CEJC 3 (2003) 260{276
Synthesis of 4-aryloxy-7-nitrobenzofurazan
Derivatives from 4-chloro-7-nitrobenzofurazan
and Various Phenoxide Anions (Including
Pharmaceuticals) in the Presence of Crown Ethers
Marioara Bem1, Miron T. Caproiu2, Dan Stoicescu3 ,
Titus Constantinescu 1, Alexandru T. Balaban4¤
1
Laboratory of Supramolecular Chemistry and Interphase Processes,
Institute \I.G.Murgulescu" of Physical Chemistry, Roumanian Academy,
Splaiul Independentei 202, 77208, Bucharest, Roumania
E-mail: titel@chim¯z.icf.ro
2
Institute \C.D.Nenitzescu" of Organic Chemistry, Roumanian Academy,
Splaiul Independentei 202 B, Bucharest, Roumania
3
SINDAN SRL Pharmaceutical Company,
Ion Mihalache Blvd. 11, 781681, Bucharest, Roumania
4
Texas A&M University at Galveston,
5007 Ave. U, Galveston, TX, 77553-1675, USA
Received 22 May 2003; accepted 14 June 2003
Abstract: 4-Chloro-7-nitrobenzofurazan reacts by nucleophilic substitution with phenoxide
anions derived from estriol (2c), ethynylestradiol (2d), phenol (3e), guaiacol (3f ), 2,6dimethoxyphenol (3g), eugenol (3h), isoeugenol (3i), the cytostatic Etoposide (4), and
Reichardt’s betaine (5) in the presence of crown ethers a¬ording the corresponding 4-aryloxy7-nitrobenzofurazan derivatives 6c, 6d, 7e { 7i, 8, and 9. The structure of these compounds
was con­ rmed by NMR spectra. Hydrophobicity/hydrophilicity parameters were investigated
by reverse phase thin-layer chromatography.
c Central European Science Journals. All rights reserved.
®
¤
Keywords: 4-aryloxy-7-nitrobenzofurazan derivatives,
nitrobenzofurazan, crown ethers, NMR spectra, RPTLC
phenoxide
E-mail: balabana@tamug.edu; Phone: (409) 741-4313; Fax: (409) 740-4787
anions,
4-chloro-7-
A.T. Balaban et al. / Central European Journal of Chemistry 3 (2003) 260{276
1
261
Introduction
Benzofurazan and benzofuroxan derivatives are routinely applied to the analytical determination of amino acids, primary or secondary amines, and polypeptides owing to the
°uorescence of the corresponding products [1-21].
For nitrobenzofurazan derivatives the IUPAC numbering requires alphabetical ordering of substituents, which leads to structural non-uniformity. For convenience, we decided
to ¯x the numbering in practically all formulas so that the nitro group is always numbered
as 7, leaving the halogen or aryloxy group numbered as 4.
The activated chlorine atom in 4-chloro-7-nitrobenzofurazan (4-chloro-7-nitrobenzo2-oxa-1,3-diazole, NBD chloride, 1) can be substituted nucleophilically by primary or
secondary amines and also by phenoxide [10,26,27,28] or tyrosine anions [22]. Some
4-substituted 7-nitrobenzofurazan and benzofuroxan derivatives were reported to possess antileukemic and immunosuppressing activity [1,22,23].Further data on benzofurazan
derivatives have been published by Boulton, Katritzky and coworkers [29-34]. 4-Chloro7-nitrobenzofurazan, or its more reactive and expensive 4-°uoro congener, are easily
accessible [35] and are also commercially available under the names of NBD chloride or
°uoride, respectively.
In a previous paper [36] it was shown that in the presence of crown ethers (CEs),
phenoxidic-type sodium salts of estrone (2a) and estradiol (2b) react with 1 a®ording
diaryl ether compounds (4-aryloxy-7-nitrobenzofurazan derivatives) 6a and 6b, respectively. Interestingly, these weakly °uorescent ethers react fast and irreversibly with amino
acids resulting in strongly °uorescent compounds. Biochemical implications are evident
because the molecular design of 4-aryloxy-7-nitrobenzofurazan derivatives 6a,b having
biologically active structural fragments will allow their use in pharmacokinetics and pharmacodynamics. Their rapid reaction with amino acids or related compounds will lead to
promising simple bioanalytical determinations of such compounds.
The present paper reports the synthesis and study of further novel 4-aryloxy-7nitrobenzofurazan derivatives with potential biomedical and bioanalytical applications.
For this purpose we investigated the reaction between NBD chloride (1) in the presence
of CE with anions of the following phenols: phenol (3e), guaiacol or 2-methoxyphenol
(3f ), 2,6-dimethoxyphenol (3g), eugenol or 4-allyl-2-methoxyphenol (3h), cis + transisoeugenol or 2-methoxy-4-propenylphenol (3i), Reichardt’s betaine or 2,6-diphenyl-4-
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A.T. Balaban et al. / Central European Journal of Chemistry 3 (2003) 260{276
263
(2,4,6-triphenylpyridinium)-phenoxide (5) [37], and the semisynthetic cytostatic Etoposide (4) [38], i.e. 9-[(4,6-O-ethylidene-¯-D-glucopyranosyl)oxy]-5,8,8a,9-tetrahydro-5-(4hydroxy-3,5-dimethoxy-phenyl)furo [3’,4’:6,7]naphtho[2,3-d]-1,3-dioxol-6(5aH)-one.
The structures of the new diaryl-ethers were con¯rmed by 1 H- and 13 C-NMR spectra,
and they are represented by formulas 6-9. The hydrophobicity/hydrophilicity and TLC
properties, as well as the reactivity towards amino acids, have been investigated.
2
Results and discussion
2.1 Synthesis of compounds 6c, 6d, 7e ¡ ¡ 7i, 8, and 9 .
When an organic or inorganic anion is involved in a supramolecular complex with a crown
ether of suitable size, the \naked anion" becomes more nucleophilic [39]. We used this
approach for the synthesis of all but the last of the title compounds. In all following
equations, subscript \s" stands for solid state, and subscript \o" stands for organic liquid
phase.
(1)
(2)
In the case of compounds 6c, 6d the solid salts could not be prepared, and were
obtained in dichloromethane solution from the alkali metal hydroxide and the phenolic derivative in the presence of crown ethers (equations 3, 4); the next stage was the
nucleophilic substitution with NBD chloride (Synthesis variant A).
¡
+
*
M+ OH¡
(s) + CE(o) ) [CE....M] OH(o)
¡
+
*
[CE....M]+ OH¡
(o) + ArOH ) [CE....M] ArO (o) + H2 O
(3)
(4)
264
A.T. Balaban et al. / Central European Journal of Chemistry 3 (2003) 260{276
For compounds 7e-7i and 8 (Synthesis variant B), it was possible to prepare the
solid salt of the phenoxidic compound Ar-O¡ M+ (where M is sodium or potassium)
in isopropyl alcohol according to eq. 5, by precipitation with petroleum ether. Then
the supramolecular complex was formed in methylene chloride solution with the corresponding crown ether (15C5 and 18C6, respectively, for sodium and potassium salts).
The choice of the cation was based on the quantitative and selective precipitation of the
potassium salt of 3f, and of the sodium salt of 3g [39]. Next, we added the equimolar
amount of 1, yielding a red Meisenheimer complex. Addition of aqueous hydrochloric acid resulted in a liquid/liquid system that allowed the isolation of the nucleophilic
substitution products from the organic phase (Synthesis variant B).
ArOH + M+ OH¡ *
) M+ ArO¡ + H2 O
(5)
For the preparation of compound 9 from the betainic phenoxide 5, the Synthesis
variant C was applied by analogy with the methylation of 5 [40], when addition of crown
ethers was not necessary.
Yield 6-9 (%)
m.p. (¸C)
A/18C6
70
235{236
6b
A/18C6
58
206{207
6c
A/18C6
37
252.5{253
6d
A/15C5
25
231{231.5
7e
B/15C5
81(M=Na+ )
45
116.5{118
7f
B/15C5
80 (M=K+ )
43
143{146
7g
B/18C6
85 (M=Na+ )
71
193{194
7h
B/18C6
72 (M=K+ )
60
135{137
7i (cis+trans)
B/18C6
85 (M=K+ )
32
146.5{148
8
B/15C5
64 (M=Na+ )
46
180{182.5
9
C
76
161.5{164
Compound
Variant/CE
6a
Yield ArOM (%)
Table 1 Yields and melting points
Among the three possible types of aromatic nucleophilic substitution (normal SN Ar
displacement of activated halogen via a Meisenheimer complex [24,25,41], substitution
of hydride, possibly in the presence of an oxidant [24,25,41], and vicarious nucleophilic
substitution of an ®-situated hydride anion in the presence of an oxidant [41-47]), the
¯rst one was involved in our case [48].
The high yields indicated in Table 1 for the aroxide salts were possible by the appropriate choice of the cation: the sodium salts of 7e,g and 8 and the potassium salts of
the remaining phenolic compounds. The yields of the nucleophilic substitution are also
A.T. Balaban et al. / Central European Journal of Chemistry 3 (2003) 260{276
265
satisfactory. Melting points of the ¯nal diaryl ether compounds are also presented. For
7i the larger temperature range for the m.p. of the cis + trans-isoeugenol derivative can
be a®ected by the ratio of the two stereoisomers.
2.2 NMR Spectra
The NMR spectra con¯rmed the structure of the synthesized compounds 10, and are
presented in the Experimental Part. In Table 2 we present the 1 H-NMR chemical shifts
for H-5 and H-6 protons from the nitrobenzofurazan moiety (the ¯rst two columns) and
for H-2’ (6’), H-3’ (5’), and H-4’ of the aryloxy moiety in the next three columns.
Since the two rings may be tilted due to steric factors when the phenol has two ortho
substituents, some of these protons may be shielded by the ring currents. Of course,
these e®ects are compounded with the electronic e®ects of substituents. The H-5, H-2’
and H-6’ protons are closest to the other ring because they are ortho to the ethereal
oxygen atom connecting the two ring systems, hence they would be more shielded by
ring currents; H-6 and H-3’ and H-5’ are meta to the ethereal oxygen and the shielding
would be lower. The 7-nitro group of the benzofurazan system exerts a strong deshielding
e®ect on H-6 ortho to it, and a weaker deshielding e®ect on H-5, meta to it. Compound
7g obtained from 2,6-dimethoxyphenol should be the most tilted, and indeed the data in
Table 2 show that the 3’ (5’) protons in the aryloxy ring of this compound are the most
shielded among all compounds with general formula 10.
2.3 Thin-layer chromatographic (TLC) characteristics of the new diaryl
ether compounds
The 11 diaryl ether compounds 6a - 6d, 7e - 7i, 8, and 9 were checked by TLC on alumina,
silica gel or silanized silica gel. Results (Rf values) are presented in Table 3. Conclusions
are that (i) in the case of silica gel as stationary phase, the Rf values are low because of
266
A.T. Balaban et al. / Central European Journal of Chemistry 3 (2003) 260{276
Compound
H-6*
H-5*
H-2’ (6’)
H-3’ (5’)
H-4’
6a
8.42
6.57
7.43
7.02
6.98
6b
8.42
6.56
7.43
7.00
6.96
6c
8.49
6.61
7.43
7.03
6.98
6d
8.35
6.50
7.36
6.93
6.88
7e
8.43
6.54
7.27
7.56
7.42
7f
8.43
6.42
7.10
7.09, 7.27
7.38
7g
8.43
6.42
-
6.72
7.29
7h
8.42
6.42
7.17
6.87, 5.91
-
7i
8.42
6.43
7.17
7.02, 7.04
-
8
8.62
6.47
9
8.51
5.74
Table 2 1 H-NMR Chemical shifts due to electronic e¬ects of substituents
* Although the numbering of the benzofurazan moiety for 7e di¬ers from that of all other
compounds (because alphabetically \phenoxy" comes after \nitro") and although in the experimental part the peaks in the NMR spectra of compound 7e obey the numbering imposed by
the IUPAC rules with a 4-nitro group, in Table 2 for uniformity the nitro group of compound
7e is assigned number 7 as in all other compounds.
strong interactions between compounds and the stationary phase; the Rf values appear
in the compound order: 6c = 9 = 8 < 6b < 6d < 6a < 7g < 7f < 7h < 7e = 7i; (ii ) in
the case of silanized silica gel, the Rf values are higher than with silica gel (the interaction
is attenuated); the Rf values increase in the same order except for 7h = 7i < 7e; (iii ) in
the case of aluminum oxide, the interactions are very weak; (iv ) strong interactions with
stationary phases are attenuated by adding methanol to the mobile phase.
2.4 Hydrophobic/hydrophilic characteristics of the new diaryl ether compounds
For biomedical applications the hydrophobicity/hydrophilicity determines how substances
interact with biomembranes and receptors, in°uencing their bioavailability and biospeci¯city. Therefore the hydrophobic/hydrophilic characteristics of the new diaryl ether compounds are of the utmost importance [49]. The octanol-water partition coe±cient and
its logarithm (log P) are the most usual parameters for estimating quantitatively these
characteristics, and they can be measured or computed.
As in previous investigations, we studied such characteristics by reverse phase thinlayer chromatography (RPTLC), and the results are presented in Table 4. The following
observations may be made: (i) for compounds having hydroxy groups, the hydrophilicity
is more pronounced, and it decreases in the series 8 > 7g > 7f > 7e > 9 > 6c > 7i >
A.T. Balaban et al. / Central European Journal of Chemistry 3 (2003) 260{276
267
Compound j Support / eluent:
A
B
C
D
E
F
6a
0.41
0.71
0.38
0.84
0.79
0.90
6b
0.15
0.25
0.16
0.52
0.38
0.61
6c
0
0.02
0
0.05
0.02
0.05
6d
0.23
0.33
0.34
0.74
0.45
0.62
7e
0.76
0.89
0.85
0.98
0.90
0.92
7f
0.71
0.85
0.82
0.98
0.89
0.92
7g
0.65
0.83
0.75
0.98
0.88
0.91
7h
0.73
0.87
0.84
0.98
0.90
0.92
7i(cis+trans)
0.76
0.89
0.84
0.98
0.90
0.92
8
0
0.02
0
0.07
0.01
0.04
9
0
0.02
0
0.03
0.02
0.04
Table 3 TLC behavior (Rf ) of compounds 6a-d, 7e-i, 8 and 9
A: silica gel GF254 (Merck), methylene chloride;
B: silica gel GF254 (Merck), methylene chloride : methanol (10 : 0.1, v/v);
C: silanized silica gel GF254 (Merck), toluene;
D: silanized silica gel GF254 Merck), methylene chloride;
E: aluminum oxide 60F254 type E plates (Merck), methylene chloride;
F: aluminum oxide 60F254 type E plates (Merck), methylene chloride : methanol (10 : 0.1, v/v).
7h > 6b = 6d > 6a (see the water-rich situation A in Table 4); (ii) the high hydrophilicity of compound 8 is due to the glycosidic residue; (iii) the hydrophilicity of compound 9
is probably due to its ionic form; (iv) among phenols 7, the most hydrophilic is 7g, devoid
of hydrophobic groups; (v) among the four estrogenic compounds 6a-d, the presence of
hydroxy groups increases the hydrophilicity leading to the ordering 6c > 6b = 6d > 6a.
Two equations (6 and 7) are proposed [50] for computing the hydrophobicity parameters presented in Table 4. Under the experimental conditions, the hydrophobic compound
6a remained on the start line, and thus its parameters could not be determined. The
correlation coe±cient (r) indicates a satisfactory relationship.
RM = log(1/Rf ¡
RM = RM 0 + bK
1)
(6)
(7)
The 4-nitrobenzofurazanic residue is relatively hydrophobic, with log P = 1.69, as
found by an improved computational method [51]. On attempting to calculate log P
values using fragmental constants [49], a relatively good correlation (r = 0.80) with experimental data for RM 0 could be obtained only for the series 7e { 7h; for the more
complex molecules 6a-d, 8, and 9 the fragmental constants do not yield satisfactory
results.
268
A.T. Balaban et al. / Central European Journal of Chemistry 3 (2003) 260{276
Compound
Rf
Hydrophobicity characteristics
A
B
C
RM 0
b
r
6a
0
0
0
0
0
6b
0.05
0.10
0.35
4.3
- 0.0505
0.97
6c
0.17
0.30
0.58
3.14
- 0.041
0.99
6d
0.05
0.15
0.41
4.63
- 0.056
0.99
7e
0.27
0.45
0.61
2.197
- 0.0297
0.99
7f
0.29
0.47
0.66
2.182
-0.030
0.99
7g
0.35
0.52
0.69
2.146
-0.031
0.99
7h
0.10
0.27
0.46
3.62
- 0.0445
0.99
7i (cis+trans)
0.12
0.31
0.48
3.35
- 0.0415
0.98
8
0.41
0.62
0.84
2.76
- 0.0435
0.99
9
0.20
0.23
0.38
1.77
- 0.0195
0.94
Table 4 RPTLC Resultsa) on the hydrophobicity characteristics (RM0 and b in eq. 7) of
compounds 6a-d, 7e-i, 8 and 9
a)
Silica gel RP-18 F254 (Merck), with: A: ethanol : water (6 : 4, v/v); B: ethanol : water (7 : 3,
v/v); C: ethanol : water (8 : 2, v/v);
RM 0 = molecular hydrophobicity (eq. 7); b = the change in the RM value caused by increasing
the concentration (K) of the organic component in the mobile phase (eq. 6); r = the correlation
coe¯ cient for parameters RM 0 and b in eq. 7.
2.5 Reactions of compounds 6a-d ;7e-i ;8 , and 9 with amino acids with
amines
It was shown in our previous paper [36] that compounds 6a and 6b react irreversibly with
amino acids yielding intensely °uorescent products. Like 6a and 6b, the new compounds
6c, 6d, 7e { 7i, 8, and 9 are weakly °uorescent, but in solution (i.e. ethanol) with
amines, amino acids (arginine, valine, lysine, proline, aspartic acid, glutamic acid, etc.)
and their derivatives (i. e. BSA, catalase, glucoxidase, etc.), they give in a short time
(cca. 5 min.) an orange-red color and an intensely yellow °uorescence (¸ em = 540 nm)
on excitation with UV light (¸ ex = 360{480 nm). The same reaction was observed
for the TLC detection (spraying with amino acids, or secondary amine solutions). This
process will be described in more detail in a forthcoming paper.
3
Conclusions
New compounds 6c,d, 7e-i, 8 and 9 were prepared starting from NBD chloride (1) and
the phenolic compounds 2c,d, 3e-i, 4 and 5 via supramolecular complexes with crown
ethers (CE), [CE. . . .M]+ A¡ (where: CE = 15C5 or 18C6; M = Na+ or K+ ; A¡ = anions
A.T. Balaban et al. / Central European Journal of Chemistry 3 (2003) 260{276
269
of compounds 2c, 2d, 3e-i, 4 and 5). This reaction involves a nucleophilic substitution
of the chloride anion in 1. The structures of the compounds were con¯rmed by NMR
spectra. The hydrophobicity/hydrophilicity properties were investigated through reverse
phase thin-layer chromatography (RPTLC), and correlations with calculated log P values
were found. The new benzofurazan derivatives 6c,d, 7e-i, 8 and 9 produce intense
°uorescence with amino acids and secondary amines.
4
Experimental part
Commercial products were employed: 1, 2a -2d, 3e, 3f, 3h, 3i (cis + trans), and
crown ethers 15C5 and 18C6, and TLC plates silica gel GF254 , silanized silica gel GF254 ,
alumina 60F254 type E and silica gel RP-18F254 (Merck) as well as the betaine 5 (Aldrich).
Compound 4 was provided by the Romanian pharmaceutical company, Sindan.
1
H-NMR and 13 C-NMR Spectra were recorded with a Varian Gemini 300BB (300
MHz) spectrometer.
4.1 General procedures for the synthesis of the diaryl ethers
4.1.1 Synthesis variant A.
The reaction conditions are the same as in the previous communication, and they have
been applied for obtaining compounds 6c and 6d. Hydrochloric acid (1N) was added
after completion of the reaction in methylene chloride, the phases were separated, the
organic layer was dried over anhydrous sodium sulfate, and the solvent was rotavaporated. Dissolution in methylene chloride, precipitation with petroleum ether, ¯ltration
and evaporation were repeated twice. Final puri¯cation was carried out by preparative
TLC: silica gel GF254 , chloroform/methanol (9:1 v/v) for 6c, three times; chloroform,
once, for 6d. For the compounds 6a and 6b elementary analysis was the same as in
the previous communication.[36] The yields and melting points for compounds 6a-c are
presented in Table 1.
Estrone 3-(7-nitrobenzofurazan) ether, 6a, described previously [36] with NMR data
for comparison with the new compounds presented now: 1 H-NMR (CDCl3 , ± ppm, J
Hz): 8.42(d, 1H, H-5, 8.2); 7.43(d, 1H, H-1’ , 8.4); 7.02(dd, 1H, H-2’ , 2.5, 8.4); 6.98(d,
1H, H-4’ , 2.5); 6.57(d, 1H, H-6, 8.2); 2.97(m, 2H, H-6’ and H-9’ ); 2.57(d, 1H, H-6’ , 9.1);
2.52{1.42(m, 12H, H-estrone); 0.96(s, 3H, H-18’).13 C-NMR(CDCl 3 , ± ppm): 220.38(Cq);
154.67(Cq); 150.67(Cq); 145.15(Cq); 145.12(Cq); 139.65(Cq); 138.98(Cq); 133.45(CH);
130.37(Cq); 127.61(CH); 120.73(CH); 117.88(CH); 107.48(CH); 50.39(CH); 47.86(Cq);
44.17(CH); 37.92(CH); 35.78(CH 2 ); 31.50(CH2 ); 29.45(CH 2 ); 26.18(CH2 ); 25.77(CH 2 );
21.56(CH 2 ); 13.82(CH3 ).
Estradiol 3-(7-nitrobenzofurazan) ether, 6b, described previously [36] (with NMR
data presented now for comparison with the new compounds): 1 H-NMR(CDCl3 , ± ppm,
J Hz): 8.42(d, 1H, H-5, 8.2); 7.43(d, 1H, H-1’ , 8.4); 7.00(dd, 1H, H-2’ , 2.5, 8.4); 6.96(d,
1H, H-4’ , 2.5); 6.56(d, 1H, H-6, 8.2); 3.77(t, 1H, H-17’ , 8.2); 2.90(m, 2H, H-estr); 2.44{
270
A.T. Balaban et al. / Central European Journal of Chemistry 3 (2003) 260{276
1.18(m, 13H, H-estradiol); 0.83(s, 3H, H-18’). 13 C-NMR(CDCl 3 , ± ppm): 154.79(Cq);
150.52(Cq); 145.18(Cq); 144.14(Cq); 139.88(Cq); 139.62(Cq); 133.50(Cq); 130.33(CH);
127.60(Cq); 120.66(CH); 117.71(CH); 107.44(CH); 81.75(CH); 50.05(CH); 44.16(CH);
43.20(Cq); 38.41(CH); 36.62(CH2 ); 30.56(CH2 ); 29.59(CH2 ); 26.89(CH2 ); 26.19(CH 2 );
23.13(CH 2 ); 13.82(CH3 ).
Estriol 3-(7-nitrobenzofurazan) ether, 6c. Anal.: calcd. for C24 H25 N3 O6 : C 63.85; H
5.58; N 9.31%; found C 63.82; H 5.56; N 9.23%;
1
H-NMR(CDCl3 + TFA, ± ppm, J Hz): 8.49(d, 1H, H-5, 8.4); 6.61(d, 1H, H-6, 8.4);
7.43(d, 1H, H-1’, 8.3); 7.03(dd, 1H, H-2’, 2.6, 8.3); 6.98(d, 1H, H-4’, 2.6); 4.51(dd, 1H,
H-16’, 8.0, 5.8, 2.0); 3.94(d, 1H, H-17’, 5.8); 2.95(m, 2H, H-6’); 1.23{2.40(m, 10H); 0.91(s,
3H, H-18’).
13
C-NMR(CDCl 3 + TFA, ± ppm): 155.20(Cq); 150.68(Cq); 145.31(Cq); 143.93(Cq);
139.82(Cq); 139.09(Cq); 134.66(CH); 129.90(Cq); 127.63(CH-1’); 120.76(CH-2’); 117.87
(CH-4’); 107.88(CH); 89.76(CH-17’); 78.94(CH-16’); 47.63(CH); 43.90(CH); 43.84(Cq13’); 37.68(CH-8’); 35.99(CH2 ); 32.59(CH 2 ); 29.35(CH2 ); 26.68(CH 2 ); 25.50(CH2 ); 12.05
(CH3 -18’).
Ethynylestradiol 3-(7-nitrobenzofurazan) ether, 6d. Anal.: calcd. for C26 H25 N3 O5 : C
67.96; H 5.48; N 9.14%; found C 67.94; H 5.44; N 9.08%.
1
H-NMR(CDCl3 , ± ppm, J Hz): 8.35(d, 1H, H-5, 8.4); 7.36(d, 1H, H-1’, 8.4); 6.93(dd,
1H, H-2’, 2.7, 8.4); 6.88(d, 1H, H-4’, 2.7); 6.50(d, 1H, H-6, 8.4); 2.87(bs, 1H, HO-17’,
deuterable); 2.83(t, 1H, H-9’, 4.2); 2.56(s, 1H, HC=C); 1.29{2.45(m, 14H-estriol); 0.86(s,
3H, Me-13’).
13
C-NMR(CDCl 3 , ± ppm): 154.78(Cq); 150.54(Cq); 145.19(Cq); 144.14(Cq); 139.86
(Cq); 139.84(Cq); 139.49(Cq); 133.49(CH-5); 127.63(CH-1’); 120.66(CH-4’); 117.75(CH2’); 107.45(CH-6); 87.34(Cq alkynic); 79.76(Cq-17’); 74.18(CH alkynic); 49.46(CH); 47.03
(Cq-13’); 43.75(CH); 38.97(CH); 38.94(CH 2 ); 32.67(CH 2 ); 29.61(CH2 ); 26.88(CH2 ); 26.27
(CH2 ); 22.80(CH2 ); 12.67(C-18’). Underlined peaks correspond to carbons in the ethynylestradiol moiety.
4.1.2 Synthesis variant B.
Phenolic compounds 3e { 3i and 4 (1 gram) in 15 mL isopropyl alcohol were treated
with the stoichiometric amount of ¯nely powdered sodium hydroxide (for 3e, 3g, 4) or
potassium hydroxide (for 3f, 3h, 3i). After stirring till the completion of the neutralization, the precipitation was completed by adding petroleum ether (30{60ºC). Filtration
through G3 glass ¯lter and washing with petroleum ether yielded the solid phenoxides
that were kept in the desiccator over calcium chloride.
Sodium salts were dissolved under stirring in methylene chloride by the addition
of equimolar amounts of crown ether 15C5; similarly, potassium salts were treated with
18C6. Then the equimolar amount of compound 1 was added and the mixture was stirred
at room temperature for one hour. Hydrochloric acid (1N) was added, the phases were
separated, the organic layer was dried over anhydrous sodium sulfate, and the solvent was
rotavaporated. Dissolution in methylene chloride, precipitation with petroleum ether,
A.T. Balaban et al. / Central European Journal of Chemistry 3 (2003) 260{276
271
¯ltration and evaporation were repeated twice. Final puri¯cation was carried out by
preparative TLC on silica gel GF254 plates from methylene chloride, using as mobile
phases: toluene (three times for 7e { 7g); toluene/n-hexane 7/3 v/v (three times for
7h, 7i); mixture of methylene chloride/methanol 10/0.2 v/v (seven times for 8) and
extraction from the silica gel strip with methylene chloride. The yields and melting
points for compounds 7e-i and 8 are presented in Table 1.
7-Phenoxy-4-nitrobenzofurazan, 7e. Anal.: calcd. for C12 H7 N3 O4 : C 56.04; H 2.74;
N 16.34%; found C 56.00; H 2.71; N 16.28%.
1
H-NMR (CDCl3 , ± ppm, J Hz): 8.43(d, 1H, H-5, 8.3); 7.56(dd, 2H, H-3’-5’, 7.3, 8.7);
7.42(tt, 1H, H-4, 1.2, 7.3); 7.27(dd, 2H, H-2’-6’, 1.2, 8.7); 6.54(d, 1H, H-6, 8.3).
13
C-NMR(CDCl 3 , ± ppm): 154.41(Cq); 152.76(Cq); 145.17(Cq); 144.14(Cq); 143.62
(Cq); 133.37(CH); 130.79(2CH); 127.28(CH); 120.91(2CH); 107.56(CH).
4-(2-Methoxyphenoxy)-7-nitrobenzofurazan, 7f. Anal.: calcd. for C13 H9 N3 O5 : C
54.36; H 3.16; N 14.63%; found C 54.33; H 3.14; N 14.57%.
1
H-NMR (CDCl3 , ± ppm, J Hz): 8.43(d, 1H, H-5, 8.4); 7.38(dd, 1H, H-4’, 1.5, 6.8,
7.5); 7.27(dd, 1H, H-5’, 1.5, 6.8, 8.0); 7.10(dd, 1H, H-6’, 1.5, 8.0); 7.09(dd, 1H, H-3’, 1.5,
7.5); 6.42(d, 1H, H-6, 8.4); 3.79(s, 3H, OMe).
13
C-NMR (CDCl3 , ± ppm): 154.00(Cq); 150.87(Cq); 144.85(Cq); 144.13(Cq); 140.99
(Cq); 130.69(Cq); 133.62(CH); 128.32(CH); 122.42(CH); 121.59(CH); 113.25(CH); 107.06
(CH); 55.85(Me).
4-(2,6-Dimethoxyphenoxy)-7-nitrobenzofurazan, 7g. Anal.: calcd. for C14 H11 N3 O6 :
C 53.00; H 3.49; N 13.24%; found C 52.98; H 3.47; N 13.19%.
1
H-NMR (CDCl 3 , ± ppm, J Hz): 8.43(d, 1H, H-5, 8.3); 7.29(t, 1H, H-4’, 8.5); 6.72(d,
2H, H-3’-5’, 8.5); 6.42(d, 1H, H-6, 8.3); 3.80(s, 6H, OMe).
13
C-NMR (CDCl3 , ± ppm): 153.97(Cq); 152.35(Cq); 144.79(Cq); 144.18(Cq); 130.30
(Cq); 129.92(Cq); 133.80(CH); 127.63(CH); 106.56(CH); 105.18(2CH); 56.15(OMe).
4-(4-Allyl-2-methoxyphenoxy)-7-nitrobenzofurazan, 7h, Anal.: calcd. for C16 H13 N3 O5 :
C 58.72, H 4.00; N 12.84%; found C 58.70; H 3.98; N 12.79%.
1
H-NMR (CDCl3 , ± ppm, J Hz): 8.42(d, 1H, H-5, 8.3); 7.17(d, 1H, H-6’, 7.9); 6.91(d,
1H, H-3’, 1.9); 6.87(dd, 1H, H-5’, 1.9, 7.9); 6.42(d, 1H, H-6, 8.3); 6.00(dd, 1H, H-8’, 16.3,
9.6, 6.7); 5.16(dquint, 1H, H-9’(trans vs. H-8’), 16.3, 1.5); 5.15(dquint, 1H, H-9’(cis vs.
H-8’), 9.6, 1.5); 3.77(s, 3H, OMe); 3.46(dquint, 2H, H-7’, 6.7, 0.9). J (H-9’)geminal º 4 J
(H-9’{ H-7’)allylic = 1.5Hz
13
C-NMR (CDCl3 , ± ppm): 154.17(Cq); 150.60(Cq); 144.84(Cq); 144.11(Cq); 140.72
(Cq); 139.22(Cq); 136.48(CH-8’); 133.67(CH-5); 130.26(Cq); 122.11(CH-6’); 121.44(CH5’); 116.68(C-9’); 113.42(CH-3’); 107.00(CH-6); 55.80(OMe); 40.05(C-7’).
(cis+trans)-4-(2-Methoxy-4-propenylphenoxy)-7-nitrobenzofurazan, 7i, Anal.: calcd.
for. C16 H13 N3 O5 : C 58.72, H 4.00; N 12.84%; found C 58.69; H 3.99; N 12.78%.
1
H-NMR (CDCl3 , ± ppm, J Hz): 8.42(d, 1H, H-5, 8.4); 7.17(d, 1H, H-6’, 7.9); 7.04(d,
1H, H-3’, 1.9); 7.02(dd, 1H, H-5’, 1.9); 6.43(dq, 1H, H-7’, 15.7, 1.2); 6.43(d, 1H, H-6,
8.4); 6.31(dq, 1H, H-8’, 15.7, 6.2); 3.79(s, 3H, OMe); 1.93(dd, 3H, H-9’, 1.5, 6.5).
13
C-NMR (CDCl3 , ± ppm): 153.72(Cq); 150.35(Cq); 144.43(Cq); 143.72(Cq); 139.32
272
A.T. Balaban et al. / Central European Journal of Chemistry 3 (2003) 260{276
(Cq); 138.03(Cq); 133.19(CH); 129.94(Cq); 129.47(CH); 127.10(CH); 121.89(CH); 118.58
(CH); 109.87(CH); 106.62(CH); 55.38(OMe); 18.06(C-9’).
Etoposide 1-(7-nitrobenzofurazan) ether, 8, Anal.: calcd. for C35 H33 N3 O16 : C 55.93;
H 4.43; N 5.59%; found C 55.90; H 4.41; N 5.53%. In the following, ETP stands for the
etoposide residue.
1
H-NMR (DMSO, ± ppm, J Hz): 8.62(d, 1H, H-5, 8.3); 6.47(d, 1H, H-6, 8.3); 7.05(s,
1H, H-arom-ETPS); 6.62(s, 1H, H-arom-ETP close to glucose); 6.46(s, 2H, H-3’-5’);
6.06(s, 2H, CH2 -formal); 5.27(d, 2H, CH2 -lactone or glucose, 5.6); 4.98(d, 1H, H-ETP,
3.0, glycosidic); 4.73(q, 1H, H-acetal, 5.3); 4.71(d, H, CH-ETPS, 5.4); 4.60(d, 1H-ETP,
7.6); 4.32(m, 2H, H-ETP-glucose); 4.09(dd, 1H, H-ETP, 4.9, 10.5); 3.63(s, 6H, OMe);
1.24(d, 3H, Me-acetal, 5.0); 2.90{3.60(m, 3H-ETPS). Some hydrogen atoms are missing
because they may be obscured by DMSO or traces of water.
13
C-NMR (DMSO, ± ppm): 170.61(Cq); 152.20(Cq); 150.75(Cq); 147.86(Cq); 146.43
(Cq); 144.58(Cq); 140.39(Cq); 131.81(Cq); 130.25(Cq); 129.04(Cq); 127.70(Cq); 110.08
(CH); 109.79(CH); 107.79(CH); 107.78(CH); 101.54(CH-acetal); 101.44(OCH2 O); 98.60
(CH); 67.87(CH 2 ); 67.35(CH 2 ); 80.12(CH); 74.40(CH); 72.72(CH); 71.71(CH); 65.78(CH);
55.98 (OCH 3 ); 43.26(CH); 37.28(CH); 20.33(CH 3 ).
4.1.3 Synthesis variant C.
The phenolic betainic compound 5 was dissolved in methylene chloride and the stoichiometric amount of compound 1 was added. After stirring at room temperature for one
hour, the solution was concentrated at reduced pressure and the product 9 was puri¯ed
by preparative TLC on silica gel GF254 plates with a mobile phase consisting of chloroform/methanol 9/1 v/v, once. The yield and melting point for compound 9 is presented
in Table 1.
1-((2,6-Diphenyl-4-(2,4,6-triphenyl-1-pyridinium)-phenoxy)-7-nitrobenzofurazan, 9.
Anal.: calcd. for C47 H31 N4 O4 Cl: C 75.14; H 4.13; N 7.46; Cl 4.73%; found C 75.11; H
4.12; N 7.40; Cl 4.71%.
1
H-NMR(DMSO, ± ppm, J Hz): 8.84(s, 2H, H-9’-11’); 8.51(d, 1H, H-5, 8.3); 8.44(dd,
2H, H-ortho-Ph-10’, 1.7, 8.2); 7.84(s, 2H, H-3’-5’); 7.58{7.76(m, 13H, H-arom); 7.28{
7.09(m, 10H, H-arom); 5.74(d, 1H, H-6, 8.4).
13
C-NMR(DMSO, ± ppm): 156.17(Cq); 155.96(Cq); 150.54(Cq); 146.16(Cq); 143.76
(Cq); 143.41(Cq); 137.81(Cq); 134.90(Cq); 133.97(Cq); 133.91(CH-5); 133.23(Cq); 133.00
(Cq); 132.56(CH-paraPh-11’); 131.15(CH-5’-3’); 130.34(Cq); 130.24(CH); 129.95(CH);
129.63(CH); 128.76(CH-ortoPh-11’); 128.44(CH); 128.33(CH); 128.29(CH); 124.83(CH9’-11’); 108.95(CH-6). One CH group is missing, probably due to overlap.
4.2 TLC behavior (Rf ) of compounds 6a-d;7e-i;8 and 9
The TLC behavior of compounds 6a-d, 7e-i, 8 and 9 (namely the conditions and Rf
values) is presented in Table 3.
A.T. Balaban et al. / Central European Journal of Chemistry 3 (2003) 260{276
273
4.3 The hydrophobicity of compounds 6a-d ;7e-i ;8 and 9
The hydrophobicity was determined by RPTLC. The conditions and the results are shown
in Table 4.
Acknowledgements
Thanks are addressed to the pharmaceutical company Sindan (Romania) for providing
the Etoposide (4) and for sponsoring in part this project. The careful observations of an
anonymous referee were much appreciated.
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