Synthesis of new 5,6-dihydrobenzo[h]quinazoline 2,4-diamino substituted and antiplatelet/antiphlogistic activities evaluation

Article abstract of DOI:10.1016/j.bmcl.2011.11.103

In pursuing our research on some 2,4-diamino-benzopyranopyrimidines and 2-amino-5,6-dihydrobenzo[h]quinazolines, previously reported as antiplatelet and analgesic/anti-inflammatory agents respectively, we designed and synthesized a new series of 5,6-dihyd

Full text of DOI:10.1016/j.bmcl.2011.11.103

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1125–1129
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of new 5,6-dihydrobenzo[h]quinazoline 2,4-diamino substituted
and antiplatelet/antiphlogistic activities evaluation
Chiara Brullo a, , Massimo Rocca a, Paola Fossa a, Elena Cichero a, Elisabetta Barocelli b, Vigilio Ballabeni b,
⇑
Lisa Flammini b, Carmine Giorgio b, Francesca Saccani b, Giuseppe Domenichini b, Olga Bruno a
a Dipartimento di Scienze Farmaceutiche, University of Genoa, V.le Benedetto XV, 3-16132 Genova, Italy
b Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, University of Parma, V.le delle Scienze 27/a-43124 Parma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 July 2011
Revised 24 November 2011
Accepted 27 November 2011
Available online 3 December 2011
In pursuing our research on some 2,4-diamino-benzopyranopyrimidines and 2-amino-5,6-dihydro-
benzo[h]quinazolines, previously reported as antiplatelet and analgesic/anti-inflammatory agents
respectively, we designed and synthesized a new series of 5,6-dihydrobenzo[h]quinazoline 2,4-diamino
substituted. The insertion of amino substituents at positions 2 and 4 of the benzoquinazoline scaffold
resulted in compounds endowed with a potent ASA-like antiplatelet activity, combined with an anti-
inflammatory activity comparable, in some cases, to that of indomethacin, used as a reference drug.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
5,6-Dihydrobenzo[h]quinazolines
Antiplatelet activity
Antiinflammatory activity
Analgesic activity
The pyrimidine ring is one of the most investigated scaffolds in
medicinal chemistry, particularly when it is fused with other het-
erocyclic moieties to give more complex molecules. Since the
pyrimidine moiety is present in nucleotides, many molecules con-
taining it show pharmacodynamic and pharmacokinetic properties
of great interest for medicinal chemists. The pharmacological
activities displayed by pyrimidine fused heterocycles range from
antibacterial,1–3 antiviral,4–6 and anti-inflammatory7–10 to antican-
cer11,12 as well as other different activities.
In the last ten years, we investigated several pyrimidine fused
heterocycles. Interesting pharmacological results, particularly con-
cerning the antiplatelet/antithrombotic activities, were obtained
with condensed coumarino-pyrimidines, which resulted in useful
leads for the development of new gastric sparing antiplatelet drugs
with combined anti ADP-TXA2 (Thromboxane A2) activity.13
In that Letter we confirmed that antiplatelet activity depended
on the concomitant contribution of both functions inserted at posi-
tions 2 and 5 of the heterocyclic system. Moreover, we demon-
strated that the presence of a carbonyl group at position 5 of the
benzopyranopyrimidine system and the simultaneous insertion
of amino substituents at positions 2 and 4 of the same scaffold
were detrimental for antiplatelet activity.
Acid), ADP and collagen-induced aggregation, together with a good
in vivo antithrombotic effect.14–16 Some of these compounds were
also found to be interesting as analgesic and/or antiphlogistic
agents.14 The basic function in benzopyranopyrimidine scaffold
was also investigated and it was concluded that it has a pivotal role
in the expression of antiphlogistic/analgesic activities without
adverse gastrolesive effects.17
In the past, the 2-aminoacyl-5,6-dihydrobenzo[h]quinazoline
2a18 showed good antiphlogistic activity, while the 2-amino ana-
logue 2b was endowed with a weak antiplatelet effect in vitro on
AA-induced aggregation, but was devoid of antiphlogistic action.19
On the contrary, compounds 2c, having different amino groups at
position 4, showed a good anti-aggregant activity in the collagen-
induced aggregation test.20
At the same time, our benzo[g]indazoles 3 and 4 showed good
anti-inflammatory/analgesic activities, in particular when an ami-
no substituent was present at position 3.21,22
On the basis of this overall picture, we designed the new basic
5,6-dihydrobenzo[h]quinazolines 5a–h ( Fig. 1) bearing different
amino groups at positions 2 and 4, in order to obtain new anti-
platelet/analgesic and anti-inflammatory compounds, devoid of
gastrolesive action (the most relevant side effects for the NSAIDs).
Compounds 5a–h were prepared according to the following
Schemes 1 and 2.
On the other hand, the previously synthesized 2-amino/cycloa-
mino-5-cycloamino-benzopyrano-pyrimidines
1 showed good
in vitro antiplatelet activity, being able to inhibit AA (Arachidonic
The starting 2-ethoxycarbonyl-3,4-dihydro-(1H)-naphthalen-1-
one23 was reacted with guanidine to obtain compound 624,25 which
was then chlorinated at position 4 to obtain derivative 7. The inter-
mediate 7 reacted with suitable aryl–amines in DMSO at 80–90 °C
⇑
Corresponding author. Tel.: +39 010 353 8373; fax: +39 010 353 8399.
E-mail address: chiara.brullo@unige.it (C. Brullo).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.103

1126
C. Brullo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1125–1129
R
N
R
O
N
C H R
6
4
N
O
N
N
N
N
N
N
N
1
1
NHC H R
R
6
4
NHC H X
6
4
1
3
2a R = N(CH )CH COOH
4
3
2
R
= NH ,
2
1
R = CH , OCH , CF , F, Cl, Br
R = H
2b R = NH ; R = H
3
3
3
X = H, Cl, Br
cycloamines
1
1
R
= CH , F
3
2
1
2c R = H; R = cycloamines
R
N
N
R = NH NHCH N(CH )
3 2
2,
3,
1
1
R
= NH(CH )nC H (n =0,1,2)
R
2
6 5
cycloamines
5a-h
Figure 1. Molecular structures of compounds 1–4 and 5a–h.
NH
2
N
N
N
A
N
H
(CH )n
2
5a-c
NH
NH
2
2
n = 0,1,2
O
O
N
N
N
N
i)
NH
ii)
2
OEt
OH
Cl
N
B
5d-f
N
6
7
Scheme 1. Reagents and conditions: (i) Guanidine, absolute ethanol, reflux, 20 h (yield: 95%); (ii) POCl3, 100–110 °C, 4 h (yield: 89%); (A) aryl-amines, DMSO, 80–90 °C, 8 h
(yields: 54–80%); (B) cycloamines, 80–90 °C, 2–3 h (yields: 53–67%).
O
S
O
CH
3
NH
HN
2
HN CH
3
N
N
N
N
N
N
i)
iii)
ii)
NH
NH
NH
8
5a
5g
iv)
H C
3
CH
N
3
N
N
NH
5h
Scheme 2. Reagents and conditions: (i) p-toluenesulfonyl chloride, pyridine, room temperature, 24 h (yield: 97%); (ii) CH3I, K2CO3, an. DMF, 60 °C, 8 h; (iii) 50% H2SO4, 120–
130 °C, 5 h (yield: 63%); (iv) formic acid, formaldehyde, 80–90 °C, 12 h (yield: 45%).
for 8 h (Method A) to give compounds 5a–c in yields ranging from
54% to 80%. To obtain compounds 5d–f, 4-chloro derivative 7 was
treated with an excess of the suitable cycloalkylamine at 80–90 °C
for 2–3 h (Method B). Compound 5a was activated with p-toluene-
sulfonyl chloride in pyridine at room temperature as sulfonyl
derivative 8, which was in turn treated with 50% H2SO4 solution
to give the desired compound 5g in good yield. The Eschweiler–
Clarke reaction on N4-Phenyl-5,6-dihydrobenzo[h]quinazoline-
2,4-diamine (5a) gave the dimethyl derivative 5h. All the compound
structures were confirmed by elemental analyses and 1H NMR data.

C. Brullo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1125–1129
1127
Table 1
Structure and in vitro antiplatelet activity expressed as potency (IC50 in
lM) of compounds 5a–h and acetylsalicylic acid (ASA) in guinea-pig PRP
R
N
N
1
R
Compd
R
R1
U46619
ADP
AA
5a
5b
5c
5d
5e
5f
5g
5h
ASA
NH2
NH2
NH2
NH2
NH2
NH2
NHCH3
N(CH3)2
NHC6H5
205 (142–302)
n.a.
n.a.
n.a.
311 (242–400)
n.a.
n.a.
307 (226–418)
336 (293–385)
341
n.a.
>500
89 (75–107)
NHCH2C6H5
NHCH2CH2C6H5
1-Pyrrolidino
1-Piperidino
4-Morpholino
NHC6H5
200 (135–295)
89 (54–148)
166 (93–295)
68 (46–100)
224 (107–468)
105 (55–200)
46 (26–81)
385 (257–576)
n.a.
n.a.
n.a.
n.a.
NHC6H5
>500
45 (30–66)
Aggregation was stimulated by 2 lM U46619, 5 lM ADP or 100 lM AA. Data are mean and (95% confidence limits). n.a. = inactive up to 500 lM.
Compounds 5a–h were screened for their in vitro antiplatelet
activity in guinea-pig platelet-rich plasma (PRP), inducing the
aggregation by U46619 (a synthetic analog of TXA2) and ADP.26,27
In vivo experiments were performed in order to evaluate their anti-
phlogistic and analgesic activities in rat paw oedema and mouse
writhing tests, respectively.28,29
The study of in vitro platelet aggregation demonstrated a vari-
able degree of antiplatelet activity for all the studied compounds
(5a–h) (Table 1). In particular, the insertion of an amino–phenyl
group (5a) or a cycloamine (5d–f) at position 4 of the 5,6 dihydro-
benzo[h]quinazoline scaffold yielded new inhibitors of platelet
aggregation induced by different stimulants (such as U46619,
ADP and AA). In detail, the 4-anilino (5a) and the 4-piperidino
(5e) derivatives showed the widest spectrum of action, inhibiting
all the aggregating stimuli.
The remaining compounds with methylene and ethylene spacers
between the phenyl and the amino group at position 4 (5b–c), as
well as the methyl- or dimethyl-amino substituted derivatives at
position 2 (5g–h), displayed a different antiplatelet profile being
effective only against AA-induced aggregation. Within this group,
compound 5h resulted as potent as ASA.
In conclusion, derivatives 5a and 5e emerged as the most active
of the series, inhibiting all the aggregating stimuli, whereas com-
pounds 5c, 5h and 5g were the most selective, inhibiting only the
Table 2
Analgesic (writhing test) and anti-inflammatory (rat paw oedema) activities of
compounds 5a–h and indomethacin (Indo) (10 mg/kg os)
Compd
mg/kg per os
Writhing test
15 ns
Rat paw oedema
5a
100
50
25
100
50
100
50
100
50
25
100
50
25
100
50
100
50
100
50
25
54**
53*
0
5b
5c
5d
62**
8
23
54**
32 ns
30 ns
19
20
42**
42*
0
5e
0
31*
41*
0
5f
19 ns
40**
0
5g
5h
54**
0
5
64**
51*
0
0
Indo
10
81**
45**
Data represent the % inhibition of writhing episodes or oedema volume, at the
indicated doses. ns = not significant.
AA-induced aggregation (IC50 89, 105 and 46 lM respectively).
*
p < 0.05.
The in vivo study demonstrated that the insertion of amino-sub-
stituents at position 4 was able to confer analgesic or antiphlogistic
properties that were completely absent in the previous unsubstitut-
ed derivative 2a (Fig. 1 and Table 2). Compounds 5b–c and 5g–h
exhibited a certain antinociceptive activity in the acetic acid writh-
ing test performed on mice, an experimental model characterized
by a local increased production of hyperalgesic PGEs.26 In particular,
compound 5h, with a dimethylamino group at position 2, was the
most active analgesic agent within this series, showing good antin-
ociceptive action (51% inhibition) at a dose devoid of any sedative
effect (data not shown). Indeed treated-mice exhibited the same
spontaneous locomotor activity as vehicle-treated mice in the
classic open field test.30
**
p < 0.01 vs vehicle treated animals.
NHNH2
N
O
N
O
9
Figure 2. Molecular structure of compound 9.
In order to explain the ability to discriminate between antinoci-
ceptive and anti-inflammatory activity, we tested cyclooxygen-
ase1–2 (COX1–2) inhibition activity of compounds 5a and 5h,
characterized by the above mentioned antiphlogistic and analgesic
activity, respectively. Data showed that 5a and 5h were able to in-
hibit both COX1 (maximal inhibition = 50.5 and 28.7, respectively)
and COX2 (maximal inhibition = 38.6 and 50.7, respectively) only
at a high concentration (1 mM). These results strengthen the
Also compounds bearing benzylamino (5b), phenylethylamino
(5c) and phenylamino (5g) substituents at position 4 showed a cer-
tain analgesic profile (62%, 54% and 54% of inhibition, respectively)
and were completely inactive as anti-inflammatory agents on
carrageenan rat paw oedema.
On the contrary, compounds 5a and 5d–f prevented rat paw
swelling induced by intraplantar carrageenan, but did not reduce
writhing responses.

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C. Brullo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1125–1129
Figure 3. Selected docking poses of compound 5a (a) and compound 9 (b) into the COX1 catalytic site (pdb code 3N8Y). The diclofenac X-ray pose is also reported (C atom:
violet). Selected docking poses of compound 5h (c) and compound 9 (d) into the COX2 catalytic site (pdb code 3MQE). The SC-75416 X-ray pose is also reported (C atom:
violet).
hypothesis that 5a and 5h, both having good antiplatelet activity
not related to COX inhibition, act with two different mechanisms
of action. Indeed, compound 5a is characterized by a high spectrum
of antiplatelet activity coupled with a good in vivo anti-inflamma-
tory effect, whereas 5h is the most active AA-induced aggregation
inhibitor in the pool of all derivatives synthesized.13–17
With the aim of gaining a better understanding of the COX1–2
enzymatic assay results, and of deriving useful guidelines for the
synthesis, a computational study on compounds 5a and 5h was
also performed. Thus, both the compounds were submitted to
docking simulations, using the X-ray data coordinates of the
COX1/diclofenac31 and COX2/SC-7541632 complexes (pdb code
3N8Y and 3MQE, respectively). 5a and 5h final docking poses were
then compared with those obtained for compound 9 (Fig. 2), a
derivative already reported by us and showing IC50 of 54 and
60 nM on COX1 and COX2, respectively.17
oxygen atoms and the COX2 residues Tyr371 and Ser516 (Fig. 3c).
According to our calculations, the dihydrobenzo[h]quinazoline core
of compound 5h appears to be properly located in correspondence
to the SC-75416 benzopyrano ring (Fig. 3c). On the other hand, the
anilino substituent of 5h is unable to detect any H-bond contact
with the key residues Tyr371 and Ser516, being engaged in
stacking with Tyr371, Trp373 and Phe504. Unfortunately, com-
p p
À
pound 5a is unable to properly occupy the enzyme binding site.
Accordingly, compound 5a (Imax = 38.6%) displays a lower Imax
value in comparison to that of compound 5h (Imax = 50.7%).
%
Interestingly, the 2-hydrazino group of compound 9 is able to
detect a H-bond with the key residue Ser516, being properly
located in the COX2 pocket (Fig. 3d).
This additional computational study confirmed that the
benzopyrano scaffold is essential for COX inhibition, being more
involved than dihydrobenzo[h]quinazoline one in the interactions
with enzyme key residues.
On the other hand, our study underlines the validity of the 5,6
dihydrobenzo[h]quinazoline scaffold as the basal core for the devel-
opment of bioactive non acidic analgesic/antiplatelet or antiphlo-
gistic agents, as compounds 5a–h are very active as anti-
aggregating agents. Indeed, the simultaneous functionalization of
positions 2 and 4 in this condensed system, promotes, on the one
hand, a prevailing anti-inflammatory activity (see compound 5a),
endowed with an efficacy quite comparable to that of the reference
drug indomethacin, and, on the other hand (see compounds 5b–c
and 5g–h), the emerging of an antinociceptive activity with a paral-
lel ASA-like antiplatelet effect, not related to the COX inhibition.
Among these latter compounds, 5h, which combines good analgesic
potency with the highest antiplatelet activity within the series, sug-
gests a more complex mechanism of action that may deserve further
investigations.
In the 3N8Y X-ray pdb, diclofenac bioactive conformation
(Fig. 3a), displays two H-bond contacts between the two oxygen
atoms of the carboxylic group and the COX1 residues Tyr385 and
Ser530, and is also engaged in
pÀp stacking and in Van der Waals
contacts with Tyr355 and Leu523 and Leu531, through the
dichloro-phenyl ring. Interestingly, on the basis of our results, com-
pound 5a (Fig. 3a) properly orients the dihydrobenzo[h]quinazoline
portion toward Tyr355, Leu523, Leu531 and is able to detect one
H-bond with the key residue Tyr385 through the aniline moiety
nitrogen atom. On the contrary, in the corresponding compound
5h the anilino group is projected toward Leu523, Leu531 and
Tyr355, and lacks any H-bond with the Ser530 or Tyr385 key resi-
dues. Accordingly, compound 5a (Imax = 50.5%) displays a higher
Imax% value in comparison with compound 5h (Imax = 28.7%).
Notably, compound 9 (Fig. 2), showing good activity as COX1
inhibitor (IC50 = 54 nM), displays hydrophobic contacts with
Tyr355, Leu523, Leu531 and one H-bond with the oxygen atom
of the Leu352 carbonyl group (Fig. 3b). This residue is not involved
in diclofenac interaction with COX1, but in any case, could increase
the inhibitor-enzyme complex stability.
Acknowledgements
The authors wish to thank Dr. R. Raggio, Mr. O. Gagliardo and
F. Tuberoni for spectral recording Financial support from Univer-
sity of Genoa is gratefully acknowledged.
As concerns the 3MQE X-ray complex, the compound SC-75416
displays two H-bond contacts between the two carboxylic group

C. Brullo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1125–1129
1129
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