Studies on beta-lactams.

Full text of DOI:10.1515/znb-1969-0909

B. G. CHATTERJEE AND R. F. ABDULLA
1120
232 V. A. K o le so v a u . Y. I. R yskin, Optics and Spectroscopy
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244 K. A. W iC k e R s h e im , J. chem. Physics 42, 579 [1965].
245 E. J u n g m a n n , k. k la R iC , S. m aRiCiC u. Z. m eiC, Sym-
posium sur les Bauxites, Oxydes et Hydroxydes d’Alumi-
nium, Zagreb 1963.
259 E. S c h w a rzm a n n u . H. M arsm ann, Z. Naturforschg.
21b, 924 [1966].
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261 a. be n O it , Spectrochim. Acta [London] 19, 2011 [1963].
Studies on beta-Lactams
basanta G. ChatteRJee and R ia z F. a bDulla
Department of Applied Chemistry, Indian Institute of Technology, Kharagpur, W. Bengal. India
(Z. Naturforschg. 24 b. 1120— 1127 [1969J ; eingegangen am 13. Mai 1969)
Since the discovery 11 of the activating effect of the N-(p-Nitro-phenyl)-group on the methine
hydrogen of halogenacetamino esters, it has been found that the p-Acetyl-phenyl and m-Nitro-phe-
nyl groups exert a similar effect. It has been shown that the delocalisation of the N-non-bonded
electrons across the orbitals of the A'-Aryl substituent is the governing factor in the cyclisation of
the above systems, and the effect of the bulk of the A-Aryl substituent is relegated to a minor
position.
Discussion
h a n and bOse was extended by ChatteRJee and
Ra O , to systems in which the methine hydrogen
atom was activated by groups other than two ester
functions7-9. It was noted that the closely related
intra-molecular alkylation of o>halogen-alkylmalon-
ic esters ocurred in poor yields even in the presence
of sodium ethoxide 10, while the base of choice in
the Bose Sh e e ha n synthesis was triethylamine.
After the presence of the beta-lactam ring and its
biological significance was established in Penicil-
lin 1, Cephalosporin and Pachysandra Terminalis 2’3,
several new methods were developed for its synthe-
sis 4. Among these, the intra-molecular alkylation of
^-substituted halogen acetamino malonates seemed
to be the most general 5-6. The technique of sh e e -
6 J. C. s h e e h a n and A. K. b O s e , J. Amer. chem. Soc. 73.
1261 [1951].
1 H. T. C la R k e , J. R. J O h n s O n and R. R O b in sO n , The Che-
mistry of Penicillin, Princeton University Press, Princeton
1947.
2 E. P. a bRa h a m and G. G. F. n e W t O n , Biochem. J. 79, 377
[1961].
7
8
B. G. C h a t t e R Je e , P. N. m O z a , and S. K. R O y . J. org.
Chemistry 28. 1418 [1963],
B. G. C h a t t e R Je e , V. V. R a O . and B. N. G. m a z u m D a R . J.
org. Chemistry 30, 4101 [1965].
3
4
5
T. K ik u c h i and S. U y eto, Tetrahedron Letters [London]
1965, 3473.
J. C. S heehan and E. J. C orey, Org. Reactions 9, 388
[1957],
J. C. S heehan and A. K. Bose, J. Amer. chem. Soc. 72,
5158 [1950].
9 B. G. C h a t t e R Je e . V. V. R a O . S. K. R O y . and H. P. S.
C h a W l a . Tetrahedron [London] 23. 492 [1967].
10 H. N. W a lb O Rsk y . J. Amer. chem. Soc. 71. 2941 [1949].
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STUDIES ON BETA-LACTAMS
1121
The effect of the group attached to the nitrogen
atom of the halogen-acetamino esters had not at-
tracted much attention until it was demonstrated
atom, but when the co-planarity was disturbed as
for example in the series (a) or (b) with bulky
or^Ao-substituents, then the activation decreased
markedly.
that an A-(p-nitro-phenyl) -group exerted
a long
range activating influence on the methine hydrogen
atom of such systems 11.
The conclusion was that in the systems (1) the
two functions attached to the carbon atom carrying
the methine hydrogen atom, played their role in
effecting cyclisation, together with the contributing
activating effect from the A-substituent, to give the
beta lactams (4).
A large number of compounds were investigated,
using various bases:
L
Base
c h /
In the work to be described attention has been
centered on firstly, whether groups other than the
nitro-group can cause activation to any marked de-
gree; secondly whether an electron withdrawing
substituent in the meta-position can cause activa-
tion; and finally to establish the role of the A-aryl
substituent as a whole more exactly.
A~\°./“ ?
\cooc2h 5
o = c - c h 2ci
1
A —( O )- N - C <
|
\— /
I
COOC2H5
0 = C - C H 2
The amino esters that were synthesised for the
study had satisfactory IR absorption characteristics,
and were characterised by microanalysis in addi-
tion (Table 1, Compounds 2). Liquids were puri-
fied and used directly for the next step as they could
not be obtained analytically pure.
(a)
H
n o 2
Ph
Cyclisation with
triethylamine
(b)
(c)
A
B
L
(a) .... None
(b) .... None
(c) .... Cyclises
N02
Cl
Ph
N02
H
Ph
The esters could all be chloroacetylated by reflux-
ing them with chloroacetyl chloride in benzene for
several hours 12.
The work indicated that in such systems, the A-
Phenyl substituent was co-planar with the three va-
lences of nitrogen and with the amide carbonyl of
the halogen acetamino-ester and also with the beta-
lactam carbonyl after cyclisation. A p-nitro-phenyl
substituent on the nitrogen of such a halogenacet-
amino ester greatly activated the methine hydrogen
The following halogen-acetamino esters were syn-
thesised (Table 2 ), by this method. They were
mostly liquids, which were purified preliminarily
and used for the next step, while two were con-
verted to their 2,4-DNPH derivatives 13, which could
be microanalysed.
c h /
A-/oJ)-N H
\cooc2h 5
d/
Compounds— (2)
B
D
L
Compd. No.
A
m. p./b. p.
Recryst. Solv.
Isopropanol
Isopropanol
H
H
H
c h 3
H
H
H
H
H
NOo
a
b*
c
d
e
CH3CO
CH3CO
CH3O
H
91-92
144-45
57-58
165—69/3 mm
77-78
C00C2H3
Ph
COOC0H5
C00C2H5
Ph
Isopropanol/petroleum
—
Isopropanol
H
Tab. 1. * Methyl ester.
11 B. G. C h a tte rje e and R ia z F. A b d u lla , Chem. Ber. 102,
2129 [1969].
13 S h rin e r, Fuson, and C u rtin , “The Systematic Identifica-
tion of Organic Compounds, 5th Ed., John W iley & Sons,
New York, p. 126.
12 Deutsches Reich Patent 264527 and 293897.
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1122
B. G. CHATTERJEE AND R. F. ABDULLA
A - / o )-N-ch /
) — '
|
0 = C - C H 2C1
x COOC2H5
W
Compounds— (3)
B
D
m. p.
A
L
Compd. No.
Recryst. Solv.
H
H
H
CH3
H
CH3CO
CH3CO
CH30
H
H
H
H
H
a
b*
c
d
e
158-159**
226-227**
COOC2H5
Ph
COOC2H5
co o c2h 5
Ph
Ethyl Acetate
Ethyl Acetate
—
—
—
—
H
n o 2
133-134
Ethanol (95%)
Table 2. *Methyl ester. ** Melting points of the 2.4-Dinitro-
phenylhydrazons.
With the weak base triethylamine in benzene so-
lution, the compound iV-(p-acetyl-phenyl),/V-chlor-
acetyl-2-phenyl-glycine methyl ester (3 b) eliminated
the elements of HC1 to afford the corresponding
beta-lactam (4 b) as a pale yellow crystalline solid
which showed the expected absorption peaks in its
IR spectrum. The compound (3 a), as expected,
cyclised much faster than the corresponding /V-phe-
nyl-substituted ester.
Certain points of difference, and others of simi-
larity, however, manifest themselves in the p-acetyl
series, as compared to the p-nitro-series of com-
pounds. Firstly, in the former, the elimination of
HC1 with triethylamine is slower than in the lat-
ter n . This is in accordance with the widely acknow-
ledged fact that the nitro-group has greater “elec-
tron withdrawing capacity” than the acetyl group.
Another interesting dissimilarity is that the beta-
As a result, it was demonstrated that the N-(p-
lactam 4 b absorbs at 5.63
(C = 0, lactam) as
acetyl-phenyl) -group also activated the methine hy-
drogen atom and that, therefore, in principle, any
electron-withdrawing group substituted on the phe-
nyl ring attached to the nitrogen atom of the halo-
genacetamino-esters would activate the methine hy-
drogen, if there was no steric disturbance of the
co-planarity of the A-aryl substituent with the three
valences of nitrogen.
against 5.5 —5.55 // in the p-nitro-phenyl series n ,
showing a greater stability of the lactam CO —N-
bond in the former relative to the latter. Surprising-
ly, in spite of this stability the beta-lactam (4 b),
formed in-situ from the halogen-acetamide (3 b) is
opened up in solution with Rexyn-201 (OH0) to
the corresponding amino acid 5:
Ph
/Ph
c h 3c o / O \ n-C-COOCH3
c h 3co- { o )- n - c h <
\COOCH3
o = c - c h 2ci
Rexyn201
--
'
j
o = c - c h 2
(4b)
(3b)
Ph
CH3CO( O )n h - c - c o o c h 3
\— /
1
h o o c - c h 2
(5)
The beta-lactam (4 a) derived from p-acetyl-
anilino-malonate was a liquid and was characterised
as a solid 2,4-DNPH derivative which showed satis-
factory microanalytical characteristics.
The amino acid V(p-acetyl-phenyl) -/3-carbmethoxy-
/?-phenyl-/?-alanine (5) was recrystallised from 95%
ethanol and characterised by its IR spectrum and
microanalysis.
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STUDIES ON BETA-LACTAMS
1123
The acetyl group in the beta-lactam 4 a and 4 b is
an attractive group to exploit. In view of the biolo-
gically interesting properties of amino-thiazoles and
the work done on them by Chatterjee and Moza 14,
the acetyl group was converted into a thiazole ring.
As distinct from the work of Chatterjee and
Moza who converted an acid function through the
alpha diazo-ketone into an alpha halogeno-ketone,
the acetyl group in 4 a was brominated. The phen-
acyl bromide was treater in-situ with a slight excess
of thiourea. 6 was generated by ammonia.
CHC03( O )N-C(COOC2H5)2
0 = C - C H 2
1. Thiourea/MeOH
Bra/AlCh
BrCH2CO( O )N - C(COOC2H5)2
--' I
I
o=c - c h 2
2. MeOH distilled
3. NH3/H20
O )-N-C(COOC2H5)2
I
h 2n
I
o = c - c h 2
(6)
The activation produced by electron withdrawing
para-substituents on the /V-phenyl group of the
systems under study and mentioned above, on the
methine hydrogen atom of those systems, leads to
the question of whether a strongly electron-with-
drawing group in a meta position could also lead
to an activation of the methine hydrogen. Here the
Surprisingly, this system (3e) also cyclised with
triethylamine, to give the desired beta-lactam. It ap-
pears that the role of the electron withdrawing sub-
stituent on the /V-aryl group is merely to deplete the
pi-orbitals of the aryl substituent of their normal
electron density, and this in some way serves to en-
hance the acidity of the methine hydrogen atom,
planarity of the /V-aryl-ZV-CO-system is not disturb- leading to cyclisation with weak bases, which with-
ed since the meta-position has little steric effect on
out this depletion of the electron-cloud would not
occur.
the
N
substituents of the halogen acetamino ester.
Ph
„Ph
/ o^ - n - c h /
Et3N
( O j- N - C - C O O C 2H5
o c - c h 2
|
XX)OC2H 5
n o 2/ ) — 0' = C —CH2C1
N0 2/
(4 e)
(3 e)
No specific commitment has yet been made as to
whether the bulk of the /V-aryl substituent played
fortunately this rule does not apply to the A'-aryl
substituent in beta-lactams. In order to demonstrate
a role in the cyclisation of compounds (3) and ana- the minor effect of bulk, relative to the importance
logs. The question of whether the driving factor in
the beta-lactam synthesis developed by Sheehan
and BoSe was derived from the bulk of the A^-aryl
substituent (forcing the carbon atoms holding the
hydrogen atom to be eliminated and the chlorine
atom to within bond forming distance) or from the
delocalisation of the Ar-non-bonded electrons into
the A'-aryl substituent was not clarified. A general
rule is that the more heavily the small ring is sub-
stitued, the stabler it is, and that, as a corollary,
heavily substitued small ring compounds are easier
of A-lone-pair delocalisation we examined the ki-
netics of elimination of three systems. We chose
chloracetanilido-malonate as
a reference system,
while in one system (3 c) the phenyl group was sub-
stituted by a p-methoxy group. In the third system
the phenyl group carried an ortho-methyl substitu-
ent (3d). Clearly if bulk governed the stability
(hence rate of cyclisation) then the fastest elimina-
tion would occur with the compound 3 d, while if
delocalisation played the key role then the decrease
in order of the rate of elimination of HC1 with base
to synthesise than their unsubstituted analogs. Un- would be from chloracetanilidomalonate, through
14 B. G. C h a tte rje e and P. N. M o za, J. Med. Chem. 9, 259
[1966].
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B. G. CHATTERJEE AND R. F. ABDULLA
Conc.
1124
Rate of elimination
Compound
2 hr.
39.8
Time
4 hr.
61.3
6 hr.
73.S
8hr.
80.3
< O /—N—CH(COO Et)2
4 g/40 ml benzene
4 ml EtaN
V— 7
I
0=C-CH2C1
CH3ö/"Ö
N—CH(COO Et)2
2
12.1
Time
3.8 g/38 ml benzene
3.8 ml EtßN
4
23.6
6
31.7
8
40.5
12
56.1
0 = C-CH2C1
3c
N —CH(COO Et)2
Time
8 hr.
19.5
18
20
5.2 g/52 ml benzene
5.2 ml EtaN
I
«C—CH2C1
36.3
41.7
3d
Table 3. Kinetics of elimination of HC1 from Compounds 3 in benzene with triethylamine at 30 °C using identical concentra-
tions.
3c (p-methoxy-substituent) to 3 d (bulk maximum,
orfAo-methyl-group, delocalisation minimum). The
table of kinetics clearly established that the order
is indeed the latter.
then we can rationalise the slow rate of elimination
as being due once again to a greatly decreased de-
localisation of the AMone pair relative to chlor-
acetanilido malonate.
As the table indicates, an electron donating sub-
stituent such as a methoxy group (CH30 —) causes
a decrease in the rate of cyclisation relative to chlor-
acetanilidomalonate and this is attributed to a de-
creased delocalisation of the /V-lone pair, resulting
in the lowering of the acidity of the methine hydro-
gen atom. With an orjAo-methyl substituent on the
Ar-phenyl ring, the rate of cyclisation decreased even
more significantly. Though the bulk of the A^-aryl
Therefore, as far as the ,/V-aryl substituent is con-
cerned its role in delocalisation of the N-\one pair
is of far greater consequence in the Sh ee ha n
Bose Synthesis than its bulk, which must be con-
sidered as having been relegated to a role of second-
ary importance. The IR absorption of the beta-
lactam carbonyl in the compounds 4 c and 4 d, oc-
curred, as expected, at a higher wavelength than for
beta-lactams in general. Thus 4d (the half ester of
substituent in this case is the most the rate of cycli- the dicarbethoxy azetidinone produced with triethyl-
sation is the slowest. Clearly bulk is not the gov- amine) showed a lactam C = 0 peak at 5.77 /< since
erning factor in effecting cyclisation. However, if
we consider that the orj/?o-methyl group causes the
/V-aryl substituent to be forced into a non-planar
the iV-lone pair was “more available” for delocali-
sation into the rr-orbitals of the lactam carbonyl.
The Table 4 shows the beta-lactams synthesised
conformation relative to the three valences of /V in the study. Those that were liquid were characte-
Compounds—(4)
I
I
d/
0= c —c h 2
m. p.
Compound
No.
M
A
B
D
l
Recryst. Solv.
a *
b
c
d
e
COOEt
Ph
COOH
COOH
Ph
COOEt
COOCH3
COOEt
COOEt
COOEt
182-183
126-127
115(d)
118-119
94-95
CH3CO
CH3CO
CH3O -
H
H
H
H
c h 3
H
H
H
H
H
Ethyl Acetate
C3H70 H/Petroleum
Benzene/Petroleum
Benzene/Petroleum
Ethanol
H
n o 2
Table 4. * Characterised as 2.4-DNPH.
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STUDIES ON BETA-LACTAMS
1125
N-[p-{5-(2-amino-thiazolyl) }-phenyl]-2-oxo-4,4'-di-
carbethoxy-azetidine (6) : 3.0 g 4a was dissolved in an-
hydrous ether (15 ml). To the solution 100 mg of anhy-
drous aluminium chloride were added followed by 0.5
ml bromine. The reaction mixture was kept at 0—5 °C
and stirred (15 min.) until the bromine color was dis-
charged. The ether along with the HBr was quickly re-
moved in a stream of dry air at 40°. The oil left was
treated with 3.5 g thiourea in 30 ml of methanol and
the mixture refluxed for 3 hours. The methanol was
distilled off and the crystalline residue left was dis-
solved in water and basified with strong ammonia
solution added dropwise to yield 2.0 g (52%) 6 as a
yellow powder. Needles from isopropanol m.p. 198
to 199°.
rised by conversion to derivatives, and the dicarb-
ethoxy beta-lactams produced from compounds 3 c
and 3 d were characterised by saponifying one of
the two ester functions to free COOH, when the
compounds could be well crystallised for analysis.
Description of the Experiments
All melting points are uncorrected and were taken
in open capillaries using a Gallenkamp m.p. apparatus.
The IR spectra have been recorded using a Perkin-
Elmer Infracord.
N-(p-acetyl-phenyl)-aminomalonic acid diethyl ester
(2 a): 5g p-aminoacetophenone and 4.5 g (3.3 ml) di-
ethyl bromomalonic ester were mixed intimately and
incubated at 20 mm pr. and at a temp, of 70° for 5
days to give on working out with benzene 4.0 g (100%)
p-amino-acetophenone hydrobromide as insoluble cry-
stals. The benzene extracts were washed successively
C18H19N305S (389.0)
Claculated C 55.52 H 4.88 N 10.79,
Found
C 56.30 H 5.26 N 10.67.
IR (Nujol) ,w2.9(N —H); 5.65 (C= 0, /Mactam) ;
5.7, 5.75 (CO, ester) ; 6.15 (C= N).
with 2
N
HC1 solution, water and dried over anhydrous
N-(p-acetyl-phenyl)- 2-phenyl-glycine-methyl ester
sodium sulfate, charcoaled and filtered. Distillation of
the benzene solution on a water bath under slightly
reduced pressure afforded 2.6 g (50%) 2 a. Recrystal-
lised from isopropanol in yellowish-brown needles m.p.
91-92 °C.
(2 b): 10 g of p-amino acetophenone and 9.0 g freshly
synthesised methyl-a-bromo-phenylacetate (b.p. 123 to
125°/15 mm.) on incubation at 60—70°/40 Torr for
48 hours, gave on working out with boiling benzene
8.0 g (100%) p-amino-acetophenone hydrobromide and
from the benzene filtrate after purification 8.0 g (80%)
2 b as a yellow residue. Yellowish flakes, from isopro-
panol m.p. 144—145 °C.
C15H19N05 (293.0)
Calculated C 61.49 H 6.48 N 4.78,
Found
IR (Nujol)
C 61.08 H 6.85 N 5.12.
2.95 (N-H); 5.7, 5.75 (CO, ester);
n
C17H17N03 (283.0)
Calculated C 72.08 H 6.01 N 4.94,
6.0 (CO, aromatic ketone).
Found
C 71.94 H 5.99 N 4.94.
N-(p -acetyl -phenyl)-N’-chloroacetyl-amino-malonic
IR (Nujol) f.i 2.99 (N —H) ; 5.75 (C= 0, ester)
6.0 (C= 0, ar. ketone).
ester (3 a)-5.45 g 2 a and 5.00 ml chloroacetyl chloride
were gently refluxed at 110 to 120° for 4 hr. (or in
50 ml of benzene for 10 hr.), cooled and poured into
7ml of ice cold isopropanol. The excess isopropanol
was distilled and the residual oil was taken in ether,
washed with potassium carbonate solution, water,
treated with sodium sulfate(anhydrous)/charcoal, fil-
tered and distilled to give 4.5 g (66%) 3 a as a brown
oil characterised as its 2,4-dinitrophenyl-hydrazone,
m.p. 157—158 (ethyl acetate).
N-(p-acetyl-phenyl) -N'-chloroacetyl-2-phenyl-glycine-
methyl ester (3b) : 5.0 g 2b and 7ml chloroacetyl chlo-
ride at 100—110 °C/2.5 hr. gave when worked out as
for 3a, 5.0 g (80%) 3b as a viscous brown oil. Charac-
terised as the 2,4-DNPH derivative m.p. 226—227°
(ethyl acetate).
C25H,2C1N507 (539.5)
Calculated N 12.97,
Found
C»3H24C1N509 (549.5)
N 12.83.
Calculated N 13.02,
Found
N 12.73.
N -(p-acetyl-phenyl)- 2-oxo-4-phenyl-4’-carbmethoxy-
azetidine (4b): To 2.5 g (3b), dissolved in 25 ml of
benzene, was added 5.0 ml of triethylamine and the
reaction mixture was stirred overnight in a stoppered
conical flask. Filtration afforded 0.9 g (95%) triethyl-
amine hydrochloride, while working out the benzene
layer as usual gave 1.7 g (76%) of 4b as a yellow
solid. Pale yellow crystals from isopropanol m.p.
126-127°.
N-(p-acetyl-phenyl)-2-oxo-4,4'-dicarbethoxy-azetidine
(4 a) : 3.60 g 3 a in 40 ml anhydrous benzene was
treated with 9.0 ml of triethylamine and the mixture
filtered after 24 hr. to give 1.4 g (100%) triethylamine
hydrochloride. The benzene layer was washed free of
base with 2N HC1 acid solution, then with water, dried
over anhydrous sodium sulfate, treated with active
charcoal filtered and distilled to yield 3.0 g (90%) 4 a
as a brown oil characterised by its 2,4-DNPH deriva-
tive, m.p. 182—183 °C (ethyl acetate).
C19H17N04 (323.0)
Calculated C 70.60 H 5.27 N 4.34,
C23H23N509 (513.0)
Found
IR (Nujol)
C 70.96 H 5.76 N 4.30.
fx 5.63 (C= 0, beta lactam); 5.7 (C= 0,
ester) ; 5.92 (C= 0, Ar. ketone).
Claculated N 13.65,
Found
N 13.62.
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STUDIES ON BETA-LACTAMS
1126
The oil was taken in ether, the ether washed with
water, dried and distilled to give 2.50 g (79.5%) N-(p-
methoxy-phenyl) -2-oxo-4-carboxy-4-carbethoxy-azetidi-
ne as an oil which solidified by tituration and cooling
under petroleum. From benzene/petroleum (40—60)
m.p. 115° (d).
N -(p-acetyl-phenyl)-ß-phenyl-ß’-carbmethoxy-ß-ala-
nine (5) : 0.90 g of halogen acetamino ester 3b, was
dissolved in 25 ml of anhydrous methanol and stirred
for 24 hr. with Rexyn-201 (OH0). The Rexyn was then
filtered out, extracted twice with 25 ml methnol at the
b.p., the filtrates added and stripped to yield 350 mg
(43.6%) of 5 as an oil which crystallised on standing
under petroleum/isopropanol, m.p. 174—175° (metha-
nol) .
C14H15N06 (293.0)
Calculated
Found
N 4,77,
N 4.45.
IR (Nujol) fi 3.85 (broad, O —H, carboxylic) ; 5.6
(C= O, beta-lactam); 5.7 (C= 0,
ester) ; 5.9 (C= 0, acid).
Ci9H19N05 (341.0)
Calculated C 67.10 H 5.58 N 4.15,
Found
IR (Nujol)
C 67.58 H 5.83 N 4.42.
n 2.93 (N-H); 3.73 (O —H); 5.69
N-(o-methyl-phenyl)-amino-malonic acid-diethyl ester
(2d): 10.7 g o-toluidine and 12.0 g diethyl bromo-
malonic ester at 40 Torr/50 —60 °C for 4 days gave
on working out with benzene 8.5 g (90%) o-toluidine
hydrobromide and 10.5 g (80%) 2d. Distillation several
times yielded pure 2 d as a pale yellow oil, b.p.
165—169 °C/3 mm pr. The oil was used directly for
the next step.
(C= O, ester); 5.78 (C= 0, acid);
6.03 (C= O, aromatic ketone).
N-(p-methoxy-phenyl)-amino-malonic acid diethyl
ester (2c): 12.0 g p-anisidine and 12.0 g diethyl
bromomalonic ester were made homogeneous by gentle
warming, cooled quickly to 30 °C and then evacuated
to 15 Torr. The mixture warmed up and was kept cooled
in an ice salt bath. After and hour the flask was al-
lowed to warm up to room temp, again and then taken
to 45—55 °C for 3 hours. Working out the cake in the
usual manner with benzene yielded 8.5 g (84%) p-ani-
sidine hydrobromide and 10.0 g (70%) 2c as brownish
large crystals. From isopropanol/petroleum (40—60)
colorless crystals m.p. 57—58°.
IR (Neat) /u 2.9 (N-H); 5.7, 5.75 (C= 0, ester).
N-(o-methyl-phenyl)- N -chloroacetyl-amino-malonic
ester (3d) : Amidification of 5.0 g 2d in 25 ml ben-
zene, with 2 g chloroacetic acid and 2 ml phosphorous
trichloride, gave on working out the solution in the
usual manner, 5.2 g (75%) 3d as a pale green fluo-
rescent oil.
C14H19N05 (281.0)
Calculated C 59.78 H 6.76 N 4.98,
IR(Neat ju 5.65 (C= 0, ester); 5.75 (C= 0, ester);
5.93 (C = O, amide).
Found
C 59.63 H 6.43 N 5.02.
IR (Nujol) fi 2.94 (N-H); 5,67, 5.77 (C= 0,
ester).
N -(o-methyl-phenyl)- 2-oxo-4,4'-dicarbethoxy-azetidi-
ne: 5.2 g halogen acetamino ester 3d in 52 ml an-
hydrous benzene was stirred with 5.2 ml triethylamine
in a stoppered Erlynm eyer flask. The solution was
filtered every two hours to keep track of the elimina-
tion (Table 3), the solution boiled with two ml excess
triethylamine to completion and filtered to give in total
2.093 g (100%) triethylamine hydrochloride. Working
out the benzene filtrate afforded the liquid beta lactam;
4.4 g (95%) ; as a white oil.
N-(p-methoxy-phenyl) -N-chloroacetyl-amino-malonic
acid diethyl ester (3c): 4.0 g 2c, in 30 ml benzene,
gave with 2.8 g chloroacetic acid and 1.8 ml PC13 , 4.5 g
3 c (80%) as a colorless glassy syrup which could not
be solidified.
lR(Neat) /x 5.7 and 5.75 (C= 0, ester); 6.0 (C= 0
amide).
N-(p-methoxy-phenyl) -2-oxo-4,4'-dicarbethoxy-azeti-
dine: 3.85 g 3c in 38.5 ml anhydrous benzene was treat-
ed with 3.85 ml triethylamine and stirred in a stoppered
vessel. The reaction was filtered every two hours to
measure the kinetics of elimination, and finally the
mixture was boiled and filtered to give a total of 1.42 g
(95.9%) triethylamine hydrochloride. Working out the
benzene filtrate in the usual manner gave 3.45 g (100%)
of the beta-lactam as an oil.
Saponification to 4d: 3.38 g N-(o-methyl-phenyl)-2-
oxo-4,4 -dicarbethoxy-azetdine were saponified in 20 ml
ethanol using 0.65 g potassium hydroxide for two
hours. The precipitated potassium salt (after ether ad-
dition) was worked out as for 4 c to give the half ester
N -(o-methyl-phenyl) -2-oxo-4-carbethoxy-4 -carboxyaze-
tidine 4 d as an oil which solidified by tituration under
light petroleum at 5 °C. Yield 1.4 g (41.7%). Very pale
yellow needles from benzene/petroleum m.p. 118-119°.
Saponification to 4c: 3.45 g TV-(p-methoxy-phenyl)-
2-oxo-4,4'-dicarbethoxy-azetidine was dissolved in 10 ml
of absolute ethanol, 0.62 g of potassium hydroxide was
added and the mixture stirred for two hours, in a stop-
pered E rlynmeyer flask. This was followed by
the addition of 50 ml of anhydrous ether, the precipi-
tated potassium salt was filtered out, dissolved in
10 ml of water chilled to 5 °C and acidified with 12 N
HC1 when the free acid 4c was deposited as an oil.
C14H15N05 (277.0)
Calculated N 5,05,
Found
N 4.78.
IR (Nujol) fx 3.8 (broad, O —H, carboxylic); 5.77
(C= 0, beta-lactam); 5.8 (C= 0,
ester) ; 6.0 (C = 0, acid).
N-(m-nitro-phenyl)2-phenyl-glycine-ethyl ester (2e) :
Incubation of 9.2 g meta-nitro aniline and 8.1 g ethyl
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H. ZAHN, W. DANHO, H. KLOSTERMEYER, H. G. GATTNER UND J. REPIN
1127
alpha-bromo phenylacetate for 4 days at 70—80 °C/
40 Torr, gave on working out with chloroform, 10 g
(100%) 2e from chloroform and 7.2 g (100%) m-nitro
aniline hydrobromide. 2 e from isopropanol gave yel-
low-orange needles, m.p. 77—78°.
mination. The filtrate was refluxed to complete the
elimination, and working out the benzene solution af-
forded 1.7 g (90%) 4e as a yellow oil, which solidified
on standing, m.p. 93—94° (95% ethanol).
C18H16O5N2 (340.0)
Calculated N 8.23,
Found
C16H16N2O4 (300.0)
Calculated N 9.33,
N 7.71.
Found
N 9.07.
IR(Nujol) fi 5.64 (C= O, beta-lactam); 5.75
(C= O, ester).
IR(Nujol) ju 2.9 (N —H) ; 5.8 (C= 0, ester)
N-(m-nitro-phenyl)-N-chloroacetyl-2-phenyl-glycine-
ethyl-ester (3e): 3.0 2e gave with 4 ml chloroacetyl
chloride in 25 ml benzene after refluxing for 4 hrs, 3.6 g
(100%) white crystals; m.p. 133—134 °C (ethanol).
The authors wish to express their highest sense of
gratitude to Professor AjAy K. Bose of Steven’s Insti-
tute of Technology, New Jersey, USA for his invaluable
help in arranging the IR spectra of so many com-
pounds to be taken. We also wish to thank Dr. M AgA r
S. M ANhAs of the same Institute. We are obliged to
Dr. j AN TrojANek of the Research Institute of Natural
Drugs in Prague, Czechoslovakia for his help in the
microanalysis, as well as Dr. H. P. S. chAw lA and
Dr. V. V. Rao of the Central Drugs Research Institute
in Lucknow-India. We acknowledge the help provided
by the National Chemical Laboratory-Poona for some
of the micro-analyses.
C18H1705N2C1 (376.5)
Calculated N 7.44,
Found
N 7.80.
IR(Nujol) /li 5.74 (C= O, ester); 5.84 (C= 0,
amide).
N-(m-nitro-phenyl)-2-oxo-4-phenyl-4'-carbethoxy-aze-
tidine (4e): 2.1 g 3e, in 35 ml dry benzene, stirred
for 18 hr. with 10 ml triethylamine to give on filtration
triethylamine hydrochloride corresponding to 66% eli-
Eine Synthese der A-Kette des Schafinsulins unter ausschließlicher Verwendung
säurelabiler Schutzgruppen*
H. Z A h N und W. d A N h o sowie H. k l o s T e r M e y e r , H. g. gA T T N e r und J. r e p iN
Deutsches Wollforschungsinstitut an der Rheinisch-Westfälischen Technischen Hochschule Aachen
(Z. Naturforschg. 24 b, 1127— 1139 [1969] ; eingegangen am 13. Juni 1969)
Die A-Kette des Schafinsulins wurde als geschütztes Derivat Boc-Gly-Ile-Val-GluCOBu^-Gln-
Cys(Trt) -Cys(Trt)-Ala-Gly-Val-Cys(Trt) -Ser-Leu-Tyr(Bu1)-Gln-Leu-GluCOBu1)-Asn-Tyr(Bu4)-Cys
(Trt) -Asn-OBzl ** aus fünf Fragmenten aufgebaut, durch BromWasserstoff in Trifluoressigsäure von
den Schutzgruppen befreit und zur Reinigung in das Tetra-5-sulfonat überführt. Nach Kombina-
tion mit B-Kette aus nativem Insulin lieferte das Präparat kristallines Insulin.
Seit den ersten Synthesen der A-Kette des Insu-
mit den beiden nativen Ketten. Da die von sä N g e r
lins im Jahre 1963 wurde die aus 21 Aminosäuren- und Th o M p s o N 2 ermittelte Sequenz der Peptidkette
sich als richtig erwiesen hatte 3, führte man die ver-
ringerte insulinbildende Potenz synthetischer Insu-
linketten auf Schädigungen der Präparate durch die
resten bestehende Peptidkette wiederholt aufgebaut.
Bei allen Synthesen wurden die Thiolgruppen der
vier Cysteinreste mit dem S-Benzylrest*, der nur
durch Einwirkung von Natrium in flüssigem Ammo- Natriumbehandlung zurück. Dieses Argument über-
zeugte umso mehr, als auch native Insulin-Ketten
nach S-Benzylierung und Deblockierung bei der
Resynthese weniger Insulin lieferten als die unbe-
handelten Ketten 4. k A T s o y A N N is 5 machte als erster
niak entfernbar ist, geschützt. Ein kritisches Litera-
turstudium zeigt, daß die synthetischen Insulin-Ket-
ten bei Kombination mit nativer Gegenkette in der
Regel weniger Insulin lieferten als Blindversuche
*73. Mitt. über Peptide; 72. Mitt. s. H.
Chem. Ber., im Druck. — Teils aus der Dissertation W.
d A N h o , Techn. Hochschule Aachen, 1967.
** Abkürzungen nach den Regeln der IUPAC-IUB-Commis-
sion on Biochem. Nomenclature, Hoppe-Seyler’s Z. physiol.
Chem. 348, 256 [1967].
k
l o s T e r M e y e r ,
2 F. S a n g e r u . E. O . P. T hom pson , Biodiem. J. 53, 353,
366 [1953].
3 H. Z a h n , H. B re m e r u . R. Z a b e l, Z. Naturforschg. 20 b,
653 [1965].
4 C.-L. Tsou, Y.-C. Du u. G.-J. Xü, Scientia Sinica 10, 332
[1961].
1 R. H. s if f e r d u. V. Du V ig N e A u d , J. biol. Chemistry 108,
753 [1935].
5 P. G. k A T s o y A N N is , Diabetes 13, 339 [1964].
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