Functionalized 1,2-dioxetanes as potentially useful biological and chemotherapeutic agents

Full text of DOI:10.1515/znb-1984-0524

Functionalized 1,2-Dioxetanes
as Potentially Useful Biological and Chemotherapeutic Agents
Waldemar Adam *a, Chrisostomos Babatsikos3, and Giuseppe Cilentob
Institut für Organische Chemie“, Universität Würzburg, D-8700 Würzburg, West Germany
and
Departamento de Bioquimicab. Instituto de Quimica, Universidade de Säo Paulo,
C.P. 20.780, Säo Paulo, Brasil
Z. Naturforsch. 39b, 679—682 (1984); received December 8, 1983
Dioxetanes, Esterification, Photoaffinity Labeling, Excited Carbonyl Products,
Chemi-energization
Hydroxymethyl-substituted 1,2-dioxetanes (3) have been converted in reasonable yields
(10—40%) into the carboxylate-substituted 1,2-dioxetanes (4) by means of the Brewster—Ciotti or
Mitsunobu esterification and into the tosylate-substituted 1,2-dioxetanes (5) by means of tosyl
chloride in pyridine. The fact that hydroxy-functionalized 1,2-dioxetane can be chemically at-
tached to carboxylic acids and sulfonic acids whit preservation of the dioxetane moiety opens up
new opportunities for biomedical applications.
illustrates, on thermal decomposition, the 1,2-diox-
In photoaffinity labeling [1] a biochemically im-
portant target molecule (T), e.g. a substrate, hor- etane (1) dissociates into two carbonyl products (2),
mone, allosteric effector, etc., is modified by attach- of which one is electronically excited (2*); usually
the chemically reactive triplet n, 7t* state is produced
[4]. Therefore, in principle two strategies could be
followed to perform dioxetane-energized photoaffin-
ity labeling. Either the carbonyl group of the biologi-
cal target molecule, e.g. a steroidal hormone, could
be masked in form of a dioxetane (alternative A,
eq. (3)) or a
ing a photolabile group. In an ideal situation the
modified substrate is still reversibly bound to its re-
ceptor (R), e.g. an enzyme or specific protein. Sub-
sequent irradiation generates
a reactive species
which now binds irreversibly to the receptor
(eq. (1)). One can in this way obtain information on
the location and structure of
hv
->T -R
T + R r* T ---R
(1)
0"
0—0
I
I
I
the receptor binding site. To the best of our knowl-
edge, photoaffinity labeling has been conducted only
in vitro and may be handicapped by the fact that the
photo-generated reactive species is largely wasted by
reaction with water.
While mainly azides and diazoalkanes are utilized
as photo-active labels [1], also electronically excited
carbonyl groups qualify [2]. This special case of
photoaffinity labeling provides us with the unique
opportunity of exploiting 1,2-dioxetanes (1) for the
purpose of “photoaffinity without light” [3], As
eq. (2)
S — C— C - R
-
-C —
R
s —c—R —
I
I
R
R
(3)
0-0
I I
o-o
I
I
I
S - X - C - CI- R
-
S - X - C - R
S
+
X -C -C -R -
I I
R
R
R
R
dioxetane with a functional handle could be attached
to the biological target molecule (alternative B,
eq. (3)). Thermal activation under physiological con-
ditions would release the electronically excited car-
bonyl group for photochemical attachment at the re-
ceptor site. In contrast to “optical” photoaffinity
labeling, such “chemical” photoaffinity labeling
could possibly be carried out in vivo because the usu-
al problems of directing the radiation to the specific
site are obviated. The potential of this unique
strategy for biological testing and for developing new
chemotherapeutic agents can hardly be overem-
phasized [3, 5],
0-0
I I
0
o*
R - C - C - R
I I
2 )
A A
R
R
R
R
R
R
2
2*
1
* Reprint requests to Prof. Dr. W. Adam.
0340-5087/84/0500-0679/$ 01.00/0
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680
W. Adam et al. ■Functionalized 1,2-Dioxetanes
A fundamental requisite for the realization of the
alternative B strategy is the availability of 1,2-diox-
etanes with functional handles, which would permit
was chosen to provide additional chemical inertness
[7] and thermal stability [8], should esterification
lead to destruction of the dioxetane. For the conver-
attaching such latent in situ photoaffinity labels to sion (3)—»(4) the Brewster—Ciotti [9] and the Mit-
the biological target molecule. In view of the labile
nature of the dioxetanes, attachment of func-
sunobu [10] esterification methods were used, while
the conversion (3)-^(5) employed tosyl chloride in
tionalized derivatives to the biological substrate de- pyridine [11], Typical esterification procedures are
mands sufficiently mild chemical transformations to
assure preservation of the dioxetane moiety. We pre-
sently demonstrate the feasibility of such functional
group manipulation of dioxetanes, with the convic-
tion that these chemical model studies will pave the
way for numerous biomedical applications.
The chemical transformation that we illustrate
here is the esterification of hydroxymethyl-substi-
tuted dioxetanes (3) with carboxylic acids and sul-
given below and the yields, physical constants and
spectral data are summarized in the Table.
As the results in the table show, the pure carboxy-
late- and tosylate-substituted dioxetanes (4) and (5),
respectively, could be obtained in reasonable yields.
In the case of the dioxetanes (4), the Mitsunobu es-
terification gave usually higher yields. In the Brew-
ster—Ciotti esterification mixtures of carboxylate (4)
and tosylate (5) were obtained. In fact, in the case of
the sluggishly reacting stearic acid, the correspond-
ing tosylate (5) was formed as major product.
Nevertheless, the fact that hydroxymethyl-func-
tionalized dioxetanes can be chemically linked to
carboxylic acids with preservation of the dioxetane
moiety, especially fatty acids such as stearic acid,
opens up promising and challenging opportunities in
biomedical research [3],
R2 R3
0
R R
0
1 1 I
'I
u
R - C - C - C H - O -C - R 4
I I
0-0
R - C - C - C H - 0 - S - R
R -C -C -C H -O H
i
i
2
ii
0
2
i i
2
0-0
0-0
3a R1=R2=R3=Me
LQ R1=R2=R3=R4=Me
5a R1= R2=R3=Me
&b R'=R2=R3=Me
” ' CH> ' 0
Experimental
,
I X U
R=n-C17H35
Preparation o f Carboxylates (4)
R
=
H
/
1
2
3
5
b
r ’+ r ^
Lc R = R = R = Me
,
Brewster—Ciotti esterification [9]: A 25 ml Erlen-
meyer flask, capped with a rubber septum, was pro-
vided with spinbar and charged with 12 mmol car-
boxylic acid and 24 mmol tosyl chloride in 25 ml
anhydrous, freshly distilled pyridine, cooled to 0 °C.
and w'hile magnetically stirring was added by means
of a syringe a precooled solution of 6 mmol hy-
droxymethyl-l,2-dioxetane (3) in 5 ml pyridine.
Reaction progress was monitored by TLC on silica
gel eluting with dichloromethane. After optimal
yields of esterified dioxetanes (4) were achieved
(usually 60 min at 13 °C), the reaction mixture was
poured on ca. 50 g crushed ice, the mixture transfer-
red to a separatory funnel and extracted with
3x20 ml methyl f-butyl ether. The combined extracts
were washed w'ith 3x10 ml 2 N-aqu. HC1 and 10 ml
water, dried over anhydr. M gS04, the solvent roto-
evaporated at 0 °C and 18 Torr and the peroxidic
residue chromatographed on activity grade III silica
gel (adsorbant to substrate ratio ca. 60:1) at —27 °C,
eluting with dichloromethane. Solid dioxetane pro-
ducts were recrystallized for final purification. Purity
was established by iodometry (KI in HOAc).
R=N° 2 - \ >
=CH3“ 0
"
Ld r1+r2=
R =H, R =N02~^~*^~
Lq r1+ r2=
R3=H; R= Me
fonic acids to result in the carboxylates (4) and the
sulfonates (5), respectively. The hydroxymethyl-sub-
stituted dioxetanes (3) were prepared according to
the synthetic sequence outlined in eq. (4) [6]. The
spiroadamantane substitution in the dioxetane (3b)
CH,0H
RJ 0
R
CH,
R^
CH.
l
5
10 2
f
RC03H
C_CH2 R,Ne 0He
(M
R2—C -0
C
C
Mitsunobu esterification [10]:
A 100 ml three-
R
I 00H
R2/ "R1
R2 / p 0 0 H
necked. round-bottomed flask, provided with spin-
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Table. Yields, physical constants and spectral data of the carboxylate and tosylate-functionalized 1,2-dioxetanes (4) and (5).
Dioxe- Methoda Yield Purity Phys. Const.
IR (CC14)
'H NMR
13C NM R
tane
(90 MHz; CDC1,)
[ppm]
(100 MHz; CDC1,)
[ppm]
[% f [%]c m.p. [°C]
[ppm]
4a
BC
16
97
97
yellow oild
1.40 (s; 3 H, CH,),
1375, 1250, 1230, 1.45 (s; 3 H, CH,),
17.89 (q),
2940, 1755, 1425,
20.84 (q, C H ,C = 0 ),
22.13 (q), 23.92 (q),
66.08 (t, C H ,),
88.86 (s), 89.07 (s),
170.76 (s; C = 0 )
1190, 1040, 865.
690
1.65 (s; 3 H, CH ,),
2.05 (s; 3H , CH ,), AB-pattern
(<3A=4.55, <3b=4.28,
7=11.7 Hz; 2H , CH,)
4be
2915, 2825, 1750, 0.85 (t. 7=6.72 Hz; 3H . C H X H ,), 14.13 (q, C H ,C H ,),
1420, 1410, 1375, 1.18-1.31 (m; 28H .
1230, 1215, 1190, (C H ,)14CH3),
BC
M
3
17
6 6 -6 7
(pale yellow
prisms)
17.94 (q), 22.16 (q),
22.65 (q), 23.89 (t),
1150
1.42 (s; 3H , CH3),
24.77 (t), 29.02 (t),
1.43 (s; 3H , CH ,),
29.18 (t), 29.38 (t),
1.60 (m; COCHjCH,),
1.66 (s; 3H , CH3),
29.64 (overlapping C H ,’s),
31.86 (t), 34.02 (t),
2.34 (t, 7=7.57 Hz;
2H , C O -C H ,), AB-pattern
(öa=4.57, öb=4.51,
7=11.49 Hz; 2H , OCH,)
65.78 (t, C H ,0 ), 88.86 (s),
89.04 (s), 173.43 (s; C = 0 )
17.94 (q), 22.16 (q),
24.01 (q), 67.26 (t, CH ,),
88.94 (s), 89.16 (s),
123.56 (d. ortho),
130.76 (d, meta),
134.69 (s, alpha),
150.47 (s, para),
4c
2950, 1740, 1540, 1.45 (s; 6H , CH ,),
1350, 1280, 1250, 1.67 (s; 3H , CH3),
BC
39
98
124-126
(yellow needles)
AB-pattern (<3A=4.83,
öB= 4.73, 7=12.0 Hz; 2H , CH,)
AA'BB'-pattern (öA= 8.38,
<3B= 8.20, 7=9.6 Hz;
1120, 1105, 865,
720
4H , p -N O ,-C 6H4- )
164.27 (s; C = 0 )
4d
2900, 2840, 1740, 1.40—2.80 (m; 14H, adamantane), 25.87, 25.99, 30.57, 31.45,
BC
M
13
36
97
97
79
1535, 1355, 1275, 4.93 (d, 7=5.1 Hz; 2H , CH ,),
1130, 1105, 1020, 5.20 (t, 7=5.1 Hz; 1H, CH),
32.61, 33.36, 35.12, 35.73,
37.34, 64.18 (t; C H ,0 ),
86.87 (d; CH-dioxetane),
91.32 (s; spiro-dioxetane),
123.63 (d, ortho),
(yellow needles)
720
8.15-8.45 (m; 4H ,
p-N O, —C6H4—)
130.85 (d, meta),
134.65 (s, alpha),
150.56 (s, para),
164.36 (s; C = 0 )
1.51—2.14 (m; 12H, adamantane), 20.83 (q; C H ,), 25.74,
4ee
BC
23
30
7 3 -7 5
(yellow needles)
2920, 2860, 1755,
25.92, 30.52, 31.38, 32.41,
33.17, 34.93, 35.68, 37.29,
62.80 (t; C H ,0 ),
1570, 1550, 1460, 2.09 (s; 3H , CH,),
2.52 (m; 1H, adamantane),
2.70 (m; 1H, adamantane),
1375, 1240, 1045,
1010, 985
ABX-pattern (<3A=4.58, <3B=4.62, 86.98 (d; CH-dioxetane),
7AX=3.78 H z ; 7bx=4.04 H z ,
7ab=12.08 H z ; 3H . OCH,CH)
91.22 (s; spiro-dioxetane),
170.79 (s; C = 0 )
99
5 5 -5 6
(yellow needles)
3060, 3040, 2980, 1.28 (s; 3H , CH ,),
1605, 1390, 1205, 1.32 (s; 3H , CH ,),
1190, 1110, 1005, 1.60 (s; 3H , CH ,),
17.98 (q),
21.70 (q, A r -C H ,),
22.11 (q), 23.86 (q),
70.60 (t; C H ,), 88.37 (s),
89.23 (s), 127.98 (d, ortho),
670
2.45 (s; 3H , CH ,),
AB-pattern (ÖA=4.64, öB=4.03,
7=9.5 Hz; 2H , CH2), A A 'B B '- 129.99 (d, meta),
pattern
(<3a=7.74, <3b= 7.32;
7= 9.6 Hz; 4H , MeC6H4- )
131.53 (s, para),
145.40 (s, alpha)
5b
30
2900, 2850, 1455, 1.2—2.3 (m; 12H, adamantane),
21.73 (q; CH3), 25.75,
25.97, 30.45, 31.45, 32.42,
33.14, 34.73, 35.70, 37.09,
67.34 (t; CH ,CO ),
99
9 4 -9 5
(yellow needles)
2.45 (s; 3H , - C 6H4-C H 3),
2.6—2.8 (m; 2H , adamantane),
d of AB-pattern (öA=4.59,
(3b=4.45,
1380, 1210, 1195,
1180, 1100, 1050,
1005, 660
86.53 (CH-dioxetane),
7ab—10.2 Hz, 7=5.1 and 6.6 Hz; 91.39 (s, spiro-dioxetane),
2H , CH,), 4.95 (dd, 7=6.6
and 5.1 Hz; 1H, CH),
AA 'BB'-pattern (<3A=7.84,
<3B= 7.41. 7=8.1;
128.02 (d, meta),
130.06 (d, ortho),
131.94 (s, para),
145.42 (s, alpha)
4H , M e -C 6H 4—)
a BC = Brewster—Ciotti esterification, Ref. [9]; M = Mitsunobu esterification, Ref. [10]; T = Tosylation, Ref. [11];
b isolated, purified yields; c by iodometry (KI in HOAc); d after silica gel chromatography at -2 7 °C, eluting with
dichloromethane; e 'H NM R measured at 400 MHz.
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682
W. Adam etal. ■Functionalized 1,2-Dioxetanes
bar and three-way stopcock, was charged with
1 mmol of triphenylphosphin. After flame-drying
under nitrogen atmosphere. 10 ml of diethyl ether
(freshly distilled from LiAlH4) and 1 mmol of freshly
distilled ethyl azodicarboxylate were added, stirred
for 5 min and at 0 °C 1 mmol of the carboxylic acid
in 15 ml ether and 1 mmol hydroxymethyl-l,2-di-
oxetane (3) in 10 ml ether were syringed into the
flask in that order. Reaction progress was monitored
by TLC on silica gel eluting with dichloromethane.
After optimal yields of esterified dioxetanes (4) were
achieved (usually 2 h at 0 °C, but for the stearate
(4b ) 12 h at 10 °C) the solvent was roto-evaporated
at 5 °C and 18 Torr, the residue triturated with 20 ml
ligroin, the combined triturates washed with
2x20 ml water and dried over anhydr. M gS04.
Roto-evaporation of the solvent at 0 °C and 18 Torr
and chromatography of the residue on activity grade
III silica gel (adsorbant to substrate ratio of 40:1) at
—20 °C eluting with dichloromethane afforded the
dioxetane products (4). Solid dioxetanes were recrys-
tallized for final purification. Purity was established
by iodometry (KI in HOAc).
Preparation o f Tosylates (5)
The same procedure as outlined for the Brewster-
—Ciotti esterification was used, except that the carb-
oxylic acid was left out. Furthermore, the solid tosy-
lates (5) were purified as described in the Brewster-
—Ciotti esterification.
Acknowledgements are made to the Stiftung
Volkswagenwerk, the Deutsche Forschungsgemein-
schaft, the Fonds der Chemischen Industrie, the
Financiadora de Estudios e Projectos („FINEP“, Rio
de Janeiro), the Conselho Nacional de Desenvol-
vimento Cientifico e Tecnologica (Brasilia), and the
Fundagäo de Ampazro ä Pesquisa de Estado de Säo
Paulo (Säo Paulo) for generous financial support.
Particular appreciation is expressed to the Stiftung
Volkswagenwerk which enabled this collaborative
research in the first place. We thank Drs. R. J. Ro-
senthal and D. Scheutzow for help with the I3C NMR
spectra.
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