Synthesis and structure-activity relationships of 3H-quinazolin-4-ones and 3H-pyrido[2,3-d]pyrimidin-4-ones as CXCR3 receptor antagonists

Article abstract of DOI:10.1002/ardp.200700037

CXC chemokine receptor-3 (CXCR3) is a G-protein coupled receptor (GPCR) predominantly expressed on activated T lymphocytes that promote Th1 responses. Previously, we described the 3H-quinazolin-4-one containing VUF 5834 (decanoic acid {1-[3-(4-cyano-pheny

Full text of DOI:10.1002/ardp.200700037

Arch. Pharm. Chem. Life Sci. 2007, 340, 281–291
S. Storelli et al.
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Full Paper
Synthesis and Structure-Activity Relationships of 3H-
Quinazolin-4-ones and 3H-Pyrido[2,3-d]pyrimidin-4-ones as
CXCR3 receptor antagonists
Stefania Storelli1, Dennis Verzijl1, Jawad Al-Badie1, Niels Elders2, Leontien Bosch1,
Henk Timmerman1, Martine J. Smit1, Iwan J. P. De Esch1, and Rob Leurs1
1 Leiden/Amsterdam Center for Drug Research (LACDR), Division of Medicinal Chemistry, Faculty of
Sciences, Vrije Universiteit Amsterdam, Amsterdam, Netherlands
2 Department of Chemistry, Vrije Universiteit Amsterdam, Amsterdam, Netherlands
CXC chemokine receptor-3 (CXCR3) is a G-protein coupled receptor (GPCR) predominantly
expressed on activated T lymphocytes that promote Th1 responses. Previously, we described the
3H-quinazolin-4-one containing VUF 5834 (decanoic acid {1-[3-(4-cyano-phenyl)-4-oxo-3,4-dihydro-
quinazolin-2-yl]-ethyl}-(2-dimethylamino-ethyl)-amide) as a small-molecule CXCR3 antagonist
with submicromolar affinity and as a lead structure for the development of CXCR3 antagonists.
More recently, the related 3H-pyrido[2,3-d]pyrimidin-4-one compounds AMG 487 and NBI-74330
have been reported as nanomolar CXCR3 antagonists and these ligands are currently under clin-
ical investigation. The aim of this study is to link the structure-activity relationship (SAR) of the
previously published class of 3H-quinazolin-4-one containing CXCR3 ligands with these novel
clinical candidates. From the modification of the lead structure VUF 5834 emerged the impor-
tance of the (4-fluoro-3-(trifluoromethyl)phenyl)acetyl and the 3-methylen-pyridine as substitu-
ents to improve the affinity at the human CXCR3 receptor, whereas other features are less
important. The described molecules serve as tool to investigate the role of the CXCR3 receptor in
various inflammatory conditions.
Keywords: Antagonist / Chemokine / CXCR3 / GPCR /
Received: February 23, 2007; accepted: March 28, 2007
DOI 10.1002/ardp.200700037
interact with CXC chemokine receptor-3 (CXCR3) to exert
their functions [4]. Like all the chemokine receptors,
CXCR3 belongs to the superfamily of the G-protein
coupled receptors (GPCR) [5]. Activation of CXCR3 via G-
proteins of the Gi-type leads to an increase of intracellu-
lar calcium and cellular migration [6, 7]. Several studies
have shown that levels of CXCL9, CXCL10, and CXCL11
are elevated in various chronic inflammatory diseases
such as rheumatoid arthritis [8, 9], multiple sclerosis
[10], transplant rejection [11], hepatitis C infected liver
[12], atherosclerosis [13], chronic skin reactions
[6, 14, 15], and chronic obstructive pulmonary disease
[16] and are related to the infiltration of CXCR3-positive
T-cells. Consequently, it is believed that CXCR3 antago-
nists provide a therapeutic approach in chronic inflam-
matory and neoplastic diseases [17–19].
Introduction
Chemokines are a family of small proteins (70–80 amino
acids) secreted by leukocytes or tissue cells that mediate
cellular recruitment to sites of inflammation or injury
[1]. Depending on the number and spacing of conserved
cysteine residues, chemokines are classified into four
major groups (CC, CXC, CX3C, and XC) [2, 3]. The IFN-c-
inducible CXC chemokines, CXCL9, CXCL10, and CXCL11
Correspondence: Rob Leurs, Leiden/Amsterdam Center for Drug Re-
search (LACDR), Division of Medicinal Chemistry, Faculty of Sciences,
Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam,
The Netherlands
E-mail: r.leurs@few.vu.nl
Fax: +31 20 5987-610
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Figure 1. Structures of reference compounds VUF 5834 1a, AMG 487 2, and NBI –74330 3.
Reagents and conditions: i) propionyl chloride, DMF, 400C; ii) p-phenetidine or p-amino-benzonitrile, PCl3, toluene, reflux; iii) Br2, CH3COONa, CH3COOH, 408C; iv)
H2N(CH2)2N(CH3)2, EtOH, reflux; v) acid chloride, NEt3, dioxane or carboxylic acid, EDCI, DCM, DMF; vi) 3-(aminomethyl)pyridine, NEt3, DMF, DMF ; vii) carboxylic acid, EDCI,
DCM.
Scheme 1. General synthesis of 3H-quinazolin-4-one analogues.
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Chemokine CXCR3 Antagonists
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Reagents and conditions: i) iso-butylchloroformate, NMM, DCM, -208C; ii) 4-ethoxy-phenylamine or 4-iodo-phenylamine, -208C; iii) iso-butylchlorofor-
mate, NMM, DCM, – 208C; iv) Zn (CN)2, Pd(P(Ph)3)4, DMF, 808C; v) TFA, DCM, rt; vi) 3-pyridinecarboxaldehyde, Na(CH3COO)3BH, DCE; vii) acid chlor-
ide, NEt3, dioxane or carboxylic acid, EDCI, DCM, DMF or carboxylic acid, EDCI, HOBT, DIPEA, DCM.
Scheme 2. General synthesis of 3H-pyrido[2,3-d]pyrimidin-4-one analogues.
Several classes of CXCR3 antagonists have recently pounds VUF 5834 1a, AMG 487 2, and NBI –74330 3 see
been reported in scientific literature [20–25]. We [24] and Fig. 1.
others [26] have previously described VUF 5834, the dec-
anoic acid {1-[3-(4-cyano-phenyl)-4-oxo-3,4-dihydro-quina- Chemistry
zolin-2-yl]-ethyl}-(2-dimethylamino-ethyl)-amide 1a, as a The 3-phenyl-3H-quinazolin-4-one scaffold was con-
submicromolar CXCR3 antagonist and we have contin- structed as reported earlier (Scheme 1) [24]. The reaction
ued to explore the SAR of this series. During our studies, of 2-amino-benzoic acid with propionyl chloride afforded
Amgen Inc. announced that the structural derivative 2-propionylamino-benzoic acid 5 and treatment of this
AMG 487 (2) was under clinical investigation [27], while intermediate with phosphorus trichloride and 4-ethoxy-
scientists from Neurocrine Biosciences Inc. described analine or 4-amino-benzonitrile afforded compounds 6a
pharmacological evaluation of patent compound NBI- and 6b, respectively, in moderate yields. Intermediates
74330 (3) [28]. These ligands are the most potent CXCR3 7a and 7b were obtained in good yields via reaction with
ligands reported to date, showing affinity values for the bromine and were used for the subsequent reaction with
human CXCR3 receptor in the nanomolar range. In this the commercially available N,N-dimethylethylene dia-
study, we describe the SAR that links the previously pub- mine (leading to 9a and 9b) or 3-picolylamine (leading to
lished 3H-quinazolin-4-one compound VUF5834 1a and 8). Reaction of 9a and 9b with decanoyl, cyclohexanecar-
these novel 3H-pyrido[2,3-d]pyrimidin-4-one containing bonyl, benzoyl, or phenylacetyl chloride afforded com-
ligands 2 and 3. For the structures of reference com- pound 1a–d and 1f whereas compounds 1e and 10 were
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obtained via coupling of 8 or 9a, respectively, with (4-tri- Indeed, the absence of the decanoyl chain as in amine 9b
fluoromethoxy-phenyl)-acetic acid and using EDCI as acti- or in the presence of alternative lipophilic acetamide
vating agent.
moieties such as cyclohexanecarbonyl 1b, benzoyl 1c, or
The 3H-pyrido[2,3-d]pyrimidin-4-one derivatives 11a–f phenylacetyl 1d resulted in a complete loss of affinity
were prepared according to Scheme 2 [26]. Boc-D-alanine (Table 1). Inspired by the Amgen patent application [32],
was activated with iso-butylchloroformate under basic we subsequently synthesized the 4-trifluoromethoxy-
conditions at –208C and then allowed to react with 2- phenyl-acetyl compound 1e which showed a threefold
aminonicotinic acid leading to 13. Because of the poor increase in CXCR3 affinity (Table 1) compared to our lead
stability of 13 [29], the crude material was immediately structure 1a. Considering that the phenyl-acetyl deriv-
reacted with either 4-ethoxy-phenylamine or 4-iodo-phe- ative 1d does not have affinity for the CXCR3 receptor,
nylamine leading to compound 14a and 14b, respec- the affinity of the 4-trifluoromethoxy-phenylacetyl deriv-
tively. Subsequent ring closure afforded compounds 15a ative 1e is striking. Clearly, the 4-trifluoromethoxy sub-
and 15b in a moderate yield.
stituent is having an enormous effect on the CXCR3 affin-
With the aim to obtain the para-cyano analogue (in our ity and it seems that this part of the ligands is able to
hand the best substituent of the 3-phenyl-3H-quinazolin- have very specific interaction with the binding site of the
4-one series [24]) of reference compound 2, intermediate receptor.
15b was treated with zinc cyanide using tetrakis(triphe-
Next, the influence of the para-substitution on the phe-
nylphosphine)palladium as catalyst [30], resulting in 15c nyl ring in position 3 of the 3H-quinazolin-4-one scaffold
in acceptable yield. The boc protecting group of 15a and was investigated. We previously described the para-cyano
15c was cleaved off using trifluoroacetic acid and the moiety (as in 1a) as the best substituent for CXCR3 bind-
amines 16a and 16b were obtained in excellent yield. ing and reported it to be substantially better than a
HPLC analysis using chiral columns showed that the first methoxy group in this position [24]. Considering the
three steps result in racemization and loss of optical para-ethoxyphenyl group in reference compound 2 [32],
activity of the material. Key intermediates 17a and 17b we synthesized the para-ethoxy substituted analogue for
were obtained via reductive amination with pyridine-3- the 3H-quinazolin-4-one series and the resulting ligand
carbaldeyde. Final compounds 11a–c and 11f were 1f showed a twofold better affinity compared to the para-
obtained by coupling the appropriate amine with the cor- cyano-substituted lead compound 1a. The differences in
responding carboxylic acids [31] whereas for compounds affinity between the lower affinity of the para-methoxy-
11d and 11e the corresponding acid chlorides were used. phenyl derivative and the improved para-ethoxyphenyl
Reference ligands 2 and 3 have been reported as enantio- derivative, again illustrates the fine optimization work
pure compounds [32]. In our hands, the synthesis route by Amgen scientists. The subsequent replacement of the
described in this patent, results in the racemic mixtures N,N-ethylendiamine group at R1 with a pyridin-3-
(compounds 11a and 11c).
ylmethyl afforded the analogue 10. With a pKi value of
7.5, this compound is the most potent CXCR3 antagonist
belonging to the 3H-quinazolin-4-one series. Moreover,
Pharmacology
The affinity for the human CXCR3 of all compounds was the development of 10 nicely illustrates the key role of
determined by the displacement of [125I]CXCL10-binding both the R1 and R2 substituents at the amino function.
to membranes from HEK-293 cells stably expressing the
Next, we explored if the SAR of the 3H-quinazolin-4-
human CXCR3 receptor (see Experimental, Section 4, for ones could be transferred to the new 3H-pyrido[2,3-d]pyri-
details).
midin-4-ones scaffold. Comparing the two series (Tables 1
and 2) shows that the introduction of an additional nitro-
gen atom in the heterocyclic scaffold did not affect the
CXCR3 affinity (compare 10 with 11a). However, the
different amino substituents at R1 and R2 again had dra-
Results and discussion
In this study, we set out to link the SAR of the previously matic effects on the CXCR3 affinity. Compound 11d
described 3H-quinazolin-4-one and the more recent 3H- shows that the decanoyl chain is tolerated, as it is for the
pyrido[2,3-d]pyrimidin-4-one CXCR3 ligand classes. In the 3H-quinazolin-4-one series, although only a submicromo-
series of the 3H-quinazolin-4-one analogues (1a–f, 9b, 10, lar affinity is obtained. In addition, the unsubstituted
Table 1), proper decoration of the amide group is phenylacetyl analogue 11e did not have affinity for the
required to achieve appreciable affinity for the receptor. CXCR3 receptor, while the slightly more functionalized
Previously, we found that the decanoyl chain of 1a was 4-trifluoromethoxy-phenylacetyl group, as in reference
necessary to obtain micromolar affinity values at CXCR3. compound 2, significantly improves affinity. Again, a
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Table 1. The pKi values of the 3H-quinazolin-4-one analogues as determined by the displacement of [125I]CXCL10 binding to the
human CXCR3 receptor.
Compound No
X
R1
R2
pKia)
1a
9b
1b
1c
1d
1e
1f
CN
CN
CN
CN
CN
CN
OCH2CH3
OCH2CH3
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(CH3)2
pyridin-3-ylmethyl
CO(CH2)8CH3
H
cyclohexanecarbonyl
benzoyl
phenyl-acetyl
(4-(trifluoromethoxy)-phenyl)acetyl
CO(CH2)8CH3
(4-(trifluoromethoxy)-phenyl)acetyl
6.26 l 0.25
a5
a5
a5
a5
6.71 l 0.04
6.56 l 0.04
7.45 l 0.13
10
a)
Binding experiments were carried out on cell-membranes fractions from HEK-293 cells stably expressing the human CXCR3
receptor. The compounds were tested for their ability to displace [125I]CXCL10 binding. The values are represented as the mean
l S.E.M. of at least three independent experiments.
Table 2. The pKi values of the 3H-pyrido[2,3-d]pyrimidin-4-one analogues as determined by the displacement of [125I]CXCL10 binding
to the human CXCR3 receptor.
Compound No
X
R1
R2
pKia)
11a
11b
11c
11d
11e
11f
OCH2CH3
CN
pyridin-3-ylmethyl
pyridin-3-ylmethyl
pyridin-3-ylmethyl
pyridin-3-ylmethyl
pyridin-3-ylmethyl
H
(4-(trifluoromethoxy)-phenyl)acetyl
(4-(trifluoromethoxy)-phenyl)acetyl
(4-fluoro-3-(trifluoromethyl)phenyl)-acetyl
CO(CH2)8CH3
phenyl-acetyl
(4-(trifluoromethoxy)-phenyl)acetyl
H
H
7.39 l 0.08
7.39 l 0.05
8.13 l 0.12
6.47 l 0.09
a5
OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
5.34 l 0.19
a5
16a
17a
H
pyridin-3-ylmethyl
a5
a)
The binding experiments were carried out on cell-membranes fractions from HEK-293 cells stably expressing the human CXCR3
receptor. The compounds were tested for their ability to displace [125I]CXCL10 binding. The values are represented as the mean
l S.E.M of at least three independent experiments.
properly substituted phenyl ring is a key determinant for sciences Inc. [27, 28]. The presence of both a proper acet-
CXCR3 binding.
amide substituent and the pyridin-3-ylmethyl group are
Compound 11c has a fivefold higher affinity for CXCR3 necessary for high CXCR3 affinity. The absence of one of
than 11a, meaning that (4-fluoro-3-trifluoromethyl-phe- the two (compounds 17a and 11f) or both (16a) causes a
nyl)-acetyl is the best substituent at R2 for the 3H-pyr- dramatic loss of affinity. Nevertheless, 11f with (4-tri-
ido[2,3-d]pyrimidin-4-ones series. These results validate fluoromethoxy-phenyl)-acetyl substituent retains
a
recent disclosures by Amgen Inc. and Neurocrine Bio- micromolar affinity for the receptor.
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Impact Ion Mass Spectra (HREIMS) were recorded on a Finnigan
MAT 900 mass spectrometer (Thermo Electron Corporation, Bre-
men, Germany).
Finally, the influence of the para-substituent on the
phenyl ring in position 3 of the 3H-pyrido[2,3-d]pyrimi-
din-4-ones was investigated. Compound 11b was synthe-
sized to determine the effect of para-cyano substitution.
For this particular scaffold, the para-ethoxy derivative
11a and para-cyano derivative 11b do not show signifi-
cant differences in CXCR3 affinity.
2-Propionylamino-benzoic acid 5
Anthranilic acid 4 (68.60 g, 0.50 mol) was dissolved in N,N-dime-
thylformamide (250 mL). Propionyl chloride (47.60 mL,
0.55 mol) was added dropwise at such a rate that the tempera-
ture of the mixture remained below 408C. The product began to
precipitate after about one half of the acid chloride was added,
and the suspension was stirred vigorously at room temperature
for an additional three hours after the addition was completed.
The resulting mixture was poured into water (2.0 L) and stirred
for an additional two hours. The precipitated product was col-
lected by filtration, washed with water and dried in a vacuum
oven at 400C under reduced pressure to afford 73.03 g (76%) of
white crystals .1H-NMR (CDCl3) d: 1.25 (t, J = 7.6 Hz, 3H), 2.55 (q, J =
7.6 Hz, 2H), 6.75 (brs, 1H), 7.07–7.14 (m, 1H), 7.55–7.59 (m, 1H),
8.02–8.14 (m, 1H), 8.72–8.76 (m, 1H), 10.98 (s, 1H).
Conclusions
This study presents the synthesis and initial SAR of
CXCR3 antagonists of the 3H-quinazolin-4-one and 3H-
pyrido[2,3-d]pyrimidin-4-one series. Currently, we are
evaluating these compounds as tools for targeting
CXCR3 in a variety of inflammatory models. Moreover,
the structural insights obtained may be used in the
design of novel CXCR3 antagonists.
We thank Dr. Smoluch for conducting (HR) MS measurements,
Dr. De Kanter for NMR analysis, and Dr. Orru for his construc-
tive help with chiral HPLC analysis.
3-(4-Ethoxyphenyl)-2-ethylquinazolin-4(3H)-one 6a
To a suspension of 2-propionylamino-benzoic acid (3.00 g,
15.52 mmol) and p-phenetidine (2.41 g, 15.52 mmol) in toluene
was added dropwise phosphorus trichloride (0.61 mL,
6.98 mmol). The resulting suspension was heated to reflux for
8 h. After cooling to room temperature, a saturated sodium car-
bonate solution was added and the mixture was stirred vigo-
rously until all solid was dissolved. The solvent was removed in
vacuum and the resulting solid was collected by filtration,
rinsed with water, dried, and recrystallized from isopropyl alco-
hol to afford the desired compound as a white solid. Yield: 2.19 g
(48%). 1H-NMR (CDCl3) d: 1.20 (t, J = 7.4 Hz, 3H), 1.42 (t, J = 7.0 Hz,
3H), 2.44 (q, J = 7.4 Hz, 2H), 4.10 (q, J = 7.0 Hz, 2H), 7.01–7.23 (m,
4H), 7.39–7.58 (m, 1H), 7.62–7.89 (m, 2H), 8.22–8.26 (m, 1H).
Experimental
Chemistry
General procedures
The solvents were dried according to standard procedures. All
reactions were performed under an atmosphere of dry nitrogen.
1H- and 13C-NMR spectra were recorded on a Bruker AC-200
(200 MHz; Bruker Bioscience, Billerica, MA, USA) spectrometer.
Flash column chromatography was carried out with J. T. Baker
silica gel (J. T. Baker, Deventer, The Netherlands). Analytical
HPLC-MS analyses were conducted using a Shimadzu LC-8A prep-
arative liquid chromatograph pump system with a Shimadzu
SPD-10AV UV-Vis detector set at 254 nm, with the MS detection
performed with a Shimadzu LCMS-2010 liquid chromatograph
mass spectrometer (Shimadzu, Tokyo, Japan). The analyses were
performed using the following two conditions. Condition I: an
Xbridge (C18) 5 lm column (100 mm64.6 mm), with the follow-
ing two solvents: solvent A: 10% buffer pH 8 – 90% H2O; solvent
B: 10% aqueous buffer pH 8 – 90% acetonitrile. Flow rate =
2.0 mL/min; gradient from 5% to 90% B in 10 min, then 5 min at
90% B, then 15 min at 5% B. The total run time was 30 min. The
buffer-pH 8 is prepared with ammonia bicarbonate in H2O (0.4%
w/v) adjusted to pH 8 with ammonia hydroxide. Condition II:
Xbridge (C18) 5 lm column (100 mm64.6 mm), with the follow-
ing two solvents: solvent A: 0.1% formic acid – 99.9% H2O; sol-
vent B: 0.1% formic acid/99.9% acetonitrile. Flow rate = 2.0 mL/
min. Gradient from 5% to 90% B in 10 min, then 5 min at 90% B,
then 15 min at 5% B. The total run time was 30 min. Purities cal-
culated are based on RP HPLC-UV peak surface area of the com-
pounds. Chiral HPLC analyses were performed with a CHIRAL
CEL OD-H column (4.6 mm6250 mm) DAIC 14325, using as
eluants 0.05% diethylamine in hexane/isopropyl alcohol (95 : 5).
Flow rate = 1.1 mL/min, T = 308C. High Resolution Electron
4-(2-Ethyl-4-oxoquinazolin-3(4H)-yl)benzonitrile 6b
Analogous to the preparation of 6a, using p-amino-benzonitrile
1
(2.22 g, 15.52 mmol). Yield: 1.50 g (35%). H-NMR (CDCl3) d: 1.18
(t, J = 7.4 Hz, 3H), 2.34 (q, J = 7.4 Hz, 2H), 7.30–7.55 (m, 3H), 7.60–
7.95 (m, 4H), 8.21–8.29 (m, 1H).
2-(1-Bromo-ethyl)-3-(4-ethoxy-phenyl)-3H-quinazolin-4-
one 7a
A mixture of 6a (2.23 g, 7.56 mmol), anhydrous sodium acetate
(756 mg) and glacial acetic acid (10 mL) was warmed 408C. A sol-
ution of bromine (7.56 mmol) in acetic acid (2 mL) was added
over a period of 3 h. After the addition was completed the mix-
ture was poured into water (150 mL) and stirred at room temper-
ature for about 2 h. The precipitate product was isolated by fil-
tration, washed with warm water to remove the trace of the ace-
tic acid, then rinsed with a small amount of isopropyl alcohol
and dried to provide the desired compound 7a as a solid. Yield:
2.71 g (96%). 1H-NMR (CDCl3) d: 1.44 (t, J = 7.0 Hz, 3H), 2.02 (d, J =
6.7 Hz, 3H), 4.08 (q, J = 7.0 Hz, 2H), 4.61 (q, J = 6.7 Hz, 1H), 6.90–
7.25 (m, 3H), 7.30–7.55 (m, 2H), 7.75–7.80 (m, 2H), 8.24–8.28
(m, 1H).
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Chemokine CXCR3 Antagonists
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nicotinic acid (3.65 g, 26.43 mmol) was added and the reaction
mixture was allowed to warm up to room temperature, stirred
overnight, and then refluxed for 2.5 h. The suspension was
cooled to room temperature, filtrated, and concentrated under
reduced pressure. The raw product was used for the next step
without any purification.
4-[2-(1-Bromo-ethyl)-4-oxo-4H-quinazolin-3-yl]-
benzonitrile 7b
Analogous to the preparation of 7a, using 6b (2.08 g,
7.56 mmol). Yield: 2.34 g (88%). 1H-NMR (CDCl3) d: 2.02 (d, J =
6.6 Hz, 3H), 4.40 (q, 1H, J = 6.6 Hz), 7.25–7.35 (m, 1H), 7.45–7.65
(m, 1H), 7.70–7.95 (m, 5H), 8.20–8.24 (m, 1H).
{1-[3-(4-Ethoxy-phenylcarbamoyl)-pyridin-2-
2-[1-(2-Dimethylamino-ethylamino)-ethyl]-3-(4-ethoxy-
ylcarbamoyl]-ethyl}-carbamic acid tert-butyl ester 14a
Compound 13 (7.70 g, 26.43 mmol) as crude material was dis-
solved in dry DCM (250 mL) cooled to –208C. 4-Ethoxyphenyl-
amine (3.27 mL, 26.43 mmol) was added and stirred for 3 h. The
reaction mixture was allowed to warm up to room temperature
overnight, and then washed with 1.0 N hydrochloric acid, satur-
ated aqueous sodium bicarbonate, and brine. The organic phase
was dried over sodium sulphate, filtered, and concentrated in
vacuum. The raw product was used for the next step without fur-
ther purification.
phenyl)-3H-quinazolin-4-one 9a
A mixture of 7a (2.82 g, 7.57 mmol) and N,N-dimetylethylendi-
amine (1.31 mL, 12.11 mmol) in ethanol (100 mL) was heated to
reflux for 18 h. The ethanol was removed in vacuo and the con-
centrate dissolved in chloroform and washed with saturated
aqueous sodium bicarbonate (3650 mL) and the combined
organic extracts dried over sodium sulfate, filtered, and concen-
trated. The crude material was purified by flash chromatogra-
phy on silica gel eluting with methanol/dichloromethane (1:9)
to afford the desired compound as white solid. Yield: 1.48 g
(52%). 1H-NMR (CDCl3) d: 1.24 (d, J = 6.4 Hz, 3H), 1.43 (t, J = 6.9 Hz,
3H), 2.20 (s, 6H), 2.35–2.75 (m, 4H), 3.48 (q, J = 6.4 Hz, 1H), 4.07 (q,
J = 6.9 Hz, 2H), 7.15-7.25, (m, 4H), 7.40–7.55 (m, 1H), 7.55-7.80 (m,
2H), 8.22–8.26 (m, 1H).
{1-[3-(4-Iodo-phenylcarbamoyl)-pyridin-2-ylcarbamoyl]-
ethyl}-carbamic acid tert-butyl ester 14b
Analogous to the preparation of 14a, using 4-iodo-phenylamine
(5.79 g, 26.43 mmol). The raw product was used for the next step
without further purification.
4-{2-[1-(2-Dimethylamino-ethylamino)-ethyl]-4-oxo-4H-
quinazolin-3-yl}-benzonitrile 9b
{1-[3-(4-Ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-
Analogous to the preparation of 9a, using 7b (2.68 g,
1
7.56 mmol). Yield: 1.28 g (47%). H-NMR (CDCl3) d: 1.23 (d, J =
d]pyrimidin-2-yl]-ethyl}-carbamic acid tert-butyl ester 15a
A solution containing the crude 14a (6.69 g, 15.62 mmol) in
50 mL DCM and N-methylmorpholine (0.86 mL, 7.81 mmol) was
stirred for 20 min at room temperature, then cooled to –208C.
Iso-butylchloroformate (1.01 mL, 7.81 mmol) was added drop-
wise. The solution was stirred for 3 h and subsequently allowed
to warm to room temperature overnight. The reaction mixture
was washed with 1.0 N hydrochloric acid, saturated aqueous
sodium bicarbonate, and brine. The organic phase was dried
over sodium sulphate, filtered, concentrated in vacuum to give a
dark yellow oil. This material was dissolved in a small amount of
DCM. While stirring, petroleum ether was added. The resulting
precipitate was collected by filtration, washed with petroleum
ether, and dried to afford 15a as a pale yellow solid. Yield: 3.47 g
6.6 Hz, 3H), 2.22 (s, 6H), 2.30–2.59 (m, 4H), 3.31 (q, J = 6.6 Hz, 1H),
7.22–7.50 (m, 3H), 7.70–7.86 (m, 4H), 8.20–8,24 (m, 1H). 13C-
NMR (CDCl3) d: 20.87, 44.69, 45.12 (2 C), 54.93, 58.83, 113.55,
117.52, 120.42, 126.88, 127.04, 127.22 (2C), 129.71, 133.60,
133.66, 134.87, 140.66, 147.13, 158.59, 161.92. HREIMS m/z:
361.18876 (calcd. for C21H23N5O, 361.1903). Anal. RP-HPLC I: tR =
10.86 min (purity 100%), II: tR = min 9.57 (purity 97%), MS (ESI)
m/z 362.1 [M+H]+.
3-(4-Ethoxy-phenyl)-2-{1-[(pyridin-3-ylmethyl)-amino]-
ethyl}-3H-quinazolin-4-one 8
A solution of 3-(aminomethyl)pyridine (0.11 mL, 1.07 mmol) and
triethylamine (0.15 mL, 1.07 mmol) in DMF (50 mL) was stirred
for 0.5 h at room temperature. Then 7a (0.40 g, 1.07 mmol) was
added and the solution was stirred overnight. The reaction mix-
ture was concentrated, dissolved in water and extracted with
DCM. The organic layer was washed with water and a saturated
aqueous sodium carbonate, then dried, concentrated, and puri-
fied via flash chromatography (DCM/MeOH, 9.5:0.5) to afford
1
(64%). H-NMR (CDCl3) d: 1.17 (d, J = 7.00 Hz, 3H), 1.29–1.50 (m,
12H), 4.06 (q, J = 7.00 Hz, 2H), 4.61–4.66 (m, 1H), 5.75–5.79 (m,
1H), 7.01–7.44 (m, 5H), 8.56-8.58 (m, 1H), 8.93–8.95 (m, 1H).
{1-[3-(4-Iodo-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-
d]pyrimidin-2-yl]-ethyl}-carbamic acid tert-butyl ester 15b
Analogous to the preparation of 15a, using 14b (6.40 g,
15.62 mmol). Yield: 3.80 g (59%) of a pale yellow solid. 1H-NMR
(CDCl3) d: 1.28 (d, J = 6.77 Hz, 3H), 1.36 (s, 9H), 4.45–4.54 (m, 1H),
5.60–5.73 (m, 1H), 6.97–7.01 (m, 2H), 7.40–7.47 (m, 1H), 7.86–
7.96 (m, 1H), 8.56–8.58 (m, 1H), 8.96–8.97 (m, 1H).
1
the desired compound as 170 mg (40%) of pale yellow oil. H-
NMR (CDCl3) d: 1.23 (d, J = 6.6 Hz, 3H), 1.40 (t, J = 7.0 Hz, 3H), 3.48
(q, J = 6.6 Hz, 1H), 3.40–3.80 (m, 2H), 4.00 (q, J = 7.00 Hz, 2H),
6.75–7.25, (m, 5H), 7.45 (m, 1H), 7.55–7.80 (m, 3H), 8.22-8.26 (m,
1H), 8.35–8.50 (m, 2H).
[1-(4-Oxo-4H-pyrido[2,3-d][1,3]oxazin-2-yl)-ethyl]-
{1-[3-(4-Cyano-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-
carbamic acid tert-butyl ester 13
d]pyrimidin-2-yl]-ethyl}-carbamic acid tert-butyl ester 15c
To a solution containing 15b (0.50 g, 1.02 mmol) and zinc cya-
nide (0.17 g, 1.52 mmol) in DMF (30 mL) was added tetrakis(tri-
phenylphosphine)palladium (0) (0.24 g, 0.2 mmol). The reaction
mixture was heated to 808C for 5 h, diluted with ethyl acetate
and filtered trough a pad of celite. The filtrate was concentrated
N-(tert-butoxycarbonyl)-D-alanine (5.00 g, 26.43 mmol) was dis-
solved in dry DCM (350 mL). N-methylmorpholine (7.30 mL,
66.07 mmol) was added and the reaction mixture was allowed to
stir for 20 min. Iso-butylchloroformate (6.9 mL, 52.85 mmol) was
added and the reaction flask was cooled to –208C. Next, 2-amino-
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S. Storelli et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 281–291
under reduced pressure. The residue was diluted with ethyl ace-
tate and washed with saturated aqueous sodium bicarbonate
(1650 mL). The aqueous layer was extracted with ethyl acetate
(3650 mL) and the combined organic layers were concentrated.
The crude material was purified by flash chromatography
(ethyl acetate/hexane, 1:1) to afford 15c as white solid. Yield:
0.24 g (60%).1H-NMR (CDCl3) d: 1.25 (d, J = 6.8 Hz, 3H), 1.31 (s, 9H),
4.42–4.44 (m, 1H), 5.53–5.56 (m, 1H), 7.34–7.94 (m, 5H), 8.53–
8.55 (m, 1H), 8.93–8.95 (m, 1H).
4-(4-Oxo-2-{1-[(pyridin-3-ylmethyl)-amino]-ethyl}-4H-
pyrido[2,3-d]pyrimidin-3-yl)-benzonitrile 17b
Analogous to the preparation of 17a, using 16b (3.38 g,
11.60 mmol) Yield: 2.52 g (57%) of a white powder. 1H-NMR
(CDCl3) d: 1.34 (d, J = 6.61 Hz, 3H), 3.35 (q, J = 6.61 Hz, 1H), 3.62–
3.88 (m, 2H), 7.15–7.32 (m, 2H ), 7.33–7.35 (m, 1H), 7.54–7.56
(m, 1H), 7.69–7.71 (m, 2H), 7.84–7.86 (m, 1H), 8.49–8.51 (m, 2H),
8.59–8.61 (m, 1H), 9.00–9.02 (m, 1H).
Decanoic acid {1-[3-(4-cyano-phenyl)-4-oxo-3,4-dihydro-
2-(1-Amino-ethyl)-3-(4-ethoxy-phenyl)-3H-pyrido[2,3-d]-
quinazolin-2-yl]-ethyl}-(2-dimethylamino-ethyl)-amide 1a
To one equivalent of 9b (0.36 mg, 1.00 mmol) and one equivalent
of triethylamine (0.14 mL, 1.00 mmol) dissolved in 25 mL diox-
ane was added one equivalent of decanoyl-chloride (0.21 mL,
1.00 mmol). The resulting mixture was stirred at room tempera-
ture for 18 h and concentrated under reduced pressure. The resi-
due was dissolved in dichloromethane and washed with satur-
ated aqueous Na2CO3 solution, then twice with water. The
organic phases were dried over anhydrous Na2SO4 and evapo-
rated. The crude material was purified by flash chromatography
with ethyl acetate/methanol (9:1) to afford the desired com-
pound as free bases. Yield 0.18 g (35%). 1H-NMR (CDCl3) d: 0.83–
0.85 (m, 3H), 1.20–1.22 (m, 16H), 1.34 (d, J = 6.9 Hz, 3H), 1.37–
1.49 (m, 2H), 1.86–2.19 (m, 7H), 3.30–3.50 (m, 2H), 4.65 (q, 0.2H),
5.18 (q, J = 6.9 Hz, 0.8H), 7.33–7.83 (m, 7H), 8.19–8.22 (m, 1H).
13C-NMR (CDCl3) d: 13.94, 16.35, 22.48, 22.23, 29.10, 29.27 (2C),
31.67, 30.09, 41.141, 45.33, 45.61 (2C), 50.58, 59.69, 113.36,
117.76, 120.66, 126.87, 127.27, 127.44, 129.33, 130.10, 133.28,
133.38, 134.66, 140.14, 146.78, 155.28, 161.89. HREIMS m/z:
515.32438 (calcd. for C31H41N5O2, 515.3260). Anal. RP-HPLC I: tR =
15.80 min (purity 95%), II: tR = 15.43 min (purity 100%), MS (ESI)
m/z 516.3 [M+H]+.
pyrimidin-4-one 16a
Compound 15a (2.16 g, 5.27 mmol) in DCM (25 mL) was treated
with trifluoroacetic acid (8.50 mL) and stirred for 4 h at room
temperature. The reaction was extracted with hydrochloric acid
1M (3650 mL), combined water layers were made basic with
aqueous NH4OH solution (25%, pH 9-10) and extracted with DCM
(36150 mL). The combined organic layers were dried over
Na2SO4 and concentrated to afford 1.64 g of 16a in quantitative
yields as a white solid. 1H-NMR (CDCl3) d: 1.31 (d, J = 6.6 Hz, 3H),
1.44 (t, J = 7.0 Hz, 3H), 1.81 (s, 2H), 3.78 (q, J = 6.6 Hz, 1H), 4.07 (q, J
= 7.0 Hz, 2H), 7.00–7.17 (m, 4H), 7.40–7.42 (m, 1H), 8.55–8.57
(m, 1H), 8.95–8.97 (m, 1H). 13C-NMR (CDCl3) d: 14.61, 22.77,
49.57, 63.71, 115.65, 115.86, 123.55, 124.27, 128.58, 129.03,
132.35, 140.65, 152.15, 156.50, 159.56, 164.54. HREIMS m/z:
310.14254 (calcd. for C17H18N4O2, 310.1430). Anal. RP-HPLC I: tR =
10.08 min (purity 99%), II: tR = min 10.44 (purity 100%). MS (ESI)
m/z 311.1 [M+H]+. Chiral HPLC: I enantiomer: tR = 14.00 min, II
enantiomer: tR = 22.00 min.
4-[2-(1-Amino-ethyl)-4-oxo-4H-pyrido[2,3-d]pyrimidin-3-
yl]-benzonitrile 16b
Analogous to the preparation of 16a, using 15c (2.06 g,
5.27 mmol). Yield: 1.53 g (100%) of a white solid. 1H-NMR (CDCl3)
d: 1.31 (d, J = 6.6 Hz, 3H), 2.39 (brs, 2H), 3.62 (q, J = 6.6 Hz, 1H),
7.23–7.70 (m, 3H), 7.70–7.87 (m, 2H), 8.51–8.53 (m, 1H), 8.96–
8.98 (m, 1H).
Cyclohexanecarboxylic acid {1-[3-(4-cyano-phenyl)-4-
oxo-3,4-dihydro-quinazolin-2-yl]-ethyl}-(2-dimethylamino-
ethyl)-amide 1b
Analogous to the preparation of 1a, using 9b and cyclohexanoyl
chloride (0.13 mL, 1.00 mmol). Yield 0.36 g (77%).1H-NMR (CDCl3)
d: 1.21–1.56 (m, 6H), 1.63–1.89 (m, 8H), 2.12–2.36 (m, 8H),
3.41–3.59 (m, 2H), 1.86–2.19 (m, 7H), 5.24 (q, J = 6.7 Hz, 1H),
7.34–7.83 (m, 7H), 8.19–8.22 (m, 1H).13C-NMR (CDCl3) d: 16.23,
25.40, 25.52 (2C), 25.61, 41.21, 41.64, 45.66 (2C), 50.18, 60.28,
113.31, 117.84, 120.67, 126.89, 127.40, 129.50, 130.13, 133.26,
133.60, 134.69, 140.10, 146.85, 155.35, 161.88, 176.36. HREIMS
m/z: 471.26212 (calcd. for C28H33N5O2, 471.2634). Anal. RP-HPLC I:
tR = 13.19 min (purity 99%), II: tR = 11.22 min (purity 100%), MS
(ESI) m/z 472.2 [M+H]+.
3-(4-Ethoxy-phenyl)-2-{1-[(pyridin-3-ylmethyl)-amino]-
ethyl}-3H-pyrido[2,3-d]pyrimidin-4-one 17a
To a solution containing 16a (3.60 g, 11.60 mmol) in dichloro-
ethane (150 mL) was added 3-pyridinecarboxaldehyde (1.21 mL,
12.76 mmol) followed by sodium triacetoxyborohydride (3.69 g,
17.40 mmol). The reaction was allowed to stir at room tempera-
ture overnight. The mixture was diluted with DCM and washed
with 1.0 M ammonium hydroxide (16150 mL). The organic
phase was dried over magnesium sulfate, filtered, and concen-
trated to afford an yellow oil that was purified by flash chroma-
tography (DCM/MeOH, 9.5:0.5). A white powder was isolated.
Yield: 3.58 g (77%). 1H-NMR (CDCl3) d: 1.29 (d, J = 6.6 Hz, 3H), 1.41
(t, J = 7.0 Hz, 3H), 2.53 (brs, 1H), 3.42–3.80 (q, J = 6.6 Hz, 1H),
3.42–3.80 (m, 2H), 4.02 (q, J = 7.0 Hz, 2H), 6.80–7.24 (m, 5H),
7.41–7.43 (m, 1H), 7.61–7.63 (m, 1H), 8.41–8.43 (m, 2H), 8.59–
8.61 (m, 1H), 8.97–8.99 (m, 1H). 13C-NMR (CDCl3) d: 14.56, 21.32,
48.77, 53.27, 63.68, 115.45, 115.58, 115.99, 122.28, 123.19,
127.46, 128.62, 128.88, 134.83, 135.52, 136.77, 148.36, 149.44,
156.12, 157.51, 159.53, 162.67, 164.66. HREIMS m/z: 386.16152
(calcd. for C23H23N5O2 – CH3, 386.1617 ). Anal. RP-HPLC I: tR =
10.75 min (purity 97%), II: tR = 10.63 min (purity 97%), MS (ESI)
m/z 402.1 [M+H]+.
N-{1-[3-(4-Cyano-phenyl)-4-oxo-3,4-dihydro-quinazolin-
2-yl]-ethyl}-N-(2-dimethylamino-ethyl)-benzamide 1c
Analogous to the preparation of 1a, using 9b and benzoyl chlor-
ide (0.12 mL, 1.00 mmol).
1
Yield: 0.10 g (22%). H-NMR (CDCl3) d: 1.47 (d, J = 6.8 Hz, 3H),
1.87(s, 6H), 2.01–2.45 (m, 2H), 3.42–3.57 (m, 2H), 5.21–5.25 (m,
1H), 6.75–7.14 (m, 8H), 7.15–7.95 (m, 4H), 8.12–8.25 (m, 1H). 13C-
NMR (CDCl3) d: 18.66, 44.33 (2C), 57.13, 52.35 58.40, 114.85 (2C),
119.75, 122.05, 127.87, 128.93 (2C), 129.92, 130.28, 130.32,
130.67, 131.64, 133.32, 133.81, 134.33, 134.71, 136.57, 141.03,
147.72, 156.22, 177.85 HREIMS: the compound is not eligible for
detecting the mass ion peak. Anal. RP-HPLC I: tR = 12.39 min
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Arch. Pharm. Chem. Life Sci. 2007, 340, 281–291
Chemokine CXCR3 Antagonists
289
(purity 100%), II: tR = 12.09 min (purity 100%), MS (ESI) m/z 466.1
for C31H29N5O3, 519.2270). Anal. RP-HPLC I: tR = 12.16 min (purity
100%), II: tR = 13.22 min (purity 100%), MS (ESI) m/z 520.2 [M+H]+.
[M+H]+.
N-{1-[3-(4-Cyano-phenyl)-4-oxo-3,4-dihydro-quinazolin-
2-yl]-ethyl}-N-(2-dimethylamino-ethyl)-2-phenyl-
acetamide 1d
N-{1-[3-(4-Cyano-phenyl)-4-oxo-3,4-dihydro-quinazolin-
2-yl]-ethyl}-N-(2-dimethylamino-ethyl)-2-(4-
trifluoromethoxy-phenyl)-acetamide 1e
Analogous to the preparation of 1a, using 9b and phenylacetyl
chloride (0.13 mL, 1.00 mmol). Yield: 0.19 g (40%). 1H-NMR
(CDCl3) d: 1.37 (d, J = 7.0 Hz, 3H), 1.77-2.62 (m, 9H), 3.33-4.66 (m,
3H), 5.18 (q, J = 7.0 Hz, 1H), 7.12–7.97 (m, 7H), 8.21–8.23 (m, 1H).
13C-NMR (CDCl3) d: 16.23, 40.14, 42.52, 45.64 (2C), 51.01, 59.73,
113.31, 117.76, 120.70, 126.88, 127.02, 127.35, 127.90, 128.03,
128.62, 128.76, 129.30, 130.06, 133.32, 133.81, 134.33, 134.71,
140.00, 146.72, 155.10, 161.85, 171.20. HREIMS m/z: 479.23178
(calcd. for C29H29N5O2, 479.2321). Anal. RP-HPLC I: tR = 12.83min
(purity 98%), II: tR = 12.31 min (purity 99%), MS (ESI) m/z 480.2
[M+H]+.
To one equivalent of 9b (0.36 mg, 1.00 mmol) and one equivalent
(4-trifluoromethoxy-phenyl)-acetic acid (0.22 g, 1 mmol) dis-
solved in 25 mL dichloromethane and two drops N,N-dimethyl-
formamide, one equivalent of 1-(3-dimethylaminopropyl)-3-eth-
ylcarbodiimide hydrochloride (EDCI) (0.19 g, 1.00 mmol) was
added. The reaction mixture was stirred overnight, then basified
with sodium bicarbonate solution and extracted with dichloro-
methane (3650 mL). The organic layers were collected, dried
over sodium sulfate concentrated, and purified by flash chroma-
tography (DCM/methanol, 9.5:0.5) to afford the desired com-
pound as free base. Yield: 0.26 g (47%). 1H-NMR (CDCl3) d: 1.36 (d,
J = 7.0 Hz, 3H), 1.88–2.48 (m, 9H), 3.34–4.74 (m, 3H), 5.13 (q, J =
7.0 Hz, 1H), 7.07–7.40 (m, 4H), 7.44–7.91 (m, 7H), 8.21–8.23 (m,
1H) 13C-NMR (CDCl3) d: 16.22, 39.39, 42.80, 45.66 (2C), 51.25,
59.93, 113.41, 117.67, 120.69, 121.01, 126.95, 127.29, 128.12,
129.49, 130.26, 131.61 (2C), 132.18, 133.10, 133.32, 133.42,
133.81, 134.75, 135.04, 140.68, 148.00, 155.41, 161.80, 170.96.
HREIMS m/z: 563.21360 (calcd. for C30H28F3N5O3, 563.2144). Anal.
RP-HPLC I: tR = 14.01 min (purity 95%), II: tR = 13.73 min (purity
100%), MS (ESI) m/z 564.2 [M+H]+.
Decanoic acid (2-dimethylamino-ethyl)-{1-[3-(4-ethoxy-
phenyl)-4-oxo-3,4-dihydro-quinazolin-2-yl]-ethyl}-amide
1f
Analogous to the preparation of 1a, using 9a (0.38 g, 1.00 mmol)
and decanoylchloride (0.21 mL, 1.00 mmol). Yield: 0.23 g (42%).
1H-NMR (CDCl3) at 278C d: 0.78–0.81 (m, 3H), 1.05–1.29 (m, 18H),
1.33–1.50 (m, 4H), 2.11–2.38 (m, 8H), 3.10–3.17 (m, 0.4H), 3.53–
3.57 (m, 1.6H), 3.98 (q, J = 7.0 Hz, 2H), 4.80 (q, J = 7.2 Hz, 0.8H),
5.13 (q, J = 7.2 Hz, 0.2H), 6.84–7.15 (m, 3H), 7.25–7.77 (m, 4H),
8.05–8.28 (m, 1H). Peak splitting due to the presence of
rotamers, as confirmed by 1H-NMR (DMSO) measurements at
908C. HREIMS m/z: 534.35434 (calcd. for C32H46N4O3, 534.3570).
Anal. RP-HPLC I: tR = 16.67 min (purity 100%), II: tR = 16.44 min
(purity 100%), MS (ESI) m/z 535.3 [M+H]+.
N-{1-[3-(4-Ethoxy-phenyl)-4-oxo-3,4-dihydro-quinazolin-
2-yl]-ethyl}-N-pyridin-3-ylmethyl-2-(4-trifluoromethoxy-
phenyl)-acetamide 10
Analogous to the preparation of 1e, using 8 (0.40 g, 1.00 mmol)
and (4-trifluoromethoxy-phenyl)-acetic acid. Yield: 0.35 g (59%).
1H-NMR (CDCl3) at 278C d: 1.16 (d, J = 7.1 Hz, 2H), 1.32-1.41 (m,
5H), 2.56 (dd, J1 = 16.1 Hz, J2 = 40.0 Hz, 0.8H), 3.21–3.78 (m, 1.2H),
3.53–4.06 (m, 2.5H), 4.48 (dd, J1 = 14.8 Hz, J2 = 46.4 Hz, 0.6H),
4.79–5.02 (m, 1.6H), 5.34 (q, J = 7.1 Hz, 0.3H), 6.55–5.85 (m,
0.5H), 6.91–7.21 (m, 9H), 7.33–7.56 (m, 3H), 7.64–7.83 (m, 1.5H),
8.11–8.46 (m, 2H). Peak splitting due to the presence of
Decanoic acid {1-[3-(4-ethoxy-phenyl)-4-oxo-3,4-dihydro-
pyrido[2,3-d]pyrimidin-2-yl]-ethyl}-pyridin-3-ylmethyl-
amide 11d
Analogous to the preparation of 1a, using 17a (0.40 g,
1.00 mmol) and decanoyl chloride (0.21 mL, 1.00 mmol). Yield:
0.22 g (40%). 1H-NMR (CDCl3) at 278C d: 0.80–0.82 (m, 3H), 1.10–
1.97 (m, 20H), 2.23–2.30 (m, 2H), 4.01 (q, J = 7.0 Hz, 2H), 4.78–
5.22 (m, 2.4H), 5.30 (q, J = 7.2 Hz, 0.6H), 6.54–6.59 (m, 0.3H),
6.90–8.19 (m, 8H), 8.42–8.55 (m, 1.7H), 8.90–9.01 (m, 1H). Peak
1
rotamers, as confirmed by H-NMR (DMSO) measurements at
908C. HREIMS m/z: 602.21506 (calcd. for C33H29F3N4O4, 602.2141).
Anal. RP-HPLC I: tR = 14.53 min (purity 100%), II: tR = 15.36 min
(purity 100%), MS (ESI) m/z 603.1 [M+H]+.
1
splitting due to the presence of rotamers, as confirmed by H-
NMR (DMSO) measurements at 908C. HREIMS Not eligible for
detecting the ion peak mass. Anal. RP-HPLC I: tR = 15.06 min
(purity 96%), II: tR = 16.28 min (purity 96%), MS (ESI) m/z 556.3
[M+H]+.
N-{1-[3-(4-Cyano-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-
d]pyrimidin-2-yl]-ethyl}-N-pyridin-3-ylmethyl-2-(4-
trifluoromethoxy-phenyl)-acetamide 11b
Analogous to the preparation of 1e, using 17b (0.40 g,
1.00 mmol) and (4-trifluoromethoxy-phenyl)-acetic acid. Yield:
0.42 g (72%). 1H-NMR (CDCl3) d: 1.27 (d, 3H, J = 7.2 Hz), 3.50–3.72
(m, 2H), 5.03–5.13 (m, 3H), 7.00–7.19 (m, 5H), 7.23–7.49 (m, 3H),
7.85–7.99 (m, 3H), 8.48–8.56 (m, 3H), 8.99–9.02 (m, 1H). 13C-
NMR (CDCl3) d: 16.23, 39.81, 46.69, 52.39, 114.13, 116.24, 117.47,
121.05, 122.77, 123.54 (2C), 129.39, 130.15 (3C), 130.35 (2C),
132.41, 132.98, 133.20, 133.77, 134.10, 136.81, 139.34, 147.20,
148.87, 156.40, 157.07, 161.48, 161.88, 172.77. HREIMS m/z:
584.17947 (calcd. for C31H23F3N6O3, 584.1784). Anal. RP-HPLC I: tR
= 12.77 min (purity 100%), II: tR = 13.70 min (purity 100%), MS
(ESI) m/z 585.1 [M+H]+.
N-{1-[3-(4-Ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-
d]pyrimidin-2-yl]-ethyl}-2-phenyl-N-pyridin-3-ylmethyl-
acetamide 11e
Analogous to the preparation of 1a, using 17a and phenylacetyl
chloride (0.13 mL, 1.00 mmol). Yield: 0.04 g (8%). 1H-NMR (CDCl3)
at 278C d: 1.20 (d, J = 7.2 Hz, 2H), 1.40 (t, J = 6.9 Hz, 3H), 2.40–2.99
(m, 0.4H), 3.45-3.55 (m, 1.6H), 4.03 (q, J = 6.9 Hz, 2H), 4.35–5.17
(m, 2H ), 5.35 (q, J = 7.2 Hz, 1H), 6.85–7.50 (m, 11H), 7.50–7.75
(m, 1H), 8.25–8.60 (m, 3H), 8.75–8.92–9.04 (m, 1H). Peak split-
ting due to the presence of rotamers, as confirmed by 1H-NMR
(DMSO) measurements at 908C. HREIMS m/z: 519.22551 (calcd.
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S. Storelli et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 281–291
N-{1-[3-(4-Ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-
d]pyrimidin-2-yl]-ethyl}-N-pyridin-3-ylmethyl-2-(4-
trifluoromethoxy-phenyl)-acetamide 11a
Pharmacology
125I-CXCL10 binding to human CXCR3 receptor
Cell membranes from HEK293 cells stably expressing the human
CXCR3 receptor were prepared as follows. Cells were washed
with cold PBS, detached using cold PBS containing 1 mM EDTA
and centrifuged twice at 1500g for 10 min at 48C. The pellet was
resuspended in cold membrane buffer (15 mM Tris pH 7.5, 1 mM
EGTA, 0.3 mM EDTA, 2 mM MgCl2) and homogenized by 10
strokes at 1100–1200 rpm using a Teflon-glass homogenizer.
The membranes were subjected to two freeze-thaw cycles using
liquid nitrogen and centrifuged at 40 000 g for 25 min at 48C.
The pellet was rinsed with cold Tris-sucrose buffer (20 mM Tris
pH 7.4, 250 mM sucrose), resuspended in the same buffer and
frozen in liquid nitrogen. Protein concentration was deter-
mined using a Bio-Rad protein assay (BioRad, USA). Membranes
were incubated in 96-well plates in binding buffer (50 mM
HEPES pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 100 mM NaCl, 0.5%
BSA) with approximately 50 pM 125I-CXCL10 (PerkinElmer Life
and Analytical Sciences, Boston, MA) and increasing concentra-
tions of antagonists for 2 h at RT. Subsequently, membranes
were harvested via filtration through Unifilter GF/C plates (Per-
kinElmer Life and Analytical Sciences) pretreated with 1% poly-
ethylenimine and washed three times with wash buffer (50 mM
HEPES pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 500 mM NaCl). Radioac-
tivity was measured using a MicroBeta (PerkinElmer Life and
Analytical Sciences) counter. Binding data were analyzed using
Graphpad Prism.
4-(Trifluoromethoxy phenyl)-acetic acid (0.44 g, 2.00 mmol), 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDCI
(0.38 g, 2.00 mmol), 1-hydroxybenzotriazole hydrate HOBT
(0.27 g, 2.00 mmol) and diisopropylethylamine (DIPEA) (2.27 mL)
were stirred in DCM (70 mL). After 30 min, 17a (0.40 g,
1.00 mmol) was added and the reaction mixture was stirred
overnight, then extracted with 1 M HCl (3650 mL), basified
with ammonia hydroxide 25% solution and extracted with DCM
(36200 mL). The combined organic layers were dried over
Na2SO4 and concentrated to obtain the crude material that was
purified by flash chromatography (EtOAc/EtOH, 9.5:0.5) to
afford the desired compounds as free base. Yield: 0.33 g (54%).1H-
NMR (CDCl3) at 278C d: 1.25 (d, J = 7.2 Hz, 0.9 H), 1.36 (t, J = 6.9 Hz,
3H), 1.43 (d, J = 7.2 Hz, 2.1H), 2.70 (dd, J1 = 16.0 Hz, J2 = 33.6 Hz,
0.6H), 3.59 (dd, J1 = 15.7 Hz, J2 = 13.5 Hz, 1.4H), 4.03 (q, J = 6.9 Hz,
2H), 4.57 (dd, J1 = 14.8 Hz, J2 = 107.3 Hz, 0.6 H), 5.05–5.12 (m, 0.3
H), 5.18 (dd, J1 = 18.4 Hz, J2 = 2.9 Hz, 1.4H), 5.37 (q, J = 7.2 Hz,
0.7H), 6.70–6.74 (m, 0.3H), 6.85–7.24 (m, 8.5H), 7.36–7.47 (m,
2H), 7.61–7.66 (m, 0.6H), 8.17–8.23 (m, 0.6H), 8.42–8.56 (m, 2H),
8.93–9.04 (m, 1H). Peak splitting due to the presence of
rotamers, as confirmed by 1H-NMR (DMSO) measurements at
908C. HREIMS m/z: 603.20902 (calcd. for C32H28F3N5O4, 603.2093).
Anal. RP-HPLC I: tR = 13.38 min (purity 99%), II: tR = 14.65 min
(purity 99%), MS (ESI) m/z 604.2 [M+H]+.
N-{1-[3-(4-Ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-
d]pyrimidin-2-yl]-ethyl}-2-(4-trifluoromethoxy-phenyl)-
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Analogous to the preparation of 11a, using 16a (0.31 g,
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