Synthesis of flavin-calix[4]arene conjugate derivatives

Article abstract of DOI:10.1002/hlca.201000260

The synthesis of two new flavin substituted calix[4]arene derivatives, 9 and 10, is described. The first flavin substituted calix[4]arene derivative 9 was synthesized by the reaction of 3-methylalloxazine (5) with 25,27-bis(3-bromopropoxy)-26,28-dihydroxy-5,11,17,23-tetra(tert-butyl)calix[4] arene (4) in high yield (92%). The other derivative 10 was prepared from 3-methylalloxazine-1-acetic acid (7) and 25,27-bis(3-cyanopropoxy)calix[4]arene (3). All new compounds were characterized by a combination of FTIR and ¹H-NMR spectroscopy, and elemental-analysis techniques.

Full text of DOI:10.1002/hlca.201000260

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Synthesis of FlavinꢀCalix[4]arene Conjugate Derivatives
by Serkan Sayin*a), Gꢀlderen Uysal Akkus¸b), Radek Cibulkac), Ivan Stiborc), and Mustafa Yilmaza)
a) Department of Chemistry, Selcuk University, TR-Konya-42075
(phone: þ 903322233873; fax: þ 903322410106; e-mail: saynserkan@hotmail.com)
b) Department of Chemistry, Afyon Kocatepe University, TR-Afyonkarahisar-03200
c
´
) Department of Organic Chemistry, Institute of Chemical Technology, Technicka 5 CZ-16628-Prague 6
The synthesis of two new flavin substituted calix[4]arene derivatives, 9 and 10, is described. The first
flavin substituted calix[4]arene derivative 9 was synthesized by the reaction of 3-methylalloxazine (5)
with 25,27-bis(3-bromopropoxy)-26,28-dihydroxy-5,11,17,23-tetra(tert-butyl)calix[4]arene (4) in high
yield (92%). The other derivative 10 was prepared from 3-methylalloxazine-1-acetic acid (7) and 25,27-
bis(3-cyanopropoxy)calix[4]arene (3). All new compounds were characterized by a combination of FT-
IR and 1H-NMR spectroscopy, and elemental-analysis techniques.
Introduction. – Flavins play an important role in living organisms [1], and they are
involved in several important photobiological and photochemical processes, such as
phototropism, phototaxis, and photodynamic action [2][3]. Since 1966, the photo-
chemistry and photophysics of alloxazine derivatives have been studied, due to the
discovery of the proton-transfer reactions in lumichrome and related compounds
[4][5]. It is well-known that substituted alloxazine (¼ benzo[g]pteridine-2,4(1H,3H)-
dione) derivatives, mainly lumichromes, are present in many foods and formed in the
normal metabolic process of ingested riboflavin [6]. Recently, alloxazines have
attracted attention due to realization of their possible involvement in a wide variety of
biological systems [6][7]. For instance, it has been shown that lumichrome may be used
to inhibit flavin reductase in living Escherichia coli cells [8].
Supramolecular science has been developed tremendously over the past 30 years
[9][10]. It is known that preorganization and cooperativeness of multifunctional
groups play a major role in biological reaction kinetics [11]. Some calix[4]arene
derivatives, which bear azo groups [12], guadinium units [13], or nucleoside hybrids
[11], are compounds which can be used as enzymes in the cell. At the same time, flavins
that are obtained by condensation between a-phenylenediamine and alloxane [14] are
involved in several biological processes [15]. These are generally redox cofactors
involved in electron redox processes [16] of enzymatic reactions [17][18]. To this end,
we considered that compounds containing both flavin and calixarene units, i.e., 9 and
10, respectively, might have interesting biological activities. Here, we report the first
synthesis of calixarene derivatives that contain flavin units.
Results and Discussion. – Synthesis. The calix[4]arenes can be functionalized with
desired groups both at the upper and the lower rim [19][20] in order to obtain
appropriate arrangements or to achieve the preorganized conformation [9].
ꢀ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

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The aim of this work was the design and synthesis of new calix[4]arene derivatives,
which are cited as flavinꢀconjugate calixarene derivatives. All new compounds were
1
characterized by means of H-NMR and IR spectroscopy, and elemental analyses. The
synthetic routes leading to new calixarene derivatives are depicted in Schemes 1 and 2.
Scheme 1. The Synthetic Route for the Preparation of 3 and 4
i) AlCl3, PhOH, toluene, r.t., 78%. ii) K2CO3, MeCN, Cl(CH2)3CN, reflux, 79%. iii) 1,3-Dibromopro-
pane, K2CO3, MeCN, reflux, 64%.
p-(tert-Butyl)calix[4]arene 1 was chosen as the starting material, and it was
transformed to the derivatives 3 and 4 according to known procedures [19][21][22] (see
Scheme 1). The flavin part of the molecules was synthesized starting from compound 5,
which is available by condensation of benzene-1,2-diamine with N-methylalloxane
according to the procedure described in [14]. Treatment of 5 with BrCH2COOEt
afforded ethyl 3-methylalloxazine-1-acetate (6; Scheme 2). Then, upon hydrolysis of 6
with HCl, the flavin-carboxylic acid 7 was obtained. Treatment of 7 with SOCl2 at 458
for 2 h yielded the Cl derivative 8. The substitution of p-(tert-butyl)calix[4]arene
derivative (4) at its bromoalkoxy chains was conducted in the presence of K2CO3 in
DMF under N2 with flavin to afford the cone conformer flavinꢀcalix[4]arene conjugate
9 in high yield (92%). The other flavinꢀcalix[4]arene conjugate 10 was synthesized by
treatment of 1-(2-chloro-2-oxoethyl)-3-methylalloxazine (8) with 25,27-bis(3-cyano-
propoxy)calix[4]arene (3) in MeCN in the presence of K2CO3 at 858 for 37 h in 42%
yield. The 1H-NMR spectra of 9 and 10 display a typical AX pattern for the H-atoms of
a CH2 bridge (ArCH2Ar) of the calixarene moiety at d(H) 4.17 and 3.5 ppm (J ¼ 12.8)

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Scheme 2. The Synthetic Route for the Preparation of 9 and 10
i) BrCH2COOEt, K2CO3, DMF, r.t., 77%. ii) HCl, 80 – 908, 55%. iii) SOCl2, 458. iv) 4, NaI, K2CO3, DMF,
N2, r.t., 92%. v) 3, MeCN, K2CO3, 858, 42%.
for 10, and 4.11 ppm (J ¼ 12.8) for 9, indicating that compounds 9 and 10 exist in the
cone conformation [23].
Conclusions. – We have synthesized two new calix[4]arene derivatives which
contain flavin units at the lower rim of calix[4]arene. The structures of flavinꢀ
calix[4]arene conjugates have been established by means of IR and 1H-NMR
spectroscopy, and elemental analyses.
It is well-known that the compounds which have N and O donor atom groups are
able to inhibit the enzyme production [24]; therefore, 9 and 10, which have several
donor atoms, potentially could inhibit the enzyme production, and their biological

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activity could be investigated in vivo and in vitro. In conclusion, 9 and 10 may be used
for numerous applications in the medicinal area.
We thank the Scientific and Technical Research Council of Turkey (TUBITAK – Grant No. TBAG
AY/396) for financial support of this work.
Experimental Part
General. Solvents were dried by storing them over molecular sieves (Aldrich; 4 ꢂ, 8 – 12 mesh).
Toluene was dried with CaH2 and stored over Na wire. DMF was dried with CaSO4 and stored over
molecular sieves. The drying agents employed during workup were Na2SO4 and MgSO4. All aq. solns.
were prepared with deionized H2O that had been passed through a Milli-Q Plus water purification
system. Starting materials and reagents were obtained from Aldrich, Lancaster, and Fluka and were used
without further purification. TLC: DC Alufolien Kieselgel 60 F254 (Merck). M.p.: Barnsted/Electro
thermal apparatus in a sealed capillaries; uncorrected. IR Spectra: Perkin-Elmer 1605 FTIR System
Spectrum BX spectrometer; as KBr pellets. 1H-NMR Spectra: Varian 400 MHz and Varian Mercury Plus
300 MHz spectrometers; chemical shifts in ppm rel. to TMS (d ¼ 0.0 ppm). Elemental analyses (C, H, N):
Perkin-Elmer 240 analyzer.
Syntheses. 5,11,17,23-Tetra(tert-butyl)-25,26,27,28-tetrahydroxycalix[4]arene (1), 25,26,27,28-tetrahy-
droxycalix[4]arene (2), 25,27-bis(3-cyanopropoxy)-26,28-dihydroxycalix[4]arene (3), 25,27-bis(3-bro-
mopropoxy)-5,11,17,23-tetra(tert-butyl)-26,28-dihydroxycalix[4]arene (4), and 3-methylalloxazine (5)
were synthesized according to literature procedures [14][19][21][22]. Ethyl (3-methylalloxazin-1-
yl)acetate (6), (3-methylalloxazin-1-yl)acetic acid (7), (3-methylalloxazin-1-yl)acetyl chloride (8), and
the final products flavinꢀcalix[4]arene 9 and flavinꢀcalix[4]arene conjugates 9 and 10, resp., were
prepared for the first time according to the procedures described below.
25,27-Bis(3-cyanopropoxy)-26,28-dihydroxycalix[4]arene (¼4,4’-{[26,28-Dihydroxypentacyclo-
[19.3.1.13,7.19,13.115,19]octacosa-1(25),3(28),4,6,9(27),10,12,15(26),16,18,21,23-dodecaene-25,27-diyl]bis-
(oxy)}dibutanenitrile; 3). Prepared according to [21]. Yield 79%. M.p. 244 – 2468. IR (KBr): 2363 (CN).
1H-NMR (CDCl3, 400 MHz): 2.39 (quint., J ¼ 8.0, 2 CH2CH2CN); 3.08 (t, J ¼ 7.2, 2 CH2CN); 3.47 (d, J ¼
12.8, 2 ArCH2Ar); 4.13 (t, J ¼ 5.6, 3 OCH2CH2); 4.21 (d, J ¼ 12.8, 2 ArCH2Ar); 6.70 (t, J ¼ 7.2, 2 arom.
H); 6.79 (t, J ¼ 7.2, 2 arom. H); 6.94 (d, J ¼ 7.2, 4 arom. H); 7.09 (d, J ¼ 7.2, 4 arom. H); 7.79 (s, 2 OH).
Anal. calc. for C36H34N2O4 (558.68): C 77.40, H 6.13, N 5.01; found: C 77.01, H 6.22, N 4.92.
25,27-Bis(3-bromopropoxy)-5,11,17,23-tetra(tert-butyl)-26,28-dihydroxycalix[4]arene (¼26,28-
Bis(3-bromopropoxy)-5,11,17,23-tetra-(tert-butyl)pentacyclo[19.3.1.13,7.19,13.115,19]octacosa-1(25),3(28),4,
6,9(27),10,12,15(26),16,18,21,23-dodecaene-25,27-diol; 4). Prepared according to [22]. Yield 64%. M.p.
277 – 2798. 1H-NMR (CDCl3, 300 MHz): 7.05 (s, 4 arom. H); 4.32 (d, J ¼ 13.3, ArCH2Ar); 4.28 (t, J ¼ 5.6,
2 CH2O); 3.83 (t, J ¼ 6.4, 2 CH2Br); 3.32 (d, J ¼ 13.3, 2 ArCH2Ar); 1.29 (s, 2 t-Bu); 0.94 (s, 2 t-Bu). Anal.
calc. for C50H66Br2O4 (890.88): C 67.41, H 7.47, Br 17.94; found: C 67.53, H 7.50, Br 17.93.
3-Methylalloxazine (¼ 3-Methylbenzo[g]pteridine-2,4(1H,3H)-dione; 5). Prepared according to
[14]. Yield: 65%. M.p. 2858. 1H-NMR ((D6)DMSO, 300 MHz): 3.29 (s, MeN); 7.74 – 7.79 (m, 1 arom. H);
7.92 (d, J ¼ 3.5, 2 arom. H); 8.18 (d, J ¼ 8.2, 1 arom. H); 12.23 (s, NH). Anal. calc. for C11H8N4O2 (228.21):
C 55.77, H 3.81, N 23.65; found: C 56.12, H 3.41, N 23.55.
Ethyl (3-Methylalloxazin-1-yl)acetate (¼ Ethyl (3-Methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-
1(2H)-yl)acetate; 6). A mixture of 5 (0.3 g, 1.315 mmol), K2CO3 (1 g, 7.303 mmol), and BrCH2COOEt
(2.195 g, 13.146 mmol) were stirred for 5 h in DMF (110 ml) at r.t. Then, the solvent was removed in
vacuo, and the yellow product was crystallized from CH2Cl2/Et2O. Yield: 0.318 g (77%). M.p. 205 – 2078.
1H-NMR ((D6)DMSO, 400 MHz): 1.31 (t, J ¼ 6.9, Me); 3.62 (s, MeN); 4.26 (q, J ¼ 7.5, CH2O); 5.20 (s,
CH2N); 7.77 (t, J ¼ 7.8, 1 arom. H); 7.89 (t, J ¼ 6.8, 1 arom. H); 7.99 (d, J ¼ 7.7, 1 arom. H); 8.36 (d, J ¼ 8.2, 1
arom. H). Anal. calc. for C15H14N4O4 (314.3): C 57.32, H 4.49, N 17.83; found: C 57.57, H 4.32, N 17.78.
(3-Methylalloxazin-1-yl)acetic Acid (¼(3-Methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-1(2H)-yl)-
acetic Acid; 7). A mixture of 6 (0.318 g, 1.012 mmol) and HCl (5 ml) was stirred at 80 – 908 for 105 min.
The mixture was cooled, and ice-water (15 ml) was added to the soln. The yellow solid that precipitated
was filtered off and washed with H2O. Reprecipitation from 2n AcOH gave 7 as a light yellow solid.

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Yield: 0.158 g (55%). M.p. 289 – 2938. 1H-NMR ((D6)DMSO, 400 MHz): 3.31 (s, MeN); 4.98 (s, CH2N);
7.85 (t, J ¼ 6.3, 1 arom. H); 8.00 (d, J ¼ 5.1, 2 arom. H); 8.26 (d, J ¼ 7.8, 1 arom. H); 13.15 (s, OH). Anal.
calc. for C13H10N4O4 (286.24): C 54.55, H 3.52, N 19.57; found: C 54.67, H 3.45, N 19.53.
(3-Methylalloxazin-1-yl)acetyl Chloride (¼(3-Methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-
1(2H)-yl)acetyl Chloride; 8). Compound 7 (1.00 g, 0.350 mmol) was added to SOCl2 (2.5 ml,
31.903 mmol), and the suspension was stirred at 458. After 2 h, the solid had dissolved, SOCl2 was
evaporated at < 458, and the crude chloride 8 obtained was used without further purification.
1,1’-{[5,11,17,23-Tetra-(tert-butyl)-26,28-dihydroxypentacyclo[19.3.1.13,7.19,13.115,19]octacosa-1(25),
3(28),4,6,9(27),10,12,15(26),16,18,21,23-dodecaene-25,27-diyl]bis(oxypropane-3,1-diyl)}bis(3-methyl-
benzo[g]pteridine-2,4(1H,3H)-dione); 9). K2CO3 (0.6 g), 5 (0.438 mmol), and NaI (0.4 g) were added to
a soln. of 4 (0.219 mmol) in 20 ml of dry DMF, and the mixture was stirred under N2 at r.t. for 47 h. The
reaction was monitored by TLC. The salts were filtered off, and the solvent from the filtrate was removed
under reduced pressure. Then, Et2O was added, the precipitates formed were filtered off, and the
received product was dried in a vacuum desiccator. Yield 92%. M.p. > 3508. 1H-NMR ((D6)DMSO,
400 MHz): 8.53 (s, 2 OH); 8.07 – 7.93 (m, 4 arom. H); 7.76 – 7.76 (m, 2 arom. H); 7.58 – 7.48 (m, 2 arom.
H); 7.10 (br. s, 8 arom. H); 4.62 (t, J ¼ 6.4, 2 CH2O); 4.11 (d, J ¼ 12.8, 2 ArCH2Ar); 4.0 (t, J ¼ 5.2,
2 CH2N); 3.35 (overlapped with DMSO, this area should correspond to 10 H-atoms belonging to
ArCH2Ar and MeN); 2.32 – 2.29 (m, 2 CH2); 1.16 (s, 2 t-Bu); 1.10 (s, 2 t-Bu). Anal. calc. for C72H80N8O8
(1185.45); C 72.95, H 6.80, N 9.45; found: C 73.02, H 6.88, N 9.32.
26,28-Bis(3-cyanopropoxy)pentacyclo[19.3.1.13,7.19,13.115,19]octacosa-1(25),3(28),4,6,9(27),10,12,
15(26),16,18,21,23-dodecaene-25,27-diyl Bis[(3-methyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-1(2H)-
yl)acetate]; 10). A mixture of 8 (0.11 g, 0.36 mmol), 3 (0.09 g, 0.164 mmol), and K2CO3 (0.10 g,
0.724 mmol) in MeCN (6 ml) was stirred at 858 for 37 h. After cooling, MeCN was removed to dryness,
and the residue was dissolved in CH2Cl2, washed with H2O (5 ꢁ 30 ml), dried (Na2SO4), and then
concentrated to a third of the original volume on a rotary evaporation. The residue was then mixed with
EtOH. Precipitated 10 was filtered off and dried under vacuum. Yield: 75 mg (42%). M.p. 3408. IR (KBr
disk): 1740 (ester C¼O). 1H-NMR ((D6)DMSO, 400 MHz): 2.32 (quint., J ¼ 6.1, 2 CH2CH2CN); 3.10 (t,
J ¼ 7.0, 2 CH2CN); 3.36 (s, 2 MeN); 3.49 (d, J ¼ 12.8, 2 ArCH2Ar); 4.07 (t, J ¼ 5.6, 2 CH2O); 4.17 (d, J ¼
12.8, 2 ArCH2Ar); 5.77 (s, 2 CH2N); 6.63 (t, J ¼ 7.5, 2 arom. H); 6.82 (t, J ¼ 7.5, 2 arom. H); 7.08 (d, J ¼ 7.6,
4 arom. H); 7.18 (d, J ¼ 7.5, 4 arom. H); 7.80 – 7.83 (m, 2 arom. H); 8.01 (d, J ¼ 2.3, 2 arom. H); 8.24 (d, J ¼
7.9, 4 arom. H). Anal. calc. for C62H50N10O10 (1095.14): C 68.00, H 4.60, N 12.79; found: C 67.97, H 4.51, N
12.93.
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