Synthesis and in vitro evaluation of substituted pyrimido[5,4-d]pyrimidines as a novel class of Antimycobacterium tuberculosis agents

Article abstract of DOI:10.1016/j.ejmech.2010.03.047

Novel pyrimido[5,4-d]pyrimidines were efficiently synthesized and evaluated for antibacterial activity against Mycobacterium tuberculosis strain H ₃₇Rv. This new structural class of compounds showed high activity against the bacilli. The activity depends on the substituents present in N-3 and C-8 of the pyrimido[ 5,4-d]pyrimidine core. Compounds having a 4-MeOC ₆H₄, a Ph or a 4-FC₆H₄ group as the substituent on C-8 and a 4′-pyridinyl, a Ph or 2′-furyl group as the substituent on N-3 were active. The highest activity was registered for compounds having 4- FC₆H₄ or 4-MeO C₆H ₄ as substituents in C-8 and a heteroaryl group as substituent in N-3. The new compounds showed high potency and promising antitubercular activity, as is the case of N-[8-[(4-fluorophenyl)amino]-4-iminopyrimido[5,4-d] pyrimidin- 3(4H)-yl]isonicotinamide with an IC₉₀= 3.58 μg/mL, and should be regarded as new hits for further development as a novel class of Antimycobacterium tuberculosis agents.

Full text of DOI:10.1016/j.ejmech.2010.03.047

European Journal of Medicinal Chemistry 45 (2010) 3234e3239
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and in vitro evaluation of substituted pyrimido[5,4-d]pyrimidines
as a novel class of Antimycobacterium tuberculosis agents
*
Ana H. Bacelar, M. Alice Carvalho , M. Fernanda Proença
Departamento de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel pyrimido[5,4-d]pyrimidines were efficiently synthesized and evaluated for antibacterial activity
against Mycobacterium tuberculosis strain H37Rv. This new structural class of compounds showed high
activity against the bacilli. The activity depends on the substituents present in N-3 and C-8 of the pyr-
imido[5,4-d]pyrimidine core. Compounds having a 4-MeOC6H4, a Ph or a 4-FC6H4 group as the
substituent on C-8 and a 40-pyridinyl, a Ph or 20-furyl group as the substituent on N-3 were active. The
highest activity was registered for compounds having 4-FC6H4 or 4-MeOC6H4 as substituents in C-8 and
a heteroaryl group as substituent in N-3. The new compounds showed high potency and promising
antitubercular activity, as is the case of N-[8-[(4-fluorophenyl)amino]-4-iminopyrimido[5,4-d]pyrimidin-
Received 2 February 2010
Received in revised form
24 March 2010
Accepted 29 March 2010
Available online 3 April 2010
Keywords:
Antimicrobial
Tuberculosis
Pyrimido[5,4-d]pyrimidine
Antimycobacterial activity
3(4H)-yl]isonicotinamide with an IC90 ¼ 3.58
development as a novel class of Antimycobacterium tuberculosis agents.
Ó 2010 Elsevier Masson SAS. All rights reserved.
mg/mL, and should be regarded as new hits for further
1. Introduction
Since mid-1985s a renewed interest in the discovery of new
antitubercular drugs led to the appearance of new classes of
compounds active against M. tuberculosis [8e10]. Nowadays several
agents belonging to the fluoroquinolone, oxazolidinone, diaryl-
quinoline, and nitroimidazo-oxazole/-oxazine classes are in various
stages of development [2e4,11]. However, new clusters of exten-
sively drug resistant tuberculosis may always appear and, currently,
there is still an urgent demand for new and more effective anti-TB
drugs possessing new modes of action.
To the best of our knowledge the pyrimido[5,4-d]pyrimidines
were never evaluated as antitubercular agents, either as a core or
incorporated as substituents in other base structures. Herein we
describe the synthesis and the in vitro activity against M. tubercu-
losis strain H37Rv of this novel structural class of pyrimido[5,4-d]
pyrimidines. These compounds showed high activity against the
bacilli.
Infection with Mycobacterium tuberculosis affects much of the
world population, despite the fact that drugs for treating tubercu-
losis (TB) were available for over half a century. Each year, it is
estimated that 9.2 million new cases appear, of which many lead to
death [1e4].
The World Health Organization (WHO) has estimated that
approximately 2 billion people worldwide are latently infected
with M. tuberculosis and approximately 10% will develop the active
disease during their lifetime. In addition, tuberculosis is a frequent
HIV co-infection and a major cause of death among people living
with HIV-AIDS. In recent years, multidrug-resistant (MDR-TB) and
extensively drug-resistant (XDR-TB) tuberculosis strains emerged
and tuberculosis can be considered one of the most significant
threats to global health [5,6].
The current TB treatment takes 6e12 months and requires
a combination of three or four drugs that were developed almost
half a century ago: isoniazid, streptomycin, rifampin and pyr-
azinamide (Fig. 1). The narrow choice of antibiotics, lengthy treat-
ment regimens, and patient noncompliance has provided
conditions for acquired antibiotic resistance that led to worldwide
emergence of strains resistant to virtually all available drugs [5e7].
According to the literature, pyrimido[5,4-d]pyrimidines can be
synthesized from the 2,4,6,8-tetrachloro derivatives by nucleo-
philic substitution of the chlorine atoms by the desired amine
[14,16,17,19]. They can also be synthesized from substituted
pyrimidines by reaction with convenient electrophiles or nucleo-
philes [18,20,21]. The 6-cyanopurines were also used as precursors
of pyrimido[5,4-d]pyrimidines upon reaction with amines
[12,13,15,17e25].
During the last few years, our research group developed
a simple and efficient method to synthesize 6-cyanopurines 1 and
their reactivity with amine nucleophiles is currently under study.
* Corresponding author. Tel.: þ351 253 604 380; fax: þ351 253 678 983.
E-mail address: mac@quimica.uminho.pt (M.A. Carvalho).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.03.047

A.H. Bacelar et al. / European Journal of Medicinal Chemistry 45 (2010) 3234e3239
3235
2. Results and discussion
N
NH
NH2
HN
2.1. Chemistry
HO
OH
OH
HN
H2N
The 6-cyanopurines 1aed (Scheme 2) used as starting materials
were prepared according the procedures reported previously
[26,27].
Considering that the product isolated from the reaction between
the 6-cyanopurine 1 and amine nucleophiles 3 depends mainly on
the nature of the purine and amine used, it was necessary to
establish the appropriate reaction conditions for the selective
formation of compounds 4. The study was carried out using purine
1a (R ¼ 4-MeOC6H4) and isoniazid 3a.
HN
NH2
O
N
H
O
O
O
Isoniazid
N
N
H
N
O
OH
O
O
H2N
OH
The first attempt was performed at room temperature, using
ethanol as solvent. Purine 1a and isoniazid 3a were combined in
a 1:1 molar ratio and after 6 days the 1H NMR spectrum of the
reaction mixture showed only the starting materials. The same
result was obtained when ethanol was replaced by dioxane. After
these observations, the reactions proceeded under reflux. After 2
days under reflux in ethanol, or after 3 days under reflux in dioxane,
complex mixtures were formed, as evidenced by 1H NMR spec-
troscopy of the isolated solids. When the reaction was performed
using base catalysis (DBU) and ethanol or dioxane as solvent, at
room temperature, the 1H NMR spectra of the solids, isolated after 6
days, showed a complex mixture and a minor component identified
as the desired product 4a. It was also observed that the percentage
of product 4a in the isolated solids decreases as the reaction time
increases. These results suggested that the product 4a was unstable
under the reaction conditions and evolved to the complex mixture
if the reaction rate is low. In order to increase the reaction rate,
DMSO was selected as solvent, the molar ratio of hydrazide was
increased to 1.5 equivalents and the base catalysis was maintained.
The reaction was performed at room temperature (tlc control).
When the starting material was completely consumed, the product
4a was isolated upon addition of water to the reaction mixture. The
same procedure was applied to the reactions of 6-cyanopurines 1
with isoniazid, acetic, benzoic and furoic hydrazides. The reactions
were carefully monitored by TLC and when the starting material
was absent in the reaction medium, all the 4aen products were
precipitated from solution by addition of water (Scheme 2). The
solids were isolated in an analytically pure form in 38e92% yield.
The derivatives 4d, 4k and 4o were very soluble in DMSO/water
and the reactions were carried out in acetonitrile (4d) or using
ethanol as solvent (4k, 4o). The reactions were complete after 7
days, 25 and 30 h, at room temperature, respectively. The products,
in suspension in the reaction medium, were simply filtered and
washed with diethyl ether.
OH
OH
Pyrazinamide
Streptomycin
H3C
CH3
CH3
O
HN
HO
OH
HO
R
CH3
OH
AcO
CH3
HO
CH3
O
O
O
H3C
O
H3C
Rifampin
R=
N
N
N
CH3
Fig. 1. Drugs currently used for TB treatment.
The pyrimido[5,4-d]pyrimidines of structure 2 were obtained from
the reaction of 6-cyanopurines 1 with ammonia, primary amines
and hydrazine. Our studies showed that these reactions are very
sensitive to the experimental conditions often leading to the
competitive formation of side-products (Scheme 1) [25,26,28].
As an extension of this work, we reacted the 6-cyanopurines 1
with hydrazides in order to obtain new derivatives 4 (Scheme 2)
with potential activity against M. tuberculosis. The new derivatives
have some structural features that led us to consider them as viable
candidates to evaluate as antitubercular agents. The pyrimidopyr-
imidine planar core is similar to the quinoline core, and several
potent quinoline-based antitubercular compounds have been
identified. Many hydrazide derivatives have also been reported as
active against M. tuberculosis. Furthermore, we selected the isoni-
azid, one of the drugs used in the treatment of tuberculosis (Fig. 1),
as one of the hydrazides (3a), to incorporate in the molecule. The
combination of these two structural subunits in a single molecule
was expected to generate new antitubercular agents.
All the compounds were fully characterized by elemental anal-
ysis, melting point, infrared (IR), and nuclear magnetic resonance
(1H and 13C NMR) spectroscopy including 1H/13C correlation tech-
niques. Typically, the 1H NMR spectra of these compounds show
three NH protons at
d > 9 ppm, confirmed by D2O exchange. The
6-H proton appears between
d
8.8 and 8.5 ppm and the 2-H proton
RHN
N
N
RHN
N
R
N
R
N
N
N
H2NR1
N
N
N
and/or
NHR1
and/or N
N
N
N
N
NH
N
NHR1
CN
R1
H2N
2
1
R1= H, Alkyl, Ar, OCH2Ph, NH2
Scheme 1. Products obtained from the reactions of 6-cyanopurines with amine nucleophiles.

3236
A.H. Bacelar et al. / European Journal of Medicinal Chemistry 45 (2010) 3234e3239
8
RHN
N
4
6
R
N
8a
N
)
i
N
N
2
H2NNHCOR1
4a
+
3a
b
N
N
HN
NH
R1
N
3
d
CN
a R1=4´-pyridine
1
b R1=Ph
O
a R=4-MeOC6H4
b R=Ph
c R=4-NCC6H4
d R=4-FC6H4
c R1=Me
4
d R1=2´-furyl
a R=4-MeOC6H4 , R1=4´-pyridinyl
b R=4-MeOC6H4, R1=Ph
c R=4-MeOC6H4, R1=2´-furyl
e R=Ph , R1=4´-pyridinyl
f R=Ph, R1=Ph
iii)
ii)
3c
3c
g R=Ph, R1=2´-furyl
h R=Ph, R1=Me
4d R=4-MeOC6H4, R1=Me
i R=4-NCC6H4 , R1=4´-pyridinyl
j R=4-NCC6H4, R1=Ph
l R=4-FC6H4 , R1=4´-pyridinyl
m R=4-FC6H4, R1=Ph
4k R=4-NCC6H4, R1=Me
o R=4-FC6H4, R1=Me
n R=4-FC6H4, R1=2´-furyl
Scheme 2. Reagents and conditions: (i) 1. DMSO, rt, DBU (cat). 2. H2O (10 mL); (ii) CH3CN, rt, DBU (cat); (iii) EtOH, rt, DBU (cat).
between
d
8.1 and 8.8 ppm depending on the substituent R1. When
The results, summarized in Table 1, show that most of the
R1 ¼ Me this proton appears at lower chemical shift and when
pyrimidopyrimidines 4 were active against Mycobacterium tuber-
culosis and the activity depends on both substituents R and R1
present in C-8 and N-3, respectively, of the pyrimido-pyrimidine
core.
R1 ¼ pyridinyl it shifts to higher
d values. The combined analysis of
the HMBC and HMQC spectra of the compounds allowed the
identification of C-2 at
157 ppm, C-4a at w134 to 139 ppm and C-8a at
128 ppm, and confirmed the structure assigned to compounds 4.
d
w148 to 149 ppm, C-6 at
d
w155 to
w124 to
d
d
When the substituent R1 is a 4-pyridinyl unit the activity
depends mainly on the nature of the R group. The compound
having R ¼ 4-NCC6H4, 4i, was weakly active, however, when
a phenyl, a 4-FC6H4 or a 4-MeOC6H4 is present as the R substituent,
the compounds 4a, 4e and 4l were active.
2.2. Pharmacology
When the substituent R1 ¼ Ph, the activity shows a minor
In vitro evaluation of the antitubercular activity was carried out
within the Tuberculosis Antimicrobial Acquisition & Coordinating
Facility (TAACF) screening program for the discovery of novel drugs
for the treatment of tuberculosis. Under the direction of the US
National Institute of Allergy and Infectious Diseases (NIAID),
Southern Research Institute that coordinates the overall program.
All the new pyrimido-pyrimidines 4 were screened for anti-
mycobacterial activity on the M. tuberculosis strain H37Rv according
to procedures previously published by the TAACF organization [29].
According to the information on the TAACF webpage [29c] the IC90
of the compounds is determined as a primary screen. Any
dependence on the nature of the substituent R. In this case, the IC90
varies from 36.09 mM (4j) to 13.48 mM (4m).
A furyl group as substituent R1 in molecule 4 led to a decrease of
activity when a 4-MeOC6H4 is the substituent R (4c). However,
when R ¼ Ph or 4-FC6H4 the compounds were active (4g and 4n).
The presence of a methyl group as the substituent R1 led to
inactive or weakly active compounds whichever the group R is (4d,
4h, 4k and 4o). The presence of a 4-NCC6H4 group as the substit-
uent R led also to inactive or weakly active compounds (4i-k).
Compounds having simultaneously the 4-MeOC6H4 group as
R and the 20-furyl group as R1 or the group 4-NCC6H4 as R and the
40-pyridinyl group as R1 were weakly active.
compound having an IC90 ꢀ 10
mg/mL is considered active for
antitubercular activity.
Table 1
Antibacterial activity of compound 4 against M. tuberculosis strain H37Rv and cytotoxic activity on VERO cells.
R1
IC90
(
m
g/ml)
IC90
(m
M)
IC50
(
m
g/ml)
EC50
(
mg/ml)
SI
a
Entry
Compound
R
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4-MeOC6H4
4-MeOC6H4
4-MeOC6H4
4-MeOC6H4
Ph
Ph
Ph
Ph
4-NCC6H4
4-NCC6H4
4-NCC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
40-pyridinyl
Ph
4.22
9.23
>100
>100
7.69
9.49
1.59
4.11
3.56
46.52
4.30
3.65
1.15
2.12
nd
0.27
0.23
nd
23.36
>261
>298
21.00
17.40
13.99
>314
>240
36.09
>311
9.12
20-furyl
Me
nd
nd
40-pyridinyl
Ph
4.37
2.35
nd
nd
nd
nd
nd
1.53
3.05
nd
0.57
0.38
nd
nd
nd
nd
nd
0.43
0.60
nd
6.22
4.86
20-furyl
Me
3.16
>100
>100
13.80
>100
3.58
5.06
3.57
28.97
44.91
17.20
2.47
42.10
0.91
2.92
2.17
18.70
9
40-pyridinyl
Ph
10
11
12
13
14
15
Me
40-pyridinyl
Ph
13.48
9.77
91.42
20-furyl
Me
nd
nd
a
Compounds having an IC90 ꢀ 10
mg/mL are considered active for antitubercular activity.

A.H. Bacelar et al. / European Journal of Medicinal Chemistry 45 (2010) 3234e3239
3237
The most active compounds were further evaluated for their
cytotoxicity (EC50) on mammalian VERO cell lines [29], by TAACF,
and showed a low Selectivity Index (SI).
1598, 1574, 1538, 1511 cmꢂ1
;
dH (300 MHz, DMSO-d6) 10.28 (s, 1H,
NH), 9.42 (br s, 2H, NH), 8.70 (s, 1H, 6-H), 8.66 (s, 1H, 2-H), 8.62
(d, J ¼ 5.7 Hz, 2H), 8.01 (d, J ¼ 5.7 Hz, 2H), 7.81 (d, J ¼ 9.0 Hz, 2H),
6.97 (d, J ¼ 9.0 Hz, 2H), 3.76 (s, 3H, OCH3); dC (75 MHz, DMSO-d6)
168.01, 156.44, 156.16, 156.00, 152.29, 149.30, 147.77, 145.14, 133.61,
131.00, 127.46, 123.92, 122.53, 113.68, 55.26.
3. Conclusion
Pyrimido[5,4-d]pyrimidines of general structure
4 having
different aryl substituents on C-8 and aryl, alkyl or heteroaryl
substituents on N-3 were efficiently synthesized. The activity of
these compounds against M. tuberculosis strain H37Rv was assessed
and most of the molecules proved to be active. Their activity
depends on both the substituents R and R1. Compounds having
a 4-MeOC6H4, a Ph or a 4-FC6H4 group as the substituent on C-8 and
a 40-pyridinyl, a Ph or 20-furyl group as the substituent on N-3 were
active. The highest activity was registered for compounds where
R ¼ 4-FC6H4 and R1 ¼40-pyridinyl or 20-furyl, and also for the
compound having R ¼ 4-MeOC6H4 and R1 ¼40-pyridinyl. These
novel molecules are new promising antitubercular hit compounds.
The presence of an isoniazid unit incorporated in N-3 was not
essential for activity. This observation may indicate that the
biological target is different from that of isoniazid. Furthermore, as
the compounds discussed herein have no structural similarity to
any other compounds active against M. tuberculosis, this may
indicate that pyrimido-pyrimidines act by a new mechanism of
action.
4.1.2. N-(4-Imino-8-(4-methoxyphenylamino)pyrimido[5,4-d]
pyrimidin-3(4H)-yl)benzamide (4b)
Yellow solid; Yield: 0.37 g, 87%; m.p. >236 ꢁC (dec) (Found: C,
61.32; H, 4.49; N, 24.81. C20H17N7O2.0.1(CH3)2SO requires C, 61.34;
H, 4.46; N, 24.82); IR (Nujol mull) nmax: 3404, 3257,1639,1590,1607,
1567, 1537, 1506 cmꢂ1
; dH (400 MHz, DMSO-d6) 10.17 (s, 1H, NH),
9.39 (br s, 2H, NH), 8.63 (s, 1H, 6-H), 8.60 (s, 1H, 2-H), 8.12
(d, J ¼ 8.1 Hz, 2H), 7.80 (d, J ¼ 9.0 Hz, 2H), 7.44 (m, 3H), 6.96
(d, J ¼ 9.0 Hz, 2H); dC (75 MHz, DMSO-d6) 169.07, 156.55, 156.16,
155.98, 152.72, 148.45, 134.45, 136.04, 131.11, 130.21, 128.21, 127.62,
126.92, 123.91, 113.73, 55.30.
4.1.3. N-(4-Imino-8-(4-methoxyphenylamino)pyrimido[5,4-d]
pyrimidin-3(4H)-yl)furan-2-carboxamide (4c)
Yellow solid; Yield: 0.09 g, 60%; m.p. >253 ꢁC (dec) (Found: C,
56.78; H, 4.41; N, 25.72. C18H15N7O3.0.3H2O requires C, 56.48; H,
4.12; N, 25.62); IR (Nujol mull) nmax: 3411, 3295, 1654, 1602, 1570,
1537, 1507 cmꢂ1
; dH (400 MHz, DMSO-d6) 10.15 (s, 1H, NH), 9.39
Further structural modifications of the identified hits are in
progress in order to enhance the efficacy of this new structural class
of compounds active against M. tuberculosis.
(br s, 1H, NH), 8.62 (s, 1H, 6-H), 8.59 (s, 1H, 2-H), 7.80 (d, J ¼ 9.2 Hz,
2H), 7.74 (d, J ¼ 1.4 Hz, 1H), 7.05 (d, J ¼ 3.2 Hz, 1H), 6.96
(d, J ¼ 9.2 Hz, 2H), 6.57 (dd, J ¼ 1.4, 3.2 Hz, 1H); dC (100 MHz,
DMSO-d6) 162.35, 156.45, 156.09, 155.96, 152.64, 150.59, 148.16,
143.68, 134.36, 131.05, 126.88, 123.80, 113.65, 112.33, 111.16, 55.23.
4. Experimental
The 6-cyanopurines
1
used in this work were prepared
4.1.4. N-[4-Imino-8-[(4-methoxyphenyl)amino]pyrimido[5,4-d]
pyrimidin-3(4H)-yl] acetamide (4d)
according to previously described procedures [26,27]. NMR spectra
were recorded with a Varian Unity Plus (300 MHz) or with a Bruker
Yellow solid; Yield: 0.11 g, 43%; m.p. ¼ 247e248 ꢁC (Found: C,
53.60; H, 4.82; N, 29.18. C15H15N7O2.0.6H2O requires C, 53.37; H,
5.08; N, 29.37); IR (Nujol mull) nmax: 3369, 3226, 3039, 1679, 1596,
Avance III NMR spectrometer (400 MHz), including the 1H and 13
C
correlation spectra (HMQC and HMBC). IR spectra were recorded
with a FT-IR Bomem MB 104 using nujol mulls and NaCl cells. The
reactions were monitored by thin-layer chromatography (TLC) with
the use of silica gel 60 F254 (Merck). The melting points were
determined with a Gallenkamp melting point apparatus and are
uncorrected. Elemental analysis was performed with a LECO CHNS-
932 instrument.
1575, 1528, 1510 cmꢂ1
; dH (300 MHz, DMSO-d6) 9.67 (s, 1H, NH),
8.49 (s, 1H, 6-H), 8.08 (s, 1H, 2-H), 7.74 (d, J ¼ 9 Hz, 2H), 6.93
(d, J ¼ 9 Hz, 2H), 3.74 (s, 3H, OCH3), 2.04 (s, 3H, CH3); dC (75 MHz,
DMSO-d6) 170.01, 156.66, 155.86, 155.51, 153.95, 149.18, 138.63,
131.38, 124.81, 123.53, 113.66, 55.24, 20.80.
4.1.5. N-(4-Imino-8-(phenylamino)pyrimido[5,4-d]pyrimidin-3
(4H)yl) isonicotinamide (4e)
4.1. General procedure for the synthesis of pyrimido[5,4-d]
pyrimidines (4aen)
Yellow solid; Yield: 0.26 g, 79%; m.p. >300 ꢁC (Found: C, 59.89;
H, 4.01; N, 30.52. C18H14N8O.0.1(CH3)2SO requires C, 59.65; H, 3.99;
N, 30.59); IR (Nujol mull) nmax: 3242, 3109, 1660, 1604, 1573, 1564,
The hydrazide 3 (1.5 equivalents) and a catalytic amount of DBU
were added to a suspension of 6-cyanopurine 1 in dimethyl sulf-
oxide, acetonitrile (for 4d) or ethanol (for 4k and 4o), under stir-
ring at room temperature. When TLC of the reactions carried out in
DMSO showed absence of the starting material, cold water (10 mL)
was added to the reaction mixture and the suspension was filtered
under vacuum after 45 mine6 h 15 min. The solid was washed
with water followed by ethanol and diethyl ether. For the reactions
carried out in ethanol or acetonitrile, when TLC showed absence of
starting material, the suspension was simply filtered under
vacuum after 25 he7 days and the solid was washed with ethanol
and diethyl ether and was identified as the pyrimido[5,4-d]
pyrimidine 4.
1525 cmꢂ1
; dH (300 MHz, DMSO-d6) 10.36 (s, 1H, NH), 9.8e9.2 (br s,
2H, NH), 8.72 (s, 1H, 6-H), 8.67 (s, 1H, 2-H), 8.62 (dd, J ¼ 1.5, 4.5 Hz,
2H), 8.02 (dd, J ¼ 1.5, 4.5 Hz, 2H), 7.95 (d, J ¼ 7.8 Hz, 2H), 7.40
(t, J ¼ 7.8 Hz, 2H), 7.17 (t, J ¼ 7.8 Hz, 1H); dC (75 MHz, DMSO-d6)
168.01, 156.57, 155.92, 152.34, 149.30, 147.93, 145.14, 138.11, 137.78,
128.53, 127.52, 124.38, 122.53, 122.23.
4.1.6. N-(4-Imino-8-(phenylamino)pyrimido[5,4-d]pyrimidin-3
(4H)-yl)benzamide (4f)
Yellow solid; Yield: 0.35 g, 92%; m.p. ¼ 313e315 ꢁC (Found: C,
63.83; H, 4.42; N, 27.19. C19H15N7O requires C, 63.86; H, 4.23; N,
27.44); IR (Nujol mull) nmax: 3303, 3228, 3108, 1657, 1599, 1564,
1525 cmꢂ1
; dH (300 MHz, DMSO-d6) 10.23 (s, 1H, NH), 9.48 (br s,
4.1.1. N-(4-Imino-8-(4-methoxyphenylamino)pyrimido[5,4-d]
pyrimidin-3(4H)-yl) isonicotinamide (4a)
2H, NH), 8.70 (s, 1H, 6-H), 8.62 (s, 1H, 2-H), 8.13 (d, J ¼ 6.0 Hz, 2H),
7.95 (d, J ¼ 7.8 Hz, 2H), 7.44 (m, 5H), 7.15 (t, J ¼ 7.5 Hz, 1H); dC
(75 MHz, DMSO-d6) 168.86, 156.60, 155.78, 152.75, 148.65, 138.21,
136.30, 134.90, 130.26, 128.51, 128.17, 127.61, 126.84, 124.23,
122.10.
Yellow solid; Yield: 0.32 g, 79%; m.p. ¼ 287e288 ꢁC (dec)
(Found: C, 51.81; H, 4.87; N, 25.47. C19H16N8O2.3H2O requires C,
51.54; H, 4.98; N, 25.32); IR (Nujol mull) nmax: 3353, 3301, 1678,

3238
A.H. Bacelar et al. / European Journal of Medicinal Chemistry 45 (2010) 3234e3239
4.1.7. N-[4-Imino-8-(phenylamino)pyrimido[5,4-d]pyrimidin-3
(4H)-yl]furan-2-carboxamide (4g)
8.02 (dd, J ¼ 1.5, 4.5 Hz, 2H), 7.96 (dd, J ¼ 5.1, 9.0 Hz, 2H), 7.24
(t, J ¼ 9.0 Hz, 2H); dC (75 MHz, DMSO-d6) 164.93, 159.14
(d, J ¼ 241 Hz), 157.02, 156.83, 154.61, 147.01, 146.93, 143.73, 134.62,
134.21 (d, J ¼ 2 Hz),128.03,124.76 (d, J ¼ 9 Hz),115.31 (d, J ¼ 23 Hz),
123.78.
Yellow solid; Yield: 0.06 g, 38%; m.p. ¼ 223e224 ꢁC (Found: C,
58.92; H, 3.97; N, 27.99. C17H13N7O2 requires C, 58.79; H, 3.77; N,
28.23); IR (Nujol mull) nmax
: 3501, 3289, 1656, 1600, 1567,
1530 cmꢂ1
;
dH (300 MHz, DMSO-d6) 10.22 (s,1H, NH), 9.47 (br s, 2H,
NH), 8.69 (s, 1H, 6-H), 8.61 (s, 1H, 2-H), 7.94 (d, J ¼ 7.8 Hz, 2H), 7.75
(d, J ¼ 1.8 Hz, 1H), 7.39 (t, J ¼ 7.8 Hz, 2H), 7.15 (t, J ¼ 7.8 Hz, 1H), 7.06
(d, J ¼ 3.3 Hz, 1H), 6.57 (dd, J ¼ 3.3, 1.8 Hz, 1H). dC (75 MHz,
DMSO-d6) 162.24, 156.57, 155.83, 152.70, 150.48, 148.38, 143.78,
138.18, 134.75, 128.49, 126.89, 124.22, 122.07, 112.46, 111.22.
4.1.13. N-(8-(4-Fluorophenylamino)-4-iminopyrimido[5,4-d]
pyrimidin-3(4H)-yl) benzamide (4m)
Yellow solid; Yield: 0.37 g, 92%; m.p. >300 ꢁC (dec) (Found: C,
60.64; H, 4.01; N, 25.88. C19H14N7OF requires C, 60.80; H, 3.76; N,
26.12); IR (Nujol mull) nmax: 3310, 3108, 1658, 1611, 1572, 1523,
1506 cmꢂ1
; dH (300 MHz, DMSO-d6) 10.32 (s, 1H, NH), 9.49 (br s,
4.1.8. N-(8-Anilino-4-iminopyrimido[5,4-d]pyrimidin-3(4H)-yl)
acetamide (4h)
2H, NH), 8.67 (s, 1H, 6-H), 8.61 (s, 1H, 2-H), 8.12 (d, J ¼ 7.8 Hz, 2H),
7.95 (dd, J ¼ 4.8, 9.2 Hz, 2H), 7.44 (m, 5H), 7.15 (t, J ¼ 9.2 Hz, 2H); dC
(75 MHz, DMSO-d6) 168.84, 158.70 (d, J ¼ 240 Hz), 156.59, 155.73,
152.73, 148.66, 136.32, 134.88, 134.59 (d, J ¼ 2 Hz), 130.25, 28.16,
127.61, 126.77, 124.14 (d, J ¼ 8 Hz), 115.11 (d, J ¼ 23 Hz).
Yellow solid; Yield: 0.20 g, 69%; m.p. ¼194e196 ꢁC (Found: C,
52.71; H, 4.90; N, 30.75. C145H13N7O.1.3H2O requires C, 53.00; H,
4.86; N, 30.56); IR (Nujol mull) nmax: 3369, 1648, 1615, 1608,
1569 cmꢂ1
; dH (300 MHz, DMSO-d6) 9.75 (s, 1H, NH), 8.56 (s, 1H,
6-H), 8.09 (s, 1H, 2-H), 7.89 (d, J ¼ 8.7 Hz, 2H), 7.36 (t, J ¼ 7.7 Hz, 2H),
7.11 (t, J ¼ 7.9 Hz, 1H), 2.05 (s, 3H, CH3); dC (75 MHz, DMSO-d6)
169.96, 156.67, 155.38, 153.91, 149.40, 138.99, 138.46, 128.48, 124.93,
123.81, 121.68, 20.78.
4.1.14. N-{8-[(4-Fluorophenyl)amino]-4-iminopyrimido [5,4-d]
pyrimidin-3(4H)-yl}furan-2-carboxamide (4n)
Yellow solid; Yield: 0.12 g, 79%; m.p. >267 ꢁC (dec) (Found: C,
55.90; H, 3.47; N, 26.47. C17H12N7O2F requires C, 55.89; H, 3.31; N,
26.84); IR (Nujol mull) nmax: 3390, 3295, 3120, 1655, 1611, 1590,
4.1.9. N-(8-(4-Cyanophenylamino)-4-iminopyrimido[5,4-d]
pyrimidin-3(4H)-yl) isonicotinamide (4i)
1572, 1532, 1505 cmꢂ1
; dH (300 MHz, DMSO-d6) 10.31 (s, 1H, NH),
9.46 (br s, 2H, NH), 8.66 (s, 1H, 6-H), 8.60 (s, 1H, 2-H), 7.94 (dd,
J ¼ 5.1, 9.2 Hz, 2H), 7.75 (d, J ¼ 0.6 Hz, 1H), 7.23 (t, J ¼ 9.2 Hz, 2H),
7.06 (d, J ¼ 3.0 Hz, 1H), 6.57 (dd, J ¼ 0.6, 3.0 Hz, 1H); dC (75 MHz,
DMSO-d6) 162.27, 158.71 (d, J ¼ 240 Hz), 156.57, 155.81, 152.70,
150.48, 148.39, 143.79, 134.73, 134.55 (d, J ¼ 2 Hz), 126.86, 124.13
(d, J ¼ 8 Hz), 115.11 (d, J ¼ 23 Hz), 112.47, 111.23.
Yellow solid; Yield: 0.19 g, 70%; m.p. >300 ꢁC (Found: C, 54.92;
H, 4.02; N, 29.95. C19H13N9O.1.8H2O requires C, 54.84; H, 4.00; N,
30.30); IR (Nujol mull) nmax: 3373, 3268, 3186, 3101, 2226 (CN), 61
1608, 1562, 1546 cmꢂ1
; dH (300 MHz, DMSO-d6) 10.72 (br s, 1H,
NH), 9.56 (br s, 2H, NH), 8.84 (s, 1H, 6-H), 8.76 (s, 1H, 2-H), 8.62
(d, J ¼ 4.5 Hz, 2H), 8.28 (d, J ¼ 8.4 Hz, 2H), 8.02 (d, J ¼ 4.5 Hz, 2H),
7.86 (d, J ¼ 8.4 Hz, 2H); dC (75 MHz, DMSO-d6) 167.95, 156.44,
155.52, 152.53, 149.31, 148.27, 145.02, 142.69, 134.23, 132.88, 127.70,
122.52, 121.69, 119.09, 105.57.
4.1.15. N-{8-[(4-Fluorophenyl)amino]-4-iminopyrimido[5,4-d]
pyrimidin-3(4H)-yl} acetamide (4o)
Gray solid; Yield: 0.17 g, 64%; m.p. >300 ꢁC (Found: C, 53.21; H,
4.02; N, 30.55. C14H12N7OF.0.2H2O requires C, 53.02; H, 3.92; N,
30.93); IR (Nujol mull) nmax: 3336, 1660, 1615, 1584, 1567, 1536,
4.1.10. N-(8-(4-Cyanophenylamino)-4-iminopyrimido[5,4-d]
pyrimidin-3(4H)-yl) benzamide (4j)
1504 cmꢂ1
; dH (300 MHz, DMSO-d6) 11.5e10.5 (br s, 1H, NH), 9.88
Yellow solid; Yield: 0.31 g, 87%; m.p. >300 ꢁC (Found: C, 62.85;
H, 3.81; N, 28.99. C20H14N8O requires C, 62.82; H, 3.69; N, 29.30); IR
(Nujol mull) nmax: 3355, 3298, 3230, 3191, 2225 (CN), 1652, 1602,
(s, 1H, NH), 8.54 (s, 1H, 6-H), 8.10 (s, 1H, 2-H), 7.91 (dd, J ¼ 4.8,
8.9 Hz, 2H), 7.20 (t, J ¼ 8.9 Hz, 2H), 2.05 (s, 3H, CH3); dC (75 MHz,
DMSO-d6) 169.92, 158.49 (d, J ¼ 240 Hz), 156.89, 155.36, 153.93,
149.41, 139.04, 134.86 (d J ¼ 2 Hz), 124.87, 123.78 (d, J ¼ 8 Hz),
115.06 (d, J ¼ 22 Hz), 20.77.
1564, 1545, 1520 cmꢂ1
; dH (300 MHz, DMSO-d6) 10.60 (s, 1H, NH),
9.58 (br s, 2H, NH), 8.81 (s, 1H, 6-H), 8.63 (s, 1H, 2-H), 8.28
(d, J ¼ 8.7 Hz, 2H), 8.11 (d, J ¼ 8.1 Hz, 2H), 7.86 (d, J ¼ 8.7 Hz, 2H),
7.46 (m, 3H); dC (75 MHz, DMSO-d6) 168.76, 156.35, 154.93, 152.77,
149.19, 143.03, 136.59, 136.53, 132.89, 130.20, 128.08, 127.64, 127.03,
121.36, 119.19, 105.12.
Acknowledgements
Antimycobacterial data were provided by the Tuberculosis
Antimicrobial Acquisition and Coordinating Facility (TAACF)
through a research and development contract with the US National
Institute of Allergy and Infectious Diseases. The NMR spectrometer
(Bruker 400 Avance III) is part of the National NMR Network and
was purchased in the framework of the National Programme for
Scientific Re-equipment, contract REDE/1517/RMN/2005, with
funds from POCI 2010 (FEDER) and Fundação para a Ciência e
a Tecnologia (FCT). The authors gratefully acknowledge the finan-
cial support by Universidade do Minho, Centro de Química and
Fundação para a Ciência e a Tecnologia (project PPCDT/QUI/59356/
2004 and a PhD grant awarded to Ana Bacelar SFRH/BD/24959/
2005).
4.1.11. N-{8-[(4-Cyanophenyl)amino]-4-iminopyrimido[5,4-d]
pyrimidin-3(4H)-yl} acetamide (4k)
Green solid; Yield: 0.22 g, 85%; m.p. >300 ꢁC (dec) (HRMS (FAB)
m/z Found: 321.1219. C15H12N8O requires: 321.1212); IR (Nujol mull)
nmax
:
3351, 3248, 2237 (CN), 1684, 1660, 1614, 1598, 1558,
dH (300 MHz, DMSO-d6) 10.16 (s, 2H, NH 8.67 (s, 1H,
1523 cmꢂ1
;
6-H), 8.11 (s, 1H, 2-H), 8.22 (d, J ¼ 8.7 Hz, 2H), 7.81 (d, J ¼ 8.7 Hz,
2H), 2.05 (s, 3H, CH3); dC (75 MHz, DMSO-d6) 169.89, 156.39, 154.93,
153.70, 149.79, 143.08, 139.79, 132.83, 125.39, 121.10, 119.19, 104.87,
20.76.
4.1.12. N-(8-(4-Fluorophenylamino)-4-iminopyrimido[5,4-d]
pyrimidin-3(4H)-yl) isonicotinamide (4l)
References
Yellow solid; Yield: 0.30 g, 84%; m.p. >293 ꢁC (Found: C, 55.07;
H, 3.77; N, 28.56. C18H13N8OF.0.9H2O requires C, 55.03; H, 3.77; N,
28.53); IR (Nujol mull) nmax: 3212, 3067, 1681, 1652, 1620, 1584,
[1] C. Dye, Nat. Rev. Microbiol. 7 (2009) 81e87.
[2] A.M. Ginsberg, Semin. Respir. Crit. Care Med 29 (2008) 552e559.
[3] H.D. Showalter, W. Denny, Tuberculosis 88 (1) (2008) S3eS17.
[4] H. Tomioka, Y. Tatano, K. Yasumoto, T. Shimizu, Expert Rev. Respir. Med. 2
(2008) 455e471.
1549, 1530, 1509 cmꢂ1
;
dH (300 MHz, DMSO-d6) 10.46 (s, 1H, NH),
9.40(brs,2H, NH), 8.62(dd,J ¼ 1.5, 4.5 Hz,2H),8.72(s,2H, 6-H þ 2-H),

A.H. Bacelar et al. / European Journal of Medicinal Chemistry 45 (2010) 3234e3239
3239
[5] R. Goldman, K.V. Plumley, B.E. Laughon, Infect. Disord. Drug Targets 7 (2007)
73e91.
[19] M. El-Araby, R.S. Pottorf, M.R. Player, Comb. Chem. High T. Scr. 7 (2004)
413e421.
[6] L. Nguyen, C.J. Thompson, Trends Microbiol. 14 (2006) 304e312.
[7] R. Shi, N. Itagaki, I. Sugawara, Mini-Reviews Med. Chem. 7 (2007) 1177e1185.
[8] L. Ballell, R.A. Field, K. Duncan, R.J. Young, Antimicrob. Agents Chemother.
(2005) 2153e2163.
[9] A. Nayyar, R. Jain, Curr. Med. Chem. 12 (2005) 1873e1886.
[10] R.P. Tangallapally, R. Yendapally, A.J. Daniels, R.E.B. Lee, Curr. Top. Med. Chem.
7 (2007) 509e526.
[11] B.E. Laughon, Curr. Top. Med. Chem. 7 (2007) 463e473.
[12] P.C. Srivastava, G.R. Revankar, R.K. Robins, R.J. Rousseau, J. Med. Chem. 24
(1981) 393e398.
[13] P.D. Cook, D.A. Berry, US Patent 4,801,698; EP 257,488A2; Chem. Abstr..
109:110861h.
[14] Y.S. Sanghvi, S.B. Larson, S.S. Matsumoto, D.F. Nord, L.D. Smee, R.C. Willis, T.
L. Avery, R.K. Robins, G.R. Revankar, J. Med. Chem. 32 (1989) 629e637.
[15] T.E. Mabry, C.D. Jones, T.S. Chou, J.M. Colacino, G.B. Grindey, J.F. Worzalla, H.
L. Pearce, Nucleosides Nucleotides 13 (1994) 1125e1133.
[16] N.J. Curtin, H.C. Barlow, K.J. Bowman, A.H. Calvert, R. Davison, B.T. Golding,
B. Huang, P.J. Loughlin, D.R. Newell, P.G. Smith, R.J. Griffin, J. Med. Chem. 47
(2004) 4905e4922.
[17] J.S. Northen, F.T. Boyle, W. Clegg, N.J. Curtin, A.J. Edwards, R.J. Griffin, B.
T. Golding, J. Chem. Soc. Perkin Trans. 1 (2002) 108e115.
[18] G.W. Rewcastle, A.J. Bridges, D.W. Fry, J.R. Rubin, W.A. Denny, J. Med. Chem. 40
(1997) 1820e1826.
[20] G.W. Rewcastle, W.A. Denny, D.H. Showalter, Curr. Org. Chem. 4 (2000)
679e706.
[21] A. Al-Azmi, J. Chem. Res. 8 (S) (2005) 530e534.
[22] H.M. Berman, R.J. Rousseau, R.W. Mancuso, G.P. Kreishman, R.K. Robins,
Tetrahedron Lett. 33 (1973) 3099e3101.
[23] J.D. Westover, G.R. Revankar, R.K. Robins, R.D. Madsen, J.R. Orgden,
J.A. North, R.W. Mancuso, R.J. Rousseau, E.L. Stephen, J. Med. Chem. 24
(1981) 941e946.
[24] T.S. Rao, G.R. Revankar, Nucleosides Nucleotides 14 (1995) 1601e1612.
[25] M.A. Carvalho, S. Esperança, T. Esteves, M.F. Proença, Eur. J. Org. Chem. (2007)
1324e1331.
[26] A. Al-Azmi, B.L. Booth, R.A. Carpenter, M.A. Carvalho, E. Marrelec, R.
G. Pritchard, M.F. Proença, J. Chem. Soc. Perkin Trans. 1 (2001) 2532e2537.
[27] M.J. Alves, B.L. Booth, M.A. Carvalho, R.G. Pritchard, M.F. Proença, J. Heterocycl.
Chem. 34 (1997) 739e743.
[28] A. Ribeiro, M.A. Carvalho, M.F. Proença, Eur. J. Org. Chem. (2009) 4867e4872.
[29] (a) L.A. Collins, S.G. Franzblau, Antimicrob. Agents Chemother. 41 (1997)
1004e1009;
(b) I. Orme, J. Secrist, S. Anathan, C. Kwong, J. Maddry, R. Reynolds,
A. Poffenberger, M. Michael, L. Miller, J. Krahenbuh, L. Adams, A. Biswas,
S. Franzblau, D. Rouse, D. Winfield, J. Brooks, Antimicrob. Agents Chemother.
45 (2001) 1943e1946;
(c) http:www.taacf.org (accessed 02.02.10).