Recognition properties of flavin analogues with bile acid-based receptors: Role of steric effects in hydrogen bond based molecular recognition

Article abstract of DOI:10.1071/CH07342

The recognition properties of 7,8-dimethyl flavin analogues by bile acid-based receptors that contain 2,6-diaminopyridine and the dioctylamide of 2,6-diaminopyridine in CHCl₃ were determined. The results show that the bile acid-based receptors bind 7,8-dimethyl flavin analogues less effectively as compared to 7,8-unsubstituted flavins reported earlier, which is contrary to the known fact that the association constants increase with increasing electron-donating capacity of the substituents at the 7 and 8 positions of the flavin analogues. CSIRO 2008.

Full text of DOI:10.1071/CH07342

Full Paper
CSIRO PUBLISHING
Aust. J. Chem. 2008, 61, 216–222
www.publish.csiro.au/journals/ajc
Recognition Properties of Flavin Analogues with Bile
Acid-Based Receptors: Role of Steric Effects in
Hydrogen Bond Based Molecular Recognition
Prosenjit Chattopadhyay,A Rekha Nagpal,A and Pramod S. PandeyA,B
ADepartment of Chemistry, Indian Institute ofTechnology Delhi, Hauz Khas, New Delhi-110016, India.
BCorresponding author. Email: pramod@chemistry.iitd.ac.in
The recognition properties of 7,8-dimethyl flavin analogues by bile acid-based receptors that contain 2,6-diaminopyridine
and the dioctylamide of 2,6-diaminopyridine in CHCl3 were determined.The results show that the bile acid-based receptors
bind 7,8-dimethyl flavin analogues less effectively as compared to 7,8-unsubstituted flavins reported earlier, which is
contrary to the known fact that the association constants increase with increasing electron-donating capacity of the
substituents at the 7 and 8 positions of the flavin analogues.
Manuscript received: 26 September 2007.
Final version: 24 January 2008.
Introduction
Flavoenzymesthatcontainacoenzymeriboflavin, oftenasflavin
mononucleotide (FMN) or flavin adeninedinucleotide (FAD),
are a diverse class of biological redox catalysts, and act in cellu-
lar redox metabolism by either single electron or simultaneous
two-electron transfer mechanisms.[1] In the process of catalyz-
ing biological redox reactions, the flavin moiety itself is reduced
uponacceptingelectronsfromasubstrateandreoxidizedbyelec-
tron transfer to another acceptor. The apoprotein is responsible
for substrate selectivity and kinetic and thermodynamic controls
of the electron-transfer process.
O
N
N
H
H
R1
O
N
N
R2
N
N
R2 ϭ Hexyl
H
O
O
N
R1
There has been a considerable argument over the reaction
pathways and mechanisms used by these flavoproteins because
of the complexity of the native enzymatic systems.[2] Synthetic
models have a deciding role to play, as they help us to under-
stand the individual interactions and reaction pathways, which
become fairly difficult with the complex flavoenzyme systems.
To mimic the function of these enzymes by chemical models
for mechanistic studies, much effort has been devoted to the
chemical synthesis of flavin analogues.[3]
2,6-Diaminopyridine derivatives bind flavin analogues by
three hydrogen bonds at C(2)=O, N(3)–H, and C(4)=O posi-
tions in aprotic solvents (Fig. 1). Consequently, there has been
considerable interest to modulate the strength of hydrogen bond
interactions in the complexes of 2,6-diaminopyridine derivatives
and flavin analogues to better understand or to better mimic the
flavoenzyme behaviour.[4]
In a systematic study, Rotello and Cooke have extensively
studied the effect of functional group variations at C7 and C8
positions of artificial flavins on hydrogen bond interactions and
the redox potential of isoalloxazine derivatives in aprotic sol-
ventsusingsyntheticreceptorsbasedon2,6-diaminopyridine.[3i]
These studies highlighted the profound electronic effect on the
hydrogen bonding. The affinity of the flavin unit to bind with a
2,6-diaminopyridine receptor increased with electron-donating
substituents at the 7 and 8 positions of the isoalloxazine ring
R1 ϭ OCHO
Fig. 1. Three point hydrogen bonding between bile acid-based acyclic
receptor and 7,8-dimethyl-N(10)-hexyl flavin.
and decreased with electron-withdrawing substituents. It is also
believed that enzyme-cofactor π-stacking interactions play a
substantial role in the modulation of flavin reactivities by the
apoenzymes. Rotello and coworkers have explored the effect of
π-stacking on flavin redox chemistry. In these studies, they used
a series of receptors to examine the effects of π-stacking on
flavin recognition and established that aromatic stacking inter-
actions between the receptors and their flavin guests effectively
modulate hydrogen bonding and hence, the redox potential.[5]
However, there have been very little studies on the role of steric
effects on the binding properties of flavin with diaminopyri-
dine model systems. Rotello and coworkers studied the binding
behaviour of 7,8-dimethyl-10-isobutyl flavin with various amide
and urea derivatives of 2,6-diaminopyridine. It was observed
that 2,6-N,Nꢀ-di(propionylamino)pyridine bound three times
stronger than did the 2,6-N,Nꢀ-di(isobutanoylamino)pyridine
receptor to the 7,8-dimethyl-10-isobutyl flavin, which sug-
gests that the increase in branching of the acyl substitutents
© CSIRO 2008
10.1071/CH07342
0004-9425/08/030216

Recognition Properties of Flavin Analogues with Bile Acid-Based Receptors
217
O
CH3
H
O
CH3
H
N
(i)
NH2
(ii)
N
NO2
1
2
3
(iii)
R1
H
N
R1
N
N
(iv)
(v)
H
H
H
NO2
NO2
5a–c
NH2
4
6a–c
(vi)
Fig. 2. Thermal ellipsoid view of compound 7c with 30% probability
factor.
R1
R1a ϭ Octyl
R1b ϭ Hexyl
R1c ϭ Butyl
N
N
O
O
N
N
H
7a–c
2.890
2.890
Scheme 1. Reagents and conditions (and yields): (i) (CH3CO)2O,
dichloroethane, triethylamine, reflux, 12 h (98%); (ii) HNO3, H2SO4, glacial
acetic acid, 0–10◦C, 0.5 h (85%); (iii) 10% NaOH, H2O, reflux, 12 h (90%);
(iv) NaH, alkyl bromide, DMF, rt, 12 h (∼70%); (v) Pd/C, MeOH, H2, rt,
4 h (∼95%); (vi) alloxan, H3BO3, glacial acetic acid, 60◦C, 12 h (40–50%).
Fig. 3. X-Ray crystal structure of 7c showing weak hydrogen bonding.
on the amino groups of the 2,6-diaminopyridine receptors
decreases their association constants with 7,8-dimethyl-10-
isobutyl flavin.[4d] The present paper throws light on the influ-
ence of the steroidal features of bile acids as well as flavin
analogues in downplaying the electronic effects with regard to
their hydrogen bonding interactions.
Table 1. Selected bond lengths [Å] and angles [◦] for compounds 7b
and 7c
Bonds
Compound
7b
7c
Bond lengths [Å]
1.363(3)
Results and Discussion
N1–C2
N1–C10A
C2–O1
N3–C2
N3–C4
N5–C4A
N5–C5A
N10–C9A
N10–C10A
1.359(4)
1.314(4)
1.219(4)
1.406(4)
1.355(4)
1.291(4)
1.367(4)
1.385(4)
1.365(4)
Synthesis of 7,8-Dimethyl Flavin Analogues
1.318(3)
1.226(3)
1.398(3)
1.362(3)
1.304(3)
1.369(3)
1.362(3)
1.362(3)
7,8-Dimethyl flavin analogues 7a–c were prepared using the
modified method of Rotello and coworkers[3i] and is outlined in
Scheme 1.The method is, in principle, applicable to the synthesis
of riboflavin analogues that possess a variety of substituents at
the N(10)-position, starting from readily available 3,4-dimethyl
aniline 1. Acetylation of 1 with acetic anhydride followed by
nitration afforded 3 in good yields.[6]
Hydrolysis of 3 gave 4, which on reaction with the corre-
sponding alkyl halide using NaH in DMF gave 5a–c. Reduction
of the nitro group in 5a–c was accomplished with Pd/C in
methanol and the crude N-alkyl-1,2-benzenediamines 6a–c were
used without further purification for the condensation with
alloxan in acetic acid to give 7a–c.[7]
Bond angles [◦]
118.0(2)
C10A–N1–C2
N1–C2–O1
O2–C4–N3
117.8(3)
122.6(3)
123.0(3)
121.5(3)
121.8(3)
The crystal structure of 7b was reported earlier.[7a] Selected
bond distances and angles for 7b and 7c are listed in Table 1. As
expected, no appreciable differences in their values are observed
in these compounds.
Crystal Structure of Flavin Analogue 7c
Recrystallization of 7c from CHCl3/MeOH afforded yellow sin-
gle crystals, and thus, X-ray crystal analysis∗ was carried out to
clarify the structural details of 7c.The thermal ellipsoid diagram
of 7c is shown in Fig. 2.
The crystal structure of 7c shows that it exists as a dimer with
intermolecular hydrogen bonding giving the trans-geometry as
shown in Fig. 3.
Modified Synthesis of Bile Acid-Based Acyclic Receptors
Recently, bile acids, because of their rigid framework, facial
amphiphilicity, and suitably oriented hydroxy groups, have
attracted considerable interest from a molecular engineering
point of view.[9] Previously, we have reported the synthesis of a
∗Single-crystal diffraction study was carried out on a Bruker SmartApex CCD diffractometer with a Mo Kα (λ = 0.71073 Å) sealed tube. The crystal structure
was solved by direct methods and refined using the SHELXTL package.[8] All hydrogen atoms were included in idealized positions, and a riding model was
used. Non-hydrogen atoms were refined with anisotropic displacement parameters.

218
P. Chattopadhyay, R. Nagpal, and P. S. Pandey
(i)
O
R3
H
R3
H
O
O
O
R2
R2
R1
R1
9a R1 ϭ OCHO, R2, R3 ϭ H
9b R1, R3 ϭ OCHO, R2 ϭ H
9c R1, R2, R3 ϭ OCHO
8a R1 ϭ OH, R2, R3 ϭ H
8b R1, R3 ϭ OH, R2 ϭ H
8c R1, R2, R3 ϭ OH
R3
R1
R2
O
N
H
(ii)
N
N
H
O
10a R1 ϭ OCHO, R2, R3 ϭ H
10b R1, R3 ϭ OCHO, R2 ϭ H
10c R1, R2, R3 ϭ OCHO
R1
R2
R3
O
O
H3C
CH3
N
H
N
N
(CH2)6
(H2C)6
(iii)
(CH2)6
H3C
COCl
H
11
12
Scheme 2. Reagents and conditions (and yields): (i) HCOOH, 60◦C, 4 h (98%); (ii) ethyl chloroformate,
triethylamine, rt, 4 h, 2,6-diaminopyridine, triethylamine, THF, reflux, 12 h (89%); (iii) 2,6-diaminopyridine,
triethylamine, THF, 12 h (90%).
R2
N
R1
1
9
6
O 1
2
N 10a
8
7
9a
5a
O
H
N
O
N
10
OH
3
5
N
N
N
H
N
4a
4
O
O
O 2
O
O
N
N
N
H
O
7a R1 ϭ Octyl
7b R1 ϭ Hexyl
7c R1 ϭ Butyl
14a R2 ϭ Octyl
14b R2 ϭ Hexyl
N
H
O
O
Fig. 5. Chemical structures of flavin analogues.
O
O
O
OH
This synthetic route is much cleaner, easy to handle, and
most importantly gives good yields. The dioctyldiamide of 2,6-
diaminopyridine[11] 12 was prepared from the reaction of the
corresponding acid chloride 11 with 2,6-diaminopyridine.
13
Fig. 4. Steroid-based cyclic receptor having 2,6-diaminopyridine unit.
Complexation Studies
new class of bile acid-based acyclic as well as cyclic receptors
that contain a 2,6-bis(acylamino)pyridine unit for recognition of
7,8-unsubstituted flavin and uracil analogues.[4m,10]
7,8-Dimethyl flavin analogues 7a–c and 7,8-unsubstituted flavin
analogues 14a,b employed in the complexation studies are
shown in Fig. 5.
We have now used a modified procedure for the synthesis
of the acyclic receptors that is outlined in Scheme 2 and starts
with the protection of hydroxy groups of the bile acids to obtain
9a–c. In the following step, formylated bile acids 9a–c were
treated with ethyl chloroformate in the presence of triethylamine
to give the corresponding anhydrides that were subsequently
condensed with 2,6-diaminopyridine to give the acyclic recep-
tors 10a–c. Compound 10b was further used to synthesize
cholaphane 13[4m] shown in Fig. 4.
The binding behaviour of the flavin analogues were deter-
mined with 2,6-diaminopyridine-based receptors using 1H NMR
titration in CDCl3. As a typical example of a NMR titration,
addition of flavin derivative 7a to the solution of bile acid-based
acyclic receptor 12 in CDCl3 resulted in the downfield shift in
the resonances of the amidic protons of the receptor. The change
in the chemical shift of the amidic protons was followed as a
function of increasing guest concentration until saturation of the
chemical shift values was reached.Analysis of the saturation data

Recognition Properties of Flavin Analogues with Bile Acid-Based Receptors
219
Table 2. Binding constants Ka (m−1) for complexation
of flavin analogues with 2,6-diaminopyridine-based
receptor 12A
bonds in complexes is related to the individual hydrogen-
bonding acidity of the donor group and the basicity of the
acceptor group involved, there are additional factors that also
influence the stability of the complex. The manifestation of
this comes from the fact that 7,8-unsubstituted flavin analogues
14a,b, as compared to the 7,8-dimethyl flavin analogues 7a–c,
show a higher affinity towards acyclic bile acid-based receptors,
whichisincontrastwiththeearlierworksofthegroupsofRotello
and Rizzo.The unexpected decrease in the binding constant, pre-
sumably, is a result of steric effects arising from the interaction
of the methyl groups at the 7 and 8 positions of the flavin with the
rigid steroidal framework, which outplay the electronic effects
of the flavin system. Moreover, the difference in the binding
affinity between 7,8-dimethyl-N(10)octyl flavin 7a and N-10-
octylisoalloxazine 14a for acyclic bile acid receptors 10a–c is
small compared to the difference in the binding affinity between
7,8-dimethyl-N(10)hexyl flavin 7b and N-10-hexylisoalloxazine
14b. This might be attributed to the large chain length of the
octyl group that creates more hindrance in binding and decreases
the influence of the 7,8-dimethyl groups. However, the binding
affinity of 7,8-dimethyl-N(10)-butyl flavin, which can have bet-
ter binding because of the smaller size of the butyl group, shows
a lower binding affinity compared to 7a and 7b, which may be
a result of the dominant steric interactions of the 7,8-dimethyl
groups with the steroidal framework. Hence, it seems that among
7a–c, 7,8-dimethyl-N(10)hexyl flavin 7b has the most suitable
chainlengthatN(10)fortheformationofasupramolecularstruc-
ture with acyclic bile acid-based receptors 10a–c. In the case of
the cyclic receptor cholaphane 13, which is already a poor recep-
tor because of its constrained geometry, there is no significant
change in the binding constant with respect to 7,8-unsubstituted
and substituted flavin analogues.
Flavin derivative
Ka [m−1
]
7a
7b
7c
14a
14b
280
300
300
145
150
ADetermined in CDCl3, at 25◦C, errors estimated to be
≤10%.
Table 3. Binding constants Ka (m−1) for complexation of flavin
analogues with bile acid-based receptorsA
Receptor
Flavin derivatives
7a
7b
7c
14a
14b
10a
10b
10c
13
240
230
230
95
400
350
310
100
150
200
200
100
250B
240B
260
600B
400B
400
110B
110B
ADetermined in CDCl3, at 25◦C, errors estimated to be ≤10%. BData from
reference [4m].
withWINEQNMR software,[12] a non-linear regression curve fit-
ting program, revealed a 1:1 complexation, which was further
confirmed by Job’s plot.The titration experiment was carried out
at least twice. The observed binding constants for the complex-
ation of receptor 12 with flavin analogues 7a–c and 14a,b are
listed in Table 2.
Conclusion
The 1H NMR data in Table 2 show that the association con-
stants are low (Ka 145–150 m−1) for 7,8-unsubstituted flavin
analogues 14a,b and bis(octanoylamino)pyridine 12. Upon
introducing a methyl substituent at the C7 and C8 positions of
the flavin analogues 7a–c, the association constants increase to
280–300 m−1. Complexation of 12 with 7,8-dimethyl flavin ana-
logues 7a–c is stronger than 7,8-unsubstituted flavin analogues
14a,b because the electron donating capacity of the methyl
groups at the C7 and C8 positions increases the electron den-
sity throughout the flavin system, which subsequently increases
the hydrogen bonding capability, because there are two hydro-
gen bond acceptors C(2)=O and C(4)=O against one hydrogen
bond donor N(3)–H. This observation concurs with the earlier
works of Rotello and coworkers[3i] and Hasford and Rizzo.[13]
The results of the titration experiments for the complexation of
bile acid-based receptors with flavin analogues 7a–c and 14a,b
are summarized in Table 3.
In conclusion, an efficient synthetic procedure for the synthesis
of 7,8-dimethyl-N(10)alkyl flavins has been developed. Binding
studies of these flavins with bile acid-based diaminopyridine
(DAP) systems shows a prominent influence of the steric factors
that is the opposite to electronic effects. Hence, it seems likely
that the steric effect also plays an important role in the regulation
of binding and electrochemical properties of flavoenzymes.
Experimental
Melting points are uncorrected. IR spectra were recorded on a
Nicolet Protégé 460 FTIR Spectrometer, using potassium bro-
midepellets. 1Hand13CNMRspectrawererecordedonaBruker
Spectrospin DPX 300. Tetramethylsilane was used as internal
reference and the chemical shifts are expressed as displacement
(δ) in ppm downfield from tetramethylsilane. High-resolution
mass spectra (ES) were recorded on a VG-Fisons ‘Autospec’
spectrometer. Column chromatography was carried out using
Spectrochem silica gel 230–400 mesh for flash chromatography.
The solid compounds were dried under vacuum in the presence
of P2O5.
Surprisingly, the electronic effects of the methyl substituents
at the C7 and C8 positions of the flavin analogues do not seem
to play a prominent role in the complexation of the flavin
analogues with bile acid-based acyclic receptors. The bind-
ing constants of the complexes of the acyclic receptors 10a–c
with 10-hexylisoalloxazine (7,8-unsubtituted flavin analogue)
N-(3,4-Dimethylphenyl)acetamide 2
14b is in the range of 400–600 m−1 [4m]
,
whereas with 7,8-
3,4-Dimethylaniline 1 (5.0 g, 41.1 mmol) was dissolved in dry
1,2-dichloroethane (25 mL) and to this acetic anhydride (6.3 g,
61.8 mmol) and triethylamine (6.2 g, 61.8 mmol) were added.
The reaction mixture was refluxed in an oil bath for 12 h.
TLC was used to monitor the course of the reaction and to
confirm the complete consumption of starting material. The
dimethyl-N(10)-hexylisoalloxazine (7,8-dimethyl flavin ana-
logue) 7b, they are in the range of 310–400 m−1. A similar
trend was observed for the complexation of 7,8-unsubstituted
and substituted-N(10)-octyl flavin with bile acid-based acyclic
receptors 10a–c. Although the strength of multiple hydrogen

220
P. Chattopadhyay, R. Nagpal, and P. S. Pandey
solvent was then evaporated under reduced pressure and the
residue was dissolved in chloroform (30 mL) and washed with
1 N hydrochloric acid (10 mL), saturated sodium bicarbonate
solution (10 mL), and brine (10 mL), respectively, dried over
sodium sulfate and concentrated. The crude product was puri-
fied by flash chromatography on silica gel (elution with ethyl
acetate/hexane, 3/7) to give 6.59 g of 2. Yield 98%, mp 97–
100◦C. ES-HRMS calc. for (C10H13NONa)+ 186.0895, found
186.0894. νmax (KBr)/cm−1 3291, 3190, 2967, 1540, 1663,
1607, 1313. δH (300 MHz, CDCl3, TMS) 7.27 (s, 1H, –NH),
7.20 (m, 2H, Ar-H), 7.05 (d, 1H, J 8.10, Ar-H), 2.23 (s, 3H,
CH3), 2.21 (s, 3H, CH3), 2.14 (s, 3H, –COCH3). δC (75 MHz,
CDCl3, TMS) 168.92, 137.04, 135.82, 132.55, 129.84, 121.65,
117.82, 29.71, 24.30, 19.82, 19.15.
(4,5-Dimethyl-2-nitrophenyl)octylamine 5a
ES-HRMS calc. for (C16H26N2O2Na)+ 301.1892, found
301.1891. νmax (KBr)/cm−1 3379, 3068, 2926, 2855, 1628,
1571, 1509. δH (300 MHz, CDCl3, TMS) 7.86 (br s, 1H,
–NH), 7.79 (s, 1H, Ar-H), 6.50 (s, 1H, Ar-H), 3.18–3.12 (m,
2H, –NCH2), 2.15 (s, 3H, CH3), 2.05 (s, 3H, CH3), 1.65–1.58
(m, 2H, –CH2), 1.40–1.17 (m, 10H, –(CH2)5), 0.78 (t, 3H, J
6.9, CH3). δC (75 MHz, CDCl3, TMS) 147.10, 144.23, 129.47,
126.26, 124.03, 114.02, 42.97, 31.71, 29.20, 29.06, 28.97, 27.01,
22.57, 20.61, 18.43, 13.99.
(4,5-Dimethyl-2-nitrophenyl)hexylamine 5b
Starting material 4 (1 g, 6.02 mmol) and hexyl bromide
(0.99 g, 6.02 mmol) were used. The product was isolated as
1.05 g of a red-orange solid 5b. Yield 70%, mp 45–48◦C. ES-
HRMS calc. for (C14H22N2O2Na)+ 273.1579, found 273.1579.
νmax (KBr)/cm−1 3375, 2925, 2857, 1627, 1568, 1505. δH
(300 MHz, CDCl3, TMS) 7.97 (br s, 1H, –NH), 7.92 (s, 1H,
Ar-H), 6.61 (s, 1H, Ar-H), 3.30–3.24 (m, 2H, –NCH2), 2.26 (s,
3H, CH3), 2.17 (s, 3H, CH3), 1.76–1.69 (m, 2H, –CH2), 1.53–
1.31 (m, 6H, –(CH2)3), 0.90 (t, 3H, J 7.0, CH3). δC (75 MHz,
CDCl3, TMS) 147.24, 144.32, 129.55, 126.35, 124.13, 114.10,
43.06, 31.49, 29.02, 26.76, 22.56, 20.70, 18.52, 13.99.
N-(4,5-Dimethyl-2-nitrophenyl)acetamide 3
To a solution of N-(3,4-dimethylphenyl)acetamide 2 (2 g,
12.3 mmol) in glacial acetic acid (10 mL) was added concen-
trated nitric acid (0.73 mL) over 15 min while maintaining the
reaction temperature below 10◦C, followed by the dropwise
addition of sulfuric acid (0.46 mL) over 15 min. The resulting
solution was stirred at room temperature for 30 min. The reac-
tion mixture was added dropwise on to crushed ice and the
yellow precipitate obtained was filtered off and washed gen-
erously with water. The crude product was purified by flash
chromatography on silica gel (elution with ethyl acetate/hexane,
1/9) to give 2.17 g of 3. Yield 85%, mp 101–104◦C. ES-HRMS
calc. for (C10H12N2O3Na)+ 231.0746, found 231.0744. νmax
(KBr)/cm−1 3341, 2959, 1705, 1620, 1583, 1502. δH (300 MHz,
CDCl3, TMS) 10.35 (s, 1H, –NH), 8.54 (s, 1H, Ar-H), 7.98 (s,
1H, Ar-H), 2.34 (s, 3H, –COCH3), 2.28 (s, 3H, CH3), 2.27 (s,
3H, CH3). δC (75 MHz, CDCl3, TMS) 167.91, 145.78, 133.12,
131.73, 131.30, 124.83, 121.61, 24.57, 19.47, 18.08.
Butyl-(4,5-dimethyl-2-nitrophenyl)amine 5c
Starting material 4 (1 g, 6.02 mmol) and butyl bromide
(0.83 g, 6.02 mmol) were used. The product was isolated as
0.95 g of a red solid 5c. Yield 71%, mp 54–56◦C (lit.[8b] mp
55–57◦C). ES-HRMS calc. for (C12H18N2O2Na)+ 245.1266,
found 245.1265. νmax (KBr)/cm−1 3372, 2925, 2862, 1626,
1569, 1507. δH (300 MHz, CDCl3, TMS) 7.98 (br s, 1H, –NH),
7.92 (s, 1H, Ar-H), 6.62 (s, 1H, Ar-H), 3.31–3.25 (m, 2H,
–NCH2), 2.26(s, 3H, CH3), 2.17(s, 3H, CH3), 1.75–1.66(m, 2H,
–CH2), 1.51–1.41 (m, 2H, –CH2), 0.98 (t, 3H, J 7.5, CH3). δC
(75 MHz, CDCl3,TMS) 144.21, 129.48, 126.23, 124.01, 114.00,
42.64, 147.11, 31.04, 20.58, 20.12, 18.40, 13.69.
4,5-Dimethyl-2-nitroaniline 4
N-(4,5-Dimethyl-2-nitrophenyl)acetamide3(2 g, 9.62 mmol)
was added to a 10% sodium hydroxide solution (25 mL). The
mixture was refluxed for 12 h. The reaction mixture was allowed
to cool to room temperature and the rust colored precipitate was
filtered off, dissolved in chloroform (30 mL), and washed thor-
oughly with water. The organic layer was dried over anhydrous
sodium sulfate and concentrated. The crude product was puri-
fied by flash chromatography on silica gel (elution with ethyl
acetate/hexane, 1/9) to give 1.43 g of 4. Yield 90%, mp 137–
139◦C. ES-HRMS calc. for (C8H10N2O2H)+ 167.0821, found
167.0822. νmax (KBr)/cm−1 3488, 3374, 1638, 1583, 1487. δH
(300 MHz, CDCl3, TMS) 7.87 (s, 1H, Ar-H), 6.59 (s, 1H, Ar-H),
5.90 (br s, 2H, –NH2), 2.23 (s, 3H, CH3), 2.18 (s, 3H, CH3). δC
(75 MHz, CDCl3,TMS) 146.65, 143.08, 130.25, 126.15, 125.64,
119.04, 20.12, 18.60.
General Procedure for Reduction
In a typical example, to a stirred suspension of 5a (1 g,
3.59 mmol) in dry methanol (30 mL), 10% Pd/C (0.2 g) was
added in a single portion. The resulting reaction mixture was
stirred at room temperature for 4 h under balloon pressure of
hydrogen. The catalyst was removed by filtration and washed
with dry methanol (10 mL). The solvent was evaporated under
reduced pressure. The resulting residue was extracted with chlo-
roform and dried over anhydrous sodium sulfate. The organic
layer on evaporation gave 0.85 g of a colourless viscous oil 6a,
which was used without further purification.
General Procedure for N-Alkylation
General Procedure for the Synthesis of
7,8-Dimethyl-10-alkyl Isoalloxazines
In a typical example, 4,5-dimethyl-2-nitroaniline 4 (1 g,
6.02 mmol) was dissolved in dry DMF (30 mL) and to this
sodium hydride (60% suspension in mineral oil, 0.24 g) was
added under a nitrogen atmosphere. Octyl bromide (1.16 g,
6.02 mmol) was then added dropwise and the reaction mixture
wasstirredatroomtemperaturefor12 h.Thesolutionwasdiluted
with ethyl acetate (40 mL) and washed four times with water,
dried over sodium sulfate, and concentrated. The crude prod-
uct was purified by flash chromatography on silica gel (elution
with ethyl acetate/hexane, 1/99) to give 1.17 g of a rust colored
viscous oil 5a (70%).
In a typical example, 1-(N-octylamino)-2-amino-4,5-dimethyl-
benzene 6a (0.50 g, 2.01 mmol) was dissolved in glacial acetic
acid (10 mL), and the contents were heated to 60◦C. To this,
a preheated (60◦C) mixture of alloxan monohydrate (0.32 g,
2.01 mmol) and boric acid (0.18 g, 3.02 mmol) in acetic acid
were added. The reaction mixture was stirred overnight at 60◦C.
Acetic acid was removed under vacuum, and the solid obtained
was flashed chromatographed on a silica gel column (elution
with methanol/chloroform, 3/97) to give 0.31 g of a bright yellow
solid 7a.

Recognition Properties of Flavin Analogues with Bile Acid-Based Receptors
221
7,8-Dimethyl-N(10)-octylisoalloxazine 7a
chloroform solution. Molecular formula C16H18N4O2, Mw
298.34, triclinic, space group P-1, a 8.152(6) Å, b 10.051(8) Å,
c 10.325(8) Å, α 64.562(12)◦, β 68.541(13)◦, γ 78.667(14)◦,
Yield 43%, mp 230–233◦C. ES-HRMS calc. for
(C20H26N4O2Na)+ 377.1953, found377.1958. νmax (KBr)/cm−1
3456, 3163, 2925, 2856, 2811, 1716, 1659, 1577. δH (300 MHz,
CDCl3, TMS) 9.03 (br s, 1H, –NH), 8.15 (s, 1H, Ar-H), 7.38 (s,
1H, Ar-H), 4.78–4.68 (m, 2H, –NCH2), 2.68 (s, 3H, CH3), 2.56
(s, 3H, CH3), 2.01–1.79 (m, 2H, –CH2), 1.56–1.28 (m, 10H,
–(CH2)5), 0.87 (t, 3H, J 6.6, CH3). δC (75 MHz, CDCl3, TMS)
159.55, 155.34, 150.00, 148.16, 136.99, 136.08, 134.92, 132.77,
131.04, 115.29, 45.31, 31.68, 29.20, 29.08, 27.10, 26.80, 22.55,
21.65, 19.47, 14.01.
V 710.2(10) Å3, T 298 K, Dcalcd 1.408 g cm−3, µ 0.095 mm−1
,
Z 2, λ 0.71073 Å, ω and ϕ scans, R1/wR2 (I ≥ 2σ(I))
0.0871/0.1593. Crystallographic data for the structure have been
deposited with the Cambridge Crystallographic Database Centre
as supplementary publication number CCDC 645150.
1H NMR Titrations and Determination of Stoichiometry
The binding tendency of receptors based on 2,6-diaminopyridine
for flavin analogues has been investigated by 1H NMR titration
experiments in CDCl3 on a Bruker DPX300 (300 MHz) spec-
trometer at 298 K. In particular, a solution [(8–12) × 10−3 m]
of the receptor in CDCl3 was titrated with aliquots from a
flavin stock solution [(40–60) × 10−3 m] in the same solvent.
The changes in the chemical shift of the amide protons were
monitored as a function of increasing flavin concentration until
saturation of the chemical shift values were reached.The associa-
tion constants Ka were calculated by the analysis of the saturation
data with WINEQNMR software,[13] a non-linear regression
curve fitting program, which also revealed a 1:1 complexation.
Every titration was repeated at least once until consistent results
were obtained. The complex stoichiometry was determined by
using Job’s method of continuous variations. A plot of the prod-
uct of the host concentration and difference in the change of
the chemical shift of the host upon addition of the guest versus
the guest mole fraction was made. In each case, this peaked at a
guest mole fraction of 0.5, which indicated the 1:1 stoichiometry
of the complex formation.
7,8-Dimethyl-N(10)-hexylisoalloxazine 7b
Starting material 6b (0.5 g, 2.27 mmol), alloxan monohy-
drate (0.36 g, 2.27 mmol), and boric acid (0.21 g, 3.40 mmol)
were used. The product was isolated as 0.33 g of a bright yellow
solid 7b.Yield 45%, mp > 240◦C (lit.[8a] mp 267◦C). ES-HRMS
calc. for (C18H22N4O2Na)+ 349.1640, found 349.1643. νmax
(KBr)/cm−1 3437, 3220, 2926, 1712, 1659, 1540. δH (300 MHz,
CDCl3, TMS) 8.50 (s, 1H, –NH), 8.07 (s, 1H, Ar-H), 7.39 (s,
1H, Ar-H), 4.65–4.72 (m, 2H, –NCH2), 2.57 (s, 3H, CH3), 2.46
(s, 3H, CH3), 1.88–1.81 (m, 2H, –CH2), 1.59–1.37 (m, 6H,
–(CH2)3), 0.91 (t, 3H, J 6.9, CH3). δC (75 MHz, CDCl3, TMS)
159.63, 155.64, 150.00, 148.26, 137.09, 136.07, 134.96, 132.76,
131.05, 115.34, 45.37, 31.41, 27.11, 26.49, 22.51, 21.69, 19.50,
13.95.
7,8-Dimethyl-N(10)-butylisoalloxazine 7c
Starting material 6c (0.5 g, 2.60 mmol), alloxan monohydrate
(0.42 g, 2.60 mmol), and boric acid (0.24 g, 3.90 mmol) were
used. The product was isolated as 0.37 g of a bright yellow
solid 7c. Yield 48%, mp > 240◦C (lit.[8b] mp > 240◦C). ES-
HRMS calc. for (C16H18N4O2Na)+ 321.1327, found 321.1320.
νmax (KBr)/cm−1 3451, 3153, 3100, 3023, 2959, 2866, 2815,
1713, 1656. δH (300 MHz, CDCl3, TMS) 8.51 (s, 1H, –NH),
8.05 (s, 1H, Ar-H), 7.39 (s, 1H, Ar-H), 4.72–4.67 (m, 2H,
–NCH2), 2.56(s, 3H, CH3), 2.44(s, 3H, CH3), 1.89–1.79(m, 2H,
–CH2), 1.60–1.49 (m, 2H, –CH2), 1.02 (t, 3H, J 7.2, CH3). δC
(75 MHz, CDCl3,TMS) 159.56, 155.19, 150.05, 148.27, 137.05,
136.05, 134.97, 132.82, 131.08, 115.28, 45.15, 29.12, 21.74,
20.16, 19.54, 13.78.
Accessory Publication
X-ray data (CIF), NMR spectra, Job’s plot figures and binding
isotherms are available from the author or, until March 2013, the
Australian Journal of Chemistry.
Acknowledgements
The authors are thankful to the Council of Scientific and Industrial Research,
New Delhi, and the University Grants Commission, New Delhi for research
fellowships to P.C. and R.N., respectively. The authors thank Shailesh Upreti
and M. Senthil Kumar for helpful discussions. The authors also thank the
Department of Science and Technology, New Delhi for funding a single
crystal diffractometer under FIST to the Department of Chemistry, IIT Delhi,
India.
General Procedure for the Modified Synthesis
of Acyclic Bile Acid-Based Receptors
In a typical example, to a solution of 9a (4 g, 9.9 mmol) and
triethylamine (1.4 mL) in dry tetrahydrofuran (THF) under a
nitrogen atmosphere was added ethyl chloroformate (1.07 g,
9.9 mmol) dropwise over a period of 10 min. The reaction mix-
ture was stirred at room temperature for 4 h. A solution of 2,6-
diaminopyridine (0.44 g, 4.03 mmol) in dry THF (15 mL) was
added and the reaction mixture was refluxed for 12 h. THF was
evaporated and the residue was extracted with chloroform. The
solution was washed with 1 N HCl, saturated sodium bicarbonate
solution, and brine, dried over sodium sulfate and evaporated.
The residue was purified by flash chromatography (elution with
EtOAc/hexane, 1/6) to give 3.88 g of 10a (89%). All the com-
pounds 10a–c gave identical melting points and spectroscopic
values similar to the compounds reported earlier.[4m]
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