Straightforward entry to pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones and their ADME properties

Article abstract of DOI:10.1016/j.bmc.2014.06.009

A straightforward synthesis of pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones was developed starting from 2-chloropyridine-3-carboxylic acid by esterification, nucleophilic aromatic substitution and amide formation in one step, and ring closure allowing their synthesis with two identical or two different group attached to nitrogen. The structural diversity of these [2,3-d]pyrimidine-2,4(1H,3H)-diones resulted in significant variation in the biopharmaceutical properties. This was reflected by the broad range in fasted state simulated intestinal fluid solubility values (12.6 μM to 13.8 mM), Caco-2 permeability coefficients (1.2 × 10^-6 cm/s to 90.7 × 10^-6 cm/s) and in vitro-predicted human in vivo intrinsic clearance values (0 to 159 ml/min/kg).

Full text of DOI:10.1016/j.bmc.2014.06.009

Bioorganic & Medicinal Chemistry 22 (2014) 3947–3956
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Straightforward entry to pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones
and their ADME properties
Martyna Jatczak a, Koen Muylaert a, Laurens M. De Coen a, Janneke Keemink b, Benjamin Wuyts b,
Patrick Augustijns b, Christian V. Stevens a,
⇑
a Research Group SynBioC, Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
b Drug Delivery and Disposition, Department of Pharmaceutical and Pharmacological Sciences, Faculty of Pharmaceutical Sciences, KU Leuven, O&N II Herestraat 49—box 921,
3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A straightforward synthesis of pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones was developed starting from
2-chloropyridine-3-carboxylic acid by esterification, nucleophilic aromatic substitution and amide for-
mation in one step, and ring closure allowing their synthesis with two identical or two different group
attached to nitrogen. The structural diversity of these [2,3-d]pyrimidine-2,4(1H,3H)-diones resulted in
significant variation in the biopharmaceutical properties. This was reflected by the broad range in fasted
Received 23 March 2014
Revised 2 June 2014
Accepted 4 June 2014
Available online 16 June 2014
state simulated intestinal fluid solubility values (12.6 lM to 13.8 mM), Caco-2 permeability coefficients
Keywords:
(1.2 ꢀ 10ꢁ6 cm/s to 90.7 ꢀ 10ꢁ6 cm/s) and in vitro-predicted human in vivo intrinsic clearance values
(0 to 159 ml/min/kg).
Pyridopyrimidines
Pyrimidinediones
Pyrimidines
ADME
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
O
O
R
O
R
O
N
N
Pyridines and pyrimidines have proven to be very valuable
building blocks because of diverse pharmacological activities. They
possess a wide range of pharmacological activities, including anti-
bacterial (nalidixic type), antitumor, antihypertensive, cardiotonic,
bronchodilator, vasodilator, antialergic, antimalarial, analgesic,
antifungal and CNS antidepressant properties.1 Pyrido[2,3-d]pyri-
mides also exhibit a variety of promising pharmacological activi-
ties, some of which are: dihydrofolate reductase inhibition,
diarrhea treatment, cyclin dependent kinase 4 inhibition and
K562 apoptose inhibition. Compounds containing pyrido[2,3-
d]pyrimidines as a central core have been identified as a new class
of fibroblast growth factor receptor (FGFR3) tyrosine kinase inhib-
itors.2 Other pyrido[2,3-d]pyrimidines were found as the main
metabolites of flupyrsulfuron in soil.3
Various methods are known for the synthesis of pyrido[2,3-
d]pyrimidine-2,4(1H,3H)-diones (Scheme 1). The most important
approach starts from 6-aminouracil4 or 6-amino-1,3-dimethylura-
cil5 (Route a) and pyridine-3-caboxylic acid derivatives6 (Route f).
Although some of these synthesis methods are useful, many of
them have major drawbacks such as the use of toxic chemicals,7
O
O
N
R
N
N
R
R
O
R
O
N
N
b
H2N
N
R
O
N
R
a
c
O
O
O
R
O
R
R
O
N
N
N
h
d
O
N
N
R
N
O
N
H
O
N
R
e
g
O
O
f
R
N
R
O
N
N
N
O
O
N
R
N
R
O
N
R
OH
NH2
N
Scheme 1. Possible pathways towards the pyrido[2,3-d]pyrimidine scaffold, R = H,
CH3.
harsh reaction conditions,8 low reaction yield9 and expensive
reagents10 or are not used commonly.11
Additionally, it is important to evaluate the biopharmaceutical
properties of these pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones in
order to foresee possible issues concerning intestinal and hepatic
⇑
Corresponding author. Tel.: +32 9 264 59 57; fax: +32 9 264 62 21.
E-mail address: chris.stevens@ugent.be (C.V. Stevens).
http://dx.doi.org/10.1016/j.bmc.2014.06.009
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

3948
M. Jatczak et al. / Bioorg. Med. Chem. 22 (2014) 3947–3956
Table 2
disposition and prevent attrition of promising compounds in later
drug development. Recently, we have reported on the biopharma-
ceutical profile of a pyrido[4,3-d]pyrimidines library, highlighting
the significant effect of the substitution pattern on the compounds’
biopharmaceutical characteristics.12 Hence, the intestinal solubil-
ity, permeability and hepatic metabolic stability were determined
for a selection of pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones.
Preparation of pyrido[2,3-d]pyrimidines 4
O
O
NR
NHR
CDI, NaH
THF/NMP, rt (16-18 h)
N
N
R
O
N
NHR
3
4
Entry
3
Time (h)
Yield (%)
2. Result and discussion
1
2
3
4
5
6
3a
3b
3c
3d
3f
18
18
16
18
16
17
4a
4b
4c
4d
4f
69
58
61
91
—
60
In view of the need to develop a pyridopyrimidine library cover-
ing a larger part of the chemical space including two different nitro-
gen substituents, a straightforward synthesis has been developed.
We report a simple and efficient protocol for the synthesis of pyr-
ido[2,3-d]pyrimidine-2,4(1H,3H)-diones based on the reaction of
2-propen-1-yl 2-chloro-3-pyridinecarboxylate 1 with alkyl and aryl
amines. Starting from commercially available 2-chloropyridine-3-
carboxylic acid and allyl alcohol using 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide hydrochloride (EDC) as coupling agent in the
presence of a catalytic amount of 4-(N,N-dimethylamino)pyridine
a
3j
4j
a
Product detected by LCMS; product was not isolated.
Under these conditions N-cyclohexyl-2-(cyclohexylamino)-3-pyri-
dinecarboxamide 3f (Table 2, entry 5), did not react at room tem-
perature nor reflux. For the ring closure, also triphosgene in the
presence of the amine was evaluated.16 Although after 30 hours
of stirring at room temperature, the desired product 4f was
formed, the isolation using column chromatography or crystalliza-
tion was not successful.
In order to develop a series of pyrido[2,3-d]pyrimidines with
two different alkyl groups, compound 2f or 2h was refluxed with
a different amine (Table 3). In the reaction of allyl 2-(cyclo-
hexyl)-3-pyridinecaboxylate 2f with allylamine, the yield was
moderate even after a longer period of reflux (Table 3, entry 1),
compared to the t-octyl derivative (Table 3, entry 4). For the
i-pentyl derivatives, longer reaction times provided the same yield
as for the t-octyl derivative (Table 3, entries 3 and 6). Reactions
with cyclopropylamine or i-propylamine did not occur even after
an extended period of reflux (4 days).
Compound 5 containing two different N-substituents were con-
secutively transformed to pyrido[2,3-d]pyrimidine 6, using the
same reaction conditions as for the ring closure of compound 4.
The yields were very moderate for allyl derivatives, 47% for cyclo-
hexyl derivative and 49% for the t-octyl derivative, respectively. All
other amines gave excellent yields (Table 4, entries 2, 3, 5 and 6).
Because of the variety of pharmacological activities of the pyr-
idopyrimidine scaffold, it is important to explore the drug-like
properties of this series of compounds. An ADME (Absorption, Dis-
tribution, Metabolism, and Excretion) study was therefore per-
formed on selected examples of the synthesized library (i.e., 2i,
3a, 3d, 3f, 4a, 4b, 4d, 4j, 5e, 6a). Key physicochemical properties
were determined using Marvin Sketch (Table 5). All compounds
were considered drug-like according to the Lipinski’s rules
(DMAP), 2-propen-1-yl 2-chloro-3-pyridinecarboxylate
1 was
obtained in 78%.13
The allyl alcohol was chosen without any specific reason,
however it might open up possibilities for later RCM reactions.
Compound 1 was further reacted with aliphatic/aromatic primary
amines leading to a mixture of compounds 2 and 3 in moderate
to good yields (Table 1). The reactions were carried out under
reflux, using the corresponding amine as solvent. Only for the
reaction with 1-adamantylamine, acetonitrile (MeCN) was used
as solvent (Table 1, entry 9). Compound 2 was obtained as single
product in the reaction with sterically hindered amines such as
cyclopropylamine, t-butylamine, t-octylamine and 1-adamantyl-
amine (Table 1, entries 5, 7, 8 and 9). Reaction with cyclohyexyl-
amine provides two products, 2-cyclohexylamino-nicotic acid
allyl ester 2f was obtained together with N-cyclohexyl-2-cyclohex-
ylamino-nicotinamide 3f (Table 1, entry 6). However, products 2
and 3 could be separated easily by column chromatography. The
reaction proceeds preferably by nucleophilic aromatic substitution
of the chlorine atom, which is more favorable than the amide
formation.14
Using 1,1-carbonyldiimidazole (CDI) as coupling reagent and
sodium hydride (NaH) as deprotonating reagent in a mixture of
THF and 1-methylpyrrolidinone (NMP) (ratio 2:3), ring closure
could be realized in 16–18 h at room temperature (Table 2).15
Table 1
Preparation of 3-substituted 2-aminopyridines 2 and 3
O
O
O
O
O
NHR
NHR
RNH2
reflux (17-259 h)
Table 3
+
Preparation of 2-aminopyridylcarboxamides 5
N
Cl
N
NHR
N
O
O
2
1
3
O
NHR´
NHR
R´NH2
Yielda (%)
reflux (42-189 h)
Entry
RNH2
Time (h)
N
NHR
N
1
2
3
4
5
6
7
8
9
10
allylamine
propylamine
butylamine
i-pentylamin
cyclopropylamine
cyclohexylamine
t-butylamine
t-octylamine
1-adamantylamine
benzylamine
19
20
23
17
68
21
259
27
117
21
2a
36
29
23
2
31
68
63
73
51b
26
3a
61
56
74
68
—
30
—
—
5
2
2b
2c
2d
2e
2f
2g
2h
2i
3b
3c
3d
3e
3f
3g
3h
3i
Entry
2
R0NH2
Time (h)
Yield (%)
1
2
3
4
5
6
7
8
2f
allylamine
189
42
186
192
43
113
88
192
5a
5b
5c
5d
5e
5f
54
55
85
—
74
68
85
—
propylamine
i-pentylamine
cyclopropylamine
allylamine
propylamine
i-pentylamine
cyclopropylamine
2h
—
56
2j
3j
5g
5h
a
After chromatography.
Reaction in MeCN.
b

M. Jatczak et al. / Bioorg. Med. Chem. 22 (2014) 3947–3956
3949
Table 4
Preparation of pyrido[2,3-d]pyrimidinediones 6
O
O
NR´
NHR´
CDI, NaH
THF/NMP, rt (15-18 h)
N
N
R
O
N
NHR
5
6
Entry
5
Time (h)
Yield (%)
1
2
3
4
5
6
5a
5b
5c
5e
5f
15
16
19
19
19
19
6a
6b
6c
6e
6f
47
91
97
49
97
86
Figure 1. Solubility (
pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones is presented as the mean + SD (n = 3).
l
M) in fasted state human intestinal fluid (FaSSIF) of the
5g
6g
concerning the molecular weight (MW), the amount of hydrogen
bond donors (HBD) and acceptors (HBA) and the partition coeffi-
cient (LogP)17 Moreover, polar surface areas were below 140 Å2
which is recommended by Veber et al.18
With respect to the evaluation of intestinal solubility and per-
meability, plain aqueous buffers are often used. However, plain
aqueous buffers insufficiently represent the in vivo conditions,
possibly resulting in too low solubilities to allow further experi-
ments. Hence, biorelevant media are more promising as media,
mimicking the in vivo environment more accurately due to the
presence of mixed micelles of taurocholate and lecithin, i.e. fasted
simulated intestinal fluid (FaSSIF).20,21 Recently, we have shown a
good correlation between solubility in FaSSIF and fasted state
human intestinal fluid (FaHIF).22 Table 5 and Figure 1 reveal a
broad range in solubility values for this series, ranging from
Figure 2. Absorption potential of the pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones in
fasted state simulated intestinal fluid (FaSSIF), estimated as the apparent perme-
ability coefficient (Papp) of the Caco-2 monolayer in presence and absence of 4 lM
P-glycoprotein (P-gp) inhibitor GF-120918 (data presented as the mean + SD, n = 3).
lower aqueous solubility. Also for the 2-aminopyridine scaffold,
the use of the 2 cyclohexyl substituents (3f) resulted in a lower
solubility compared with compounds containing aliphatic chains
on the same position (3a, 3d, 5e). These observations suggest that
aliphatic chains are preferred if solubility issues are encountered.
Similar as for the intestinal solubility, a broad range was
observed for the intestinal permeability of the compounds using
FaSSIF as apical medium (Table 5 and Fig. 2). With exception of
compound 2i, the Papp values of the pyrido[2,3-d]pyrimidine-
12.6 lM for compound 3f to 13.8 mM for compound 3a. To gener-
ally describe ‘solubility’ the United States Pharmacopoeia (USP)
uses different solubility expressions based on parts of solvent (in
this case FaSSIF) required for one part of solute.19 The selected
compounds could be classified as practically insoluble (2i, 3f, 4j,
5e, 6a) to slightly soluble (3a). The use of aliphatic chains as sub-
stituent on the pyrido[2,3-d]pyrimidines scaffold (4a, 4b, 4d)
resulted in a higher solubility compared with compound 4j con-
taining benzyl groups. The presence of ring structures as substitu-
ent generates a higher lipophilicity of the compound leading to a
2,4(1H,3H)-diones were significantly higher than that of
a
paracellular marker atenolol (5.3 ꢀ 10ꢁ6 cm/s) and lower than that
Table 5
Properties of pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones
a
b
e
Compound
MW (g/mol)
HBd
HBa
cLogPc
PSAd (Å2)
pKa
FaSSIF solubility (lM)
Classificationi
Papp (ꢀ10ꢁ6) (cm/s)
Drug-likef
2i
<500
3128
2171
2772
3012
2431
2471
3032
3431
2892
2851
65
1
2
2
2
0
0
0
0
610
3
65
4.3
2.0
3.8
4.2
1.7
2.0
3.4
3.7
3.9
2.7
6140g
51.2
54.0
54.0
54.0
53.5
53.5
53.5
53.5
54.0
53.5
4.9
5.3
5.4
5.3
1.0
1.1
1.0
1.0
5.3
1.0
20.7 5.3h
13801.7 937.8
509.2 55.9
12.6 2.4
1054.9 111.1
3265.2 75.6
384.7 55.2
53.7 0.5
207.8 17.2
43.1 19.7
PI
SS
1.2 0.5
46.8 1.8
19.8 1.3
9.7 2.0
90.7 15.0
63.0 6.2
13.9 1.7
30.5 1.2
14.7 1.3
32.0 0.9
3a
3d
3f
4a
4b
4d
4j
3
3
3
3
3
3
3
3
VSS
PI
VSS
VSS
VSS
PI
5e
6a
2
0
PI
PI
3
Abbreviations: MW, molecular weight, HBd, hydrogen bond donors, HBa, hydrogen bond acceptors, cLogP, computational partition coefficient, PSA, polar surface area, pKa,
acid dissociation constant, FaSSIF, fasted state simulated intestinal fluid, Papp, apparent permeability, Clint,hep,human, human hepatic intrinsic clearance.
a
Calculated with Marvin Sketch.
Calculated with Marvin Sketch.
Calculated with Marvin Sketch.
Calculated with Marvin Sketch.
b
c
d
e
Calculated with Marvin Sketch.
f
Lipinski’s rule of five.17
g
Veber et al., 2007.18
h
Standard deviation (SD), n = 3.
i
Solubility classification according to the United States Pharmacopoeia (USP, 2007): PI, practically insoluble, VSS, very slightly soluble, SS, slightly soluble.19

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M. Jatczak et al. / Bioorg. Med. Chem. 22 (2014) 3947–3956
of a transcellular marker indomethacin (93.3 ꢀ 10ꢁ6 cm/s). In liter-
ature, a Papp value of 10 ꢀ 10ꢁ6 cm/s has been reported to result in
a fraction absorbed in humans of at least 90%.23 Hence, for 9 out of
10 compounds the permeability was relatively high.
The permeability of the pyrido[2,3-d]pyrimidine-2,4(1H,3H)-
diones was also determined in the presence of 4 lM of the P-glyco-
was used as a positive control compound for hepatic metabolism
as it is known to be an extensively metabolized compound.27,28
Compounds 5e, 4d, 3f are more extensively metabolized compared
to verapamil which could be useful when the metabolite shows
biological activity. The metabolism of 2i is comparable to verapa-
mil. Five of the compounds are more stable than verapamil (4j,
3a, 3d, 6a, 4b). Since the biological activity of these compounds
still needs to be evaluated, compounds showing intermediate
metabolism (2i, 4j, 3a, 3d and 6a) are the most promising. Parent
concentrations remained stable in incubations performed in the
absence of NADPH and glucose-6-phosphate, indicating chemical
stability of the compounds under the incubation conditions used.
protein (P-gp) inhibitor elacridar (GF120918) to explore whether
P-gp has a modulatory effect on the absorption of the selected
compounds.24 None of the compounds showed
a significant
increase in Papp when elacridar was included in the medium; this
is in contrast with the 3.2-fold increase which was observed for a
known P-gp substrate indinavir.25 Hence the intestinal absorption
of these compounds is not expected to be modulated by P-gp-med-
iated efflux transport in the intestine.
3. Conclusions
It is important to note that solely the free concentration of the
compounds is able to permeate across the epithelial membrane.
Compounds with a high lipophilicity are readily incorporated in
micelles present in FaSSIF and are therefore less available for
absorption.26 Figure 3 clearly illustrates the significant correlation
between Papp and cLogP in FaSSIF.
The Clint of the pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones,
determined in human liver microsomes (HLM), was clearly com-
pound dependent, as depicted in Figure 4. Scaled values for intrin-
sic hepatic clearance in human (Clint,hep,human) varied between 0
and 159 ml/min/kg. For compound 4a, no metabolism was
observed under the conditions used. A possible explanation is that
the metabolism of this compound is not mediated by cytochrome
P450 enzymes (CYP) and can therefore not be determined with
liver microsomes as in vitro drug metabolism model. Verapamil
In summary,
a new synthesis of pyrido[2,3-d]pyrimidine-
2,4(1H,3H)-diones was developed using 2-chloropyridine-3-
carboxylic acid and amines, all of which are inexpensive and
commercially available materials. Compounds 2 and 3, obtained
without solvent (in case of solid amines solvent is required) or
catalyst, could be used for the synthesis of pyrido[2,3-d]pyrimi-
dine-2,4(1H,3H)-diones. The selective nucleophilic aromatic
substitution with sterically hindered amines at C1 (t-octylamine,
t-butylamine, 1-adamantylamine) is more difficult and needs
longer time of reactions than with other amines (e.g., allylamine,
butylamine, i-pentylamine). Amines with a sterically hindered
C2, allows a synthesis of N-alkyl 2-(alkylamino)-3-pyridinecarbox-
amides in good yields. The cyclopropylamine is an exception from
this rule, in the reaction with cyclopropylamine only compound 2e
was obtained. The synthesis of precursor 5 to pyrido[2,3-d]pyrimi-
dine-2,4(1H,3H)-diones with different groups attached to nitrogen,
required longer time reactions and yields are lower than in case
two identical alkyl group.
The biopharmaceutical profiling of a selection of pyrido[2,3-
d]pyrimidine-2,4(1H,3H)-diones reveals a broad range of struc-
ture-dependent solubility, permeability and hepatic metabolism
values. Concerning permeability and hepatic metabolism, all com-
pounds, except for 2i, show acceptable drug-like properties; the
outcome of the hepatic metabolism study depends on the biologi-
cal activity of the parent compound and its metabolites. The
selected compounds suffer from poor solubility in FaSSIF. Formula-
tion strategies could be necessary to achieve a sufficient solubility;
however, extra costs associated with the development of enabling
formulations should be taken into consideration.
Figure 3. Relationship between the intestinal permeability (Papp) of the pyrido[2,3-
d]pyrimidine-2,4(1H,3H)-diones and their in silico predicted lipophilicity (cLogP).
4. Experimental section
4.1. Chemicals
Hanks’ balanced salt solution (HBSS), Dulbecco’s modified
Eagle’s medium (DMEM), phosphate buffered saline (1ꢀ, PBS),
penicillin-streptomycin (10,000 IU/ml), nonessential amino acid
medium (100ꢀ),
L-Glutamine, fetal bovine serum (FBS) and 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were
obtained from Lonza (Verviers, Belgium). Simulated intestinal fluid
(SIF) powder was purchased from Biorelevant (Croydon, UK).
D-a-tocopheryl polyethylene glycol 1000 succinate (TPGS1000)
was obtained from Eastman Chemical Company (Kingsport, TN),
acetic acid from Chem-lab (Zedelgem, Belgium). Elacridar
(GF120918) was provided by GSK (London, UK). Water was
purified with a Maxima system (Elga Ltd, High Wycombe Bucks,
UK). Dimethyl sulfoxide (DMSO) and magnesium chloride were
obtained from Acros Organics (Geel, Belgium). Sodium hydroxide
pellets and nicotinamide adenine dinucleotide phosphate
tetra-sodium salt were obtained from Merck (Darmstadt, Germany).
Figure 4. Mean Clint values ( SD n = 3) for selected pyrido[2,3-d]pyrimidine-
2,4(1H,3H)-diones determined in a pool of human liver microsomes (HLM) from
45 donors.

M. Jatczak et al. / Bioorg. Med. Chem. 22 (2014) 3947–3956
3951
Sodium acetate trihydrate, methanol and sodium chloride were
purchased from VWR International (Leuven, Belgium). Verapamil
4.3.2.1. Allyl 2-(allylamino)nicotinate (2a).
EtOAc/PE (1/4),
36%; light yellow liquid; IR: 3372 (NH), 1658 (C(O)O), 1591, 1579
(C@C), 1508 (C@C), 1241 (CAN), 1126 (CAO). 1H NMR (300 MHz,
CDCl3): 4.18 (t, J = 5.50 Hz, 2H, CH2), 4.78 (d, J = 5.50 Hz, 2H,
CH2), 5.28 (qd, J = 11.56, 1.10, J = 14.86, 1.10, J = 8.81, 1.10,
J = 18.71, 1.10, Hz, 4H, 2ꢀCH2), 6.01 (ddq, J = 5.50 Hz, 2H, 2ꢀCH),
6.54 (dd, J = 12.66, 4.95 Hz, 1H, py), 8.07 (s, 1H, NH), 8.16 (dd,
J = 9.36, 1.65 Hz, 1H, py), 8.29 (dd, J = 6.05, 1.65 Hz, 1H, py); 13C
(CDCl3): d 66.7, 119.3, 122.2, 126.9, 131.4, 140.4, 150.1, 152.0,
164.2; MS: m/z (%) 219 [M+1]+ (100), 220 (12); HRMS calcd for
hydrochloride,
D-glucose 6-phosphate sodium salt (G6P) and
sodium phosphate monobasic monohydrate were obtained from
Sigma–Aldrich (St. Louis, MO).
4.2. Media
Caco-2 cell culture medium consisted of DMEM supplemented
with 10% FBS, 1% nonessential amino acid, and 100 IU/ml penicil-
lin-streptomycin (DMEM+). Transport medium consisted of HBSS
containing 25 mM glucose and was buffered with 10 mM HEPES
to pH 7.4 (HBSS+). Fasted state simulated intestinal fluid (FaSSIF)
was prepared by dissolving SIF powder in a phosphate buffer
(2.24 mg/ml), according to the manufacturer’s preparation
protocol.
C
12H15N2O2 [M+1]+ 219.1128; found 219.1135.
4.3.2.2. N-Allyl 2-(allylamino)-3-pyridinecarboxamide (3a). EtOAc/PE
(1/4), 61%; orange liquid; IR: 3327 (NH), 1626 (C(O)NH), 1577
(NH), 1504 (C@C), 1257 (CAN). 1H NMR (300 MHz, CDCl3): 4.03
(t, J = 5.50 Hz, 2H, CH2), 4.13 (t, J = 5.50 Hz, 2H, CH2), 5.17 (td,
J = 12.11, 1.65, J = 12.11, 1.65, J = 18. 71, 1.10 Hz, 4H, 2ꢀCH2),
5.95 (dqd, J = 5.50 Hz, 2H, 2ꢀCH), 6.23 (s, 1H, NH), 6.48 (dd,
J = 12.66, 4.95 Hz, 1H, py), 7.59 (dd, J = 9.36, 1.65 Hz, 1H, py),
8.20 (dd, J = 6.05, 1.10 Hz, 1H, py), 8.20 (s, 1H, NH); 13C NMR
(CDCl3): d 42.3, 43.3, 109.9, 110.6, 115.4, 116.9, 134.0, 135.2,
135.3, 151.9, 157.9, 168.3; MS: m/z (%) 218 [M+1]+ (100), 219
(20); HRMS calcd for C12H16N3O [M+1]+ 218.1288; found 218.1297.
4.3. General
All the reagents were commercially available and used without
further purification. Tetrahydrofuran (THF) was distilled from
sodium, dichloromethane (CH2Cl2) was distilled from calcium
hydride and immediately prior to use. TLC was carried out on glass
plates coated with silica gel (Merck, Kiesegel 60F254, precoated
0.25 mm). 1H (300 MHz/400 MHz) and 13C (75 MHz/100.6 MHz)
NMR spectra were recorded in CDCl3 as solvent, with a Jeol Eclipse
FT spectrometer and a Bruker Avance III Nanobay 400 MHz spec-
trometer at room temperature. Low-resolution mass spectra were
recorded using a direct inlet system with an Agilent 1100 series
LC/MSD type SL with a UV detector and mass spectrometer with
Electrospray Ionisation Geometry (ESI 70 eV) using a quadrupole
detector. IR spectra were recorded with a Perkin–Elmer Spectrum
One FTIR spectrometer with an ATR (Attenuated Total Reflectance)
accessory in neat form. Melting points were measured using Kofler
bench, type WME Heizbank of Wagner & Munz.
4.3.2.3. Allyl 2-(propylamino)-3-pyridinecarboxylate (2b). EtOAc/PE
(1/4), 29%; yellow liquid; IR: 3373 (NH), 1685 (C(O)O), 1593,
1580 (C@C), 1513 (C@C), 1242 (CAN), 1121 (CAO). 1H NMR
(300 MHz, CDCl3): 1.01 (t, J = 7.15 Hz, 3H, CH3), 1.67 (dq, J = 7.15,
7.15 Hz, 2H, CH2), 3.47 (dd, J = 19.81, 6.60 Hz, 2H, CH2), 4.77 (d,
J = 5.50 Hz, 2H, CH2), 5.29 (dd, J = 11.56, 1.10 Hz, 1H, @CH2), 5.39
(dd, J = 17.06, 1.1 Hz, 1H, @CH2), 6.00 (dq, J = 5.5, 5.5 Hz, 1H, CH),
6.50 (dd, J = 13.21, 4.95 Hz, 1H, py), 7.97 (s, 1H, NH), 8.14 (dd,
J = 9.36, 1.65 Hz, 1H, py), 8.29 (dd, J = 6.60, 1.65 Hz, 1H, py); 13C
NMR (CDCl3): d 11.8, 22.8, 42.9, 65.3, 105.5, 110.7, 118.3, 132.3,
140.1, 153.9, 158.9, 167.3; MS: m/z (%) 221 [M+1]+ (100), 222
(14); HRMS calcd for
221.1293.
C
12H17N2O2 [M+1]+ 221.1285; found
4.3.1. 2-Propen-1-yl ester 2-chloro-3-pyridinecarboxylic acid 1
To a cold solution of 2-chloropyridine-3-carboxylic acid (0.5 g,
0.003 mol) and allyl alcohol (0.184 g, 0.003 mol) in 40 ml of dry
CH2Cl2, EDC (0.67 g, 0.0035 mol) and DMAP (0.04 g, 0.0003 mol)
were added. The reaction mixture was stirred at room temperature
for 16 h. Then, H2O was added and the organic layer was washed
with NaHCO3 aqueous saturated solution and a saturated solution
of NaCl. The organic layer was dried over MgSO4, filtered and con-
centrated under vacuum. The product was purified by column
chromatography, using EtOAc as eluent, to obtain 0.49 g (78%) of
1 as colorless liquid. IR: 1733 (C(O)O), 1578, 1560 (C@C), 1402
(C@CH2), 1297, 1287, 1269, 1242 (C(O)O), 1063, 1051 (C@CH2);
1H NMR (300 MHz, CDCl3): 4.87 (d, J = 5.5 Hz, 2H, CH2), 5.34 (dd,
J = 10.5, 1.1 Hz, 1H, CH2), 5.45 (dd, J = 17.34, 1.1 Hz, 1H, CH2),
6.04 (ddt, J = 6, 11, 11, 6 Hz, CH), 7.35 (dd, J = 7.71, 4.95 Hz, 1H,
py), 8.2 (dd, J = 7.71, 2.2 Hz, 1H, py), 8.53 (dd, J = 4.4, 1.65 Hz, 1H,
py); 13C (CDCl3): d 66.65, 119.29, 122.19, 126.85, 131.41, 140.41,
150.12, 152.02, 154.78, 164.18; MS: m/z (%) 198 [M+1]+ (100),
199 (12), 200 (20); HRMS calcd for C9H8ClNO2 [M+1]+ 198.0316;
4.3.2.4. N-Propyl 2-(propylamino)-3-pyridinecarboxamide (3b). EtOAc/
PE (1/4), 56%; yellow liquid; IR: 3325 (NH), 1625 (C(O)NH), 1577
(NH), 1509 (CAH), 1459 (CAH), 1257 (CAN). 1H NMR (300 MHz,
CDCl3): 0.98 (dt, J = 7.71, 7.15 Hz, 6H, 2ꢀCH3), 1.53–1.73 (m, 4H,
2ꢀCH2), 3.37 (ddt, J = 7.15, 6.05 Hz, 4H, 2ꢀCH2), 6.32 (br, NH),
6.42 (dd, J = 4.95, 7.15 Hz, 1H, py), 7.55 (dd, J = 7.71, 1.65 Hz, 1H,
py), 8.08 (br, NH), 8.19 (dd, J = 4.40, 1.10 Hz, 1H, py); 13C NMR
(CDCl3): d 11.5, 11.8, 22.7, 22.9, 41.6, 42.9, 110.1, 135.1, 151.7,
158.1, 168.6; MS: m/z (%) 222 [M+1]+ (100), 223 (14); HRMS calcd
for C12H20N3O [M+1]+ 222.1601; found 222.1609.
4.3.2.5. Allyl 2-(butylamino)-3-pyridinecaboxylate (2c). EtOAc/PE
(1/4), 23%; yellow liquid; IR: 3373 (NH), 2957, 2929, 2871
(C@CAH), 1685 (C(O)O), 1593, 1580 (C@C), 1513 (CAH), 1241
(CAH), 1121 (CAN). 1H NMR (300 MHz, CDCl3): 0.96 (t,
J = 7.15 Hz, 3H, CH3), 1.64 (tt, J = 7.15 Hz, 2H, CH2), 1.45 (qt,
J = 7.15 Hz, 2H, CH2), 3.51 (dd, J = 19.26, 6.60 Hz, 2H, CH2), 4.77
(d, J = 5.50 Hz, 2H, CH2), 5.29 (dd, J = 11.56, 1.10 Hz, 1H, @CH2),
5.39 (dd, J = 18.71, 1.65 Hz, 1H, @CH2), 6.00 (dq, J = 5.5, 5.5 Hz,
1H, CH), 6.50 (dd, J = 12.66, 4.95 Hz, 1H, py), 7.94 (s, 1H, NH),
8.14 (dd, J = 9.36, 1.65 Hz, 1H, py), 8.29 (dd, J = 6.60, 1.65 Hz, 1H,
py); 13C NMR (CDCl3): d 13.9, 20.4, 31.7, 40.8, 65.3, 105.3, 110.7,
118.3, 132.3, 140.1, 153.9, 158.9, 167.3; MS: m/z (%) 235 [M+1]+
(100), 236 (14); HRMS calcd for C13H19N2O2 [M+1]+ 235.1441;
found 235.1439.
found 198.0319. Literature: M. H. Sherlock, CH 534129
19730413.
A
4.3.2. General procedure for 2 and 3
The mixture of 2-propen-1-yl ester 2-chloro-3-pyridinecarb-
oxylic acid (0.5 g, 0.003 mol) and amine (ꢂ5 ml) as solvent were
refluxed. The reaction mixture was cooled to room temperature,
precipitate was filtered off and washed with EtOAc (2ꢀ). Solvent
was removed under vacuum and product was purified by column
chromatography. The synthesis of compound 2i was carried out
in acetonitrile (20 ml) as solvent.
4.3.2.6. N-Butyl 2-(butylamino)-3-pyridinecarboxamide (3c). EtOAc/PE (1/
4), 74%; orange liquid; IR: 3324 (NH), 2957, 2929, 2871 (C@CAH), 1624

3952
M. Jatczak et al. / Bioorg. Med. Chem. 22 (2014) 3947–3956
(C(O)NH), 1577 (C-C), 1510 (CAH), 1258 (CAN). 1H NMR (300 MHz, CDCl3):
0.95 (dt, J = 7.15, 7.15 Hz, 6H, 2ꢀCH3), 1.29–1.49 (m, 4H, 2ꢀCH2), 1.59 (tt,
J = 7.15 Hz, 4H, 2ꢀCH2), 3.40 (qt, J = 7.15 Hz, 4H, 2ꢀCH2), 6.13 (br, NH),
6.44 (dd, J = 12.66, 4.95 Hz, 1H, py), 7.51 (dd, J = 9.36, 1.65 Hz, 1H, py), 8.05
(br, NH), 8.20 (dd, J = 6.05, 1.65 Hz, 1H, py); 13C NMR (CDCl3): d 13.8,
14.01, 31.6, 31.7, 39.6, 40.8, 110.0, 134.9, 151.8, 151.1, 168.3; MS: m/z (%)
250 [M+1]+ (100), 251 (17); HRMS calcd for C14H24N3O [M+1]+ 250.1914;
found 250.1924.
J = 4.40 Hz, 1H, CH), 5.96 (br, NH), 6.40 (dd, J = 12.11, 4.95 Hz, 1H,
py), 7.51(dd, J = 9.36, 1.65 Hz, 1H, py), 8.11 (d, J = 7.15 Hz, 1H,
NH), 8.18 (dd, J = 5.50, 1.10 Hz, 1H, py); 13C NMR (CDCl3): d 24.9,
25.0, 25.6, 26.0, 33.2, 33.3, 48.6, 48.8, 109.8, 135.1, 151.8, 157.5,
167.7; MS: m/z (%) 302 [M+1]+ (100), 303 (20); HRMS calcd for
C
18H28N3O [M+1]+ 302.2227; found 302.2227.
4.3.2.12. Allyl 2-(t-butylamino)-3-pyridinecaboxylate (2g). EtOAc/PE
(1/7), 63%; yellow liquid; IR: 3352 (NH), 1686 (C(O)O), 1591,
1528 (C@C), 1514 (CAH), 1243 (CAN), 1127 (CAO). 1H NMR
(400 MHz, CDCl3): 1.50 (s, 9H, 3ꢀCH3), 4.75 (dd, J = 2.92, 1.36 Hz,
1H, ACH2A), 4.76 (dd, J = 2.92, 1.56 Hz, 1H, ACH2A), 5.28 (dq,
J = 10.39 Hz, 1H, @CH2), 5.38 (dq, J = 17.61, 1H, @CH2), 6.01 (qt,
J = 5.57 Hz, 1H, ACH@), 6.46 (dd, J = 12.48, 4.68 Hz, 1H, py), 8.05
(s, 1H, NH), 8.12 (dd, J = 7.80, 2.08, 1H, py), 8.25 (dd, J = 4.66,
2.06, 1H, py); 13C NMR (CDCl3): d 29.2, 51.4, 65.1, 105.5, 110.2,
118.2, 132.3, 139.8, 153.2, 158.7, 167.5; MS: m/z (%) 235 [M+1]+
(100), 236 (14); HRMS calcd for C13H19N2O2 [M+1]+ 235.1441;
found 235.1447.
4.3.2.7. Allyl 2-(i-pentyl)-3-pyridinecaboxylate (2d). EtOAc/PE (1/5), 2%;
yellow liquid; IR: 3372 (NH), 2954, 2927, 2870 (@CAH), 1686 (C(O)O),
1593, 1581 (C@C), 1513 (CAH), 1242 (CAN), 1124 (CAO). 1H NMR
(400 MHz, CDCl3): 0.96 (d, J = 6.60 Hz, 6H, 2ꢀCH3), 1.56 (dt, J = 6.73 Hz,
2H, CH2), 1.69–1.79 (m, 1H, CH), 3.52 (dt, J = 7.29 Hz, 2H, CH2), 4.77 (d,
J = 5.60 Hz, 2H, CH2), 5.29 (dq, J = 10.52 Hz, 1H, @CH2), 5.39 (dq,
J = 17.25 Hz, 1H, @CH2), 6.02 (qt, J = 5.00 Hz, 1H, CH), 6.49 (dd, J = 12.48,
4.76 Hz, 1H, py), 7.89(s, 1H, NH), 8.14 (dd, J = 9.72, 2.00 Hz, 1H, py), 8.29
(dd, J = 6.72, 1.96 Hz, 1H, py); 13C NMR (CDCl3): d 22.6, 25.4, 38.5, 39.2,
65.2, 105.5, 110.6, 118.2, 132.1, 139.9, 153.7, 158.8, 167.3; MS: m/z (%)
249 [M+1]+ (100), 250 (18); HRMS calcd for C14H21N2O2 [M+1]+
249.1598; found 249.1608.
4.3.2.13. Allyl 2-(t-octylamino)-3-pyridinecaboxylate (2h). EtOAc/
PE (1/10), 73%; orange liquid; IR: 3353 (NH), 1686 (C(O)O), 1592
(C@C), 1515 (CAH), 1243 (CAN), 1126 (CAO). 1H NMR (300 MHz,
CDCl3): 0.95 (s, 9H, 3ꢀCH3), 1.54 (s, 6H, 2ꢀCH3), 1.97 (s, 2H,
CH2), 4.75 (d, J = 5.50 Hz, 2H, CH2), 5.28 (dd, J = 11.56, 1.10 Hz,
1H, @CH2), 5.38 (dd, J = 18.71, 1.10 Hz, 1H, @CH2), 6.00 (qd,
J = 5.5, 1H, CH), 6.43 (dd, J = 12.11, 4.40 Hz, 1H, py), 8.06 (s, 1H,
NH), 8.12 (dd, J = 9.91, 2.20, 1H, py), 8.24 (dd, J = 6.05, 1.65, 1H,
py); 13C NMR (CDCl3): d 30.1, 31.6, 31.8, 50.7, 55.3, 65.2, 105.4,
110.1, 118.2, 132.3, 139.9, 153.2, 158.7, 167.5; MS: m/z (%) 291
[M+1]+ (100), 292 (20); HRMS calcd for C17H27N2O2 [M+1]+
291.2067; found 291.2077.
4.3.2.8. N-(i-Pentyl) 2-(i-pentylamino)-3-pyridinecarboxamide
(3d). EtOAc/PE (1/5), 68%; yellow liquid; IR: 3327 (NH), 2954,
2928, 2869 (@CAH), 1624 (C(O)NH), 1577 (NH), 1510 (CAH),
1257 (CAN). 1H NMR (300 MHz, CDCl3): 0.94 (dd, J = 3.30,
0.55 Hz, 6H, 2ꢀCH3), 0.96 (dd, J = 3.30, 0.55 Hz, 6H, 2ꢀCH3), 1.52
(dt, J = 7.71, 7.71 Hz, 4H, 2ꢀCH2), 1.61–1.78 (m, 2H, 2ꢀCH), 3.43
(dt, J = 7.15, 7.15 Hz, 4H, 2ꢀCH2), 6.02 (br, NH), 6.45 (dd,
J = 12.66, 4.95 Hz, 1H, py), 7.50 (dd, J = 9.36, 1.65 Hz, 1H, py), 8.01
(s, 1H, NH), 8.21 (dd, J = 6.60, 1.65 Hz, 1H, py); 13C NMR (CDCl3):
d 22.6, 22.7, 38.2, 38.5, 39.4, 110.0, 134.9, 151.9, 158.1, 168.2;
MS: m/z (%) 278 [M+1]+ (100), 279 (19); HRMS calcd for
C
16H28N3O [M+1]+ 278.2227; found 278.2237.
4.3.2.14. Allyl 2-(1-adamantylamino)-3-pyridinecaboxylate (2i). EtOAc/
PE (1/7), 51%; white solid; mp. 94 °C; IR: 3338 (NH), 2905, 2848
(@CAH), 1686 (C(O)O), 1588 (C@C), 1511 (CAH), 1252 (CAN),
1130 (CAO). 1H NMR (300 MHz, CDCl3): 1.63–1.79 (m, 6H,
3ꢀCH2), 2.1 (s, 3H, 3ꢀCH), 2.19 (s, 6H, 3ꢀCH2), 4.75 (d,
J = 5.50 Hz, 2H, CH2), 5.27 (dd, J = 11.56, 1.10 Hz, 1H, @CH2), 5.38
(dd, J = 18.16, 1.10 Hz, 1H, @CH2), 6.00 (qt, J = 5.50 Hz, 1H, ACH@),
6.44 (dd, J = 12.66, 4.95 Hz, 1H, py), 7.97 (s, 1H, NH), 8.11 (dd,
J = 9.36, 1.65 Hz, 1H, py), 8.21 (d, J = 6.60, 2.20 Hz, 1H, py), 8.39
(d, J = 4.40 Hz, 1H, py); 13C NMR (CDCl3): d 29.8, 36.8, 41.9, 52.2,
65.2, 105.4, 110.3, 118.2, 132.4, 139.9, 153.2, 158.9, 167.6; MS:
m/z (%) 313 [M+1]+ (100), 314 (24); HRMS calcd for C19H25N2O2
[M+1]+ 313.1911; found 313.1914.
4.3.2.9. Allyl 2-(cyclopropyl)-3-pyridinecaboxylate (2e). EtOAc/PE
(1/8), 31%; yellow liquid; IR: 3370 (NH), 1686 (C(O)O), 1579
(C@C), 1500 (CAH), 1251, 1230 (CAN), 1128 (CAO). 1H NMR
(300 MHz, CDCl3): 0.52- 0.62 (m, 2H), 0.86 (dd, J = 18.16, 6.05 Hz,
2H, CH2), 2.89 (tt, J = 3.85, 3.30 Hz, 1H, CH), 4.76 (d, 1H,
J = 4.95 Hz, 2H, CH2), 5.29 (d, J = 10.46 Hz, 1H, @CH2), 5.39 (d,
J = 17.06 Hz, 1H, @CH2), 5.99 (dq, J = 5.50, 5.50 Hz, 1H, ACH@),
6.58 (dd, J = 12.11, 4.40 Hz, 1H, py), 8.05 (s, 1H, NH), 8.15 (d,
J = 7.71 Hz, 1H, py), 8.39 (d, J = 4.40 Hz, 1H, py); 13C NMR (CDCl3):
d 7.2, 23.8, 65.4, 106.1, 111.5, 118.4, 132.2, 139.9, 154.0, 159.8; MS:
m/z (%) 219 [M+1]+ (100), 220 (14); HRMS calcd for C12H15N2O2
[M+1]+ 219.1128; found 219.1135.
4.3.2.15. Allyl 2-(benzylamino)-3-pyridinecaboxylate (2j). EtOAc/PE
(1/3), 26%; colorless liquid; IR: 3371 (NH), 3085, 3063, 3028,
2926, 2875 (@CAH), 1685 (C(O)O), 1592, 1578 (C@C), 1509
(CAH), 1242 (CAN), 1131 (CAO). 1H NMR (300 MHz, CDCl3): 4.76
(dd, J = 5.50, 0.55 Hz, 2H, CH2), 4.77 (s, 2H, CH2-Bn), 5.29 (dd,
J = 11.56, 1.10 Hz, 1H, @CH2), 5.38 (dd, J = 19.26, 1.65 Hz, 1H,
@CH2), 6.00 (qt, J = 5.50 Hz, 1H, CH), 6.56 (dd, J = 12.66, 4.95 Hz,
1H, py), 7.21–7.43 (m, 6H, Bn), 8.17 (dd, J = 9.36, 1.65 Hz, 1H, py),
8.30 (dd, J = 6.60, 1.65 Hz, 1H, py), 8.29 (s, NH); 13C NMR (CDCl3):
d 44.9, 65.4, 105.7, 111.4, 118.4, 127.1, 127.6, 127.6, 128.6, 132.2,
139.5, 140.1, 153.9, 167.3; MS: m/z (%) 269 [M+1]+ (100), 270
4.3.2.10. Allyl 2-(cyclohexyl)-3-pyridinecaboxylate (2f). EtOAc/PE
(1/6), 68%; colorless liquid; IR: 3359 (NH), 1685 (C(O)O), 1592,
1578 (C@C), 1507 (CAH), 1241 (CAN), 1106 (CAO). 1H NMR
(300 MHz, CDCl3): 1.10–2.13 (m, 10H, cyclohexyl), 4.09 (tt,
J = 3.85 Hz, 1H, CH), 4.77 (d, J = 5.50 Hz, 2H, CH2), 5.29 (dd,
J = 11.56, 1.10 Hz, 1H, @CH2), 5.39 (dd, J = 18.71, 1.62 Hz, 1H,
@CH2), 6.00 (qt, J = 5.50 Hz, 1H, ACH@), 6.47 (dd, J = 12.66,
4.95 Hz, 1H, py), 7.96 (d, J = 6.60, NH), 8.14 (dd, J = 9.91, 2.20 Hz,
1H, py), 8.27 (dd, J = 6.60, 1.65 Hz, 1H, py); 13C NMR (CDCl3): d
24.9, 25.9, 48.8, 65.2, 105.3, 110.5, 118.3, 132.3, 140.2, 153.9,
158.2, 167.4; MS: m/z (%) 261 [M+1]+ (100), 262 (15); HRMS calcd
for C15H21N2O2 [M+1]+ 261.1598; found 261.1607.
(18); HRMS calcd for
269.1296.
C
16H17N2O2 [M+1]+ 269.1285; found
4.3.2.11. N-Cyclohexyl 2-(cyclohexylamino)-3-pyridinecarboxa-
mide (3f). EtOAc/PE (1/6), 30%; white solid; mp. 168 °C; IR: 3355,
3260 (NH), 2924, 2851 (@CAH), 1617 (C(O)O), 1576, 1552 (C@C),
1503 (CAH), 1258 (CAN). 1H NMR (300 MHz, CDCl3): 1.10–2.10
(m, 20H, cyclohexyl), 3.88 (tt, J = 3.30 Hz, 1H, CH), 4.02 (tt,
4.3.2.16. N-Benzyl 2-(benzylamino)-3-pyridinecarboxamide (3j). EtOAc/
PE (1/3), 56%; white solid; mp. 98 °C; IR: 3398, 3297 (NH), 3083,
3058, 3027, 2924, 2852, (@CAH), 1614 (C(O)N), 1573 (N-H),
1519, 1504 (C@C), 1260 (CAN). 1H NMR (300 MHz, CDCl3): 4.53
(d, J = 5.50 Hz, 2H, CH2), 4.70 (d, J = 5.50 Hz, 2H, CH2), 6.45 (dd,

M. Jatczak et al. / Bioorg. Med. Chem. 22 (2014) 3947–3956
3953
J = 12.66, 4.95 Hz, 1H, py), 6.49 (br, NH), 7.20–7.40 (m, 10H, 2ꢀBn),
7.54 (dd, J = 9.36, 1.65 Hz, 1H, py), 8.20 (d, J = 4.95 Hz, 1H, py), 8.50
(br, NH); 13C NMR (CDCl3): d 43.9, 44.9, 109.8, 110.8, 127.0, 127.6,
127.8, 127.9, 128.6, 128.9, 135.3, 138.0, 139.7, 152.1, 157.9, 168.3;
MS: m/z (%) 318 [M+1]+ (100), 319 (20); HRMS calcd for C20H20N3O
[M+1]+ 318.1601; found 318.1608.
4.3.3.5. N-Propyl 2-(t-octylamino)-3-pyridinecarboxamide (5f). EtOAc/
PE (1/7), 68%; white solid; mp. 73 °C; IR: 3348 (NH), 3276 (NH),
2954, 2871 (C@CAH), 1622 (C(O)N), 1581, 1553 (C@C), 1512
(CAH), 1263 (CAN). 1H NMR (300 MHz, CDCl3): 0.95 (s, 9H,
3ꢀCH3), 1.52 (s, 6H, 2ꢀCH3), 0.96 (t, J = 8.26 Hz, 3H, CH3), 1.60
(dq, J = 7.15, 7.15 Hz, 2H, CH2), 1.95 (s, 2H, CH2), 3.33 (dd,
J = 19.81, 6.60 Hz, 2H, CH2), 6.09 (s, 1H, NH), 6.36 (dd, J = 12.11,
4.40 Hz, 1H, py), 7.48 (dd, J = 9.36, 1.65 Hz, 1H, py), 8.10 (s, 1H,
NH), 8.15 (dd, J = 6.05, 1.65 Hz, 1H, py); 13C NMR (CDCl3): d 11.5,
22.9, 30.1, 31.6, 31.8, 41.6, 50.6, 54.9, 109.4, 110.1, 134.8, 151.1,
157.9, 168.9; MS: m/z (%) 292 [M+1]+ (100), 293 (20); HRMS calcd
for C17H30N3O [M+1]+ 292.2383; found 292.2388.
4.3.3. General procedure for 5
The mixture of allyl 2-(alkylamino)-3-pyridinecaboxylate (0.5 g,
0.003 mol) and amine (ꢂ5 ml) as solvent were refluxed. The reac-
tion mixture was cooled to room temperature, precipitate was fil-
tered off and washed with EtOAc. Solvent was removed under
vacuum and product was purified by column chromatography.
4.3.3.6. N-(i-Pentyl) 2-(t-octylamino)-3-pyridinecarboxamide
(5g). EtOAc/PE (1/10), 85%; yellow solid; mp. 68 °C; IR: 3286
(NH), 2955, 2870 (C@CAH), 1620 (C(O)N), 1578 (C@C), 1515
(CAH), 1264, 1223 (CAN). 1H NMR (300 MHz, CDCl3): 0.95 (s, 9H,
3ꢀCH3), 1.52 (s, 6H, 2ꢀCH3), 1.41–1.58 (m, 2H), 1.59–1.73 (m,
1H), 1.95 (s, 2H), 3.38 (dt, J = 6.05, 6.05 Hz, 2H, CH2), 6.09 (br,
NH), 6.35 (dd, J = 12.11, 4.95 Hz, 1H, py), 7.48 (dd, J = 7.71 Hz, 1H,
py), 8.13 (d, J = 6.60 Hz, 1H, py), 8.15 (br, NH); 13C NMR (CDCl3):
d14.3, 22.6, 26.0, 30.1, 31.6, 31.8, 38.2, 38.5, 50.6, 54.9, 109.4,
110.1, 134.8, 151.1, 157.9, 168.9; MS: m/z (%) 320 [M+1]+ (100),
321 (24); HRMS calcd for C19H34N3O [M+1]+ 320.2696; found
320.2703.
4.3.3.1. N-allyl 2-cyclohexylamino-3-pyridinecarboxamide (5a). EtOAc/
PE (1/8), 54%; white solid; mp. 96 °C; IR: 3357 (NH), 3285 (NH),
1620 (C(O)N), 1573, 1551 (C@C), 1503 (CAH), 1252 (CAN). 1H
NMR (300 MHz, CDCl3): 1.19–1.49 (m, 4H, cyclohexyl), 1.53–1.80
(m, 2H), 1.92–2.07 (m, 2H), 4.03 (t, J = 5.50 Hz, 3H, CH2 from CH2-
ACH@ and CH-cychex), 5.19 (dd, J = 9.91, 1.10 Hz, 1H, @CH2),
5.26 (dd, J = 17.61, 1.10 Hz, 1H, @CH2), 5.92 (qt, J = 5.50 Hz, 1H,
ACH@), 6.11 (br, NH-all), 6.43 (dd, J = 12.11, 4.95 Hz, 1H, py),
7.55 (dd, J = 9.36, 1.65 Hz, 1H, py), 8.12 (d, J = 6.05, NH-cychex),
8.20 (d, J = 3.30 Hz, 1H, py); 13C NMR (CDCl3): d 24.9, 26.0, 33.2,
42.2, 48.8, 109.2, 109.9, 116.9, 134.1, 135.2, 152.2, 157.5, 168.4;
MS: m/z (%) 260 [M+1]+ (100), 261 (18); HRMS calcd for
4.3.4. General procedure for 4 and 6
C
15H22N3O [M+1]+ 260.1757; found 260.1761.
To a solution of N-alkyl-2-(alkylamino)-3-pyridinecarboxamide
in a mixture of dry THF and NMP (2:3 ratio), NaH (3 equiv) was
added and CDI (3 equiv). The reaction mixture was stirred at room
temperature for 15–19 h. After this time, solid was filtered off and
washed with THF (2ꢀ), filtrate was concentrated under vacuum.
The desired product was isolated by column chromatography.
4.3.3.2. N-Propyl 2-cyclohexylamino-3-pyridinecarboxamide
(5b). EtOAc/PE (1/6), 55%; white solid; mp. 119 °C; IR: 3360
(NH), 3300 (NH), 2923, 2852 (C@CAH), 1620 (C(O)N), 1573
(C@C), 1500 (CAH), 1252 (CAN). 1H NMR (300 MHz, CDCl3): 0.97
(t, J = 7.71 Hz, 3H, CH3), 1.18–1.50 (m, 4H, cyclohexyl), 1.60 (dq,
J = 14.31, 7.15 Hz, 2H, CH2), 1.68–1.80 (m, 2H, cyclohexyl), 1.95–
2.08 (m, 2H, cyclohexyl), 3.35 (dd, J = 19.81, 6.60 Hz, 2H, CH2),
4.00 (tt, J = 3.85 Hz, 1H, CH from cyclohexyl), 6.13 (br, NH), 6.41
(dd, J = 12.66, 4.95 Hz, 1H, py), 7.52 (dd, J = 9.36, 1.65 Hz, 1H, py),
8.09 (d, J = 6.60 Hz, 1H, NH from NH-cyclohexyl), 8.18 (dd,
J = 6.60, 1.65 Hz, 1H, py); 13C NMR (CDCl3): d 11.5, 22.9, 24.9,
26.0, 33.2, 41.6, 48.8, 109.8, 109.8, 135.1, 151.9, 157.5, 168.6;
MS: m/z (%) 262 [M+1]+ (100), 263 (18); HRMS calcd for
4.3.4.1. 1,3-Diallyl-pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione
(4a). EtOAc/PE (1/3), 69%; white solid; mp. 91 °C; IR: 2986,
2923, 2853 (C@CAH), 1708 (NC(O)N), 1657 (C(O)N), 1595
(C@C), 1482, 1462, (C@C). 1H NMR (300 MHz, CDCl3): 4.70 (t,
J = 5.5 Hz, 2H, CH2), 4.99 (t, J = 5.50 Hz, 2H, CH2), 5.26 (td,
J = 5.50, 0.55 Hz, J = 3.30, 0.55 Hz, J = 17.06, 0.55 Hz, 4H, 2ꢀ@CH2),
5.97 (dqd, J = 5.50 Hz, 2H, 2ꢀCH), 7.22 (dd, J = 12.11, 4.40 Hz, 1H,
py), 8.47 (dd, J = 8.81, 1.10 Hz, 1H, py), 8.67 (dd, J = 5.50, 1.10 Hz,
1H, py); 13C NMR (CDCl3): d 43.9, 44.5, 110.9, 117.8, 118.3, 119.1,
131.6, 132.0, 137.9, 150.5, 150.6, 154.3, 160.9; MS: m/z (%) 244
[M+1]+ (100), 245 (13); HRMS calcd for C13H14N3O2 [M+1]+
244.1081; found 244.1084.
C
15H24N3O [M+1]+ 262.1914; found 262.1918.
4.3.3.3. N-(i-Pentyl) 2-cyclohexylamino-3-pyridinecarboxamide
(5c). EtOAc/PE (1/7), 85%; orange oil; IR: 3320 (NH), 2954, 2928,
2854 (C@CAH), 1626 (C(O)N), 1576 (C@C), 1507 (CAH), 1257
(CAN). 1H NMR (400 MHz, CDCl3): 0.95 (d, J = 6.65 Hz, 6H, 2ꢀCH3
i-pentyl), 1.24–1.35 (m, 4H, 2ꢀCH2, cyclohexyl), 1.38–1.46 (m,
2H), 1.46–1.52 (m, 2H, CH2 i-pentyl), 1.57–1.65 (m, 1H, i-pentyl),
1.70–1.78 (m, 2H, cyclohexyl), 3.41 (dt, J = 5.95 Hz, 2H, i-pentyl),
3.98–4.06 (m, 1H cyclohexyl), 5.93 (br, NH), 6.41 (dd, J = 12.40,
4.80 Hz, 1H, py), 7.49 (dd, J = 9.40, 1.80 Hz, 1H, py),8.08 (br, NH),
8.19 (dd, J = 6.55, 1.75 Hz, 1H, py); 13C NMR (CDCl3): d ; MS: m/z
(%) 290 [M+1]+ (100), 291 (20); HRMS calcd for C17H28N3O
[M+1]+ 290.2227; found 290.2238.
4.3.4.2. 1,3-Dipropyl-pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione
(4b). EtOAc/PE (1/4), 58%; yellow liquid; IR: 1709 (NC(O)N), 1659
(C(O)N), 1597 (C@C), 1484, 1461, 1346 (CAH). 1H NMR (300 MHz,
CDCl3): 0.98 (t, J = 7.15 Hz, 3H, CH3), 1.00 (t, J = 7.15 Hz, 3H, CH3),
1.68–1.83 (m, 4H, 2ꢀCH2), 4.05 (dd, J = 15.41, 7.71 Hz, 2H, CH2),
4.31 (dd, J = 15.41, 7.71 Hz, 2H, CH2), 7.19 (dd, J = 12.11, 4.40 Hz,
1H, py), 8.46 (dd, J = 9.36, 1.65 Hz, 1H, py), 8.65 (dd, J = 6.60,
1.65 Hz, 1H, py); 13C NMR (CDCl3): d 11.3, 11.4, 21.1, 21.2, 43.4,
44.1, 110.9, 118.7, 137.7, 150.8, 154.1, 161.4; MS: m/z (%) 248
[M+1]+ (100), 249 (17); HRMS calcd for C13H18N3O2 [M+1]+
248.1394; found 248.1405.
4.3.3.4. N-Allyl 2-(t-octylamino)-3-pyridinecarboxamide (5e). EtOAc/
PE (1/8), 74%; white solid; mp. 65 °C; IR: 3314 (NH), 3270 (NH), 2949,
2903, 2860 (C@CAH), 1620 (C(O)N), 1577 (C@C), 1513 (CAH), 1261
(CAN). 1H NMR (300 MHz, CDCl3): 0.95 (s, 9H, 3ꢀCH3), 1.52 (s, 6H,
2ꢀCH3), 1.95 (s, 2H, CH2), 4.02 (t, J = 5.5 Hz, 2H, CH2), 5.22 (qd,
J = 12.66, 1.65, J = 19.26, 1.65 Hz, 2H, CH2), 5.91 (qt, J = 5.50 Hz, 2H,
CH2), 5.98 (s, 1H, NH), 6.39 (dd, J = 12.66, 4.95 Hz, 1H, py), 7.51 (dd,
J = 10.46, 2.20 Hz, 1H, py), 8.18 (dd, J = 6.60, 2.20 Hz, 1H, py); 13C
NMR (CDCl3): d 30.1, 31.6, 31.8, 42.2, 50.6, 55.0, 109.4, 116.8, 134.1,
134.8, 151.4, 157.9, 168.7; MS: m/z (%) 290 [M+1]+ (100), 291 (20);
HRMS calcd for C17H28N3O [M+1]+ 290.2227; found 290.2228.
4.3.4.3. 1,3-Dibutyl-pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione
(4c). EtOAc/PE (1/3), 61%; yellow liquid; IR: 2958, 2930, 2871
(@CAH), 1710 (NC(O)N), 1663 (C(O)N), 1598 (C@C), 1484, 1462,
1431, 1405, 1348 (CAH). 1H NMR (300 MHz, CDCl3): 0.98 (dt,
J = 3.85 Hz, 6H, 2ꢀCH3), 1.42 (qt, J = 7.15 Hz, 4H, 2ꢀCH2), 1.63–
1.76 (m, 4H, 2ꢀCH2), 4.08 (t, J = 7.71 Hz, 2H, CH2), 4.35 (t,
J = 7.71 Hz, 2H, CH2), 7.19 (dd, J = 12.11, 4.40 Hz, 1H, py), 8.45
(dd, J = 9.36, 1.65 Hz, 1H, py), 8.65 (dd, J = 6.60, 1.65 Hz, 1H, py);

3954
M. Jatczak et al. / Bioorg. Med. Chem. 22 (2014) 3947–3956
13C NMR (CDCl3): d 13.8, 13.9, 20.2, 20.3, 29.9, 30.1, 41.8, 42.5,
110.9, 118.7, 137.6, 150.8, 150.9, 154.0, 161.3; MS: m/z (%) 276
[M+1]+ (100), 277 (18); HRMS calcd for C15H22N3O2 [M+1]+
276.1707; found 276.1716.
8.45 (dd, J = 9.72, 2.00 Hz, 1H, py), 8.64 (dd, J = 6.72, 2.00 Hz, 1H,
py); 13C NMR (CDCl3): d 22.5, 24.9, 25.9, 26.0, 33.1, 38.2, 38.4,
48.7, 109.7, 109.8, 135.0, 151.8, 157.4, 168.5; MS: m/z (%) 316
[M+1]+ (100), 317 (23); HRMS calcd for C18H26N3O2 [M+1]+
316.2020; found 316.2028.
4.3.4.4. 1,3-Di(i-pentyl)-pyrido[2,3-d]pyrimidine-2,4(1H,3H)-
dione (4d). EtOAc/PE (1/5), 91%; white solid; mp. 48 °C; IR:
2954, 2928, 2881, 2867 (@CAH), 1702 (NC(O)N), 1656 (C(O)N),
1592 (C@C), 1483, 1466, 1460, 1351 (CAH). 1H NMR (300 MHz,
CDCl3): 0.99 (t, J = 5.50 Hz, 12H, 4ꢀCH3), 1.52–1.66 (m, 4H,
2ꢀCH2), 1.66–1.77 (m, 4H, 2ꢀCH2), 4.07 (d, J = 6.60 Hz, 1H, CH),
4.10 (d, J = 6.60 Hz, 1H, CH), 4.19 (dd, J = 12.66, 4.95 Hz, 1H, py),
8.45 (dd, J = 9.36, 1.65 Hz, 1H, py), 8.65 (dd, J = 6.05, 1.65 Hz, 1H,
py); 13C NMR (CDCl3): d 22.5, 22.6, 26.3, 26.4, 36.5, 36.5, 40.6,
40.9, 110.9, 118.6, 137.5, 150.7, 150.8, 154.0, 161.2; MS: m/z (%)
304 [M+1]+ (100), 305 (18); HRMS calcd for C17H25N3O2 [M+1]+
304.2020; found 304.2022.
4.3.4.9. 3-Allyl-1-(t-octyl)-pyrido[2,3-d]pyrimidine-2,4(1H,3H)-
dione (6e). EtOAc/PE (1/8), 49%; yellow liquid; IR: 2986, 2950,
2868 (C@CAH), 1715 (NC(O)N), 1668 (C(O)N), 1598, 1579 (C@C),
1435 (CAH). 1H NMR (300 MHz, CDCl3): 0.98 (s, 9H, 3ꢀCH3), 1.87
(s, 6H, 2ꢀCH3), 2.40 (s, 2H, CH2), 4.63 (d, J = 5.50 Hz, 2H, CH2),
5.21 (dd, J = 13.21, 2.20, J = 19.81, 2.20 Hz, 2H, @CH2), 5.92 (qt,
J = 5.50 Hz, 1H, CH), 7.15 (dd, J = 12.38, 4.95 Hz, 1H, py), 8.39 (dd,
J = 9.36, 1.65 Hz, 1H, py), 8.61 (dd, J = 6.05, 1.65 Hz, 1H, py); 13C
NMR (CDCl3): d 31.4, 31.9, 32.1, 43.9, 51.3, 68.1, 112.6, 117.5,
118.4, 132.0, 137.0, 151.4, 151.6, 152.7, 161.6; MS: m/z (%) 204
[M+1]+ (100), 205 (13); HRMS calcd for C10H10N3O2 [M + 1]+
204.0768; found 20774.
4.3.4.5. 1,3-Dibenzyl-pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione
(4j). EtOAc/PE (1/3), 60%; white solid; mp. 120 °C; IR: 3088,
3065, 3028 (@CAH), 1711 (NC(O)N), 1658 (C(O)NH), 1592 (C@C),
1484, 1465, 1450 (CAH). 1H NMR (300 MHz, CDCl3): 5.25 (s, 2H,
CH2), 5.54 (s, 2H, CH2), 7.14 (dd, J = 12.66, 4.95 Hz, 1H, py), 7.18–
7.33 (m, 5H, Bn), 7.43–7.55 (m, 5H, Bn), 8.43 (dd, J = 9.36,
1.65 Hz, 1H, py), 8.61 (dd, J = 6.05, 1.65 Hz, 1H, py); 13C NMR
(CDCl3): d 45.2, 45.5, 110.9, 119.2, 127.7, 127.8, 128.5, 128.6,
128.7, 129.2, 136.7, 137.0, 137.9, 150.8, 151.3, 154.2, 161.3; MS:
m/z (%) 344 [M+1]+ (100), 345 (23); HRMS calcd for C21H18N3O2
[M+1]+ 344.1394; found 344.1397.
4.3.4.10. 3-Propyl-1-(t-octyl)-pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione
(6f). EtOAc/PE (1/10), 97%; light yellow liquid; IR: 2956, 2874
(C@CAH), 1715 (NC(O)N), 1667 (C(O)N), 1598, 1580 (C@C), 1430
(CAH). 1H NMR (300 MHz, CDCl3): 0.97 (t, J = 8.26, 3H, CH3), 0.98
(s, 9H, 3ꢀCH3), 1.69 (qd, J = 7.71, 7.71 Hz, 2H, CH2), 87 (s, 6H,
2ꢀCH3), 2.40 (s, 2H, CH2), 3.98 (t, J = 7.71 Hz, 2H, CH2), 7.14 (dd,
J = 12.66, 4.95 Hz, 1H, py), 8.37 (dd, J = 9.36, 1.65 Hz, 1H, py),
8.59 (dd, J = 6.60, 2.20 Hz, 1H, py); 13C NMR (CDCl3): d 11.4, 21.3,
31.4, 31.9, 32.1, 45.5, 51.3, 67.9, 76.7, 77.1, 77.5, 112.8, 118.4,
136.9, 151.5, 151.8, 152.7, 161.9; MS: m/z (%) 206 [M+1]+ (100);
HRMS calcd for C10H12N3O2 [M+1]+ 206.0924; found 206.0924.
4.3.4.6. 3-Allyl-1-cyclohexyl-pyrido[2,3-d]pyrimidine-2,4(1H,3H)-
dione (6a). EtOAc/PE (1/8), 47%; white solid; mp. 141 °C; IR:
2925, 2852 (C@CAH), 1709 (NC(O)N), 1658 (C(O)N), 1589 (C@C),
1485, 1447, 1408 (CAH). 1H NMR (300 MHz, CDCl3): 1.16–2.00
(m, 8H, cyclohexyl), 2.59 (dt, J = 11.56 Hz, 2H, cychexyl), 4.68 (d,
J = 4.95 Hz, 2H, ACH2ACH@), 5.21 (dd, J = 10.46, 1.10 Hz, 1H,
@CH2), 5.29 (dd, J = 17.06, 1.10 Hz, 1H, @CH2), 5.39 (br, CH-cyclo-
hexyl), 5.94 (qt, J = 5.50 Hz, 1H, ACH@), 7.19 (dd, J = 12.11,
4.95 Hz, 1H, py), 8.46 (dd, J = 9.36, 1.65 Hz, 1H, py), 8.65 (dd,
J = 6.05, 1.65); 13C NMR (CDCl3): d 25.4, 26.6, 29.0, 29.8, 43.8,
55.4, 111.1, 118.1, 118.7, 131.8, 137.9, 151.2, 153.6, 161.2; MS:
m/z (%) 286 [M+1]+ (100), 287 (16); HRMS calcd for C10H10N3O2
[M+1]+ 204.0768; found 204.0773. Literature: K. Noda, A. Nakaga-
wa, T. Motomura, S. Yamasaki, DE 2334266 A1 19740131.
4.3.4.11. 3-(i-Pentyl)-1-(t-octyl)-pyrido[2,3-d]pyrimidine-2,4(1H,3H)-
dione (6g). EtOAc/PE (1/7), 86%; yellow liquid; IR: 2922, 2854
(C@CAH), 1708 (NC(O)N), 1656 (C(O)N), 1588 (C@C), 1449 (CAH). H
1
NMR (400 MHz, CDCl3): 0.97 (s, 3H, CH3), 0.98 (s, 9H, 3ꢀCH3), 0.99 (s,
3H, CH3), 1.50–1.55 (m, 1H, CH), 1.60–1.72 (m, 1H, CH), 1.87 (s, 6H,
2ꢀCH3), 2.40 (s, 2H, CH2), 4.03 (dt, J = 4.98, 7.80 Hz, 2H, CH2), 7.14
(dd, J = 12.36, 4.68 Hz, 1H, py), 8.37 (dd, J = 9.76, 2.04 Hz, 1H, py),
8.59 (dd, J = 6.68, 2.04 Hz, 1H, py); 13C NMR (CDCl3): d 22.5, 26.4,
31.3, 31.8, 32.0, 36.6, 40.7, 51.2, 67.8, 112.7, 118.3, 136.8, 151.4,
151.6, 152.6, 161.7; MS: m/z (%) 234 [M+1]+ (100), 235 (6); HRMS calcd
for C12H15N3O2 [M+1]+ 234.1237; found 234.1237.
4.4. Intraluminal solubility profiling
4.3.4.7. 3-Propyl-1-cyclohexyl-pyrido[2,3-d]pyrimidine-2,4(1H,3H)-
dione (6b). EtOAc/PE (1/6), 91%; white solid; mp. 133 °C; IR: 2925,
2852 (C@CAH), 1709 (NC(O)N), 1656 (C(O)N), 1589 (C@C), 1448
The solubility of the pyrido[2,3-d]pyrimidines was experimen-
tally determined in the biorelevant medium FaSSIF, possessing
mixed micelles of taurocholate and lecithin. The thermodynamic
solubility was determined by adding an excess of compound to
1
(CAH). H NMR (300 MHz, CDCl3): 0.98 (t, J = 7.15 Hz, 3H, CH3), 1.22–
1.55 (m, 4H, cyclohexyl), 1.60–1.79 (m, 4H, 2ꢀCH2), 1.81–1.97 (m,
2H, cyclohexyl), 2.59 (dt, J = 11.56 Hz, 2H, CH-cyclohexyl), 4.02 (t,
J = 7.15 Hz, 2H, CH2), 7.17 (dd, J = 12.11, 4.40 Hz, 1H, py), 8.46 (dd,
J = 9.36, 1.65 Hz, 1H, py), 8.64 (dd, J = 6.04, 1.65 Hz, 1H, py); 13C NMR
(CDCl3): d 11.7, 21.2, 25.5, 26.6, 28.9; 30.4, 43.4, 111.2, 118.6, 137.8,
151.2, 153.4, 161.5; MS: m/z (%) 288 [M+1]+ (100), 289 (18); HRMS
calcd for C16H22N3O2 [M+1]+ 288.1707; found 288.1713. Literature: K.
Noda, A. Nakagawa, T. Motomura, S. Yamasaki, DE 2334266 A1
19740131.
the medium (0.8 mg/300 ll). This suspension was shaken for
24 h at 175 rpm and 37 °C (KS 4000i Control incubator shaker,
Staufen, Germany) and afterwards centrifuged at 20,817g for
15 min (5804 Centrifuge, Eppendorf, Hamburg, Germany) to
remove undissolved drug. The supernatant was analyzed with
HPLC and UV detection.
4.5. Intestinal permeability
4.3.4.8. 3-(i-Pentyl)-1-cyclohexyl-pyrido[2,3-d]pyrimidine-
2,4(1H,3H)-dione (6c). EtOAc/PE (1/7), 97%; white solid; mp.
118 °C; IR: 2922, 254 (C@CAH), 1708 (NC(O)N), 1656 (C(O)N),
1588 (C@C), 1484, 1449, 1411, 1381 (CAH). 1H NMR (300 MHz,
CDCl3): 0.98 (s, 3H, CH3), 0.99 (s, 3H, CH3), 1.26–1.38 (m, 1H, CH-
cyclohexyl), 1.39–1.52 (m, 4H, 2ꢀCH2-cyclohexyl), 1.57 (dt,
J = 7.89 Hz, 2H, CH2 i-pentyl), 1.65–1.75 (m, 4H, 2ꢀCH2-cyclo-
hexyl), 1.84–1.94 (m, 2H, CH2-cyclohexyl) 4.07 (dt, J = 5.20,
5.20 Hz, 2H, CH2 i-pentyl), 7.18 (dd, J = 12.44, 4.72 Hz, 1H, py),
Caco-2 cells were obtained from American Type Culture Collec-
tion (Manassas, VA) and were grown in DMEM+ at 37 °C in an
atmosphere of 5% CO2 and 90% relative humidity. Cells were pas-
saged every 3 to 4 days (at 80–90% confluence) at a split ratio of
1:6. For transport experiments, cells were seeded at a density of
90,000 cells/cm2 on Costar Transwell membrane inserts (3
lm
pore diameter, 12 mm diameter; Corning Inc., Corning, NY) and
were used for experiments 17 to 18 days after seeding. Only mon-
olayers with transepithelial electrical resistance values higher than

M. Jatczak et al. / Bioorg. Med. Chem. 22 (2014) 3947–3956
3955
Table 6
and incorporated in the following equation to obtain scaled Clint
values for hepatic metabolism in human (Clint,hep,human):
Conditions of the HPLC analysis of pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones
analogues
a
0:693
incubation volumeðmlÞ
Compound
Mobile Phase
(Methanol/25 mM
acetate buffer pH 5.5)
UV/Fluo detection
(nm)
tR (min)
Scaled Clint;hep;human
¼
ꢀ
in vitro t1=2
mg microsomes
45mg microsomes
g liver
20g liver
ꢀ
ꢀ
kg bodyweight
2i
3a
3d
3f
4a
4b
4d
4j
95/5
340
11
60/40
80/20
90/10
80/20
80/20
90/10
80/20
90/10
90/10
40/60
70/30
80/20
60/40
Ex:330/Em:369
340
4.7
4.9
5.3
4.9
7.2
8.6
10.1
5.6
5.7
2.8
4.4
5.1
9.7
Ex:256/Em:420
Ex:243/Em:366
Ex:243/Em:366
Ex:222/Em:361
340
4.7. HPLC analysis
The HPLC system used to analyze the pyrido[2,3-d]pyrimidine-
2,4(1H,3H)-diones samples consisted of a Waters 2790 Alliance
series separations module and a Novapak C18 column under radial
compression (Waters, Milford, MA). UV absorbance was monitored
using a Waters 2487 detector and fluorescence using a FP-1520
intelligent fluorescence detector (Jasco, Tokyo, Japan). The
observed peaks were integrated using Empower Pro (Empower 2)
software. The calibration curves of the pyrido[2,3-d]pyrimidine-
2,4(1H,3H)-diones were linear over the concentration range of
5e
6a
350
Ex:245/Em:363
Ex:271/Em:302
318
Ex:256/Em:290
Ex:275/Em:315
Atenolol
Indomethacin
Indinavir
Verapamil
a
Retention time.
0.39
l
M to 100
l
M. The assessment of intraday and interday
M and 0.125 M for each
reproducibility at concentrations of 1.25
l
l
150
X
cm2 were used. Transport studies were performed using a
pyrido[2,3-d]pyrimidine-2,4-dione in 50:50 microsomal buffer/
ACN, resulted in a RSD below 2.1 (n = 5). The deviation from the
theoretical concentration was lower than 3.2%. The different HPLC
methods of the pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones are
listed in Table 6.
previously described method.29 HBSS+ (pH 7.4) containing 0.2%
D-
a-tocopheryl polyethylene glycol 1000 succinate was used in the
basolateral compartment to create sink conditions; in the apical
compartment, FaSSIF (pH 6.5) was used. The experiment was initi-
ated by adding the incubation medium, containing the pyrido[2,3-
Acknowledgments
d]pyrimidine-2,4(1H,3H)-diones (30
GF120918 (4 M), to the donor compartment. Due to solubility
issues of compound 2ai in DMSO, an initial concentration of
20 M was used in order to obtain a non-toxic DMSO concentra-
lM) in absence or presence of
l
The authors are indebted to the IWT-Vlaanderen for the finan-
cial support to the SBO project ‘‘Synthesis of novel and drug-like
compound libraries for screening across a broad range of therapeu-
tic areas’’.
l
tion below 1%. Samples were shaken at 300 rotations per minute
(rpm) for 1 h at 37 °C (Thermostar, BMG Labtech, Offenburg, Ger-
many). Afterwards, analysis of the samples was done with HPLC
References and notes
and UV detection. The apparent permeability coefficient (Papp
)
was calculated according to the following equation:
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D
Q
1
Papp
¼
ꢀ
Dt
A ꢀ Cdonor
where Q is the cumulative amount of drug appearing in the acceptor
compartment, A is the surface area of the Transwell membrane, and
Cdonor is the drug concentration in the donor compartment.
4.6. Human hepatic in vitro intrinsic clearance
Test compounds (8 lM) were incubated with a pool of human
liver microsomes (HLM) from 45 donors (KaKy-Cell, Plobsheim,
France) (0.5 mg microsomal protein/ml), NADPH (1 mM), glu-
cose-6-phosphate (3 mM) in a total volume of 400 ll phosphate
buffer (0.1 M) containing 3 mM MgCl2 at a pH of 7.4. Incubations
were conducted at 37 °C at 350 rpm (Thermostar, BMG Labtech,
Offenburg, Germany). Reactions were commenced with the addi-
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30 min, aliquots (75
(75 l) to quench the reaction. Incubations in the absence of
NADPH and glucose-6-phosphate were performed as a negative
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l
l
with human liver microsomes in a concentration of 0.25 mg micro-
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the linear regression from the log percentage remaining versus
incubation time was used to calculate the elimination rate con-
stant k = ꢁslope⁄(ln10). Finally, in vitro t1/2 is calculated from k

3956
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