)B2 H6, THF (5)HO, H202

0 0, too:COMe♦ XI = X:D 0) x =

10(-HO 103 105 ax-Ho 104 106

- 42 - a Biological Activity

Norman and coworkers' have summariaed'the structure/

activity relationship of calciferols in their "blueprint":

1. A lac-hydroxyl substituent or its pseudoeauivalent

(for example the 3p. -hydroxyl in 5,6-trans-cholecalciferol

(107) or 25-hydroxyisotachysterol3 (10:3) is required for

activity in anephric animals. •

2. Active analogues do not require a hydroxyl located in

what geometrically corresponds to the 3/3 -hydroxyl. These

include lc< -hydroxy-3-deoxycholecalciferol (89a),

25-hydroxydihydrotachysterol (100), 5,6-trans-

cholecalciferol (107) and isotachysterol (10 ). 3

3. Active analogues do not require a 19-methylene (for example isotachysterol (108) ). 3

4. The lo< -hydroxyl must occupy equatorial as opposed to

axial orientation for optimisation of biological activity.

That ring A of calciferol exists in two conformations in

solution has recently been demonstrated. 58

•

107 108

- 43- • The followinc ad:Iitions have been made to the

"blue print": 14 1. The 2 -hydroxyl is inhibitory. 44 2. The 40, -hydroxyl is inhibitory.

3. 24R, 25-Dihydroxycholecalciferol (29a)

is more active than its 24S epimer (29b).57

• 4. 26-nor-25-hydroxycholecalciferol (102b) and 26,27-bis-nor-25-hydroxycholecalciferol

(102c) are less active in intestinal calcium

transport and bone calcium mobilisation than

25-hydroxycholecalciferol (28). Omission of

the side chain results in total loss of activity.55

•

-44- a STEUC=AL ELUCIDATION CF THE TOXI3TEROLS2

In addition to the photoisomers of ergocalciferol (1)

described in the introduction several ill defined compounds

formed during the photolysis of ergosterol (4) or ergocalciferol

(1) have been reported. However their structures, mode of

formation and biological activity have hitherto remained unknown.

• The apparently most important of these elusive compounds were

the toxisterols2. Overirradiation of ergosterol (4) has been

reported to give compounds characterised by a chromophore at 59 60 1 250nm ' , toxicity6 and an ability to raise serum calcium • 1 2 levels. Westerhof and Keverling Buisman isolated three I crystalline compounds "toxisterols A, A and B" but were 2 unable to suggest structures. Barton and coworkers6 isolated

two compounds "toxisterol diene I" and "toxisterol diene II. 2 • 2 "Diene I" was shown by degradation to have structure (109).

• 109

Ozonolysis of "diene I" gve the ketoacid (110) and the

ketone (111). Ketone (111) gave the azobenzene (112)

with 2,4-dinitrophenyLhydrazine,3-methylnhenol toluene-

-Sulphonate (113) with toluene-4-sallthonyl chloride,

_ 145 _ • tribromourcinol (114) with N-bromosa ccimmide and

3-methylphenol (115) with acid or base.

• 110

N.B.S.

111

or HO- -11oC6114SO2C1 114 3 112

OH 0502

However structure (109) is unattractive since its formation

involves a questionable methyl migration. Structure (116)

was suggested for "toxisterol2 diene

116 • We ha7e foun:-1. that the irradiation of er-ostcrol (4)

in quartz with a high pressure mercury arc. lamp in

methanol, ethanol or iso-pronanol solution gave a mixture

of compounds with an intense chromophore which absorbed at

242,252 and 260 nm. The yield of this material was

increased on irradiation in the presence of citric acid.

However irradiation in cyclohexane or diethyl ether failed

to produce the clromonhoric species. Significantly photolysis

in t-butanol resembled that in cyclohexane or diethyl ether

except in the presence of water when the 242,252, 260nm.

absorption was aF7ain obtained.

Irradiation in pyrex or with a medium pressure mercury

arc lamp gave similar results but longer photolyses times

were required. All photolyses were conducted at room

temperature to minimise formation of ergocalciferol (1)

and products derived therefrom.

Ergosterol (4) was irradiated in ethanol containing

5 water and 0.4% citric acid (Fig.I). The course of the

reaction was followed by the decrease in the ergosterol

chromophore and concomitant formation of the absorption at

242, 252, 260 nm. The product mixture was reneatedly

chromatographed on alumina to gi7e (in order of increasing • polarity) fractions Ai, A2, B1, B2, B3/34, C, D and E.

The complexity of the photolysate is illustrated by

ig. II - VITI.

-47- • • •

FIGURE 1 PHOTOLYSIS OF ERGOSTEROL IN ETHANOL

Before photolysis

8hours 40minutes e

c 2,6hours 15minutes ban bsor A

I I I I I 26 0 225 250 275 300 325 350 400nm. •

FIGURE II FIGURE III Ergosterol/Ethanol' Fraction A crude photolysate EtOAc ; petrol 1:3, ;2.:9"

B1 B22 B31/B32 B

D11 D12/D13

FIGURE IV FIGURE V

Fraction A,, developed twice Fraction A21 1O%AgNO3/Si02 developed twice

PhH : Et 220 petrol = petrol = 3 : 197

1 : 19 221 ------A • 222 • 223 %.%" A224 A A111 225 A A112 226 All3 A114 A115 Al2 A13 FIGURE VI FIGURE-VII Fraction B1 Fraction B /B benzoate 3 4 Developed thrice 10%AgNO3/S102, developed twice 2 Et20 PhH : petrol = :c petrol = 1 : 9 7 : 13

Bu. B31 benzoate BI2 B13 B benzoate 32 Bbenzoate4

".."1 •••=ile

FIGURE VIII

Fraction11-D12 325-dinitrobenzoate Developed thrice

3,5-dinitrobenzoate -1

D11/D12 325- dinitrobenzoate-10 12

Note all t.l.c. carried out on silica unless otherwise stated. 0 PHOCF-2AGNENTA"'TC. T'TZC7-)7'

Preliminary results suggested the Al fraction consisted of 65 a mixture of hydrocarbons. Argentation chromatography

gave eight compounds (in order of increasing, polarity)

A111, A112, A113, A114, A1151 Al2, A13 and A14. Spectral and analytical data (see Experimental) suggested the

compounds had the following structures in which the

steroidal C and D rings remained intact: Ai12 (117), A114 (118), AI15 (119) and A13 (120).

117 11 8

120

The remaining compounds A A were either 111' 113' '12 and A14 formed in too small an amount to warrant isolation or were

inhomogeneous. Structures of the photofragments (117),

(118)(119) and (120) were all confirmed by synthesis

(see below).

- • Oxidation of er:,ocalciferol (1) in benzene with

potassium permanganate in aqueous sulphuric acid gave

Grundmann's ketone (121)65isolated by chromatography on

alumina. Since Grundmann's ketone (121) was easily 66 epimerised at C-14 giving ketone (122) recovery from

its semicarbazone (123) was not possible. The -parent 67 cis-hydrindan-1-one is more stable than its trans-isomer.

0 1 21

NH2NHCONH2, CH2 (0Me)2, Hcl Me0H or Me0H NaNO2' HOAc

NNHCONH2 123

Treatment of Grundmann's ketone (121) with

methylenetriphenylphosDhorane - in THE gave diene (120)

in 74: yield. Formation of the exocyclic methylene

compound (120) was con:T:istent with the infra red s7ectrum

-49- • (887cm '-1), n.m.r. (T 5.27, 5.57 (2H, ABq, J=2Hz) ),

mass spectrum and analysis. The product was identical

in all respects with 43. An attempt was made to prepare

a crystalline derivative of diene (120). Arylnitrile

oxides function as 1,3-dipoles and are used to prepare

isoxazolines from olefins and isoxazoles from acetylenes.

Functionalisation of the 23-methylene in (124) by 69 1,4,6-trimethylbenzonitriIe oxide has been reported. • Reaction of diene (120) with 2,4,6-trimethylbenzonitriie

oxide gave a single compound probably isoxazoline (125).

Since the compound could not be induced to crystallise it

was not examined further. However the structure formed

from 0‘. attack was consistent with the low field 1404. -H

triplet at 7.16 in the n.m.r. spectrum and steric

considerations.

•

- 50 - •

•

Sodium borohydride reduction of Grundmann's ketone (121)

gave the e-,:pected -alcohol* (126) as the major (84) product.

Presumably the minor product was the 80( -alcohol (127).

T.l.c. indicated that ebimerisation prior to reduction did not

take place. Formation of the 8 p- alcohol (126) was

• consistent with the shift of the 18-methyl to lower field in

the n.m.r. spectrum (1: 9.32 - 9.04) on reduction. The W H

value (6Hz) of the 8-hydrogen was consistent with it being 70 equatorial. Esterification gave the known 3,5-dinitrobenzoate

(128).71

•

* Footnote: Steroid numbering is retained for the photofragments.

51 Dehydration of alcohol (126) with methane sulphonic anhydride in pyridine gave diene (118) as the exclusive product isolated in 82;:. yield. The structure was consistent with spectral data, analysis and was the expected product from the favoured trans-diaxial

A -IL was identical with the synthetic elimination. 1.1. 4 material.

126 R = H 127

128 R = C 8

72 Xanthates (via the Chugaey reaction) esters73, 74 76 amine N-oxides 1 sulehoxides75, and selenoxides etc., on pyrolysis give the olefin formed by svn elimination.

Since xanthate (129) was readily prepared from the V - alcohol (126) its pyrolysis was investiated. Pyrolysis at 250° for 5 hours gave diene (119). Since sulphur by products were eresent silver nitrate chromatography was required to obtain eure (119) in 57 yield. Formation of the cis disubstituted olefin was consistent with the infra red spectrum (675(S.) ) and the 18-methyl (I.,- 9.30) and vinyl signals (1C 4.43) in the n.m.r. spectrum.Diene (119)

was identical with A115.

- 52 - •

SMe 129 Isomerisation of diene (120) with sulphuric acid in

aqueous THF gave the more stable tetrasubstituted olefin (117)

in quantitative yield. The structure of the product was

consistent with absence of vinyl signals in the n.m.r. and A 22 infra red spectra (other than those of the system),

a downfield shift of the 18-methyl ( 1; 9.43-9.13) and the

presence of a vinyl methyl ( I; 8.48). The optical rotation o 804) decrease (+48 - +120) also supported A formation

(since diene (118) has a low positive rotation). A.112 was identical with the synthetic diene (117).

•

120 117

- 53 - • It was necessary to synthesisokene (130) since it

was thought Ala may have had this structure. Enimerisation 66 of Grundmann's ketone (121) gave ketone (122) which on

sodiuM borohydride reduction gave two alcohols (131) and

(132). It was possible to separate the isomers by p.l.c.

and they were characterised as thir 3,5-dinitrobenzoate

derivatives (133) (134). Since the cis hydrin4ne system

is flexible and therefore configuration dos not imply

conformation it was not possible to distinguish between

(131) and (132). Dehydration of the alcohol mixture (131)

and (132) gave diene (118) as the major and diene (130) as

the minor product. Silver nitrate p.l.c. gave the required

diene (130) in 23 yield. The structure was consistent

with the low field 18-methyl (t 9.13) compared with the

1404 -epimer (119) ( 1:9.30)70 , the A 2 '9 vinyl multiplet -1 ( 1,4.47), the infra red spectrum (796cm ) and negative

rotation. Although the n.m.r. spectrum and t.l.c. behaviour

of A. 12and diene (130) were very similar the infra red and

mass spectra and o-otical rotation showed them to be different.

Clearly Ai2 has an additional double bond but its low

availability ruled out further study.

•

0 122 131 13 2 80-H 13 3 80(-H, 3, 5-dinitrobenzoa.te

1 3 4 8p-H, 3, 5-dinitrobenzoate - •

•

130 118

Initially the preparation of diene (119) from toluene-

4-sulphonylhydrazone (135a) and methyllithium77 was

investigated. Treatment of Grundmann's ketone (121)

with toluene-4-sulphonylhydrazide in methanol ga7e a

product. The infra red spectrum n.m.r. spectrum and

analysis were in accord with structure (135a). However

the m.p. and optical rotation showed a large variation from

4 sample to sample and mutarotation in solution was observed. This may be explained by syn/anti isomerisation. The mass

spectrum also showed a variation. In some samples the

molecular ion (444) was absent; the highest mass ion being

54P,. 3utyllithium treatment of the rroduct gave diene (130)

as the major and diene (119) as the minor :roducts.- • Epimerisation of Grundmann's ketone (121) and

subsequent formation of the toluene-h-sulphonylhydrazone

gave a solid Similar to that prepared directly from

ketone (121). Methyllithium treatment of this product

gave diene (130) as the sole hydrocarbon. Clearly

Grundmann's ketone (121) was being epimerised during the

reaction with toluene-4-sul2honylhydrazide. A mass ion

• measurement indicated the ion at :;41-8 was du=: to the azine (136), presumably formed in the mass spectrometer prior to

ionisation. The formation of azines in the preparation 78 of toluene-k-sulphonylhydrazones has been reported.

1 3 6

•

Chromatography of the A2 fraction gave two ethoxyvinyl

ethers A and A T.1.C. indicated the presence of 222 223' other compounds including vinyl ethers in A2. However these

were not stable to repeated p.l.c. or were present in

intractable mixtures. The intractable mixtures were treated

5 6 • with hydrochloric acid/chloroform but this was not

satisfactory in the isolation of 1:erived carbonyl

compounds. The structures of vinyl ethers A222 and

A were confirmed by synthesis. 223 Grundmann's ketone 79 (121) and ethoxymethylenetrinhenylphosphorane gave two

vinyl ethers (137) and (138) each in 25% yield. Formation

of triphenylohosthine from the decomposition of excess glide

• hindered isolation but it was removable with iodomethane in

light petroleum. The structures of vinyl ethers (137) and

(138) were consistent with the n.m.r. spectra and the intense -1 C = C stretch in the infra red s-ectra (1682 cm ) Itwas

not possible to distinguish structures (137) and (133).

The synthetic materials were identical with A and A223. 222

Photofragmentation of olefins is a rare process and • go has previously been observed only in the gas phase .

Presumably the hydrocarbons are formed by a radical process.

The vinyl ethers A222 and A could conceivably be formed 223 via vinyl carbonium ion (139) or more plausibly by ethanol

addition :rior to fragmentation and a subsequent radical

pathway. If vinyl carbonium ion (139) is the intermediate

solvent capture prior to symmetrisation is in accord with the

isolated yield ratio of A222 and A223. However the 2nd • 11 pathway via (140) and (141) is of lower energy. Ethanol addition is not required to form the hydrocarbon photofragments

since they were also formed in cyclohexane alone.

- 57 -

•

1 4 0 141

• •

■ B Toxisterols 2

Chromator;raphic behaviour and sr,ectral data showed

fraction B to be identical with "toxisterol diene I" 1 2 63 previously reported . However t.l.c. indicated the

fraction was not homogeneous in contrast to previous 63 Repeated chromatograrhy gave a minor B and reports. 11 two major toxisterols2 B12 and B13 ( Xmax. 23, 252, 262 nm.). • Both B and B were homogeneous by t.l.c. and n.m.r. 12 13 spectroscopy. Spectral and analytical data for both

compounds was consistent with structures (142) or (143).

•

- 59 - a It was not possible to distinguish between the

oxa—bicyclo-/2,2,17 (142) and oxa:bicyclo-/3,1,17 (143)

systems. Although system (142) is more attractive \ degradation by ozonolysis suggested (143)63. Itis

conceivable that methyl migration took place during

ozonolysis. Alternative degradation sequences were 81 examined. Lythgoe et al , have oxidised ergocalciferol

(1.) to Grundmann's ketone (121) and dieneal (144) by • regioselective hydroxylation, /giving (145)7 followed by A 6 lead tetraacetate cleavage. .The LA system of

ergocalciferol can also be selectively hydroxylated using 82 lead tetracetate in glacial acetic acid.

HO KMn04' Me2CO,

1 145

(L.T.A. = Pb(0Ac)4)

144 •

Oxidation of toxisterol B with potassium permanganate 2 12 in aqueous acetone followed by lead tetraacetate cleavage

failed to give any ring A ketones or aldehydes.

- 6o - • 3-Chloroperbenzoic acid oxidation of toxisterol2

B gave a crystalline monoepoxide, toxisterol B epoxide-1. 12 2 12 Spectral data and anal:'sis suggested intact ring A cyclic

ether, 66 22 and A 5 or A 7 systems. The epoxide function

was either 5o( , 604. , 5ts , 61s or 70( , 8(24 The 7(3

SP epoxides would be expected to be too congested to be

formed and the similarity in 18-methyl shifts of B and 12

• its epoxide supported this exclusion. It was not possible to distinguish the 16 possible structures. Paraperiodic

acid83 treatment of B epoxide-1 in THF gave a polar 12 product(s) of high molecular weight. The expected

carbonyl compounds were not formed. An intractable

mixture of polar compounds were obtained from B12 epoxide

and trifluoroacetic acid in aqueous THF.

Toxisterol B epoxide-1 is currently being studied 2 21 byI7ray crystallography.

Repeated chromatography of fractions B2 and B3/B4 gave

three toxisterols characterised by a single chromophore at 2 250-253 nm. Toxisterol2 B21 was isolated as its crystalline

3,5-dinitrobenzoate. The ester was identical with the 62 previously reported "toxisterol2 A 3,5-dinitrobenzoate" 63 and "toxisterol - diene-II 3,5-dinitrobenzoate". • 2 Toxisterol B was obtained as a crystalline solid and 2 72 toxisterol B isolated as a benzoate by silver nitrate 2 31 chromato;raphy. Analyses and mass spectra of the three

toxisterols and dcrived esters indicated the compounds to 2 be isomeric with ergocalciferol (1). The n.m.r., infra red

and ultra violet spectra indicated the presence of the

heteroannular diene-CH = CH-4 =

- El - • 13 C n.m.r. spectra ,:ere particularly helpful in structural 13 elucidation. The C n.m.r. spectra of a series of model

compounds (ergosterol (4), ergocalciferol (1),

stigmasterol (146), dihydroergocalciferol-II (147),

dihydroergocalciferol-IV Suprasterol2-I (12),

suprasterol -II (13) and tetrahydrosuprasterol -II (149) ) 2 2 were recorded.

•

R 147R = Me, = H R' 148R = H, R' = Me

146

HO..

H 149

13 C L;hifts -,:ere assigned unaLbiuously by comparison with

the spectra of the derived 3,5-dinitrobenzoates and with

lanthanide shift reagents (Eu(fod)3). Spectra of model

- 62 - • compounds and assignment rationale will be described 8 4 13 elsewhere . The C n.m.r. snectra of toxist'frols, and B indicated the presence of an "extra" B21, B22 31 quaternary carbon atom at C 54.11, 52.74 and 53.21

respectively. Formation of three spirosteroids of

structure (150). differing in configuration at C-4 and C-8

was in agreement with all spectral data.

•

150

The confi,:urtions at C-4 were assigned by coupling

constants in the 7;roton n.m.r. between 3-H and 4-H.

In toxisterol2 B21 3,5-dinitrobenzoate the 3-proton

(15 4.71, which must bey( ) was coupled to 2 trans ,:rctons

= 10Hz. and 1 cis proton (J = 2.5Hz.). The 4-H

(IC 7.00) was coupled to 1 trans proton ( J = 10 Hy.) and must therefore have been p . By comparison of the 3c< ..-H/4.-HcouplingconstantsandW,values k-H was found

to be consistent with an cK configuration in toxisterol 2 B and a configuration in toxisterol2 B31. 22

- 63 - • The configuration at the spiran centre C-8 ra-313 assigned

by observation of the nuclear Overhauser effect (N.O.B.)

between 7-H and 18-methyl. B21 -I 5-dinitrobenzoate

exhibited a strong effect but the N.O.E. was not observed

in either B or B benzoate. Hence toxisterols B 22 31 21' B and B have structures (151), (152) and (153) 22 31 respectively.

•

151 152

(a) R =H (a) R = H (b) R = C (b) R = N2Ph 0

• (d) R = COPh RO (c) R = C NO2

(f) R = OAc

(g) R = C 0 153 (h) R = CS Me 2 (a) R = H (i) R = SiMe , (d) R = SiMe 3 (b) R = COPh - 6 - (c) R = C(0) nH2Ph • Additional crystalline esters of toxisterol B (151a) 2 21 62 were 1;re:ared., In contrast to earlier reports

B 4-phenylazobenzoate (151g) was obtained as orange 21 62 crystals identical with "toxisterol2 A 4-phenylazobenzoate".

B and B 4-chenylazobenzoates (152b) and(153c) could 22 31 ' not be obtained crystalline.

The structure of toxisterol B 3,5-dinitrobenzoate 2 21 (151b) was confirmed by anMray crystallographic study.85

The spirosteroids (151a), (152a) and (153a) may be

considered to arise from preergocalciferol (7) via

hydrogen shifts.

From the mother liquor of toxisterol B 2 21 3,5-dinitrobenzoate (151b) a 2nd compound B 23 3,5-dinitrobenzoate was isolated. Although homogeneous

on t.l.c. the n.m.r. srJectrum indicated the Iresence of

a mixture of compounds. B.), was not the 4th srirosteroid.

- 65 - • Three additional benzoates were isolated during

chromatography of toxistcrol B benzoate (153b). 2 31 The two major compounds B benzoate and B 32 4 benzoate had the triple chromophore absorbing at 241, 250, 259 rim.

This suggested the dihydrotachysterol2 function (154).

154

Toxisterol B was probably isomeric with ergocalciferol (1) 2 32 and may have contained a cyclopropane ring. As the compound

was an oil and it was not possible to remove all impurities

its structural determination was abandoned. Toxisterol B4

was an ethanol addition coml:,ound (m/e 546 (M) ).

However, for reasons outlined later, its structural

determination was not undertal:en. The corresponding

• 4-phenylazobenzoates could not be obtained■-•• crystalline.

Dehydration of toxisterol2 B21 (151a) with hydrogen

chloride in chloroform gave a crystalline derivative.

The n.m.r. spectrum indicated 3 aryl protons, an aryl methyl,

an aryl methylene and intact side chain. The u.v. and i.r. -1 spectra (778 cm , 1,2,3-trisubstituted benzene) surported

formation of aromatic compound (1"5).

_ 66 _ •

;

155 156

Likewise dehydration of toxisterol B (153a) gave the 2 31 aromatic isomer (156). The 13-methyl of (156) was at

lower field (1: 9.05) than in isomer (155) (7C 9.17).

This is in accord with the expected deshielding by the aromatic ring on the p face in (156). Dehydration of B (152a) gave a compound that was -probably toxisterol2 22 (156). However impurities detected by n.m.r. spectrum

depressed the melting point by 50. Attempts to dehydrate 86 B (152a) with phosphorus oxychloride in byridine , 22 - + carbethoxyamidosul-p:uryltriethylammonium (Et02CNS02NEt3) 37 in benzene or with chlorotristriphenylphosphinerhodium (I) • in chloroform88 gave imoure (156).

A minor hydrocarbon was formed during the recording 13 C n.m.r. srectrum of toxisterol B benzoate (157b). of the 2 31

- 67 - The comound was more polar on t.l.c. than (156).

Analysis an'l the mass spectrum inr1icated C ,11 as the 40 molecular formula. The n.m.r., i.r. and u.v. spectra suggested that the compound was the indene (157).

The 18-methyl shift C 8.73) was consistent with additional unsaturation. Presumably the compound was formed by aerial oxidation and dehydration.

157 n attempt was made to confirm the C-4 stereochemistry of B (151a) by pyrolysis of its xanthate (151h) to the 21 tetraene (158). Pyrolysis at 210° under argon gave three components: a tetraene methane thiol (m/e 426), a tetraene (m/e 378) and a pentaene (m/e 376, presumably resulting from dehydrogenation by sulphur compounds).

Pyrolysis of xanthate (151h) in the presence of dimethyl acetylenedicarboxylate in an attempt to prepare (159) by a

Diels Alder/Retro-Diels Alder route gave an intractable mixture of compounds.

- 68 - 4

• 151h 158

Photolysis of toxisterol2 B21 (151a) in cyclohexane

slowly gave a complex mixture of ultraviolet inactive

compounds (Fig. IX).

-69- • • • 0

FIGURE IX PHOTOLYSIS OF TOX/STEROL 82,1 IN CY CLOHEXA NE

Before photolysis

10 minutes

30minutes

70 minutes

rn 180 minutes han r Abgn

• I 275 300 325 350 400nm. 200 225 250 C/D TU;:ISTEROLS, L

Preliminary examination of the C toxisterols 2 indicated a complex mixture of compounds with the

dihydrotachysterol2 chromoDhore (154). The compounds

were not examined further.

Crystallisation of the CD toxisterols gave 2 ergosterol (D2) an'. an oil CD1 T.l.c. examination of the

D toxisterols indicated three components (in order of 1 increasing polarity) D , D12 and D13. Chromatography 11 gave a partial separation into and D12/D13. D11/D12 T.l.c. of the derived D 12 - 3,5-dinitrobenzoates 11/D indicated twelve compounds which were only partially

seT)arated by chromatography. The most polar column

• fraction gave toxistrol D /D 3,5-dinitrobenzoate-10 2 11 12 as an amorphous solid from acetone/methanol. Analysis and

the mass spectrum showed the compound to be an ethanol

addition product. The ultraviolet spectrum consisted of

the dihydrotachysterol2 chromo:hore ( X max. 2.3, 20.5, 259 nm). The n.m.r. spectrum indicated an intact side chain,

transoid diene,an ethoxy group and a 19-methyl singlet.

Thus D 5 5-clinitrobenzoate-10 was assigned the 11, D12 '

• structure (160). •

- 70- 0 c-d

(61NO 02N 2

160

The stereochemistry was tentatively assigned by com)arison

to dihydroergocalciferol-II (147a) and dihydroergocalciferol-IV

(148a). Hydrogenation of ergocalciferol (1) using

chlorotristriphenylphosphinerhodium (I) gave both Po 89 dihydroergocalciferol-II-' (147a) and dihydroergocalciferol-IV

(148a) as the only products.

• •

H PhH, (Ph P) 1412C1,, 2, 3 3

1 RO""

147 148

•

The shift and peak width WH'of the 3o( -H in the derived

esters (147b) and (148b) were considerably different.

inen the ester group and 19-methyl were trans as in (147b)

the 30( -proton occurred at lower field ( -C 4.96) and lower

peak width (W = 6.5HZ.) than when cis as in (147b) H • (7 5.07, = 20Hz.). This is consistent with the 3o( -proton being rseudoaxial when the 19-methyl and ester

were trans but pseudo equatorial when cis. S i nce the

30( -proton in toxisterol2 D /D12 3,5—Jinitrobenzoate-10 11- occurred at 5.07, W.„7 = 20Hz. the ethoxy-7roup must have

been o( . That 6-H (1: 4.12), 7-H ('-r- 3.58) and 14-H

/D12 (1: 7.65) occurred at lower field in D11 3,5-dinitrobehzoate-10 than (147b) or (143b) cuEgested

- 72 -

deshielding by the ethoxy-group and that D12 D11, 3,5-dinitrobenzoate-10 had structure (160). In the

n.m.r. spectrum the etLexy-methylene cost,,d of a

double quartet presumably arising by transmission of

C-10 asymmetry through_ the oxygen atom; the • ethoxy-met.y1 was a triplet (from the o-Terlap of

2 double doublets). 12 3'5- dinitrobenzoate-10 11' D • was acid labile being converted into a triene with A max.

230, 290, 302 nm: presumably isotachysterol 2 3,5-dinitrobenzoate (24b).

•

RO'

160 24b

• NO 2 Two additional compounds were isolated from D /D 11 12 3,5-dinitrobenzoate: ergocalciferol 3,5-dinitrobenzoate-(Ib)

(D /D 3,5-dinitrobenzoate-5) and an unknown ester 11 12 D 3,5-dinitrobenzcate-4.D11/D12 3,5—nitrobenzoate-4H 11/12 was a hir:h sharp me-tine point soli,: whi h lacked a chromorhore

and the only vinyl signals in the n.n.r. snectrum were due to A22 the LA sy The mass spectrum indicated m/e 5914, 592, 590

- 73-

as the highest mass ions. Possibly the compound was a

steroidal dimer and this was supported by the complex n.m.r.

spectrum. T.l.c. indicated that )5 compounds with the

acid labile dihydrotachysterol chromophore remained in 2 3,5-dinitrobenzoate mixture. D11/D12 Presumably these were ethanol addition compounds. T.1.6., mass and

ultraviolet spectra indicated :3;, 2 ethanol addition

• compounds in fraction D12/D13. Since ethanol addition to

polyenes during photolysis is well known and the C/D

toxisterols wore complex mixtures further investigations

were not carried out. Since fractions C and D composed

the bulk weight of the ergosterol photolysate the origin of

the intense chromophore at 242, 252, 260 nm. has been shown

due to ethanol addition.

• T.l.c. and mass spectral analysis of fraction E indicated

polymeric material and ethanol and water addition sterols.

Photolysis of ergosterol (4) (Fig.X) in c7clohexane and

repeated chromatography gave hydrocarbon photofragments Ai ,

toxisterols2 B12, B13, B21 and B22. Toxisterols2 B31 B11, (153a) was not detected. In addition, by chromatography

of the corresponding esters, suprasterol2-II 3,5-dinitrobenzoate

(162b), lumisterol 3,5-dinitrobenzoate (6b), ergocalciferol • 2 3,5-dinitrobenzoate (Ib) and the unknown D113

3,5-dinitrobenzoate-1 and D 3,5-dinitrobenzoate-11 were 13 isolated. 3,5-dinitrobenzoate-1 was identical with 13 D11/D12 3,5-dinitrobenzoate-4 from the ergosterol/ethanol

rhotolysate. J 13 3,5-dinitrobenzoate-11 exhibited

m/e 594, 592, 590 as the highest mass ions and also may have

been dimeric. Absorbance 1— 2 0 i 25 FIGURE X it 25 0

PHOTOLYSIS OFERGOSTEROL 2 7 5 300 I

INCYCLOHEXANE 325 1 350 v

480 ntn. ir

•

H 1 6 2b

•

0 II CO

• •

0 11 , ccc

- 75 - •

In addition to the photofragments a minor crystalline

hydrocarbon was also isolated. The mass Spectrum and

analysis suggested C40 H as the molecular formula. 64 The n.m.r. spectrum showed the presence of the 18-methyl

group (consistent with a A 7 system), intact side chain and an additional vinyl singlet ( I 4.64). Formation of

a homoannular diene was indicated by the ultraviolet spectrum.

The compound was assigned structure (161).

•

Since the photofragments were formed in such low • concentration dimerisation prior to loss of ring A

giving (161) would be more plausible than photofragment

dimerisation.

- 76 - a

PITCTM=. 77:(7CCALC:=0T. (1 ) T_:?r;=,CI7-7CL (7)

Photolysis of errocalciferol (1) was investi-'ated

firstly to prepare suprasterol2-I (163a) and suprasterol2-II 2 13 (162a) reauired as C n.m.r. models and secondly to

investigate the reorted formation of toxisterols from 2 91 ergocalciferol 2 (1). Irradiation of ergocalciferol (1) in petroleum at -60° (to prevent thermal reactions) gave

suirasterol2-I (163a) and suprasterol2-II (162a).

In addition minor less polar u.v. active sterols (X max.

240, 270 nm.) (presumably vinylallenes (14) and (15),

vinylcyclobutenes (16) and (17) and the derived 5,6-trans- 2 ergocalciferol2 (18) ) were formed.

a •

14,15 16,17 18 - 77 - FIGURE XI PHOTOLYSIS OF ERGOCALCIFEROL IN PETROLEUM AT -600

1—

before • photolysis

ban 60 bsor

A 80minutes

12 15 2 5 2 0 300 3125 350 400nm• • No toxisterols wire detected by t.l.c. or u.v. 2

92 Pfordte has described the formation of

"lumicalciferol2" on irradiation of ergosterol (4),

lumisterol (6) or ergocalciferol (1) in benzene at o 36-81 . ,.he compound formed was thought to be tricyclic

with four double bonds. In our opinion "lumicalciferol " 2 and suprasterol2-I (163a) are the same compound. Pfordte

did not report the isolation of suprasterol2-I (163a) and

his tabulated data was incorrect. He has confused

Suprasterol -I (163a) and hexahydrosuprasterol -I (164) 2 2 which was referred to as "suprastanol I "by ',:indaus93 2 B . The suDrastero12-I (163a) and its 3,5-dinitrobenzoate we

have prepared are identical with "lumicalciferol" and its

3,5-dinitrobenzoate (m.D and /C4 7D). Both suprasterol2-I

• (163a) and its allophanate (163c) have physical properties 94 in agreement with data reported by Setz .

• •

1 6 3c OCONHCONII 2 •

Hydrogenation of suorasterol -II (162) in ethyl 2 acetate gave tetrahydrosuprasterol2-II (165) required 13 as a C n.m.r. model. •

(a) R = H

(b) R =

165

NO2

• • Ergocalciferol (1) on fluorenone or acetone sensitised

photolysis reached equilibrium with a less polar compound

presumably 6-trans-ergocalciferol (IC). Havinga has o5 described '- the equilibration of ::re2rgocalciferol (7) and

tac]rlystcrol, (19) via the triplet state but the data suggested

pseudoequilibrium with slow formation of a-.1ditional compounds.

-79-

Preercocalciferol (7a) on fluorenone sensitised

irradiation reached equilibrium with tachysterol (XIX), 2 no other compounds were detected by t.l.c.

(a)R = H 19 (b)R = =0

02 N O0 2

Preparative photolyses of er7osterol (4) at room • temperature resulted in formation of ergocalciferol (1)

and thus low temp?rature irradiation was investigated.

The low solubility of ergosterol in petroleum crecluded

preparative scale photolysis. T.l.c. indicated

attainment of pseudoequilibrium between ergosterol (it),

preergocalciferol (7a), tachysterol2 (19) and lumisterol 2 (6) and the slow formation of additional compounds.

The yield of spirosteroids was not noticeably increased.

An attembt was made to isolate the 4th spirosteroid

(166). Since rhotolysis

- 8o - •

166 in ethanol gave a complex mixture due to ethanol addition

and toxisterol B (153a) was not formed in cyclohexane a 2 31 preparative photolysis in t -butanol was carried out.

Two previo-:sly undetected compounds (one a t -butanol

addition compound with m/e 470 (M ) ) were isolated;

neither was the 4th spirosteroid (166).

Since all photofragments and toxisterols2 were formed

in low yields and may have been present in starting

ergosterol (4) a chromatographic check was undertaken .

The ergosterol (4) was found not to be contaminated.

Photolysis of ergosterol in cyclohexane under air gave

little or no hydrocarbon photofragments clisproving the

hypothesis that their formation resulted from aerial

• oxidation during photolysis.

92 Three com,Doun-7s "substance von Hess" , "suprasterol a oc; 9? ri III and "Linsert's com7;ound" " have been described

in the literature but their structures remain undetermined.

Havinga has suggested the identity of "Lin.7ert's compound" 2 and vinylallene (167) . •

167

That " urrasterol2 III" was only sparingly soluble in

petroleum, diethyl ether or carbon tetrachloride but very

soluble in ethanol or methanol must be regarded with

scepticism. Such solubility behaviour, atypical of

ergocalciferol photoisomers, suggests a product(s) of

aerial oxidation.

q8 Recently Havinga- has described the isolation and

structural determination of two ethanol addition compounds

(168) and (169) from the photolysis of 7-dehydrocholesterol

(5). A bicyclo /5, 1, 07 isomer (170),a product (171)

derived from the photochemically allowed antarafacial /1, 57 • • hydrogen shift and two spirosteroids (172) have also been isolated. The authors were unable to report stereochemical

elucidation of (172). •

0 OH 170 168 R = 1,1e, R' = Et0

169 R = EtO, = Me

171 172 All the isolated toxisterols, photofragments and

crude photolysis resins were examined for antirachitic

activity and toxicity. None of the pure compounds were • active. Variable antirachitic activity in the crude • photolysate probably indicated variation in the

ercocalciferol (1) concentration. Although not implying

toxicity the term toxisterol was retained (for compounds

with X max. 250 nm.) for historical reasons. • AN IN117,TIGA7ION CF STTTOID ONOTRON =NIS=

Attempted Imnrovement in the Prenaration of Cholesta-5, 7-lien-3p -ol.

Cholesta-5,7-lien-3 p -ol '(5) is prepared from

cholesterol (173a) by allylic oxidation followed by

elimination. 1

•

173a 5

However the product is contaminated with cholest-4,

6-dien-3 g -ol (174) and cholest-2,4,6-triene (175).99

• •

174 175

Since much recent work has been directed towards the

synthesis of hydroxylated ehnlecaloiferolo it is desirable

9 IL a to improve the introduction of the A 5 '7 system.

Iron in the oxidation state of zero is known to readily

form stable diene tricarbonyl complexes. For example

ergosterol iron tricarbonyl (176) has recently been 100 prepared from ergosterol (4) and iron pentacarbonyl.

If cholest-5-ene-3p ,7-diols (177) were dehydrated

in the presence of an iron carbonyl then it is conceivable

that the A 5'7 complex (178) could be formed as the exclusive product. Oxidation of cholesterol acetate (173b)

with sodium chromate gave 7-ketocholesterol acetate (179).

Lithium aluminium hyjride reduction gave the amorphous

mixture of cholest-5-ene-3P. ,7-diols (177). The formation

of diols (177) was confirmed by the preparation of

cholest-3-ene-3p ,7V -diol 3-benzoate (130a) and • cholest-3-ene-3(3 , 7(53 -diol dibenzoate (180b) as

crystalline derivatives.

• a NaOAc, Ac20 LiA1H Na Cr0, Et 0 RO 2 AcO 2 AcOH, R = Ac

173 179 (a)R = H (b)R = Ac (c)R = Ph

- E5 •

(CO), THF, 4-MeO

.• HO OH HO ile(C0)3

177 178

(a) 7c4-H (b) 713-H

180 (a) R = H (b) R = COPh a

Attermted dehydration of (177a) and (177b) in di-::-butyl

ether or THE in the tresence of toluene-4-sullthonic acid

and iron rentacarbonyl or the more reactive diiron

nonacarbonyl gave an intractable mixture of products.

- 86- • The acid c.talysed carbon monoxide exchane of iron o 101 pentacarbonyl at -20 has been reported addition

of trifluoroacetic acid to diols (177a) (177b) and iron

pentacarbonyl at -15° gave a complex mixture of products.

That exT:osterol (4) formed ergosterol iron tricarhonyl

(176) on reflux withdiiron nonacarbonyl. and toluene-4-

sulphonic acid in TIIF indicated complexation was feasible

in the presence of acid.

HO Fe(CO) 3

4 176

Since 7-ketocholestcrol acetate (179) was available

the dehydration of the Jlerived cholest-5-ene-3p ,7-diol

3-acetates (131a) and (181b) was examined. Sodium

borohydride reduction of 7-ketocholesterol acetate (179)

gave cholest-5-ene-3p ,7-diol 3-acetates (181a) and (131b).

The major 7 p isomer was obtained on repeated crystallisation

or by p.l.c. Dehydration with carbethoxyamidosulphuryltrietliy1- + 87 ammonium (Et0 CNS0 Na ) in benzene gave cholest-4,6-dien- 2 2 3 3 p -ol acetate (182) as the major product. Cholest-5,7-dien-

3 p -ol acetate (183) was not fored. •

Ac0 OH

181

(a)7o(-0H

(b) -OH 183

The classical synthesis of 7-6ehydro-cholesterol

benzoate (184a) by 7;yr:,lysis of 3 p , 7 cs, -benzoate (135) was 102 first described by Windaus It was thus of

PhCO 2 PhCO2

185 184

•

interest to examine the Pyrolysis of the corresponding

xanthate (186). Xanthate (186a), prepared from

cholest-5-ene-3 , 7 -Hiol 3-acetate (121b), was found

to be very thermally labile and readily converted into

dithiolcarbonate (127a). Analysis was consistent with • isomerisation. Formation of the dithiolcarbonate (187a) -1 was indicated by the infra red spectrum (1680 cm ) and

S-methyl in the n.m.r. spectrum. Inversion at C-7 was

consistent with coupling constants J, peak widths W and H C-18 and C-19 methyl shifts in the n.m.r. spectrum.

Rearrangement of allylic xanthates, usully accompanied

by double bond migration, has been reported.103

186 187 (a) R = Ac

• (b) R = CS2Me During preparation of xanthate (186a) excess methyllithium

gave the bis xanthate (1C6b). This compound was also

thermally labile presumably giving dithiolcarbonate (187b).

S •

_ e9 - THE DIY.ERT::::A7I0:! 07 fl7 LYT, 7)ERT7ATIV7 72,Y T701.T. C=CNYLS

The dehydrobromination of 7o( -bromo_cholesterol

benzoate (138) in the presence ofdiiron nonacarbonyl was

examined as a synthesis of (184b). Since,iron carbonyls 2+ react with amines to give carbonylate salts (Fe(amine) x - 104 Fe (CO) 2 X= 4,6; y,z = 1,4; 1,3; 3,11; 4,13) a Y z

• hindered amine was required for dehydrobromination. N-Bromosuccinimide bromination of cholesterol benzoate (173c) 105 and fractional crystallisation gave 70( -bromocholesterol

benzoate "(188). Attempted dehydrobromination by heating

(183) to reflux in benzene or THE containing (LUron

nonacarbonyl and di-iso-Pronylethylamine gave two iron free

products X and Y. Analyses and mass spectra indicated both

X and Y to be dimeric benzoates. Although the molecular • ions (m/e 978) were absent peaks due to loss of one and two

moles of benzoic acid (356, 734) were present. The high

melting points of X and Y were as expected for dimeric

structures. The ultra violet spectra indicated only the

benzoate chromophore. Both X and Y were concluded to be

7-7 dimers. Since the n.m.r. soectrum of X indicated a

single vinyl peak CC 4.87 (2H) ) it probably had structure

(189). The n.m.r. spectrum of I was more complex with two • vinyl peal:s ( 15 !:.4O (1H), 4.67 (11) ) and probably had

structure (190).

PhCO

188 00189 190 Presumably X and Y were formed via the intermediacy of 106 -allyl iron tricarbonyl bromides. Cholesterol

benzoate (173c) was not formed during the reduction.

Iron pentacarbonyl has been reported to dimerise 107 geminal dihalides to give tetraarylethylenes.

•

X = H, Cl, Me, But, Me0 Y = CI, Br

lo8 A German patent describes the preparation of

tetrasubstituted butanes by reductive dimerisation with

• zerovalent iron or nickel compounds".

R R XCH CH' ' 11... CHCH CH CH/ 2 RR'/ 2 2

R = H, alkyl R' = CN, CO2R, COR, CONR2

The preparation of ethane from iodomethane and cyanogen

• from cyanogen chloride by iron pentacarbonyl reduction has 109 been reported. Alper described the formation of

1,4-diketones, ketones and veto-eDoxides on iron 110 1:entacarbonyl reduction of 0( -bromoThetones. Bruce

has also found hydro enolysis accompanied dimerisation in 111 the preparation of tetraarylethanes.

F-) 2 CH3r Fe'(Ce)5,(cF ) Cg + ( (C.F C114.- (C6 o 5 2 -2 b 5;2 2

-91 - • Japanese workers have described the Diels Alder reactions

of intermediate (191) formed from reduction of

0,1,, 0( -dibromoketones and have used the reaction in

synthesis of tropinones.112,113

191

Iron pentacarbonyl has also been used for rreparation of 114 thiolsulphonates from sulphonyl chlorides,

104, (3 -diketones from acid anhydrides115 and for the • 116 polymerisation of 1,4-dichlorobut-Z-ene.

Migration of carbon-carbon double bonds by iron

carbonyls are well documented processes. Although 117 monoolefins have been isomerised the reaction is most

often employed for the conjugation of dienes.

Cycloocta-1,5-diene gave cycloocta-1,3-diene on treatment

with iron pentacarbonyl followed by oxidative removal of 118 • iron. Likewise _hexa-1,5-diene gave hexa-1,

3E-diene and hexa-23,4E-diene. Allylic and homoallylic

alcohols were found to be isomerised to ketones or 119 al dehydes. Labellin7 studies indicated that

- a 2 Fe(CO) EtC0C9H19

H Fe(CO) c H 5 A > 9 CHO • 19

the reaction proceeded by complexation, migration of the

o(-hydrogen and deco=lexation. This was confirmed by

the rearrangement of (192); the epimer (193) being inert

since the 04. -hydrogen was on the opposite face to 120 complexation. •

192 • •

93 •

•

193

•

194 195

• •

- 94 - • The synthesis of 7-dehy:lrocholesterol iron tricarbonyl

(195) from cholest-4,6-dien-3p -ol (194) by double bond

migration and complex tion was thus investigated. For

convenience of preparation compounds in the ergosterol

series were examined.

Cppenauer oxidation of ergosterol (4) :Jnd acid catalysed

isomerisation of the intermediate ergosterone (196) gave 121 isoergosterone (197).

•

4 196 197

Sodium borohydride reduction followed by benzoylation gave

ergosta-4,6,22-trien-3 p -ol benzoate (198b). .Formation

of (198b) was consistent with spectral and analytical data 122 and hydrolysis to the known alcohol (198a). - • •

(a)R = H (b)R = PhCO R = me si (c) 3

198

-95- • However when err;osta-4,6,22-trien-3 -ol benzoate (19b)

was heated to reflux in benzene with diiron nonacarbonyl a

nonpolar iron free compound Z was obtained. Z was also

formed in toluene, Tin?, 1,2-dimethoxyethane or di-n-butyl

ether in the presence of iron pentacarbonyl, diiron

nonacarbonyl ortriiron ,Thodecacarbonyl at 50-130°.

Z- was formed in the presence of acid scavengers (styrene

oxide, propylene oxide). T.l.c. behaviour, analysis and • the mass spectrum indicated the comPound to be a dimeric

6 hydrocarbon of molecular formula C H36. The ultra violet and n.m.r. spectra were consistent with retention sf the

4,6,2 triene system. Comparison of the infra red spectrum

vinyl C-H deformations of Z and ergosta-4,6,22-trien-3p -ol

(198a) confirmed this. Hence Z was probably the 3-3 dimer

(199). That the n.m.r. spectrum exhibited

•

• 199 200

two 19-methyls, four overlappin7 doublets for 21-methyl and

2P;-methyl and a broad 1 -methyl su,:ported a 3p -304

link or a mixture of 2 isomers. Compound Z was also the

exclusive product on iron pentacarbonyl reduction of

• ergosta-1!,6,22-trien-3 p -ol trimetllylsily1 ether (198c). The

dimerisation of benzoates nd trimethylsilylethers by iron

carbonyls is without precedent. In contrast to the 119 expected rearrangements ergesta-4,6,22-trien-3 (3 -ol.

(198a) gave dimer Z. Ergosta74,22-dien-3-one (20) was

not formed. As predicted by these results cinnamyl

alcohol trimethylsilylether (201b) gave (albeit in poor

• yield) 1,6-diphenylhexa-1E,5E-diene (202) on diiron nonacarbonyl reduction.

P PhCH===CI-T- CHTOSiMe3 (PhCH===CH CH2)2

201b 202

This novel dLierisation would be of interest if it was

applicable to the dimerisation of farnesyl benzoate (203)

to give squalene (204).

203

• 204 As a model system preparation of tetraene (205) from

geraniol (206a) derivatives was examined.

OH 206a 205

- 7 - Reaction of geraniol benzoate (206b) with diiron

nonacarbonyl in benzene under reflux gave a mixture of

iron containing species. After removal of iron with

ferric chloride the mixture was separated by

chromatography giving three fractions R, S and T.

The least Polar fraction R consisted of.a mixture of

dimeric material containing iron. Fraction S consisted

of polar polymeric material lacking benzoate functions. • The most polar fraction T was a single benzoate, isomeric

with the starting material. Spectral data indicated the

compound to be 2,6-dimethylocta-2,4E-dien-3-ol benzoate

(207b) and this was confirmed by saponification to the

known alcohol (207a). The reaction of geraniol (206a)

•

OR OR 206 207

(a)R = H (a) R = H

(b)R = PhCO (b) R = PhCO

(c)R = SiNe3

• or its trimethylsilylether (206c) with diiron nonacarbonyl

both gave complex mixtures of comnounds. Presumably the

diversity of reactivity of Jiene alcohols their esters

with iron carbonyls does not depend on electrophilicity of

the allyl function but on diene flexibility. If both

double bonds can be complexed simultaneously (either with

the 1r systems parallel and diequatorial or orthogonal

-98- • and equatorial/axial in the trigonal bipyrimidal

intermediate) the double bond migr-1tion is.favoured.

Otherwise dimerisation is the predominant pathway.

This hypothesis is consistent with isomerisation of

cycloocta-1,5-diene, cyclohexa71,4-diene, acyclic dienes

and is also a2plicable to the allylic and homoallylic

alcohol rearrangement which give carbonyl compounds • (where the 04 -hydrogen functions as the 2nd ligand).

- 39- •

The synthesis of preerecalciferol iron tricarbonyl

(203a) was investizated since its synthesis re:juired

development of low temperature .comrlexing technii.:ues and

would possibly be a useful interediate in elaboration

at C-1. Ergocalciferol (4) has been complexed by reaction

withdiiron nonacarbonyl or the enone complex (209) at 600 123 in benzene solution.

210 2 09 (a) p(-Fe(C0)3

(b) p-Fe(C0)3

with ergocalciferol (1) and is known to b less reactive 124 tcards dieno 'Lines than er7ccalciferol (1) :hotolytic

com7lexation was examined. Eetal carbonyl cos .loxes not

realY1 7 available by ther7a1 means have b-:,en fre-I-ently 125 ore ared b‘, irra7lat4 on of

ergocalciferol (1) and -;,= •cntacrb.Dnyl in 71F solution

usinc, a rr-sz-,ur mercury arc lam:- in pyrex aHoar7Itus

gave ergalciferol iron trca.1.1-Denyl (210) 1_7etical

_ 100 - 123 that -nreyared thermally. The rate of for::;ation and

yield of complex (210) was much increased in quartz

a_paratus; no photo roducts of ergocalciferol (1) were

formed. Photolysis of preergocalciferol (7) in Tiff/iron

pentacarbonyl gave little of the required complex (208a)

since the rate of complexation was slower than the rate of

thermal isomerisation and subsenuent complexation.

However photolysis with tri iron dodecacarbonyl in a dimethoxyethane gave a yellow complex that was probably

isomer (2C8a). Models suggested that this is the least

hindered isomer. This was- in accord with the reported 126 conformation of :;reergocalciferol.

7 208 (a) R= Not (a) R= H • (b) R = Ac (c) R = CO (b) R = Ac

Since the complex cochromatographed with preergocalciferol

(7a) and was decomposed with 4-r2heny1-1,2,4-triazoline-3, 0 127 5-dione at -78 difficulty was experienced in its

purification. Thermal equilibration of unreacted

preergocalciferol (7a) with ergocalciferol (1) and removal

by p.l.c. was hin::_ered, by partial -com osition of the

- 1C1 • complex on p.l.c. to give more preergocalciferol (7a).

The formation of (208a) w s consistent with the high

resolution mass ion, infra red and n.m.r. spectra and

t.l.c. Acetylation of preergocalciferol iron

tricarbonyl (208a) or photolysis of preergocalciferol

acetate (7a) and trilron dodecacarbonyl in

dimethoxyethane gave a yellow oil that was probably r preergocalciferol acetate iron tricarbonyl (208b). Since the compound decomposed slowly on p.l.c. it was not

possible to purify it.

Abstraction of hydride from cyclohexadiene iron

tricarbonyl with triphenylmethyl tetrafluoroborate has

been used to generate the stable cationic complex (211).

Subsequent nucleophilic attack on (211) gave the derived • 128 trans substituted cyclohexadiene iron tricarbonyl (212).

--Fe(C0)3

ArMMe Fe(C0)3 3 = Si, Sn BF4

211 - -Fe(CO )3 Zn/Cu

Fe(CO) 3

- 102 -

•

(0C)3Fe BF4 0 R (Me0)2 RCd2 or 1(Ke0) P -- Fe(CO) • (0C)Fe 3 3 R2Zn 2 Me0H

-Fe(C0) 3

(0C)3FE7

• • If such a sequence could be applied to preergocalciferol

acetate iron tricarbonyl (207,13) then functionali.sation of

C-1 might be possible. Reaction of ergocalciferol acetate

iron tricarbonyl (210c) and triphenylm.-Ahyl tetrafluoroborate

in dichloromethane at reflux gave a mixture of iron free 123 comoounds. Preergocalciferol acetate iron tricarbonyl

(208b) reacted slowly with triphenylmethyl tetrafluoroborate

in dichloromethane at -10° but more rapidly at room temperature.

Quenching with tetra-n-propylammonium acetate gave a mixture

of iron free steroids. The reaction was therefore not

examined further. Presumably the site for hydride

abstraction must be coplanar with the complexed diene system

and close to the iron for cationic complex formation to take

place. Neither ergocalciferol acetate iron tricarbonyl

(210c) nor preergocalciferol acetate iron tricarbonyl (208b).

were able to meet these requirements.

Molybdenum hexacarbonyl reacts thermally or photochemically

with trienes or benzenes to give the corresponding molybdenum

tricarbonyl complex. Complexes of cycloheptc.triene (213), 129 130 313 cyclooctatriene (214) cyclooctatetraene (216) 132 133 cyclononatetraene (217) and oestrone methyl ether (213)

have been reported. • •

10 tf - •

mo(co) 3

213

No(CO)2 2 • o(CO)3

214 215

mo(co) 3 • 216

mo (co ) 6 L.

No(co) 3 217

• •

NeO

218

- 105 -

• Preoaration of ergocalciferol (219) or preergocalciferol

molybnum tricarbonyl (220) was thus of interest to

investigate

HO"

219 220

As a model system cycloheptatriene molybdenum tricarbonyl

(213) was prepared thermally, photochemically and by room

temperature reaction with the yellow compound (presumably

mo(c0) (THF) ) prepared by photolysis of molybdenum 6-x x 125 hexacarbonyl in THF. However reaction of ergocalciferol

(1) or preergocalciferol (7) gave transient coloured

materials that rapidly deposited molybdenum. Triene • • complexation by M9(0)3 presumably requires an all cis triene. Hindrance between the 9-H and 19-methyl group

in preergocalciferol (7) rules out planarity; as A 500 6,P(9) is r:er-oendicular to the diene. Ergocalciferol

(1) is also revented from taking un the required conformation

because of C-19/C-9 congestion. Both diene and mono ene 134 complexes of molybdenum are known to be often labile.

- ic6 - Although the synthesis of cholesta-5,7-di.-:n-3 p -ol (V)

has not been improved a novel dimerisation reaction of

allyl bromides, benzoates and trimethylsilyl ethers has

been investiated. Preergocalciferol iron tricarbonyl

(208a) has been prepared. Functionalisation of the

derived complex (203b) by hydride removal and subsequent

nucleophilic attack was unsucessful. That ergocalciferol

iron tricarbonyl (210a)/(210b) was readily available

photolytically suggests a possible direct synthesis by

photolysis of ergosterol (IV) in the presence of iron

carbonyls.

•

- 1c7 - • EXP2PI=AL

Melting points were determined using a Kofler hot

stave. Ultraviolet spectra were recorded in ethanol,

unless otherwise stated, on a Unicam SP800A ultraviolet

sectrophotometer. N.m.r. srectra were recorded in acid

free deuterochloroform with tetramethylsilane as an internal

reference on a Varian T60 instrument, XL 100 (off resonance • 13C multiplicity in parenthesis) or using the HR220 Service

at P.C.M.U., Harwell. Infra red srectra were recorded on

a Unicam SP200 or Perkin Elmer 257 instruments. Optical

rotations were recorded in chloroform, unles otherwise

stated, on a Perkin Elmer 141 Polarimeter.

Thin layer chromatrograrhy, both prerarative and

• analytical, was carried out under an atmosphere of carbon dioxide using GF254 silica plates unless stated to the

contrary.

Ultraviolet inactive compounds on t.l.c. 'ere

visualised with anisaldehyde:su?.rhuric acid: methanol

1: 1: 1F and warming (PANS rea7ent) charring with sulT)huric

acid or with a hot wire. In general column chromatography

was carried out on neutral Fra.,:e III alumina. S

The fol*owing grades of solvent were employed:

'zetroleum redistilled b.:). 40-60° fraction;

benzene, toltlene sodium dried analar reagents;

triethylamine, di-iso-pro7::ylethylamine, pyridine ...

redistilled from potassium hydroxide and stored over

4; molecular sieve; .

- 0 methanol, ethanol, acetone, chloroform, carbon tetr.lchloride

AaalaR or srectroscopic;

diethyl ether, THF, dioxan, dimethoxyethane, dilyme

di-n-butyl ether ... freshly distilled from sodium,

sodium hydride, lithium aluminium hydride or sodium/

beni'ophenone ketyl; t-butanol, carbon disulphide,

dichloromethane)dimethylsulphoxide (DM30), iodomethane,

2,4,6-trimethylpyridine, cyclohexane, :Ityrene epoxide, • 135 butan-2-one ... purified according to standard procedures.

The fol owing reagents were recrystallised before use:

ergosterol (acid free chloroform/methanol), fluorenone

(ethanol), methane sulphonic anhydride (diethyl ether),

cinnamyl alcohol (diethyl ether/petroleum) and

m-chloroperbenzoic acid (extracted with pH 7.5 buffer). 135

• Unless stated to the contrary all photolyses were

carried out in quartz apparatus at room temperature degassing

with nitrogen prior to and during irradiation with a

Phillips IPK 125'i type 57203 3/00 high pressure mercury arc

lamp.

Organic extracts were dried over anhydrous sodium

sulphate. Solvents were removed under reduced pressure

• below 40°. repeated procedures are described in full in the 1st instance only.

Microanalyses and mass spectral measurements were

carried out by the resT)ective laboratories, Imperial College.

- 109 - • PTICTflLY:117 OF =C:31=1, (4) T."

Ergosterol (4) (6.0g.) was dissolved in a solution of

citric acid (0.9g.) in ethanol (2280m1.)-water (120m1.)

and irradiated. The photolysis was monitored by ultraviolet

spectra. 'dhen the ergosterol absorption ( X max. 271, 292

294nm.) reached a minimum (22-25 hours) the irradiation was

stopped. The photolysis was carried out on six occasions. • Sodium carbonate (0.7g.) in water (100m1.) was added and

theethanol removed to leave an oily residue which was

extracted into ether, washed with 10c: aoueous sodium

carbonate, wa t er, brine and dried (Na29-SO,). The ether

was- removed to leave the crude toxisterol re-sin (40g. in

all) which was chromatographed on alumina (120g.)

(petroleum: diethyl ether = 1 : 0, 9 : 1, 4 : 1, 7 : 3)

• giving four crude fractions A, BC, CD and E (in increasing

order of polarity).

n n T 7'P.fCmTO:T A

The total A fraction was separated by p.l.c.

(2 40 X 20 X 0.1 cm silica plates developed once in diethyl

ether: petroleum = 3 : 197) to give A (372 mg.) and A l 2

♦ (4o4 mg). The A fraction was further separated by l • repeated p.l.c. (40 X 20 X 0.1 cm then 20 X 20 X 0.04 cm

10 silver nitrate/silica plates multiply developed in

benzene: petroleum = 1 : 19 - 1 : 24) to give (in order of

increasing polarity) A111 (29 mg..), A112 (5 me.), A112 mg.), A114 (36 mg.), (14 mg.), A113 (2': A115 Al2 (8 mg.), A (109 mg.) and A (49 m .) 13 114 g

- '110- • was essentially solvent residue 15 :.73 (m.),9.10 (m.), A111 A was identical with diene (117) (i.r., 'n.m.r. 112 (with the exception of non steroidal impurities at 15 8.75), m.s. and t.l.c. (105: silver nitrate/ , 24 5° silica benzene; Petroleum = 1 : 19), /C42D • = 5 o 24.5° o - 24.5 °, = + 9-8 , / o( 7 46nm = 10.6 + 9.2 .°S7 2436nm '/5°c° 757nin= + 16.0 o (C , = 0.499) ).

1:4.80 (22-H, 23-H), 8.43, 3.75, 8.92, 9.03, A112/A113 9.12, 9.22, 9.32 (methyl peaks), m/e 290, 274, 259, 163, 149, 109.

was identical with diene (118) (i.r., n.m.r., m.s., A114 t.l.c. (10i;: silver nitrate/silica, benzene: o ,- , 4o oo ,- .724 petroleum = 1 : 19, /01..4/ D = + 9.o , /04../57pnm= pop ° /71 7".4 = + 11.6o 10.5°, /O4J7 c2;/4:6nm = + ' i `4-'436nm 0 lu. (C = 0.276) ).

was identical with diene (119) (i.r., n.m.r., m.s., A115 t.l.c. (10(,:: silver nitrae/silica, benzene: petroleum = nno ,22 ° = 350, / 38o, 1:19), /-64. / D 0/ nm - 22 722° = + of 7 = 4- (C=0.133) ). 546nm 43°, -/ - 436nm 76° cochromatographed with diene (130) (10 silver nitrate/ silica, benzene: petroleum = 1 : 19) but was not the same compound, /7K2 21 '5 °=-106°, -111°, • = = - 130°, r.C..7 /24:1e a = 262° (C= 0.49)

max. (C22) 3070 3040 (w), 3020 (w), 2960. (.) 2930 (s), 2370 (s.), 1625 (w.), 1370 (s.), 1000 (w.),

970 (s.), 950 (w.), 390 (s.), 363 (w.), 318 (s.), 75n (w.), 740 (w.), 730 (w.) cm,1 s 4.43 (2H, 4.77 (2:1, n., 22-H, 23-H), 3.93, 9.03, 9.13, 9.23 (12H, side chain Me), 9.13 (3H, s., 18-Me), I1/e 272

(e), 267, 229, 201, 146.

A13 was identical with diene (120) (i.r., n.m.r., m.s., t.l.c. (10 silver nitrate/silica, benzene: o 22.5 = o 7 22.5o. petroleum = 1 : 19) /-0(./ -to , / / 0 6' o' - D am 22 5° + 49°, /-t,.4 7 Mm = 56°, /-0( 7 436nm = + 97°

(C = 0.721) ). •

T.l.c. (10 silver nitrate/silica, 2 developments in

benzene : petroleum = 1 : 24) indicated A 14 to be essentially decomposed material.

The A fraction was separated by p.l.c. (two 40 x 20 x 2 0.1 cm silica plates developed once in diethyl ether : e petroleum = 1 : 19) to give A21 (78 mg.), .1122 (S9 mg.), A23 (62 mg.) and A24 (40 mg.) all as oils.

SEPA2,ATION OF FRACTION A21

p.l.c. of A (3 40 x 20 x 0.04 cm 105L silver nitrate/ 21 silica plates developed twice in benzene : Petroleum = 1:19)

gave A (39 mg.) and A mg.), 4.78 l 21 (22 (2H, m., 22-H, 23-H), 6.52 (2H, a., J = 7 Hz., OCH2CH3), 8.30 (3H, t., • • J = 7 Hz., - OCH CH )' 8.75, 8.93, 9.03, 9.13, 9.22 (side chain methyls, 18-Me and 8-Ne?), Me (M+). Repeated 7,.1.c. (silica) resulted in extensive decomposition

to an oil (12 mg.), *0 max. (CHC13) 2920 (s.), 2870 (s.), 1780 (,%), 1710 (s.), 1580 (m.), 1460 1370 (s.), 1090 (m.), 970 (s.)cm-1. The resulting oil and 2,4-

dinitrorhenyThydrazine (1 m7.) in ethanol (5 ml.)/concentrated

• - 11 2 -

• hydrochloric acid (0.03 ml.) were heated to reflux

(5 minutes). The bulk of the ethanol (4 ml.) was

removed under vacuum and the residue taken up in diethyl

ether (10 ml.), washed with Ovate - (2 x 10 ml.), saturated

brine (10 ml.), dried and evaporated to leave a yellow oil.

T.l.c. (silica, dichloronethane) indicated one major and

five minor dinitrophenylhydrazone derivatives.

Chromatography on alumina (2 g.) (eluent benzene) gave an • oil that failed to crystallise.

SEPA'2ATION OF FRACTION A,),

P.l.c. of A (2 40 x 20 x 0.1cm silica plates 22 developed three times in diethyl ether : petroleum =

gave (in order of increasing polarity) A /A 3 : 197) 221 222

• (10 mg.), A222 (3 mg.), A223 (29 mg.), A223/A224 (12 mg.),

A (20 mg.). 224/A225/A226

A /A There was insufficient A present to warrant 221 222 221

isolation.

A222 was identical with the less Polar vin71 ether (137 or 133) (t.l.c., n.m.r.). An attemt to purify the

comtound by'p.l.c. (silica) resulted in its decomposition.

• A was identical with the more Polar vinyl ether 223 (137 or 133) (i.r., u.s., t.l.c. (silica, diethyl

ether : petroleum = 3 : 197), m.p. 47 - 49°, m.m.p. = 4_ k = 56°, 46 - 7 , /1 / ilK-/ 58°, 2-1 o - 6 = + To( = + 111 I0.; 7546nm 65 - -7 436nm 25 cc = 0.203, cyclohaxane) ). (silica, dichloromethane) indicated

A was immediately hydrolysed by 5 trifluoroacetic acid 223 in dietlly1 ether giving a more Polar carbonyl comtound.

- 113 - • A224/A2 5/A226 were not redily separated by p.l.c.

and the oil was treated with hydrochloric acid (6N, 1m1.)/

chloroform (4ml.) for 3 hours at room temperature.

The mixture was diluted with diethyl ether and washed

with saturated -;.queoa sodium bic.:rboniate, saturZated

brine and dried. Evaporation gave an oil; t.l.c.

(silica, diethyl ether : petroleum = 3 : 197) indicated

• the formation of both less polar and more polar products,

max. (dId 7) 2920 (s.), 260 (s.), 1715 (n.), 1660 (w.), 1600 (w.), 1460 (s.), 1370 (s.), 1030 (n.), 975(8.)cm.-1

A /A 25 24 were both intractable mixtures of compounds.

•

• •

• - 114 - • p7:A717r:TI 0-7 (1=-):"ATA'S 1:,;;TOT•V;- (121) :-Irundmann's ketone

(121) (32;:, from ergocalciferol) Ire-oared according to a

standard procedure.' was isolated by chromatography on

alumina (30 g.) (eluent diethyl ether : petroleum = 3 : 17)

and purified by subsequent chromatography on alumina (10 g.)

(eluent diethyl ether : petroleum = 1 :.24). The ketone

obtained consisted of 1 major spot on t.l.c. (silica,

dichloromethane) with a minor (ca 1;:) less polar comr.ound,

max. (CHC13) 2940 (s.), 2830 (s.), 1708 (s.), 1455 (s.),

1384 ( s.), 1355 (m.), 970 ( s.) cm:1, 1: 4.30 (2H,

22-H, 23-H), 3.90, 9.02, 9.14, 9.22 (12H, 4 overlapping

doublets, side chain methyls), 9.32 (3H, s., 13-Me),

m/e 276 (M+), 233, 215, 173, 151, 150, 133, 109.

65,136 The ketone was also isolated as its semicarbazone

• (123) (26 from er7ocalciferol (I) ) as a cream coloured 65 amorphous solid m.p. 218-220° (lit. 2220) from methanol,

13 max (CHC1 ) 3530 (n.), 3390(.71.), 2940 (s.), 2870 (s.), 3 1690 (s.), 1565 (s.), 1450 (s.), 1370 (n.), 970 (III.) cm71

lr.". 1.83 (111, 5., rapid excham:e, with D20, N-H), 4.47

(2H, broad s., slow exchange with D20, NH2), 4.81

(2H, M., 22-H, 23-H), 8.92, 9.00, 9.10, 9.22 (12H, side

chain methyls), 9.3? (3H, s., 18-Me), rie 333W), 318,

• 290, 274, 259, 143, 133 (base), impurity at 359. •

Attelpted hydrolysis of the semicarba zone (123) with 137 dimethoxymethane and methanolic hydrochloric acid or 138 sodium nitrite in glacial acetic acid gave 3rundmann's 66 ketone (121) and its C-14 eimer (122) (t.l.c., n.m.r.

evidence).

- 115 - • C. m- ' (120)

n-Butyllithium (0.73m1., 2N in hexane) was added to a suspension of methyltriphenylphosphonium bromide

(m.p. 232.5-234°, (lit.68 232-233) ) (0.52 g.) in THE

(25m1.) and the yellow solution stirred for 4. hours under nitrogen. Grundmann's ketone (121) (8P mg.) in THE (5m1.) was added and the mixture stirred overnight at room • temperature. Diethyl ether (30m1.) was added and the

solution washed with aqueous ammonium chloride (10r,

3 x 30m1.), saturated brine (2 x 20 ml.) and dried. The residue after evaporation was chromatographed on alumina (15 g.) (eluent Petroleum) to give diene (130)

(88 mg.). Subsequent purification by p.l.c:- (one 40 x 20 x 0.1 cm silica plate developed once in diethyl e• ether : petroleum = 1 : 19) gave diene (130) (65 mg., 74%) as an oil, \ max. (CHC13) 2930 (8.), 2870 (. .), 1648 (1.), 1458 (m.), 1370 On.), 973 (.111.), 887 (.m.)cm71,

154.82 (2H, 1M., 22-H, 23-H), 5.27 (1H, d., J=2Hz.,7-H),

5.57 (1H, d., J=2Hz., 7-H), % '9- (3H, d., J=7Hz., 21-Me or 28-Me), 9.09 (3H, d., J=7Hz., 21-Me or 28-Me), 9.16, .9.18(6H, 2 d., J=7Hz., 26-Me , 27-Me), 9.43 (3H, s., 18-Me),

m/e 274(11), 259,231, 176, 175, 161, 149 (base), 147,

• 135, 134, 133, 121, 109, 107, 103, 101, 81, 79, 69, 67, ,-04 7 22.5° = ,10 /7„, 7 22.5° /-04 722.5° = + 48° -/D ' / 578nm. 546nm = 58°, /;<_74;L.I'm = + 100° (C=0.628), (Found: C, 87.52.; H,12.31; C20 H34 requires C, 87.50; H,12.0.

- 116- • ATTNP= '- A CRYSTATLIN-: 1ETIV\TI71 C7 TT= (130).

A solution of diene (130) (12 mg.) and 2, 4, 6-

trimethylbenzonitrile oxide (7.3 mg., 1 equivalent) in

THF (8 ml.) was heated to reflux under nitrogen until t.l.c.

(silica, chloroform : benzene = 3 : 17) indicted complete

reaction (1 week). No further reaction took place when the

reflux was continued after the addition of nitrile oxide

(7.1 mg., 1 equivalent). P.l.c. (1 20 x 20 x 0.04 cm silica

plate developed once in chloroform : benzene = 3 : 17) gave

(in order of increasing polarity) unreacted nitrile oxide

and a product (15 mg., 79;1) which was probably the

isoxazoline (125) as an oil, 157.10 (2H, s., aromatic H),

4.80 (2H, 22-H, 23-1), 7.16 (1H, d., J=11Hz., 14 c< -H),

7.73 (3H, s., 4 -Me), 7.76 (6H, s., 2 -Me, 6 -Me), 8.94, 9.02, 9.13, 9.22 (1211, side chain methyls), 9.33 (3H, s., 18—Me).

Diene (130) was recovered unchanged when treated with

2-nitrobenzonitrile oxide (prepared in situ from

p -nitrobenzohydroximoyl chloride m.p. 123.5-124.5° 139 0 (lit. 125 ) and triet.nylamine) in diethyl ether at room

temperature.

•

• - 117 - RE:T.CTION 07 :17:UNDI':._;- K1=17, (121)

Sodium borohydride (108 mg.) in water (2 ml.) was

added to a solution of Crundmann's ketone (121) (102 mg.)

in dioxin (2 ml.)/(THF (3 ml.) at 0°. After 6-`21- hours

t.l.c. (silica, dichloromethane) indicated complete reaction

and the formation of a major (less polar) and a minor (more

polar) alcohol. Diethyl ether (30 ml.) and saturated brine • (20 ml.) were added and the aqueous phase extracted with

diethyl ether (30 ml.). The combined ethereal solutions

were washed with saturated brine (3 x 20 ml.) and dried

(Na After removal of solvents the alcohols were 2 s04) searated by p.l.c. (one 40 x 20 x 0.1cm silica plate

developed once in diethyl ether : petroleum = 3 : 7) to

give the major 8p alcohol (126) (86 mg. 84) and the minor

• alcohol (6 mg., 6) both as colourless oils.

8(3 - AL7,C:7CI, (126), t) .pax. (CHC13) 3430 (it.), 2930 (s.),

2870 (s.), 1590 (a.), 1490 (s.), 1457 (s.), 1365 it.),

1293 (Tn.), 1162 (a.), 1110 - 1040 (broad m.), 970 (s.)cm.T1

1;4J',0 (2H, M., 22-H, 23-H), 5.93 (id, n., WH=6Hz.,

80( -H), 8.97, 9.03, 9.12, 9.23 (12H, side chain methyls),

9.04 (3H, S., 18-Me).

111 6 Alternatively the reduction was carried out using 140 sodium berohyd:.-icle in :liglyme. The re _action was followed by t.l.c.; no e,imrisation- of Orundann's ketone (121) was

observed nrior to reduction and the n17ohols obtnined were

identical to those obtained from the -crevious reduction.

• - 118 - • However total reoval of diglyme (under high vacuum

followed by partition between water and petroleum was

difficult.

The crude alcohol (126) from the reduction of

Grundmann's ketone (121) (139 mg.) converted into its

3,5-dinitrobenzoate (129) was obtained as pale yellow glistening

plates m.p. 140.5-141.5° ( lit.71 146°) (116 mg., 47(A from

acetone/methanol (recrystallised twice), 1) max. (CHC13)

3090 (W.), 2930 (m.), 2370 (m.), 1727 (s.), 1623 (m.),

1595 (w.), 1548 (s.), 1455 (m.), 1348 (s.), 1288: (M.),

1268 (m.), 1170 (w.), 1152 (11.), 970 (M.), 940(w.),

910 (w.)cm., 1: 0.79 (3H, M., aromatic-H), 4.49 (IH, m.,

tiles = 6Hz., 8 0(. -H), 4.77 (2H, M., 22-H, 23-H), n.90, 9.02,

9.10, 9.20 (12H, side chain methyls), 8.87 (3H, s., 13-Me), 71)22o= 70o, m/e 472 (M4.), 347, 346, 345, 260, • ibz 22° o 22o 82o /7 722 o 154 7 = + 148 - 578nm . 72 , - 546nm ' -/ 436nm (C = 0.289), (Found: C, 65.83; H, 7.55; N, 6.10; C-26-36 H N206 requires C, 66.06; H, 7.68; N, Alcohol

(126) (47 mg., 94) was obtained from the saronification

of the 3,5-dinitrobeozoate (123) (26 mg.).

P=.12AATIO:1 CF DI NT., (11).

• (126) (47 • A mixture of alcohol mg.), pyridine (2 ml.) and methane sulphonic anhydride (91 mg.) was stirred at room

temperature (1R- hours) until t.l.c. (silica, diethyl ether :

petroleum = 1 : 1) inijicated complete conversion to the more 0 polar mesylate. The temrerature was increased to 60+5

(8 hours) then 80+5° (2 hours) when t.l.c.

complete diene formation. Water (1 ml.) was added and

- 119 - • after 15 minutes the mixture extracted with diethyl ether

(3 x 5 ml.). The ether extract was washed with saturated aqueous sodium bicarbonate (3 x 5 ml.), saturated brine

(5 ml.) and dried. After removal of solvents diene (112)

(36 mg., PZ:) isolated by p.l.c. (one 40 x 20 x 0.1cm silica plate developed once in diethyl ether : petroleum =

1.: 49) was obtained as an oil, max. (film) 2950 (s..), 2920 (s.), 2260 (s.), 1455 (s.), 1430 (4.), 1330 (M.), 1367 (s.), 1200 (w.), 1105 (W.), 1015 (M.), 920 (s.),

950 (w.), 930 (W.), 928 (w.), 910 (w.), Boo 1) max (CHC1 ) 1600 (w.)-c- , T.4.74 (1H, m., 8-H), 4.78 - 3 (2H, m., 22-H, 23-H), %98 (3H, d., J=7Hz., 21-Me or

28-Me), 9.08 (311, d., J=7Hz., 21-Me or 23-Me), 9.16, 9.18

J = 7Hz., 26-Me & 27-Me), 9.09 (3H, s., 18-Me),

/-04!_717= 9.1°1°47 C,nm 9'9°' /-c5 -7 f=46nm=

41 11.10 (C=0.362), m/e 260 +10.5°, /-°(..7 36nm = (e), 245, 231, 217, 189, 176, 161, 149, 147, 136, 135, 134, 133

119, 109 107, 105, 95, 93, 91, 83, 79, 77, 69, 67, 57, 55, 53, (Found: C, 87.86; H, 12.29; C101132 requires C, 87.61;

FREFAATI N 07 sfA7',TATE (129)

Sodium hydride (41 mg., 80% in mineral oil) was washed

• with petroleum (sodium dried, 10 x 2 ml.) and added as a THE slurry (2 x 1m1.) to a solution of alcohol (126) (35 mg.)

and imidazole (4 mg.) in THE (5 ml.). A pink colour was

produced and rapidly discharged. The mixture was heated to

reflux under nitro7en (5-,±I hours) ';7hen carbon disulphide was

added and reflux continued (l 'hour). Iodomethane (0.5 ml.) was added to the orang :7olution and reflux continued (;,L hour)

1 - 120 - Diethyl ether (20 ml.) was added and the reaction mixture

washed with saturated brine (3 x 10 ml.), dried and

evaporated to leave a red oil. Chromatography on alumina

(grade H, 5g.) (eluent diethyl ether : petroleum = 1 : 24)

and p.l.c. (one 40 x 20 x 0.1 cm silica plate developed

once in diethyl ether : petroleum = 3 :'97) gave xanthate

(129) (39 mg., 84) as a colourless oil.

• Alternatively the sodium hydride was replaced by

methyllithium but purification of the xanthate was more

difficult because a side product (t 7.27 (3H, s.),

8.39 (3H, s.) ) probably methyl dithioacetate was close in polarity to the xanthate, '? max. (CHC1,) 2930 (s.),

2870 (s. ), 1600 (w.), 1460 (s.), 1370 (ill.), 1342 (w.),

1314 (w.), 1295 (w.), 115o (8.), 1043 (s.), 980 (s.), • 948. (w.), 930 (w.), 908 (W.), 870 (m.)cm71, 15 4.03 (1H, = 6Hz., 3 0K -H), 4.32 (2H, M., 22-H, 23-H),

7.43 (3H, S., S-pie), 8.93, 9.03, 9.13, 9.23 (12H, side chain methyls), 9.03 (3H, S., 18-Ne), M/e (50°) M4- (absent),

260 (M+-COS-MeSH), 6o (cos), 43 (MeSE), 47 (CH2=H), (Found: C, 68.51; H, 9.67; C21H360S2 requires

C, 68.4o; H, 9.85).

• PYROLYSI:7, OF XAI.11'HATE (129)

+ o Xauthate (129) (76 mg.) was r:yrolysed (250 10 )

under argon at one atmosphere for 5 hours collecting the

distillate on a cold finger. Diene (119) (30 mg., 55) isolated from the distillate by p.l.c. (3 20 x 20 x 0.04cm

10 silver nitrate/silica plates developed twice in benzene

• - 121 - • retroleum = 1 : 19) was obtained as an oil, A) max. (film)

3010 (m.), 2950 (3.), 2920 (s.), 2'65 (s.), 145P. (S.),

1370 (s.), 1225 (w.), 980 (s.), 675 (s.)c;71.1 1) max (CHC13)

1632 (w.)cm:1, 'IC 4.43 (2H, m., 2-H, 9-H), 4.80 (2H, m.,

22-11, 23-1I), 8.97 (3H, d., J=6.8Hz., 21-Me or 28-Me),

9.08 (3H, d., J=6.9Hz., 21-Me or 28-Me), 9.16 (3H, d. J=6.6Hz., 26-He or 27-M2), 9.18 (3H, d., J=6.7Hz., .721° o 26:77570:n2m7-.M:),419.30/ (ZH,7 S., 18-Me), / = + 39 , o o f:, .7 21436nm 4' 86° ' 546nm = ' = (C=0.590), m/e 260 (M+), 217, 199, 175, 161, 147, 136, 135, 134, 133, (Found: C, 87.89; H, 12.36; C H requires 19 32 C, 87.61; H, 12.39;.).

Pyrolysis of the xanthate at 220:1 10° at 0.1mm. resulted in the xanthate distilling unchanged.

P:EPARATION DI: NE (117)

A solution of diene (120) (21 mg.) in THE (5 ml.) and aqueous sulphuric acid (30: v/v, 1.5 ml.) was heated

to reflux (3-2: hours) under nitrogen when t.l.c. (10';:: silver nitrate/silica, benzine : petroleum = 1:19) injicted complete

isomerisation. Diethyl ether (20 ml.) and saturated aqueous sodium bicarbonate (20 ml., dropwise) were added and the • • organic phase washed (saturated aqueous sodium bicarbonate

(3 x 20 ml.), saturated brine (2 x 10 ml.) ) and dried.

Evaporation gave diene (117) (21 mg.,107.) an oil which

was TAarified by p.l.c. (one 20 x 20 x 0.04 cm.10;7: silver

nitrate/silica Plate developed once in benzene: petroleum

= 1:19), V max (CS,) 2960 (s.,), 2930 (s.), 2P;70 (s.),

1370 (3.), S'70 ,s.)cm., 1.4.77 •(2H, m., 22-H, 23-H), .4P,

• - 1P2 • (3H, S., 7-ne), 8.93, 9.03, 9.13, 9.23 (12H, side chain methyls), 9.13 (311, s., 18-Me) /oc, / ' = + 12.4°, 'o D o /---/ 7 23o= 12.8° o 7 23 = +14.214.2°° , /--/ 7 3 / 578 ', ! -/ 546nm '1/4.-/ 436nm = + 19.6? (C=1.08), _rr/e 274 (M+), 259, 245, 231, 203, 139, 175, 162, 161, 149 (base), 133, 123,. 107, 93, 81, 69, (Found:

C, 87.37., H, 12.28; C20H34 requires C; 87.50; H, 12.50;).

EranEIRISA2IC:: OF G=NDEANE'S K7TON: (121)

The epimerisation of Crunimann's ketone (121) (67 mg.)

in deuterochloroform (0.5 ml.) containing trifluoroacetic

acid (0.02 ml.) was followed by n.m.r. until equilibrium was

reached (3 days). The base catalysed epimerisation of

ketone (121) 5 mg.) in d4-methanol (0.5 ml.)/sodium deuteroxide in D70 (40%, 1 drop) was also followed by n.m.r.

(rapid reaction). Both the acid and base catalysed products • were identical on t.l.c. (silica,- dichloromethane) consisting of the less polar 1413 -H ketone (122) ( >9%) and

Grundmann's ketone (121) ( 4:%). Separate neutralisation,

extraction with ether, evaporation and chromatograrhy on

alumina (grade H, 3g.) (eluent diethyl ether : petroleum = 66 1:24) gave ketone (122) (60 mg., 33';':) contaminated with

ketone (121), 1) max. (CHC1 ) 2920 (s.), 2870 (s.), 1705 (s.), 3 1462 (s), 1372 (s.), 1320 (a.), 1160 (w.), 1110 (w.), • 975 (s.), 958 (w.), 885 (w.)cm:1, 't 4.80 (2H, m., 22-H, 23-H), S.93, 9.03, 9.07, 9.13, 9.23 (12H, side chain methyls), F.93 (3H, s., 18-Ye), m/e 276 (1:), 253, 215, 179, 178, 152, 151, 133, 125, 123, 111, 109, 107, 105, 78.

4 - T23 -

• SO7IUY1 BOR=DTID-:: CF KET= (122)

Sodium lio_tohydride reduction of ketone (122). (5 mg.) in aqueous THF/dioxan and repeated p.l.c. on silica (multiple development in diethyl ether : petroleum =

1:3, 7:13) gave the less polar alcohol (131 or 132) (29 mg., 34(A as an oil, 15, 4.74 (2H, n., 22-H, 23-H), 6.14 (1H, M., Wu = 16Hz., 8-H) 8.97, 9.00, 9.10, 9.23 (12H, side chain methyls), 9.10 (31„ 18-Me) and the

more polar alcohol (131 or 132) (36 mg., 42) as an oil, T...4.8o (2H, r., 22-H, 23-H), 6.63 (1H, m., WH = 16Hz., 8-H), 8.95, 9.03, 9.10, 9.23 (12H, side chain methyls), 9.10 (3H, s., 13-Ne).

Both alcohols converted into their 3,5-dinitrobenzoates were obtained as oils. T.l.c. (silica, developed 3 times / more in diethyl ether : petroleum = 1:9) indicated the/p olar alcohol gave the less polar ester (and vice versa) and p.l.c. via the 3,5-dinitrobenzoates would also be difficult. Each ester was nurified by chromatography on alumina (2g.)

(eluent benzene); more polar ester (133 or 134) 74° o 2 ° o &_7; = - 13°, L'_7 nm= -15 ' /°.L7546nm -17°,

/ 042.., "34° = -33° (C = 1.74 ), To.87 (3H, m., aromatic H), 4.50 (1H, m., 8-H), 4.67 (2H, m., 22-H, 23-H), 3.73, n.37,

• 8.95, 9.07, 9.15, 9.23 (15H, side chain ,-,nd 18-methyls); ° less nolar ester (133 or 134), /oc../-,24D = +45°, -0( 24° 50 7 24° /-04 7 = + 42° / 7 = = 573 nm • - - 546 rim ' 49‘ -/ L 76nm + 97° (C = 0.533), 1.) max. (01C13) 3100 (m.,), 2930 (s.,) 2870 1728 (s.), 1630 (M.), 1550 (s.), 1460 (m.), 1370 (M.), 134" (s.), 12F0 (s.), 1170 (w.), 975 (h.), -1 960 (w.), 917 (w.)on., '1,0.87 (37, a., aromatic H),

- 124 - • 4.77 (2H, n., 22-H, 23-H), 4.97 (1H, WH = 16Hz.,

8-H), 8.90, 9.03, 9.13, 9.23 (12H, side chain Me), 8.95 (3H, s., 18-Me).

In a subsequent reduction the alcohol mixture converted

into the 3,5-dinitrobenzoates and purified by p.l.c. (one 20 x 20 x 0.4 cm silica plate developed once in diethyl ether : petroleum = 1:9, esters not resolved) was obtained • as a yellow oil, /C,.(_7 3.)3'5 °.= + 21°, / 64_7 Pe° = + 22° , o o 23.5 /7, 7 23.5 = + 23 /-IN! = + 42° (C = 0.228) (s-‘‘-/ 46nm 7 hence the ester mixture contained 57,58% dextrorotatory

isomer), m/e 472 (M+),457, 429, 347, 260, 245, 217, 195, 176, 163, 162, 161, 149, 135 (base), 133, 125, 124, 109,

107, 97, 95, 93, 83, 81, (Found: C, 65.91; H, 7.84; N, 5.94; C2036N206 requires C, 66.06; H, 7.68; N,5.94 • Alcohols (131/132) (67 mg., 91';0) from the hydrolysis of the

mixed esters (133/134) (125 mg.) were obtained as an oil.

P=PARATION OF DI7::2 (130)

A solution of Mixed alcohols (131/132) (55 mg.) and

methane sulphonic anhydride (77 mg.) in pyridine (1 ml.)

was stirred at room temperature (50 minutes). Toluene

(2 ml.) was added and the mixture heated to reflux until

• t.l.c. (silica, diethyl ether : petroleum = 2:23) indicated

complete dehydration (170 minutes). Work ur, chromatoTraphy

on alumina (2 g., eluent petroleum) and p.l.c.

• (2 20 x 20 x 0.04 cm 10c: silver nitrate/silica plates developed twice in benzene : petroleum = 1:19) gave diene

(118) (23 mg. identical (t.l.c., n.m.r.) to that

- 125- • previously prep -.red and liene (130) (14 m:-2;., 23: ),

-*max (CS2 ) 3020 (a ) 2950 (s.), 2920 (s.), 2.70 165o (m.), 1370 (s.), 972 (s.), 880 (J.), 810 ('.), 796 (LI.), 744 (w.), 710 (q.)cm:1, 1:4.47 (2H, rP.., S-H,

9-H), 4.80 (2H, 22-H, 23-H), 8.97, 9.07, 9.15, 9.17, 9.27 (12H, side chain methyls), 9.13 (31I, s., 18-Me), o o o /z5 7 22 -oe 7 22 370, 7 2 _39o, - -32°, i 57Flam !s'-/ 546nm 69° Az 7 - (C == 0.244), m/e 260 (M+), 245, 217, • /c..4-36:6rm = 189, 161, 136, 135, 134, 133, 119, 109, 107, 105, 93, 91, (Found: C, 87.32; 11, 12.31; H requires C, 87.61; 9 32 - H 12.39).

P1:11;PATIO CT 3:1::=in ::ETCNE TOLUTE- r - SIF,PHCTLLET,)2AL:01:::; (135a)

A solution of Grundmann's ketone (121) (101 and toluene-n- • sulphonylhydraide_inMe0H(3 ml.) was stirred at room temperature until t.l.c. (silica, chloroform) in-licated complete reaction (6 hours). Chromatography on alumina (10 g.) (eluent ether : -oetroleum = 1:19, 3:7) gave the title corrxound (135a) (151 ms., 93::) as an oil, ),) max. (CHC13) 3235 (.1t.), 3210 (Jr..), 2930 (s.), 2870 (s.), 1630 (s.), 1600 (m.), 1490 (v.), 1460 (s.), 1380 :s.), 1340 (s.), 1310 (w.), 1290 (w.), 1160 (s.), • 4 1120 (m.), 1090 (m.), 1005 kw.), 1000 (w.), 970 (p.), -1 910 (_ti.)cm., 1; 2.14, 2.71 (511, A3g., J=FHz., aromatic H, 11-H), 4.80 (211, 22-H, 23-H), 7.57 (3H, s., aromatic re), 9.00,9.10, 9.20 (1511, site c]iain and 1:-methyl ), + 9.60 (ir=rit, (150°) L1,4 (r ), 2:9 (ba se,

Ne-'""674" SO2 ) 260 ), 245, 217, 135, 548 (impurity),

- 126- a 24° 2 (:) ,-_, 724° 0 -..( ct.< 7 . + = + 26 , / 7 24° = + 29 , - - D -, 1 ... "'"'-'578nm -. 54onm ° 20 ro(_ 7-436nm . . +49° (C = 0.824).

In a subsequent prenaration the toluene-p sulphonylhydrazone was obtained as white needles m.p. 115-117° from diethyl ether/petroleum /oC. 7 24.° -32°, o - D 24o 24 L 47- 578nm = -34°' /°4]546nm =-38°' 1" 7 1=4: nm -81o (C = 0.524), j5,(CC14) 2.20, 2.73 (5H, ABq., J=8Hz aromatic H and N-H), 4.83 (2H, m., 22-H, 23-H), 7.57 (3H, aromatic Me), 8.95, 9.03, 9.12, 9.20, 9.53 (15H, methyl neaks).

evaporated. TheZmother liquor was recrystallised three times from

diethyl ether/petroleum to E7ive white needles m.n. 102.5-105°, o o 22 i-iv 7 22(3 ro< 7 D = + 15.2°, = +15.9°, /-04.! o(7-/ ../., = o /--‘"-/ 578nm _,,6nm , ,o /-,,,i 7 22 _ + 17.5° / '''.-/ 4.46nm - + 24.6° (C .=. 0.709), which after 30 minutes in chloroform solution increased to + 28°, o o o +29 , +33 and +57 respectively. The n.m.r. spectrum was identical with the oil from the 1st preparation, We (175°) 548, reak at 444 absent, 282, 162, 156, (Thund: C, 70.05; H, 8.89; N, 6.20; 0, 7.46; Calculated for

C N S C, 70.21; H, 9.07; N, 6.30; S, 7.22%) 2oEH40 2 02

Both the laevorotatory and dextrorotatory solids were

identical on t.l.c. (silica, chloroform, multiple development).

- 127 - PEACTTON T3= 7,7 (17.7 p SULETCNYLMIGIT:: (135a) ,',ED 7,UTY:LI7=.

Butyllithium (0.37 ml., 2N in hexane) was injected

into a solution of toluene- p -sulohonylhydrazone (135a)

(83 mg.) in diethyl ether (2 ml.) under nitrogen. The

solution became yellow in colour and gas evolution took

place. After 10 minutes water (5 ml.) and -7.iethyl ether

(10 ml.) were added. The organic chase was washed with

water (3 x 10 ml.), saturated brine (10 ml.) and dried.

After removal of the solvents, the residue was purified by

p.l.c. (one 4C x 20 x 0.1cm silic plate developed once in

diethyl ether: petroleum = 3:197, followed by one

20 x 20 x 0.04cm 10 silver nitrate/silica developed twice

in benzene: petroleum .= 1:19) to give the more polar diene

• (130) (17 mg., 35;'::) and the less polar diene (119) (8 mg., 16) identical (t.l.c., n.m.r.) with samples from

unambiguous syntheses.

CD7' TnUENE- 2 -J177, (135b) T OLU=7- n -:717= , ==27=A1 (135b) (6fl mg., 71Y) from

ketone (122) (60 mg.) and toluene- F -sulphonylhydrazide

(47 mg.) was obtained as white needles m.p. 99-102° from

diethyl ether/petroleum. A subseouent recrystallisation

• from aqueous ethanol gave white needles m.p. 100-104°, o - 3 = ,,o 723 = + 21° /-o< 7 3 ° = 24o, /- 0 (- /D ' 578nm ' '~46nm 23° + 33° (0 ( 14)4. /7 436nm = 1.048) after 2 hours the o o o 0 rotations had increased to + 29 , + 31 , 4- 35 and + 60

resectively, -1) max. (nujol) 3200 (s.), 1622 (w.),

1595 (m.), 1460 (s.), 1400 (14.), 1380 (.5.), 1335 (5,),

* - 128- • 1290 ( !..), 1210 ('.'. ), 1188 (w.), 1170 (o.), 1090 (rd.),

1030 (n.), 1015 (w.), 970 (m.), 950 (w.), 927 (.),

91 0 (w.), 9i0 (s.), 710 (w.), 690 (n.)cm:1, 1:2.131

2.70 (5H, ABq., J=8Hz., aromatic H, N-H), 4.80 (2H,

22-H, 23-H), 7.57 (3H, s., S-Me), 9.02, 9.08, 9.12, 9.20

(15H, methyl peaks), /e (1500) 548, 529, 364, 276, 262,

246, (High resolution mass measurement 548.5085

Calculated for C H N (azine (136) ) 548.5069). • 38 64 2

REACTION 7-3ETTEN ToLTTE- -SITTPHONYLITTDRAZONE

(135b) AND NETTTYLLITHIUM

T.l.c. (10% silver nitrate/silica, benzene: petroleum

1:19) indicated diene (130) was the only steroidal product

formed on treatment of toluene- p. -sul'ohonylhydrazone (135b)

with methyllithium in diethyl ether. •

PRET=AIZATION OF 7=OXYlIETHYL=.7=1YLIT.031=HONIUM CHLO=E

The title compound (25.3g., 93) prepared as for the

79 methoxy-analogue from triphenylphosrhine (20g.) and

chloromethyl ethyl ether (7.6g.) was obtained as a white

solid. Crystallisation from chloroform/ethyl acetate

(twice) gave white prisms m.p. .04.5 -205, -0 max (AUjol)

• 1585(.), 1483 (s.), 1440 (s.), 1325 (m.), 118' (w.), •

1155 (m.), 1 115 (s.), 1090 ( 5.), 1010 ( w.), 1000 (s.),

930 (w.), 910 (n.), 265 (m.), 796 ( m.), 770 ( 5.), 752 ( .3.),

(15H, M., aromatic H), 725 ( s.), 695 ( 3.)cm:1, "r; 2.24

4.07 (2H, d., JP../1 = 4Hz., 0H7), 6.06 (2::, q., J=71iz.,

-212 CH3), :8.83 (3H, t., J=7112., -CH22113), (Found:

C, 70.68; 6.04 ; 11 C1 OP rerm i res C, 70.66; H, 6.22). 21 22

• - 129 -

• CF VT=I:T77'.3 (137) L.TD (137;)

"3utyllithium (0.92 ml., 2N in hexane) was injected / methyl into a suspension of e-L:ox*rir,henylphosphonium chloride

(0.70 g.) in THF (10 ml.) under nitrogen and the bright

red ylide solution stirred for 1 hour at room temperature.

Grundmann's ketone (121) (69 mg.) in THF (3 x 2 ml.) was

added, the mixture stirred overnight and worked up to give • a red oil. Extraction with petroleum (10 x 5 ml.), and chromatography on alumina (3 g.) (eluent petroleum) gave

an oil (220 mg.). P.l.c. (one 40 x 20 x 0.1cm silica plate developed once in diethyl ether : petroleum = 3:197)

gave vinyl ethers (137) (138) both contaminated by

triphenyl:hosphine. The triphenylphosphine was removed by treatment with iodomethane in petroleum and filtration

• through alumina. The more polar vinyl ether gave a white solid (17 mg.) from acetone/methanol; p.l.c. of the mother

liquor gave additional solid (3 mg., 25;7:: overall).

Repeated (3 times) recrystallisation from acetone/methanol

gave white plates m.p. 46-42.5°, o o n4o 21 oo - 21 = + 610 f-04. 7 = + 690 /-c47 ! -/ 546nm Do = 5° ("

• 1432 (s.), 1372 (s.), 136^ (6.), 1322 (W.), 1255 (s.), 1212 (M.), 1160 (S.), 1120 (s.), 1040 (m.), 1020 (w.), 990 (W.), 970 (w.), 970 (s.), nSo (w.), FL';o (w.), 24o On.), 225 (.,790 (w.)cm:,1 7: E.40 (111, s., 7-H), 4.2o (2H, m., 22-H, 23-H), 6.35 (2H, q., j=711z., C72-0H3), r%rs0 (3H, t.,

J=7Hz., -CH_CH-), 3.93, 9.03, 9.13, 9.23 (12H, side chain He),

9.43 (3H, S., 17-He), m/e 312 (MI.), 304, 275. 272, 259, 229,

• _ 130- 201, 193, 149, 147 (base), 135, 133, 105, (Found:

C, 83.16; Ii, 11.923C27,H23 0 requires C, 82.94;

H, The less Polar vinyl ether (20 rig., 255.) o o 22 22 was obtained as an oil, /-0< = + 49° /-0Z -7 D ' 7 578nm

51°, /_)6nm4. = 58°, /-6c7&nm + 102° (C = 0.352, cyclohexane), 'V max. (film) 2950 (s.),

2930 (s.), 2870 (s.), 1685 (M, 1460 (..%), 1380 (-ts.), 1298 (m.), 1238 (111,1 1210 (.m.), 1190 (M.), 1150 (s.),

1135 (s.), 1112 (m.), 1048 (m.), 1018 (W.), 970 (s.), 880 (w.), 863 (w.), 151:-.14 (1H, s., 7-H), 4.8o (2H, rt., 22-H, 23-H), 6.40 (2H, q., J=?Hz., -CH2CH3),

8.83 (3H, t., J=7Hz., -CH2c11.3), 2.93, 9.03, 9.13, 9.23, (12H, side chain methyls), 9.32 (3H, S., 18-Me).,

m/e 318 (1.1 +), 304, 275, 272, 259, 229, 201, 193, 149, 147 (base), 135, 133, 105, (Found: C, 83.15; H, 11.78; a 0 requires C, 82.94; H, 12.03). C22H38

SEPA:-ZATION OF B TOXI372ROLS 1

The BC toxisterols were separated by chro:1,1tography on alumina (elaent diethyl ether : petroleum = 0:1 - 1:9)

B B /B to give (in order of increasing polarity) B1' 2' 2 3' (1.35 g.) and C all as oils. B3/B4

• S Repeated p.l.c. of the B1 toxisterols (silica,

40 x 20 x 0.04cm plates developed 6 times in diethyl ether:

petroleum = 3:47, 20-25 mr;.- per plate gave (in order of (24 mg.), B12 (155 m:.) and increasing -colarity)311/312 B13 (54 mg.) all as oils. Attem2ted p.l.c. on 10 silver nitrate/Tilica resulted in decomposition.

- 131-

• 24 4 B12 /-04.04. = + PO° / - --,D-7D $ -C< -/ -157o nnm = + 830, 24 24 0 = 96° /-0 7- n roc -7 54 6nm - - 436nm = 102 (C = 0.292, cyclohexane), V max. (film) 2960 (s.),

2930 (s.), 2870 (s.), 162' (w.), 1460 s.), 1320 (s.),

1370 (s.), 1318 (M.), 1300 (W.), 1285 (M.), 1265 (we ),

1215 (m.), 1200 (w.), 1185 (w.), 1150M), 1110 (.),

1080 (w.), 1020 (m.), 1000 (rn.), 970 (s.), 950 (M.),

(w.), 365 (m.), 850 (w.), 240 (w.), 330 (W.), • 925 ( 11.), 795 (m.)cm:,1 Amax. (cyclohexane) 243 (22,000), 252 (26,000),

262nm. (19,000), Y (CC14) 4.05, 1 -.20 (minor), 4.62 (2H, ABq.,

broadened at -4.05, J=11Hz., 6-H, 7-H), 4.83 (2H, rn., 22-H,

23-H), 5.52 (1H, t., J=5.511z., 5c< -H), 2.49 (311, 3., / 9.03 19-Me), 2.98X(6E,2A., J=7Hz., 21-Me &- 28-Me), 9.16 (3H,

d., J=6.5Hz., 26-Me or 27-Me), 9.13 (311, d., J=6.5Hz.,

26-Me or 27-Me), 9.44 (3H, s., 12-Me), 'We 596(M), 331,

353, 271, 133 (base), (=Found: C, 84.54; H, 11.07;

C H 28 44 0 requires 0,84.77; H, 11.19).

B 24° 7 240 13 /74 / = + 115°, / /off 7 24 - D ro< = + 121° o - - 578nm ' !-'-' 546nm 24 + 140° /C.( 7 268° (C = 0.807, cyclohexme), 1 436nm = )„imax. (film) 2960 (s.), 2930 (s.), 2270 (s.), 1710 (W.),

1620 (w.), 1460 (s.), 1380 (.s.), 1370 (s.), 1318 (m.),

1300 (w.), 1285 (w.), 1255 (w.), 1215 (in.), 1195 (M.),

• 1150 (w.), 1115 (M.), 1085 (W.), 1065 (W.), 1020 (W.),

1000 (w.), 975 (s.), 950 (W.), 925 (w.), 880 711.), '70 (P.),

855 (m.), 240 (m.), 790 (w.), 760 (W.)cm:,/ )% max. (cyclohexane) 2_43 (712,000), 252 (26,0("0), 262nm (1:.,oco).,

vocio 3.91, ''.35 (2H, ABq., J=1211z., 6-H, 7-11), 4.84

(2H, m., 22-H, 23-H), 5.55 (1H, t., J=5.5Hz., 3o( -H), 8.53

- 132- • (3H, s., 19-Me), 3.93 (3H, d., J=6.5Hz., 21-Me or 27-Me),

9.09 (3H, d., J=6.5Hz., 21-Me or 23-Me), 3.17 (3H, d., J=6.5Hz., 26-Me or 27-Me), 9.13 (3H, d., J=6.5Hz., 26-Me or 27-Me), 9.34 (31I, s., 18-Me), M/e 396 (e),

381 ,. 353, 271, 133 (base),(Found: C, 84.57; H, 11.10; 0 requires C, 84.77; H, C28H44

EPDXIDATICN CY TOXI:3T::.:201, B12 •

3-chloro:)erbenzoic (21 mg.), toxisterol B12 (43 mg.), diethyl ether (10 ml.) and sodium bicarbonate (101 mg.) were stirred for 4 days at 00. The mixture was treated with cyclohexene (1.0 ml.) for 1 hour, filtered through alumina (5 g.) (elu,:nt diethyl ether : petroleum = 1:4). P.l.c. (four 20 x 20 x 0.04 cm silica plates developed

• once in diethyl ether : petroleum = 3:7) gave (in order of increasing polarity) B12 (4 mg.), toxisterol B12 epoxide-1

(15 mg., 37) and B12 epoxide-2 (14 mg., 34:,).

Bi, El)OXIDE-1 was obtained as white needles m.p. 6 23° 140.5 - 142° (lit. ' 137-139°) from methanol / / = 0 ' - D 7 2-I-z° = no, / 7 23 690, IC< % . Po° o - 578nm 23 0:exane). 1) max. o(- 7-1-36nm 1 = 139° ( C "110: C5:611 • (nujol) 1218 (ii.), 1202 (m.), 1157 (m.), 1110 (S.), 1020 (m.), • 970 (s.), 950 (M.), 910 (M.), 870 (s.), 860 (M.), 838 ( .)cm:1 1,4.?0 (2H, M., 22-H, 23-H), 4.98 (1H, d., J--r.Hz., vinyl

6-H or 7-H), (iP, 3:K -::), 6.72 (1H, d., epoxide 6-H or 7-H), 7.52 (211, Tn., 4 a:-H, 4 , 8.50

(311, S., 19-Me), 8.93, 9.03, 9.12, 9.22 (1211, side chain Me), + 9.32 (311, S., 17-Me), m/e 1-12 (M )1 397, 394, 334, 363, 369, 354, 161, 137, 136, 135, 134, 133, (Found: C, 71.33; II, 1.461

ar -1.33- • requires C, 81.4n; H, )„ C28F44' 02

TOXIT2EPLI, Bi,'0XIDE-2- was an oil, '1.4.83-5.57

(m., vinyl 3 01,E), 8.27, 8.50, 8.53, 8.93, 9.00, 9.10,

9.20, 9.27, (Methyl peaks). T.l.c. (silica, diethyl ether :

petroleum = 3:7, developed twice) inicated two major and

one minor components.

• Treatment of toxisterol B ePoxide-1 (1.6 mg.) with 12 - THF (1.0 ml.)/water (0.30 ml.)/trifluoroacetic acid (0.10 ml.)

gave a mixture of more polar compounds.

ATTENPTED 0X:IDATIVT:; CLEAVAGE OF TOXIS=01, B12 7„7. 0]:IDE-1

Periodic acid (10 mg.) in THF (0.40 ml.) was added to

• B epoxide-1 (16 mg.) in THF (3.0 ml.) under nitrogen. 12 After 2 hours at room temperature a white solid had

precipitated and t.l.c. (silica, diethyl ether : petroleum =

3:7) indicated icsence of starting material and carbonyl

compounds and formation of polar product(s). Additional

periodic acid (0.40 ml., THF solution) was added after six

hours. T.l.c. in.licat',id no carbonyl compound formation

after 23 hours (total). Diethyl ether (20 ml.) was added

the solution was washed with saturated aqueous sodium

bicarbonate (10 ml.), saturated brine (2 x 10 ml.) and dried.

va gave an oil (29 mg. soluble in benzene but

insoluble in petroleum, m/e 556, 554, 552, 45?, 442, 440, 42g, 426, 424, 410, 409, 408.

Ring A ketones or aldehydes';!re not detected in the

oxidation of toxictorol 3 using ersocaiciferol (I) 12 P i oxic7ative degrade ';ion.

• _134 - • 7.ROLS .021:::.;ATTO::- •OF B2 7=37

was converted into a mixture Toxic:sterol fraction B2 141 , of 3,5-dinitrobenzoates wnicn gave yellow needles of 3,5-dinitrobenzoate(151b) (190 mg.) m.p. Toxisterol B21 173-174° from :acetone/methanol (111.62 173-174°), 20° 0 ,o /704 7 = -1.3° /-o‘ 7 " -1.4° /-- o4- 7546nm = - - D o - - 578nm - 0 -21 -3'0°' /7)( -7 43 6nm = ° (C = 0.989), 1)max. (nUjol) 1730 (,$.), 1630 (a.), 1600 (4.), 1553 (.s.), 1350 (s.), 1330 (w.), 1315 (w.), 1275 (s.), 1175 (s.), 1078 (s.), 1035 ( 1005 ( w.), 990 ( w.), 970 (;s.), 930 (34) , 735(s.), 922 (.m.), 900 965 (JO , 825 (.m.), 765 ( s.) ,725 ( s.)cm:; 'max. (cyclohexane) 232 nm. (28,000). -"C.. 0.72, 0.83 t., J = 7Hz., 6-H, 7-H) (3H, tri., aromatic-H), 3.68 (24 4.71 (1H, dt., J1=10Hz., J2 = 2.5Hz., 3 c{ -H) , 4.81 (1H, dd., J1 = 15.5Hz., • J = 7.5Hz., 22-H or 23-H), 4.99 (1H, dd. = 15.5Hz., 2 ' J1 J2 = 8Hz., 22-H or 23-H), 7.00 (1H, d., J = 10Hz., 4 p -H), 8.28 (3H, -S.,19-Me), 9.06 (3H, d., J = 7Hz., 21-Me or 28-Me), 9.13 (3H, d., J = 7Hz., 21-Me or 2-Me), 9.15 (31I, d., J = 6.5Hz., 26-Me or 27-Me), 9.17 (3H, d., J = 6.5_'z.,z., 26-he or 27-he), 9.10 (3H, -S., 18-e),M 8- (13C) 161.97 (.%, C=0), 148.82 (s., 3 -c, 5 -0), 140.37 (d., 7-C), 134.82 (.s., 1/-0), 134.34 10-0), 135.32 (d., 22-0), 132.05 (d., 23-C), 129.54 (a., 2/-0), 6/-0), 129.23 (a., 6-0\, • 123.53 ( s., 5-C), 122.39 (d., 4 -C), 76.51 (d., 3-C), 57.80 (d., 17-C), 56.12 (d., 14-C), 53.94 (s., 7-C), 50.76 (d., 4-0), 43.72 (3., 13-C), 42.62 (d., 24-C), 40.30 (t., 12-C), 40.10 (d., 20-C), 35.81 (t., 1-C), 33.08 (a., 25-C), 31.04 (t., 9-C), 29.17 (t., 2-C), 27.43 (t., 16-C), 22.45 (t., 15-C), 20.63 (q.,-21-C), 19.89 (q., 27-C), 19.86 (t., 11-0), 19.68 (q., 26-C), 12.36 (q., 19-C), 17.33

• - 135 - • (q., 28-C), 14.07 (q., 13-0), m/e 590 (N ), 378 (base),

253, 159, 157, (Found: C, 71.22; H, 7.64; N, 4.64;

C H 0, requires C, 71.16;. H, 7.85; N, 35 46 N24

P.l.c. of the mother liquor gave B,3 3,5-dinitrobenzoate

(100 mg.) as an oil, 1: 0.80 (3H, M., aromatic H), 3.87

(111 , M.), 4.58 (1H, M., 3 0( -H) , 4.82 (2H, m., 22-H, 23-H),

8.78, 8.95, 9.05, 9.13, 9.23, 9.62 (methyls peaks).

Saponification gave B (65 mg.) as an oil, ›N max. (ethanol) 23

242, 251, 261nm.

Toxisterols B /B cave toxisterol B (152a) (30 mg.) 2 3 ' 22

as white needles m.p. 107.5-109.5° from petroleum (two

19° 7 10° crystallisations), D = + 169°, /-0.< ./ 5 8nm =

nm= + 205°, ro(74:1 nm = + 396° • + 177°, raj ;4: (C = 0.697), 1) max. (CS2) 3550 (al.), 2950 (s.), 2930 (a.),

2870 (S.), 1370 (s.), 1210 (P.), 1175 (M.), 1090 (M.),

1065 (M.), 1010 (M.), 990 (s.), 97o (s.), 86o (m.), 800 (m.),

760 (a.)cm7; A max. (ethanol) 253nm. (22,000).

T3.82, 4.63 (2H, ABq., J = 5.5Hz., 6-H 7-H), 4.83

(2H, M., 22-H, 23-H), 5.39 (1H, M., WH = 19Hz., 3o( -H),

7.37 (1H, M., WH -= 9Hz., 4 0( -H) 8.25 (3H, s., 19-2Ie),

• 9.00 (3H, d., J = 6.5Hz., 21-Me or 2P,-Me), 9.09 (3H, d., • J = 6.5Hz., 21-Me or 28-Me), 9.16 (3H, d., J = 6.5Hz.,

26-Me or 27-Me), 9.17, (3H, d., J = 7Hz., 26-Me or 27-He),

9.23 (311, s., 17-ne), g(13C) 147.32 (d., 7-0), 138.52

8 (3., 10-C), 135.70 (d., 22-C), 131.88 (d., 23-C), 125.9

(d., 6-C), 122.30 (s., 5-C), 66.92 (d., 3-C), 57.27

(d., 14-C), 57.27 (d., 17-C), 52.74 (s., 8-C), 44.57

• • (d., 4-c), 42.89 (a., 24-c), 42.21 (3., 13-c), 40.67 (t., 12-c), 40.03 (d., 20-c), 33." (t., 9-C), 33.12 (d., 25-0), 31.28 (t., 2-0), 27.16 (t., 16-C), 26.31 (t., 1-C), 21.71 (t., 15-C), 21.31 (t., 11-C), 20.87 (q., 21-0, 19.99(q., 27-C), 19.67(q., 26-C), 19.08(q., 19-C), 7.70 (q., 2:7-C), 12.47 (q., 12-C), m/e 396 (11 ), 378, 253, (Found: C, 34.68; H, 10.99; C281144 0 requires C, 84.77;

H, 11.19-).

• HYDROLYSIS OF TOXISTEROL B 5-DINITROBITZOATE (151b) 21 3'

Potasium hydroxide (115 mg.) in methanol (2.0 ml.)

was added to a stirred solution of toxisterol B21 3,5-dinitrobenzoate (151b) (217 mg.) in diethyl ether

(20 ml.) under nitrogen. The solution immediately became blood red in colour; the colour changing to purple as the • reaction nroceeded. After 110 minutes solid carbon

dioxide (1 g.) was added and when effervescence had

subsided diethyl ether (20 ml.) was added. The solution

was washed with water (3 x 20 ml.), saturated brine (20 ml.)

and dried. Evaporation and chromatography on alumina (5 g.)

(eluent diethyl ether: petroleum = 1:5) gave toxisterol B 21 1° (151a) (144 mg., 98) as an oil, /o‹..7 = - 157°, 21° 21° 21° ° / <-7 . 78nm = - 166 , / C)4_7 546nm = 193°' /;(.../7 436nm= • -386° (C = 1.235), 1) max. (film) 3400 (M.), 2960 (S.), • 2930 (s.), 2870 (s.), 1460 (s.), 1370 (s.), 1260 (M.), 1200 (w.), 1110 (w.), 1040 (M.), 1015 (M.), 990 (m.),

970 (s.), 930 (w.), 865 225 (w.), 760 (s.)cm:1 A max. (cyclohexane) 250nm (21,000). 3.72 (2H, 8., 6-H, 7-H), 4.83 (2H, M.,22-H, 23-H), 6.18 (1H, M., Ww= 35Hz., 3 ac-H), 7.55 (1H, d., J=10Hz., 4 is -H), 8.33

(3H,S 9-M,.), 9.00 (3H, d., J=5.5Hz., 21-Ne or 28-ne),

- 137 - • 9.09 (311, d., J=6.51Iz., 21-He or 28-Me), 9.10 (3H, s., 18-Me), 9.17 (3H, d., J=7Hz., 26-Ne or 27-Me), 9.18

(3H, d., J=7Hz., 26-Me or 27-Me), S (13C) 140.69 (d., 7-C), 135.76 (.s., 10-C), 135.76 (d., 22-C), 131.76 (d., 23-C), 129.29 (d., 6-c), 122.75 (.s., 5-c), 69.83 (d., 3-C), 55.78 (d., 14-C), 54.11 (s.,.8-C), 54.05 (d., 4-C), 54.04 (d., 17-C), 43.85 (s., 13-C), 42.84 • (d., 24-C), 40.01 (d., 20-C) 39.92 (t., 12-C), 35.22 (t., 1-C), 33.86 (t., 2-C), 33.10 (d., 25-c), 31.52 (t., 9-C), 27.64 (t., 16-C), 22.30 (t., 15-C), 20.62 (q., 21-C), 20.12 (t., 11-C), 19.98 (q., 27-C), 19.67 (q., 26-C), 18.38 (q., 19-C), 17.64 (q., 28-C), 14.20 (q., 1S-C), r:/e 396 (M+), 378, 376, 271, 253, (Found: C, 84.56; H, 11.02; C28 11440 requires C, 84.77;

Hi 11.19%).

PREPARATICN OF ADDITIONAL TOXISTEROL B21 ESTERS

(151a) (30 mg.) Esterification of toxisterols B21 with (a) 3,5-dinitro-4-methylbenzoyl chloride, (b) benzoyl chloride, (c) 4-nitrobenzoyl, chloride, (d) acetic anhydride in benzene/pyridine (as for preparations of 3,5-iinitrobenzoates) until t.l.c. (silica, diethyl ether : petroleum = 9:11)

• indicated completion and work up gave (a) toxisterol B • 21 3,5-dinitro-4-ethylbenzoate (151c) (44 mg., 961 as long

white needles m.p. 171.5-172° from acetone/Methanol, X max. (cyclohexane) 223nm (31,C00), m/e 604 (n+), 378 (base), 253,

157, (Found: C, 71.53; H, 8.26; N, 4.52; C.;6H1,8N2Oh requires C, 71.48; H, 8.01; N, 4.63=;.), (b) toxisterol B21

benzoate (151d) (35 mC., 92) as long white needles m.p.

- 138- 106-108° from acetone/methanol, A max. (cyclohexane)

229(21,000), 235(21,000), 250(20,000), 279nm (1,100),

m/e 500 (11+)$ 378, 253, 157,(Found: C, 83.91; H, 9.57; requires C, 83.94; H, 9.67 (c) toxisterol B21 21 4-nitrobenzoate (151e) (37 mg., 90) as yellow needles m.p.

102-103.5° from acetone/methanol, m/e 545 (M-4-), 378 (base),

253, 157, (Found: C, 76.92; H, 8.65; N, 2.39; C351147N04 • requires C, 77.01; H, 8.68; N, 2.56%), (d) toxisterol B21

acetate (151f) (29 mg., 87;..) as large colourless prisms ,m.p. 103.5-105° from acetone/methanol, X max. (cyclohexane)

250nm (20,000), m/e 438 (11+), 378 (base), 253, 157, (Found:

C, 82.15; H, 10.45; 030114602 requires C, 82.12; H, 10.58%).

Crystals of toxisterol B22 (152a) and derivatives of

toxisterol B (151a) fork.ray crystallographic study were 21 prepared by the slow cooling (>40 hours) of saturated

solutions from ca. 40° to 0°.

P3EPA2ATIO:: 07 7CXI=OL B. 4-H7.,7:7:1,A7LOTZCATE (151).

A solution of toxisterol B (151a) (4.4m,7.) and 21 142 4-phenylazobenzoy1 chloride (15m-.) in pyridine (1. ml.) was stirred at room temperature until t.l.c. (silica, • • diethyl ether: petroleum = 1:3) indicated complete reaction

(2 hours). Benzene (15 ml.) was added and the solution washed with saturated aqueous sodium bicarbonate (3 x 5 ml.), 5N hydrochloric acid (2 x 5 ml.), saturated aqueous sodium bicarbonate (5 ml.), saturated brine (5 mi.) and filtered

through alumina (5 g.) (eluent benzene). Evaporation cave the title compound (1517) (6.0 mg., 901 as an orange solid.

- 139-

• Four recrystallisations from acetone/methanol gave large

orange needles m.p. 124.5-126° (lit.62 "toxisterol A/

4-phenylazobenzoate" 125.5-126.5°).

SEPA2ATION OF =1ST:20LS B...../B4

Chromatography of toxisterols B3/B4 (1.35 g.) on silica (MFC, 100 g.) (eluent diethyl ether: petroleum =

3:97, 1:19, 7:93, 3:22) gave additional toxisterol B22

(152a) (20 as white needles m.p. 107.5-109.5° from petroleum and unresolved toxisterols B3/B4. Separation

of toxisterol B3/B4 3,5-dinitrobenzoates by chromatography on MFC silica or repeated p.l.c. was only partially

successful. However the B3, B4 benzoate mixture (0.94 g.)

was separated by p.l.c. (0.55 g. on three 40 x 20 x 0.1 cm 15% silver nitrate / Silica plates developed twice in benzene:

petroleum = 7:13, 0.39 g. on five 40 x 20 x 0.1 cm silica plates developed twice in diethyl ether: petroleum = 1:24)

to give (in order of increasing polarity) toxisterol B31

benzoate (153b) (411 mg.), toxisterol B32 benzoate (165 m.),

toxisterol B33 benzoates (29 mg.), toxisterol B4 benzoate benzoate (72 mg.) all as oils. (154 mg.), toxisterols B32/4

22° = ISTL:1,1 3:1 BZ0ATE= (153b), / 04 _/ D = + 133°, • o „o o 22 o 22 /-4-7 578 nm = 139°1 1--°4 -7 56.nm = 159 = + 280° (c = 0.439), I) max. (film) 3060 (rTi.), 2950 (a.),

2930 (s.), 2270 (a.), 1725 (S.), 16C0 ( M.), 1582 (m.),

1455 ( .1370 (a.), 1320 (.m.), 1275 (a.), 1215 (.ra.),

1175 (.M.), 1115 ( a.), 1070 ( =M. ), 1040 ( .m.), 970 ( 3.) , 910 01.), 275 ( 7.), 850 (w.), S30 (w.), 80-7) (w.), 780 (.s.),

- 140 - • 760 (S.), 725 (s.), 690 (m.)C1111., )\ max. (cz,-clohexane) 228(25,000), 235(22,000), 250 (17,000), 279 nm (1,800),

1.92, 2.52 (5H, m., aromatic H), 3.62, 3.91 (2H, A:3g., J=6Hz., 6-H, 7-H), 4.33 (3H, m., 22-H, 23-H, 3 a( -H),

7.86 (1H, d., J=10Hz., 4 (3 -H) , 2.31(3H, s., 19-Me), 9.02 (311, d., J=7Hz., 21-Me or 28-Me), 9.11 (3H, d., J=7Hz., 21-Me or 28-Me), 9.20 (3H, d., J=7Hz., 26-Me or

• 27-Me), 9.22 (3H, d., J=7Hz., 26-Me or 27-Me), 9.32 (311, S., 13-Me), S (13C) 166.12 (5., C=0), 140.79 (d., 7-C), 137.68 (S., 10-C)f 136.03 (d., 22-C), 132.79 (d., 4/.-C), 131.33 (d., 23-C), 130.81 (s., 1/-C), 129.53

(d., 2/-C, 6/-C), 128.70 (d., 6-C), 128.42 (d., 3 -C,

5 -C), 121.27 (s., 5-C), 73.89 (d., 3-C), 56.25 (`1., 4-C), 54.29 (d., 17-C), 53.1P (S., °-C), 42.56 (d., 14-C), 44.64

(s.,13-C), 42.97 (d., 24-C), 39.99 (d., 2c-C), 39.67

(t.,12-C), 35.61 (t., 9-C), 33.15 (d., 25-C), 31.08 (t., 2-C), 29.66 (t., 1-C), 29.53 (t., 16-C), 26.10

( q., 19-C), 23.94 (t., 15-C), 21.94 (q., 21-C), 20.03

( q., 27-C), 19.68 (q., 26-C), 13.37 (q., 13-C), 17.68 (q., m/e 500 (e), 378 (base), 253, 157 (minor

impurity at 546, 424), (Found: C, 23. H,1 9.39; C35H4802 requires C, 83.94; H, 9.67,).

• TCXTS'7:20L B I3=0::77,, kmax. 231, 250, 259 nm., • 32 152.00, 2.60 (5H, M., aromatic H), 3.72, 4.20 (2H, ABq., J=11Hz.), 4.76 (3:1, n., 22-H, 23-H, 3o( -H), 8.96, 9.02,

9.10, 9.20 (12H, side cl-.ain metyls), 9.10 (3i1, s., 1%-Me?)

9.54 (1H, s.), 9.68 (1H, s.), ./e 500, 378, 253, weak ions

at 424, 546. A small Quantity of toxisterol B,2 was obtained on analytical separation of toxisterols B3/B4,

_141 - • Xmax. (liet-1,y1 ether) 242, 250.5, 259nm; chromophore

stable to acid.

:=7=L B33, B7,NZOLTE, Xmax. (ethanol) 230, 245,. 255nm, n.m.r. spectrum indicated inhomogeneity.

TOXI3TEI1OL B4 B Xmax. 235, 241, 250, 259nm;

chromophore acid labile being converted into 229, 279, • 289, 301nm, 1.-,(CC1" 4) 2.02, 2.49 (5H, m., aromatic H),

3.67, 4.27 (2H, ABq., J=12Hz), 4.80 (3H, m., 22-H, 23-H, 3 of-H), 6.68 (21I, m., -OCH 2 CH ), 8.87 (3H, t., -OCH 3 CH ) 9.10, 2 3 ' a.95, 9.Q4.2 (1511, methyl peaks), 546 (M+), 500,

424 (most intense ion > 200), 385, 378, 253, 209, 133, 119, 105 (base).

HYDRCLYSI:S OF TOXIS=OL B,1 BENZOATE (153b)

Potassium hydroxide (169 mg.) in methanol (2 ml.) was

added to a stirred solution of toxisterol B 31 benzoate (153b) (19 mg.) in diethyl ether (5 ml.) under nitrogen.

After 3 days at room temperature work up (as for 3,5-

dinitrobenzoate hydrolysis) and p.l.c. (one 20 x 20 x0.04cm

silica plate developed once in diethyl ether: petroleum = 3:7) gave toxisterol 3_ (153a) (12 mg., FOY) as an oil, • )1 nA o ,40 • /_, 7 c' = 16. ° /7., 7 = +17.4o ,_, 7.21 ! '91/41 D ' 10 '"c..1 578 nm -/ 546nm 17.4 °, /7,0( -7 )onmi.,-2,r = +8.5° (c = 0.980), 1) max. (film) 3350 (s.,), 3050 (ri:), 2960 (s.), 2920 (s.), 2C70 (s.), 1455 (s.), 1370 (s.), 120 (m.), 1120 (m.), 1035 (m.), -1 970 (s.), 780 (c.), 760 (m.)cm., j.max. (c:;clohexane) 250 nm (17,000), rt 3.65, 3.95 (1-2H, ABq., J=7Hz., 6-H, 7-1'-),

• - 142 - • 4.80 (211, m., 22-H, 23-H), (;.24 (1H, m., = 34Hz.,

3 d\-11), 8.33 (311, s., 19-lie), 9.02, 9.13, 9.22

(12H, side chain methyls), 9.10 (3H, s., 18-Me), g(13c) 140.72 (d., 7-C), 137.97 (s., 10-c), 136.18 (d., 22-c), 131.85 (d., 23-c), 128.71 (d., 6-c), 120.72 (s., 5-c), 69.90 (d., 3-c), 59.73 (d., 4-c), 54.20 (d., 17-c), 53.21 (s., 8-c), 48.31 (d., 14-c), 44.86 (s., 13-c), 42.93 (d., 24-c), 40.17 (d., 20-c), 40.10 (t., 12-c) , 35.62 (t., 9-c), 34.00 (t., 2-c), 33.18 (d., 25-c), 31.44 (t., 1-c), 29.51 (t., 16-c), 26.03 (q., 19-c), 24.00 (t., 15-c), 21.98 (q., 21-c), 20.03 (q., 27-c), 19.67 (q., 26-c), 18.35 (q., 18-c), 17.70 (., 28-c), M/e 396 (Hi), 378, 37C, 271, 253, (Found: C, 84.43; II, 10.95; C2814440 requires C, 84.77; H, 11.19%).

PREPARATION OF TOXISTEROL B32/134 4-PHENYLAZOB:MOATES

The title compounds (62 mg.) prepared from B32/B4 benzoate (52 mg.) and isolated by chromatography on alumina (eluent benzene) were obtained as a red oil that failed to crystallise from acetone methanol.

DEHYDRATION OF TOXI3T_:ROL B21 (151a)

A solution of toxisterol B (151a) (16 mg.) in • 21 chloroform (2 ml.) saturated with dry hydrogen chloride was stood overnight at room temmerature under nitrogen (solution in colour). Diethyl ether (20 ml.) was added, the solution washed with saturated aqueous sodium bicarbonate (2 x 20 ml.), saturated brine (20 ml.), dried, evalorated- and chromatograhed on alumina (2 g.) (eluent petroleum).

-143,-

• substituted Crystallisation from acetone/methanol gave the benzene (155) (13 mg., 89A as white plates m.p. 35-86.5 or white needles 7 22.5° =_,,o ■/7:54. 7 22.5° ,00 m.p. 33.89-5° ! -/ D _/ 578nm _.../0 1 7.22.5° = 1-_, 7 22.5° ! "-/ 546nm -66°, `'s-/ 46nm = -121° (c = 0.914), Vmax. (CS2) 3090 (m.), 3060(m.), 3020 (m.), 2970 (s.), 2920 (s.), 2370 (s.), 1370 (s.), 1340 (b.), 1230 (m.), 1160 (m.), 1120 (m.), 1070 (m.), 1025 (m.), 970 (s.), 935 (m.), 920 (w.), 832 (w.), 860 (w.), 778 (s.), 740 (w.), 720 (m.), 710 (m.)cm:I, '9. max. (CHC13) 1598 (m.), 1460 (s.)cm.T1 Xmax. (cyclohexane) 262sh. (477), 265 (570), 273.5nm (585), 152.92 (1H, m., 2-H), 3.07 (2H, m., 1-H, 3-H), 4.81 (2H, m., 22-H, 23-H), 7.22 (21I, t., J=7Hz., with smaller J ca.1Hz., 6 00:-H, 6 f3-H), 7.77 (31I, s., 19-Me), 8.97 (3H, d., J=6.5Hz., 21-Me or 28-Me), 9.09 (3H, d., J=6.5Hz., 21-Me or 28-Me), 9.17 • (3H, s., 10-Me), 9.17 (3H, d., J=7Hz., 26-Me or 27-Me): 9.18 (3H, d., J=6.5Hz., 26-Me or 27-Me), We 378 (111-) 363, 355, 318, 307, 230, 253, 251, 211, 209, 205, 197, 171, 170,

157 (base), 145, 144, 143, 142, 131, (Found: C, 33.5P'; H, 11.01; C28Hh2 requires C, 38.81; H, 11.19).

DEHYDRA:I0H CF TOXIZT-201, B71 (153a).

4 Dehydration of toxisterol B (153a) (42 mc.) with • 31 chloroform (2 ml.) saturated with hydrogen chloride (solution emerald green in colour) and p.l.c. (one 40 x 20 x 0.1cm silica plate developed once in diethyl ether: petroleum = substituted 1.99) gave the4benzne (17-:6) (20 mg., 50;-,i) as white needles o m.p. 67-69.5° from acetcne/meti=o1, 3 = + 6.0o 0 0 0 /-,,4 7 23 ,-. + 6.o 23 = 6 ,o /J7, 7 23 - 57Fnm °' / -04- 75461-in ' 436nm= + 11.2° (c = 0.537), )) max. (C:2) 7°60 Cm.), 3030 (m.),

to, _ 11:4 — 3010 (m.), 2950 (s.), 2920 (s.), 2270 (s.), 2850 (m.), 1370 (s.), 1305 (w.), 1245 (w.), 1135 (s.), 1075- (m.),

970 (s.), 940 (w.), 775 (s.), 705 (m.)cm:1 Vmax. (0H01 ) 1598 (m.), 1460 (s.) cm.-1 3 \max. (cyclohexane) 262sh. (612), 265.5 (712), 273.5nm (670), '15 2.91(1H, m., 2-H), 3.06 (2H, m., 1-H, 3-H), 4.79 (211, m., 22-H, 23-H),

7.21 (2H, t., J=7Hz., 6(3 -H), 7.77 (3H, s., • 19-Me), C.94 (311, d., J=7Hz., 21-He or 2^.-Me), 9.05 (3H, s., 18-Me), 9.09 (311, d., J=7Hz., 21-Me or 28-Me), 9.16 (3H, d., J=6.5Hz., 26-e or 27-Me), 9.18 (31I, d., J=6.5Hz.,

26-1Ie or 27-Me), q/e 378 (M+), 363, 355, 312, 307, 220, 253, 251, 211, 2C9, 205, 197, 171, 170, 157, (base), 145, 144, 143, 142, 131, (Found: C, 33.68; H, 11.09; C28H42 requires C, 2%81; H, 11.195':)

• DEHYDIIATION OF TOXIST7:201, B22 (152a)

Dehydration of toxisterol B22 (152a) (18 mg.) with chloroform (1 ml.) saturated with hydrogen chloride (solution copper sulphate blue in colour) and p.l.c. (one 20 x 20 x 0.04cm silica plate developed once in diethyl ether: petroleum = substituted 1:99) gave thef,benzene (156) (7 ms., 41) as white needles o -04.. 722o _ o m. p. 62-64°, /04 7.3°, / + 7.5 o -/ 578nm = 22 8.40, E.04.7 22 o • /-.; 7 13.1 °(C = 0.358), • - - 546nm 436nm = identical (i.r., n.m.r. (impurities at 3.13, 8.78, 9.28), m.s., t.l.c. (10, silver nitrate/silica, benzene: petroleum

1:19))with the rroduct from toxisterol(153a).331 The impurities were not removed on p.l.c. (10 silver nitrate silica, benzene : petroleum = 1:19) or by repeated crystallisation from acetone/methanol.

• - 145 - Attempted dehydration of toxisterol B22 (152a) with 86 Phosphorus oxychloride in pyridine or carbethox7amidosulphuryltri- 87 ethylammonium in benzene gave very impure benzene (156).

Treatment of toxisterol - B (152a) with 22 chlorotristriphenylPhosphinerhodium (I) in chloroform88

under reflux in an attempted to migrate the double bonds

prior to dehydration gave a mixture of steroidal components. •

PREPAATION C2 I:':1)=7; (157)

Indene (157) (15 mg.) formed durinr7 the recording of the 13 C n.m.r. spectrum of toxisterol B 31 benzoate (153b) (210 mr;.) in CDC13 and isolated by p.l.c. (one 40 x 20 x 0.1 cm silica

plate developed once in diethyl ether : petroleum = 9;91) o was obtained as large slender prisms m.p. 96.5-97 from acetone/methanol, Vmax. (CHC1 ) 2930 (s.), 3- 2370 (s.), 1600 (m.), 1460 (s.), 1370 (s.), 1100-1070 (bs.), 970 (m.), 910 (s.)cm:1, )\ max. 262nm (6,900), T.2.94 (3H, m.,

1-H, 2-H, 3-H), 3.19, 3.27 (2H, m., 6-H, 7-H), 4.79 (2H, m.,

22-H, 23-H), 7.60 (3H, s., 19-Ye), E.73 (3H, s.,

8.93, 9.03, 9.12, 9.23 (12H, side chain methyls), m/e 376

(11), 361, 333, 305, 291, 273, 277, 251 (base), 209, 195,

169, 157, 155, 143, 141, 131, 107, (Found: C, 89.12;

H, 10.52; C28H requires C, r a 40 '9.29; H, 10.71). •

T.l.c. (10c: silver nitrate/silica, benzene : petroleum =

1:19) inlicated indone (157) to be more polar than the

cochromatora:hing benzenes (156) and (1,75).

• _ 146- • P:2EPA2,A7IC.1 rinouzsic, o: TOXI3ROL B?1 =!"-Ar: (1r--,1h)

n Butyllithium (0.20 ml., 2N in hexane) was added to a

stirred solution of toxisterol B21. (151a) (26 mg.) and an-hydrous redistilled di-iso-rronylamine (0.5 ml.) in THE (5 ml.). After 40 minutes carbon disulphide (0.50 ml.) was added followed 110 minutes later by iodomethane (0.5 ml.). Work up after 31- hours, chromatography on alumina (5 g.) (eluent Petroleum) and p.l.c. (one 40 x 20 x 0.1cm silica plate developed once in diethyl ether : petroleum = 1:49) .3-methyl1 xanthate (151h) as a colourless gave toxisterol, 21B, 210 -721 o 721 „a9 gum, /t"( 7 D = -100°, /1)4;7 78nm = -106 , /o( 546 = -1c) ' o 21 (c = 0.501), 'Orix. (film) 2950 (s.), CX-7 436nm = -299° 2930 (s.), 2870 (s.), 1460 (s.), 1372 (s.), 1250 (s.), 1218 (s.), 1055 (s.), 1005 (w.), 98' (w.), 970 (s.), 942 (w.), 900 (m.), 862 (m.), 842 (w.), 832 (w.), 800 (w.), 765 (s.), 678 (m.)cm:1 N) max. (CHC13) 16n0 (m.) cm:1,

15 3.68 (2H, m., 6-H, 7-H), 4.28 (1H, broad m., 30( -H), 4.80 (2H, m., 22-H, 23-H), 7.43 (3H, s., S-e), 8.30 (31, s., 19-Me), 3.95, 9.03, 9.07, 9.12, 9.22 (15H, side chain and 18-methyls), 9.12 (1 -Me),m/e 486 (m, very weak), 378, 363, 335, 308, 281, 280, 253, 251, 211, 210, 199, 197, 171, 169, 160, 159, 158, 157, 147, 146, 145, 144, 143, 142, • 131, (Found: C, 74.14; H, 9.41; C30H46S20 requires C, 74.00; IT 9.53%i).

o Pyrolysis of xanthate (151h) (6 mg.) at 220-10 under argon at 1 atmos:-here for 4 hours gave three compounds all -more polar than benzene (155) (t.l.c., 10;:. silver nitrate/

silica, benzene: = 1:19), 1426, 378, 376.

- 147: - • Pyrolysis of xanthnte (151h) (15 mg.) in

dimethyl acetylene dicarboxylate (1.0 ml.) under.reflux

under argon for 31 hours gave a resin and several steroidal

components.

FHOTOLYSI:, OF TOXISTOL B (151a). 21

A solution of toxisterol B21 (151a) (12 mg.) in • cyclohexane (10 ml.) was irradiated. Ultraviolet spectra and t.l.c. (silica, diethyl ether : petroleum = 3:7) indicated slow decrease in intensity of the chromophore

and formation of a complex mixture of products.

SEPA7TIOT: CF C TOXIT=LS

T.l.c. (silica, multiple development in dichloromethane)

indicated the C toxisterols consisted of • )7 components. Separation on an analytical scale gave (in order

of increasing polarity) C1/C2/C3 ( X max. (diethyl ether)

242, 249, 252, 275, 285, 300nm.), C4 (242, 250, 259nm.),

C5/C6 (242, 242, 259nm.), C7 (242, 24, 260nm.). Separation was not possible on a preparative scale.

OF CD TOXIS=OIS

• Bulk ergosterol (2g.) was removed from the CD S toxisterols (16 g.) by trituration with ethanol at -20°,

filtration and evanoration to give fraction CD 1 (14g.) Chromatography of CD1 (7 g.) on alumina (210 g.) (eluent

diethyl ether : petroleum .= 1:9, 3:17, 17:83, 1:4, 3:7)

gave (in order of increasing polarity) toxisterols C (1g.

toxisterols Di /D12 (3g.) and t.7.-,xistrols D /D13 (3g.). 12

• - 143 - 3,5-Dinitrobonzoyl chloride (1.6g.), toxist rols

D /D (2.06 g.), 11 12 toluene (50 ml.) and pyridine (5 ml.) were stirred together at room temperature for 8 hours.

The reaction mixture was washed with saturated aqueous

sodium bicarbonate (3 x 20 ml.), water (20m1.), saturated

brine (20 ml.) and dried. Toluene/pyridine were removed

to leave 3 9-dinitrobenzoate as a yellow oil. D11/D12 • T.l.c. (silica, diethyl ether : 17,etroleum = 1:9) indicated

the presence of 12 esters. Small samples were separated

by analytical t.l.c. to give in order of increasing polarity)

D /D 3,5-dinitrobenzoate-1 ( )s,max. (diethyl ether) 236, 11 12 3,5-dinitrobenzoate-2 (241nm.), 250, 259nm.), D11/D12 3,5-dinitrobonzoate-3 (240, 286sh., 306sh.nm.), D11/D12 - D /D 3,5-dinitrobenzoate-5 (236, 260sh.nm.) D /D 11 12 , 11 12 /D12 3,5-dinitrobenzoate- 3,5-dinitrobenzoate-6/7 (238nm.), D11 8/9 (231, 241 sh., 250 sh., 259 sh. nm. the compounds were

acid labile being converted into 225, 280, 290, 302nm.),

dinitrobenzoate-11/12 (243, 250, 259 sh.nm. the D11/D12 3'5- compounds were acid labile being converted 280, 290, 302nm).

Chromatogra]:hy on alumina (100 g.) (eluent diethyl ether:

7Detroleum = 1:39, 1:24, 3:37) gave 3 fractions. The most

polar fraction gave toxisterol D11/D12 3,5-dinitrobenzoate

• -10 (160) (720 mg.) as a pale yellow gel drying to an • amorphous solid m.p. 125.5-126.50 from acetone/methanol, o „o = *L7° 20° /7X-7 16 7 = 50° im" 7 = ' 572nm 546nm 5C°, 20 16<-7 436nm= + 112° (c = 1.42), V max. (nujol) 1719 (s.), 1630 (m.), 1552 (s.), 1468 (s.), 1380 (m.), 1345 (s.),

1228 (s.), 1175 (s.), 1020 (m.), 990 (m.), 740 (s.) X max. (diethyl ether: ethanol = 1:/1) 243 (45,000),

- 149 - • 250.5 (44,000), 259 (29,000), 0.75, 0.90 (311, m., aromatic-H), 3.58, 4.12 (211, ABq., J=11Hz., 6-H., 7-H),

4.82 (2H, n. , 22-1I, 23-H), 5.07 (IH, m. , 'J11 = 20Hz.,

3 c4.-H), 6.63, 6.82 (211, dq., J= 7Hz., -00E20E3), 6.96, 7.20 (211, m., 4o( -H, 4 P' -H), 7.65, (1H, t., J=12Hz., 14 o4(-H), r',.63 (311, s., 17-Ne), R.87 (3H, t., J= 7 1Iz., -OCH2CH3), /8.98(3H, d., J = 6.5Hz., X21-Me or 22-Me), 9.10 (3H, d., J=7.0Hz., 21-He or 28-Me),

• 9.17 (3H, d., J=6.8Hz., 26-Me cr 27-Ne), 9.19 (3H, d., J=6.8Hz., 26-Me or 27-Me), 9.44 (3H, s., 18-Me), /e 636 (111-), 621, 590, 466, 465, 425, 378, 254, 253, 133, 119, 105, 81, 77, (Found: C, 69.70; H, 8.33; N, 4.32; C37H52N207 requires C, 69.77; H, S.24; N, 4.4(Z).

D11/D12 3,5-dinitrobenzoate-10 (160) was acid sensitive being converted into a less polar ester with • X max (diethyl ether: ethanol = 1:4) 280 (34 000), 290 (41,000), 302nm (30,000).

The medium polarity fraction gave D11/D12 3,5-

dinitrobenzoate- (43 mg.) as white needles m.p. 208-209.5 from benzene/ethanol, \) max. (najol) 1722 (s.), 1635 (m.), 1560 (s.), 1478 (s.), 1390 (m.), 1355 (s.), 1295 (s.), 1180 (m.), 990 (m.), 740 (s.)cm:1, 'Amax. (diethyl ether) • 225nm., 15 0.80-0.95 (3H, m., aromatic-H), 4.85 (3H, m., • 22-H, 23-H, 3o. -11), 2.95, 9.05, 9.C9, 9.14, 9.21, 9.45 (methyl -,3-17s), "We 59f., 592, 590, 54q, 494, 467, 466, 465, 255, 109, 107, 105, 69.

Repeated r.l.c. of the mother liquor from D11/D12 3,5-dinitrobenzoate-4 (silica, diethyl ether : petroleum)

_ 150 - • gave 3 5-dinitroben7oate-5 (Ib) was yellow nris:ns D11/D12 ' p.p. 146-147° from acetone/meti-lanol. D /D 3-,5- 11 12 dinitrobonzoate-5 was identical (m.n., m.m.p., t.l.c., u.v.)

with ergocalciferol 3,5-dinitrobenzoate (Ib).

T.l.c. (silica, diethyl ether: petroleum = 7:17)

and m.s. (71./e 634 (11+), 592, 590, 485, 363) indicated

• toxisterol D 3,5-dinitrobenzoates consisted of >2 13 ethanol addition compounds.

OF T TC=EROL3

A sample of the E Toxisterols (3.6g.) was separated by

7-).1.c. (one 20 x 20 x 0.04 cm silica Plate developed twice

in ethyl acetate: petroleum = 1:1 and once in petroleum) to give (in order of increasing polarity E1 (We 792, 456, 442), We 792, 456, 446), L3 (14/e 520, 506, 4'L2) and E4 (N/e 4g6, 442, 414).

HYDP=A7ICIT OF ...20CCALCIF=OL (1)

A solution of ergocalciferol (1) (306 mg.) and 143 chlorotristrinhenylphosphinerhodium (I) (79 mg.) in

benzene (36 ml.) was stirred under hydrogen overnight.

After removal of benzene the residue was extracted with • petroleum, filtered through celite and separated by p.l.c.

(two 40 x 20 x 0.1cm silica plates developed twice in

diethyl ether : netroleum = 1:1) to give in order of

increasing polarity) dihyJroergocalciferol-II (147a)

(154 mg., 50..) as an oil and dihydroergoc7,1ciferol-IV (147a) C9 (82 mg., 27-) as white plates m.p. 61° (lit. 61.5-63°)

from methanol.

- 151 - • A subsequent hydrog-::nation, searation and estrific.•tion .;ave dihyroocolciferol-II 3,5- dinitrobenzoate (147b) (23c;: overall) as pale yellow needles m.p. 164.5-165.5° (lit:9 164-166°) from acetone/metanol, 1.7; 0.87, 0.9 (311, m., aromatic H), 3.98, ''.20 (2H, ABq., J=11Hz., 6-H, 7-H), 4.69 (1H, m.,

We 6.5Hz., 36■ 4.86 (2H, m., 22-H, 23-H), 6.86, 7.25 (2H, m., 404.-H, 4 ?, -II), 7.33 or 7.54 (111, m., 10-H), .81 (3H, d., J=7.211z., 19-Me), 8.97 (311, d., J=6.6Hz., 21-Me or 22-Ne), 9.07 (3H, d., J=6.21iz., 21-Me or 2 ,-ne), 9.15 (3H, d., J=6.8 z., 26-Me or 27-Me), 9.16 (3H, d., 6.6Hz., 26-Ne or 27-Me), 9.44 (3H, s., 18-Me) and dihydroergocalciferol-IV 3,5-dinitrobenzoate (142b) 89 (69 mg., 21(,) as orange prisms m.p. 174-175° (lit. 175-75.5°) from acetone/methanol, 1; 0.86, 0.90 (3H, m., • aromatic))3.91, 4.26 (1H, d., J=11Hz., 6-H, 7-H), 4.86 (2H, m., 22-H, 23-H), 5.07 (1H, m., Wu = 20Hz., 3o( -H), 6.92, 7.35 (211, m., 4‘,C -H , 4(3. -Ii), 7.43 or 7.52 (111, m., 10-H), 8.83 (311, d., J=7.0llz., 1, Me), 8.95 (3M, d., J=6.6Hz., 21-Me or 2n-Me), 9.06(3H, (I., J=6.9Hz., 21-Me or 23-Me), 9.14 (3H, d., J=6.2Hz.,26-M.2 or 27-Me), 9.15 (3H, d., J=6.911z., 26-He or 27-Me), 9.41 (311, s., 18-Me).

0 • T'HOTOLYSIT CF =:03=C1, (4) I:I C'"= J.XANE

Ergoaterol ('') (20 g.) in c:,-clohex7me was irradiated

until the starting ch-omo7aho-e had reached a minimum

(72 hours). Sol7ent was evaorated and the residue

chromatograrhed on alumina (600 g.) (eluent diethyl ether:

;petroleum = 0:1, 1:9, 1:0) to give three fractions A

(..5C :7.), 5/ (0.20 - and C' (17: all as oils.

• _ 152 - co Fraction A gave white needles m.p. 107-2170 from

petroleum (4 crystallisation). T.l.c. (10c:: silver

nitrate/silica, benzene: petroleum = 2:23) indicated

the presence of two compounds. P.l.c. (four 20 x 20 x

0.04 cm. silver nitrate/silica plates developed twice

in benzene: petroleum = 2:23) and 3 crystallisations from

benzene/ethanol gave the major more polar compound (20 mg.)

• as white glistening plates that started to sublime at 200°, m.p. 203°, resolidified 205°, 2nd m.p. 210-215°, clear 2y) o /,/ 7 24 24° = + 210 578nm = + 218° 225°, /X ' I , /0104._7546nr,71 - 7 2h° + 250°, nr1 0 436nm= 450 / -7365nm = 795 (CS ) = 0.303), Vmax. 2 2955 (s.), 2920 (s.), 2(870 (s.), 1380 (s.), 1370 (s.), 1030 (w.), 975 (s.), 95o (w.), 35o (s.), 770 (w.), 700 (w.)cm.71 >,max. (cyclohexane) 263.5 (9,000) , 273 (12,600), 284 (13,100), 296nm (7,600), -C4.64 (2H, s., 7-H, 7 -H), 4.8o (4H, m., 22-H, 22 -H, 23-H, 23 -H), 7.70-3.90 (28H, methylene envelope), 8.93, 9.03, 9.12,

9.22 (24H, side chain methyls), 9.37 (6H, s., 1c2-Me,

18/-Me), m/e 544 (n+), 542, 419, 323, 293, 1r9, 197

(Found: C, 88.00 ; II, 11.78' c40H64 requires C, 88.15 ; H, 11.85 ;':); and the less polar minor compound (5 mg.)

as a white solid, 4.43, 4.80 (vinyl protons), 9.951 9.03, 9.12, 9.1-2, 9.38 (methyl si-nals). T.l.c. (10;', silver

• nitrate/silica, benzene: petroleum = 1:19) -indicated the hydrocarbon presence of all the/photofragments isolated from the

ergosterol/ethanol photolysis.

Chromatography of fraction B on alumina eluent

(diethyl ether: petroleum = 0:1-1:9) gave toxisterol Bi,

(0.4 g.) (identical on t.l.c. with toxisterol B 3 Ind B - 11' 12 - 13

_ 153 - • and toxisterol B2 (0.45 g.). Fraction B gave 2 toxisterol B g.) white needles' from 22 (152a) (0.13 petroleum. The esterified mother liquor gave toxisterol

3,5-dinitrobenzoate (151b) (0.18 g.) as yellow needles B21 from acetone/methanol.

o Fraction D was triturated with ethanol at -20 to

• remove bulk ergosterol (4) and a fraction of the residue D , (3 g.) chronatographed on alumina (100 g.) (eluent diethyl ether: petroleum = 3:7, 1:0) to give D 11/D 12 (0.95 g.), D'13 (0.90 g.) and D. 14 (0.55 g.). D 11/D 12 (0.40 g.) was converted into its mixture of 3,5-dinitrobenzoates.

Chromatography on silica (:;'C, 100 g.) (eluent diethyl ether:

petroleum = 1:99 - 1:9) and subsequent p.l.c. (silica plates

multiply developed in diethyl ether: petroleum = 1:9) • gave (in orrer of increasing polarity) D 11 3,5-dinitrobenzoate-1,

D' 3,5-dinitrobenzoate-2 and D.12 3,5-dinitrobenzoate-1. 11

11 3,5-dinitrobenzoate-1 was an inhomogeneous oil (n.m.r.). • 5-dinitrobenzoate-2 gave a mixture of white needles •11 '' 0 0 m.p. 134-135.5 and yellow spheres m.p. 128-139 from

acetone/methanol. Repeated recrystallisation from acetone/ methanol gave D 3,5-dinitrobeazoate-21 as white needles • 11 o o 20 m.p. 135-136°, /-o-(2 D = + 108°, /-o.(2 (- 811m = + 113°, o 20o 2 o 1- ".`"-( 7,4 6nm = 4- 131°, ra(-7436nm= 4. 232 (c = 0*345' acetone) identical (t.l.c., n.m.r., m.m.p. /-0(_7D , m/e)

with su3rasterol II 3,5-dinitrobeuzoate (162b).

• - 154 - • 3 5-dinitrobenzoate gave yellow i)risms m.p. 140-*1o •1 2 ' . from acetone/methanol, / 0(../ ;01 2-7 = +200 (C = 2.75_5), Xmax. 229 (21,000), 268sh (9,700), 2P0 (8,600), 291 sh. nm. (4,700). '15 0.80 (311, m., aromatic-H), 4.50 (3H, m., 30( -H, 6-H, 7-11), 4.80 (211, m., 22-H, 23-H),

7.40 (2H, m., 40(- H, 4 F -H), 8.93, 9.03, 9.10, 9.20 (12H, side chain methyls), 9.17 (3H, s., 19-Me), 9.33 • (31i, s., 1-Me), m/e 590 (Mt), 465, 378, 253. (lumisterol 3,5-dinitrobenzoate (66) m.p. 139-1410,

coz _7 .`" = 20°).

Fraction D (0.90 g.) was converted into its mixture 13 of 315-dinitrobenzoates. Crystallisation from acetone gave 145.5-147° •13 3,5-dinitrobenzoate-2 as yellow prisms m.p. . • 13 3,5-dinitrobenzoate-2 was identical with ergocalciferol • • 3,5-dinitrobenzoate (Ib) (m.m.p., t.l.c., u.v., n.m.r.). P.l.c. of the mother liquor (four 40 x 20 x 0.1cm silica

plates developed four times in diethyl ethers : petroleum =

1:9) gave additional D 13 3,5-dinitrobenzoate-2 and 13 3,5-dinitrobenzoate-1 as white needles m.p. 20r';-209.50 D / from benzene/ethanol. D 3,5-dinitrobenzoate-1 was 13 /D identical (m.p., t.l.c., u.v., n.m.r.) with D11 12 • 3,5-dinitrobenzoate-4 from the ergosterol/ethanol photolysis. • The mother liquor from D 13 3,5-dinitrobenzoate-1 gave

D 13 3,5-dinitrobenzoate-11 as pale yellow needles m.p.

143-1470 from benzone/etilanol ancl acetone/methanol o o -721 o - -721 (reeated cr-:stallisation)' roc:- -/ D = -27 ' -/c< - 57(Jrim/ = 7 o -)o o 21 210 -72° , .40‘. / 546nm = -34 ' 436nm = (acetone, c = C.L15), 'We 594-, 592, 390.

Fraction D 14, was a rolar intractable resin.

• - 155-

• 0: :1;.CCALCI=CTI, (1 )

Ergocalciferol (1) (600 m::.) in petroleum

(unsaturated hydrocarbon free b.p. 33-400, sodium dried,

120 ml.) was cooled to -650 (ethanol/cardice in quartz

beaker) and irradiated by an external high pressure

mercury arc iamn. After the starting chromorphore had

disappeared (4-6 hours) the solution was allowed to warm • up to 0° and petroleum removed. r.l.c. (twelve

40 x 20 x 0.1 cm silica plates developed twice in diethyl

ether: petroleum = 11:9) gave (in order of increasing

polarity) minor steroids (56mg., Xmax. (diethyl

ether 240, 270 nm.), suprasterol 2 II (162a) (23T mg., o 144 o 32':,) as whits plates m.p. 110-112 (lit. 109-110 )

from acetone and suprasterol2 I (163a) (119 mg., 20.) as o 4 o (1- 2 white needles m.p. 101-102 (lit.14 , lumicalciferol' o m.p. 102-103 ) from acetone.

Esterificution gave suprasterol2 II 3,5-dinitrobenzoate 144 (162b) as white needles m.p. 139-141° (lit. 140-141°)

from ncetone/metanol, -15 0.78, 0.87 (311, s., aromatic-11), 4.50 (1H, m., 30/, -H), 4.26 (211, m., 22-H, 23-H), 9.04, 9.13

(6H,Zd., J=71Iz., 21-Me & 23-Me), 9.19 (3H, d., J=711z., 26-11e or 27-Me), 9.21 (311, d., J=7Hz., 26-Ne or 27-ne), • • 9.22 (3H, s., 13-Me), suprasterol2 II 3,5-dinitro-4- 0 met::ylbcnzoate (162c) as white feedles m.p. 170-171 o o ,21 (lit. 170-172 ), from acetone/methanol, -/- 0(- /D = 11,o, 721 o + 110°, 76( _ 51,_6rim = + 133 , o - '1 _,0 (acetone, c = 0.650) (1 it.144 /- - 7436nm /-o< 108°) and suprasterol I 7, 5-diritobenzoate -7D + 2 -D (163b) as feathery orange needles m.ip. 147-1,4P°

• • (lumicalciferol 3,5-dinitrobenzoate lit.92 m.p. 146-147°) o 2 0 , o , from acetone/methanol, /-a(2 p = -q4 (c = 1.03)

(lumicalciferol 3,5-dinitrobenzoate lit.92 /0 -Z = -1=2.70), 1-; 0.78, 0.87 (3H, m., aromatic H), 4.67 (1H, m., 304. -H), 4.84 (211, m., 22-H, 23-H), 8.99 (311, 0-, J=6.5Hz., 21-He

or 28-Me), 9.08 (31I, d., J=7IIz., 21-Me. or 28-He), 9.16

(311, d., J=6.5Hz., 26-Me or 27-Me), 9.18 (3H, d., J=7Hz.,

• 26-Me or 27-Me), 9.21 (3H, s., 13-Me), M/e 590 (M+), 465, 378, 330, 260, 253, 161, 136, 135, 134, 119, 118 (base), 117, (Found: C, 71.08; H, 7.78; I, 4.62; (C3 1-140206

requires C, 71.16; II, 7.85; N, 4.74'„).

p 07 3=A=OL, I 1:TI0PTTA"Am7, (163c) 145 Cyanic acid gas from the depolymerisation of

cyanuric acid at 400° was carried by a stream of nitrogen o into toluene at -20 . The solution was stored at -20°

under nitrogen (stable for over 1 month). The cyanic acid content was estimated by adding pyridine (1 drop) to an

aliquot (4.0 ml.) and filtering off the cyanuric acid

after 1 hour (0.31g/4n1.).

Cyanic acid in toluene (4 ml.) followed by pyridine

(1 drop) were added to a solution of sunrasterol2 I (163a)

• (40 mg.) in toluene (1 ml.). After 1 hour the reaction • mixture was chromatographed on alumina (25g.) (eluent ethyl acetate) to give the title compound (163c) (45mg., 92;;) as

white plates 222.5-225° (lit. 219° 144, 225-226°94 0 062 /,-,1 721 = ..5co, 223-226 ) from ethyl acetate, ,... 'D o o o 21 21 21 7 = / - 578nm = -61° ' /c 4 - 7546nm = -71°, (°<-7436nm o he 144 06°94 o62 -142 (c = 0.259) (lit. _ , -5 , -39 ),.m/ 482 (11+), 465, 396, 378, 253, 205, 136, 118 (base).

_ 157 - • F.Y.DMG=ATTOrT C7 0TJASTE2OL, II (162a)

A solution of suprasterol II (162a) (240 mg.) and 2 glacial acetic acid (0.1m1.) in ethyl acetate (10 nil.) was

stirred with Adams catalyst (2.1 mg.) under hydrogen until t.l.c. (silica, developed three times in diethyl ether:

petroleum = 9:11) indicated absence of starting material

(3 days). Diethyl ether (20 ml.) was added and the solution washed with saturated aqueous sodium bicarbonate

(2 x 20 ml.), saturated brine (20 ml.), dried and filtered

through celite. The residue, after evaporation, gave

tetrahydrosuDrasterol2 II (165a) (136m1., 77;.') as white

needles m.p. 94-100° from acetone. Recrystallisation 146 o twice gave a sample m.p. 99-103 (lit. 99-102 ). X6..14 m., = 12Hz., 3c< -H), 9.11, 9.16, 9.23, ri 9.24 (12H, d., J=7Hz., side chain rnethyls), 9.25 (311, s., 18-Me).

Esterification gave tetrahydrosuprasterol II 2 3,5-dinitrobenzoate (165b) (79.) as white needles m.p. 146 o 112-114° (lit. 110-112 ) from acetone/methanol. /7:4 7 12;00 „o Pn = -24°, /-o< ------7 57(mm0 = -25° , 1,;( 7 46 7 20° 6- 5 nn -29°, ./. .v/ 436nm = -50° (c = 0.228) (lit.'" -23.7°).

• m/e 594 (:-1), 332 (base), 261, 255 • .147 148 :2REPAATIC OY T:2=-ITo'..1,077=' (7a) '

A solution of ergocalciferol 3,5-dinitrobenzoate (Ib)

(2 73,7 ) 4 1 toluene (25:11 .) was heated to reflux under argon for seven hours. Tolupne was removed under high vacuum .

and the residue crystallised from acetone/methanol.

_ 153 • The crystalline solid (ergocalciferol 3,5-dinitrobenzoate

(Ib)) was reequilibrated by reflux in tollaene (25r:il.).

After removal of solvent and crystallisation the combined

mother liquors were eva-oorated, dissolved in butan-2-one/

methanol and cooled to -200. After filtration and

evaporation the oil was chromatoc:raT)hed' on alumina (150g.)

(eluent diethyl ether: petroleum = 49:1, 39:1) to give

preergocalciferol 3,5-dinitrobenzoate (7b) (372mg., 13`). 148 as pale yellow needles m.p. 100-1030 (lit. 103-1040) from acetone/methanol, 1: 0.83, 0.95 (3H, m., aromatic H),

4.02, 4.35 (all, ABq., J=13Hz., 6-H, 7-H), 4.50 (1H, m.,

9-H), 4.82 (3H, m., 22-H, 23-H, 3CK -H), 3.28 (31 , broad s.,

19-11e), 3.93, 9.04, 9.13, 9.22 (12H, side chain methyls),

9.30 (311, s., 18-Ile).

Saponification with potassium hydroxide in methanol/

diethyl ether at -20° for 2-;1: hours gave Freergocalciferol(7a)

(249ms., 100) as an oil (less polar on t.l.c. than

ergocalciferol (I) ), 154.11, 4.45 (2H, ABq., J=13Hz.,

6-H, 7-H), 4.57 (111, m., 9-H), 4.87 (21, m., 22-H, 23-H), 6.20 (1H, m., 3o( -H), 8.38 (311, s., 19-lie), 8.93, 9.03,

9.13, 9.23 (12H, siJe chain Me), 9.30 (3H, s., 18-Me)

• Preergocalci'ferol (7a) (24) wan alternatively prepared from the thermal equilibration of ergocalciferol (1) in

toluene under reflux and isolated by chromatography on

alumina.

• - 159 - I:: 7;2:7.0IT (7a) 7TY,CAT,CT71:;2CL (1)

A solution of fluorenone (36 mg.) and

.,reergocalciferol- (40 ms.) in toluene (20 ml.)

(in pyrex arparatus) was irradiated by an external

750W tungsten lamp (10 cm away). After 12 hours t.l.c.

indicated equilibration with a sinsle less polar, air

sensitive sterol ( X max. 260, 270, 278, 291nm.) which

was 1;resumably tachysterol (19)95; B /B toxisterols 2 3 were not formed'.

T.l.c. indicated erg)ocalciferol (1) reached equilibrium

with a sinzle less polar sterol ( X max. 272nm.), Presumably: .9 5,6-trans-ergocalciferol (18)1/ ' on fluorenone or acetone

sensitised irradiation.

LOW 7 lITTOLYSI C2 -IZGOSOL (4) Al;D P=RG:C=7.77,ROL (7a)

T.l.c. (silica, diethyl ether: netroleum = 1:1) and

ultraviolet spectra:in'.icatod the photolysis of ergosterol o (4) or preersocalciferol (7a) in ehanol at -60 reached 1 . The formation of toxisterols a pseudoequilibrium B1/B2 was not increased. • 4

PHOM1= Ci --]:GC:=?OL (4) IN t-3?7NOL

A solution of erTestr'rol (h) (3.07.) 1n t-butanol

(1200m1.) was irradiated for 49 hours (reaction followed

by u.v.). Eval:,oration and chromatography on alumina and

toisterols 3 (24mc.), toxist rols B /B p.l.c. 1 21/322 31 (4mL;..) and a -,-,reviouoly undetecte compound (int2rmediate in

• 60_ • polarity between B rune B 1 2) (517z.), X max. (r:.iethyl ether) 235-240, 250sh., 260sh., 282sh.nm., m/e 470, 430, 414, 413,

412, 411, 410, 396, 271.

Samples of to::isterols B21 (151a), B22 (152a) B31 (153a) and the fraction from ergosterol/cyclohexane B21/B22/B31 photolyses were converted intc their respective 150 • trimethylsilyl ethers . P.l.c. (one 20 x 20 x 0.04cm 10% silver nitrate/alumina plate developed once in diethyl

ether: benzene : petroleum = 1:2:17) of B 21/B /B - 22 31 trinethyl silyl ethers gave (in order of incre-ising. polarity)

B -trimethylsilyl ether (151i)' B -trimethylsilylether 21 22 (152c), B -trimethylsilylether (153d) and a minor trimethyl- 31 silylether ( X max. (diethyl ether) 225nm.

• CHROMA=APHl CH7,CH OF TIGGS7.:ROL (4)

Chromatography of ergosterol (4) (7.0g.) on alumina

failed to detect any photofragments 'or toxistrirols.

PHOTOLYSI- ':.-iR.00:7_701, (4) III. 77:: -fRT7,71:7CE 07

Photol- ois of ergosterol (4) (6g.) in cyclohexane

(2500m1.) under air until the starting chromonhore had

disa,.)peared (1?) hours), chromatogranhy on alumina and

p.l.c. gave hydrocarbons (21mg.) (the n.m.r. spectrum

indicated little or no photofrarments) and toxiterols 2

B (87m7.). 1 /B2 -

(127b)

A solution of cholesterol (173a) (2''c.) in -,;.y-idine

(100 ml.) - acetic anhy:',ride (20m1.)-was left for 72 hours o at 0 , -rollred into water, the solid filtered off and washed

_ 161 - • with water. The title compound (173b) (20.5g., 9Z1 0 151 was obtained as white plates u.n. 113.5-114.9 (lit.

114-115°) from acetone, ^C4.60 (ill, broad s., WH = 10Hz.,

6-H), 5.40 (1H, m., WH = 22Hz., 304. -II), 7.96 (3H, s.,

0-Ac), %97 (311, s., 19-11e), 9.08, 9.20 (911, side chain

methyls), 9.32 (31I, s., 18-Mc).

2H=ATTOTT OF 7-T:7]Th=1.=101, ACIT= (179) S Sodium chromate (17.5g.) was added over half an hour

to a susnension of cholesterol acetate (173b) (20g.) in

acetic acid (140m1.) and acetic anhydride (80m1.) containing o sodium acetate (12g.). The mixture was heated (50-60 )

for 48 hours, cooled, poured into water (1400m1.) and

stirred for 1 hour at 10°. The solid was filtered off,

dissolved in diethyl ether, washed with water, saturated

brine and filtered through alumina (360g.).(eluent diethyl

ether). Evaporation and crystallisation from ethanol gave

the title compound (179) (11.0g., 55A. Recrystallisation

from etIlanol gave white elongated plates m.p. 156-158°

(lit.152 157-159°), 7-042 j)2c) 105° (c = 1.22)

(lit.153 - 103°).

PREP:,,RATIOU CF (177) • A solution of lithium aluminium hydride (0.20g.) in • diethyl ether (20 ml.) was added droawise to a cooled

solution (0°) of 7-hetochblesterol acetate (179)(1.50g.)

in diethyl ether (30m1.). The mixture was heated to re flux

for 1:21- hours, cooled and celite (2g.) and saturated aqueous

sodium sulphate (dropwise) added. After filtration and

evaporation the title comnounds were obtained as a ce.1

to an amorhous coli.; from aqueous ethanol.

0 - 162 - -46 Benzoylation of diols (177) (25mg.) (benzoyl

chloride (21m g.) - pyridine (0.5m1.)-toluene (2.0m1.),

24 hours at room temperature) and o.l.c. (one 20 x 20 x

0.04cm silica plate developed twice in diethyl ether: petroleum = 7:13) have cholest75-ene-3 , 7 (1 -diol 3-benzoate (180a) as white plates m.p. 191.5-194° 191-194o, (Lit. 154 ) from diethyl ether. Benzoylation

S of diols (177) (37mg.) (pyridine (1.0n1.) -benzoyl chloride (0.10m1.), 72 hours at room temnerature) and 7.1.1.c. gave

cholest-5-ene-3p , 7(3 -diol dibenzoate (180b) (31mg., 55) o 154 as white needles, m.p. 173.5-173 (lit. 171-173°) from

ethanol.

ATTE. IIED 12==i01: OF 7-DEHYDROCROLST7R0T IRON ?I'7C==1, (170)

(1)A solution of cholest-5-ene-3 (2) , 7-diols (177)

(119mg.) and iron pentacarbonyl (2.0m1.) in di-n-butyl

ether (6m1.) was heated to reflux under argon overnight.

3(CO) ) The solution rapidly became green in colour (Fe 12 T.l.c. (silica, diethyl ether : netroleum = 1:1) indicated

cholest-5-ene-3 , 70( -diol (177a) reacted faster than its

C-7 epiner (177b) and the formation of 5 non polar r:oducts.

Addition of toluene-n-sulnhonic acid nonohydrate resulted in

more raid reaction Eiving a mixture of 2:.:37 comronents.

(2) 'Irifluor o acetic acid (0.05:11. ) was added to a o, cooled s .3penslon (-15 , of diols (177) (10mg.) in iron

pcntacarbonyl (1.5ml.) under argon. After 15 minutes the oran-e solution was cuc,nched with trioth"lamino (0.1m1.)

and solvents removed under vacuum. T.l.c. (silica, 2ietl_yl : -e:roleum = 1:1 o: the C co .po::ents.

- 163 -.

• 0 Reaction at room temperature instead of -15 gave a dark

blue solution. After quenchinz (EL') t.l.c. indicated

the presence of only non polar comi)onents, however the

n.m.r. spectrum indicated a complex mixture.

155 (3) Diiron nonacarbonyl (206mg.), cholest-5-ene-

3 p , 7-diols (177) (59 mg.), toluene-n-sulnhonic acid

monohydrate (9mg.) and THF (25ml.) were heated to reflux a under argon. The solution rapidly became burgundy in

colour. T.l. c. indicated the formation of .1) 6 components

including non polar components, sterol iron complexes were o not formed. 2eaction in toluene at 60-70 instead of THF

(solution green in colour) also gave a complex mixture.

T,7CTICI.T 07 - (1 ) , Th7T'sCIT 71- 7==VT.

T.l.c. diethyl ether: petroleum = 7:13)

indicated ergosterol iron tricarbonyl (176) was formed on

heating ergosterol (4) (192mg.), diiron nonacarbonyl (173mg.)

and toluene-p-sulphonic acid monohydrate (20 .3mr- 0. ) - (50 ml.) to reflux under argon overnight.

EC 7.=_OTICII 07 7.-::2TC:=7.77:::01, ACI7,T7E (179) Sodium borohydride (1.5g.) in water (10m1.) was added

• to 7-keto-cholesterol acetate (179) (5.0g.) in THF (30m1.)- a dioxan (10m1.) and the mixture stirred overnight at 00.

.jork u-c and chromatography on silica (IETC, 350r.) (eluent

'iethvl e h r : 1:etroleum = 1 : 9 -3: 2) gave cholest-5-ene-

3(3 7-diol 3-acetates (131) (4.75c., 95Y) as an oil.

A sample separated by p.l.c. (1 0 x 20 x 0.1cm silica plates

developed reeatedly in diethyl ether : Petroleum = 1 : 4,

3:7) gave t:13 major. (le... 7.olar)cl-Lolest-_-7-ene-3r) -diol

• - 164 - 3-acetate W1b) as white needles m.p. 110.5-112° 156 (lit. 110-1120) -/-- = -6.3° (c = 1.364) ' 156 0 (lit. -5.0 ) from diethyl ether or petroleum and the

minor (more polar) cholest-5-ene-3 , 70( -diol 3-acetate 156 139o), (181a) as white needles m.p. 136-139° (lit.

/-047 lDi8° = -87° (c = 0.396) (lit.156.-87.5°) from petroleum then methanol. T.l.c. (silica, developed

• twice in diethyl ether : petroleum = 2 : 3) indicated

repeated crystallisation of the diol mixture from petroleum gave mainly the 7 p ej.mer (131b), m.p. 110.5-112°, ,7o ro(7 - D = -12.4° (c = 2.25).

DEHYDA?:.C:- 0F CHGT.73T-5-17IIE-313 , 7-DIOL 3-..:; 7A773 (181) 87 (Carbethoxyamidosulphuryl) triethylammonium

in benzene (5m1.) was added to the title compounds (131) • (85mg.) in benzene (5m1.). The mixture was stirred overnight at room temperature under argon, filtered through alumina (2g.) (eluent benzene (25m1.) ) and evaporated to

give an oil. 15 4.07, 4.36 (2:1, ABo., cT = ?Hz., 6-H, 7-H),

4.70 (211, m., 3 o( -H, 4-H), 7.96 (3H, s., 0-Ac), 2).98 (3H, s., 19-Me), 9.3, 9.20 (OH, side chain mothyls), 9.27

(3H, s., 18-Me), \ sax. (diethyl ether) 23F., 245nm. P.l.c. (one 20 x 20 x 0.1cm silica plate developed once in

diethyl ether : petroleum = 7 : 13) gave an aJorphous solid,

m.7). 66-69° (cholasta-4, 6-dien-3p) -ol acetate (182) m.p. 157 lit. 78-79°) from acetone/methanol (3 crystallisations).

(177,c) Cholesterol (173c) (20g.),-2yridine (150m1.) and benzoyl chloride (7.04g., 1.1 eclivalents) were stirred to7ether o overniTht at 0 , TToured into wa.,er (41.) 7nCI the solid

• • filtered off. Crystallia7.tion from acetone/ethyl acetate

gave the title com7lound (24.6g., 97 ) as souare ,;!hite elates

m.p. 150-151° (lit.151 150-151°) , /-0(..7 2° = -15.2°

(c = 0.998).

(-.1 7,44- 17- ::CCUCJ:7;Sr::',110L 3NNZCATI] (1P8)

A solution of cholesterol benzoate (173c) (3.0g.) and

NBS (1.50 g.) in carbon tetrachloride (30 ml.) was heated

to reflux for 15 minutes, cooled, succinimide filtered off

and washed with carbon tetrachloride. The combined

filtrate and washings were evaporated and crystallised from

acetone (20m1.). Recrystallisation from acetone/ethyl

acetate (1 : 1, 10m1.) then benzene (1.5m1.)/acetone (4m1.)

gave the title compound (188) (0.75g.) as elongated plates - 72 ° m.p. 141.5-143° (lit.105 140°), /coc_i p 2 = -182° (c = 0.903)

(lit.105 -172°). Separation of 70( -bromocholesterol

benzoate and unreacted cholesterol benzoate by triangular 158 crystallisation from petroleum gave material with m.p. o o 21 o 140-142 / 04. 7 ' D -175 (c = 1.30).

R2ACTIC1:- N1NAC!.,T3G1YL 70‹ -7,C.1:0CNOT-:37-72CL =NAT:7, (188)

70( -Bromocholesterol benzoate (128) (224mg., 0.5mmole), • e di-iso---Dropylethylamine (71mg., 0.55mmole) and diiron

nonacarbonyl (1.82., 5mmole) in THF (30m1.)- were heated to

reflux under nitrogen for 3-7 hours. The solution became

burgundy in colour on reflux commencing. TIYF was removed

under vacuum, t'he residue extracted with diethyl ether :

petroleum = 1 : 9 (100m1.) and filtered through silica

(MFC, 30g.) to give a clear F7reen solution. IvaDoration

and e.l.c. (two 2fl x 20 x 0.1cm silica :dates developed once

• -166- • in diethyl ether : petroleum = 1:9) cave "H" (less polar) (80m L:.) and "Y" (more polar) (133n1c.).

"X" was obtained as white needles m.p. 193-202o, 0 clear 215 from benzene/acetone (recryatallised three times), /-04 7 22 = 7 22o ,no +20°, /-04 +21°,' /7:4 7 = +24° / Do / 573nmo / 546nm ' 7 22 = /04... ,7365nm2 = 4100° !`"'`../ 436nm -) , (c = 0.956), a l) max. (nujol) 1715 (s.), 1605 (m.), 1588 (m.), 1315 (m.), 1270 (s.), 1178 (m.), 1112 (s.), 1070 (m.), 1028 (a.), 1005 (w.), 980 (w.), 940 (w.), 930 (v.), 830 (w.), 787 (w.), 710 (s.), 688 (w.)=-1., Xmax. (cyclohexane) 228 (26,000), 265 sh. (1,900), 272 (2,500), 2S0nm (2,100). 1:: 1.95, 2.50 (10H, a., PhCO2-), 4.87 (2H, m., W7 = 6Hz., 6-H), 5.16 (2H, a., WH = 20Hz., 30‹ -H), 7.63 (4H, m., 4-H), 8.93 (6H, s., 19-Me), 9.07, 9.18 (1CH, side chain methyls), 9.28 (6H, s., 18-11e), m/e M+ absent, 356 (M+ - PhCO2H),

734 (M+ - 2PhCO2H), (Found: C, 83.35; H, 9.95; C68H9804 requires C, 83.37; H, 10.09A.

"Y" was obtained as white needles m.r. 190-1960 (from ,20 0 0 acetone, then twice from benzene/acetone), / 6(2 D = -9.7 0 ,04 = _10.0o / 720 = _10.„o 720, = -/ 57,2nm. ' 546nm , /01K,- -/ 42)onm /04. 7 o -9.8o' -/ 365nm = +9.0 (c = 1.17), )) max. (nujol) • 1720 (s.), 1605 (m.), 1585 (m.), 1310 (m.), 1285 (m.), S 1270 (s.), 1250 (m.), 1170 (a.), 1110 (s.), 1070 (a.),

1023 (s.), 995 (m.), 990 (a.), 940 (w.), -30 (w.), 260 (w.), 245 (':!.), 710 635 (w.)cm-1., X max. (c:clohexane) 230 (25,000), 265sh. (2,100), 272 (2,r_;00), 280 (2,200)nm. 1: 1.96, 2.50 (10H, m., Ph002-), 4.40 (1H, m., W, = 6Hz )

4.67 (1H, m., W. = 10Hz.), 5.10 (2H, a., = 23Hz., 34K -H),

07 r 7 • •

• - 167 - TVe 114- absent, 856 (11+ phco2H), 734 (114- -2PhCO ) ,

490, 4r, 366, 247, 122, (Found: C, 3.43; H, 9.99;

C681198°4 requires C, 83.37; H, 10.09A.

Reduction of 7o( -bromocholesterol benzoate (188)

(142mg.) with diiron nonacarbonyl (0.588.) in benzene

(20m1.)/di-iso-propylethylamine (33mg.) by reflux for

2 hours under nitrogen and c.l.c. (one 20 x 20 x 0.1cm

silica plate developed once in diethyl ether : petroleum=

1:9) gave "X" (35mg.) and "Y" (90mg.) identical with the compounds previously described.

:REPA11TION A7.31:S=N7 CF C:i0U:3T-.`:-EITE-3 7f -DIOL 3-ICE?= 7-S-Y,2TYL XAN=TE (186a)

Nethyllithium (2i in hexane, 0.26m1.) was added to a

solution of cholest-5-ere-3p , 7p -di of 3 acetate (1 °ib)

(222mg., 0.5mmole) in THE (10m1.) under nitrogen at -78°. After 15 minutes stirring carbon disulahide (1m1.) was

added and the solution allowed to warm up to room temrerature.

After 1 hour iodomethane (1m1.) was added and 1 hour later

THE was removed under high vacuum. The residue in diethyl

ether was chromatographed on alumina (5g.) (eluent dietityl

ether : petroleum = 3 : 17 (100m1.) ). LvaTloration and

0 trituration with acetone/methanol gave the title compound (16a)

as rosettes of white needles (270mg., 100), 1) max.

(carbon tetracilloride) 2930 (s.), 2270 (s.), 1740 (s.),

1670 (w.), 1640 (w.), 1470 (s.), 1440 (m.), 1320 (s.),

1370 (s.), 1335 (w.), 1320 (w.), 1240-1210 (s.), 1175 (m.), 1160 (m.), 1140 (m.), 1070-1030 (s.), 960 (m.), 905 (m.), -1 885 (w.), 260 (m.)cm ., 15 4.07 (1:!, d., 7:4 -H),

-168- • 4.50 (1H, broad s., = 6Hz., 6-H), 5.37 (III, m., = 24Hz., 30k -H), 7.40 (3H, s., S-re), 7.93 (3H, s. 0-Ac), 8.87 (3H, s., 19-Me), 9.07, 9.17 (9H, side chain methyls), 9.27 (3H, s., 13-Me). Attempted crystallisation from warm acetone/methanol and p.l.c. (four 40 x 20 x 0.04cm silica plates developed twice in diethyl ether. : petroleum 7 : 93) dithiolcarbonate (197a) (134mc.) as white needles m.p. -07.5o • 129.5-131.5° from acetone/methanol, /Mac / = +160°,) „o o - D / c4 7 = +169° = +196°, /-0( 717* - 578nm , re( - 746nm 1(' - 436nm = +371° (c = 0.531). )) max. (nujol) 1730 (s.), 1630 (s.), 1310 (w.), 1235 (s.), 1150 (w.), 1130 (w.), 9n0 365 (s.), 760 (m.)cm-1., et 4.67 (1H, broad s., WH = 6Hz., 6-H), 5.38 (111, m., Wu = 24Hz., 3c& -H), 6.02(1H, broadened d., J = 6Hz., WH = 14Hz., 7 13 -H), 7.58 (3H, s., S-Me), 7.98 (3H, s., 0-Ac), 9.0 (3H, s., 19711e), 9.08, 9.13 (9H, side chain methyls), 9.32 (3H, s., 18-Me), We 144. absent, 474 (M+ - AcOH), 366 (MI- - MeSCOSH, base), 247, 159, 157, 147, 145, 143, 133, 131, 119, (Found: C, 69.81; H, 9.29; S, 12.04; C311150S203 requires C, 69.59; H, 9.43; S, 12.00;:). and a minor less polar compound contaminated with dithiolcarbonate (187a) (40 mg.), X max. (diethyl ether) 250nm., ^C 4.40-7.00 (several multiplets), 7.58 (S-Me), 7.95, 7.98 (0-Ac), 8.97, 9.00 (19-Me), 9.08, 9.18 11, • (side chain methyls) 9.32 (18-Me).

During prearation of xanthate (136a) excess methyllithium gave the non 7)olar bis-xanthate (126b) as a by Iroduct, m.p.

125-128° bulk (as white needles from acetone), /54.08 (1H, 5H z., 70( -K), 4.53 (2H, 04.-H, 6-H), 7.43 (6H, s., 3-Me), 8.75 (3H, s., 19-Me), 9.08, 9.18 (9H, side

- 169- • chain methyls), 9.2c (31I, s., 13-Me), (round: C, 63.77; H, 8.51; C T4 - 0 requires C, 63.85; H, 8.65';). 31-50°4 2 Partial rearrangement (ca. 50c,) of the bis-xanthate

(186b) took place on storage presumably giving

dithiolcarbonate (187b), "1", 3.90-4.80 (3H, m., 30( -H,

6-H, 7-H), 7.43 (474H, s., S-Me), 7.58 .(1.6H, s., S-Me), 8.85, 8.97 (3H, 2s., 19-Me), 9.08, 9.18 (9H, side chain

• methyls), 9.28, 9.33 (311, 2s., 18-Me).), "d max. (nujol) 1680 (s.), 146-1450 (s.), 1375 (s.), 1210 (s.), 1050 (s.), 865 (s.)cm-1.

Pyrolysis of dithiolcarbonate (186a) at 1700 under argon for hours F.-aye a complex mixture of products.

Starting material (20mg., 91) m.p. 131.5-133.5° (from o acetone/methanol), m.m.p. 129.5-131.5 was recovered • unchanged when dithiolcarbonate (187a) (22mg.) in iodomethane 0 (1.0m1.) was heated overnight at 807100 (sealed tube).

N.m.r. indicated dithiolcarbonate (187a) (20mg.) in carbon

tetrachloride (0.50m1.) failed to react with methyl

fluorosulphonate (26mg.) over 12 days at room temerature.

7-1- m'Cil OF Tc:0200:;T=O1E (197). A solution of ergosterol (4) in toluene (100m1.)/ • cyclohexanone (40g.) was distilled collecting 10m1. of • distillate. Aluminium isopronoxide (8.0g.) in toluene (100m1.) was rapidly added and the solution heated to reflux

for 2 hours. After cooling the toluene was washed with

aqueous sulphuric acid (4c':., 2 x 400m1.), saturated aqueous

sodium bicarbonate (2 x 10Cm1.), dried and toluene/

c:.ciollexanone removed (90-100°, Cyclohxane h conder,.3aticr_ pro,Jucts were removed (150-1700, 10 'mm.),

- 170 -

the residue extracted with diethyl ether and filtered

through alumina (50g.). Evaporation and crystallisation from acetone/petroleum gave ergosterone (196) (5.6g., 56). Hydrogen chloride was bubbled through a solution of

ergosterone (5.6g.) in chloroform (10m1.) at 0° for 1 hour. The chloroform solution was washed with saturated

aqueous sodium bicarbonate (2 x 100m1.), water (2 x 50m1.),

s0) and evaporated. The residue in ether was • dried (Na2 filtered through alumina (30g.), evaporated, crystallised

from petroleum and recrystallised twice from ethyl acetate/

methanol to give isoergosterone (197) (2.59g., 46) as 121 needles m.p. 106-107° (lit. 106-108°) /C X 718° 121 -21° (c = 1.07) (lit. -24.5) X max. (ethanol) 284nm. 121 (30,000) (lit. 284nm. (23,000) ), 3.85 (2H, s., 6-H 7-H), 4.30 (11, s., 4-H), 4.77 (2H, m., 22-H, 23-H). 8.87 (3E, s., 19-Me), 8.92, 9.02, 9.10, 9.20 (12H, side chain methyls), 9.20 (3H, s., 13-ne).

1,7=3TIC:1 07 HET!G=A-4, 67 22-'217:7-3p -ol 7:7177,07E (1?8b).

Sodium borohydride (0.30g.) in water (4m1.) reduction

of isoergostrone (197) (2.0g.) in T2Y (10m1.)-dioxan (5m1.) 0 overnight at 0 and 4,:f hours•at room temperature, work up

and benzoylation gave the title comound (1flCh) (2.25g., ci) as a gel drying to an amorphous solid. Crystallisation"

from dichloromethane/methanol gave white needles m.p. 5o 123.5-125.5°, /o(...7; ?*-5o = -134-1 2),° ,, 10K -71 ' 1‘.0 ,, n m _,_o 19.5 o /- -oC 19.5 = /-o 7 o = 314 7546nm ,- - /c)6nm '/ - - 3o5nm. -574° (c = 0.947), ).) max. (nujol) 1710 (s.), 1600 (w.), 1585 (w.), 1330 (w.), 1310 (m.), 1270 (s.), 1170-1140 (m.), 110C (c.), 1070 (a.), 1025 (a.), 970 (s.), 930 (m.),

- 171 - • 86o (s.), 945 (s.), 760 (m.), 705 (s.), 685 (w.)cm71.,

Q.51.91, 2.43 (5H, m., PhCO2-), 4.04, 4.37 (2F, AT3q., J = 10Hz., 6-H, 7-H), 1;.45 (1H, m., 3o11.4. -II), 4.58

(1H, broad s., WH = 6Hz., 4-H), 4.82 (PH, m,, 22-H, 23-H),

8.93 (3H, s., 19-Ne), P.93, 9.03, 9.12, 9.23 (12H, side t chain methyls), 9.25 (31I, s., 18Ne), m/e 500 (M ), 378 (M - PhCOOH, base), 253 (373 - allylic cleavage),

• (Found: C, 83.80; H, 9.46; C3 H4802 requires C, 83.94;

HY7:::0T_:`.{;'",I ,] CT :"--:`1C'::::7A-4, 6 222-77.7-EN-3 -ol (199b)

.Hydrolysis of benzoate (19%) gave trien-3p -01 (1n8a) 22 (400m5., n3;..) as white needles•m.p. 122-126° (lit.

118-120°) from acetone/methanol, X max. (ethanol) 233(12,000), 239 (19,000), 242 (15,000)nm., 1) max. ( S2) 1020 (s.), 975 (s.), 935 (m.), 910 (w.), 880 .(w.), 860 (s.), 830 (w.), 760 (5.), 660 (w.)cm-1.

REACTIO1: ERG0STA-4, 6,22-T2IEH-3 ei -ol =0 T_, (198b) !D DI7Pc; FONACAI:',RC:NYL

Benzoate (198b) (200mg.), diiron nonacarbonyl (0.54g.)

and benzene (10m1.) were heated to reflux under argon.

The solution rapidly became green (Fe3(C0) 12- ) in colour. • After 220 minutes the mixture was cooled, filtered through • alumina, evaporated and chromatographed on alumina (60g.) (eluent petroleum : 7Tiethyl ether = 1: 0 (20m1.), 99 : 1 (200m1.), 197 : 3 (100m1.; ). Evaporation gave "Z"

(115mE., 76.) which was purified by r.-.c.. (two 20. x x 0.1cm silica plates developed once in diethyl ether : petroleum =

1 : 39)and 3 recrystallications from butan-2-one/benzene to • Dro, ,n0 o nive white needles m.r. 151-1.5 bulk 154-197 , /0( 7 . +157°

- 172 - ,e0 260 0 ?6° :4 7 , = +165° / = — 57jnm ' - 1,46nm- = 195 , %-°(-736nm -,2 ° + 4100, /o< / r = + 866° ( c = 0.233), .1) max. (00 ) - 3o5nm 2 3020 (m.), 2950(s.), 2870 (s.), 1380 (s.), 1370 (s.), 1020(w.), 972 (s.), 935 (w.), 880 (s.), 865 (s.),

770 (m.), 660 (w.)cm-1., X max. (cyclohexane) 234 (41,000), 243 (46,000), 251nm (37,C00), 14.17, 4.50 (4H, ABq., J = 10Hz., 6-H, 7-H), 4.69 (21J, broad s., 4 H), 4.82 (4i1, m., 22-H, 23-H) 8.98, 9.00 (6H, 2s., • - 19-Me), 9.07, 9.09, 9.10, 9.13 (12H, 4 overlapping doublets, 21-Me, 2$-Ne), 9.16 (6H, d., J = 6.5Hz., 26-Me or 27-Me),

9.18 (6H, d., J = 6.5Hz., 26-Me or 27-Me), 9.26 (6H, broad s.,

12-Me), m/e 758 (11+), 756, 379 (base), 348, 125, 117,

(Found: C, 0F.35; H, 11.32;-; 056H86 requires C, 88.57; H, 11.43Y).

Zu was also formed from benzoate (198b) and diiron 159 nonacarbonyl,triiron do decacarbonyl or iron pentacarbonyl in toluene, THF, dimethoxyethane or di-n-butyl ether at

50-130° with or without propylene epoxide or styrene erioxide

rresent.

PREPAP.A1'.10:•: 7.7) 7.-;7,`TICTICI",- '2' !.".: G A ••• 6,22—TPIEN-3 f, -ol T,YL ( 198c )

• Chlorotrimethylsilane (0.6ml.) was added to a solution • of ergosta-4,6,22-trien-3 -ol (19$a) (200mg.) in pyridine

(5 ml.)/hexamety:disilazane (1'11.). After z hour solvents were ror:oved under high vacuum, the residue extracted with toluene (4 x 2m1.), filtered throuh alumina and evaporated

to give the title com-nound (19$c) (234mg., 99;',) an a white

solid, 't (carbon tetrac:iloride) 4.13, 4.50 (2H, A3q., J=11Hz.,

r, 5.73

- 173 - 8.93, 9.00, 9.10, 9.20 (12H, side WH = 20Hz., 50k -H), chain methyls), 9.00 (31, s., 19-Ee), 9.25 .(311, s.,

1S-Ne), 9.37 (9H, s., Sine,).

The product and iron pentacarbonyl (1.0m1.) in

di-n-butyl ether (6m1.) were heated to reflux under argon

overnight. After cooling the reaction mixture was filtered

through alumina (eluent diethyl ether) and evaporated to

leave a yellow semisolid (211mg.), V max. -(chloroform)

2920 (s.), (s.), 2040 (a.), 1930-1910 (s.), 1460 (s.), 1370 (s.), 975 (s.), 390 (m.), 875 (m.), 340 (w.)cm-1.

The n.m.r. spectrum was vary similar to that of "Z".

The semisolid and feriic chloride (1.3g.) in THE (10m1.)/ ethanol (20 ml.) was heated to reflux (30 minutes),

evaporated and extracted with petroleum (50 ml.) and water

(50 ml.). The petroleum solution was washed with water (2 x 20 ml.), dried, filtered through alumina and eva-oorated

to give an oil (172m.). Crystallisation from butan-2-one

gave white needles. The product was identicl with "Z"

(m.o. 147-169° (bulk 152-157°), i.r., n.m.r., t.l.c.).

REDUCTIC, -01 (193a)

T.l.c. (silica, diethyl ether : petroleum = 1 : 19) • indicted reduction of the title compound (IIc) (124mg.) by

reflux with diiron nonac-_rbonyl .(0.007.) in benzene (10m1.)

gave only chimer "Z".

- 174 - -=CTIC7o CI=YL c i I7fITYLCILYT, (2011).

The title compound (2C1b) (251mg., 93A prepared

from cinnamyl alcohol (201a) (175mg.) was used without o 25 160 further nurification, n = 1.5092 (lit. 1.5105),

'U max. (film) 3080 (w.), 3060 (w.), 3022 (m.), 2950 (m.),

2840 (w.), 1600 (w.), 1495 (m.), 1450 (m.), 1380 (m.),

• 1252 (s.), 1120 (broad s.), 1060 (broad s.),.965 (s.), -1 880 (s.), 840 (s.), 750 (m.), 730 (m.), 692 (m.) cm 1,2.75 (511, m., Ph), 3.34-4.03 (2H, m., -cH=c11-), 5.77

(2H, d., J = 4Hz., -CH2-), 9.37 (9H, s., SiNe3).

Trimethylsilyl ether (201b) (123mg.), diiron

nonacarbonyl (1.1g) and benzene (10m1.) were heated to

reflux under nitrogen for 9,2 hours. After coolin,7 the reaction mixture was filtered thrOugh silica (1IFC)

(eluent benzene), evaporated and heated to reflux with

ferric chloride (0.5g.) in THF (5 )/ethanol (5m1.) until

the green colour was discharged (40 minutes). After cooling

diethyl eth.:.r (50 ml.) w-s added and the solution washed with

water (2 x 20m1.), saturated brine (20m1.), dried and

evaorated. Chromatograrhy on alumina (5g.) (eluent retroleum (30m1.) ) gave a yellow oil (42mg.), 15 (carbon S • tetrachloride) 2.76(h), 3.27-4.23 (vinyl protons), 6.55, 7.36, 7.62, .13, 8.,(.1'2, 9.12 (alirhatic rrotons). Crystallisation from 3:etrolcum at -20° gave 1,6-

dinhenylhexa-1E,=-7E-diene (202) (12mg.,

2ecvystThsation fro':. :7ethanol :;a7e off white Tolates

(lit.161 n 0) (C,]7) 500 (T.1.), 3060(m.), 30P0 (m.), 2 20 (m.), P7.40 (), 1960 (w.), 1240 (w.),

175 - • 1285 (w.), 1560 (.), 1200 (w.), 1650 (w.), 1350-1,700 (w.),

1C72 (m.), 1030 (m.), 9'0 (m.), 969 (s.),

920 (w.), 910 (w.), 745 (s.), 692 (s.)cm-1., v 2.85 (10H, m., Ph-), 3.30-3.90 (4H, m., -CH=CH-), 7.65 (4H, m., -0712-). The mother liquor had ') max. (film) 3080 (w.), •

3060 (u.), 3025 (m.), 2970 (m.), 2920 (m.), 2070 (u.), 2040 (s.), 1960 (s.), 1600 (m.), 1500 (s.), 1450 (s.),

• 1375 (m.), 1125 (m.), 1060 (s.), 965 (s.), 790 (s.), 695 (s.) cm-I., m'e 234 (11'), 128, 127 (base), 126, 125, 71.

PRA'-,.---_`-' 0:. 72,==..7 (206b) A solution of geraniol (206a) (commercial, 5.0g.),

benzoyl chloride (5.0g., 1.1eq.) and pyridine (20m1.) was

stood overnight at room temperature. ,fork ulo and chromatography on alumina (150g.) (eluent diethyl ether :

petroleum = 1 : 19) gave the title compound (206b)

(7.8g., 93r,) as an oil, jr; (CC14) 1.89, 2.46 (511, m.,

PhCO2 -)' 4.5`0 (111, t., J = 7.5Hz., 2-H), 4.88 (IH, m., 6-H), 5.20 (2:1, d., J = 7.5Hz., 1-H), 7.87 (4H, m., 5-H),

8.20 (3H, s., 3-le), 8.31, 8.37 (6H, 2s., Ile2C=).

1:777.CTIO 02 0=:ICT, T=CA117: (206b) • Geraniol benzoate (206b) (0.93g.), benzene (25ml.) and • diiron nonacarbonyl (6.6c.) were heated to reflux under

nitro gen overnight. The mixture was cooled, filtered

through alumina washin;-. through with ethc:r and evarorated.

The residue an ferric chloride hexahy -at- (2g.) 4 n ethanol (20m1 .)/THF (20 ml.) was heated to reflux unThr nitrogen for

P.- hours worked u T.l.c. (,'lica, diethyl ether :

7,2troleum = tnd the -,7:istence of iron com niexes.

- 176- • The crude products and ferric chloride hexahy:srate, (2E.)

in ethanol (30m1.) were heated to reflux under nitrogen for 7=lz hours. Work up, chromatography on alumina

(100g.) (eluent petroleum) and repeated p.l.c. (silica, multiple development in diethyl ether; petroleum = 3 : 47) gave (in order of increasing polarity).

"R" (113mg.), "0" (62mg.), "T" trans-2,6-dimethylocta-2, 4-dien-8—ol benzoate (207b) (108ms.). "R" was obtained as a yellow oil, 'IC, 4.83, 8.00, 8.30, 8.37, 8.75, 4.87, 9.07, Ve 416, 388, 360, 332, 304, 276, 248, 243, 137. "3" was obtained as a colourless oil, 1:3.10, 4.50-5.03, 5.63, 6.67, 7.40, 7.73, 8.40, 8.73, 8.83, 9.13, Ve 530, 394, 274. "T" trans-2,6-dimethylocta-2,4-dien-8-ol

benzoate (207b), 'N) max. (film) 3060 (w.), 3020 (w.),

2960 (m.), 2920 (m.), 2870 .(m.), 1720 (s.), 1602 (w.), 1585 (w.), 1450 (m.),. 1375 (m.), 1315 (m.), 1275 (s.), 1175 (m.), 1110 (s.), 1070 (m.), 1030 (m.), 965 (m.), 715 (s.), 690 (w.)cm-1., -1r 2.07, 2.57, 2.63 (5H, m., PhCO2-), 3.80 (1H, m., 4-H), 4.23 (IH, d., J = 10Hz., 3-H), 4.57 (11'_, dd., JI = 15Hz., J2 = 7Hz., 5-H), 5.66 (211, m., 8-H), 7.59 (1H, m., 6-H), 8.13 (2H, m., 7-H), 8.27 2s., Me2C.) 8.68 (311 , d J=7Hz., 6-Me), 8.43, (6H, ' 5.14, 9.02 (innurities), 111./e 258(M ), 136, 121 (base), 105, • • (Found: C, 78.85; H, 8.74; C17H2202 requires C, 79.02;

H, 8-59).

Saponification of "T" (44mg.) gave trans-2,6-dimethylocta-2, 164 4-dien-S-ol (207a) (27ms., 100,:.) as an oil which was

purified by p.l.c. (one 20 x 20 x 0.1cm.silica plate developed once in diethyl ether : retroleum = 3 : 7) ,

- 177 - • \.) max. (CO2) 3620 (m.), 3350 (broad n.), 3080 (w.), 3020 (w.), 2960 (s.), 2920 (s.), 2870 (s.), 1660 (w.),

1375 (s.), 1120 (h.), 1050 (s.), 990 (m.), 965 (s.),

885 (m.), 870 (m.), 785 (w.), 760 (w.)cm-1, A max. (ethanol) 231 (25,000), 236nm. (26,000)..

T.l.c. (silica, diethyl ether : petroleum = 1 : 19)

indicated a similar mixture of compounds were formed on

reduction of geraniol benzoate (206b) by reflux with triiron

dodecacarbonyl in dimet.oxyethane overnight.

PUR:::=TC: =a:7 =c:: o =AHICL (206a) Geraniol (206a) was purified via its calcium chloride 5o (11_12mm.)162. complex and distillation b.p. 11 1.m ,

(silica, diet.ly1 ether : petroleum = 7 : 13) indicated formation of both polar and non polar compounds on

reduction of the title compound (206a) by reflux with

triiron dodecacarbonyl in dimethoxyethane for 100 minutes.

1=7,EF=TIC AHT) , 11)1:CTICE OF :=NIOL 7=7,THYLTLvUT7HR (206c) The title comr)ound (206c) (510mg., 93) prepared from geraniol (206a) (urified, 379mc.) was obtained as a colourless

oil, 1) max. (film) 2960 (s.), 2920 (s.), 2360 (s.),

• 1670 (w.), 1450 (m.), 1330 (m.), 1250 (s.), 1105 (s.), • 1065 (s.), 330 (s.), 340 (5.), 750 (s.), 688 (w.)cm-1.,

1;4.7:7.. (17, t., J = 6.5Hz., 2-:), 4.90 (1E, m., 6-H) 5.94

(2H, d., j = 1-7), 7.937, (4H, m., 4-:, 5-7), i.32

(3::, s., ?.40 s Ne2c=), 9.93 s., c=e3)-

Trimetylsily1 ether (206:) (331m:.), diiron nonacarbonyl

(2.4,.) benzen,, .(1'2,m1.) ','ere hcatc,d to reflux unde r for 2'+. hour:.:., cooled, filtered t:.: of alumina wasing. through

- 17P

• with benzene and evaporated. The residue was extracted

with petrcleum (sodium dried, 5 x 1m1.), filtered and

evaporated. T.l.c. (silica, petroleum) and n.m.r.

indicated the formation of both trimethylsilylethers and

hydrocarbon iron tricarbonyls..

A2A2'I0ih C7 Ei:GCCALCITE2OL T2IC.1230NYL (210).. o • A cooled (0 ) solution of ergocalciferol (I) (37mS..) and iron pentacarbonyl (1m1.) in THE (9m1.) was degassed

with nitrogen prior to and luring irradiation through pyrex

with a hi_ h pressure mercury arc lamp overnight. The

burgundy solution was filtered trough alumina washing

through with diethyl ether to give a clear green solution.

Evaporation, extraction with petroleum (5 x mml.), filtration,

evaporation and p.l.c. (one 20 x 20 x 0.1cm silica plate

developed three times in diethyl ether : petroleum =

7 : 13) gave the title comeound (210) (33mg., 28(A and

ergocalciferol (I). The complexes (210) were identical 123 (t.l.c. and n.m.r.) with authentic sam.ples Crysallisation

twice from methanol gave isomer (210b) as yellow elongated o plates m.p. 124.5-126.5 , recryrtallisation from petroleum

gave pale yellow feathery needles m.F. 134.5-135.5 123 loo 19° = 134°), /- 04. 7 469° rip( 757.' . +501° - - D • - 8nm o o • -713 = +609 /04. - +6nm (c = 0.325, ethanol).

Photolysis of ergocalciferol in iron pentacarbonyl/

netroleum was unsatisfactory because of lieht cut out by

large a.: can of aiiron nenacarbonyl f.)re,:e • erocalciferol

iron tricarbcnyl (21C) was formed.

• - 179 - T.l.c. (silica, diethyl ether : petroleum = 2 : 3)

and n.m.r. indicated >90Y conversion to ergocalciferOl

iron tricarbonyl (210) on prolonged irradiation in quartz

apparatus.

PTITAA7I0::3 017 -1=E2GOCATCIT= )

A cooled (01.50) slurry of triiron dodecacarbonyl (4g.)

S and preergocalciferol (7a) (331mg.) in dimethoxyethane (20m1.) in a auartz test tube was mechanically stirred under nitrogen

and irradiated for 8 hours through a quartz dewar with a high

pressure mercury arc lamp. The mixture was filtered through

alumina washin;; through with diethyl ether, evaporated,

extracted with petroleum (5 x lml.) and filtered. After

evaporation the residue in Petroleum was stood at room

temperature overnight, evaporated and separated by p.l.c.

(4 40 x 20 x 0:1cm silica plates developed three times in

diethyl ether : petroleum = 3 : 7) to give (in order of

increasing polarity) triiron dodecacarbonyl, minor comround

(16mg.), -=er7ocalciferol iron tricarbonyl

preerrrocalciferol (7a) (92ms.), ergocalciferol iron

tricarbonyl (210) an' ergocalciferol (1) (46m:.)..

An attempt to remove preergocalciferol (7a) from the crude

preergocalciferol iron tricarbonyl (209a) by repeated • thermal equilibration with ergocalciferol (1) and

failed due to the ease of decomposition of complex (208a)

ving r;reergocalciferol (7a).

An attemt to isolate rcergocalciferol iron tricarbonyl

(2Ca) from the crude 7hotolysate by removal of 7:reer2ocalciferol

(7a)/ercocalciferol (1) win 1 equivalent of 4-rheny1 -1,2,4- ,o 127 is ;ol 5-dione in iichThror.Inth:.ne at failed.

S - 180 - Although excess Cookson's reagent was quenched at -70

with ergosterol rIcetate t.l.c. (silica, diethyl -ether

petroleum = 1 : 1) after warming up to room temperature

indicated (in order of increasing polarity) triiron

dodecacarbonyl, ergosterol acetite, minor unknown sterol

iron tricarbonyl, calciferol iron tricarbonyl (210) and

polar compounds only.

T.l.c. (silica, diethyl ether : petroleum = 2 : 3)

indicated complexation of preergocalciferol (7a) by

irradiation in iron pentacarbonyl/TNF was slow relative

to thermal isomerisation followed by complexation.

Photolysis at -70° with triiron dodecacarbonyl/dimethoxyethane

was unsatisfactory due to the low solubility of the carbonyl

at this temperature. max. \ (CS2) 3605 (m.), 2960-2920 (s.), 2870 (s.), 2C38 (s.), 1960 (s.), 1370. (s.), 1120 (m.),

1040 (m.), 975 (m.), 820 (w.), 740 (w.)cm-1, T; 4.43, 4.62

(1:1, m., * ), 4.77 (2H, m., 22-H, 23-H), 6.04 (1H, m.,

30( -7), 8.27, 8.88, 8.92, 9.02, 9.12, 9.23, 9.27*, 9.42*

(methyl 7eaks), (peaks resulting from preergocalciferol (?a) + or ergocalciferol (1) indicated thus*), m/e 536 (M ), 50P,

420, 452, 396 (base), 363, 337, 271, (Found: 536. 2602;

0. Fe(CO) requires 536. 2589). C28H44 • 3 • I=P=771:-N 0? PP7:R0CCATCT177,20J (7b)

A solution of preergocalciferol (7a) (150mg.) in

toluene (5m1.), pyridine (2m1.) and acetic anhydride o (0.5s1.) was stood at 0 for 2 days. Diethyl ether (50m1)

was added and the solution washed with water (2 x 30m1.),

saturated brine (20 ml.), dried and evaporated. . P.1.c. (three 20 x 20 x 0.1cm silica plates developed once

in diethyl ether : ,petroleum = 1 : 19) gave the title coonnd

(7b) (62mg. 37>J) as an oil, ') max. (CS2) 2950 (s.), 2920 (s.), 2870 (s.), 1740 (s.), 1375 (m.), 1365 (m.), 1245 (s.), 1102 (w.), 1030 (m.)., 975 (m.), 950 (w.),

750 (w.)cm-1., v4.07, 4.40 (2H, = 14Hz., 6-H,

7-H), 4.52 (1H, m., '; -I11 = 10 Hz., 9-H), 4.83 (2H, m.,

• 22-H, 23-H), 5.09 (11I, m., 300 -H), 7.98 (3H, s., 0-Ac), 8.35 (31I, s., 19-ne), 8.92, 9.02, 9.12, 9.22 (12H, side chain

methyls), 9.30 (3H, s., 18-Ne), Ve 433 (e), 378, 253, (Found: C, 82.02; H, 10.35; C30114602 requires C, 82.12; H, 10.58).

Pr7:7A2ATI0N OF P=RC;COALCIFOL ACF,TATE IRON =CATH3ONYL (208b)

Photolysis of preergocalciferol acetate (27b) (185mg.) • and triiron dodecacarbonyl (4s.)•ih dimethoxyethane (20m1.)

at 0-50 under nitrogen for 30 hours gave a yellow oil. P.l.c. (four 20 x 20 x 0.1cm silica plates developed three

times in :iethyl -ther: -cetroleum = 1:19) gave in order of

increasing polarity) T:reer7ocalciferol acetate (7b) (71mg.)

and creer[7ociciferol acetnte iron tricarbonyl (208D)

(77 mg., 52;..) as a yellow oil. The complex cochromato=aphed with ergocalciferol acetate. It was not Possible to purify • • the complex further because of its facile decomposition giving preergocalciferol acetate (7b). Acetylation of preergocalciferol iron tricarbonyl (208a) and c.l.c. gave

the sa-:le (n.m.r., t.l.c.) com ound, 1) max. (,)2) 2970-2920

(5.), 277 .), (s.), 1960 ([3.), 1740 (e., 1370 (s.),

1245 (5.), 1120 (n.), 1030 (a.), (s.), 910 (w.), -1 ' 740 ('::.)c m ., L 3.94, 4.23, ( *), 4.7 (2H, m., 22-H, 2.7-1), 5.0- (111, 3o( -H) (2H, m., viny1-7e(C0) ) 3

0 • 7.97 (311, s., 0-Ac), 8.30 (311, s., 19-Y,e), 8.93, 9.02,

9.12, 9.22 (1211, side chain methyls), 9.27, 9.30* (3H, 2s., 13-Me), (peaks resulting from preercocalciferol

acetate (7b) indicated thus*), F/e 578 (M+), 522, 494,

438, 373 (base), 253.

ATT=PTZD .j=l'LlTION C7 I7'.2=COCAMCI777:CL T17:IC=301= (20r"b) • ACETATE A solution of tetra-n-pronylammonium iodide (0.61g.)

in water was aded to silver acetate (0.33g) in water (10m1.)

and the mixture warmed, cooled, filtered and water removed

by repeated azeatrope with toluene. The residue of

tetra-n-propylammonium acetate in dichloromethane (10m1.)

was stored over activated 4A molecular sieve.

Preergocalciferol acetate iron tricarbonyl (20,:b)

(78mg.) in dichloromethane (3 x 1ml.) was added dropwise

to a stirred solution of triphenylmetyl tetrafluoroborate

(90ms.) in dichloromethane (10m1.) at -10° under argon.

T.l.c. (aliquiot quenched with diethyl ether) (silica,

diethyl ether : petroleum = 1 : 9) ind icated little reaction

after 3 hours. After a further 2 hours at room tem erature

t.l.c. indicated absence of starting material. Tetra-n-

• propylarmonium acetate in dichloromethane (10m1.) was added; the dark yellow colour being instantly discharged. The

solution war diluted with dichloromethane (100m1.) washed

with saturated aqueous sodium bicarbonate (20 ml.), water

( x 100m1. and rigid, Eaioration have an oil l:et t.l.c.

and n.'m.r. of in:ic.te:1 a mixture of several steroids

(in addition _3C9 B7,1)y rroducts. lbo iron cc' exec

or c ounTh of the ox-,ctoj -'iacr,tate 7o1 rit: wor.: rro.7ent.

• M 7,3ICYL (217)

(1)A solution of ::olyblenum hex-tcarbcnyl (`'5m-.) in

cyclohentatriene (10m1.) was heated to reflux under -rgon

for 4. hours. The solvent was removed under vacuum and

the residue chromato7raphed on silica (1:10C, 5g.) to -ive

molybdenum hexacarbonyl (eluent petroleum) and cyclohep,tatriene

molybdenum tricarbonyl (213) (eluent petroleum : diethyl ether o 163 = 4 : 1) as red plates (3nmg., m.. n 100-101.5 (lit.

100.5-1.5°) from petroleum, N max. (cyclohexane) 255(12,000),

324(9,300), 331(3,500), 495nM.(560).

(2)A solution of cycloheptatriene (inns.) and molybdenum

hexacarbonyl (264mg., 5 equivalents) in dichloromethane

(2.0m1.) was degassed with argon nrior to and during

irradiation (Pyrex arparatus) with a high pressure mercury

arc lamn for 5 hours at room temperature. Evaporation and

chromatography gave cycloheptatriene molybdenum tricarbonyl

(213) (14mg., 26).

,(3) A cooled (-10°) solution of molybdenum hexacarbonyl

(124mg.) in TH7 (10m1.) under argon in Pyrex apparatus was

irradiated with a high pressure mercury arc lamp. Tlir was

removed from the red solution (0°, lmm.) to give a yellow

solid th t rapidly darkened. Cycloheptatriene (Smg.) in

dichloromethane (4.0m1.) was added and the dark mixture

stirred at room teerature for 4 hours. Evaroration and

chrcmatocraz,hy gave cycloheptatriene molybdenum tricarbonyl

(213) (3mE., 33).

a -154 - ATT.1;:IPMA =L.=:= C.H=GOCALCi::MOL YkL173.;;TUM T2ICM0= (71.20) rir2TCA-'730= (219) T.l.c. (silica, diethyl ether : petroleum = 7 : 13)

indicated preergocalciferol (7a) was unchanged on irradiation

(Pyrex apparatus, high pressure mercury. arc lamp or tungsten

lamp) in petroleum (saturated hydrocarbons only, 40-60°), o o THE or dichloromethane at -10 to 0 in the presence of • molybdenum hexacarbonyl. A brown steroid free solid

(probably molybdenum) was formed. Attempted preparation

of ergocalciferol molybdenum tricarbonyl (219) by reaction

(THF) complex in dichloromethane with the yellow lio(CO)x 6-x •or carbon tetrachloride gave red solutions that rapidly

deposited molybdenum. T.l.c. (silica, diethyl ether:

petroleum = 3 : 7) indicated a yc,llow compound

• cochromatograohing with ergocalciferol, the colour was

rapidly discharged on standing,

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150. 1.1. Makita and J. H Wella, Analyt. 73iochem.,

1963, 5, 523.

151. "Dictionary of Orzanic Com-rounds" 4th Edn.,

IL:yr° and ::;ottiswoode- Ltd., London, 1965, r. 702

152. G. J. Kent and E. S. Wallis, J. Orr:. ChoLl., 1959, Ph,

153. 0. 'iinterseiner and H. ::oore, J. Arner Chen. Soc.,

1(717n ?2 1(127

154. C. . 3no1-,7)ee and E. C. Kowf.::an, J. Chem. Sec. C,

9F;1.

• - 202 -

155. E. H. 73.1.-.7!ve and Hubei, Ynor7. Synn., 1966,

VII:, 17%

156. H. B. Henbest an E. W. H. Jones, J. Ch:' Sc., 1948, 1792.

157. V. L. Petrow, J. Cher. Soc., 1940, 66 •

152. S. Berstein, L. J. Binori, L. Dorfman, i. J. Sax.

and Y. Subba Row, J. Or. Chem., 1949, 14, 433.

159. .1. NcFarlane and C. Wilkinson, Inorg. Synth.,

1966, VIII, 121.

160. W. Gerrard and K. D. Kilburn, J. Chem.Soc., 1956, 1536.

161.. A. Hon.-?on, Annelen, 1957, 603. 115.

162. H. A. Jones and J. . Wood, Int. Enp;. Chem., 1942.

3k. 1'7.

163. H. J. Dauben Jr. an', L. R. Homier, J. Amer. Cherl. Soc.,

195r, 90, 5570.

41,

164. H . H. _,sc 9inasi, J. Cr-'. Chem., 1970, 35, 1097. Photofragmentation Reactions of Ergosterol • By ANTHONY G. M. BARRETT, DEREK H. R. BARTON,• RICHARD A. RUSSELL, and DAVID A. WIDDOWSON (Chemistry Department, Imperial College, London SW7 2AY)

• Reprinted from

Journal of The Chemical Society Chemical Communications 1975

The Chemical Society, Burlington House, London W1V OBN 102 J.C.S. CHEM. COMM., 1975

Photofragmentation Reactions of Ergosterol By ANTHONY G. M. BARRETT, DEREK H. R. BARTON,• RICHARD A. RUSSELL, and awn) A. WIDDOWS02.1_ (Chemistry Department, Imperial College, London SW7 2A1-) Summary The photochemical formation of de-An-ergost- FRAGMENTATIONS during vapour-phase photolyses are 22-ene derivatives from ergosterol is described. documented.' We report the isolation and structural •

J.C.S. CHEM. COMM., 1975 103

determination of the de-An-ergost-22-ene derivatives (Ia)- triphenylethoxymethylenephosphorane). The room tem- (Ic), (Ha), (IIb), and (IIIa). perature acid isomerisation (tetrahydrofuran-H2O-H,S0, 100; 21; 9) of the diene (Ia) gave the diene (IIb) (100%). Reduction (NaBH4) of the ketone (IV) gave de-AB-ergost- C9H17 C 9 I-117 22-en-8$-ol (Va) (oil, 84%) isolated as its 3,5-dinitrobenzoate (Vb), m.p. 141-142°, (lit,* 146°), 1a]1,2 + 70°, (all [a], in CHC1, unless stated otherwise). Dehydration (methane sulphonic anhydride-pyridine) of (Va) gave the diene (Ha) (82%) and pyrolysis of the xanthate (Vc) (oil) gave the diene (II1a) (55%). An attempt to synthesise 4 (111a) from the ketone (IV) via its toluene-p-sulphonyl- R1=R2=H LID R =H MI) a; 14ct-H hydrazone (m.p. 116-117°, 93%) and methyl-lithium gave the. C-14 epimer (IIIb) (oil, 40%), alternatively prepared Ri=0Et,R2-r.H 131 R=Me 1,1 1411-H b; from the ketone (IVb)s by NaBH4 reduction and dehydra- c; R1= H,R2= OE t tion (17%, overall, [a]t2 32°).

TABLE C91-117 C91117 Structure Yield/mg M.p. [a]p (CHC1,) (Ia) 76 Oil +48° (Ib)b 29 47-49° +56° (cyclohexane) go b , e 8 Oil +49° (cyclohexane) (Ila) 36 Oil +9-8° 0 RO (IIb) 5 Oil +12.4° (Ma) 14 Oil +35°

(IV a; 14a-H ry) a; R H (VI) a All new compounds gave satisfactory analytical and spectral 2 2 b1 14 [3-H b; R = CLIO) C6H3 -3,51NO ) data. b Stereochemistry may be reversed. Tentative assign- R CUS) SMe ment (n.m.r., t.l.c. only).

• Irradiation of ergosterol (36 g) as before2 and chromato- The photolysis of ergosterol in cyclohexane gave the graphy over alumina (Grade III, neutral), silica (Merck dienes (Ia), (Ha), (IIb), and (Ilia). GF2,) and silica-AgNO, (10%) p.l.c. gave photofragments We favour vinyl carbonium ion (VI) intermediacy in the (Ia)—(IIIa) as in the Table. The structures of these com- formation of the vinyl ethers (Ib) and (Ic) and a radical pounds were confirmed by synthesis. pathway for hydrocarbon fragment formation. Wittig reactions of 'Grundmann's ketone' (IVa.)3 gave the diene (Ia) (74%) (with triphenylmethylenephosphorane) and the vinyl ethers (Ib) (25%) and (Ic) (25%) (with (Received, 28th November 1974; Com. 1445.) s R. Srinivasan, J. Amer. Chem. Soc., 1960, 82, 5063. 2 A. G. M. Barrett, D. H. R. Barton, C. H. Carlisle, P. F. Lindley. M. Pendlebury, L. Phillips, R. A. Russell, and D. A. Widdowson, J.C.S. Chem. Comm., preceding communication. A. Windaus and W. Grundmann, Annalen, 1936, 524, 295. 4 H. H. Inhoffen, G. Quinkert, S. Schutz, G. Friedrich, and E. Tober, Ber., 1958, 91, 781. K. Dimroth and H. Jonsson, Ber., 1941, 74, 520.

• •

Structures of the Toxisterols24 X-Ray Crystal Structure of Toxisterol2-A 3,5 -Dinitrobenzoate

By ANTHONY G. M. BARRETT, DEREK H. R. BARTON,• MARTYN H. PENDLEBURY, LAWRENCE PHILLIPS, RICHARD A. RUSSELL, and DAVID A. WIDDOWSON (Chemistry Department, Imperial College, London SW7 2AY)

and CHARLES H. CARLISLE and PETER F. LINDLEY• (Crystallography Department, Birkbeck College, Malet Street, London WC1E 7HX)

Reprinted from

Journal of The Chemical Society Chemical Communications 1975

The Chemical Society, Burlington House, London W1V OBN

101 J.C.S. CHEM. COMM., 1975

•

Structures of the Toxisterols2.t X-Ray Crystal Structure of Toxisterol2-A 3,5-Dinitrobenzoate

By ANTHONY G. M. BARRETT, DEREK H. R. BARTON,* MARTYN H. PENDLEBURY, LAWRENCE PHILLIPS, RICHARD A. RUSSELL, and DAVID A. WIDDOWSON (Chemistry Department, Imperial College, London SW7 2AY)

and CHARLES H. CARLISLE and PETER F. LINDLEY* (Crystallography Department, Birkbeck College,•3Ialet Street, Loncbm WC I E 711X ) Summary The structures of toxisterol,-A, -B, and -C have 0.4% citric acid at room temperature for 22---25 h gave a been determined by spectroscopic and chemical evidence resin. Repeated chromatography over alumina (Grade 11) supplemented by an X-ray crystallographic study of gave (from 40 g of resins toxisterol,-A as 3.5-dinitro- toxistero12-A 3,5-dinitrobenzoate. benzoate (100 -R (50 ingi awl -C as benzoate (410 mg)]. I.Ve have not yet located the fourth possible stereo- TOX/STEROLS are substances of undetermined structure and isomer. of reputed biological activity originally described about 'H N.m.r., u.v., and i.r. data. which need not he detailed 50 years ago.' Only one toxisterul (toxisterol2-A) has been here, showed that all three i0xisterols2 had the 22(23-trans- properly characterised before.' All toxisterols have a u.v. double bond intact and contained a cyclic heteroannular maximum near 250 nm. We report the isolation. character- diene system of the type ('H -CH- CC -(Me) -. Since the isation (Table) and structure determination of three such C__ 18 methyl group was also present in all compounds the compounds, toxisterolsz-A, -B, and -C. vinylic methyl was presumed to he the original C _,2 methyl.

I TABLE A i_ot.f. A„,„„inm Toxisterols2 NI.p. (C1-1Cl,) ft) Other derivatives A(V) •. • • Oil —157° 250(21,000)a p-Phenyla7.1,betizoateb tn.p. 124 126' Acetate .. .. 103-105° 250120,0001, Benzoate ni p. I 14 - IHs' 3,5-Dinitrobenzoated .. 173-174' —1 232(25.014401 p-Nitrobelizoate in.p 1412- 104' B (V1) . . 107-- 109' — 169 253(22,000), C (V11) Oil -L 17 Benzoate . Oil —133 2522 (ti(( 12.751(01 141 01;j: 235(22,000t 250(17,000) 4 In cyclohexane. b Lit.,' in.p. 125-126'. C EtOH. d Lit.,' m.p. 171--172`. Irradiation (Phillips HPli. 125 \V type 57203 B.:01)) of The "C n.m.r. spectra were especially helpful and showed ergosterol (I) in degassed ethanol containing .5",, water and that an 'extra' quaternary carbon was present. t The subscript 2 indicates that the compounds are derived from ergosterol rather than cholesterol (subscript 3).

J.C.S. CHEM. COMM., 1975 102

that these compounds were 1,2,3-trisubstituted benzenes with one of the substituents being methyl. Accepting the data at their simplest level of interpretation and using the plausible hydrogen atom transfer depicted in the Scheme, then structure (IV) seems reasonable for the toxisterols,. This permits four stercoisomers about C-4 and C-8. The relationship of the configuration at C-3 (un- HO changed) and that at C-4 was established by 1H n.m.r. spectroscopy. The configuration at the C-8 spiran centre was determined by the Nuclear Overhauser effect in the proton n.m.r. spectrum between the C-18 methyl group and the C-7 vinyl hydrogen (observed for the A isomer, but not for the B and C isomers). Toxisterols,-A, -B and -C can therefore be represented by formulae (V), (VI), and (VII) respectively. The complete Structure of toxisterol,-A was also determined independ- ently by an X-ray crystallographic study of the derived 3,5-dinitrobenzoate. Crystal data: C35H45OeN2, monoclinic, a = 6.358(1), b = 21.808(3), c = 12.259(2) A, p = 102.81(1)°, space group P21 (Cl, No. 4), De = 1.18 cm-3, Z = 2. t.t.(Cu — K2 6.6 cm-1). X-Ray intensity data were collected using Ni-filtered Cu radiation on a Hilger-Watts Y290 automated four-circle diffractometer. 1856 Independent reflections for which I > 3o. (I) were measured over the range.0 < 0 < 70°, and were corrected for Lorentz and polarisation effects; no 28 correction was made for absorption. Several attempts to 26 determine the structure using direct methods of phase determination were unsuccessful. The structure was eventually solved with extreme difficulty using a vector verification technique' to locate the 3,5-dinitrobenzoate unit, followed by iterative Fourier syntheses to find the remaining atoms. The structure has been refined using a partial full-matrix method to give a conventional R value of 0.056 with all non-hydrogen atoms treated anisotropically. The figure is a stereo-drawing of the sterol portion of the E11 11 molecule viewed along the crystallographic y axis, and

LI. 1 ) 4** / VA. I r ti ■' .t• I . FIGURE. A stereo-drawing of toxisterolt-A, showing the configuration at the spiro carbon atom C-8. The shaded atom at C-3 is the oxygen atom of the 3.5-dinitrobenzoate group which has been omitted for clarity. Treatment of toxistero12-A with CHC13-HCl afforded clearly shows the spiro-configuration at C-8. The methyl smoothly a benzene derivative (II). mn.p. :ac r, hydrogen atoms have been placed in calculated. positions —550 (all in CHC12). Treatment of toxisterol2-B and but the remainder were located from a difference Fourier -C in the same way gave a different benzene (III). m.p. synthesis. (19-71°, 6.() The n.m.r. and i.r. data showed (Receieed, 28th November 1974: Con:. 1444.) I P. Westerhoi and J. .1. levering Buisinan. Rec. Tray. chim.. 1956, 75, 1245: and references there cited. 2 1'. 13. Bratill. J. Flurnstra, and J. I. Leenhout,. Phihrs fe,;. 1969, 24, 85.