Synthesis and biological activities of 2,4-diaminopteridine derivatives

Article abstract of DOI:10.1002/ardp.200800192

Substituted 2,4-diaminopteridine derivatives 10a-10l were prepared in moderate to good yield. Their structures were confirmed by ¹H-NMR and MS spectroscopy, as well as by elemental analysis. Their inhibitory properties against inducible nitric oxide synthase (iNOS) were evaluated in vitro. Biological tests indicated that compound 10a, 10d, 10e, 10h, 10i, and 10l showed potent inhibitory activities similar to that of methotrexate (MTX), while the activities of compound 10b, 10c, 10f, 10g, 10j, and 10k are stronger than MTX. Two compounds, i. e., 10b (IC₅₀ = 18.85 μM) and 10i (IC ₅₀ = 24.08 μM) were further studied for their effect on septic shock in rats and immunologically liver injured mice (in vivo). The results demonstrated that 10b and 10i had the capacity to increase the blood pressure in septic shock and showed notable protective activities on immunological hepatic injury.

Full text of DOI:10.1002/ardp.200800192

274
Arch. Pharm. Chem. Life Sci. 2009, 342, 274–280
Full Paper
Synthesis and Biological Activities of 2,4-Diaminopteridine
Derivatives
Fei Ma, Gang Lꢀ, Wei-Fen Zhou, Qiu-Juan Wang, Yi-Hua Zhang, and Qi-Zheng Yao
School of Pharmacy, China Pharmaceutical University, Nanjing, P. R. China
Substituted 2,4-diaminopteridine derivatives 10a–10l were prepared in moderate to good yield.
Their structures were confirmed by 1H-NMR and MS spectroscopy, as well as by elemental anal-
ysis. Their inhibitory properties against inducible nitric oxide synthase (iNOS) were evaluated in
vitro. Biological tests indicated that compound 10a, 10d, 10e, 10h, 10i, and 10l showed potent
inhibitory activities similar to that of methotrexate (MTX), while the activities of compound
10b, 10c, 10f, 10g, 10j, and 10k are stronger than MTX. Two compounds, i. e., 10b (IC50
=
18.85 lM) and 10i (IC50 = 24.08 lM) were further studied for their effect on septic shock in rats
and immunologically liver injured mice (in vivo). The results demonstrated that 10b and 10i had
the capacity to increase the blood pressure in septic shock and showed notable protective activ-
ities on immunological hepatic injury.
Keywords: Inhibitor of nitric oxide synthase / Synthesis / Tetrahydrobiopterin /
Received: December 9, 2008; accepted: December 12, 2008
DOI 10.1002/ardp.200800192
ing site of NOS may be an ideal target for selective phar-
macological intervention [4].
Introduction
Some structural analogs of BH4 have been shown to
inhibit the various forms of NOS, for example, the 4-ami-
nopteridine nucleus, and the substitution pattern at the
2-, 4-, 5-, 6-, and 7-position was systematically varied [5].
Encouraged by these findings, we designed and synthe-
sized a series of analogues (10a–10l) of 2,4-diamino pteri-
dine, and determined the effects that twelve derivatives
inhibited inducible nitric oxide synthase (iNOS). In addi-
tion, compounds 10b and 10i were tested for their
capacity of increasing the blood pressure in septic-shock
rats and their protective effects on immunological hep-
atic injured mice.
Nitric oxide (NO) is a mediator with protean functions. It
is involved in signalling in the cardiovascular, gastroin-
testinal, genitourinary, respiratory, and nervous systems,
and disordered NO generation has been implicated in a
wide range of diseases [1]. Nitric oxide synthase (NOS) cat-
alyzes the oxidation of a guanidino N-atom of L-arginine
to NO with concomitant formation of L-citrulline [2]. A
critical aspect of NOS function is the requirement for the
cofactor tetrahydrobiopterin (BH4). Maintenance and sta-
bilization of NOS dimers is dependent on BH4, and BH4
also plays a direct role in the multistep oxidation of argi-
nine through the N-hydroxy-L-arginine intermediate and
the subsequent generation of NO [3]. Thus, the BH4 bind-
Results and discussion
Correspondence: Qi-Zheng Yao, School of Pharmacy, China Pharma-
ceutical University, Nanjing 210009, P. R. China.
E-mail: qz_yao@yahoo.com.cn
Synthesis
Compounds 10a–10l were prepared according to
Scheme 1. The general principle for the synthetic
approach of 2-amino-4-arylamino / isopropylamino-6-
arylpteridines was based on the condensation of 2,5,6-tri-
amino-4-arylamino / isopropylaminopyrimidine deriv-
atives 6a–6d with different substituted phenylglyoxal-
Fax: +86 25 86634730
Abbreviations: aminoguanidine (AG); ceral ligation and perforation
(CLP); cyclophosphamide (CY); glutamate pyruvate transaminase (GPT);
glutamic oxalacetic transaminase enzyme (GOT); hepatic necrosis (HN);
inducible nitric oxide synthase (iNOS); methotrexate (MTX); nitric oxide
(NO); nitric oxide synthase (NOS)
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Arch. Pharm. Chem. Life Sci. 2009, 342, 274–280
2,4-Diaminopteridine Derivatives
275
Reactions and conditions: a) NaNO2, 22.0% HCl, Urea, 0 – 58C, 0.5 h; b) 4-chloro-2,6-diaminopyrimidine, CH3COONa, H2O, r.t., 16 h, 81%; c)
DMF, 0 – 58C, 5 h, 86 – 93%; d) (1) EtOH, 5% HCl, zinc powder, 708C, 0.5 h; (2) 708C, 1 h, 78 – 85%; e) 1. SeO2, 1,4-dioxane / H2O (25 : 1),
508C, 0.5 h; 2. reflux, 30 h, 62 – 68%; f) MeOH, H2O, 3.6% HCl, pH 3 – 4, 608C, 5 h, 56 – 64%; g) MeOH, N2, reflux, 4 h, 30 – 62%.
Scheme 1. The synthesis of substituted 2,4-diaminopteridine derivatives.
monoximes 9a–9c under refluxing MeOH [6]. p-Chloroa- steps were in the range of 86–93% and 78–88%, respec-
niline 1 was diazotized with NaNO2 and 22% HCl, and tively.
subsequent coupling with 2,6-diamino-4-chloropyrimi-
Acetophenone derivatives 7a–7c were treated with
dine 2 gave 3 in 81% yield [7]. In 3, the Cl-atom of the pyr- SeO2 in 1,4-dioxane / H2O (25 : 1) to provide the corre-
imidine ring is sufficiently activated to be nucleophili- sponding 8a–8c in 62–68% yield; then, 8a–8c treated
cally displaced by acyclic or arylic amines 4a–4d in dehy- with acetone oxime [10] in MeOH (pH 3–4) at 508C led to
drated alcohol to form 5a–5d, which, on reduction with 9a–9c in 56–64% yields [8]. Finally, boiling a solution of
Zn powder, generated 6a–6d [9]. The yields of these two the 2,5,6-triamino-4-arylamino / isopropylaminopyrimi-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 274–280
Table 1. Structures and yields of substances 10a–10l.
Compound
R3
NR1R2
Yield (%)
10a
10b
10c
10d
10e
10f
10g
10h
10i
H
H
H
H
Me
Me
Me
Me
MeO
MeO
MeO
MeO
HNCH(Me)2
62
35
37
32
59
32
51
30
33
35
34
40
HNC6H4COOEt
MeNC6H4COOCH3
HNC6H4COGluEt2
HNCH(Me)2
HNC6H4COOEt
MeNC6H4COOCH3
HNC6H4COGluEt2
HNCH(Me)2
10j
10k
10l
HNC6H4COOEt
MeNC6H4COOCH3
HNC6H4COGluEt2
Figure 1. Effect of compound aminoguanidine (AG), 10b, and
10i on septic-shock rats.
Table 2. Inhibitive activities (IC50 in (M) of Pteridine derivatives
10a–10l against iNOS.
teridine derivatives 10i–10l had significantly more
inhibitive activity than the former two series.
Pharmacological results suggest that modification at
the 4-amino substituents of pteridine by alkyl / benzoate
appeared crucial for their bioactivities. The compounds
with [p-(ethoxylcarbonyl)phenyl]amino or [p-(methoxy-
carbonyl)phenyl]methylamino functional groups in the
4-position displayed more potent inhibitive activities
than those with isopropylamino or {p-[(N-L-glutamate)car-
bonyl]phenyl}amino groups. In the structure of the mur-
ine iNOS oxygenase domain, the BH4 is axially co-oridi-
nated to the haem and hydrogen bond bridges from the
BH4 N(3) (directly) and O(4) (through a H2O molecule)
through heme propionate to the L-Arg a-amino group
[11, 12]. The cavity and the electron of the pterin with
aminobenzoate may perhaps more easily interfere with
hydrogen bonds between the BH4 and the heme propio-
nate.
Compound
IC50 (lM)
Compound
IC50 (lM)
10a
10b
10c
10d
10e
10f
31.01 l 1.90
18.85 l 0.82
18.22 l 1.71
42.37 l 1.82
28.14 l 1.73
16.85 l 0.52
24.59 l 1.43
10g
10h
10i
10j
10k
10l
16.77 l 0.65
37.21 l 0.63
24.08 l 0.81
14.02 l 1.42
15.08 l 1.20
33.77 l 2.40
MTX
a)
Methotrexate (MTX) used as a positive control.
Inhibition of BH4 (2 lM) stimulated NOS total activity at an
inhibitor concentration of 100 lM.
b)
The data were the mean l SD obtained from three independ-
ent experiments.
dines hydrochloride 6a–6e with 9a–9c gave the corre-
sponding 2-amino-4-arylamino/isopropylamino-6-aryl
Based on the in-vitro inhibitive activities of BH4 deriv-
atives against iNOS, compound 10b and 10i were selected
for the determination of septic shock in rats and immu-
nological liver injured mice. Septic shock was induced by
topical application of ceral ligation and perforation (CLP)
[13]. 12 h after laparotomy, all CLP animals demonstrated
reduced activity, piloerection, and exudation around the
eyes and nose. Generalized peritonitis was confirmed
post mortem in all CLP animals. As shown in Fig. 1, 10b
and 10i demonstrated a similar effect as aminoguanidine
(AG) in elevating the MBP (mean blood pressure) signifi-
cantly (compare control group P a 0.01, Fig. 1), while the
MBP of the control rats tended to decline.
Immunological liver injury was induced by tail-vein
injection of 0.25 mg/10g Corynebacterium parvum (CP),
and ten day later, the mice became aggressive, irritable,
they hit the cage, and nibble on the forage and trough;
they are restless. [14]. Mucous membranes swelled all
over the body and multi-ulcer developed, especially in
pteridines 10a–10l in yields in the range of 30–62% (see
Table 1) [9].
The structures of the newly synthesized compounds
1
were characterized by H-NMR and MS spectroscopy, as
well as by elemental analysis.
Pharmacology
All of the prepared compounds 10a–10l were evaluated
for their inhibitive activities against iNOS according to
the reported methods [10], and the results are listed in
the Table 2.
The data in Table 2 indicated that 10e and 10i exhib-
ited potent inhibitory activities similar to that of Metho-
trexate (MTX), while the activities of 10b, 10c, 10f, 10g,
10j, and 10k are stronger than that of MTX [10]. As com-
pared with 6-phenyl-pteridine derivatives 10a–10d, the
corresponding 6-(4-methylphenyl)pteridine derivatives
10e–10h showed a slight increase in inhibitive activities
against iNOS, respectively, while 6-(4-methoxyphenyl)p-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 274–280
2,4-Diaminopteridine Derivatives
277
Figure 4. Effect of compound CY, AG, 10b, and 10i on immuno-
logical liver injury in mice.
Figure 2. Effect of compound cyclophosphamide (CY), AG, 10b,
and 10i on immunological liver injury in mice. ** P a 0.01 and
P a 0.01.
##
reported by our group [10]. The inhibitory activities on
iNOS of these compounds was evaluated in vitro. Within
the series of compounds, 10b, 10c, 10e, 10f, 10g, 10i, 10j,
and 10k showed potent inhibitive activities against iNOS
similar to or better than that of MTX. Compound 10b and
10i showed similar effects as AG on rats with septic shock
and immunological liver injury in mice. Our results may
provide some guidance for the development of novel
pteridine compounds as nitric oxide synthase inhibitor.
Further, structure-activity relation studies and mechanis-
tic studies on this new class of pteridine compounds are
currently in progress.
The authors have declared no conflict of interest.
Figure 3. Effect of compound CY, AG, 10b and 10i on immuno-
logical liver injury in mice. ** P a 0.01 and ## P a 0.01.
Experimental
the footpad and the tail. Treatment with cyclophospha-
mide (CY), AG, and compounds 10b and 10i alleviated
these symptoms, while injection with lipopolysaccharide
(LPS) promoted death. Serum glutamate pyruvate transa-
minase (GPT), glutamic oxalacetic transaminase enzyme
(GOT), nitric oxide (NO) were assayed by commercially
available kits. Significantly elevated NO, GPT, and GOT
levels were reduced and immunological liver injury was
alleviated by treatment with 10b and 10i (control group P
a 0.01, Figs. 2, 3 ,4); these two compounds showed similar
effects as CY and AG.
Chemistry
Chemicals were bought from Shanghai Chemical Reagent Com-
pany (Shanghai, China) and were used without further purifica-
tion. Melting points were determined with the capillary tube
method, and the thermometer was uncorrected. Reaction
kinetics was checked using silica gel 60 F254 plates (250 lm;
Qingdao Ocean Chemical Company, China). Mass spectra were
obtained with a Hewlett-Packard 1100 LC-MS (Agilent, Palo Alto,
CA, USA). 1H-NMR spectra were run on a Bruker ARX-300 instru-
ment (Bruker Bioscience, Billerica, MA, USA), TMS as the internal
standard. Elemental analysis was performed by a Elementar
Vario EL III instrument (Heraeus GmbH, Hanau, Germany) for C,
H, and N and the results are within l0.5% of the theoretical val-
ues.
Conclusions
2,6-Diamino-4-chloro-5-(p-chlorophenyl)azopyrimidine 3
A solution of p-chloroaniline 1 (15.24 g, 0.12 mol) in 6 mol/L HCl
(79 mL) was cooled to 0–58C. Then, NaNO2 (8.30 g, 0.12 mol) in
H2O (30 mL) was added dropwise with stirring. After the addition
was completed, the solution was stirred for 15 min and checked
by iodine-starch-paper to give a blue color. Urea (3.00 g) was
In search of novel pteridine compounds, three series of
2,4-diamino-pteridine derivatives were designed and syn-
thesized. Compounds 10a–10g, 10i, 10j, and 10k are new
compounds, while 10h and 10l had already been
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F. Ma et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 274–280
added to destroy the excess HNO2. The diazonium salt solution 2
was then poured into a solution of 2,6-diamino-4-chloropyrimi-
dine (15.60 g, 0.11 mol) in H2O (300 mL) and stirred for 30 min.
NaOAc (42.00 g) was added, and the mixture was stirred at room
temperature for 16 h. The yellow precipitate was collected by fil-
tration and washed with H2O to afford 3 (26.40 g, 81%). M.p.:
268–2708C (dec.); ESI-MS: 282.1 [M – H]– .
centrated under reduced pressure. The precipitate was stirred
with 10% HCl-ether (15 mL) for 1 h, then the solution was poured
over and dried to afford 6a–6d.
2,5,6-Triamino-4-isopropylamino-pyrimidine
dihydrochloride 6a
Yield: 83%. M.p.: 233–2368C. ESI-MS: 205.2 [M + Na]+, C7H14N6
(182.23).
General procedure for the synthesis of 2,6-diamino-4-
substituted-5- (4-chlorophenyl)azopyrimidine 5a–5d
A mixture of 3 (4.00 g, 14.20 mmol) and acyclic or arylic amines
4a–4d (33.90 mmol) in DMF (15 mL) was heated at 708C for 5 h.
Then, the ice-water (20 mL) was added to the reaction and the
final mixture was cooled at room temperature. The obtained
solid was filtered, washed with H2O, and crystallized from EtOH
to afford 5a–5d.
2,5,6-Triamino-4-[(4-ethoxylcarbonylphenylamino]-
pyrimidine dihydrochloride 6b
Yield: 88%. M.p. 285–2878C. ESI-MS: 289.2 [M + H]+, C13H16N6O2
(288.31).
2,5,6-Triamino-4-[(4-methoxylcarbonylphenyl)-
methylamino]pyrimidine dihydrochloride 6c
Yield 78%. M.p.: 275–2778C. ESI-MS: 289.4 [M + H]+, C13H16N6O2
(288.31).
2,6-Diamino-4-isopropylamino-5-(4-
chlorophenyl)azopyrimidine 5a
1
Yield: 86%. M.p.: 221–2238C. H-NMR (DMSO-d6) d: 1.22 (d, J =
(S)-Diethyl2-[4-(2,5,6-triaminopyrimidin-4-
6.8 Hz, 6H), 4.08–4.28 (m, 1H), 5.52 (s, 2H), 6.17 (br, 2H), 7.45–
7.47 (dd, J = 6.6 Hz, J = 6.6 Hz, 4H). ESI-MS: 306.1 [M + H]+. Anal.
calc. for C13H16Cl1N7 (305.77): C, 51.07; H, 5.27; N, 32.07. Found:
C, 51.25; H, 5.47; N, 32.30.
ylamino)benzamido]pentanedioate dihydrochloride 6d
Yield: 82%. M.p.: 254–2578C. [a]20 + 22, c = 1 in water; ESI-MS:
D
446.5 [M + H]+, C20H27N7O5 (445.47).
General procedure for the synthesis of compounds 9a–9c
Selenium dioxide (36.96 g, 0.33 mol) was added to a solution of
H2O (10 mL) in dioxane (250 mL), and the mixture was heated to
50(C for 0.5 h. Then, 7a–7c (0.30 mol) was added, the solution
was heated to reflux for 30 h. After cooling down, the solution
was filtered to remove elemental selenium and the yellow-
orange filtrate was concentrated under reduced pressure. The
residue was recrystallized from H2O to obtain pure 8a–8c. Ace-
tone oxime was prepared according to the method described
elsewhere [10]. To a stirring mixture of 8a–8c (0.10 mol) in
MeOH (20 mL) was added H2O (70 mL). After addition of acetox-
ime (8.76 g, 0.12 mol), the mixture was acidified to pH 3 to 4
with 3.6% (w / v) aq. HCL solution. Stirring was continued at 608C
for 5 h. After cooling down in an ice-bath, the solution was fil-
tered and the residue was washed with water (3610 mL) to
afford 9a–9c.
2,6-Diamino-4-[(4-ethoxylcarbonylphenyl)amino]-5-(p-
chlorophenyl)azopyrimidine 5b
Yield: 88%. M.p.: 285–2878C. 1H-NMR (CDCl3) d: 1.34 (t, J = 6.0 Hz,
3H), 4.35 (q, J = 6.0 Hz, 2H), 5.48 (s, 2H), 6.15 (br, 2H), 7.45–7.47
(dd, J = 6.8 Hz, J = 6.8 Hz, 4H), 7.63–7.66 (m, 4H). ESI-MS: 412.2 [M
+ H]+. Anal. calc. for C19H18N7O2Cl1 (411.84): C, 55.41; H, 4.41; N,
23.81. Found: C, 55.25; H, 4.47; N, 23.38.
2,6-Diamino-4-[(4-methoxylcarbonylphenyl)-
methylamino]-5-(p-chlorophenyl)azopyrimidine 5c
Yield: 85%. M.p.: 268–2788C. 1H-NMR (CDCl3) d: 3.34 (s, 3H), 3.89
(s, 3H), 5.45 (s, 2H), 6.10 (br, 2H), 7.41–7.44 (dd, J = 7.8 Hz, J =
7.8 Hz, 4H); 7.63–7.67 (m, 4H). ESI-MS: 412.5 [M + H]+. Anal. calc.
for C19H18N7O2Cl1 (411.84): C, 55.41; H, 4.41; N, 23.81. Found: C,
55.35; H, 4.40; N, 23.68.
2-Oxo-2-phenylacetaldehyde oxime 9a
Yield: 64%. M.p.: 127–1298C. ESI-MS: 150.1 [M + H]+, C8H7N1O2
(149.15).
(S)-Diethyl2-{4-[2,6-diamino-5-[2-(4-chlorophenyl)-
diazenyl]pyrimidin-ylamino]benzamido}pentanedioate
hydrochloride 5d
1
Yield: 93%. M.p.: 178–1808C. H-NMR (DMSO-d6) d: 1.17 (t, J =
2-Oxo-2-(4-methylphenyl)acetaldehyde oxime 9b
Yield: 56%. M.p. 98–1008C. ESI-MS: 164.5 [M + H]+, C9H9N1O2
(163.17).
7.0 Hz, 3H), 1.22 (t, J = 7.0 Hz, 3H), 2.08–2.23 (m, 2H), 2.40–2.43
(m, 2H), 3.07 (bs, 2H), 4.04 (q, J = 7.0 Hz, 2H), 4.58–4.61 (m, 1H),
7.36 (d, J = 8.5 Hz, 2H), 7.53 (bs, 1H), 7.71–7.76 (m, 4H), 7.87 (d, J =
8.5 Hz, 2H), 8.16 (bs, 2H), 8.80 (bs, 1H). [a]2D0 + 24, c = 1 in water;
ESI-MS: 591.3 [M + Na]+.
2-Oxo-2-(4-methoxyphenyl)acetaldehyde oxime 9c
Yield: 64%. M.p.: 119–1008C. ESI-MS: 202.1 [M + Na]+, C9H9N1O3
(179.17).
General procedure for the synthesis of 2,5,6-triamino-4-
substituted-pyrimidine dihydrochloride 6a–6d
General procedure for the synthesis of compounds
10a–10l
Compounds 6a–6d (3.00 mmol) were dissolved in dry MeOH
(15 mL). Then 9a–9c (4.50 mmol) in dry MeOH (15 mL) was
slowly added under N2 atmosphere and the solution was stirred
To a stirring mixture of 5a–5d (4 mmol) in 20 mL alcohol, 5%
HCl (w / v) solution (15 mL) was added and the mixture was
heated at 708C, then Zn powder (2.62 g, 40 mmol) was portion-
wise added over 30 min. After adding, the mixture was heated at
708C for 1 h. The solution was filtered, and the filtrate was con-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 274–280
2,4-Diaminopteridine Derivatives
279
at reflux (ca. 658C) for 4 h. Then, the solution was evaporated to
dryness in vacuo. The residue was purified by column chromatog-
raphy (silica gel; CHCl3 / MeOH 20 : 1, v / v) to afford compounds
10a–10l.
2-Amino-4-[(p-methoxylcarbonylphenyl)methylamino]-6-
(4-methylphenyl)pteridine 10g
Yield: 51%, yellow solid. M.p.: 273–2758C. 1H-NMR (CDCl3) d: 2.45
(s, 3H), 3.68 (s, 3H), 3.97 (s, 3H), 5.41 (bs. s, 2H), 7.23 (d, J = 8.0 Hz,
2H), 7.37 (d, J = 8.0 Hz, 2H), 8.03 (d, J = 8.0 Hz, 2H), 8.11 (d, J =
8.0 Hz, 2H), 9.08 (s, 1 H). ESI-MS: 401.1 [M + H]+. Anal. calc. for
C22H20N6O2 (400.43): C, 65.99; H, 5.03; N, 20.99. Found: C, 65.87;
H, 5.29; N, 20.85.
2-Amino-4-isopropylamine-6-phenylpteridine 10a
Yield: 62%, yellow solid. M.p.: 269–2718C. 1H-NMR (CDCl3) d: 1.42
(d, J = 6.5 Hz, 6H), 4.56 (m, 1H), 6.22 (bs, 2H), 7.52 (m, 1H); 7.59 (m,
3H), 8.02 (m, 2H), 9.23 (s, 1H). ESI-MS: 281.1 [M + H]+. Anal. calc.
for C15H16N6 (280.32): C, 64.27; H, 5.75; N, 29.98. Found: C, 64.37;
H, 5.69; N, 29.89.
(S)-Diethyl2-[4-(2-amino-6-(4-methylphenyl)pteridin-4-
ylamino)benzamido]-pentanedioate 10h
Yield: 30%, yellow solid. M.p.: 235–2378C. 1H-NMR (CDCl3) d: 1.23
(t, J = 7.0 Hz, 3H), 1.31 (t, J = 7.0, 3H), 2.10–2.38 (m, 2H), 2.41 (s,
3H), 2.47–2.55 (m, 2H), 4.12 (q, J = 7.0 Hz, 2H), 4.25 (q, J = 7.0 Hz,
2H), 4.77–4.80 (m, 1H), 5.88 (bs. s, 2H), 7.21–7.35 (m, 3H), 7.87-
7.96 (m, 7H), 9.05 (bs. s, 1H), 9.19 (s, 1H). [a]2D0 +19 c = 1 in acetic
acid; ESI-MS: 558.3 [M + H]+. Anal. calc. for C29H31N7O5 (557.60): C,
62.46; H, 5.60; N, 17.58. Found: C, 62.06; H, 5.59; N, 17.52.
2-Amino-4-[(4-ethoxylcarbonylphenyl)amino]-6-
phenylpteridine 10b
Yield: 35%, yellow solid. M.p.: 275–2778C. 1H-NMR (CDCl3) d: 1.41
(t, J = 6.9 Hz, 3H) 4.39 (q, J =6.9 Hz, 2H), 6.06 (bs, 2H), 7.41–7.51
(bs, 3H), 7.64 (d, J = 8.1 Hz, 2H), 7.67 (d, J = 8.1Hz, 2H), 8.13 (d, J =
8.1 Hz, 2H), 9.23 (s, 1 H), 9.32 (s, 1H). ESI-MS: 387.2 [M + H]+. Anal.
calc. for C21H18N6O2 (386.40): C, 65.27; H, 4.70; N, 21.75. Found: C,
65.47; H, 4.69; N, 21.89.
2-Amino-4-isopropylamine-6-(4-methoxyphenyl)-
pteridine 10i
Yield: 33%, yellow solid. M.p.: 298–3008C. 1H-NMR (CDCl3) d: 1.42
(d, J = 6.5 Hz, 6H), 3.91 (s, 1H), 4.55 (m, 1H), 5.91 (bs, 2H), 7.39 (d, J
= 8.8 Hz, 2H), 7.48 (bs, 1H), 7.97 (d, J = 8.8 Hz, 2H), 9.17 (s, 1H). ESI-
MS: 311.1 [M + H]+. Anal. calc. for C16H18N6O1 (310.35): C, 61.92; H,
5.85; N, 27.08. Found: C, 61.77; H, 5.69; N, 28.29.
2-Amino-4-[(4-methoxylcarbonylphenyl)methylamino]-6-
phenylpteridine 10c
Yield: 37%, yellow solid. M.p.: 268–2708C. 1H-NMR (CDCl3) d: 3.68
(s, 3H), 3.96 (s, 3H), 5.36 (bs. s, 2H), 7.16–7.56 (m, 7H), 8.11 (d, J =
8.0 Hz, 2H), 9.10 (s, 1H). ESI-MS: 387.2 [M + H]+. Anal. calc. for
C21H18N6O2 (386.40): C, 65.27; H, 4.70; N, 21.75. Found: C, 65.17;
H, 4.51; N, 22.12.
2-Amino-4-[(p-ethoxylcarbonylphenylamino]-6-(4-
methoxyphenyl)pteridine 10j
Yield: 35%, yellow solid. M.p.: 262–2648C. 1H-NMR (CDCl3) d: 1.40
(d, J = 7.0 Hz, 3H), 3.89 (s, 3H), 4.37 (q, J = 7.0 Hz, 2H), 5.60 (bs, 2H),
7.05 (d, J = 7.5 Hz, 2H), 7.86 (d, J = 7.5 Hz, 2H), 7.98 (d, J = 7.5 Hz,
2H), 8.07 (d, J = 7.5 Hz, 2H), 9.11 (bs, 1H), 9.24 (s, 1H). ESI-MS:
417.3 [M + H]+. Anal. calc. for C22H20N6O3 (416.43): C, 63.45; H,
4.84; N, 20.18. Found: C, 63.55; H, 4.69; N, 20.14.
(S)-Diethyl2-[4-(2-amino-6-phenylpteridin-4-
ylamino)benzamido]pentanedioate 10d
Yield: 32%, yellow solid. M.p.: 228–2318C. 1H-NMR (CDCl3) d: 1.21
(t, J = 7.0 Hz, 3H), 1.32 (t, J = 7.0 Hz, 3H), 2.09–2.11 (m, 2H), 2.45–
2.53 (m, 2H), 4.07 (q, J = 7.0 Hz, 2H), 4.18 (q, J = 7.0 Hz, 2H), 4.55–
4.68 (m, 1H), 5.78 (bs. s, 2H), 7.18-7.31 (m, 3H), 7.85-7.91 (m, 7H),
9.01 (bs. s, 1H), 9.14 (s, 1H). ESI-MS: 544.2 [M + H]+. [a]2D0 + 20, c = 1
in acetic acid; Anal. calc. for C28H29N7O5 (543.57): C, 61.87; H,
5.38; N, 18.04. Found: C, 61.65; H, 5.41; N, 18.22.
2-Amino-4-[(p-methoxylcarbonylphenyl)methylamino]-6-
(4-methoxyphenyl)pteridine 10k
Yield: 34%, yellow solid. M.p.: 240–2428C. 1H-NMR (CDCl3) d: 3.70
(s, 3H), 3.87 (s, 3H), 3.96 (s, 3H), 5.66 (bs. s, 2H), 6.75 (d, J = 8.4 Hz,
2H), 7.16 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 8.12 (d, J =
8.4 Hz, 2H), 9.06 (s, 1H). ESI-MS: 439.1 [M + Na]+. Anal. calc. for
C22H20N6O3 (416.43): C, 63.45; H, 4.84; N, 20.18. Found: C, 63.53;
H, 4.69; N, 20.13.
2-Amino-4-isopropylamine-6-(4-methylphenyl)-pteridine
10e
Yield: 59%, yellow solid. M.p.: 318–3208C. 1H-NMR (CDCl3) d: 1.42
(d, J = 6.5 Hz, 6H), 4.01 (s, 1H), 4.55 (m, 1H), 5.91 (bs, 2H), 7.39 (d, J
= 8.1 Hz, 2H), 7.51 (bs, 1H), 7.91 (d, J = 8.1 Hz, 2H), 9.19 (s, 1 H). ESI-
MS: 295.1 [M + H]+. Anal. calc. for C16H18N6 (294.35): C, 65.74; H,
5.52; N, 28.75. Found: C, 65.57; H, 5.69; N, 28.89.
(S)-Diethyl2-[4-(2-amino-6-(4-methoxyphenyl)pteridin-4-
ylamino)benzamido]-pentanedioate 10l
Yield: 40%, yellow solid. M.p.: 237–2398C. 1H-NMR (CDCl3) d: 1.23
(t, J = 7.0 Hz, 3H), 1.30 (t, J = 7.0 Hz, 3H), 2.16–2.29 (m, 2H), 2.39–
2.48 (m, 2H), 3.71 (s, 3H), 4.12 (q, J = 7.0 Hz, 2H), 4.24 (q, J = 7.0 Hz,
2H), 4.70–4.75 (m, 1H), 6.50 (bs. s, 2H), 7.21 (bs. s, 1H), 7.45–7.60
(m, 3H), 7.85–8.16 (m, 6H), 8.79 (bs. s, 1H), 9.12 (s, 1H). [a]2D0 +19,
c=1 in acetic acid; ESI-MS: 574.2 [M + H]+. Anal. calc. for
C29H31N7O6 (573.60): C, 60.72; H, 5.45; N, 17.09. Found: C, 60.53;
H, 5.51; N, 17.21.
2-Amino-4-[(p-ethoxylcarbonylphenylamino]-6-(4-
methylphenyl)pteridine 10f
Yield: 32%, yellow solid. M.p.: 262–2648C. 1H-NMR (CDCl3) d: 1.38
(d, J = 7.0 Hz, 3H), 2.94 (s, 3H), 4.34 (q, J = 7.0 Hz, 2H), 5.96 (bs, 2H),
7.26 (d, J = 4.6 Hz, 2H), 7.84 (d, J = 7.3 Hz, 2H), 7.87 (d, J = 7.3 Hz,
2H), 8.00 (d, J = 7.3 Hz, 2H), 8.99 (bs. s, 1H), 9.16 (s, 1H). ESI-MS:
401.2 [M + H]+. Anal. calc. for C22H20N6O2 (400.43): C, 65.99; H,
5.03; N, 20.99. Found: C, 65.54; H, 4.74; N, 21.29.
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280
F. Ma et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 274–280
divided into six groups: Group 1, control; Group 2, model (HN);
Group 3, HN + cyclophosphamide (CY); Group 4, HN + aminogua-
nidine (AG); Group 5, HN + compound 10b; Group 6, HN + com-
pound 10i [14]. On the first day, a dose of 0.5 mg Corynebacterium
parvum was injected into each mouse of group 2 to group 6 via
the tail vein. On the 5th, 7th, and 9th day, the mice in the
treated groups were injected with cyclophosphamide, amino-
guanidine, compound 10b, or compound 10i (30 mg/kg, i.p.),
respectively. On the 10th day, a dose of 10 lg LPS was injected
into each mouse. For the control group, we injected normal sal-
ine. 12 h later, mice were sacrificed and blood samples were col-
lected. The serum content of glutamic-pyruvic transaminase
(GPT), glutamic-oxalacetic transaminase (GOT), and nitric oxide
(NO) were determined. All data were expressed as mean l S.D.
Differences between the groups were compared with Student's t-
test.
Pharmacology
Enzyme activity
The inhibitory activities of compounds 10a–10l against the
NOS-II (i-NOS) enzyme were evaluated in vitro according to the
manufacturer's (Jiancheng, Nanjing, China) instructions, with
2,4-diamino-biopterin (ABP) as the positive control. NOS-II was
incubated for 20 min at pH = 7.2 and 378C in a mixture contain-
ing 2 lM tetrahydro-L-biopterin, 50 nM CaM, 1 mM CaCl2, 5 lM
FAD, 10 lM FMN, 250 lM 3-[(3-cholamidopropyl)-dimethylam-
monio]-2-hydroxy-1-propanesulfonat, 50 mM triethanolamine
(TEA), 1 mM NADPH, 7 mM GSH, and 50 lM L-arginine. Then, the
reaction was stopped by adding ice-cold acetate buffer (pH = 5.5)
and the optical density was measured at 530 nm. The IC50 values
were calculated according to the Logit method after getting the
inhibitory rate. NOS-II and tetrahydro-L-biopterin was purchased
from from Sigma-Aldrich (St. Louis, MI, USA). All other chemicals
used were of analytical grade or higher purity. IC50 values were
expressed in lM. The inhibitory activities of 2,4-diamino bio-
pterin derivatives 10a–10l againist NOS-II were summarized in
Table 2.
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www.archpharm.com