成蹊大学理工学研究報告
J. Fac. Sci. Tech., Seikei Univ.
Vol.44 No.２ (2007) pp.53-64（特別研究費に係る論文）

Synthesis of New 2,4-Pteridinediones and Their Application to Fluorescence
Derivatization Reagents
Akira KATOH*1, Kayo KAMEO*2, Ryota SAITO*3, and Yuriko MATSUMURA*4
ABSTRACT: 3-(p-Chlorocarbonyl)phenyl-1-methyl-2,4(1H,3H)-pteridinedione (6) was synthesized
through 4 steps from commercially available methyl 3-amino-2-pyrazinecarboxylate.
3-(p-Chlorocarbonyl)phenyl-6-(p-methoxy)phenyl-2,4(1H,3H)-pteridinedione (11) was also synthesized
through 5 steps from methyl 3-amino-2-pyrazinecarboxylate. The derivatization of phenethylamineand cholesterol with pteridinedione (6) was carried out to give the derivatized products (12) and (13).
The derivatization of phenethylamine with pteridinedione (11) was also carried out to give thederivatized product (14), which emitted a strong fluorescence. Absorption and emission maxima of
the derivatized products (12) and (13) were observed at around 330 and 380 nm in MeCN, respectively.
Absorption and emission maxima of the derivatized product (14) were observed at 291 and 369 nm, and474 and 473 nm, respectively. Pteridinedione (11) was demonstrated to be applicable to a new
fluorescence derivatization reagent for amines.
Keywords: 2,4(1H,3H)-pteridinedione, synthesis, fluorescence derivatization reagent, amine(Received September 15, 2007)

Introduction

Since 1,2,3,4-tetrahydroisoquinoline was found to be oneof

There are many bioactive substances such as carboxylic

inductive

substances

pharmacological

and

for

Parkinson’s

biochemical

studies

disease,ontheacids, amines, amino acids, alcohols, and ketones in urine

derivatives has been intensively performed.

or blood, although the quantity of each substance is very

occurring 2-phenylethylamine is a trace amine in bodysmall.

fluids and thought to be a neuromodulator in the central

Determining the quantity of these trace substances

Naturally

seemed to be indispensable for elucidation of many kinds

nervous system.

of physiological functions in the body, diagnosis ofobserved in patients with neurogical diseases such as

sickness, explication of cause of disease, and medical

schizophrenia and depression.

treatment.

Knowing accurate movement of drug in thename of three kinds of amines, viz., adrenaline,

body is required for effective treatment of each patient.

noradrenalin, and dopamine which contain the catechol

From these backgrounds, the importance of quantitativeskeleton.

analysis of biologically active substances and drugs has

neurotransmitter and intercellular transmitter.2)

increased year by year.1)

An abnormal amount in urine is
Catecholamine is generic

They are adrenocortical hormones and act as

There are many kinds of alcohols like cholestanol,

Various kinds of amines are found in biologicalcholesterol, and so on.

Cholestanol is a metabolite ofmaterials, and play physiologically important roles at tracecholesterol.

levels. Parkinson’s disease is a disease of nervous

concentration of cholestanol raises although that ofsystem characterized by a slowly spreading tremor,cholesterol is almost constant.

muscular weakness and rigidity, and a peculiar gait.

quantitative analysis of these compounds serves to

In case of crebraltedoxanthma (CTX), thediagnose CTX.
*1: Professor, Department of Materials and Life Science,
katoh@ st.seikei.ac.jp*2: Graduate student*3: Lecturer, Department of Chemistry, Faculty of Science, Toho
University*4: Research Assistant, Department of Materials and Life Science

The simultaneous

Therefore, the determination of thesesubstances was required for biomedical and clinicaltreatment.3)－53－

As mentioned above, a highly sensitive and selective

amines have been developed.

However, these reagentsdetermination of biologically active substances is quite

have some disadvantages.

important for their biological and clinical investigation.

naphthalene-2,3-dicarbaldehyde (NDA) was limited to

Unfortunately, biological substances have the following

primary amines.

disadvantages; 1) they are high boiling points and polar(PIB-Cl) was not suitable for aromatic amines.

compounds, and 2) they usually absorb light at shortersame time, the stability of the derivatized compounds iswavelength region below 250 nm.

not so high, and thus the decomposition was sometimes

These two factors

For example, the use of the(2-Phthalimidyl)benzoyl chloride

cause the low-sensitive determination by means ofobserved during analysis.

conventional

nitrobenzo-2-oxa-1,3-diazolegaschromatography(GC),

GC-mass

spectrometry, and ion-exchange chromatography.

At theIn cases of 7-chloro-4(NBD-Cl)

and

9-

There

(2-acridone)oxyethylcarbonylimidazole(AOCD),

are several high-performance liquid chromatography

Stokes' shifts are not so large.

In a case of(HPLC)(9-fluorenyl)methyl

methods

withelectrochemical

andspectrophotometric detection.

However, these detections

have a limit in sensitivity.

At present, one of popular(FMOC-Cl),

itsemission wavelength is short.
A variety of fluorescence derivatization reagents such as
DMEQ-CON312),

methods for the detection of biological substances is
HPLC method with fluorescence detection.

chloroformate

their

Accordingly,

DACB-NCO

15)

MOC-CON313),

MOC-COCl14),

andfor alcohols have also been developed.

various fluorescence derivatization reagents with highHowever, these reagents also have some disadvantages; 1)

sensitivity have been used for HPLC determination of

they are unstable toward moisture, 2) the reaction of thembiological substances as depicted below.

with alcohols requires heating for complete derivatization,and 3) they are expensive.

Thus, the development of

more efficient and cheap fluorescence derivatization
reagents is still desired.

baioactive
substance
R-NH2
R-OH
no fluorescence

F.D.R

In our laboratory, synthesis of 3-(aryl)substituted-+

2,4(1H,3H)-pteridinediones (A) and their UV-VIS and
Fluorescence
Derivatization
Reagent

fluorescence spectroscopic characteristics have beenreported.16)

Compound (A, R=H) showed two absorption

bands at 320 and 390 nm in MeCN, the latter having asmall molar absorption coefficient, and at 273 and 371 nm
in EtOH.
R-X

F.D.R.

Similar spectra were obtained in case ofpteridinedione (A, R=OCH3).

X-NH, O

(Table 1)

It is well knowthat 3-substituted 2,4(1H,3H)-pteridinedione exists intautomeric equilibrium between lactam (A) and lactim (B)Fluorescence derivatization products

as shown in the next page.

Then, the date of

3-substituted 2,4-pteridinediones were compared with
The following points are required for a highly sensitivethose of model compounds for these two tautomers,

and selective fluorescence derivatization reagent; 1) it

1,3-dimethyl-2,4(1H,3H)-pteridinedione

(C)

and

17)

The

emits strong fluorescence at region of longer wavelength,2-methoxy-3-methyl-4(3H)-pteridinedione (D).

2) the difference in the excitation and emission wavelength

absorption bands around 320 nm in MeCN and 270 nm inis large, 3) the reaction proceeds rapidly and quantitatively

EtOH were observed on the spectra of model compounds

under mild conditions, and 4) the derivatization reagent(C) and (D), respectively, suggesting that 3-(aryl)

and the resulting product are stable and the molecular size

substituted 2,4(1H,3H)-pteridinediones predomi- nantlyof the derivatization reagent is small.

exist in the lactam from (A) in MeCN and in the lactim

In the past, a variety of fluorescence derivatization
4)

5)

6)

from (B) in EtOH.

pteridinediones showed two emission bands at around 380

forand 460 nm in MeCN.

reagents such as OPA , NDA , Dansyl-Cl , NBD-Cl ,
8)

9)

10)

11)

DMEQ-COCl , FMOC-Cl , AEOC , and AOCD

In the fluorescence spectra,

7)

－54－

On the other hand, pteridinedines

showed a emission band at around 470 nn when thepteridinediones

bearingthep-(chlorocarbonyl)phenyl

excitation wavelength of 320 nm was applied.

group at C-3 position, and 3) the evaluation of thepossibility of synthetic compounds as new fluorescence

Table 1

UV and fluorescence properties ofpteridinediones (A)UV/λmax nm (ε)

R
H
OCH3

derivatization reagents for amines and alcohols.
O

Fluorescence/λmax nm

in MeCNin EtOHin MeCNin EtOH

320(3900)

273(10300)

376

467

389 (260)

371 (3300)

450

317(7900)

274(19500)

377

382 (360)

374 (6000)

471

O

O
R2

N

Cl

O
N

N

N

N
O
R1
R2=H, aryl

469

Cl
N

N

N

OR1

Results and Discussion
From these results, it was suggested that pteridinediones
have the possibility as new fluorescent derivatization

Synthesis of N-alkylated pteridinediones

reagents if pteridinediones having a reactive group at theAsolutionofcommerciallyavailable

methylpara position of the phenyl group at C-3 position and high3-amino-2-pyrazinecarboxylate and ethyl 4-isocyanato-solubility in organic solvents are synthesized.

benzoate in dry pyridine was refluxed for 9 h to give

R

O
N
N

N

N
N
H

O

N

N
N
Me

Me
O

of
OH

N

(A)

N

(1) in 73% yield. (Scheme 1)

N

N

O

3-(p-ethoxycarbonyl)phenyl-2,4(1H,3H)-pteridinedione

R

O

(B)

spectroscopies.

Meppm, respectively, in DMSO-d6.

N(C)

The absorption bands

due to NH and two C=O stretching vibrations wereOMe

observed at 3426, 1717 and 1677 cm-1, respectively.

(D)

O
N

The chlorocarbonyl and sulfonyl chloride groups arewidely used as reactive groups on the derivatization
reaction of amines and alcohols.

The signals assignable to 6-H and 7-H on

the pteridinedione ring were observed at δ 8.59 and 8.72
NN

3-(p-ethoxycarbonyl)phenyl-2,4-(1H,3H)-pteridinedi-one (1) was carried out by means of 1H-NMR and IRONThe structural assignment

N

O

OCH3

+ O=C=NOC2H5NH2It has already been

refluxin dry pyridine

reported that 9-fluorenylmethyl chloroformate having aOchlorocarbonyl group reacts with primary and secondaryOamines in the borate buffer solution (pH 8) and theNreaction completes within 2 minutes at room temperature.

N

Chlorocarbonyl, carbonyl azide, isocyanate, and carbonyl

OC2H5
N

N
H

O
(1)

nitrile groups are also used as reactive groups inderivatization reagents for alcohols.

reagents, the chlorocarbonyl group has been widely andO

conventionally used for a long time.

N

We have reported the application of heterocyclic
compounds to fluorescence derivatization reagents.18)

NAs

synthesis of N-alkylated and O-alkylated pteridinedione2)

synthesisof

6-(aryl)NNRO

a: R=Meb: R=Bn
c: R=Pr

Scheme 1

derivatives bearing the p-(chlorocarbonyl)phenyl group at
position,

OC2H5(2a-c)

extensive studies, we would like to describe here 1) theC-3

in dry DMF
O

RX/NaHAmong thesesubstituted－55－

compounds (3-5) was carried out by means of 1H-NMR

Pteridinedione (1) was allowed to react with CH3I in thepresence of NaH in dry DMF at 80 ºC for 4 h to giveand IR spectroscopies.

3-(p-ethoxycarbonyl)phenyl-1-methyl-2,4(1H,3H)-

signals assignable to 6-H and 7-H on the pteridinedione

pteridinedione (2a) in 96% yield as yellow powders.

ring were shifted to upper magnetic field at δ 8.36 andThe

In the case of compound (3), thestructural assignment of compound (2a) was carried out by

7.74 ppm in CDCl3, respectively.

means of 1H-NMR, IR, and UV spectroscopies.

due to COOH stretching vibration were observed at 2845

The

The absorption bands

signal observed at δ 3.76 ppm in CDCl3 was assignable to(broad) and 1679 cm-1.

N-CH3 or O-CH3 protons, because compound (1) produced

signals due to protons of the ethyl ester were observed at δ

ambient anion when was treated with NaH.

The structure

4.36 and 1.39 ppm, and two doublet signals owing to 5-Hof the product was determined by comparison of UV-VIS

and 6-H on the pyrazine ring were observed at δ 8.00 andspectrum of the product with the model compounds.

7.99 ppm in CDCl3, respectively.

The

In the case of compound (4), theThe absorption bands

absorption bands around 320 nm in MeCN and 270 nm indue to NH and two C=O stretching vibrations were

EtOH were observed on UV spectra of model compounds

observed at 3316, 1736 and 1695 cm-1, respectively.

(C) and (D), respectively.

17)

In

Actually, the absorption

the case of compound (5), the signals assignable to protons

bands of compound (2a) were 329 nm in MeCN and 331of the ethyl ester group of the starting material (2a) could

nm in EtOH, respectively.

not be observed in CD3OD.

Therefore, the structure of

The absorption bands due tocompound (2a) was assigned to be N-methylated product.

the COOH stretching vibration were observed at 3077

The alkylation of pteridinedione (1) with benzyl bromide(broad) and 1718 cm-1.

and propyl iodide were carried out in the similar manner to

The conversion of the carboxyl into chlorocarbonyl

give pteridinedione (2b) in 64% as white crystals and (2c)group was attempted.

in 74 % yield as white powders, respectively.

SOCl2

was

A solution of pteridinedione (5) in

refluxed

for8

using two solvent systems [MeOH:DMF(1:1) or MeOH].

SOCl2

stirred at room temperature for 4 h, but the desired
3-(p-carboxy)phenyl-1-methyl-2,4(1H,3H)-pteridinedione

O

Instead, the ring-openingO

products (3) and (4) were isolated.

N

N
O

O

OH

N

NH
CH3

N

OC2H5

N
H
NH
CH3

N

(3)

O

N
O
CH3

(6)

vibration of the COCl group was observed at 1770 cm-1.
OH

An attempt to synthesize the O-alkylated pteridinone

N
N
O
CH3

N

The absorption band typical for the C=O stretching
O

N

Cl

Scheme 2

(4)

N

3-(p-

(5)

(1:1) mixture was added 1M NaOH and the mixture was

N

give

e (6). (Scheme 2)

At first, to a solution of compound (2a) in MeOH:DMF

O

tochlorocarbonyl)phenyl-1-methyl-2,4(1H,3H)-pteridinedion

The hydrolysis of pteridinedione (2a) was carried out(5) could not be obtained.

hAt first, synthesis of O-alkylated pteridinone wasattempted according to Scheme 3.
(5)

The chlorination of

pteridinedione (1) was carried out using three methods

On the other hand, to a solution of compound (2a) in(method A: thionyl chloride; method B: phosphorus

MeOH was added 1M NaOH and the mixture was stirred

oxychloride; method C: a mixture of phosphorus

at room temperature for 4 h to give the desired productoxychloride and phosphorus pentachloride).

(5) in 58% yield.

of method A, a solution of pteridinedione (1) and SOCl2 in

The structural assignment of－56－

In the casedry dioxane and DMF was stirred at 90 ºC for 5 h, but the

C=O stretching vibrations were observed at 1739 and 1693

starting material (1) was recovered.

cm-1.

(1)

Br

O

O

OCH3

N
(7)

OC2H5

O
N

NH2

N

Chlorination

OCN

+

OC2H5

O
in dry pyridine

N
N

N

O

Cl

O
Br

MeONa
O

N

OC2H5

O
N

N

N

N
N
H

O

(8)

Scheme 4

N

N

OC2H5

OCH3

The

Suzukicouplingofcompound

(8)

with

p-methoxyphenylboronic acid was attempted according to

Scheme 3

Scheme 5.
In the case of method B, a solution of pteridinedione (1) in
POCl3 was refluxed for 4 days.
product could not be obtained.

B(OH)2

Again, the desired

In the case of method C,(8)a solution of pteridinedione (1), POCl3, and PCl5 in CHCl3+

was refluxed for 2 days, and then heated at 120 ºC for 40 h
in a sealed tube.

OCH3

However, the desired product again

could not be obtained.

Methods I-IV

Next, the preparation of O-alkylated pteridinone wasOattempted according to the reported method.19) However,
any trials are unsuccessful.

H3CO

From these results, theOsynthesis of O-alkylated pteridinone was abandoned.

N

Synthesis of 6-(aryl) substituted pteridinedioneN

A solution of commercially available methyl 3-amino-

OC2H5
N

N
H

O

(9)

Scheme 52-pyrazinecarboxylate and an excess of Bu4NBr3 in a
mixture of dry CHCl3 and pyridine was refluxed for 15 h
to give methyl 3-amino-6-bromopyrazine-2-carboxylate

Four methods were applied to optimize the Suzuki(7) in 49% yield as yellow powders.

coupling as summarized in Table 2.

A solution of

However, the yieldcompound (7) and ethyl p-isocyanatobenzoate in dryof pteridinedione (9) could not be improved.

pyridine

structural assignment of compound (9) was carried out by

wasrefluxed

for

11

h

to

give

3-(p-

The

ethoxycarbonyl)phenyl-6-bromo-2,4(1H,3H)-pteridinedione

means of 1H-NMR and IR spectroscopies.

(8) in 58% yield as brown crystals. (Scheme 4)

The

assignable to 3’’, 5’’-H and 2’’, 6’’-H on the benzene ring

signal typical for 7-H proton on the pteridinedione ringand protons of the methoxy group at C-6 position were

was observed at δ 8.70 ppm in CDCl3.

observed at δ 8.08 and 7.04, and 3.89 ppm, respectively, in

The absorption

The signals

band due to C-Br stretching vibration was observed at 517

CDCl3.

cm-1.

observed at δ 9.03 and 8.64 ppm, respectively.

Further, the absorption bands due to two kinds of－57－

The signals due to 7-H and N-H protons wereTwo

signals due to ethyl ester were also observed at δ 1.41 and(11) which was used for the next reaction without further

4.42 ppm.

purification.

In IR spectrum, the absorption bands due to

The absorption band typical for the C=O

stretching vibration of COCl was observed at 1796 cm-1.

three kinds of C=O stretching vibrations were observed at
1735, 1712, and 1690 cm-1.

Measurement of UV-VIS and fluorescence spectra ofpteridinediones

Table 2 Reaction conditions and yields of compound (9)MethodBase

Conditions

Yield(%)

I

Na2CO3

dioxane/reflux (10 h)

20II

Na2CO3benzene / reflux (15 h)

0

III

K3PO4

DMF / 80-130 ºC (6930

UV-VIS and fluorescence spectral data of compounds(2a-c and 9) are summarized in Table 3.

maxima of compounds (2a-c) were observed around 330
nm both in MeCN and in MeOH.

K2CO3

DMF/120 ºC (7 days)

Emission maximum ofcompound (2b) in MeOH was observed at about 30 nmh)IV

Absorption

longer wavelength than that in MeCN.

25

However, in acase of this compound, its fluorescence intensity was
remarkably decreased.

To a solution of pteridinedione (9) in MeOH was added
1M NaOH.

Table 3

UV-VIS and fluorescence spectral data ofcompounds (2a-c) and (9)The reaction mixture was stirred at room

temperature for 5 days and at 60 ºC for another 3 days toComgive the corresponding carboxylic acid (10) as yellow

pd.

UVa)/ λmax nm (ε)

Fluorescenceb)/ λmax nm

in MeCNin MeOHin MeCNin MeOH2a330 (7810)

330 (7320)

381

382

2b

328 (8010)

329 (7370)

385

412

observed at 3060 (broad) and 1692 cm-1, respectively.

2c332 (7880)

331 (7670)

382

391

(9)

9

290(23800)

293 (23140)

474

495

369 (8500)

373 (7570)

474

497

solids.

(Scheme 6)

The signals assignable to theprotons of ethyl ester of the starting material (9)disappeared in DMSO-d6. The absorption band due to the
O-H and C=O stretching vibrations of COOH wasa) Concentration: 1.0 x 10-4 M

1 M NaOH

b) Concentration: 1.0 x 10-6 M

O
H3CO

O
N
N
SOCl2

H3CO

were observed at around 290 and 370 nm both in MeCN

N

and in MeOH.

N
H

O

refluxN

MeCN and in MeOH.
than that in MeCN.

Cl

The difference in the fluorescenceintensity between in MeCN and in MeOH was found to beOlarge.

Compound (9) showed the strongest emission

band at 474 nm in MeCN when the excitation wavelength(11)of 290 nm was applied.

Scheme 6

Application of pteridinediones to fluorescence

The conversion of –COOH into –COCl was attempted
as follows.

Emission maximum of compound(9) in MeOH was observed at 20 nm longer wavelength

O

N
N
H

From this result, it was suggested thatcompound (9) exists in the lactim tautomer like D both in

(10)

O

N

On the other hand, absorption maxima of compound (9)OHderivatization reagents of phenethylamine

A solution of pteridinedione (10) in SOCl2

was refluxed for 4 h to give 3-(p-chlorocarbonyl)

A solution of pteridinedione (6) and phenethylamine inphenyl-6-(p-methoxy)phenyl-2,4(1H,3H)-pteridinedione

dry MeCN was stirred at room temperature to give the－58－

product (12) as pale yellow powders in 80% yield. From

The signals due to the phenethyl group were observed at

TLC monitoring, it was found that the derivatization

δ 2.95, 3.63, and 7.26-7.33 ppm in CD3OD.

completed within a few minutes.

The

absorption band due to the N-H and C=O stretching

signals due to the phenethyl group were observed at δ 2.88,

vibrations of amide were observed at 3425 and 1687 cm-1,

3.52, and 7.26-7.34 ppm in CD3OD.

respectively.

(Scheme 7)

The absorption banddue to the N-H and C=O stretching vibrations of amide

NH2

were observed at 3305 and 1636 cm-1, respectively.

(6)

(11)

+

in dry DMF

NH2

+

The

O
H3CO

in dry CH3CN

O
N
N

O
O
N

N
H

N
H
N

N
H

O

(14)

Scheme 9

N

N

N
O
CH3

Measurement of UV-VIS and fluorescence spectra of
the derivatized products
UV-VIS and fluorescence spectral data of the(12)

Scheme 7

derivatized

products(12),summarized in Table 4.

To a solution of pteridinedione (6) in dry CH2Cl2,

(13),

and

(14)

are

Absorption and emission

pyridine, and DMF was added cholesterol, and then the

maxima of compounds (12) and (13) were observed atreaction mixture was stirred at room temperature toaround 330 and 380 nm in MeCN, respectively.

give the product (13) as white solids.

From TLC

absorption and emission maxima of compounds (12)analysis, it was found that the derivatization completedand (13) were the almost same as compound (2a)within 10 minutes. (Scheme 8) The signals assignable

regardless of the ester or amide linkage.

The

to protons of the cholesteryl group were observed atTable 4

δ 0.70, 0.86, 0.92, 0.99-2.01, 2.47, 4.89, and 5.44 ppm
in CDCl3.

The absorption bands due to ester andamide C=O stretching vibrations were observed at 1731

UVa)/ λmax nm (ε)

Fluorescenceb)
λmax nm12329 (7266)

383

CH3

13

330 (6900)

381

CH3

14

291 (16130)

474

369 (5192)

473

Compd.

-1

and 1695 cm , respectively.
cholesterol(6)

UV-VIS and fluorescence spectral data of thederivatized products (12), (13), and (14) in
MeCN

H3C
H3C

in dry CH2Cl2 / pyridine/ DMF
H3C
O
O
N
N

a) Concentration: 1.0 x 10-4 M

Ob) Concentration: 1.0 x 10-6 M

N
O
N
CH3

From these results, pteridinedione (6) is applicable to a

(13)

new fluorescence derivatization reagent for amines andScheme 8

alcohols, although the emission maximum is fairly shortwavelength.

A solution of pteridinedione (11) and phenethylamine in

On the other hand, the absorption maxima

dry MeCN and DMF was stirred at room temperature toof compound (14) were observed at 291 and 369 nm.

give the product (14) as yellow solids.

The emission maximum of compound (14) were observed(Scheme 9)－59－

The fluorescence intensity of

2’,6’-N-Ph), 8.59 (1H, d, J = 2.4 Hz, C6-H), and 8.72 ppm

compound (14) was apparently stronger than those of(1H, d, J = 2.4 Hz, C7-H); IR (KBr): 3426 (νN-H), 1717

compounds (12) and (13).

The Stokes' shift of compound(νC=O), and 1677 cm-1 (νC=O).

EA Calcd for C15H12N4O4:(14) was remarkably larger than those of compounds (12)C, 57.69; H, 3.80; N, 17.94%.

Found: C, 57.47; H, 4.09;

and (13).

N, 17.97%

at around 470 nm.

From these results, pteridinedione (11) was

demonstrated to be applicable to a new fluorescence

3-(p-Ethoxycarbonyl)phenyl-1-methyl-2,4(1H,3H)-pteri

derivatization reagent for amines.

dinedione (2a)A solution of compound (1) (489 mg, 1.6 mmol) in dry

Experimental

DMF (50 mL) was added dropwise to a solution of NaH

Melting points were recorded on a Melting(73 mg of 60 weight% dispersion in paraffin oil, 1.8

Point Apparatus SMP3 in open capillaries and aremmol) in dry DMF (10 mL), and then the mixture was

uncorrected.

stirred at room temperature for 1 h.

General.

IR spectra were recorded on a JASCO

CH3I (426 mg, 3.0

FT/IR-470 plus Fourier Transform Infrared spectrometer.

mmol) in dry DMF (5 mL) was added dropwise to the

UV-VIS spectra were recorded on a JASCO V-550

mixture, and the reaction mixture was stirred at 80 ºC for

UV/VIS spectrophotometer.

4.5 h.

Fluorescent spectra wererecorded on a JASCO FP-777 and JASCO FP-6500
spectrofluorometers.

After addition of EtOH (0.5 mL), the solvent wasevaporated off under reduced pressure, and then the

1

H-NMR spectra were recorded onorganic materials were extracted with CHCl3 (100 mL x 2).

JEOL JNMLA400D NMR spectrometer in CDCl3,

The organic layer was washed with H2O (100 mL x 4),

DMSO-d6, and CD3OD are reported in ppm (δ) downfieldsaturated NaCl aqueous solution (50 mL), and then dried

from internal Me4Si.

over anhydrous Na2SO4.

Thin-layer chromatography (TLC)

After evaporation of the solvent,was performed on silica gel 60F-254 with a 0.2 mm Layer

the residue was purified by column chromatography on

thickness.

Column chromatography was carried out withsilica gel with CHCl3 : acetone : EtOH (100 : 10 : 1)

KANTO CHEMICAL silica gel 60N (spherical, neutral,

mixture to give the pure product (2a) as pale yellow

63-210 µm).

powders.

Flash column chromatography was carriedout with Merck silica gel 60 (40-63 µm).

Yield: 493 mg (96%); mp: 238-239 ºC;

1

Recycle GPC

H-NMR (δ, CDCl3, 400 MHz): 1.41 (3H, t, J = 7.2 Hz,

was carried out with a JAI LC-9201 equipped with aCH3), 3.76 (3H, s, 2-N-CH3), 4.42 (2H, q, J = 7.2 Hz, CH2),

MDL-101 by using columns packed with a JAIGEL-1H

7.39 (2H, d, J = 8.8 Hz, 3’,5’-N-Ph), 8.23 (2H, d, J = 8.8

and JAIGEL-2H. High performance liquid chromatography

Hz, 2’,6’-N-Ph), 8.65 (1H, d, J = 2.2 Hz, C6-H), and 8.71

(HPLC) was carried out with JASCO 800-PU, a 821-FP

ppm (1H, d, J = 2.2 Hz, C7-H); IR (KBr): 1726 (νC=O),

equipped with a JASCO 807-IT integrator by using a1691(νC=O), and 1282 cm-1 (νC-O).

column packed with a Crestpak C18T-5.

C16H14N4O4•0.5H2O: C, 57.31; H, 4.51; N, 16.71%.

Combustion

EA Calcd foranalyses were performed on a Perkin Elmer 2400 Series II

Found: C, 57.22; H, 4.26; N, 16.50%

CHNS/O analyzer.

The following products (2b and 2c) were prepared3-(p-Ethoxycarbonyl)phenyl-2,4(1H,3H)-pteridine-acording to the similar manner to compound (2a).

dione (1)3-(p-Ethoxycarbonyl)phenyl-1-benzyl-2,4(1H,3H)-pteri

A mixture of methyl 3-amino-2-pyrazinecarboxylate

dinedione (2b) as white crystals; yield: 258 mg (64%);(529 mg, 3.5 mmol) and ethyl p-isocyanatobenzoate (950

mp: 215-216 ºC; 1H-NMR (δ, CDCl3, 400 MHz): 1.41 (3H,

mg, 5 mmol) in dry pyridine (100 mL) was refluxed at 135

t, J = 7.1 Hz, CH3), 4.41 (2H, q, J = 7.1 Hz, CH2), 5.56

ºC for 9 h.

(2H, s, 1-N-CH2Ph), 7.31-7.35 (3H, m, 1-N-CH2Ph), 7.39

After evaporation of the solvent, ether wasadded to the residue for washing.

The collected crude(2H, d, J = 8.8 Hz, 3’,5’-N-Ph), 7.53-7.56 (2H, m,product was recrystallized from MeOH to give the pure

1-N-CH2Ph), 8.22 (2H, d, J = 8.8 Hz, 2’,6’-N-Ph), 8.65

product (1) as yellow crystals.

Yield: 480 mg (45%); mp:(1H, d, J = 2.2 Hz, C6-H), and 8.73 ppm (1H, d, J = 2.2 Hz,

280-285 ºC; 1H-NMR (δ, DMSO-d6, 400 MHz): 1.34 (3H,

C7-H); IR (KBr): 1726 (νC=O), 1687 (νC=O), 1273 (νC-O),t, J = 7.1 Hz, CH3), 4.36 (2H, q, J = 7.1 Hz, CH2), 7.52

747 and 702 cm-1 (δC-H).

EA Calcd for C22H18N4O4: C,(2H, d, J = 8.3 Hz, 3’,5’-N-Ph), 8.09 (2H, d, J = 8.3 Hz,

65.66; H, 4.51; N, 13.92%.

Found: C, 65.66; H, 4.49; N,－60－

13.88%MHz): 1.39 (3H, t, J = 7.1 Hz, CH3), 3.80 (3H, s, N-CH3),

3-(p-Ethoxycarbonyl)phenyl-1-propyl-2,4(1H,3H)-pteri

4.36 (2H, q, J = 7.1 Hz, CH2), 7.54-7.99 (4H, m, -NH-Ph),

dinedione (2c)as white powders; yield: 264 mg (74%);

7.99 (1H, d, J = 2.2 Hz, C5-H), and 8.00 ppm (1H, d, J =1

mp: 184-186 ºC; H-NMR (δ, DMSO-d6 at 120 ºC, 400

2.2 Hz, C6-H); IR (KBr): 3316 (νN-H), 1736 (νC=O), 1695

MHz): 0.95 (3H, t, J = 7.5 Hz, 1-N-CH2CH2CH3), 1.37

(νC=O), and 770 cm-1 (δC-H).

(3H, t, J = 7.1, Hz, CH3), 1.77 (2H, sext, J = 7.3 Hz,[Method B]: To a solution of compound (2a) (103 mg, 0.3

1-N-CH2CH2CH3), 4.23 (2H, t, J = 7.3 Hz, 1-N-mmol) in MeOH (360 mL) was added 1M NaOH (10 mL,

CH2CH2CH3), 4.39 (2H, q, J = 7.1 Hz, CH2), 7.50 (2H, d,

10 mmol), and the mixture was stirred at room temperature

J = 8.5 Hz, 3’,5’-N-Ph), 8.08 (2H, d, J = 8.5 Hz,for 4 h.

2’,6’-N-Ph), 8.62 (1H, d, J = 2.2 Hz, C6-H), and 8.78 ppm

was added H2O (50 mL) and CHCl3 (50 mL).

(1H, d, J = 2.2 Hz, C7-H); IR (KBr): 1728 (νC=O), 1692

aqueous layer was adjusted about pH 2 with 1 M HCl,

-1

After evaporation of the solvent, to the residue
The(νC=O) and 1273 cm (νC-O).

EA Calcd for C18H18N4O4: C,

extracted with CHCl3 (200 mL x 2), and then the CHCl3

61.01; H, 5.12; N, 15.81%.

Found: C, 60.94; H, 4.91; N,layer was dried over anhydrous Na2SO4. After evaporation

15.70%of the solvent, the residue was recrystallized from AcOEt

3-(p-Carboxy)phenyl-1-methyl-2,4(1H,3H)-pteridine-di

to give the pure product (5) as pale yellow crystals.

one (5)

Yield: 53 mg (58%); 1H-NMR (δ, CD3OD, 400 MHz):[Method A]: To a solution of 3-(p-ethoxycarbonyl)-3.72 (3H, s, 2-N-CH3), 7.47 (2H, d, J = 8.5 Hz, 3’,5’-N-Ph),phenyl-1-methyl-2,4(1H,3H)-pteridinedione (100 mg, 0.31

8.18 (2H, d, J = 8.5 Hz, 2’,6’-N-Ph), 8.59 (1H, d, J = 2.4mmol) in MeOH:DMF (1:1) mixture (50 mL) was added

Hz, C6-H), and 8.81 ppm (1H, d, J = 2.4 Hz, C7-H); IR

1M NaOH (0.6 mL, 0.6 mmol), and the mixture was(KBr): 3077 (νO-H), 1718 (νC=O), 1663 (νC=O), and 779 cm-1

stirred at room temperature for 2.5 h.

(δC-H).

An additional 1 M

EA Calcd for C14H10N4O4: C, 56.38; H, 3.38; N,

NaOH (0.6 mL, 0.6 mmol) was added to the mixture and18.78%.

the reaction mixture was stirred at room temperature for

3-(p-Chlorocarbonyl)phenyl-1-methyl-2,4(1H,3H)-

another 3 h.

pteridinedione (6)After evaporation of the solvent, to theresidue was added H2O (50 mL) and CHCl3 (50 mL).

Found: C, 56.25; H, 3.14; N, 18.55%.

A solution of compound (5) (30 mg, 0.1 mmol) in

The aqueous layer was adjusted about pH 2 with 10%

SOCl2 (3 mL, 0.3 mmol) was refluxed for 8 h.

citric acid and extracted with CHCl3 (200 mL x 2), whichsolvent was evaporated under reduced pressure to give thewas washed with H2O (100 mL) and then dried overproduct (6) as pale yellow powders; IR (KBr): 1770 (νC=O,

anhydrous Na2SO4. After evaporation of the solvent, the-1COCl), 1727 (νC=O), and 1683 cm (νC=O)

residue was recrystallized from AcOEt to give the pure

Methyl 3-amino-6-bromopyrazine-2-carboxylate (7)product (3) as yellow powders.

The

On the other hand, theTo a solution of commercially available methyl

CHCl3 layer was evaporated off under reduced pressure

3-amino-2-pyrazinecarboxylate (1.53 g, 10 mmol) andand the residue was purified by column chromatography

Bu4NBr3 (6.79 g, 14 mmol) in dry CHCl3 (25 mL) wason silica gel with CHCl3:acetone:EtOH (100:10:1) mixture

added dry pyridine (2 mL), and then the mixture wasto give the product (4) as yellow powders.

refluxed for 15 h.

However,

To the reaction mixture was added

compound (5) could not be obtained.

H2O (100 mL), and then the aqueous layer was extracted

3-(Methylamino)pyrazine-2-carboxylic acid (3): Yield:with CHCl3 (100 mL x 4).

1

The combined organic layers

23 mg (48%); H-NMR (δ, CDCl3, 400 MHz): 3.10 (3H, d,were washed with 10% NaHSO3 (30 mL), saturated NaCl

J = 4.9 Hz, CH3), 7.74 (1H, d, J = 2.2 Hz, C5-H), 7.90 (1H,

aqueous solution (30 mL), and then dried over anhydrous

br, NH), and 8.36 ppm (1H, d, J = 2.2 Hz, C6-H); IR

Na2SO4.

After evaporation of the solvent, the residue-1(KBr): 3365 (νN-H), 2845 (broad, νO-H), and 1679 cm

was purified by column chromatography on silica gel with

(νC=O).

EA Calcd for C6H7N3O2: C, 47.06; H, 4.61; N,

CHCl3:acetone:EtOH (100:10:1) mixture to give the pure27.44%.

Found: C, 47.25; H, 4.57; N, 27.19%.

product (7) as yellow powders.

Yield: 1.14 g (49 %);12-[(p-Ethoxycarbonyl)phenyl]amido-3-(methylamino)pH-NMR (δ, CDCl3, 400 MHz): 3.98 (3H, s, CH3) and1

yrazine (4): Yield: 11 mg (12 %); H-NMR (δ, CDCl3, 400

8.29 ppm (1H, s, C5-H); IR (KBr): 3416 and 3363 (νN-H),－61－

1701 (νC=O), and 516 cm-1 (νC-Br).

mg, 0.01 mmol, 5 mol%), triphenyl phosphine (7.2 mg,

EA Calcd for

C6H6BrN3O2: C, 31.06; H, 2.61; N, 18.11%.

0.03 mmol), p-methoxyphenylboronic acid (34 mg, 0.2

Found: C,

31.19; H, 2.37; N, 17.82%mmol), and 2.0 M Na2CO3 (0.22 mL), and the reaction

3-(p-Ethoxycarbonyl)phenyl-6-bromo-2,4(1H,3H)-

mixture was refluxed for 15 h under argon atmosphere.

pteridinedione (8)The same work-up as method I, but compound (9) could
not be obtained.

A solution of compound (7) (1.05 g, 4.5 mmol) andethyl p-isocyanatobenzoate (1.77 g, 9.2 mmol) in dry
pyridine (60 mL) was refluxed for 11 h.

[Method III]: To a solution of compound (8) (157 mg, 0.4

Aftermmol) in dry DMF (6 mL) was added bis(triphenyl-

evaporation of the solvent, ether (100 mL) was added tophosphone)palladium(II) dichloride (15 mg, 0.02 mmol,

the residue for washing. The collected crude product was

5 mol%), triphenyl phosphine (11 mg, 0.04 mmol),recrystallized from MeOH to give the product (8) as brown

p-methoxyphenylboronic acid (62 mg, 0.4 mmol), andcrystals.

1

K3PO4 (196 mg, 0.9 mmol).

Yield: 782 mg (58 %); H-NMR (δ, CDCl3, 400

The reaction mixture wasMHz): 1.41 (3H, t, J = 7.1 Hz, CH3), 4.42 (2H, q, J = 7.1

stirred at 80 ºC for 5 h, at 90 ºC for 15 h, at 100 ºC for 8

Hz, CH2), 7.38 (2H, d, J = 8.1 Hz, 3’,5’-N-Ph), 8.23 (2H, d,h, at 110 ºC for 5 h, at 120 ºC for 12 h, and at 130 ºC for

J = 8.1 Hz, 2’,6’-N-Ph), and 8.70 ppm (1H, s, C7-H); IR

1 day under argon atmosphere in which the temperature(KBr): 1739 (νC=O), 1693 (νC=O), 1292 (νC-O), 869 (δC-H),of oil bath was gradually elevated in order to complete-1

and 517 cm (νC-Br).

the reaction.

3-(p-Ethoxycarbonyl)phenyl-6-(p-methoxy)phenyl-2,4(1the pure product (9) in 30 % yield.
[Method IV]: A solution of compound (8) (201 mg, 0.5

H,3H)-pteridinedione (9)mmol), bis(triphenylphosphone)palladium(II) dichloride[Method I]: A solution of compound (8) (196 mg, 0.5
To the(18 mg, 0.026 mmol, 5 mol%), triphenylphosphine (14bis(triphenylphosphone)

mg, 0.05 mmol), p-methoxyphenylboronic acid (85 mg,mmol) in dioxane (20 mL) was degassed.
mixture

was

added

The same work-up as Method I afforded

palladium(II) dichloride (19 mg, 0.026 mmol, 5.3

0.56 mmol), and K2CO3 (2.5 mg, 0.02 mmol) in drymol%), triphenylphosphine (15 mg, 0.05 mmol),DMF (6 mL) was stirred at 120 ºC for 7 days underp-methoxy- phenylboronic acid (77 mg, 0.5 mmol), andargon atmosphere.

2.0 M Na2CO3 (0.6 mL), and then the reaction mixture

afforded the pure product (9) in 25 % yield.

The same work-up as Method I

was refluxed for 10 h under argon atmosphere.

After

3-(p-Carboxy)phenyl-6-(p-methoxy)phenyl-2,4-cooling, the precipitate was filtered off.

After

(1H,3H)-pteridinedione (10)evaporation of the filtrate, H2O (30 mL) was added to

To a solution of compound (9) (51 mg, 0.12 mmol) in

the residue, the aqueous layer was extracted with CHCl3

MeOH (100 mL) was added 1 M NaOH (5 mL, 5 mmol),(40 mL x 5), and the organic layer was dried overand the mixture was stirred at room temperature for 5 days.

anhydrous Na2SO4.

After evaporation of the solvent,An additional 4 M NaOH (3 mL, 12 mmol) was added to

the residue was purified by column chromatography on

the mixture, and then the reaction mixture was stirred atsilica gel with CHCl3:acetone:EtOH (100:20:4) mixture

room temperature for 3 days.

to give the product (9) as yellow solids.

solvent, to the residue was added H2O (10 mL) and CHCl3

Yield: 40 mg

1

After evaporation of the(20 %); H-NMR (δ, CDCl3 400 MHz) : 1.41 (3H, t, J =(10 mL).

The aqueous layer was adjusted about pH 2

7.1 Hz, CH3), 3.89 (3H, s, OCH3), 4.42 (2H, q, J = 7.1

with 1 M HCl, extracted with CHCl3 (50 mL x 4). At thisHz, CH2), 7.04 (2H, d, J = 8.8 Hz, 2”,6”-Ph), 7.42 (2H,stage, the yellow solids in the aqueous phase wered, J = 8.3 Hz, 3’,5’-N-Ph), 8.08 (2H, d, J = 8.8 Hz,

collected by suction filtration, and they were found to be3”,5”-Ph), 8.24 (2H, d, J = 8.3 Hz, 2’,6’-N-Ph), 8.64

the desired product (10).

Yield: 11.5 mg (25 %);(1H, br, NH), and 9.03 ppm (1H, s, C7-H); IR (KBr) :1

3518 (νN-H), 1735 (νC=O), 1690 (νC=O), 1275 (νC-O)and

7.12 (2H, d, J = 9.0 Hz, 2”,6”-Ph), 7.50 (2H, d, J = 8.8 Hz,

840 cm-1 (δC-H).[Method II]: To a solution of compound

3’,5’-N-Ph), 8.08 (2H, d, J = 9.0 Hz, 3”,5”-Ph), 8.13 (2H,

(8) (78 mg, 0.2 mmol) in benzene (18 mL) was addedd, J = 8.8 Hz, 2’,6’-N-Ph), 9.30 (1H, s, C7-H), and 12.4

bis- (triphenylphosphone)palladium(II) dichloride (8.0ppm (1H, s, OH); IR (KBr): 3416 (νN-H), 3060 (broad,

H-NMR (δ, DMSO-d6, 400 MHz): 3.83 (3H, s, OCH3),－62－

νO-H), 1692 (νC=O), and 833 cm-1 (δC-H).

phenethylamine (2 drops), and then the reaction mixture

3-(p-Chlorocarbonyl)phenyl-6-(p-methoxy)phenyl-

was stirred for 9 h at room temperature.

2,4(1H,3H)-pteridinedione (11)evaporation of the solvent, the residue was washed withAfter

H2O to give the derivatization product (14) as yellow

A solution of compound (10) (11.5 mg, 0.03 mmol) in
The solvent was

solids.; 1H-NMR (δ, CD3OD, 400 MHz): 2.95 (2H, t, J =evaporated off under reduced pressure to give the product7.3 Hz, -CONHCH2CH2-Ph), 3.63 (2H, q, J = 7.3 Hz,(11) as orange powders; IR (KBr): 3425 (νN-H), 1796 (νC=O,-ONHCH2CH2-Ph), 3.80 (3H, s, OCH3), 7.08 (2H, d, J =SOCl2 (1 mL) was refluxed for 4 h.

-1COCl), 1744 (νC=O), and 839 cm

9.0(δC-H).

Hz,

2”,6”-Ph),

7.26-7.33

(5H,

m,

-CONH-

3-(p-Phenethylamide)phenyl-1-methyl-2,4(1H,3H)-

CH2CH2-Ph), 7.48 (2H, d, J = 8.8 Hz, 3’,5’-N-Ph), 7.93

pteridinedione (12)(4H, d, J = 8.8 Hz, 2’,6’-N-Ph), 8.14 (2H, d, J = 9.0, 3”,5”-

To a solution of compound (6) (10 mg, 0.03 mmol) inPh), and 9.19 ppm (1H, s, C7-H) ; IR (KBr): 3425 (νN-H),

dry acetonitrile (30 mL) was added phenethylamine (37.61735 (νC=O), 1687 (νC=O), 1609 (νC=C), and 836 cm-1 (δC-H).

mg, 0.3 mmol), and the reaction mixture was stirred for 24
h at room temperature.

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After filtration of the solvent, thefiltrate was evaporated off under reduced pressure to givethe solid (13 mg) as pale yellow powders.

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The solid was

Ohkura, M. Kai, and H. Nohta, Bunseki Kagaku, 43,

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259 (1994); c) K. Imai: Advances in Chromatography,

product (12) as pale yellow powders.

Yield: 10 mg

27, p. 215. Marcel Dekker Inc., New York (1987); d) Y(80%); H-NMR (δ, CD3OD, 400 MHz): 2.88 (2H, t, J =Ohkura and H. Nohta: Advances in Chromatography,

7.3 Hz, -CONHCH2CH2-Ph), 3.52 (2H, q, J = 6.4 Hz,

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1

3.76 (3H, s, 1-N-CH3), 7.39 (2H, d, J = 8.5 Hz, 3’,5’-N-Ph),

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－64－