4. ANÁLISIS Y DESARROLLO DE LOS REQUISITOS DE LA NORMA
4.5 Responsabilidad de la dirección
T h is approach w as m ore su ccessfu l. W e decid ed to approach the azirid in e (1 1 9 ) by the sy n th etic pathw ay o u tlin ed in Schem e 6.10.
The synthesis o f azido acetate (1 2 3 ) is given in Scheme 6.14. F irs tly eth y l 3 -m e th y l-2 ,3 -e p o x y -b u ta n o a te (eth y l 3,3- d im e th y l g ly c i d a te) 195 (130) w as synthesised by a Darzens co n d en satio n o f acetone and e th y l ch lo ro acetate. The resu ltin g ep o x id e (1 3 0 ) w as reg io se lectiv ely opened using
T iI (0'P r )2(N3)2l196,197 to p ro d u ce the azido alcohol (124). T his regioselective rin g opening w as first achieved by Dr A. D achs. The azid o alcohol w as acetylated d ire c tly to p ro d u ce the azido acetate
T i[ (0'Pr>2(N3)2 ] 196,197 to produce the azido alcohol (124). This regioselective ring opening was first ach iev ed by Dr A. D achs. The azido alcohol w as acetylated directly to p ro d u ce the azido acetate (123), w hich w as purified by flash ch ro m ato g rap h y (Schem e 6 .1 4 ). u yj A * , A „ P o t a s s i u m t e r t - b u t o x i d c i e r f - b u t a n o l (130), 69% yield T i[(O iP r )2(N 3)2]
COQEt | (Ac)2Q, pyr,. pyf . - >I_ _ L r CC OH DMAP (cat.), CH2Q2 COOEt (123), 60% yield (based on (130)) (124) >95% yield, by !H n.m .r Schem e 6.14 6 .5 SYNTHESIS OF RACEMIC F.THY1. 3.3-DIMETHYL-1H-AZIRIDINE-2-CARBOXYLATE (1211
Follow ing the procedure o f Zwanenberg el. a l V 9 racem ic azido alcohol (124) w as converted to aziridine (1 2 5 ) (Scheme 6.15).
= T - ^ C N , COOEt P P h j, b e n z e n e . reflux, 24 hrs. rac. (124) \ q C O O E t rac. (1 2 5 ), 47% afte r F.C. Scheme 6.15
6 .6 ENZYM E CATALYSED RESOLUTION OF A ZIPO ACETATE (123)
Nine com m ercial lipases were screened for their a b ility to hyd ro ly se racem ic azido acetate (123). The e n a n tio se le c tiv ity of the reso lu tio n was established by determ ining th e E valu e for a given re s o lu tio n. 22 The results from the eight e n z y m e s worked- up are g iv en in Table 6.2.
Table 6 .2 Lipase screen for the stereo selectiv e h y d ro ly sis of azido a c e tate (1231 OA< • ¿ 0 2Et > r - ^ N3 rac. (1 2 3 ), ca. 40 m g C o n d tio n s: OH V j,__ L -c ° : N , ( - ) - ( 1 2 4 ) OAc \ I ^ CQ2Et
V
( + ) - ( 1 2 3 )Lipase ( c a . 20 mg), phosphate buffer, pH 7, lOOmM (2 m l), R.T.
L ipase e x T im e / h r s c %ee ( 1 2 4 ) % ee ( 1 2 3 ) E P s e u d o m o n a s flu o r e s c e n s 7 0 .3 4 92 4 8 39 C h r o m o b a c te riu m visc o su m 30 0 .1 9 76 1 9 9
P o rcin e pancreatic lip ase 4 0 .0 4 95 4 41
M u co r m eh ei 48 0 .2 3 67 2 0 6
C a n d id a cylindracea 2.5 0 .4 7 >98 8 8 5 8 4
R h izo p u s languinosa 24 0 .3 4 81 4 2 30
P é n ic illiu m ro q u efo rti 3 3 6 0 .1 9 87 2 0 17
A s p e r g illu s niger 2.5 0 .5 2 77 8 3 2 0
The ee o f the product azid o alcohol (124) was e sta b lish ed by firstly c o n v e rtin g the alcohol to its corresponding (R )-M o sh er's ester ( 1 3 1 ) ,103 followed by *H n.m .r. (400 M Hz) an a ly sis (see Section 6 .7 ).
ester ( 1 3 1 ),103 follow ed by JH n.m .r. (400 M Hz) analysis (s e e Section 6 .7 ).
The ee o f th e azid o acetate (123) w as measured directly by ! H n.m .r. (4 0 0 M Hz) in th e presence o f the chiral solvating a g e n t ((S )-(+ )-2 ,2 ,2 -triflu o ro -l-(9 -a n th ry l)e th a n o l, 1 m ol e q .174) (see Section 6.8).
The best enzym e w as the lipase from C a ndida cy lin d ra cea ( e x B io cataly sts L td). T h a t reaction w as scaled-up (Schem e 6 .1 6 ).
The p ro d u ct azid o alco h o l (-)-( 124) was converted in to a z irid in e (125) as in Schem e 6.15. The aziridine (125) was isolated b y flash ch ro m ato g rap h y (S ch em e 6.17). H ow ever, *H n.m .r. an a ly s is show ed an o th er co m p o u n d was p resen t. The im purity c o u ld possibly be the am in o -alco h o l (1 3 2 ). The am ino-alcohol c o u ld have arise n from inco m p lete co n version o f the o x ap h o sh o lid in e (1 3 3 ) to a z irid in e, and subsequent hydrolysis o f in te rm e d ia te (1 3 3 ) (S ch em e 6 .1 7 ). n3 ( - ) - ( 1 2 4 ) 31% yield, >98%ee, n3 ( + M 1 2 3 ) 69% yield, 42% ee, rac (1 2 3 ), 2 .0 4 g , 8.9 m m ol [a f D4= + 13.6 ° [a f D4 = - 1 3 .5 ° (c 1.03, CHCI3) (c 0.98, C H C I3) S ch em e 6.16
H (-> - ( 1 2 4 ) OH ,C 0 2Et H i>rt2 ( 1 3 2 ) [a j,4 - -3 0 .4 ° (c 0.75, CHC13) Schem e 6.17
The problem with th is reactio n may be circum vented by simply d istillin g the reactio n m ix tu re. 180 This w ould probably com pletely co n v e rt o x ap hosholidine (133) into aziridine (125). The route to azirid in e (125) and d eterm in atio n o f the absolute configuration re q u ire s fu rth e r in v e stig a tio n .
6 .7 DETERMINATION OF THE %EE OF AZ1DO ALCOHOL (124)
The % ee was determ in ed by converting the azido alcohol (124) in to its co rresp o n d in g (R )-M osher's ester (1 3 1 ).103 *H n.m.r. (400 M H z) analysis rev ealed tw o w ell resolved sets o f signals. One set w as attrib u tab le to th e m ethoxy protons o f the (R )-M osher's ester and resonated at 8 = 3.65 p .p.m . and 3.54 p.p.m respectively. The o th e r set o f singlets w as attrib u tab le to the m ethine proton at C-2 (o f th e original az id o alcohol (124)). The tw o diastereom eric p ro to n s resonated a t 6 = 4.89 p.p.m and 4.85 p.p.m . respectively. Thus an internal ch e ck on th e % ee was established. Some exam ples of the o f th e *H n.m .r. (400 M Hz) spectra are given o v erleaf in Figure 6.1 (a) and (b) and Figure 6.2 (a) and (b).
FIGURE 6.1 (a) and (b) 'H n.m.r. (400 MHz) spectra of the
s i i r i i ¿l i j't
FIGURE 6.2 (a) and (t>) ' H n.m.r. (400 MHz) spectra of the Moshers ester (131) (> 98%de) of fO-Ethvl.3-methvl-.l-
6 .8 DETERMINATION OF THE %EE OF THE RESIDUAL AZIPO A CETA TE (123)
The ee o f the azido acetate (123) was established d ire ctly at ! H n.m.r. (4 0 0 M Hz) in the presence o f one mol eq. o f the chiral s o lv a tin g ag en t ( (S )-(+ )-2 ,2 ,2 -triflu o ro -l-(9 -a n th ry l)e th a n o l in a C6D6 so lu tio n. 174 A ll o f the proton resonances o f a z id o acetate (123) w ere split, except for the acetate singlet. T h e largest splitting (> 0 .0 2 p.p.m .) were observed for the m e th in e proton at C-2 and the tw o m ethyl group protons at C-3 and C -4 . Five sets of singlets w ere b aseline resolved. Som e ex am p les a r e given in Figures 6.3 (a) and (b). Figures 6.4 (a) and (b), an d Figures 6.5 (a) and (b ) overleaf.
6.9 SUMMARY
( 1 ) D ire c t reso lu tio n o f azirid in e-2 -carb o x y lic e s te r (121) was n o t totally su ccessfully. O nly t r a n s - ( \ 2 l) w a s hydrolysed. ( 2 ) R esolution o f azido acetate (123) w as su ccessfu l.
( 3 ) T h e lipase from C andida cy lin d ra cea re s o lv e d azido acetate (1 2 3 ) w ith high degree o f stereo selectiv ity (E >100). (4 ) A route from azido alcohol (124) to azirid in e (1 2 5 ) was
estab lish ed . H ow ever, som e work still n eed s to be carried o u t to m ake the reaction reproducible. T h e ab so lu te co n fig u ratio n has not been estab lish ed .
FIGURE 6,3 fa) and (b) *H iun.r. £4QQ MHz) spectra of racemic
Ethvl .3-methyl-3-azido-2-(acetoxv)butanoate in the presence of (SW -M -2.2.2-Trifluoro-l-(9-anthrvl)ethanol (1__mol equivalent),(a) Methine region, and
FIGU RE 6.4
(a) and
(t>) ‘ H n.m.r. (400 MHz) spectra o f (+ )-E thvl.3- m eth v l-3 -a zid o -2 -(accto x v )b u tan o ate (88% ee) in the p re se n c e of(SH+)-2.2.2-Trifluoro-l-(9-amhryl)ethanol (I—
molequivalent);
FIGURE 6.5 (a) and (b) >H n.m.r. (4QQ MHz) spectra of the methvl region of E th y lJ- m ethvl-3-azido-2-(acetoxv)butanoatg— (121)
in the presence of (S)-(+)-2.2.2-TrifluQrQ; 1 -(9 -anthrvU ethanol (1 mol eq u iv a le n t); (a) Racemic (123), and
C H A P T E R S E V E N -E X P E R IM E N T A L D E T A IL S
7.1 INTRODUCTION
T h e ex p e rim en tal d e ta ils are re p o rted a c co rd in g to th e in stru ctio n s for auth o rs o f T he Jo u rn a l o f The C hem ical S o ciety. 198 C h e m ic a ls w ere e ith e r p u rifie d fo llo w in g lite ra tu re m e th o d s, 199 o r p u rc h a s e d as the highest a v a ila b le g ra d e .
N u c le ar m a g n e tic re so n an ce s p e c tra w ere re co rd e d u s in g the fo llo w ing i n s t r u m e n t s :
B ru ker W H -400 (o p e ratin g freq uency ! H n.m .r. = 4 00 M Hz), P erk in -E lm er R -3 4 (o p e ratin g frequency *H n.m .r. = 2 2 0 MHz), B ru ker W M -200 (o p e ratin g frequency *H n.m .r. = 20 0 M Hz), B ru ker 300 (o p e ratin g frequency *H n.m .r. = 300 M H z), and Jeol G X -270 (o p e ratin g frequency *H n.m .r. = 270 M H z).
A ll are fo u r ie r tran sfo rm s p ec tro m eters (e x c e p t fo r th e P erk in -E lm er R - 3 4 , w hich is a co n tin uo u s w ave spectrom eter). 13C n .m .r. w ere recorded at an o peratin g freq u en cy o f 100 M Hz. C hem ical s h ifts arc reported in 5 relative to trim e th y ls ila n e = 0 .0 0 ppm ). M ultip licities o f *H n.m .r. sig nals are abbreviated as follow s: s, sing let; b r s, broad sin g le t; d, doublet; t, triplet; q, q u arte t; m , m u ltip let. A ll o th er m u ltip lets are w ritten in full (c.g. q u i n t e t ) .
T . l.c. were run o n glass plates p re-coated w ith 0.20 m m o r 0.25m m M erck
silica gel 60 p 2 5 4 - D etection used o ne o r m ore o f the follow ing m e t h o d s: 200*201
P h o sp h o m o ly b d ic acid (7 g /l), fo llow ed by h e a tin g .
Cerium s u lp h a te (2.3g) in w a te r (160m l) and H2S O4 (48m l) follow ed by
h e a t i n g .
N inhydrin (0 .2 % ) in n -b u tan o l : acetic acid; 93 : 3, and heating. 2 ,4 -D in itro p h e n y lh y d ra z in e (0 .4 g ) in 2M HC1 (1 0 0 m l) and heating .
50% H2S O4 and charring at 2 00 ° C .
B rom ocresol green (0.04g) in eth anol (100m l), to w h ich w as added 0.1M NaOH un til th e blue co lo u r disappears.
P a ra -A n is a ld e h y d e ( lm l) in H2S O4 (1m l) and ethanol (18m l), heat. E x posure to iodine vapour.
PLC (p re p a ra tiv e lay e r ch ro m a to g rap h y ) w ere run o n 20cm x 20cm glass plates, p re -c o a te d with e ith e r 1mm o r 2mm M erck silic a gel 6 0 p 2 5 4 -
FC (F lash C h rom ato g raph y ), all colum ns contained silic a to a height o f 15-
20 cm . F C w as perform ed acco rd ing to the m ethod o f S till et. al202 The
follow ing so lv en ts w ere glass d istilled for use in FC and PLC: Light petroleum e th e r (b.p. = 40 -60 ° C ). ethyl acetate, d ieth y l ether.
O ptical ro ta tio n s w ere m ea su red on eith er an O p tica l A c tivity L im ite d , M odel A A -1 0 0 0 p o larim eter (in a 2 dm pathlen gth c e ll), o r a P erk in -E lm er. M odel 2 4 1 -C polarim eter (p ath len gth = 1 dm). C o n cen tratio n c = g / 100m l, w av elength = 589 nm . M eltin g points are u n correc ted. G as-liquid ch ro m a to g ra p h y (g .l.c .) u sin g p ack ed colum ns (1.8 m in length) u sed eith er a P Y E 104 o r a P Y E 204 instrum ent. G a s-liq u id chrom atog raphy (g .l.c .) u sin g ca p illa ry co lu m n s w ere run on a C arlo-E rba F ractov ap 2 4 5 0 series in stru m en t. N itrogen w as th e c a rrie r gas at a flow ra te o f 30 m l/m in. Infra red s p e c tra were reco rd ed on eith er a P erk in -E lm er S80-B o r a
P erk in -E lm er 1720X m ac h in e . Mass sp ec tru m w ere recorded on e ith e r a K ratos M S 80 or a F inn ig an 4000 instrum ent.
7 .2 EXPERIMENTAL DETAILS FOR CHAPTER TWQ,
A ssig n m en ts o f 13C s p e c tra use the n u m b erin g system in the follo w in g m od el o f the carbon sk eleto n :
3 -1 4 -1 2 -H v d ro x v eth v llp h e n o x v lD rQ D c n e— ( 1 6 ) - To a stirred so lu tio n of 2 -[4 -h y d ro x y p h e n y l]eth an o l ( I S ) (1 0.0g, 7 2 m m o l) in AR grade aceton e (3 0 m l), w as added a n h y d ro u s potassium ca rb o n ate (1 5.9g, S 7m m ol) and
allyl b rom ide (7.5m l, 86m m o l). The su sp en sio n was boiled u n d er reflux for
18 h ours, (g.l.c., SE30{ 10% }, 158 °C , Rt (15) = 4m in. Rt (16) = 6m in). the
re actio n m ix tu re w as d ilu te d with w ater (4 0 m l), ex tracted with d ich lo ro m eth a n e (2 x 40 m l), w ashed with aq u e o u s 5% w /v NaOH (2 x 40m l),
d rie d (M g S Û4), filtered an d evaporated u n d e r reduced p ressure. K ugelro h r
d is tilla tio n (oven te m p e ra tu re 110 °C /0 .1 m m H g ) yielded (16) as a
c o lo u rle ss liquid (9.66g, 7 5 % y ield), hom ogen eou s by g.l.c and *H n.m .r.; ôh
(2 20 M Hz; solvent CDCI3; standard Me4Si) 1.52 (1H. bs. O-//), 2.81 (2H . l. J 7Hz.
ArCZ/2), 3.83 (2H. bt. C //2O H ), 4.53 (2H. d. J6Hz, C H
2
OAt).5.28 (1H. d , J10.3H z, C //H = C H (cis)), 5.42 (1H. d. J 19.1Hz, C //H = C H (tra ns)). 6.08 (1H. m, C //= C H 2). O II 1( 26.88 (2H, d. /o rth o 8.3 Hz, A r-/ / ) , and 7.15 (2H . d. / ortho 8.3H z. A r-//); 6C
(100M Hz; solvent C D C I3) 37.9 (C -8). 63.3 (C -3), 68.5 (C -9), 114.5 (C -5), 117.1
(C -l), 129.6 (C -6), 130.6 (C -2 ), 133.1 (C -7), and 156.9 (C -4). m /z (E l) 178 (M +.
31% ), 147 ((M -C H2O H )+ . 100), and 107 (56); HRM S, found (m /z): 178.0989,
C1 1O2H14 requires: 178.0993.
L i-)-3 -i4 -[2 - H v d ro x v e th v llD h e n o x v l - 1.2 -e D Q x v p ro p a n e ( 1 7 ) . - T o a stirred so lu tio n o f 2 -[4 -h y d ro x y p h e n y l]eth an o l (1 5 ) (4 0g , 0 .2 8 9 m o l) in d ry b utan-
2 - one (100m l) w as added an hydrous po tassiu m ca rb o n ate (3 7 .6g , 0.37 6m ol)
and fresh ly d is tille d ep ib ro m o h y d rin (2 7 .3 m l, 0 .3 1 9 m o l). The su sp en sio n w as boiled under reflux for 24 hours, (g .l.c ., SE30{10% }, 2 00 ° C , R , (1 5 ) = 4 m in , Rt (17) = 9 m in ). T h e suspension w as cooled, filte re d and evaporated u n der reduced p ressure to yield (17) as a yellow oil w h ic h solid ified on stand in g. R e c ry s ta llis a tio n (e th y l ac e ta te /lig h t p etro le u m (b .p . 4 0 -6 0 ° C ) ) yield ed (17) as a w hite so lid (51.6g, 92 % y ield ), h o m og en eo us by g .l.c. and *H n.m .r.; vm ax (nujol m ull) 3 215 (b. OH ), 1 620 (m , Ar). 1 515 (s, Ar), 1 300
(m ), 1 250 (s, epoxide), 910 (m , epoxide), and 830 cm’ 1 (m ); 6h (220 MHz;
solvent CDCI3: standard Me4S i) 1.53 (1H, bs. O-//), 2.72-2.83 (4H . m . AtC H
2
and term inal ep o x id e p ro to n s), 3.37 (1H , m , m ethine e p o x id e p roto n), 3.83 (2H. t. / 7.5 Hz. C //2O H ), 3.96 (1H. dd. 7gem -11 Hz and Janli6 Hz. A rC //H (a n ti)).
4.22 (1H. dd, /g e m -11H z and J syn 3 Hz. A rO C //H (syn)). 6 .89 (2H, d. / ortho 9 Hz. A r - / /) , and 7.17 (2H , d. / ortho 9H z. A r-//); m /z (El) 194 (M + . 70% ), 176 ((M- H20 ) + . 6). 163 (96). 133 (19). and 107 (100); m /z (Cl NH3) 2 12 ((M +N H 4 )+,
16%); HRMS, found (m /z): 194.0944, C1 1O3H14 requires: 194.0943.
3 - i 4 - i2 - A c e to x v e th v llD h e n o x v lp ro p e n c ( 1 8 ) . - T o a stirred solution o f 3-[4- [2 -h y d ro x y eth y l)p h e n o x y Ip ro p en e (1 6 ) (l.O g , 5 .6 m m o l) in d ry p y rid in e (15m l), at 0 ° C u n d er n itro g en , w as added slow ly dry a c e ty l ch lo rid e (0.4m l, 5.6m m ol) and th e m ix ture w as allow ed to com e to room tem perature during
18 hours, (g .l.c ., S E 3 0 { 3 % ), 150 ° C , R t (16) = 6.5m in, R t (1 8) = ll.S m in ). The su sp en sio n w as filte re d and ev a p o rate d u n der re d u ced p re s s u re . The
re sid u e w as d is s o lv e d in dich lo ro m eth a n e (20m l), w ashed w ith an aqueous
satu ra ted s o lu tio n o f so d iu m h yd ro g e n ca rb o n ate (20m l), d ilu te su lp h u ric acid (2 0 m l), an d w a te r (1 0 m l), d ried (MgSCXt), filtere d and evaporated at re d u ced p re s s u re . K u g e lro h r d is tilla tio n (o ven te m p e ra tu re 115 °C /0 .3 m m H g ) yield ed (1 8) as a colo urless liqu id (0.5 7g , 46 % yield),
hom ogeneous by g .l.c . and 1H n.m .r. pure; 8h (220 M Hz; so lv en t CDCI3;
standard Me4S i) 2.08 (3H. s, C ( 0 ) C / / j) , 2.92 (2H, t. J 7.3 Hz, A r C « 2 ), 4.30 (2H, t.
J 7.3 Hz, AiC H2C H2 ). 4.58 (2H. d. J 5 Hz, C //2OAr), 5.35 (1H, d, J 10 Hz, C //H = C H («'*)). 5.48 (1H , d. J 18 Hz C H //= C H (irons)), 6.13 (1H . m , C H2= C H ). 6.93 (2H, d.
J ortho 9 Hz, A r- / / ) , and 7.20 (2H . d. 7 0rtho 9 Hz. A r-H); 8q (100M H z; solvent C D C I3) 20.5 ( C - l l ) . 34.0 (C -8), 64.8 (C -3), 78.5 (C -9), 114.4 (C -5), 117.1 (C -l), 129.3 (C -6), 129.6 (C -2), 133.2 (C -7), 157.0 (C -4), and 170.4 (C -10); m /z (E l) 220 ( M + , 2.6% ), 161 (1 8). 147 (15), 160 ((M -acetate)+ , 100), 119 (46). and 107 (17); HRM S, found (m /z): 220.1099; C i3 0 3H i6 requires: 220.1098. ( l ) - 3 - l 4 - [ 2 - A c e t o x v e t h v l l D h e n o x v l l . 2 - e D o xv pro Da ne M9 1. M e th o d A .- T o a s tirre d so lu tio n o f 3 -l4 -(2 -a c e to x y e th y lJ p h e n o x y ]p ro p e n e (1 8 ) (0.2 0g, 0 .9 1 m m o l) in d ry dichlo ro m eth ane (2m l) w as added m e ru -c h lo ro p e rb e n z o ic acid (0 .2 1 g . 1.2m m ol). T he re a c tio n m ix tu re w as