π…π interactions of flavins, 5. Syntheses, structures and physical properties of flavin systems...

H. A. Staab, J. Kanellakopulos, P. Kirsch, C. Krieger 1827

~ a a . 7 1 : Interactions of Flavins, 5[’1

Syntheses, Structures and Physical Properties of Flavin Systems with Covalent Bonding to 7c Donors and 7c Acceptors (Quinones) Heinz A. Staab”, Johannes Kanellakopulos, Peer Kirsch, and Claw Krieger

Abteilung Organische Chemie, Max-Planck-Institut fur medizinische Forschung, JahnstraDe 29, D-69120 Heidelberg, F.R.G. Telefax: (internat .) + 49(0)622 11486 2 19

Received May 5, 1995

Key Words: Isoalloxazinophanes I Flavins I x Donors I x Acceptors I Quinones ~~ ~ ~ ~~ ~ ~~ ~

To study 71-71 interactions of flavins of different redox states with n-electron donors and acceptors, the syntheses of compounds in which such 71 systems are linked to flavins by using the ”cyclophane concept“ were attempted and partially achieved. The syntheses of the metacyclo-isoalloxazinophanes 1 and 2 are reported as well as the X-ray structure analysis of 2. The conversion of 1 or 2 to the benzoquinono-iso-

~~ ~~ ~

alloxazinophanes 3 or 4, respectively, was not achieved, obviously due to the specific sterical shielding of the methoxy groups in the isoalloxazinophanes. Benzoquinone-isoalloxazine systems in which the two components are linked by only one triethylene chain were obtained. Spectroscopic and electrochemical properties related to intramolecular interactions are reported.

The manifold functions with which flavin enzymes are able to cope in biochemical redox reactions121, depend on the versatility of their redox states. Flavoquinones, flavose- miquinones and flavohydroquinones, due to the specific flavin structure, each may be influenced by protonation and deprotonation as well as by steric effects and hydrogen bonds modifying the respective redox potentials131. This versatility in redox-equivalent transfer made the flavin family, far beyond their actual biochemical functions, an especially interesting group of organic compounds which continues to raise crucial questions pertinent to organic chemistry in general.

In preceding papers we reported on the syntheses and intramolecular interactions between two flavin units in isoalloxazinophanes and other covalently linked bis(isoal1- oxazines)L41 and on pyridinio-isoalloxazinophanes as model systems for active-site complexes of flavoenzymes interacting with nicotinamide[’]. The present paper deals with the syntheses and structures of flavin systems linked to x- electron acceptors and donors. In this context, flavin-quinone systems were of interest since flavins as well as quinones (e.g., ubiquinones, coenzymes Q) are well-known for their participation in biological electron-transfer chains.

To study chemical and physical interactions as functions of, for example, distance, orientation, polarity of specific groups, we made extensive use in the past of the “cyclophane concept” which means the interacting substructures are kept in relatively rigid, well-defined distances and orien- tations. This concept permits the variation of structural parameters selectively one by one as was demonstrated, for example, by our recent work on the structure dependence of photoinduced electron-transfer reactions in porphyrin-

quinone cyclophane~[~]. In the present case we are trying to apply this “cyclophane concept” to the interactions of flavins with x-electron donors and acceptors of type 1 to 4.

1 : R = H

2: R = M e 3 : R = H

4: R = M e

Syntheses of 16,19-Dimethoxy[4]metacycIo[3f(lO,6)- isoalloxazinophanes 1 and 2

The assembly of the metacyclophane segments within 1 and 2 started from 1,3-bis(bromomethyl)-2,5-dimethoxybenzene from which, by double malonic ester condensation, decarboxylation and lithium aluminium reduction, 1,3- bis(3-hydroxypropyl)-2,5-dimethoxybenzene (5)l6] was obtained which with phosphorus tribromide (toluene, 5 h at 55 - 60 “C) yielded 1,3-bis(3-bromopropy1)-2,5-dimethoxybenzene (6)[6371. The one-sided reaction of 6 with triphenylphosphane to the monosubstituted phosphonium salt 7 always yielded, despite the application of a wide variety of reaction conditions and solvents, mixtures of mono- and disubstituted phosphonium salts together with the corresponding alkenes formed by elimination. By extended application of separation and chromatographic techniques 7 (m.p. 61.5-62°C) was finally obtained pure and fully characterized in 1.5% yield.

As a more suitable precursor for the further reaction se- quence to 1, it was possible, however, to obtain the

Liebigs Ann. 1995,1827- 1836 0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1995 0947-3440/95/1010- 1827 $10.00+.25/0

1828 H. A. Staab, J. Kanellakopulos, P. Kirsch, C. Krieger

monoacetylation product 8L6] from 5. Using rather specific reaction conditions (see Experimental), 8 was isolated in 46% yield with 1,3-bis(3-acetoxypropy1)-2,5-dimethoxybenzene (9; 24% yield). By conversion of the 3-hydroxypropyl group of 8 into the corresponding 3-bromopropyl group (tetrabromomethane, triphenylphosphane[*I; dichloromethane, OT), 10r'l was obtained in good yield (-70%). From 10 there are two ways to reach phosphonium salts which seemed suitable for the linkage to the isoalloxazine unit: 13 (m.p. 106- 107 "C)['] was isolated in almost quantitative yield from the reaction of 3-(3-acetoxypropyl)-l-(3-bromopropyl)-2,5-dimethoxybenzene (10) with triphenylphosphane in acetonitrile (30 h, reflux). Alternatively, the monoacetoxy compound 10 underwent basic hydrolysis to split off the acetyl group to form 1-(3-bromopropy1)-3-(3- hydroxypropyl)-2,5-dimethoxybenzene (11) (98 % yield)L61. In acetonitrile 11 reacts with triphenylphosphane (30 h, reflux) to yield the phosphonium salt 12 (97%; m.p. 143-145°C). Both 13 and 12 were expected to be likely candidates for introducing the metacyclophane segment via Wittig reactions into precursors of an isoalloxazine unit, thus paving the way to the flavinophanes. 3-Acetylamino-2- nitrobenzaldehyde (for its preparation see preceding paper of this series[']) was a suitable partner for 12 or 13 in their Wittig reactions.

OMe

5: X ' . X 2 - OH 10: X'mOAc, X2=Br

11: X ' -OH, X 2 4 r

12: X'-OH. X2-PPh,Br

e e 6: X'.X2 - Br 7: X ' -B r . X Z - P P h 3 B r

8: X '=OAc , X2=OH a e 9: X'.X2 = O A c 13: X'=OAc. X2-PPh,Br-

The Wittig reaction of the ylide of 13 (THF, sodium hexamethyldisilazane, - 40 "C) with 3-acetylamino-2-nitrobenzaldehyde (THF, 3 h at -3O"C), after conventional work-up and chromatography (silica; acetone/petroleum ether, b.p. 40"C, 2:3), yielded 14 (ZIE isomers, total yield 47%)r6l. The ZlE ratio was determined as 81:19 by 'H- NMR spectroscopy. Starting from the corresponding phosphonium salt 12 the Wittig reaction with 3-acetylamino-2- nitrobenzaldehyde under similar conditions yielded 3 1 YO of the Z/E isomers 15['] which also were obtained from 14 (methanol, ammonia; 10 h, 0°C). For the preparation of the macrocyclic system 17, the 3-hydroxypropyl compound 15 was converted to the corresponding 3-bromopropyl system 16 (tetrabromomethane, triphenylphosphane, dichloromethane; yield 55-80%).

For the cyclization of 16 to (132)-1-acetyl-7,1O-dimethoxy-20-nitro-l-aza[4.4]metacyclophan-13-ene (17), a mixture of 2 and E isomers of 16 at high dilution (about 0.01 moles in 1 liter of toluene) was added dropwise over 36 h into a boiling mixture of toluene, aqueous sodium hydroxide solution and tetra-n-butylammonium bromide in a cy-

clization apparatus[']. The reaction was monitored by DC (silica, dichloromethane/diethyl ether, 9 : 1, for details see Experimental). The macrocycle 17r61 was obtained in varying, yet acceptable yields of 35-54% as a pale-yellow crystal powder of m.p. 211.5"C.

f X R

14: X-OAc; 15: X=OH; 16: X = B r 17: R=Ac; 1 8 R=H

The first target compound, 16,19-dimethoxy[4]metacyclo[3](10,6)isoalloxazinophane (l)[lo1, was obtained in three more steps: The N-acetyl group was removed by basic hydrolysis (potassium hydroxide, ethanol/water, 80 "C, under argon) to yield 18 (89%, m.p. 144-145.5"C)@]. Cata- lytic hydrogenation (Pdkharcoal, acetic acid) followed by condensation with alloxane monohydrate in the presence of boric acidlacetic acid (8 h at 55"C, under argon and with exclusion of light) yielded 1 as yellow microcrystals, (m.p. 330°C) in 90% yield after work up (see Experimental) and crystallization from trichloromethaneldiethyl ether. El- emental analyses, 'H-NMR, mass spectral and other spectroscopic data support structure 1 which was then con- firmed by the subsequent synthesis of 2 and the N-methyl derivative of 1, as well as the X-ray structure analysis of 2.

16,19-Dimethoxy-3-methyl[4]metucyclo[3j (10,6)- isoalloxazinophane (2)

As will be shown below, the demethylation of the two methoxy groups of 1 and the oxidation to the corresponding quinone-flavinophane 3 raised unexpected difficulties. These problems were attributed in part to the low solubility of 1. From earlier experience with common flavin systems, it was to be expected that N-methylation in position 3 of 1 would increase the solubility in most organic solvents.

For the synthesis of 2, a ten-fold excess of iodomethane was added to a mixture of 1 with potassium carbonate in dimethyl formamide; the reaction was kept at 20°C for 6 h (and monitored by DC). Conventional work-up (see Exper- imental) and recrystallization from dichloromethaneln-hexane lead in 93% yield to a crystalline product (m.p. 262°C) for which all analytical and spectroscopic data were in full agreement with structure 2. From dichloromethanelcyclo- hexane 2 crystallized in orange-yellow prisms for which an X-ray structure analysis was solved. The data were collected by an Enraf-Nonius CAD 4 diffractometer (graphite-mono- chromated Mo-K, radiation, 0 - 2 0 scan). For details of crystallographic data and data collection parameters see reference 1 1.

The comparison of the molecular structure of 2 with that of simple 10-methylisoalloxazine show no significant differ- ences of bond lengths and bond angles; obviously, the isoalloxazine unit in spite of the cyclophane bridging is not perceivably affected in its planarity and in its steric and

Synthesis and X-Ray Structure Determination of

Liebigs Ann. 1995, 1827-1836

r - z Interactions of Flavins, 5 1829

electronic properties. This fact is an essential precondition for accepting these flavinophanes as models for intermo- lecular 7c*-n interactions of flavins.

Figure 1 shows the structure of 2 in a side-view from which the planarity of the isoalloxazine part as well as the insertion of the cyclophane part can be seen. The dimethoxybenzene unit is arranged facing the central 1,4-pyrazine part of the isoalloxazine. The mean distance between the planes of these two functional components is 343 pm. The transannular distances, thus, are in the typical range of charge- and electron-transfer interactions. Figure 1. Side view projections of the crystal structure of 2 (mole- cule A) along the N(5)-N(10) axis and in the approximated plane

of the isoalloxazine unit

Q

0119'1 Cf16")

C(211

The ring planes of the two cyclophane ring systems are not exactly parallel, the C(14)-C(17) axis of the dimethoxybenzene unit is inclined against the isoalloxazine plane by about 13". The crystal structure of 2 reveals two independent sets of very similar conformations in the unit cell: The conformation of the first type (A) is shown in a top view perpendicular to the isoalloxazine plane in Figure 2A. The major difference between A and the second type, B (Figure 2B), is that in A the methoxy group on C(16) is bent towards C(4) whereas in B this methoxy group is turned into the opposite direction towards N(3)["1. The set of A molecules is disordered by B molecules in the order of about lo%, and similarly the B set is disordered by A molecules. This disorder effect in the crystals of 2 is not surpris- ing since - as the comparison of Figure 2A and 2B show - the two conformers A and B are very similar in shape and steric requirements.

Attempts to Synthesize Benzoquinono-isoalloxazinophanes 3 and 4

The original intention of this study was to compare flavinophanes with n-electron donors like 1 and 2 to those linked in an analogous geometry to .n acceptors, especially the biochemically interesting quinone-flavin systems 3 and 4. It was evident from the beginning that the direct oxidative demethylation and ether cleavage of the methoxy groups of 1 and 2 followed by the oxidation of the hydro- quinones, in these cases, would be sterically strongly hindered. Attack of the methoxy groups from the "bottom side" should be blocked by the extended isoalloxazine plane and, in addition, the methoxy groups of 1 and 2 are sub- jected to the steric shielding by the cyclophane bridges. On

Liebigs Ann. 1995, 1827-1836

the other hand, the successful preparation of benzoquinono- phanes from corresponding dimethoxy-substituted precursors in sterically extremely crowded environment[I2] indi- cated that the synthesis of 3 and/or 4 was feasible.

Figure 2. Top view projections of the crystal structure of 2 in the A conformation (Figure 2A) and of the B conformation

(Figure 2B)

NIlI

The oxidative demethylation of 1 with silver(I1) oxide (acetonitrile/water, presence of pyridine-2,6-dicarboxylic acid N-oxide, 0 "C, 1 h)[131 produced, besides the predomi- nant starting material 1, an oxidation product (DC: silica, trichloromethanelmethanol, 10: 1, Rf = 0.23) in 8- 10% yield. Extension of the reaction time led to decomposition. Based on MS (mlz: 416 [M+]) and 'H NMR (360 MHz, CDC13, 305 K; one methyl signal at 6 = 3.03, two single- proton singlets at 6 = 5.76 and 5.49), the hemiacetal structure 19 is suggested for the product obtained from the oxidation of l [ l4] . Attempts to cleave the hemiacetal under various conditions (azeotropic distillation with dioxane, benzene, acetone to remove methanol; reaction with acetic acid, trifluoroacetic acid, etc.) did not succeed. Solvent re- moval always led eventually to decomposition of the product.

Various other oxidizing agents used in order to achieve the oxidative demethylation of 1 to the corresponding quinone 3, did not succeed either (silver oxide in dioxane/phos-


phoric acid and in dioxanelnitric acid; chlorotrimethylsil- anehodium iodide in acetonitrile; ammonium nitrate in acetonitrile/water in acetic acid; manganese oxide in dichloromethane, etc.).

The stepwise conversion of the 1 ,Cdimethoxybenzene unit of 1 into the corresponding quinone was planned as follows: first, demethylation to the hydroquinone, then, in a second step, oxidation to the quinone. The demethylation was tried with boron tribromide (dichloromethane, -78 "C, 5 h). The reaction product was chromatographed on silica (trichloromethane/methanol, 10: 1; Rf = 0.42) and crystallized from methanol/diethyl ether to yield a product (m. p. 267-269°C) which, according to MS (mlz = 432 [Mf], 100%) analysis, was a monodemethylation product of 1. The complete assignment of 'H-NMR signals (500 MHz, [D6]DMSO; 303 K), through comparison with 1, showed that only the sterically less hindered methoxy group on C(19) was cleaved to yield 20. Increasing the reaction time, the temperature, and using higher concentrations and greater excess of the boron tribromide solutions also did not succeed but led to increasing decomposition. This fail- ure to achieve the demethylation of the two methoxy groups in 1 depends, in addition to the specific steric situation, on the low solubility of this isoalloxazinophane and on its in- stability at higher temperatures. As mentioned before, the solubility, and probably also the stability, of the flavinophane system might be increased by the methyl-substitution on N(3) of the isoalloxazine unit.

In fact, the reaction of 2 with 12 equivalents of a 1 M

solution of boron tribromide in dichloromethane (5 h, - 30 "C) yielded, after chromatography (silica, trichloromethane/methanol, 15 : I ) , the monodemethylated product in which the sterically less-hindered methoxy group was cleaved off. MS (including high-resolution) and 'H-NMR (500 MHz, [D,]DMSO, 303 K) spectroscopy confirm structure 21 (see Experimental). In contrast to the experiments on 1, however, in this case it was possible, by increasing the reaction time (8 d, -25"C), to succeed with the second demethylation and to obtain the 16,19-dihydroxy compound 22 in the excellent yield of 89% (after filtration through silica from trichloromethane/methanol and MPLC). The structure of 22 (dark-red crystal powder) is supported by MS (including high-resolution) data and its 'H-NMR (500 MHz, [D6]DMS0, 303 IS) spectrum in which all the peaks may be assigned (for details see Exper-

zinophane 4 appeared to be in easy reach. There exists a great variety of well-established reactions to oxidize hydro- quinones to quinones under mild conditions which are adapted to a wide range of specific structural factors. Un- fortunately, this seemingly trivial reaction, and last step in the series of steps comprising this long synthesis, failed, although a wide repertoire of classical rnethods[l5], as well as a series of newer oxidation methods (ceric ammonium nitrate-impregnated silica in dichlorornethane[l6], electrochemical oxidation with lead dioxide[181, 2,3- dichloro-5,6-dicyano- 1,4-benzoquinone and silver oxide among others), were brought to bear. Under some of these oxidation conditions no reaction occured; this was most probably due to the relative low solubility of the starting material. In other cases, DC-monitoring showed that, first, a relatively homogeneous product seemed to be present which then, especially upon evaporation of solvents, decom- posed into complex mixtures of three to six products. A partial or full explanation for these unexpected difficulties in the oxidation of 21 to 4 may be that the oxidized states of either the quinone or the isoalloxazine segment, due to the cyclophane-enforced close proximity of these units, in- teract with each other (e.g., by electron transfer) leading to side reactions which are not yet understood. So far, the elucidation of this problem was hindered by the small amount of 21 which prevented more extensive experimental studies.

Open-chain Fluvin-Benzoquinone Systems In comparison with the cyclophane-type compounds dis-

cussed above, intramolecular flavin-p-benzoquinone systems, in which the two components are linked by a single trimethylene chain, were of interest as reference systems. This comparison should prove whether the difficulties in the demethylation, as well as the oxidation reactions, are indeed the result of the very specific steric cyclophane structure of the isoalloxazinophanes dealt with in this paper. On the other hand, of course, open-chain flavin-quinone systems like 25 and 26 lack the well-defined rigid orientation of the two interacting groups which was our primary interest regarding the interaction of flavins and quinones.

POMe imental).

0 0

23: R = H ; 2 4 R = Me 25: R = H; 26: R = Me

For the synthesis of 23 and 24, 1-(3-bromopropy1)-2,5- 19 20: R=H. x1 =OH. X2=OMe dimethoxybenzene reacted with 4,5-dimethyl-2-nitro-N-tri-

21 : R=Me, X'=OH, X2=OMe fluoroacetyl-aniline to yield N-[3-(2,5-dimethoxyphenyl)- 22: R=Me, x' =OH. X2=OH propyl]-4,5-dimethyl-2-nitro-aniline. Hydrogenation of the

nitro group (Pdkharcoal, acetic acid) and condensation with alloxane hydrate (boric acid/acetic acid, 55 "C, 18 h), after separation and purification, yielded 10-[3-(2,5-dimeth-

Liebigs Ann. 1995, 1827-1836

With the synthesis of the hydroquinono-isoalloxazinophane 2, the preparation of the benzoquinono-isoalloxa-

T.T Interactions of Flavins, 5 1831

oxyphenyl)propyl]-7,8-dimethyl-isoalloxazine (23). In contrast to the corresponding cyclophanes, demethylation of 23 to the respective hydroquinone (boron tribromide, dichloromethane, -70°C; yield 80%; purple crystals, m.p. 254"C)r61 and oxidation with DDQ to 25 (methanol, 3 h, room temp.; yield 70%, yellow microcrystals, m.p. 240 "C, dec.)r61 was successful. The same applies to the N(3)-methylated ana- logues 24 and 26: Compound 23 was methylated (iodomethane, potassium carbonate, DMF; 3 h, room temp. under argon, exclusion of light; yield 64%, m.p. 245 "C, dec.). The demethylation of 24 to the corresponding hydroquinone compound succeeded with excellent yield (boron tribromide, dichloromethane, 22 h, room temp.; 95% yield, dark red-brown crystal powder, m.p. 235"C)f61 as did the oxidation of 24 to the flavin-quinone compound 26 (DDQ, methanol, 3 h, room temp.; light-sensitive, mustard-yellow microcrystals, m.p. 200 "C, dec.)r61. As reference compounds for 23 and 24, the corresponding, in the side-chain unsub- stituted 10-(3-phenylpropyl)isoalloxazines were synthesized analogously (for synthetic details and characterization see Experimental).

Preliminary Results on Absorption and Fluorescence Spectra Absorption of donor-substituted metacyclo-isoalloxazin-

ophanes 1,2,21 and 22, as well as the openchained systems 23 and 24 (lop4 to lop5 M solutions in various trichloromethane/methanol/buffer systems), did not show any ob- servable charge-transfer absorption. The flavin-quinone cyclophanes 25 and 26, show absorption spectra which are well-approximated by the addition of the absorptions of the flavin and the benzoquinone moieties. Surprisingly, thus, the absorption data do not show significant evidence for intramolecular r - n interactions between the flavins in their existing oxidation state and the donor as well as acceptor subunits of these compounds.

On the other hand, for all flavin-donor systems of the open-chain series like 23 and 24, an effective quenching of the fluorescence was observed (in methanol or methanol/ dichloromethane, 1 : 1)L2O1. The corresponding flavin-quinone systems show a reduction of more than 90% of the fluorescence intensities as compared to the reference compound with a phenyl unit in place of the benzoquinone of 25 and 26. The mechanism of this quenching process is not yet clear. An interesting explanation would be that the fluorescence quenching occurs by a photoinduced electron- transfer reaction from the excited flavin to the quinone which would be analogous to the initial elementary steps of biological photosynthesis. Further spectroscopic experiments on these topics are planned, as are electrochemical studies by cyclic voltammetry which needs further elucidation as well.

Experimental Melting points: Biichi SMP 20 and 512; Bock-Monoskop M,

(m.p. >240 "C); melting points are uncorrected. - UVNis-absorption spectra: Cary 2300 (Varian). - Emission spectra: Fluorolog F112XE, Spex. - IR: Beckman IR-4240 (KBr), Perkin-Elmer FT- IR 1760X (KBr). - MS: Du Pont CEC 21-492; Finnigan MAT 212 (usually only peaks with ZreI > 15% are listed). - 'H NMR:

Bruker Physik HX 360, AM 500 (internal reference tetramethylsil- ane). - Microanalysis: Elemental Analyser 1106 Carlo Erba. - Analytical TLC: DC Micro Cards Polygram SIL G/UV254r Mach- erey-Nagel. - CC: Silica gel SiliTech 63-200 pm, ICN Biomed- icals. - Flash chromatography: Silica gel 60, 35-70 pm, Amicon. - MPLC: Gilson (column h = 38 cm, d = 2.8 cm); flow rate 30 ml . min-'; detection at h = 254 nm; silica gel 60, 20-45 pm.

1,3-Bis(3-bromopropyl)-2,5-dimethoxybenzene (6): a) To 127 g (500 mmol) of 1,3-bis(3-hydroxypropyl)-2,5-dimethoxybenzene (5)c7' in 600 ml of toluene, a solution of 33.8 ml (368 mmol) of phosphorus tribromide in 50 ml of toluene was added dropwise without increasing the internal temperature >50 "C. After 5 h stirring at 55 to 60 "C, the solution was poured off from the phospho- rous acid that had formed. The cooled solution was washed wlth 100 ml each of ice water, saturated sodium hydrogen carbonate solution and, again, water. This solution was dried over sodium sulfate and chromatographed on silica with toluene (Rf = 0.59) to yield 6 (b.p. 1 1O0C/0.O05 mbar; 80 g, 40% yie1d)m. - b) To a mixture of 20.0 g (78.7 mmol) of 5t71 and 65.3 g (197 mmol) of tetrabromomethane in 300 ml of dichloromethane, 61.9 g (236 mmol) of triphenylphosphane was added in small portions. After the reaction mixture was stirred for 12 h at room temp., the solvent was evaporated in vacuo and the residue chromatographed on silica with toluene yielding 12.5 g (42%) of 6 as a colourless

3-(3- (3-Bromopropyl) -2,S-dimethoxyphenyl]-propyl-triphen- ylphosphonizim Bromide (7): 5.00 g (13.2 mmol) of6 in methylcyclohexane and 2.33 g (8.8 mmol) of triphenylphosphane were heated under reflux for 140 h. After the reaction mixture was cooled, the solvent was removed in vacuo, and the residue was triturated with diethyl ether to yield a crystalline product (5.65 g, 72% referred to triphenylphosphane). Further purification of the product to yield DC-pure 7 was possible only with heavy losses: 84.7 mg (1.5%) 7, m.p. 61.5-62°C. - 'H NMR (360 MHz, CDCI3, 303 K): 6 = 1.99 (mc,2H),2.12("quint",J=7.5Hz,2H),2.70(t,J=7.5Hz,2H), 3.03 (t, J = 7.3 Hz, 2H), 3.39 (t, J = 6.7 Hz, 2H), 3.63 (s, 3H), 3.76(~,3H),3.94(mc,2H),6.57(d,J=3.lHz, lH) ,6 .73(d ,J= 3.1 Hz, lH), 7.64-7.82 (m, 15H). - C32H35Br702P (642.41): calcd. C 59.83, H 5.49, Br 24.88; found C 59.88, H 5.45, Br 24.81.

1 -(3-Acetoxypropyl) -3-(3-hydroxypropyl)-2,5-dimethoxybenzene (8): To a solution of 75.0 g (295 mmol) of 5 in 1 1 of dry tetrahydrofuran at -4O"C, 202 ml (323 mmol) of a 1.6 M n-butylllithium solution in n-hexane was added with stirring. After the reaction mixture was stirred for 30 min, 22.6 ml (310 mmol) of acetyl chloride was added carefully with continuous stirring and cooling. After a further 15 min, the reaction mixture was slowly warmed to room temp. When the solvent was evaporated in vacuo, an oily residue was left which was diluted with 500 ml of dichloromethane. The precipitated lithium chloride was filtered off; the filtrate was distilled in vacuo and chromatographed in portions from ethyl acet- atelpetroleum ether 40" (3:l) on silica. As the first fraction, 23.9 g (24% yield) of 1,3-bis(3-acetoxypropyl)-2,5-dimethoxybenzene (9) (Rf = 60) was obtained. The main product, 40.1 g (46%) of 8 with Rf = 0.37, was isolated as a light-yellow oil of b.p. 162°C (0.01 bar). - MS: mlz (%): 296 (100, M+). 177 (22), 175 (24) and others. - 'H NMR (360 MHz, CDC13, 303 K): 6 = 1.85 ("quint", J = 6.7

(br.s, l H ) , 2 . 6 8 ( t , J = 7 . 7 H z , 2 H ) , 2 . 7 2 ( t , J = 7 . 3 H z , 2 H ) , 3 . 5 7 (t, J = 6.0 Hz, 2H), 3.71 (s, 3H), 3.75 (s, 3H), 4.11 (t, J = 6.5 Hz,

CI6Hz4O5 (296.36): calcd. C 64.84, H 8.16; found C 64.94, H 7.96.

3- (3-Acetoxypropyl)-I - (3-bromopropyl)-2,5-dimethoxybenzene (10): To a solution of 40.0 g (135 mmol) of 8 and 56.0 g (169 mmol)

Hz, 2H), 1.96 (tt, J1 = 6.5 Hz, J2 = 7.7 Hz, 2H), 2.06 (s, 3H), 2.27

2H), 6.57 (d, J 3.1 Hz, lH), 6.59 (d, J 3.0 Hz, 1H). -

Liebigs Ann. 1995, 1827-1836

1832 H. A. Staab, J. Kanellakopulos, I? Kirsch, C. Krieger

of tetrabromomethane in 300 ml of dichloromethane, 53.0 g (202 mmol) of triphenylphosphane was added in portions with stirring at 0°C. After the reaction mixture was stirred for 36 h at room temp., the solvent was evaporated under reduced pressure (bath temperature below 25°C). On addition of 400 ml of diethyl ether, the triphenylphosphane oxide that formed precipitated and was filtered off. The solvent was evaporated from the filtrate, and the residue was chromatographed in portions on silica gel from ethyl acetateln-hexane (1 :3). With Rr = 0.33 product 10 (b.p. 152°C 0.015 mbar) was obtained as a colourless oil (31.6-35.6 g; 65-73‘Yo yield). - MS: m/z (‘YO): 358 (100, M’), 283 (23 ) , 162 (24). - ‘ H

2.15 (mc, 2H), 2.67 (t, J = 7.8 Hz, 2H), 2.75 (t, J = 7.5 Hz, 2H), 3 .42( t , J= 6 .6Hz,2H) ,3 .69(~ ,3H) ,3 .74(~ ,3H) ,4 .10( t , J= 6.5 Hz, 2H), 6.58 (s, 2H). - C16H2SBr04 (359.26): calcd. C 53.49, H 6.45, Br 22.24; found C 53.64, H 6.70, Br 22.49.

1 - (3-Acetoxypropyl) -2,5-dimethoxy-3- (3-triphenylphosphon- iopropy1)benzene Bromide (13): To a solution of 32.0 g (89.1 mmolj of 10 in 350 ml of dry acetonitrile, 70.1 g (267 mmol) of triphenylphosphane was added, and the mixture under exclusion of moisture was heated to reflux for 30 h. After the solvent was evaporated, the oily residue was triturated with diethyl ether until the product formed a crystalline white powder which was removed by vacuum filtration, washed with diethyl ether and dried: 54.2 g (98%) of 13 (m.p. 106-107°C). - ‘H NMR (500 MHz, CDCI,, 303 K): 6 = 1.90 (mc, 2H), 1.99 (mc, 2H), 2.04 (s, 3H), 2.64 (t, J = 7.8 Hz, 2H), 3.01 (t, J = 7.3 Hz, 2H), 3.63 (s, 3H), 3.76 (s, 3H), 3.78 (mc, 2H), 4.07 (t, J = 6.6 Hz, 2H), 6.58 (d, J = 3.1 Hz, lH), 6.69 (d, J = 3.1 Hz, IH), 7.66-7.79 (m, 15H). - C34H38Br04P (621.55): calcd. C 65.70, H 6.16, Br 12.86; found C 65.69, H 6.22. Br 12.87.

NMR (360 MHz, CDCI,, 303 K): 6 = 1.95 (mc, 2H), 2.03 (s, 3H),

1 - (3- Bromopropylj-3- (3-hvdroxypropyl) -2,j-dimethoxybenzene (11): To 35.0 g (1 10 mmol) of 10 in 200 ml of tetrahydrofuran, a solution of 31 g of potassium hydroxide in 80 in1 of water was added, and the mixture was stirred for 16 h at room temp. [DC- monitoring, ethyl acetateln-hexane (1 : 3): &(starting material) =

0.33, Rdproduct) = 0.081. Tetrahydrofuran was removed with a rotary evaporator; the remaining aqueous phase was extracted three times with 30 ml each of dichloromethane, the extracts dried with sodium sulfate, filtrated and the solvent evaporated (an analytically pure product was obtained by bulk-tube distillation). 34.2 g (98%) of 11, b.p. 152”C/0.015 mbar. - ‘H NMR (360 MHz, CDC13, 303 K): 6 = 1.85 (“quint”, 2H), 2.16 (“quint”, 2H), 2.45 (br. s, 1 H), 2.71 (t, J = 7.5 Hz, 2H), 2.76 (t, J = 7.5 Hz, 2H), 3.43 (t, J = 6.6 Hz, 2H), 3.58 (t, J = 6.1 Hz, 2H), 3.71 (s, 3H), 3.75 (s, 3H), 6.59 (d , J=3 .2Hz, lH) ,6 .60(d ,J=3 .4 , lH) . -Cl4HZ1BrO3(3I7.22): calcd. C 53.01, H 6.67, Br 25.19; found C 53.18, H 6.81, Br 25.12.

1 - (3-Hydroxypropyl) -2,5-dimethoxy-3-(3-triphenylphosphon- iopropy1)benzene Bromide (12): Preparation from 30.0 g (94.6 mmol) of 11 and 74.5 g (284 mmolj of triphenylphosphane following the procedure mentioned for 13 (see above). Yield 53.2 g (97%0), m.p. 143-145°C. - ’H NMR (500 MHz, CDC13, 303 K): 6 = 1.83 (mc, 2H), 1.99 (mc, 2H), 2.67 (t, J = 7.5 Hz, 2H), 2.70 (s, lH), 2.99 (t. J = 7.1 Hz, 2H), 3.53 (t, J = 6.1 Hz, 2H), 3.63 (s, 3H), 3.73 (m, 2H), 3.75 (s, 3H), 6.60 (d, J = 3.0 Hz, lH), 6.68 (d, J = 3.0 Hz, 1 H), 7.79-7.66 (m, 15H). - C32H36Br03P (579.51): calcd. C 66.32, H 6.26, Br 13.79; found C 66.57, H 6.36, Br 13.89.

(ZIE)-4-[3-(3-Acetoxypropyl) -2,5-dimethoxyphenyl]-l-(3- acetylaniino-2-nitroph~nyl)-l-butene (14): To a solution of 32.0 g (51.5 mmol) of the phosphonium salt 13 in 300 ml of dry tetrahydrofuran at -4O”C, 56.6 ml of a 1 M solution of sodium hexamethyldisilazane in tetrahydrofuran was added slowly. After the re-

action mixture was stirred for 40 min at this temperature, a solution of 11.3 g (54.5 mmol) of 3-acetylamino-2-nitrobenzaldehyde[’] in 120 ml of tetrahydrofuran was added. After stirring for another 3 h at -3O”C, the reaction mixture then was left overnight at room temp. The reaction mixture was hydrolyzed by the addition of 29 ml of 2 N aqueous hydrochloric acid, the tetrahydrofuran was distilled off, and the aqueous phase was extracted with diethyl ether. After the combined ethereal extracts were dried with sodium sulfate, the solvent was evaporated under reduced pressure, and the oily residue was flash-chromatographed on silica from acetonelpetroleum ether 40 (2:3): 12.1 g (total yield 47%) of the ZIE mixture was obtained which according to ’H NMR had a Z:E ratio of 81:19. - MS (data for 2 isomer): mlz (%): 470 (80, M+), 251 (39, 191 (loo), 189 (20), 179 (80), 177 (46j, 175 (26), 165 (49), 163 (21), 160 (23), 151 (24), 147 (26), 146 (20), 145 (20), 135 (41), 131 (26), 106 (17), 105 (19), 91 (23). - ‘H NMR (500 MHz, CDC13, 303 K, data for 2 isomer): 6 = 1.94 (m, 2Hj, 2.06 (s, 3H), 2.19 (s, 3H), 2.36 (m, 2H), 2.64-2.69 (m, 4H), 3.63 (s, 3H), 3.77 (s, 3H), 4.10 (t, J = 6.5 Hz, 2 H), 5.87 (dt, J = 1 1.5 and 7.5 Hz, 1 H), 6.45 (d, J = 1 1.5 Hz, lH), 6.49 (d, J = 3.0 Hz, lH), 6.56 (d, J = 3.0 Hz, lH), 6.94 (d,J=7.6Hz,1H),7.42(“t”,J=8.0Hz,1H),8.18(d,J=8.1 Hz, 1 H), 8.50 (br. s, 1 H). - C25H30N207 (470.52): calcd. C 63.82, H 6.43, N 5.95; found C 63.77, H 6.19, N 6.04.

(Z1E)-I- (3-Acetylamino-2-nitrophenyl) -4-[3- (3-hydroxypropyl)- 2,5-dimethoxyphenyl]-l-butene (15): a) Hydrolysis of 14: To a solution of 7.0 g (14.9 mmol) of 14 (ZIE mixture) in 100 ml of water- free methanol at O T , 100 ml of methanol saturated with ammonia was added. After the reaction mixture was stirred overnight, the solvent was removed in vacuo, and the remaining yellow oil was chromatographed on silica from acetone/benzene (1 : I); the fraction of Rf = 0.30 (5.67 g, 89”h yield) agreed in all spectroscopic data with the product prepared by procedure b. - b) Wittig reaction of 12: At -78”C, 5.00 g (8.63 mmol) of 12 and 3.60 g (18.98 mmol) of sodium hexamethyldisilazane were suspended in 75 ml of dry tetrahydrofuran. The suspension was stirred for 40 niin at -40°C; then a solution of 1.97 g (9.49 mmol) 3-acetylamino-2-nitrobenzaldehyde in 25 ml of tetrahydrofuran was added slowly. After 3 h at room temp. the reaction was quenched with concentrated aqueous ammonium chloride solution. The organic phase was separated, the aqueous solution was diluted with water to a volume 30 ml and extracted three times with 20 ml each of diethyl ether. The combined organic layers were dried over sodium sulfate; the solvents were removed at reduced pressure to yield an oil which was purified by chromatography on silica from ethyl acetate (Rf = 0.42): 1.15 g (31%) 15 as a yellow oil. - MS: mlz (%): 428 (67, M+), 209 (29),

K, Z isomer): 6 = 1.82 (m, 2H), 2.21 (s, 3H), 2.37 (m, 2H), 2.65-7.72 (m, 4H), 3.55 (t, J = 6.1 Hz, 2Hj, 3.64 (s, 3H), 3.72 (s, 3Hj, 5.87 (dt, J = 11.5 and 7.5 Hz, lH), 6.43 (d, J = 11.5, lH), 6.49 (d, J = 3.0 Hz, IH), 6.57 (d, J = 3.0 Hz, lH), 6.94 (d, J = 7.6 Hz, 1 H), 7.40 (“t”, J = 8.0 Hz, 1 H), 8.04 (d, J = 8.0 Hz, 1 H), 8.58 (br. s, 1H). - C23H28N206: calcd. C 64.47, H 6.59, N 6.54; found C 64.45, H 6.95, N 6.20. - MS-HR: calcd. 428.1947, found 428.1957.

(Z1E)-I -(3-AcetyIumino-2-ni~rop~enyf-4-[3-(3-bron?opropyl)- 2,5-dinzethoxyphenyl]-l-butene (16): To a solution of 6.00 g (14.0 mmol) of 15 and 5.80 g (17.5 mmol) of tetrabromomethane in 300 ml of dichloromethane at 0°C and under exclusion of moisture, 5.50 g (21.0 mmol) of triphenylphosphane was added in small portions. After 30 h of stirring at room temp., the solvent was distilled off in vacuo leaving an oily residue to which 100 ml of diethyl ether was added. The precipitate of triphenylphosphane oxide was filtered off, and the solvent of the filtrate was evaporated. The resi-

179 (26), 165 (loo), 135 (30). - ‘H NMR (500 MHz, CDC13, 303

Liebigs Ann. 1995, 1827-1836

r - n Interactions of Flavins, 5 1833

due was chromatographed on silica (ethyl acetatehgroin 40, 1 : 1): Z isomer Rf = 0.38; E isomer Rf = 0.35), honey-yellow oil; yield variable (best result 5.6 g, 80%). - MS: rnlz (YO): 490 (62, M+), 271 (53), 192 (62), 191 (loo), 187 (33), 177 (61), 165 (24), 161 (19),

Z isomer): 6 = 2.15 (mc, 2H), 2.20 (s, 3H), 2.38 (“quint”, J = 7.6 Hz, 2H), 2.68 (t, J = 7.7 Hz, 2H), 2.74 (t, J = 7.5 Hz, 2H), 3.42 (t, J = 6.6 Hz, 2H), 3.64 (s, 3H), 3.73 (s, 3H), 5.87 (dt, J = 11.4 and 7.5 Hz, 1 H), 6.46 (d, J = 11.4 Hz, 1 H), 6.49 (d, J = 3.1 Hz, lH), 6.58 (d, J = 3.1 Hz, lH), 6.94 (d, J = 7.6 Hz, lH), 7.43 (“t”, J = 8.0 Hz, 1 H), 8.19 (d, J = 8.2 Hz, 1 H), 8.46 (br. s, 1 H). - CZ3Hz7BrN205 (491.38): calcd. C 56.22, H 5.54, N 5.70, Br 16.26; found C 56.11, H 5.29, N 5.56, Br 16.07.

( 1 3 2 ) -1 - Acetyl- 7,1O-dimethoxy-20-nitro-l -aza-[ l . 4]metacyclophan-13-ene (17): A solution of 6.20 g (12.6 mmol) of 16 (ZIE = 9:l) in 1.1 1 of toluene was added over 36 h, by using a precision dilution to a boiling solution of 430 ml of toluene, 210 ml of 2.5 percent sodium hydroxide solution and 2.70 g (8.4 mmol) of tetra-n-butylammonium bromide. The reaction mixture was stirred under reflux for 6 h more until no further starting material was to be observed (DC: silica, dichloromethaneldiethyl ether, 9:l). The organic phase was separated, and the aqueous layer was extracted with 100 ml of toluene. The combined organic phases were shaken with concentrated aqueous ammonium chloride solution and water, dried over sodium sulfate and distilled in a rotary evaporator. To the brown oil thus obtained, 40 ml of diethyl ether was added whereupon fine crystals precipitated. After 12 h stand- ing at - 18 “C, this precipitate was separated by filtration and chromatographed on silica (Rf = 0.35) from dichloromethaneldiethyl ether (9:l) to produce 17 as a pale-yellow crystal powder of m.p. 21 1.5 “C. The yield was varying (best result 2.8 g corresponding to 54%). - MS: mlz (YO): 410 (100, M+), 165 (16). - ‘H NMR (500 MHz, CDCl,, 303 K): 6 = 1.25 (mc, IH), 1.75 (mc, IH), 1.97 (dt, J = 2.5 and 13.4 Hz, lH), 2.02 (s, 3H), 2.21 (m, IH), 2.27 (mc, lH), 2.37 (“t”, J = 12.1 Hz, IH), 2.83 (mc, lH), 2.90 (mc, IH), 2.97 (m, lH), 3.47 (s, 3H), 3.78 (s, 3H), 4.57 (mc, lH), 5.85 (dt, J= l1 .4and4.0Hz, lH) ,6 .13(d ,J= 11.4Hz,IH),6.31(d,J= 3.0 Hz, 1 H), 6.51 (d, J = 3.0 Hz, 1 H), 7.09 (d, J = 7.7 Hz, 1 H),

C&2&05 (410.47): calcd. C 67.30, H 6.38, N 6.82; found C 67.15, H 6.61, N 6.74.

(13Z)-7,lO-Dimethoxy-20-nitro-l -aza(4. 4]rnetacyclophan-l3-ene (18): After addition of 40 g of potassium hydroxide and 28 ml of water to a solution of 2.60 g (7.06 mmol) of 17 in 175 ml of ethanol, the solution was heated under argon in a 80°C bath. After the cleavage of the N-acetyl group was complete (DC-monitoring; silica, dichloromethaneldiethyl ether, 9: l), 80 ml of water were added to the solution and the reaction mixture was extracted with diethyl ether. The combined organic phases were washed with concentrated aqueous solution of ammonium chloride and water, dried over sodium sulfate, and the solvents were distilled off in vacuo. The red reside was chromatographed from dichloromethaneldiethyl ether on silica and crystallized from n-hexane; coral-red crystals, m.p. 144-145°C; yield 2.31 g (89%). - MS: rnlz (%): 368 (100, M+), 322 (22), 165 (20), 161 (25), 147 (29), 145 (35), 144 (23), 135 (17), 91 (16). - ‘H NMR (500 MHz, CDCI,, 303 K): 6 = 1.84 (mc, lH), 2.02-2.10 (m, 2H), 2.37-2.50 (ni, 3H), 2.79 (m, IH), 2.97 (m, 1 H), 3.44 (s, 3H), 3.46 (m, IH), 3.79 (s, 3H), 3.87 (m, 1 H), 5.64 (dt, J = 11.2 and 5.4 Hz, 1 H), 6.26 (d, J = 7.0 Hz, 1 H), 6.29 (d, J = 3.1 Hz, 1 H), 6.53 (d, J = 3.1 Hz, IH), 6.78 (d, J = 8.5 Hz, lH), 6.96 (d, J = 11.2, IH), 7.17 (dd, J = 7.2 and 8.5 Hz, IH), 7.69 (br. s, 1 H). - C21H24N204 (368.43): calcd. C 68.46, H 6.57, N 7.60; found C 68.75, H 6.78, N 7.50.

145 (23), 135 (28), 91 (16). - ‘H NMR (500 MHz, CDC13, 303 K

7.20 (d, J = 7.7 Hz, lH), 7.45 (“t”, J = 7.7 Hz, 1H). -

16, I9-Dimethoxy[4]metacyclo[3] (105) isoalloxazinophane (1): 600 mg (1.63 mmol) of 18 in 60 ml of acetic acid in the presence of palladium (10% on charcoal) at 20°C under normal pressure was hydrogenated and was filtered immediately with a reversible frit under argon into a solution of 667 mg (4.17 mmol) of alloxane monohydrate and 960 mg (15.52 mmol) of boric acid in 40 ml of acetic acid. The reaction mixture was stirred for 8 h under argon with the exclusion of light at 55°C. Then the acetic acid was distilled off by rotary evaporator, and from the remaining red oil, the boric ester was removed by concentrating the reaction mixture three times with absolute methanol. To the remaining solid, 30 ml of diethyl ether was added; after stirring overnight the residue was filtered from trichloromethanelmethanol (10: 1) through silica. Evaporation of the solvents left a yellow microcrystalline powder (654 mg, 90% yield) which consisted of 1 in about 95% purity and was used without further purification for the following reactions. For the characterization, the product was recrystallized from trichloromethaneldiethyl ether: m.p. 330 “C (Bock-Monoskop). - MS: mlz (%): 446 (100, M+), 269 (23), 255 (30), 91 (15), 77 (15). - ’H NMR (500 MHz, CDC13, 303 K): 6 = 1.00 (br. m, 1 H), 1.60 (m, IH), 1.83-1.92 (m, 3H), 2.40-2.62 (m. 4H), 2.68 (m, IH), 2.90 (m, lH), 3.23 (s, 3H), 3.65 (s, 3H), 3.82 (dt, J = 12.9 and 4.3 Hz, 1 H), 4.37 (mc, 1 H), 5.73 (mc, 1 H), 6.20 (d, J = 3.1 Hz, 1 H), 6.42 (d, J = 3.1, lH), 7.31 (d, J = 7.1 Hz, IH), 7.56 (d, J = 8.6 Hz, lH), 7.68 (dd, J = 7.4 and 8.6 Hz, IH), 8.30 (s, IH). - C25H26N404 (446.49): calcd. C 67.24, H 5.87, N 12.55; found C 67.46, H 6.01, N 12.67.

16,19-Dimethoxy-3-methyl[4]metacyclo[3]; 10,6) isoalloxazinophane (2): To 200 mg (0.448 mmol) of 1 and 620 mg (4.48 mmol) of potassium carbonate in 20 ml of dimethylformamide, 280 pl (4.48 mmol) of iodomethane was added, and the reaction mixture was stirred for 6 h at room temp. (DC-monitoring). The solvent was distilled off in vacuo, the residue was dissolved in dichloromethane and separated from insoluble products by filtration. The filtrate was washed twice with 10 ml each of water and dried over sodium sulfate. When the solvent was evaporated, an orange-yellow substance was obtained which after filtration through silica with trichloromethanelmethanol (10: I), was determined to be pure according to DC: 192 mg (93%) of orange rhombs of m.p. 262°C (dec., Bock-Monoskop). For the solution of the crystal structure of 2 by X-ray diffraction see above. - MS: mlz (YO): 460 (100, M+),

0.97(br.m, lH),1.59(mc, IH), 1.78-1.92(m,3H),2.41 (m, lH), 2.47-2.57 (m, 3H), 2.72 (m, lH), 2.92 (m. lH), 3.23 (s, 3H), 3.52 (s, 3H), 3.59 (s, 3H), 3.82 (“dt”, J = 10.4 and 3.2 Hz, IH), 4.34 (mc, lH), 5.73 (mc, lH), 6.17 (d, J = 3.1 Hz, lH), 6.35 (d, J = 3 . 1 H ~ , l H ) , 7 . 2 7 ( d , J = 7 . 3 H z , l H ) , 7 . 5 3 ( d , J = 8 . 6 H z , l H ) , 7.65 (dd, J - 7.2 and 8.6 Hz, 1 H). - C26H28N404 (460.53): calcd. C 67.81, H 6.13, N 12.17; found C 67.81, H 6.09, N 12.11.

16-Hydroxy-l9-methoxy[4]metacyclo[3] ( 1 0,6) isoalloxazinophane (20): 250 mg (0.56 mmol) of 1 was added under exclusion of moisture at -78°C to 7.5 ml (7.5 mmol) of a 1 M solution of boron tribromide in dichloromethane. The reaction mixture was stirred for 5 h at this temperature and then a further 12 h at room temp. Under ice-cooling aqueous sodium hydrogencarbonate was added carefully for the hydrolysis. The solvents were removed by a rotary evaporator, and the orange-brown residue was filtered through silica from trichloromethanelmethanol (10: 1; Rf = 0.42) to remove the inorganic components. After the solvents were evaporated, the red residue was washed with diethyl ether and filtered to yield 189 mg (78%) of the mono-demethylated analytically pure product 20 (m.p. 267-269”C, Bock-Monoskop). There was no in- dication of an additional cleavage of the sterically more strongly

283 (17), 269 (21). - ‘H NMR (500 MHz, CDC13, 303 K): 6 =

Liebigs Ann. 1995, 1827-1836


hindered 19-methoxy group. - MS: m/z (%): 432 (100, M+), 417 (23), 269 (16), 253 (17). - 'H NMR (500 MHz, [D,]DMSO, 303 K):F=0.88(m,lH),1.45(m,lH), 1.63-1.71(m,2H),2.28-2.55 (m, 6H; partial coincidence by DMSO signal), 2.78 (m, 1 H), 3.13 (~,3H),3.51("d",J=12.2Hz,1H),4.65(m,1H),5.19(m,1H), 6 . 0 1 ( d , J = 3 . 0 H z , l H ) , 6 . 1 1 ( d , J = 3 . 0 H z , 1 H ) , 7 . 2 7 ( d , J = 7.1 Hz, 1 H), 7.68 ("t", J = 7.9 Hz, 1 H), 7.92 (d, J = 8.5 Hz, 1 H), 8.68 (s, 1 H), 11.16 (s, 1 H). - C24H24N404 (432.48): calcd. C 66.65, H 5.59, N 12.95; found C 66.43, H 5.33, N 12.89.

303 K): 6 = 2.34 (s, 3H), 2.40 (s, 3H), 8.07 (s, lH) , 8.50 (s, lH), 11.35 (br. s, 1 H). - CIOH9F3N2O3 (262.29): calcd. C 45.81, H 3.46, N 10.68; found C 45.87, H 3.49, N 10.59. - c) N-[3-(7,5- Dimethoxyphenyl)propyl]-4,5-dimethyl-2-nitro-aniline: To a re- fluxing suspension of 1 .O g (3.8 1 mmol) of 4,5-dimethyl-2-nitro-N- trifluoroacetyl-aniline and 1.6 g (1 1.6 mmol) of potassium carbonate in 50 ml of dry DMF, 20 g (7.72 mmol) of 1-(3-bromopropyl)- 2,s-dimethoxybenzene was added in portions. After 3 h the solvent was evaporated, the residue was stirred with 70 ml of methanol at 20°C to split off the trifluoroacetyl group, 300 ml of water were

of dichloromethane. The extracts, dried over anhydrous magnesium

1 6 ~ H ~ d r o x ~ ~ 1 9 ~ m e t ~ z o ~ ~ y - 3 ~ m e t h ~ 1 ~ 4 ~ m e t ~ c ~ c 1 o ~ 3 ~ ( 1 0 ~ 6 ) i s o a 1 1 ~ added, and the mixture was extracted four times with 50 ml each oxuzinophane (21): To 20'o mg (43'4 pmol) Of at -300c' 0'43 m1 (430 w o l ) of 1 M solution of boron tribromide in dichloromethane sulfate, were evaporated to dryness and chromatographed on silica was added' After stirring at -300c, the reaction mixture was gel from cyc]ohexane/ethy] acetate (2:]), The fraction ofRf 0.59 warmed to room temp'' with pH buffer and extracted after crystallization from methylcyclohexane yielded 1 .O g (760/0) of with dichloromethane. Separation of the organic phase, drying Over the wanted product: orange m.p. 85-860C, - MS: mlz = 'Odium "Ifate and evaporation Of the in a rotary evapor- 344 (100, M+), 179 (37), 178 (27), 164 (28), 163 (23), 162 (61), 161

(24), 152 (23), 151 (15), 147 (42), 135 (25), 121 (40), 120 (IY), 119 (23), 107 (21), 106 (28), 105 (16), 91 (42), 79 (33), 78 (17), 77 (39). - 'H NMR (500 MHz, CD2C12, 303 K): 6 = 1.95-1.97 (mc, 2H),

ator yielded a yellow powder which was chromatographed on silica from trichloromethane/methanol (lo:') from which (with Rf iz

0.50) 14 mg (72%) of 21, the monodemethylated 2, was obtained (m.p. l6OoC, dec.). Due the amounts not be purified (500 MHz, [D6iDMS0, 303 K): 1.63-1.72 (m> 3H)3 2.27-2.36 (m> 2H), 2.51-2.37 (m> 3H)9 2.77

21 2.15 (', 3H), 2.23 (s, 3H), 2.70 (t, J = 7.4 Hz, 2H), 3.28 (t, J = 6.6 Hz, 2H), 3.71 (s, 3H), 3.76 (s, 3H), 6.60 (s, 1 H), 6.67-6.71 (m, ' 7 3 , 6.78 (d, J = 8.6 Hz, 1 H), 7.87 (s, 1 H), 8.00 (br, s, 1 H). - C19H24N204 (344.41): calcd, C 66.26, H 7.02, N 8.13; found C

from the starting 2. - I H NMR = 0.83 (mc, H), 1.44 (mc3

(m, 1H). 3.13 (S, 3H), 3.29 (% 3H), 3.53 (m, 1H), 4.64 (m, IH), 5.28 (m, 1 H), 5.99 (d, J = 2.9 Hz, 1 H), 6.02 (d, J = 2.9 Hz, 1 H), 7.30 (d, J = 7.1 Hz, 1 H), 7.71 (dd, J = 7.4 and 8.6 Hz, 1 H), 7.91 (d, J = 8.7 Hz, 1 H), 8.59 (s, I H). - C25H26N404 (MS-HR): calcd. 446.1954, found 446.1934.

66.27, H 7.17, N 7.95.

d) 10-[3-(2,5-DimethoxYPhenYl)PrOPYll-7,8-dimethYlisoa~~oxazine (23): 5.0 g (14.52 mmol) of the compound described above in 250 ml of acetic acid was hydrogenated (SO0 mg of 5% palladium on charcoal) until the hydrogen uptake had ceased. The diamine solution was directly filtered under argon into a suspension of 4.76 g (29.73 mmol) of alloxane hydrate and 7.35 g (1 18.9 mmol) of boric acid in 200 ml of acetic acid. The reaction mixture was stirred in the dark at 55°C for 18 h. The crude product obtained by the evaporation of the solvent was adsorbed on 15 g of florid and chronlatographed on silica gel with trich]oromethane/methanol (lo:]). The eluate of Rf = 0.59 was concentrated in vacuo to about 50 ml and treated with 400 ml of diethyl ether. The precipitate was removed with vacuum filtration: 4.35 g (71 "/), bright red crystals, m,p. 260°C (dec.), For analysis, 23 was recrystallized from acetic acid, - MS: (%) = 420 (27, M+), 405 (18), 256 (loo), 91 (21), 77 (19). - IH NMR (500 M H ~ , CDCI~, 303 K): 6 = 2.17-2.19 (mc, 2 ~ ) , 2.38 (s, 3 ~ ) , 2.41 (s, 3H), 2.85 (t, J = 6.84 Hz, 2H), 3.76 (s, 3 ~ ) , 3.78 (s, 3 ~ ) , 4.67 (br. t, 2H), 6.77-6.84 (m, 3H), 6.94 (s, 1 H), 8.01 (s, 1 H), 8.33 (br. s, 1 H). - C23H24N404 [MS-HR ( ~ + ) 1 : calcd. 420.1798, found 420.1732.

16,19-Dihydrox~-3-methyl/4/mPtac~c~o[3/ (10,6/isoalloxaz- inoPhane (22): Encouraged by the results on 21, we attempted the cleavage of the methoxY groups of 2 again at longer reaction times and somewhat higher temperature. To 150 mg (325 pmol) of 2 at -78% 0.43 ml (430 Wol) of a 1 M boron bromide solution in dichloromethane was added. This time the reaction mixture was stirred for 8 d at -25°C. After hydrolysis (see above), all Solvents were distilled off by a rotary evaporator. The inorganic salts were removed by filtration on silica with trichloromethane/methaiiol (10: 1). The fraction with Rf- 0.42 was further purified by dissol- ution of the raw product in portions of 10 ml each of trichloro- methaneimethanol (25:1), separation of the insoluble components and subsequent MPLC (conditions see above): 125 mg (89%) of a dark-red crystalline powder, m.P. 300°C (dec.1. - MS: d z (%I: 432 (100, M+), 269 (561, 266 255 (171, 243 (431, 242 (48). - 'H NMR (500 MHz, [D61DMS03 303 K): 6 = 0.68 (mc, 1f% 1.31 (rnc, lH), 1.58-1.64 (m, 2H), 1.78 (m, lH), 2.06 (m, IH), 2.18 (mc, lH), 2.46-2.61 (m, 3H), 2.77 (m, lH), 3.29 (s, 3H), 3.54 (m, 1 H), 4.52 (mc, IH), 5.45 (mc, IH), 5.91 (d, J = 2.9 Hz, lH), 5.92 (d, = 2.9 Hz, IH), 6.55 (s, 1 7,29 (d, 7.1 Hz, lH), 7,69 ("t", J = 7.3 and 8.5 Hz, lH), 7.85 (d, J i= 8.6 Hz, lH), 8.16 (s, 1 H). - CZ4Hz4N4O4 (MS-HR): calcd. 432.1797, found 432.1775.

10-(3- (2,5-Dihydroxyphenyl)propyl]- 7,8-dimethylisoalloxazine: To a suspension of 700 mg (1.66 mmol) of 23 in 150 ml of dry dichloromethane, 2.41 ml (24.97 mmol) of boron tribromide was added at -70°C. The mixture then was warmed to room temp. and was stirred for 20 h. After hydrolysis by addition of 5 ml of water and evaporation of the volatile components in vacuo, the residue

10-(3- (2,5- Dimetho.uyphenyl)propyl]- 7,8-dimethyl-isoalloxazine (23): a) For the synthesis of 23, one of the components, I-(3- bromopropyl)-2,5-dimethoxybenzene, was prepared in a conventional, yet slightly modified four-step synthesis from 2,s-dimeth- oxycinnamic acid['']. - b) 4,5-Dimethyl-2-nitro-N-tri$uoroacetyl- uniline: 15.84 g (95.3 mmol) of the corresponding aniline and 25 ml (572 mmol) of trifluoroacetic anhydride were heated for 30 min to 50°C. The crude product was chromatographed on silica gel (cyclohexane/ethyl acetate, 1 : 1, Rf = 0.72) and recrystallized from ethanol/water (7:3) to yield 23.6 g (940/), light yellow needles of m.p. 81-82°C. - MS: m/z (Y'n): 262 (49, M+), 216 (IOO), 196 (loo), 118 (17), 104 (23). 91 (22), 77 (23). - 'H NMR (360 MHz, CDC13,

was adsorbed on 15 g of florid and chromatographed on silica gel with trichloromethane/methanol (10: 1). The eluate (Rf = 0.22) was concentrated to approximately 50 ml. The crystalline product was obtained by dropwise addition of diethyl ether and dried at 90"CI 1 mbar yielded 519 mg (80%) of the demethylation product of 23: deep purple crystals, m.p. 245°C (dec.). - MS: m/z (YO): 394 (2, M + 2), 392 (2, M+), 243 (17), 242 (loo), 172 (23), 171 (64), 170 (15), 156 (33, 150 (15), 148 (22), 147 (49, 123 (61), 91 (15), 77 (15). - 'H NMR (500 MHz, [D,]DMSO, 303 K): F = 1.96-1.98 (mc, 2H), 2.39 (s, 3H), 2.43 (s, 3H), 2.66 (t, J = 6.8 Hz, 2H), 4.65 (br. t, 2H),6.45(dd,J=8.8and2.8Hz, l H ) , 6 . 6 2 ( d , J = 8 . 8 H z , IH), 6.64 (d, J = 2.8 Hz, lH) , 7.42 (s, IH), 7.89 (s, IH), 8.57 (s, lH),

Liebigs Ann. 1995, 1827-1836

r-n Interactions of Flavins, 5 1835

8.60 (s, IH), 11.29 (br. s, 1 H). - C21H20N404 . 0.5 H20 (401.42): calcd. C 62.83, H 5.27, N 13.96; found C 62.83, H 5.07, N 13.77.

exclusion of light. The mixture was then diluted with 200 ml of trichloromethane and filtered in the dark through a silica gel frit

10- (3- (I, 4- Benzoquinon-2-yl)propy’opyll-7,8-dimethylisoalloxazii~e (25): A suspension of 100 mg (0.26 mmol) of the preceding hydroquinone analogue of 25 and 289 mg (1.27 mmol) of DDQ in 50 ml of dry methanol was stirred for 3 h at room temp. in the dark. The mixture then was diluted with 200 ml of trichloromethane and immediately filtered through a silica gel frit (h = 5 cm, d = 5 cm). The crude product was eluted with trichloromethanelmethanol (4: I), the solvents were evaporated under reduced pressure, and additional purification was achieved by chromatography on silica gel (h = 20 cm, d = 5 cm; trichloromethanelmethanol, 10: 1). Con- centration of the eluate (Rf = 0.45) to about 20 ml and addition of 300 ml of diethyl ether, caused precipitation of the product which after MPLC (silica gel, same eluent) yielded 70 mg (70%) of 25 as light-sensitive, mustard-yellow microcrystals, m.p. 240°C (dec.). - MS: rnlz (YO) = 392 (1, M + 2), 242 (IOO), 172 (17), 171 (71), 156 (46), 123 (37). - ‘H NMR (500 MHz), [D$methanoUCDCl3, 1:1, 303 K): 6 = 2.11-2.13 (mc, 2H), 2.49 (s, 3H), 2.61 (s, 3H), 2.69 (t, J = 7.7 Hz, 2H), 4.82 (br. t, 2H), 6.71-6.83 (m, 3H), 7.42 (s, I H), 7.89 (s, 1 H). - C21H18N404 (390.40): calcd. C 64.61, H 4.65, N 14.35; found C 64.39, H 4.68, N 14.17.

10- (3-(2,5-Dimethoxyphenyl)propy1]-3,7,8-trimethylisoalloxazine (24): A mixture of 2.00 g (4.76 mmol) of 23, 6.57 g (47.6 mmol) of potassium carbonate, 2.96 rnl (47.6 mmol) of iodomethane and 100 ml of dry DMF was stirred for 3 h at room temp. under argon and with exclusion of light. The volatile components were evaporated under reduced pressure, and the residue was dissolved in 100 ml of dichloromethane. This solution was twice washed with water, dried over anhydrous magnesium sulfate, and the filtrate evaporated to dryness. Chromatography over silica gel (h = 15 cm, d = 5 cm; trichloromethane/methanol, 40: 1) yielded 1.33 g (64%) of 24, with Rf = 0.75, as an orange crystalline powder, m.p. 245°C (dec.). - MS: mlz (YO): 434 (19, M+), 419 (16), 270 (IOO), 213 (18). - ‘H NMR (500 MHz, CDC13, 303 K): 6 =

2.16-2.18(mc,2H),2.37(s,3H),2.40(s,3H),2.84(t,J= 7.0Hz, 2H), 3.53 (s, 3H), 3.74 (s, 3H), 3.77 (s, 3H), 4.67 (br. t , 2H), 6.75-6.83 (m, 3H), 6.93 (s, IH), 8.02 (s, 1H). - C24H26N404 (443.50): calcd. C 65.00, H 6.14, N 12.63; found C 64.77, H 6.06, N 12.54.

10- (3- (2,5-Dihydroxyphenyl)propyl]-3,7,8-trimethylisoalloxazine: A suspension of 460 mg (1.06 mmol) of 24 in 150 ml of dry dichloromethane was treated at -70°C with 1.53 ml(15.9 mmol) of boron tribromide. The reaction mixture was stirred for 22 h at room temp. in the dark. After hydrolysis by careful addition of 5 ml of water, the volatile components were evaporated in vacuo. The residue was adsorbed on 15 g of florisil and chromatographed on silica gel (trichloromethane/methanol, 10: 1). The fraction with Rf = 0.20 yielded 410 mg (95%) of a dark-red microcrystalline powder, m.p. 235°C (dec.). - MS: rnlz (YO): 406 (14, M+), 270 (50), 257 (33), 256 (IOO), 242 (27), 241 (22), 227 (19), 213 (16), 199 (53), 185 (19), 172 (17), 171 (49), 170 (29), 158 (28), 157 (26), 156 (56), 150 (54), 148 (19), 147 (30), 123 (49), 122 (18), 166 (18), 110 (23), 103 (20), 94 (27), 91 (21), 82 (32), 81 (20), 80 (24), 79 (22), 77 (33). - ‘H NMR (500 MHz, [D6]DMS0, 303 K): 6 = 1.99-2.01 (mc, 2H), 2.39 (s, 3H), 2.43 (s, 3H), 2.66 (“t”, J = 7.2 Hz, 2H), 3.27 (s, 3H), 4.58 (br. t, 2H), 6.44-6.47 (in, lH), 6.60-6.80 (m, 2H), 7.44 (s, IH), 7.93 (s, IH), 8.58 (s, IH), 8.62 (s, 1H).

10-[3- (I,4-Benzoquinon-2-yl)propyl]-3,7,8-trimethylisoalloxazine (26): A suspension of 100 mg (0.25 mmol) of the aforementioned 1,4-dihydroxyphenyl compound and 279 mg (1.23 mmol) of DDQ in 50 ml of dry methanol was stirred for 3 h at room temp. with

(h = 7 cm, d = 5 cm). The crude product was eluted with trichloromethanelmethanol (10: I) with Rf = 0.68. After the eluate was concentrated by evaporation to about 20 ml, 200 ml of petrol ether (b.p. 40°C) was added, the precipitated product was separated by vacuum filtration and purified by MPLC (silica gel; trichloromethanelmethanol, 1 O : l ) to yield 30 mg (300/0) of light-sensitive yellow microcrystals of m.p. 200°C (dec.). - MS: rnlz (YO): 406 (2, Mf + 2), 257 (19), 256 (IOO), 227 (20), 199 (54), 171 (54), 170 (27), 156 (63), 147 (26). - ‘H NMR (500 MHz, [D]trichloromethane, 303 K): 6 = 2.09-2.11 (mc, 2H), 2.45 (s, 3H), 2.57 (s, 3H), 2.67 (t, J = 7.6 Hz, 2H), 3.52 (s, 3H), 4.77 (br. t, 2H), 6.66 (d, J = 2.3 Hz, IH), 6.75 (dd, J = 2.3 and 10.1 Hz, lH), 6.78 (d, J = 10.1 Hz, 1 H), 7.38 (s, 1 H), 8.08 (s, 1 H). - C22H20N404 (404.41): calcd. C 65.34, H 4.98, N 13.85; found C 65.31, H 4.96, N 13.80.

~~

“1 Part 4: H. A. Staab, M. F. Zipplies, T. Miiller, M. Storch, C. Krieger, Chem. Be% 1994,127, 1667-1680.

12J With regard to the function of flavins “at the crossroad of biological redox chemistry” see C. Walsh, Acc. Chem. Res. 1980, 13, 148- 155; T. C. Bruice, ibid. 1980,13, 256-262; further references therein and in references 1 and 3.

1’1 See, for example, M. A. SliRin, Charge-Transfer Interactions of Biomolecules, Academic Press London-New York I97 1, Chap- ter 7.

L41 H. A. Staab, P. Kirsch, M. F. Zipplies, A. Weinges, C. Krieger, Chem. Ber. 1994, 127, 1653- 1665; further references therein.

Is] H. A. Staab, G. Voit, J. Weiser, M. Futscher, Chem. Ber. 1992, 125, 2303-2310; C. Krieger, M. Dernbach, G. Voit, T. Carell, H. A. Staab, ibid. 1993, 126, 811-821; H. A. Staab, M. Tercel, R. Fischer, C. Krieger, Angew. Chem. 1994,33, 1531 - 1534; An- gew. Chem. Znt. Ed. Engl. 1994, 33, 1463-1466; H. A. Staab, C. Krieger, C. Anders, A. Riickemann, Chem. Ber. 1994, 127, 23 1-236; further references to preceding papers on time-re- solved spectroscopy of this group of compounds may be found in: H. Heitele, F. Pollinger, T. Haberle, M. E. Michel-Beyerle, H. A. Staab, .I Physic. Chem. 1994, 98, 7402-7410.

L6J Elemental analysis, NMR, MS and other spectroscopic data confirm the structure suggested.

L7l W. R. K. Reibei, Ph. D. Thesis Heidelberg 1982, detailed pro- cedures not yet published; see also w. J. Moran, E. C. Schreiber, E. Engel, D. C. Beln, J. L. Yamins, J. Am. Chem. Soc. 1952, 74, 127. Cf. J. Hooz, S. S. H. Gilani, Can. .I Chem. 1968, 46, 86.

L91 F. Vogtle, Chemiker-Ztg. 1972, 96, 396-403. (“1 Concerning the “phane nomenclature” see: F. Vogtle, P. Neu-

mann, Tetrahedron 1970, 26, 5847-5863; in the present paper for the isoalloxazinophanes a modified version is used in which the usual atom numbering of isoalloxazines is retained in order to more easily make comparisons among the structures (see also references 1 and 4.)

[’I1 Crystallographic data for 2 (C26H28N404, molecular mass 460.54); a = 1888.6(3), b = 1290.7(2), c = 1864.2(3) pm; space group Pna2’ (no. 33 Internat. Tables); Z = 8; Dx = 1.346 g . ~ m - ~ ; 4452 symmetry-independent reflectionb of which 2581 reflections with 1 3 3.00(4 were classified as observed. Struc- ture solution by direct method (MULTAN 82); refinement in- cluded all non-hydrogen atoms with anisotropic, the hydrogen atoms with isotropic temperature factors; R = 0.044. - Further details of the crystal structure investigation are available on re- quest from Fachinformationszentrum Karlsruhe, Gesellschaft fur wissenschaftlich-technische Information m.b.H., D-76344 Eggenstein-Leopoldshafen, on quoting the depository number CSD-59034, the names of authors and the journal citation.

[I2] See, for example, H. A. Staab, V. Schwendemann, Angew Chem. 1978, 90, 805-806.

[I3] W. Syper, K. Kloc, J. Mlochowski, Z. Szula, Synthesis 1979, 521-523; J. S. Swenton, Acc. Chem. Res. 1983, 16, 74-81.

[I4] For heniiacetal formation at the oxidative demethykation of 1,4- dimethoxybenzenes see C. D. Snyder, H. Rapoport, .I Am. Chem. Sue. 1972, 94, 227-231.

Liebigs Ann. 1995, 1827-1836


J. Cason, Organic Reactions, 1948, Vol. IV, 305-361; H. J. Un- [I9] [19a] S. M. Bloom, Polaroid Corgor., US-Patent 3590692, July denvood, W. L. Walsh, Org Synth. Coll. Vol. I1 (1945), 6, 1971; Appl. July 1, 1968.- 1 J. Kanellakopoulos, unpub- 553-554. lished results.

c2O] See also K. Maruyama, T. Otsuki, Chem. Lett. 1983, 847-850. [I6] A. Fischer, G. N. Henderson, Synthesis 1985, 641 -643.

[I8] C. R. H. J. De Jonge, H. M. van Dort, L. Vollbracht, Tetru- "'1 See V. D. Parker, Electrochim. Actu 1973, 18, 519. [95137]

hedron Lett. 1970, 22, 1881-1884.

Liebigs Ann. 1995, 1827-1836

π…π interactions of flavins, 5. Syntheses, structures and physical properties of flavin systems...

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