The success of imatinib, a BCR-ABL inhibitor for the treatment of chronic myelogenous leukemia, has created a great impetus for the development of additional kinase inhibitors as therapeutic agents. However, the complexity of cancer has led to recent interest in polypharmacological approaches for developing multikinase inhibitors with low toxicity profiles. With this goal in mind, we analyzed more than 150 novel cyano pyridopyrimidine compounds and identified structure-activity relationship trends that can be exploited in the design of potent kinase inhibitors. One compound, 8-cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7x), was found to be the most active, inducing apoptosis of tumor cells at a concentration of approximately 30-100 nM. In vitro kinase profiling revealed that 7x is a multikinase inhibitor with potent inhibitory activity against the CDK4/CYCLIN D1 and ARK5 kinases. Here, we report the synthesis, structure-activity relationship, kinase inhibitory profile, in vitro cytotoxicity, and in vivo tumor regression studies by this lead compound.
The success of imatinib, a BCR-ABL inhibitor for the treatment of chronic myelogenous leukemia, has created a great impetus for the development of additional kinase inhibitors as therapeutic agents. However, the complexity of cancer has led to recent interest in polypharmacological approaches for developing multikinase inhibitors with low toxicity profiles. With this goal in mind, we analyzed more than 150 novel cyanopyridopyrimidine compounds and identified structure-activity relationship trends that can be exploited in the design of potent kinase inhibitors. One compound, 8-cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7x), was found to be the most active, inducing apoptosis of tumor cells at a concentration of approximately 30-100 nM. In vitro kinase profiling revealed that 7x is a multikinase inhibitor with potent inhibitory activity against the CDK4/CYCLIN D1 and ARK5 kinases. Here, we report the synthesis, structure-activity relationship, kinase inhibitory profile, in vitro cytotoxicity, and in vivo tumor regression studies by this lead compound.
Cancer
is now believed to result from perturbations in cell cycle
that result in unlimited proliferation and an inability of a cell
to undergo differentiation and/or apoptosis.[1−5] The cell cycle is typically divided into four phases,
G1, S, G2, and M, and it is apparent that the
order and timing of each phase is critical for accurate transmission
of genetic information. Consequently, a number of biochemical pathways
have evolved to ensure that initiation of a particular cell cycle
event is dependent on the accurate completion of another. These biochemical
pathways have been termed “checkpoints.”[1,2,5]When cells proliferate,
mitogenic growth factors bind to their
cognate receptors and initiate a cascade of events that culminate
in the expression and assembly of different kinase holoenzymes that
are composed of a regulatory subunit, called a cyclin, and a catalytic
subunit, termed a cyclin-dependent kinase (CDK).[1−3,5] CDKs are serine/threonine kinases that are inactive
when they are under-phosphorylated and monomeric.[4] The primary mechanism of CDK activation is association
with its partner cyclin. In the mammalian cell cycle, CDK4/6 associate
with D-type cyclins and control progression through the G1 phase when the cell prepares to initiate DNA synthesis.[5,6] Activation of CDK4/6/cyclin D complexes contribute to hyperphosphorylation
of the retinoblastoma (RB) family of proteins, which results in the
release of associated protein factors.[7,8] One key RB-binding
partner is the E2F-1 transcription factor, which appears to activate
the transcription of genes whose products are required for S-phase
progression. E2F-1 and other members of the E2F family are known to
bind to pRB and heterodimerize with DP-1 and -2, an interaction that
is required for the DNA-binding capacity of E2F family proteins.[1−8] Once the cell has made the G1/S transition, cyclin E/CDK2
phosphorylates the remaining residues on the RB family proteins that
are critical for E2F activation. Activation of E2F-mediated transcription
allows the cell to transit into S phase and to initiate DNA replication,
which is controlled, in part, through cyclin A/CDK2. Cyclin A/CDK2
ultimately forces the cell through the G2 phase prior to
the assembly of the cyclin B/CDK1 complex and the initiation of mitosis.[5,8]There is considerable evidence showing that a vast majority
of
humantumors exhibit deregulation of the CDK4/6-cyclin D-RB pathway.[1,9,10] For example, CDK4/6 is hyperactivated
in a number of humancancers as a result of overexpression of positive
regulators such as cyclin D or deletion and/or epigenetic alterations
of substrates such as RB.[1,9,10] In addition, mutations and chromosomal translocations in the CDK4
locus have also been described. One prominent example is the CDK4R24C mutation that results in insensitivity of CDK4 to INK4
family inhibitors and was first described in patients with familial
melanoma.[11,12] Finally, CDK4/6 amplification or overexpression
has been observed in a wide spectrum of tumors, including gliomas,
sarcomas, lymphomas, melanomas, carcinomas of breast, squamous cell
carcinomas, and leukemias.[13]Because
cyclin D, CDK4, and CDK6 activities are upregulated in
a variety of tumor types, several groups have focused their efforts
on the development of small molecule CDK4 inhibitors. Experimental
evidence indicating that CDK4 is dispensable for development[14−17] suggests that inhibitors of this kinase might be both nontoxic and
effective in the treatment of cancers that are dependent on CDK4 activity
for proliferation. The first generation of CDK inhibitors, flavopiridol
(23)[18] and roscovitine (CYC202)
(24)[19] (see Chart 1), were potent CDK4 inhibitors but were nonselective
and inhibited multiple kinases including CDK1 and CDK7 and caused
severe toxic side effects in clinical trials.[20,21] Several other pan-CDK inhibitors have since entered clinical trials,
but the therapeutic efficacy of these molecules has been modest due
to dose-limiting toxicity and poor pharmacokinetics. Several early
trials have since been discontinued.[22,23]
Chart 1
CDK Inhibitors
In an attempt to overcome the
toxicity profile of pan-CDK inhibitors,
small molecules belonging to additional chemical classes such as oxindoles
(25),[24] triaminopyrimidines
(26),[25] diarylureas (27),[26] thioacridones (28),[27] aminothiazoles (29),[28] indolocarbazoles (30),[29] and pyrido[2,3-d]pyrimidines
(31)[30−32] (see Chart 1) have been developed
that are specific for individual CDKs. Some of these compounds exhibited
a high degree of selectivity toward CDK4/6 by targeting the ATP binding
site of CDK4/6-cyclin D complexes. Of these, one CDK4/6 selective
compound, PD-0332991, which is a pyrido[2,3-d]pyrimidine
derivative, is highly specific for CDK4 and CDK6, inhibiting these
two kinases with IC50 values of 0.011 and 0.015 μM,
respectively, with little or no inhibitory activity against a large
panel of kinases including other CDKs and a wide variety of serine,
threonine, and tyrosine kinases.[30,31] PD-0332991
has been extensively studied for its efficacy in tissue culture model
systems as well as in mouse xenograft models of colorectal cancer,
mantle cell lymphoma (MCL), and disseminated myeloma.[31−38] PD-0332991 causes G1 arrest in cultured tumor cell lines
and inhibits tumor growth in xenograft models of RB-positive humantumor cell lines derived from breast, ovarian, lung, colon, prostate,
brain, and blood such as multiple myeloma and mantle cell lymphoma.[31−38] Therapeutic doses of PD-0332991 resulted in a reduction of both
phosphorylated RB and the proliferative marker Ki-67 in the tumor
tissue as well as downregulation of E2F-target genes.[37,38] On the basis of these promising results, this compound entered clinical
trials in 2004 and results from phase I and phase II trials indicate
that the side effects are tolerable.[39−42] Phase I studies with palbociclib
(PD-0332991) indicated that the clinical response was mostly cytostatic
where disease stabilization was observed in a significant number of
patients (http://clinicaltrials.gov). However, few partial
or complete remissions were observed in phase I and II clinical trials
where PD-0332991 was used as a single agent. However, combination
studies have yielded more promising results.[43−46] This drug is currently being
evaluated in phase III trials in combination with letrozole for the
treatment of ER+/HER2– advanced breast cancer.[45,46] These studies suggest that reduction of tumor burden in patients
whose tumors express high levels of cyclin D/CDK4 might require inhibition
not only of CDK4 but of other aberrantly activated proteins. With
this in mind, our goal was to identify potent inhibitors that possess
sufficient cross-reactivity with a small number of kinases, such that
the combined inhibition of these kinases will result in a more effective
treatment of these tumors. Here, we describe the synthesis, structure–activity
relationship (SAR), cytotoxic properties, kinase inhibition profile,
and mechanism of action of 7x. 7x, which
is a member of the pyridopyrimidine series of compounds, is a potent
CDK4/6 inhibitor that exhibits cross-reactivity with a small number
of kinases that play critical roles in mitogenic signaling. Mice treated
with 7x did not exhibit signs of toxicity and tumor formation
in 7x-treated xenograft nude mouse models was inhibited
over an 18-day period. Together, these studies indicate that pyridopyrimidine
compounds might represent a safe and effective chemotype to treat
tumors whose cell cycle progression is altered as a result of CDK4/6-RBhyperactivity.
Chemistry
To generate an ATP-competitive
kinase inhibitor library, we used
pyrido[2,3-d]pyrimidines (7) as the
backbone because this class of compounds have been shown to possess
kinase inhibitory activity.[47,48] To facilitate the synthesis
of a large number of compounds, we developed a unique and simple method
which is summarized in Scheme 1.
Scheme 1
Synthesis
of Pyrido[2,3-d]pyrimidines (7)
Reagents and conditions (i) X-NH2, Et3N, THF, rt, 1–3 h, 80–95%; (ii)
LiAlH4, THF, −10 °C to rt, 1 h, 80–86%;
(iii) MnO2, CHCl3, rt, 24 h, 70–90%;
(iv) CNCH2CO2H or NO2CH2CO2Et or 10 or 13 or 16, BnNH2, AcOH, 100 °C, 6 h, 62–73%; (v) m-CPBA, CH2Cl2, rt, 3 h, 87–94%;
(vi) Z-H, toluene, 100 °C, 3–8 h, 40–65%.
Synthesis
of Pyrido[2,3-d]pyrimidines (7)
Reagents and conditions (i) X-NH2, Et3N, THF, rt, 1–3 h, 80–95%; (ii)
LiAlH4, THF, −10 °C to rt, 1 h, 80–86%;
(iii) MnO2, CHCl3, rt, 24 h, 70–90%;
(iv) CNCH2CO2H or NO2CH2CO2Et or 10 or 13 or 16, BnNH2, AcOH, 100 °C, 6 h, 62–73%; (v) m-CPBA, CH2Cl2, rt, 3 h, 87–94%;
(vi) Z-H, toluene, 100 °C, 3–8 h, 40–65%.The commercially available compound 4-chloro-2-methylsulfanyl-pyrimidine-5-carboxylic
acid ethyl ester 1 was treated with acyclic and cyclic
amines in the presence of triethylamine (Et3N) to yield 2a–2j. The ester group in compounds 2a–2j was then reduced with lithium aluminum
hydride (LiAlH4) to obtain the corresponding alcohols 3a–3j, which when further oxidized with
manganese dioxide (MnO2), yielded the corresponding pyrimidine
carbaldehyde 4a–4j. The aldehyde 4a–4j were converted to pyridopyrimidines 5a–5s by Knoevenagel condensation, with
each aldehyde treated with active methylene compounds (cyanoacetic
acid or nitro-acetic acid ethyl ester or 10 or 13 or 16) in the presence of benzylamine to generate
their corresponding intermediates 5a–5s. This approach permitted the introduction of cyano,nitro, sulfonyl,
and carboxamide groups at the C-6 position of the
pyrido[2,3-d]pyrimidines. To replace methylsulfide
at the C-2 position with substituted aryl/heteroaryl
amines, it was oxidized to methyl sulfoxides 6a–6s using m-chloroperbenzoic acid (m-CPBA). The methyl sulfoxide was then substituted with
different aryl/heteroaryl amines to achieve the desired pyridopyrimidine
compounds 7a–7ap (Scheme 1).Alternatively, the compounds 5b–5j were also prepared by alkylation of 5a with different
alkyl iodides in the presence of sodium hydride (NaH) in N,N-dimethylformamide (DMF) at 50 °C as shown
in Scheme 2.
Scheme 2
Synthesis of 8-Alkyl/cycloalkyl-2-methylsulfanyl-7-oxo-7,8-dihydro[2,3-d]pyrimidine-6-carbonitrile (5b–5j) from 2-Methylsulfanyl-7-oxo-7,8-dihydro[2,3-d]pyrimidine-6-carbonitrile (5a)
Reagents
and conditions: (i)
X-I, NaH, DMF, 50 °C, 1 h, 64–75%.
Synthesis of 8-Alkyl/cycloalkyl-2-methylsulfanyl-7-oxo-7,8-dihydro[2,3-d]pyrimidine-6-carbonitrile (5b–5j) from 2-Methylsulfanyl-7-oxo-7,8-dihydro[2,3-d]pyrimidine-6-carbonitrile (5a)
Reagents
and conditions: (i)
X-I, NaH, DMF, 50 °C, 1 h, 64–75%.Later, we evaluated the role of NH proton at the C-2 position of 7x in cytotoxicity assays by acylating
and benzoylating 7x with acetic anhydride and 4-trifluoromethylbenzoyl
chloride to obtain 7aq and 7ar respectively
(Scheme 3).
Scheme 3
Acylation and Benzoylation of NH Group
of 8-Cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-
oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile
(7x)
Reagents and conditions: (i)
AC2O, 120 °C, 3 h, 64%; (ii) 4-CF3PhCOCl,
NaH, DMF, rt, 3 h, 62%.
Acylation and Benzoylation of NH Group
of 8-Cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-
oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile
(7x)
Reagents and conditions: (i)
AC2O, 120 °C, 3 h, 64%; (ii) 4-CF3PhCOCl,
NaH, DMF, rt, 3 h, 62%.The active methylene
compounds, arylmethanesulfonylacetic acids
(10a–10c), 4-chlorophenylsulfonylacetic
acid (13), and N-arylmalonamic acids
(16a–16b) were prepared as shown
in Schemes 4, 5, and 6 as per the reported procedures.[49−51]
Scheme 4
Synthesis of Arylmethanesulfonyl Acetic Acids
Reagents
and conditions: (i)
HSCH2CO2H, NaOH, MeOH, HCl, rt, 3 h, 90–95%;
(ii) TBAF solution in THF, rt, 2 h, 50%; (iii) 30% H2O2, AcOH, rt, 24 h, 85–90%.
Scheme 5
Synthesis of 4-Chlorophenylsulfonyl Acetic Acids
Reagents and conditions: (i)
ClCH2CO2H, NaOH, MeOH, HCl, rt, 3 h, 98%; (ii)
30% H2O2, AcOH, rt, 24 h, 80%.
Scheme 6
Synthesis of N-Arylmalonamic Acids
Reagents
and conditions: (i)
ClCOCH2CO2Me, Et3N, DCM, rt, 3 h,
90%; (ii) 10% NaOH, HCl, rt, 1 h, 78–80%.
Arylmethyl
bromides (8a–8c) were
treated with thioglycolic acid in the presence of sodium hydroxide
to produce corresponding arylmethylsulfanyl acetic acids (9a–9c). Deprotection of tert-butyldimethylsilyl
ether (TBDMS) group in 9c with tetrabutylammonium fluoride
(TBAF) yielded 9d.[49a] The
oxidation of 9a–9b and 9d with 30% H2O2 in acetic acid yielded the corresponding
arylmethylsulfonyl acetic acids (10a–10c) (Scheme 4).
Synthesis of Arylmethanesulfonyl Acetic Acids
Reagents
and conditions: (i)
HSCH2CO2H, NaOH, MeOH, HCl, rt, 3 h, 90–95%;
(ii) TBAF solution in THF, rt, 2 h, 50%; (iii) 30% H2O2, AcOH, rt, 24 h, 85–90%.As
shown in Scheme 5, 4-chlorothiophenol (11) was treated with chloroacetic
acid in the presence of NaOH and subsequent oxidation with 30% H2O2 to produce 4-chlorophenylsulfanyl acetic acid
(12) and 4-chlorophenylsulfonyl acetic acid (13), respectively.
Synthesis of 4-Chlorophenylsulfonyl Acetic Acids
Reagents and conditions: (i)
ClCH2CO2H, NaOH, MeOH, HCl, rt, 3 h, 98%; (ii)
30% H2O2, AcOH, rt, 24 h, 80%.The reaction of aromatic amines (14a–14b) with methyl-3-chloro-3-oxopropionate in the presence
of triethylamine generated 3-anilino-3-oxopropionic acid methyl esters
(15a–15b), which, when subjected
to hydrolysis, resulted in the formation of 3-anilino-3-oxopropionic
acids (16a–16b) (Scheme 6).
Synthesis of N-Arylmalonamic Acids
Reagents
and conditions: (i)
ClCOCH2CO2Me, Et3N, DCM, rt, 3 h,
90%; (ii) 10% NaOH, HCl, rt, 1 h, 78–80%.Commercially unavailable bicyclic amines were prepared in two steps
as shown in Scheme 7.[30] Aromatic nucleophilic substitution
of halogen in 1-nitro-4-fluorobenzene (17) and 5-bromo-2-nitropyridine
(18) by N-methylpiperazine was achieved
under heating conditions. The resulting nitro compounds 19 and 20 were reduced to corresponding amines 21 and 22 with Pd/C in the presence of hydrazine hydrate
in methanol.
Scheme 7
Synthesis of N-Methylpiperazine Arylamines
Reagents and conditions: (i) N-methylpiperazine, acetonitrile, 100 °C, 5 h, 70%;
(ii) N-methylpiperazine, tetra-n-butyl ammonium iodide, DMSO, 80 °C, overnight, 65%; (iii) 10%
Pd/C, NH2NH2·H2O, rt, 4 h, 75%.
Synthesis of N-Methylpiperazine Arylamines
Reagents and conditions: (i) N-methylpiperazine, acetonitrile, 100 °C, 5 h, 70%;
(ii) N-methylpiperazine, tetra-n-butyl ammonium iodide, DMSO, 80 °C, overnight, 65%; (iii) 10%
Pd/C, NH2NH2·H2O, rt, 4 h, 75%.
Structure–Activity Relationships (SARs)
It is evident from Table1 that the nature
of substituents X (N-8 position), Y (C-6 position), and Z (C-2 position) in the general
structure (7) (Scheme 1) specify
the cytotoxicity activity of the molecules on the cancer cells. Hence,
by varying X, Y, and Z using various combinations of atoms or groups,
we were able to generate compounds with excellent cytotoxicity. We
initially kept X and Y constant, where X = C5H9 and Y = CN, and varied the substitutions at the Z position. In our
initial attempts, we have included simple anilines at the Z position
and placed C5H9 and CN at the X and Y positions,
respectively. The cytotoxicity data from Table1 shows that 2-pyridine (7m) and 2-methoxy-6-quinoline
(7s) at the Z-position showed better activity when compared
to benzyl (7a), chlorophenyl (7b), cyanophenyl
(7c), hydroxy phenyl (7d), methoxyphenyl
(7e–7l), substituted pyridyl (7n), indolyl (7o, 7p), and substituted
quinolines (7q, 7r). Encouraged by these
results, we then attached bicyclic amines, such as substituted morpholino-aniline
(7t), morpholino-pyridine (7u), piperazino-pyridine
(7v), and pyridyl-piperazine (7w), which
can bring both potency and water solubility to the molecule. These
morpholino-anilines, morpholino-pyridines, and piperazino-pyridines
easily form water-soluble salts of hydrochlorides, lactates, and citrates
from hydrochloric acid, lactic acid, and citric acid. Although the
salts of bicyclic amines (7t, 7u, 7v, and 7w) showed enhanced water solubility
when compared to the salts of monocyclic amines (7m and 7s), the cytotoxicity properties of these compounds was decreased
by several fold when compared to 7m and 7s. We further tested the effect of incorporating additional bicyclic
amines at the Z position that might enhance the molecules’
cytotoxicity properties. Substituted piperazino-anilines could readily
be placed at the Z position, and all of the compounds tested, 7x (Table 1), 7aq, and 7ar (Table 4), showed enhanced cytotoxic
activity against these cancer cell lines. However, of these three
compounds, 7x showed superior cytotoxic activity against
both leukemic (K562) and prostate (DU145) cancer cell lines when compared
to any of the molecules listed in Table 1 and
Table 4.
Table 1
In Vitro Cytotoxicity
of Pyrido[2,3-d]pyrimidines (7a–7x) with
Variables at C-2 Position
Table 4
In Vitro
Cytotoxicity of 7aq and 7ar Compounds
Following optimization
of the Z position with N-methylpiperazino-aniline,
we then focused our efforts on varying
the X position using different substituents. The Y and Z positions
were kept constant using cyano and N-methyl-piperazino-aniline
groups on the pyridopyrimidine ring. To understand the significance
and role of the alkyl group at the X position of the ring with respect
to the cytotoxic properties of the molecule, we replaced the N-cyclopentyl group of 7x with hydrogen (7y), methyl (7z), ethyl (7aa), n-propyl (7ab), isopropyl (7ac), butyl (7ad), pentyl (7ae), cyclopropyl
(7af), and cyclohexyl (7ag) moieties (Table 2). The cytotoxic activities
of the resulting molecules were then tested using our panel of cancer
cell lines, and all were found to be substantially less active than
the original compound (7x). These data clearly show that
a cyclopentyl group at X position is the most optimal substituent,
as compound 7x showed the highest level of cytotoxicity
when compared to other molecules (7y–7ag). Once we identified suitable substituents for the X and Z positions,
we then focused our efforts on optimizing the Y position of the pyridopyrimidine
ring. Because 7x, with a cyano group at Y position, is
an active compound, we further explored the possibility of enhancing
the antiproliferative activity by replacing the cyano group with other
chemical moieties. As the cyano group is an electron withdrawing group,
we considered replacing it with nitro (7ah), sulfonyl
(7ai–7an), and carboxamide (7ao and 7ap) groups (Table 3), all of which are electron
withdrawers and are therefore similar to the cyano group with respect
to that property. All of the resulting compounds were then tested
in cytotoxicity assays using K562 and DU145 cells. The results of
these studies showed that all of the molecules were several-fold less
active than 7x, suggesting that the cyano group at the
Y position of the pyridopyrimidinone ring is critical for its activity.
Furthermore, the polarized nitrogen atom of the moiety might also
be interacting with the key amino acids of the enzymatic pocket of
the target kinase. Because SAR analysis clearly shows that 7x is best in this class, we performed all subsequent in vitro and
in vivo biological studies using this compound.
Table 2
In Vitro Cytotoxicity of Pyrido[2,3-d]pyrimidines (7y–7ag)
with Variables at N-8 Position
Table 3
In Vitro
Cytotoxicity of Pyrido[2,3-d]pyrimidines (7ah–7ap)
with Variables at C-6 Position
Biological Results and Discussion
In Vitro Antitumor Activity
of Compound 7x
We next tested the cyototoxic
activity of the most active compound
(7x) against a panel of humantumor cell lines. The results
of this study, which are listed in Table 5, show that treatment
with 7x induces growth arrest of most tumor cell lines,
with GI50 values ranging from 0.025 to 2 μM (selected
data is shown in Table 5). The evidence of
growth inhibition across multiple tumor cell types suggests that this
compound inhibits cellular proliferation by blocking key signaling
pathways that are required for growth. A comparison of growth inhibitory
activities of 7x and PD-0332991 for a panel of breast
cancer cell lines is given in Supporting Information
Table 1.
Table 5
Evaluation of 7x against
a Panel of Human Tumor Cell Lines
cell line
tumor
type
GI50 (μM)
DU145
prostate (AR−)
0.05
K562
CML
0.1
BT474
breast (ER+)
0.25
SK-BR-3
breast (ER−)
0.15
Granta-519
mantle cell lymphoma
0.025
Z138C
mantle cell lymphoma
0.025
N87
gastric carcinoma
0.9
SNU-5
gastric carcinoma
0.1
MIA-Paca-2
pancreatic
0.25
SK-OV3
ovarian
0.75
U87
glioblastoma
0.1
MCF-7
breast (ER+)
0.15
Raji
Burkitt’s
lymphoma (B-cell)
0.25
Jurkat
acute T cell leukemia
0.15
U266
multiple myeloma
0.2
N417
SCLC
0.25
Hela
cervical
0.75
A549
NSCLC
0.2
BT-20
breast (ER−)
0.1
SNU-398
hepatocellular carcinoma
0.2
SNU-449
hepatocellular
carcinoma
0.75
SNU-475
hepatocellular carcinoma
0.3
A431
epidermoid
0.25
DLD-1
colorectal
0.1
SW-480
colorectal
0.09
MDA-MB-468
breast (triple negative)
2
Colo-205
colorectal
0.1
HCC70
breast
1.5
HCC1428
breast
0.3
MDA-MB-231
breast (triple negative)
0.25
MDA-MB-157
breast
(triple negative)
1
2008
ovarian
1.5
2008/1714
resistant ovarian
1.5
MES-SA
sarcoma
0.25
MES-SA/DX5
resistant sarcoma
0.25
HCT15
colorectal
0.3
CAPAN-1
pancreatic
0.5
HFL
normal fibroblast
5.0
It is interesting
to note that this compound exhibited highest
growth inhibitory activity against two mantle cell lymphoma cell lines,
both of which are known to exhibit a chromosomal translocation that
results in the overexpression of cyclin D1 and an associated increase
in CDK4 activity. Because of its excellent potency, the kinase inhibition
profile of 7x was subsequently tested against a series
of 285 functional kinases (Reaction Biology Corp.), the results of
which are provided in Supporting Information Table
2. Interestingly, this study revealed that 7x is
a multikinase inhibitor, with the highest inhibitory activity against
CDK4, CDK6, ARK5, FGFR1, PDGFRβ, and PI3K-δ, all of which
are intimately associated with the growth, survival, and metastasis
in humantumor cells.[52,53] The kinome inhibition map of
365 kinases encoded by the human genome, as well as the IC50 values for selected kinases (as compared to the CDK4/6 inhibitor
PD-0332991), are shown in Figure 1 and Table 6, respectively.
Figure 1
Kinome inhibition
map of compound 7x: The kinases
targeted by 7x are indicated. Red circles represent kinases
targeted below 20 nM. Yellow, amber, green, and blue circles represent
kinases inhibited between 20–50, 50–100, 100–150,
and 150–250 nM, respectively. The human kinome map is adapted
with permission from Reaction Biology Corp. (http://reactionbiology.com).
Table 6
Kinase Inhibition
Profile of 7x and PD-0332991
kinase
7x IC50 (nM)
PD-0332991
IC50 (nM)
CDK4/cyclin D1
3.87
5.36
CDK6/cyclin D1
9.82
3.76
ARK5
4.95
>5000
FLT3
12.22
>10000
FYN
11.09
>10000
FMS
10.00
>10000
PDGFRβ
26.00
>10000
FGFR1
26.00
>10000
ABL
53.32
>10000
PI3K-δ
144
>10000
Kinome inhibition
map of compound 7x: The kinases
targeted by 7x are indicated. Red circles represent kinases
targeted below 20 nM. Yellow, amber, green, and blue circles represent
kinases inhibited between 20–50, 50–100, 100–150,
and 150–250 nM, respectively. The human kinome map is adapted
with permission from Reaction Biology Corp. (http://reactionbiology.com).
Molecular Modeling of 7x
Docking Prediction of 7x to CDK6 Suggest Different
Binding than PD-0332991
To explain the difference in inhibitor
potency between 7x and PD-0332991, binding of 7x to the kinase domain of CDK6 was predicted by molecular docking
and energy minimization based upon the X-ray cocrystal structure of
CDK6–Vcyclin–PD-0332991. CDK6 was used instead of CDK4
given the high amino acid similarity between these two CDKs and because
CDK6 and not CDK4 has been crystallized in the presence of PD-0332991.
In addition, a CDK4 small-molecule inhibitor X-ray cocrystal structure
is not available to date. Prediction shows that 7x may
bind to the CDK6 active site in a different orientation than PD-0332991
(Figure 2A). This change in binding may be
mainly achieved by new interactions formed by the cyano (CN) group
of 7x, which is substituted for an acetyl group (COCH3) in PD-0332991. The nitrogen of the cyano group is in close
contact with several residues of CDK6, in particular at a hydrogen
bonding distance with the side chain ε-amino group of Lys43
and main chain α-nitrogen of Ala23 (Figure 2B). Similar interactions in this binding mode might not be
energetically favorable for PD-0332991 due to the perpendicular orientation
seen in the cocrystal structure of the carbonyl group (CO) plus its
lower hydrogen bond acceptor potential when compared with the cyano
group in 7x (Figure 2C). Moreover,
the presence of the methyl group and the negative charges contributed
by the side chain carbonyl groups of Glu61 and Asp163 might not favor
the stabilization of the acetyl group in a position that is similar
to the one predicted for the cyano group in 7x. The presence
of a rigid and more electron withdrawing cyano group instead of an
acetyl group may be the main reason for the stabilization of the molecule
in this new orientation and also may explain the higher potency observed
for 7x with another closely related member of the CDK
family.
Figure 2
Model of 7x binding to CDK6. Small molecule 7x binding was predicted by docking and energy minimization
using the X-ray crystal structure of CDK6–Vcyclin–PD-0332991
(2EUF) as a
reference. Representations of the superimposition of X-ray crystal
structure (CDK6/PD-0332991) and predicted lowest energy binding (CDK6/7x) were prepared using PyMOL. (A) Ribbon representation of
CDK6 (green) bound to PD-0332991 (red) and 7x (cyan).
Small molecules are shown as sticks. (B,C) Close up view showing proximal
residues of CDK6 to 7x (blue) and PD-0332991 (pink),
respectively. Hydrogen bonds are shown as a dotted back lines.
Model of 7x binding to CDK6. Small molecule 7x binding was predicted by docking and energy minimization
using the X-ray crystal structure of CDK6–Vcyclin–PD-0332991
(2EUF) as a
reference. Representations of the superimposition of X-ray crystal
structure (CDK6/PD-0332991) and predicted lowest energy binding (CDK6/7x) were prepared using PyMOL. (A) Ribbon representation of
CDK6 (green) bound to PD-0332991 (red) and 7x (cyan).
Small molecules are shown as sticks. (B,C) Close up view showing proximal
residues of CDK6 to 7x (blue) and PD-0332991 (pink),
respectively. Hydrogen bonds are shown as a dotted back lines.Positioning of PD-0332991 in the
ATP binding pocket of CDK6 is
mainly given by hydrogen bond interactions between N3 and N2–H
to the backbone of Val101 and C6-acetyl group to
the main chain amide of Asp163. Similar to what is observed in the
X-ray structure of CDK6 and PD-0332991, 7x docking prediction
shows multiple residues with distances under 4.5 Å that may be
involved in van der Waals interactions that aid in the stabilization
of the small molecule. It is worth emphasizing that the difference
in potency and mode of action in PD-0332991 and 7x might
be due to a change in binding orientation inside the ATP binding pocket
of CDK6 and further explained by gain and loss of interactions. In
this regard, 7x does not present the same hydrogen bond
interactions described above for PD-0332991 but, as predicted by the
docking, present new ones implied by the presence of the cyano group
in 7x and residues Ala23 and Lys43 of CDK6. This slightly
deeper binding of 7x into the ATP binding site of CDK6
when compared with the binding of PD-0332991 may explain a difference
in potency in the CDK family observed for the two compounds, where 7x may interfere more efficiently with ATP binding and make
it a better inhibitor of the CDK kinase activity.Docking calculations
of 7x with ABL, FGFR, and FMS
were performed to see the interactions of the cyano group with the
amino acids in the ATP binding site of these kinases (given in Supporting Information). Results of these docking
studies show a great variability of binding between kinases, and because
crystal structures of these kinases in complex with compound with
similar structure to 7x are not available for reference
and comparison, docking simulations become hypothetical.
Inhibition
of CDK4 Kinase Activity and RB by 7x
To further
validate the results from RBC corporation (Figure 1) and molecular modeling studies, we independently
tested the inhibitory activity of 7x in an in vitro kinase
assay using recombinant CDK4 (Figure 3A). Our
results showed that 7x is a potent inhibitor of CDK4
with an IC50 of 3.87 nM, with little inhibitory activity
against CDKs 1, 2, 5, 8, and 9 (data not shown). Flavopiridol, a pan-CDK
inhibitor, and PD-0332991, a highly selective CDK4/6 inhibitor that
is currently in clinical trials, were used as positive controls.[40,54] These assays showed that PD-0332991 showed a similar level of CDK4
inhibition, with an IC50 of 5.36 nM. It is now well established
that the retinoblastoma family of proteins (pRb, p107, and p130) are
primary targets of CDK4. RB is hypophosphorylated in quiescent cells
and becomes phosphorylated on Ser780 and Ser795 by CDK4/CDK6 during mid to late G1. The hypophosphorylated
form of pRB associates with several cellular proteins and its phosphorylation
results in the disassociation of RB from its binding partners.[55−57] To determine whether 7x inhibits the activity of pRB
in vivo, two humanbreast carcinoma cell lines, MCF-7 (Figure 3B) and MDA-MB-231 (Figure 3C), were incubated with increasing concentrations of 7x for 24 h and the levels of phosphorylated RB (Ser780)
determined by Western blot analysis. The results of this study (Figure 3) show that 7x inhibits RB phosphorylation
at Ser780 as well as PD-0332991, confirming that CDK4 and
CDK6 are targets of this compound (Figure 3).
Figure 3
(A) Inhibition of CDK4/cyclin D1 activity by 7x: 10
ng of recombinant CDK4/cyclin D1 complex was incubated with the indicated
concentrations of 7x, flavopiridol (FP), or PD-0332991
for 30 min at room temperature. Kinase reactions were initiated by
the addition of the substrate mixture (5 μM ATP, 10 μci
γ-ATP, 10 mM MgCl2, and
1 μg recombinant RB substrate) and incubated at 30 °C for
20 min. The reactions were terminated by the addition of 2× Laemmli
sample buffer and heated at 95 °C for 3 min. Proteins were resolved
by 12% SDS-PAGE and the resulting gel subjected to autoradiography.
(B) 7x inhibits RB phosphorylation at serine 780: An
estrogen-dependent breast cancer cell line, MCF-7, and the triple
negative (C) human breast carcinoma cell line, MDA-MB-231, were treated
with increasing concentrations of 7x or PD-0332991 (control)
for 24 h. Western blot analysis was performed using antibodies directed
against phosphorylated (Ser780) and nonphosphorylated forms
of the retinoblastoma protein. Both 7x and PD-0332991
inhibit RB phosphorylation at Ser780, a known substrate
of CDK4.
(A) Inhibition of CDK4/cyclin D1 activity by 7x: 10
ng of recombinant CDK4/cyclin D1 complex was incubated with the indicated
concentrations of 7x, flavopiridol (FP), or PD-0332991
for 30 min at room temperature. Kinase reactions were initiated by
the addition of the substrate mixture (5 μM ATP, 10 μci
γ-ATP, 10 mM MgCl2, and
1 μg recombinant RB substrate) and incubated at 30 °C for
20 min. The reactions were terminated by the addition of 2× Laemmli
sample buffer and heated at 95 °C for 3 min. Proteins were resolved
by 12% SDS-PAGE and the resulting gel subjected to autoradiography.
(B) 7x inhibits RB phosphorylation at serine 780: An
estrogen-dependent breast cancer cell line, MCF-7, and the triple
negative (C) humanbreast carcinoma cell line, MDA-MB-231, were treated
with increasing concentrations of 7x or PD-0332991 (control)
for 24 h. Western blot analysis was performed using antibodies directed
against phosphorylated (Ser780) and nonphosphorylated forms
of the retinoblastoma protein. Both 7x and PD-0332991
inhibit RB phosphorylation at Ser780, a known substrate
of CDK4.
Effect of 7x on Cell Cycle Progression of Human
Tumor Cells
We next examined the effect of 7x treatment on MCF-7 (Figure 4A) and MDA-MB-231
(Figure 4B) cell cycle kinetics. For these
studies, cells were treated with 7x or PD-0332991 for
24 h and subjected to flow cytometric analysis to determine the distribution
of cells in various phases of the cell cycle. At time 0, the majority
of cells were in the G1 phase of the cell cycle, with smaller
percentages of the population in the S and G2 phases (data
not shown). While there was no significant change in the profile of
cells treated with DMSO throughout the course of the experiment, an
accumulation in the G1 phase was evident following treatment
with 7x and PD-0332991 (Figure 4). Prolonged treatment of cells with 7x (greater than
48 h) resulted in the appearance of a sub-G1 population,
which is indicative of cells undergoing apoptosis. This population
was absent in PD-0332991 treated cells.
Figure 4
Effect of 7x on cell cycle progression: MCF-7 (A)
and MDA-MB-231 (B) human breast carcinoma cell lines were treated
with increasing concentrations of 7x or PD-0332991 (control)
for 24 h. The cells were then harvested, fixed, and stained with propidium
iodide prior to flow cytometric analysis. The percentage of cells
at each phase of the cell cycle was calculated and represented as
% cells in each phase in the bar graph.
Effect of 7x on cell cycle progression: MCF-7 (A)
and MDA-MB-231 (B) humanbreast carcinoma cell lines were treated
with increasing concentrations of 7x or PD-0332991 (control)
for 24 h. The cells were then harvested, fixed, and stained with propidium
iodide prior to flow cytometric analysis. The percentage of cells
at each phase of the cell cycle was calculated and represented as
% cells in each phase in the bar graph.
7x Activates Programmed Cell Death
Because
flow cytometric analysis indicated that cells treated with 7x for longer periods of time might be undergoing apoptosis, we next
determined the levels of poly-ADP ribose polymerase (PARP) cleavage,
which is a marker of apoptosis. These studies show a dose-dependent
accumulation of the ∼89 kDa cleaved PARP polypeptide in cells
treated with 1 μM 7x (Figure 5). PARP cleavage was not observed in cells treated with PD-0332991,
suggesting that 7x exhibits a strong pro-apoptotic activity
that is not seen with PD-0332991.
Figure 5
7x treatment induces apoptosis
of breast carcinoma
cell lines: MCF-7 (A) and MDA-MB-231 (B) human breast carcinoma cell
lines were treated with increasing concentrations of 7x or PD-0332991 (control) for 24 h. Total cell lysates were subjected
to Western blot analysis using a PARP-specific antibody. The cleaved
protein, which is expressed in 7x treated cells, is indicative
of apoptosis.
7x treatment induces apoptosis
of breast carcinoma
cell lines: MCF-7 (A) and MDA-MB-231 (B) humanbreast carcinoma cell
lines were treated with increasing concentrations of 7x or PD-0332991 (control) for 24 h. Total cell lysates were subjected
to Western blot analysis using a PARP-specific antibody. The cleaved
protein, which is expressed in 7x treated cells, is indicative
of apoptosis.
Inhibition of the PI3Kinase/AKT
Pathway by 7x
Kinase inhibition assays performed
with 7x show inhibition
of two important growth factor receptors, PDGFRβ and FGFR1,
with an IC50 of 26 nM (Table 6).
Because these kinases are involved in the activation of the PI3Kinase/AKT
survival pathway, we next examined the ability of 7x to
inhibit PI3Kinase/AKT activity in cells treated with this compound.
We accomplished this by examining the phosphorylation status of AKT
(Figure 6), PI3Ks (see Supporting Information Table 3), and mTOR (data not shown)
proteins, which are well established modulators of cell survival.
The results presented in Figure 6 shows that 7x (but not PD-0332991) inhibits the phosphorylation of AKT,
which plays a critical role in the survival of tumor cells. This observation
might explain the differential effects of 7x and PD-0332991
on their ability to induce apoptotic death of breast tumor cells (Figure 5).
Figure 6
7x treatment inhibits AKT phosphorylation:
MDA-MB-231
human breast carcinoma cells were treated with increasing concentrations
of 7x or PD-0332991 or LY294002 (2-morpholino-8-phenyl-4H-chromen-4-one) (PI3Kinase inhibitor) for 24 h. Total cell
lysates were subjected to Western blot analysis using antibodies directed
against phosphorylated (Ser473) and nonphosphorylated forms
of AKT. GAPDH was used as a loading control.
7x treatment inhibits AKT phosphorylation:
MDA-MB-231humanbreast carcinoma cells were treated with increasing concentrations
of 7x or PD-0332991 or LY294002 (2-morpholino-8-phenyl-4H-chromen-4-one) (PI3Kinase inhibitor) for 24 h. Total cell
lysates were subjected to Western blot analysis using antibodies directed
against phosphorylated (Ser473) and nonphosphorylated forms
of AKT. GAPDH was used as a loading control.
Pharmacological Safety and in Vivo Efficacy of 7x
We next carried out studies to determine the maximum tolerated
dose of 7x in mice. CD-1 female mice (n = 3) received single doses of 7x 100 or 200 mg/kg intraperitoneally
and were monitored over a 7 day period. We observed no signs of toxicity
or weight loss, with a survival rate of 100%. We next injected mice
with 200 mg/kg of 7x (ip) for 5 consecutive days and
again monitored them for signs of toxicity. One hundred percent of
the mice survived for more than 10 days after injection (data not
shown). To determine the efficacy of 7x in vivo using
tumor xenograft models, MDA-MB-231 cells were orthotopically implanted
into the mammary fat pads of 6–8 week old female nude mice.
Once the tumors reached an average volume of 100 mm3, either
placebo or 7x (50 mg/kg body weight) was administered
on alternate days (Q2D) via IP injection. The results of this study
(Figure 7A) showed that 7x administered
on this schedule led to a dose-dependent inhibition of tumor growth
over a 21 day period. A decrease in tumor weight was also observed
at the end-point of the study (data not shown). No overt signs of
toxicity were observed in the 7x treated groups (body
weights shown in Figure 7B), indicating that
the compound is well-tolerated. In vivo pharmacokinetic studies with 7x exhibited favorable cytotoxicity, brain penetration, and
better half-life.[58]
Figure 7
In vivo efficacy of 7x against subcutaneous breast
tumor xenografts: MDA-MB-231 cells were orthotopically implanted into
the mammary fat pad of 6–8 week old female nude mice (n = 11 per group). Treatment was started when the average
tumor volume reached 100 mm3. 7x (lactate
salt dissolved in PBS) or vehicle was administered intraperitoneally
every other day (Q2D). Tumor volumes (A) and body weights (B) were
recorded every 2 days. All values represent mean ± SEM.
In vivo efficacy of 7x against subcutaneous breast
tumor xenografts: MDA-MB-231 cells were orthotopically implanted into
the mammary fat pad of 6–8 week old female nude mice (n = 11 per group). Treatment was started when the average
tumor volume reached 100 mm3. 7x (lactate
salt dissolved in PBS) or vehicle was administered intraperitoneally
every other day (Q2D). Tumor volumes (A) and body weights (B) were
recorded every 2 days. All values represent mean ± SEM.
Conclusion
In
this article, we describe the synthesis of pyrido[2,3-d]pyrimidine analogues that induce apoptotic death of a
wide variety of humantumor cell lines at nanomolar concentrations
while exhibiting little or no in vivo toxicity. Structure–function
studies described here suggest that the cytotoxic activity of pyrido[2,3-d]pyrimidines is dependent on the nature and position of
substituents at C-2, C-6, and N-8 positions. Compound 7x, with a 4-(4-methyl-piperazin-1-yl)
phenylamine group at C-2 position, cyano group at C-6 position, and cyclopentyl at N-8 position
showed optimum biological activity. The biochemical and biological
studies presented here show that this compound is a potent inhibitor
of CDK4 and CDK6 kinases and in this aspect is comparable to PD-0332991,
a dual CDK4/6 inhibitor that is currently in clinical trials. However,
unlike PD-0332991, 7x inhibits other kinases such as
ARK5, FGFR, and PDGFRβ, which are known to play critical roles
in proliferation and survival signaling in tumor cells. As has been
stated in the Introduction, considerable evidence
implicates the deregulation of the CDK4/Rb pathway in tumor cell growth,
and up-regulation of this pathway is observed in greater than 90%
of all humantumors. However, clinical trials with PD-0332991 suggest
that reduction of tumor burden in humantumors that have increased
levels of cyclin D/CDK4 activity might require inhibition of not only
CDK4 but also other signaling pathways.[44−46] Because most kinase
inhibitors are promiscuous in their target specificity and invariably
bind to more than one kinase, we took advantage of this property to
develop multitargeted kinase inhibitors that can inhibit multiple
signaling pathways that activated in a given tumor type. Our approach
to kinase inhibitor design, which incorporates tumor cell growth inhibition
as an integral parameter, led to the development of 7x. This compound inhibits other pathways that are deregulated in tumor
cells, such as the ARK5 and PI3K/AKT pathways, in addition to inhibition
of CDK4/RB. As a result, tumor cells treated with 7x underwent
apoptosis, an effect that is not seen in PD-0332991-treated cells.
The low toxicity profile and the potent tumor inhibitory activity
observed in nude mouse xenograft assays highlight the potential value
of this compound as a safe and targeted therapy for humancancers
that overexpress cyclin D/CDK4 complexes.
Experimental
Section
Chemistry: General Methods
All reagents and solvents
were obtained from commercial suppliers and used without further purification
unless otherwise stated. Solvents were dried using standard procedures,
and reactions requiring anhydrous conditions were performed under
N2 atmosphere. Reactions were monitored by thin layer chromatography
(TLC) on preloaded silica gel F254 plates (Sigma-Aldrich) with a UV
indicator. Column chromatography was performed using Merck 70-230
mesh silica gel 60 Å. Yields were of purified product and were
not optimized. Melting points were determined using an Electro-thermal
Mel-Temp 3.0 micromelting point apparatus and are uncorrected. 1H NMR spectra were obtained using a Bruker AVANCE 300 and
400 MHz spectrometer. Chemical shifts are reported in parts per million
(δ) downfield using tetramethylsilane (SiMe4) as
internal standard. Spin multiplicities are given as s (singlet), d
(doublet), t (triplet), dd (double doublet) bs (broad singlet), m
(multiplet), and q (quartet). All LC/MS data were gathered on an Agilent
1200 LC with Agilent 6410 triple quadrupole mass spectrometer detectors.
The compound solution was infused into the electrospray ionization
source operating positive and negative modes in methanol:water:TFA
(50:50:0.1% v/v) at 0.4 mL/min. The sample cone (declustering) voltage
was set at 100 V. The instrument was externally calibrated for the
mass range m/z 100–1000.
The purity of the final compounds was determined by HPLC and is 95%
or higher unless specified otherwise. Zorbax Exlipse XDB C18 (150
mm × 4.6 mm, 5 μm particle size) using gradient elution
of acetonitrile in water, 20–90%, for 25 min at a flow rate
of 1 mL/min with detection at 235 nm wavelength. For all samples 0.00154%
AcONH4 was added to water. The active methylene compounds 10,[49]13,[50] and 16(51) and amino compounds (21 and 22)[30] were prepared as per the reported procedures.
General
Procedure for the Synthesis of 4-Alkyl/cycloalkylamino-2-methylsulfanyl-pyrimidine-5-carboxylic
Acid Ethyl Ester (2)
4-Chloro-2-methylsulfanyl-pyrimidine-5-carboxylic
acid ethyl ester 1 (107 mmol) was dissolved in THF to
which triethylamine (322 mmol) and alkylamine (117 mmol) was added
and stirred for overnight at room temperature. The precipitated salts
were filtered and the solvent evaporated in vacuo. The resultant oil
was dissolved in ethyl acetate and washed with sodium bicarbonate
then dried over Na2SO4. The salts were filtered,
and the solvent was evaporated in vacuum to obtain the product.
Starting from 4-chloro-2-methylsulfanyl-pyrimidine-5-carboxylic
acid ethyl ester 1 and ammonium hydroxide, 90% of 2a was obtained as solid according to the method described
for the synthesis of 2; mp 130–131 °C. 1H NMR (300 MHz, CDCl3) δ 8.58 (s, 1H), 8.10
(bs, 2H), 4.30 (q, 2H), 2.45 (s, 3H), 1.25 (t, 3H). MS found (M +
H)+ (m/z), 214.10; calcd
for C8H11N3O2S m/z, 213.06.
Starting from 4-chloro-2-methylsulfanyl-pyrimidine-5-carboxylic
acid ethyl ester 1 and methylamine (40 wt % solution
in water), 80% of 2b was obtained as solid according
to the method described for the synthesis of 2; mp 82–83
°C. 1H NMR (300 MHz, CDCl3) δ 8.61
(s, 1H), 8.18 (bs, 1H), 4.33 (q, 2H), 3.09 (d, 3H), 2.55 (s, 3H),
1.37 (t, 3H). MS found (M + H)+ (m/z), 228.10; calcd for C9H13N3O2S m/z, 227.07.
Starting from 4-chloro-2-methylsulfanyl-pyrimidine-5-carboxylic
acid ethyl ester 1 and ethylamine (70 wt % solution in
water), 92% of 2c was obtained as liquid according to
the method described for the synthesis of 2. 1H NMR (300 MHz, CDCl3) δ 8.59 (s, 1H), 8.18 (bs,
1H), 4.26 (q, 2H), 3.50 (q, 2H), 2.51 (s, 3H), 1.35 (t, 3H), 1.25
(t, 3H). MS found (M + H)+ (m/z), 242.10; calcd for C10H15N3O2S m/z, 241.09.
Starting from 4-chloro-2-methylsulfanyl-pyrimidine-5-carboxylic
acid ethyl ester 1 and propylamine, 89% of 2d was obtained as liquid according to the method described for the
synthesis of 2. 1H NMR (300 MHz, CDCl3) δ 8.60 (s, 1H), 8.26 (bs, 1H), 4.25 (q, 2H), 3.47–3.54
(m, 2H), 2.52 (s, 3H), 1.65–1.61 (m, 2H), 1.35 (t, 3H), 1.00
(t, 3H). MS found (M + H)+ (m/z), 256.10; calcd for C11H17N3O2S m/z, 255.10.
Starting from 4-chloro-2-methylsulfanyl-pyrimidine-5-carboxylic
acid ethyl ester 1 and isopropylamine, 95% of 2e was obtained as liquid according to the method described for the
synthesis of 2. 1H NMR (300 MHz, CDCl3) δ 8.58 (s, 1H), 8.15 (bs, 1H), 5.70–5.67 (m,
1H), 4.24 (q, 2H), 2.49 (s, 3H), 1.26 (t, 3H), 1.21 (d, 6H). MS found
(M + H)+ (m/z), 256.10;
calcd for C11H17N3O2S m/z, 255.10.
Starting from 4-chloro-2-methylsulfanyl-pyrimidine-5-carboxylic
acid ethyl ester 1 and butylamine, 95% of 2f was obtained as liquid according to the method described for the
synthesis of 2. 1H NMR (300 MHz, CDCl3) δ 8.61 (s, 1H), 8.25 (bs, 1H), 4.43 (q, 2H), 3.59–3.52
(m, 2H), 2.53 (s, 3H), 1.65–1.60 (m, 2H), 1.46–1.35
(m, 5H), 0.96 (t, 3H). MS found (M + H)+ (m/z), 270.20; calcd for C12H19N3O2S m/z, 269.12.
Starting from 4-chloro-2-methylsulfanyl-pyrimidine-5-carboxylic
acid ethyl ester 1 and amylamine, 95% of 2g was obtained as liquid according to the method described for the
synthesis of 2. 1H NMR (300 MHz, CDCl3) δ 8.62 (s, 1H), 8.25 (bs, 1H), 4.35 (q, 2H), 3.58–3.51
(m, 2H), 2.53 (s, 3H), 1.67–1.62 (m, 2H), 1.40–1.34
(m, 7H), 0.93 (t, 3H). MS found (M + H)+ (m/z), 284.20; calcd for C13H21N3O2S m/z, 283.14.
Starting from 4-chloro-2-methylsulfanyl-pyrimidine-5-carboxylic
acid ethyl ester 1 and cylcopropylamine, 95% of 2h was obtained as liquid according to the method described
for the synthesis of 2. 1H NMR (300 MHz, CDCl3) δ 8.59 (s, 1H), 8.48 (bs, 1H), 4.27 (q, 2H), 2.95–2.89
(m, 1H), 2.51 (s, 3H), 1.34 (t, 3H), 0.84–0.79 (m, 2H), 0.61–0.58
(m, 2H). MS found (M + H)+ (m/z), 254.10; calcd for C11H15N3O2S m/z, 253.09.
Starting from 4-chloro-2-methylsulfanyl-pyrimidine-5-carboxylic
acid ethyl ester 1 and cyclopentyl amine, 90% of 2i was obtained as liquid according to the method described
for the synthesis of 2. 1H NMR (300 MHz, CDCl3) δ 8.60 (s, 1H), 8.25 (bs, 1H), 4.49–4.54 (m,
1H), 4.30 (q, 2H), 2.52 (s, 3H), 2.00–2.10 (m, 2H), 1.50–1.79
(m, 6H). 1.35 (t, 3H). MS found (M + H)+ (m/z), 282.20; calcd for C13H19N3O2S m/z, 281.12.
Starting from 4-chloro-2-methylsulfanyl-pyrimidine-5-carboxylic
acid ethyl ester 1 and cyclohexyl amine, 85% of 2j was obtained as liquid according to the method described
for the synthesis of 2. 1H NMR (300 MHz, CDCl3) δ 8.60 (s, 1H), 8.22 (bs, 1H), 4.30 (q, 2H), 4.09–4.14
(m, 1H), 2.51 (s, 3H), 1.94–2.27 (m, 2H), 1.73–1.81
(m, 2H), 1.59–1.64 (m, 2H), 1.30–1.41 (m, 4H). MS found
(M + H)+ (m/z), 296.10;
calcd for C14H21N3O2S m/z, 295.14.
General Procedure for the
Synthesis of (4-Alkyl/cycloalkylamino-2-methylsulfanyl-pyridine-5-yl)-methanol
(3)
Lithium aluminum hydride (53.3 mmol) was
suspended in THF under nitrogen atmosphere and cooled with dry ice.
The compound 2 (35.5 mmol) was dissolved in THF and added
dropwise to the cooled LiAlH4 solution while keeping the
reaction temperature bellow −10 °C. The reaction was brought
to room temperature and stirred for 1 h. The reaction was quenched
by the addition of water (5 mL), 15% NaOH (10 mL), and then water
(15 mL) again. The white solid that precipitated was filtered and
the filtrate evaporated in vacuo to afford the product.
Starting from 2a, 85% of 3a was
obtained according to method described for the synthesis of 3; mp 157–159 °C. 1H NMR (300 MHz,
DMSO-d6) δ 7.85 (s, 1H), 6.70 (bs,
2H), 5.30 (bs, 1H), 4.25 (s, 2H), 2.56 (s, 3H). MS found (M + H)+ (m/z), 172.00; calcd for
C6H9N3OS m/z, 171.05.
Starting from 2b, 80% of 3b was obtained according to method described for the synthesis
of 3; mp 145–146 °C. 1H NMR (300
MHz,
CDCl3) δ 7.64 (s, 1H), 5.93 (bs, 1H), 4.50 (s, 2H),
3.05 (d, 3H), 2.53 (s, 3H). MS found (M + H)+ (m/z), 186.00; calcd for C7H11N3OS m/z, 185.06.
Starting from 2c, 84% of 3c was
obtained according to method described for the synthesis of 3; mp 155–157 °C. 1H NMR (300 MHz,
CDCl3) δ 7.63 (s, 1H), 6. 55 (bs, 1H), 4.10 (s, 2H),
3.33 (q, 2H), 2.52 (s, 3H), 1.17 (t, 3H). MS found (M + H)+ (m/z), 200.10; calcd for C8H13N3OS m/z, 199.08.
Starting from 2d, 83% of 3d was obtained according to method described for the synthesis
of 3; mp 120–121 °C. 1H NMR (300
MHz,
CDCl3) δ 7.58 (s, 1H), 6.00 (bs, 1H), 4.48 (s, 2H),
3.42–3.37 (m, 2H), 2.49 (s, 3H), 1.55–1.68 (m, 2H),
0.97 (t, 3H). MS found (M + H)+ (m/z), 214.10; calcd for C9H15N3OS m/z, 213.09.
Starting from 2e, 85% of 3e was obtained according to method described for the synthesis
of 3; mp 127–129 °C. 1H NMR (300
MHz,
CDCl3) δ 7.60 (s, 1H), 5.58 (bs, 1H), 4.52–4.41
(m, 3H), 2.53 (s, 3H). 1.31 (d, 6H). MS found (M + H)+ (m/z), 214.10; calcd for C9H15N3OS m/z, 213.09.
Starting from 2f, 83% of 3f was
obtained according to method described for the synthesis of 3; mp 105–107 °C. 1H NMR (300 MHz,
CDCl3) δ 7.65 (s, 1H), 5.92 (bs, 1H), 4.50 (s, 2H),
3.54–3.48 (m, 2H), 2.52 (s, 3H), 1.64–1.57 (m, 2H),
1.45–1.37 (m, 2H), 0.96 (t, 3H). MS found (M + H)+ (m/z), 228.20; calcd for C10H17N3OS m/z, 227.11.
Starting from 2g, 86% of 3g was obtained according to method described for the synthesis
of 3; mp 110–112 °C. 1H NMR (300
MHz,
CDCl3) δ 7.64 (s, 1H), 5.93 (bs, 1H), 4.50 (s, 2H),
3.53–3.46 (m, 2H), 2.51 (s, 3H), 1.66–1.59 (m, 2H),
1.40–1.33 (m, 4H), 0.95–0.92 (m, 3H). MS found (M +
H)+ (m/z), 242.20; calcd
for C11H19N3OS m/z, 241.12.
Starting from 2h, 82% of 3h was obtained according to method described for the synthesis
of 3; mp 135–137 °C. 1H NMR (300
MHz, CDCl3) δ 7.69 (s, 1H), 6.08 (bs, 1H), 4.48 (s,
2H), 2.92–2.86 (m, 1H), 2.54 (s, 3H), 0.87–0.80 (m,
2H), 0.59–0.56 (m, 2H), MS found (M + H)+ (m/z), 212.10; calcd for C9H13N3OS m/z, 211.08.
Starting from 2i, 84% of 3i was obtained according to method described for the synthesis
of 3; mp 150–151 °C. 1H NMR (300
MHz, CDCl3) δ 7.65 (s, 1H), 5.80 (bs, 1H), 4.52 (s,
2H), 4.45–4.50 (m, 1H), 2.50 (s, 3H), 2.00–2.15 (m,
2H), 1.62–1.78 (m, 4H), 1.40–1.49 (m, 2H). MS found
(M + H)+ (m/z), 240.10;
calcd for C11H17N3OS m/z, 239.11.
Starting from 2j, 84% of 3j was obtained according to method described for the synthesis
of 3; mp 175–177 °C. 1H NMR (300
MHz, CDCl3) δ 7.59 (s, 1H), 5.80 (bs, 1H), 4.45 (s,
2H), 3.95–4.08 (m, 1H), 2.51 (s, 3H), 1.99–2.08 (m,
2H), 1.61–1.78 (m, 4H), 1.24–1.43 (m, 4H). MS found
(M + H)+ (m/z), 254.10;
calcd for C12H19N3OS m/z, 253.12.
General Procedure for the
Synthesis of 4-Alkyl/cycloalkylamino-2-methylsulfanyl-pyrimidine-5-carbaldehyde
(4)
The compound 3 (20.8 mmol)
was dissolved in chloroform to which manganese dioxide (MnO2) (119 mmol) was added and stirred for overnight, and an additional
portion of MnO2 (31.3 mmol) was added and stirred for 12
h. The solids were removed by filtration through a Celite pad and
washed with chloroform. The chloroform was evaporated in vacuum to
get the product.
Starting from 3a, 72% of 4a was
obtained according to the procedure described for the synthesis of 4; mp 186–188 °C. 1H NMR (300 MHz,
CDCl3) δ 9.80 (s, 1H), 8.45 (s, 1H), 8.20 (bs, 1H),
5.74 (bs, 1H), 2.55 (s, 3H). MS found (M + H)+ (m/z), 170.00; calcd for C6H7N3OS m/z, 169.03.
Starting from 3b, 75% of 4b was
obtained according to the procedure described for the synthesis
of 4; mp 98–99 °C. 1H NMR (300
MHz, CDCl3) δ 9.71 (s, 1H), 8.59 (bs, 1H), 8.30 (s,
1H), 3.14 (d, 3H), 2.58 (s, 3H). MS found (M + H)+ (m/z), 184.10; calcd for C7H9N3OS m/z, 183.05.
Starting from 3c, 72% of 4c was
obtained according to the procedure described for the synthesis
of 4; mp 60–61 °C. 1H NMR (300
MHz, CDCl3) δ 9.72 (s, 1H), 8.64 (bs, 1H), 8.29 (s,
1H), 3.56 (q, 2H), 2.50 (s, 3H), 1.18 (t, 3H). MS found (M + H)+ (m/z), 198.10; calcd for
C8H11N3OS m/z, 197.06.
Starting from 3d, 70% of 4d was obtained according to the procedure described for the
synthesis
of 4; mp 50–51 °C. 1H NMR (300
MHz, CDCl3) δ 9.69 (s, 1H), 8.63 (bs, 1H), 8.28 (s,
1H), 3.53–3.57 (m, 2H), 2.52 (s, 3H), 1.61–1.73 (m,
2H), 0.86 (t, 3H). MS found (M + H)+ (m/z), 212.10; calcd for C9H13N3OS m/z, 211.08.
Starting from 3e, 90% of 4e was obtained as low melting solid according to the procedure
described for the synthesis of 4; mp 54–55 °C. 1H NMR (300 MHz, CDCl3) δ 9.69 (s, 1H), 8.48
(bs, 1H), 8.29 (s, 1H), 4.51–4.40 (m, 1H), 2.55 (s, 3H), 1.30
(d, 6H). MS found (M + H)+ (m/z), 212.00; calcd for C9H13N3OS m/z, 211.08.
Starting from 3f, 90% of 4f was obtained as thick liquid according to the procedure
described
for the synthesis of 4. 1H NMR (300 MHz, CDCl3) δ 9.70 (s, 1H), 8.63 (bs, 1H), 8.29 (s, 1H), 3.63–3.56
(m, 2H), 2.53 (s, 3H), 1.70–1.60 (m, 2H), 1.49–1.39
(m, 2H), 0.97 (t, 3H). MS found (M + H)+ (m/z), 226.10; calcd for C10H15N3OS m/z, 225.09.
Starting from 3g, 87% of 4g was
obtained as thick liquid according to the procedure described
for the synthesis of 4. 1H NMR (300 MHz, CDCl3) δ 9.70 (s, 1H), 8.63 (bs, 1H), 8.29 (s, 1H), 3.61–3.55
(m, 2H), 2.55 (s, 3H), 1.70–1.74 (m, 2H), 1.40–1.34
(m, 4H), 0.95–0.91 (m, 3H). MS found (M + H)+ (m/z), 240.10; calcd for C11H17N3OS m/z, 239.11.
Starting from 3h, 80% of 4h was obtained as low melting solid according to the procedure
described for the synthesis of 4; mp 69–70 °C. 1H NMR (300 MHz, CDCl3) δ 9.61 (s, 1H), 8.
49 (bs, 1H), 8.23 (s, 1H), 2.98–2.92 (m, 1H), 2.50 (s, 3H),
0.85–0.78 (m, 2H), 0.60–0.57 (m, 2H). MS found (M +
H)+ (m/z), 210.10; calcd
for C9H11N3OS m/z, 209.06.
Starting from 3i, 87% of 4i was obtained as solid according to the procedure described
for the synthesis of 4; mp 41–42 °C. 1H NMR (300 MHz, CDCl3) δ 9.65 (s, 1H), 8.60
(bs, 1H), 8.25 (s, 1H), 4.49–4.54 (m, 1H), 2.52 (s, 3H), 2.01–2.12
(m, 2H), 1.50–1.82 (m, 6H). MS found (M + H)+ (m/z), 238.10; calcd for C11H15N3OS m/z, 237.09.
Starting from 3j, 90% of 4j was obtained as thick liquid according to the method described
for the synthesis of 4. 1H NMR (300 MHz, CDCl3) δ 9.62 (s, 1H), 8.55 (bs, 1H), 8.15 (s, 1H), 4.01–4.09
(m, 1H), 2.51 (s, 3H), 1.97–2.05 (m, 2H), 1.60–1.80
(m, 4H), 1.22–1.44 (m, 4H). MS found (M + H)+ (m/z), 252.10; calcd for C12H17N3OS m/z, 251.11.
General Procedure for 8-Alkyl/cycloalkyl-2-methylsulfanyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine (5)
A mixture of 4-alkylamino-2-methylsulfanyl-pyrimidine-5-carbaldehyde 4 (4.2 mmol), 1.2 equiv of active methylene compound, and
a catalytic amount of benzylamine was taken in to acetic acid and
refluxed for about 6 h. After completion of the reaction (checked
with TLC), the reaction mixture was cooled to room temperature and
the precipitated product was filtered. In some cases, the reaction
mixture was diluted with hexane to get solid out. The solid was washed
with saturated NaHCO3 and water and dried over vacuo. The
crude product was recrystallized in 2-propanol to get pure product
(5).
Starting
from 4a and cyanoacetic acid, 65% of 5a was
obtained
according to the method described for the synthesis of 5; mp 328–330 °C. 1H NMR (300 MHz, DMSO-d6) δ 13.12 (bs, 1H), 8.94 (s, 1H), 8.71
(s, 1H), 2.56 (s, 3H). MS found (M + H)+ (m/z), 219.10; calcd for C9H6N4OS m/z, 218.03.
Starting
from 4b and cyanoacetic acid, 67% of 5b was
obtained according to the method described for the synthesis of 5; mp 292–294 °C. 1H NMR (300 MHz,
DMSO-d6) δ 8.95 (s, 1H), 8.79 (s,
1H), 3.61 (s, 3H), 2.51 (s, 3H). MS found (M + H)+ (m/z), 233.10; calcd for C10H8N4OS m/z, 232.04.
Starting
from 4c and cyanoacetic acid, 70% of 5c was
obtained according to the method described for the synthesis of 5; mp 244–245 °C. 1H NMR (300 MHz,
CDCl3) δ 8.71 (s, 1H), 8.15 (s, 1H), 4.51 (q, 2H),
2.61 (s, 3H), 1.37 (t, 3H). MS found (M + H)+ (m/z), 247.10; calcd for C11H10N4OS m/z, 246.06.
Starting
from 4d and cyanoacetic acid, 70% of 5d was
obtained according to the method described for the synthesis of 5; mp 230–231 °C. 1H NMR (300 MHz,
CDCl3) δ 8.70 (s, 1H), 8.14 (s, 1H), 4.44–4.39
(m, 2H), 2.60 (s, 3H), 1.83–1.76 (m, 2H), 1.02 (t, 3H). MS
found (M + H)+ (m/z),
261.10; calcd for C12H12N4OS m/z, 260.07.
Starting
from 4e and cyanoacetic acid, 70% of 5e was
obtained according to the method described for the synthesis of 5; mp 200–202 °C. 1H NMR (300 MHz,
CDCl3) δ 8.68 (s, 1H), 8.10 (s, 1H), 5.89–5.80
(m, 1H), 2.67 (s, 3H), 1.65 (d, 6H). MS found (M + H)+ (m/z), 261.10; calcd for C12H12N4OS m/z, 260.07.
Starting
from 4f and cyanoacetic acid, 70% of 5f was
obtained according to the method described for the synthesis of 5; mp 220–222 °C. 1H NMR (300 MHz,
CDCl3) δ 8.70 (s, 1H), 8.14 (s, 1H), 4.45 (t, 2H),
2.65 (s, 3H), 1.77–1.71 (m, 2H), 1.49–1.41 (m, 2H),
1.00 (t, 3H). MS found (M + H)+ (m/z), 275.10; calcd for C13H14N4OS m/z, 274.09.
Starting
from 4g and cyanoacetic acid, 70% of 5g was
obtained according to the method described for the synthesis of 5; mp 160–161 °C. 1H NMR (300 MHz,
CDCl3) δ 8.71 (s, 1H), 8.15 (s, 1H), 4.43 (t, 2H),
2.66 (s, 3H), 1.78–1.73 (m, 2H), 1.42–1.38 (m, 4H),
0.93 (t, 3H). MS found (M + H)+ (m/z), 289.10; calcd for C14H16N4OS m/z, 288.10.
Starting
from 4h and cyanoacetic acid, 71% of 5h was
obtained according to the method described for the synthesis of 5; mp 158–159 °C. 1H NMR (300 MHz,
CDCl3) δ 8.70 (s, 1H), 8.14 (s, 1H), 2.99–2.97
(m, 1H), 2.65 (s, 3H), 1.27 (bs, 2H), 1.02 (bs, 2H). MS found (M +
H)+ (m/z), 259.10; calcd
for C12H10N4OS m/z, 258.06.
Starting
from 4i and cyanoacetic acid, 71% of 5i was
obtained according to the method described for the synthesis of 5; mp 209–210 °C. 1H NMR (300 MHz,
CDCl3) δ 8.68 (s, 1H), 8.10 (s, 1H), 5.89–5.98
(m, 1H), 2.64 (s, 3H), 2.23–2.35 (m, 2H), 2.09–2.16
(m, 2H), 1.83–1.96 (m, 2H), 1.63–1.76 (m, 2H). MS found
(M + H)+ (m/z), 287.10;
calcd for C14H14N4OS m/z, 286.09.
Starting
from 4j and cyanoacetic acid, 71% of 5j was
obtained according to the method described for the synthesis of 5; mp 239–240 °C. 1H NMR (300 MHz,
CDCl3) δ 8.65 (s, 1H), 8.08 (s, 1H), 5.42 (bs, 1H),
2.62 (s, 3H), 1.90–1.95 (m, 2H), 1.67–1.76 (m, 6H),
1.34–1.45 (m, 2H). MS found (M + H)+ (m/z), 301.10; calcd for C15H16N4OS m/z, 300.10.
Starting from 4i and nitro-acetic acid ethyl ester, 70% of 5k was obtained according to the method described for the synthesis
of 5; mp 182–84 °C. 1H NMR (300
MHz, CDCl3) δ 8.79 (s, 1H), 8.42 (s, 1H), 6.05–6.00
(m, 1H), 2.67 (m, 3H), 2.34–2.30 (m, 2H), 2.15–2.10
(m, 2H), 1.99–1.91 (m, 2H), 1.78–172 (m, 2H). MS found
(M + H)+ (m/z), 307.10;
calcd for C13H14N4O3S m/z, 306.08.
Starting from 4i and methyl sulfonyl acetic
acid,
73% of 5l was obtained according to the method described
for the synthesis of 5; mp 190–191 °C. 1H NMR (300 MHz, CDCl3) δ 8.76 (s, 1H), 8.49
(s, 1H), 5.88–6.00 (m, 1H), 3.34 (s, 3H), 2.65 (s, 3H), 2.28–2.41
(m, 2H), 2.09–2.16 (m, 2H), 1.84–1.99 (m, 2H), 1.67–1.73
(m, 2H). MS found (M + H)+ (m/z), 340.20; calcd for C14H17N3O3S2m/z, 339.07.
Starting from 4i and benzene sulfonyl acetic
acid,
70% of 5m was obtained according to the method described
for the synthesis of 5; mp 184–185 °C. 1H NMR (300 MHz, CDCl3) δ 8.87 (s, 1H), 8.70
(s, 1H), 7.54–7.80 (m, 5H), 5.69–5.72 (m, 1H), 2.66
(s, 3H), 2.20–2.33 (m, 2H), 1.99–2.11 (m, 2H), 1.78–1.88
(m, 2H), 1.61–1.69 (m, 2H). MS found (M + H)+ (m/z), 402.10; calcd for C19H19N3O3S2m/z, 401.09.
Starting from 4i and 4-chlorobenzene sulfonylacetic acid 13, 70% of 5n was obtained according
to the method described for the synthesis of 5; mp 222–223
°C. 1H NMR (300 MHz, CDCl3) δ 8.78
(s, 1H), 8.65 (s, 1H), 8.05–8.09 (m, 2H), 7.49–7.54
(m, 2H), 5.75–5.81 (m, 1H), 2.63 (s, 3H), 2.21–2.28
(m, 2H), 1.95–2.07 (m, 2H), 1.77–1.83 (m, 2H), 1.64–1.69
(m, 2H). MS found (M + H)+ (m/z), 436.10; calcd for C19H18ClN3O3S2m/z, 435.05.
Starting from 4i and 4-hydroxy-3-methoxy-phenylmethane
sulfonylacetic acid10c, 65% of 5p was
obtained according to the method described for the synthesis of 5; mp 180–181 °C. 1H NMR (300 MHz,
CDCl3) δ 8.48 (s, 1H), 8.12 (s, 1H), 6.52–6.72
(m, 3H), 5.73–5.90 (m, 1H), 4.55 (s, 2H), 3.69 (s, 3H), 2.49
(s, 3H), 2.12–2.28 (m, 2H), 1.89–2.02 (m, 2H), 1.68–1.81
(m, 2H), 1.48–1.61 (m, 2H). MS found (M + H)+ (m/z), 462.10; calcd for C21H23N3O5S2m/z, 461.11.
Starting from 4i and N-(4-fluoro-phenyl)-malonamic acid 16b,
65% of 5s was obtained according to the method described
for the synthesis of 5; mp 243–242 °C. 1H NMR (300 MHz, CDCl3) δ 11.65 (bs, 1H),
8.87 (s, 1H), 8.83 (s, 1H), 7.69–7.76 (m, 2H), 6.98–7.10
(m, 2H), 6.01–6.13 (m, 1H), 2.66 (s, 3H), 2.31–2.42
(m, 2H), 2.09–2.20 (m, 2H), 1.89–2.00 (m, 2H), 1.72–1.81
(m, 2H). MS found (M + H)+ (m/z), 399.10; calcd for C20H19FN4O2S m/z, 398.12.
General Procedure for the Synthesis of Compounds (5b–5j) Form 2-Methylsulfanyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (5a), (Scheme 2)
The compound 5a (5 mmol)
was taken into DMF and stirred at 50 °C for 10 min until obtaining
the clear solution, and then NaH (5 mmol) was added and stirred about
5 min. The reaction mixture was brought to room temperature and alkyl
iodides (6.5 mmol) were added. After 1 h stirring, the reaction was
quenched with water and filtered the obtained product. The crude product
was purified with flash column chromatography using 10–30%
ethyl acetate in hexanes. All physical and spectral data matched with
the compounds, which were prepared according to Scheme 1.
General Procedure for the Synthesis of 8-Alkyl-2-methylsulfinyl-6-substituted-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidines (6)
A solution of 8-alkyl-2-methylsulfanyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine 5 (3.5 mmol) and 3-chloro-benzenecarboperoxoic
acid (m-CPBA) (4.4 mmol) in CH2Cl2 was stirred at room temperature for about 4 h. After completion
of the reaction, the reaction mixture was washed with saturated NaHCO3, and the organic layer was dried over Na2SO4 and concentrated to produce the corresponding methylsuloxide 6 and was purified with flash chromatography.
Starting
from 5a and 3-chloro-benzenecarboperoxoic acid, 90% of 6a was obtained according to the method described for the
synthesis
of 6. 1H NMR (300 MHz, DMSO-d6) δ 13.10 (bs, 1H), 8.93 (s, 1H), 8.72 (s, 1H),
2.92 (s, 3H). MS found (M + H)+ (m/z), 235.10; calcd for C9H6N4O2S m/z, 234.02.
Starting
from 5b and 3-chloro-benzenecarboperoxoic acid, 92% of 6b was obtained according to the method described for the
synthesis of 6. 1H NMR (300 MHz, DMSO-d6) δ 8.92 (s, 1H), 8.88 (s, 1H), 3.62
(s, 3H), 2.91 (s, 3H). MS found (M + H)+ (m/z), 249.10; calcd for C10H8N4O2S m/z, 248.04.
Starting
from 5c and 3-chloro-benzenecarboperoxoic acid, 90% of 6c was obtained according to the method described for the
synthesis of 6. 1H NMR (300 MHz, CDCl3) δ 8.73 (s, 1H), 8.17 (s, 1H), 4.50 (q, 2H), 2.92 (s,
3H), 1.37 (t, 3H). MS found (M + H)+ (m/z), 263.10; calcd for C11H10N4O2S m/z, 262.05.
Starting
from 5d and 3-chloro-benzenecarboperoxoic acid, 92% of 6d was obtained according to the method described for the
synthesis of 6. 1H NMR (300 MHz, CDCl3) δ 8.72 (s, 1H), 8.13 (s, 1H), 4.42–4.38 (m,
2H), 2.94 (s, 3H), 1.84–1.76 (m, 2H), 1.02 (t, 3H). MS found
(M + H)+ (m/z), 277.10;
calcd for C12H12N4O2S m/z, 276.07.
Starting
from 5e and 3-chloro-benzenecarboperoxoic acid, 89% of 6e was obtained according to the method described for the
synthesis of 6. 1H NMR (300 MHz, CDCl3) δ 8.67 (s, 1H), 8.12 (s, 1H), 5.87–5.79 (m,
1H), 2.95 (s, 3H), 1.63 (d, 6H). MS found (M + H)+ (m/z), 277.10; calcd for C12H12N4O2S m/z, 276.07.
Starting
from 5f and 3-chloro-benzenecarboperoxoic acid, 90% of 6f was obtained according to the method described for the
synthesis of 6. 1H NMR (300 MHz, CDCl3) δ 8.69 (s, 1H), 8.15 (s, 1H), 4.43 (t, 2H), 2.96 (s,
3H), 1.75–1.70 (m, 2H), 1.50–1.43 (m, 2H), 1.02 (t,
3H). MS found (M + H)+ (m/z), 291.10; calcd for C13H14N4O2S m/z, 290.08.
Starting
from 5g and 3-chloro-benzenecarboperoxoic acid, 93% of 6g was obtained according to the method described for the
synthesis of 6. 1H NMR (300 MHz, CDCl3) δ 8.72 (s, 1H), 8.14 (s, 1H), 4.44 (t, 2H), 2.93 (s,
3H), 1.77–1.72 (m, 2H), 1.43–1.37 (m, 4H), 0.92 (t,
3H). MS found (M + H)+ (m/z), 305.10; calcd for C14H16N4O2S m/z, 304.10.
Starting
from 5h and 3-chloro-benzenecarboperoxoic acid, 91% of 6h was obtained according to the method described for the
synthesis of 6. 1H NMR (300 MHz, CDCl3) δ 8.70 (s, 1H), 8.14 (s, 1H), 2.96–2.93 (m,
1H), 2.94 (s, 3H), 1.25 (bs, 2H), 1.03 (bs, 2H). MS found (M + H)+ (m/z), 275.10; calcd for
C12H10N4O2S m/z, 274.05.
Starting
from 5i and 3-chloro-benzenecarboperoxoic acid, 94% of 6i was obtained according to the method described for the
synthesis of 6. 1H NMR (300 MHz, CDCl3) δ 8.69 (s, 1H), 8.13 (s, 1H), 5.98 (bs, 1H), 2.93
(s, 3H), 2.20–2.33 (m, 2H), 2.07–2.15 (m, 2H), 1.82–1.94
(m, 2H), 1.62–1.74 (m, 2H). MS found (M + H)+ (m/z), 303.10; calcd for C14H14N4O2S m/z, 302.08.
Starting
from 5j and 3-chloro-benzenecarboperoxoic acid, 92% of 6j was obtained according to the method described for the
synthesis of 6. 1H NMR (300 MHz, CDCl3) δ 8.69 (s, 1H), 8.11 (s, 1H), 5.43 (bs, 1H), 2.95
(s, 3H), 1.92–1.96 (m, 2H), 1.66–1.79 (m, 6H), 1.33–1.45
(m, 2H). MS found (M + H)+ (m/z), 317.10; calcd for C15H16N4O2S m/z, 316.10.
Starting from 5k and 3-chloro-benzenecarboperoxoic acid, 91% of 6k was obtained according to the method described for the synthesis
of 6. 1H NMR (300 MHz, CDCl3) δ
8.73 (s, 1H), 8.40 (s, 1H), 6.03 (bs, 1H), 2.94 (m, 3H), 2.35–2.32
(m, 2H), 2.16–2.09 (m, 2H), 1.99–1.91 (m, 2H), 1.76–171
(m, 2H). MS found (M + H)+ (m/z), 323.10; calcd for C13H14N4O4S m/z, 322.07.
Starting from 5l and 3-chloro-benzenecarboperoxoic
acid, 92% of 6l was obtained according to the method
described for the synthesis of 6. 1H NMR (300
MHz, CDCl3) δ 8.78 (s, 1H), 8.51 (s, 1H), 6.00 (bs,
1H), 3.34 (s, 3H), 2.95 (s, 3H), 2.28–2.40 (m, 2H), 2.10–2.15
(m, 2H), 1.86–1.98 (m, 2H), 1.66–1.71 (m, 2H). MS found
(M + H)+ (m/z), 356.10;
calcd for C14H17N3O4S2m/z, 355.07.
Starting from 5m and 3-chloro-benzenecarboperoxoic
acid, 90% of 6m was obtained according to the method
described for the synthesis of 6. 1H NMR (300
MHz, CDCl3) δ 8.86 (s, 1H), 8.70 (s, 1H), 7.55–7.80
(m, 5H), 5.71 (bs, 1H), 2.95 (s, 3H), 2.19–2.31 (m, 2H), 1.99–2.10
(m, 2H), 1.80–1.87 (m, 2H), 1.60–1.70 (m, 2H). MS found
(M + H)+ (m/z), 418.10;
calcd for C19H19N3O4S2m/z, 417.08.
Starting from 5n and 3-chloro-benzenecarboperoxoic
acid, 91% of 6n was obtained according to the method
described for the synthesis of 6. 1H NMR (300
MHz, CDCl3) δ 8.81 (s, 1H), 8.68 (s, 1H), 8.02–8.06
(m, 2H), 7.50–7.57 (m, 2H), 5.77 (bs, 1H), 2.94 (s, 3H), 2.20–2.26
(m, 2H), 1.96–2.08 (m, 2H), 1.78–1.82 (m, 2H), 1.64–1.69
(m, 2H). MS found (M + H)+ (m/z), 452.10; calcd for C19H18ClN3O4S2m/z, 451.04.
Starting from 5s and m-chloroperbenzoic acid, 87% of 6s was obtained according
to the method described for the synthesis of 6. 1H NMR (300 MHz, CDCl3) δ 11.60 (bs, 1H),
8.86 (s, 1H), 8.81 (s, 1H), 7.71–7.77 (m, 2H), 6.96–7.09
(m, 2H), 6.03–6.13 (m, 1H), 2.96 (s, 3H), 2.33–2.42
(m, 2H), 2.13–2.21 (m, 2H), 1.92–2.00 (m, 2H), 1.71–1.80
(m, 2H). MS found (M + H)+ (m/z), 415.10; calcd for C20H19FN4O3S m/z, 414.12.
General Procedure for 8-Alkyl/cycloalkyl-2-(aryl/heteroarylamino)-6-substituted-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine (7)
The mixture of 8-alkyl-2-methylsulfinyl-6-substituted-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine 6 (1.65 mmol) and aryl/heteroaryl
amines (2 mmol) in toluene was stirred at 100 °C for overnight.
The reaction mixture was cooled, and solid was collected by filtration.
The crude product was washed with toluene and purified by flash chromatography
to get pure product.
Starting
from 6i and benzylamine, 51% of 7a was obtained
according to the method described for the synthesis of 7; mp 211–212 °C. 1H NMR (CDCl3,
300 MHz) δ 8.45 (s, 1H), 8.35 (s, 1H), 7.36–7.42 (m,
5H), 5.80–5.86 (m, 1H), 2.48 (s, 2H), 2.10 (bs, 2H), 1.91 (m,
2H), 1.66 (m, 2H), 1.54 (m, 2H). MS found (M + H)+ (m/z), 346.20; calcd for C20H19N5O m/z, 345.16.
Starting
from 6i and 4-chlorophenylamine, 50% of 7b was obtained according to the method described for the synthesis
of 7; mp 272–273 °C. 1H NMR (CDCl3, 300 MHz) δ 8.63 (s, 1H), 8.02 (s, 1H), 7.58–7.55
(m, 2H), 7.40–7.37 (m, 2H), 5.89–5.83 (m, 1H), 2.30–2.27
(m, 2H), 2.05 (bs, 2H), 1.90–1.87 (m, 2H), 1.71–1.67
(m, 2H). MS found (M + H)+ (m/z), 366.20; calcd for C19H16ClN5O m/z, 365.10.
Starting
from 6i and 4-aminobenzonitrile, 55% of 7c was obtained according to the method described for the synthesis
of 7; mp 285–287 °C. 1H NMR (CDCl3, 300 MHz) δ 8.61 (s, 1H), 8.03 (s, 1H), 7.62–7.59
(m, 2H), 7.45–7.43 (m, 2H), 5.92–5.89 (m, 1H), 2.31–2.28
(m, 2H), 2.15 (bs, 2H), 1.89–1.86 (m, 2H), 1.75–1.72
(m, 2H). MS found (M + H)+ (m/z), 357.10; calcd for C20H16N6O m/z, 356.14.
Starting
from 6i and 2-methoxyphenylamine, 54% of 7e was obtained according to the method described for the synthesis
of 7; mp 159–160 °C. 1H NMR (CDCl3, 300 MHz) δ 8.60 (s, 1H), 8.31–8.33 (m, 1H),
7.99 (s, 1H), 7.10–7.16 (m, 1H), 6.96–7.06 (m, 2H),
5.89–5.98 (m, 1H), 3.90 (s, 3H), 2.28–2.36 (m, 2H),
2.04–2.10 (m, 2H), 1.85–1.96 (m, 2H), 1.64–1.75
(M, 2H). MS found (M + H)+ (m/z), 362.20; calcd for C20H19N5O2m/z, 361.15.
Starting
from 6i and 4-methoxyphenylamine, 54% of 7g was obtained according to the method described for the synthesis
of 7; mp 238–239 °C. 1H NMR (CDCl3, 300 MHz) δ 8.57 (s, 1H), 7.97 (s, 1H), 7.42–7.47
(m, 2H), 6.92–6.97 (m, 2H), 5.83 (bs 1H), 3.84 (s, 3H), 2.21–2.30
(m, 2H), 1.85–2.05 (m, 4H), 1.61–1.74 (m, 2H). MS found
(M + H)+ (m/z), 362.30;
calcd for C20H19N5O2m/z, 361.15.
Starting
from 6i and 2,4-dimethoxyphenylamine, 52% of 7h was obtained according to the method described for the synthesis
of 7; mp 201–202 °C. 1H NMR (CDCl3, 300 MHz) δ 8.57 (s, 1H), 7.96 (s, 1H), 7.19–7.23
(m, 1H), 6.51–6.56 (m, 2H), 5.85–5.91 (m, 1H), 3.91
(s, 3H), 3.85 (s, 3H), 2.30 (bs, 2H), 2.03 (bs, 2H), 1.86–1.90
(m, 2H), 1.66–1.69 (m, 2H). MS found (M + H)+ (m/z), 392.20; calcd for C21H21N5O3m/z, 391.16.
Starting
from 6i and 3,4-dimethoxyphenylamine, 50% of 7i was obtained according to the method described for the synthesis
of 7; mp 215–216 °C. 1H NMR (CDCl3, 300 MHz) δ 8.58 (s, 1H), 7.98 (s, 1H), 7.16–7.26
(m, 1H), 6.90–6.98 (m, 1H), 6.84–6.87 (m, 1H), 5.84–5.95
(m, 1H), 3.91 (s, 6H), 2.20–2.29 (m, 2H), 1.84–2.13
(m, 4H), 1.61 (bs, 2H). MS found (M + H)+ (m/z), 392.20; calcd for C21H21N5O3m/z,
391.16.
Starting
from 6i and 3,5-dimethoxyphenylamine, 52% of 7j was obtained according to the method described for the synthesis
of 7; mp 150–151 °C. 1H NMR (CDCl3, 300 MHz) δ 8.60 (s, 1H), 7.99 (s, 1H), 7.26 (s, 1H),
6.91–6.88 (m, 1H), 6.31–6.30 (m, 1H), 5.88–5.94
(m, 1H), 3.82 (s, 6H), 2.23–2.36 (m, 2H), 2.05–2.18
(m, 2H), 1.83–1.93 (m, 2H), 1.62–1.64 (m, 2H). MS found
(M + H)+ (m/z), 392.20;
calcd for C21H21N5O3m/z, 391.16.
Starting
from 6i and 3,4,5-trimethoxyphenylamine, 52% of 7k was obtained according to the method described for the
synthesis of 7; mp 169–170 °C. 1H NMR (CDCl3, 300 MHz) δ 8.59 (s, 1H), 8.01 (s,
1H), 6.91 (s, 2H), 5.93–5.99 (m, 1H), 3.90 (s, 6H), 3.87 (s,
3H), 2.23–2.32 (m, 2H), 2.03–2.14 (m, 2H), 1.85–1.92
(m, 2H), 1.52–1.58 (m, 2H). MS found (M + H)+ (m/z), 422.30; calcd for C22H23N5O4m/z, 421.18.
Starting
from 6i and 5-fluoro-2-methoxy-phenylamine, 50% of 7l was obtained according to the method described for the
synthesis of 7; mp 241–243 °C. 1H NMR (CDCl3, 300 MHz) δ 8.65 (s, 1H), 8.02 (s,
1H), 7.16–7.19 (m, 1H), 6.76–6.89 (m, 2H), 5.85–8.97
(m, 1H), 3.94 (s, 3H), 2.33–2.36 (m, 2H), 2.05–2.13
(m, 2H), 1.90–1.98 (m, 2H), 1.71–1.75 (m, 2H). MS found
(M + H)+ (m/z), 380.20;
calcd for C20H18FN5O2m/z, 379.14.
Starting
from 6i and 2-aminopyridine, 48% of 7m was
obtained according to the method described for the synthesis of 7; mp 284–286 °C. 1H NMR (CDCl3, 300 MHz) δ 8.74 (s, 1H), 8.42–8.43 (m, 1H),
8.29–8.32 (m, 1H), 8.08 (s, 1H), 7.75–7.81 (m, 1H),
7.07–7.12 (m, 1H), 5.87–5.93 (m, 1H), 2.30–2.33
(m, 2H), 2.11–2.14 (m, 2H), 1.92–1.98 (m, 2H), 1.68–1.74
(m, 2H). MS found (M + H)+ (m/z), 333.20; calcd for C18H16N6O m/z, 332.14.
Starting
from 6i and 2-amino-4-cyanopyridine, 40% of 7n was obtained according to the method described for the synthesis
of 7; mp 285–287 °C. 1H NMR (DMSO-d6, 300 MHz) δ 11.29 (s, 1H), 8.96 (s,
1H), 8.65 (s, 1H), 8.59–8.61 (m, 1H), 8.49 (s, 1H), 7.54–7.56
(m, 1H), 5.72–5.80 (m, 1H), 2.18–2.25 (m, 2H), 1.83–1.93
(m, 4H), 1.56–1.68 (m, 2H). MS found (M + H)+ (m/z), 358.20; calcd for C19H15N7O m/z, 357.13.
Starting
from 6i and 4-aminoindole, 40% of 7o was
obtained according to the method described for the synthesis of 7; mp 320–322 °C. 1H NMR (CDCl3, 300 MHz) δ 11.15 (bs, 1H), 10.36 (bs, 1H), 9.80 (s,
1H), 8.53 (s, 1H), 7.05–7.29 (m, 4H), 6.50 (bs, 1H), 5.62 (bs,
1H), 2.06 (bs, 2H), 1.35–1.60 (m, 6H). MS found (M + H)+ (m/z), 371.30; calcd for
C21H18N6O m/z, 370.15.
Starting
from 6i and 5-aminoindole, 42% of 7p was
obtained according to the method described for the synthesis of 7; mp 192–193 °C. 1H NMR (DMSO-d6, 300 MHz) δ 11.09 (bs, 1H), 10.57 (bs,
1H), 8.80 (s, 1H), 8.51 (s, 1H), 7.10–7.37 (m, 4H), 6.36 (s,
1H), 5.73–5.87 (m, 1H), 2.18–2.28 (m, 2H), 1.70–1.89
(m, 4H), 1.45–1.57 (m, 2H). MS found (M + H)+ (m/z), 371.20; calcd for C21H18N6O m/z, 370.15.
Starting
from 6i and 4-morpholin-4-yl-phenylamine, 53% of 7t was obtained according to the method described for the
synthesis of 7; mp 294–296 °C. 1H NMR (CDCl3, 300 MHz) δ 8.55 (s, 1H), 7.98 (s,
1H), 7.43–7.48 (m, 2H), 6.93–6.99 (m, 2H), 5.82–5.89
(m, 1H), 3.87–3.92 (m, 4H), 3.15–3.22 (m, 4H), 2.22–2.31
(m, 2H), 1.80–1.91 (m, 4H), 1.59–1.68 (m, 2H). MS found
(M + H)+ (m/z), 417.10;
calcd for C23H24N6O2m/z, 416.20.
Starting from 6i and 5-morpholin-4-yl-pyridin-2-ylamine,
40% of 7u was obtained according to the method described
for the synthesis of 7; mp 240–242 °C. 1H NMR (DMSO-d6, 300 MHz) δ
10.30 (bs, 1H), 8.57 (s, 1H), 8.31 (s, 1H), 7.87–7.87 (m, 1H),
7.84–7.88 (m, 1H), 6.71 (d, 1H), 5.80 (bs, 1H), 3.88–3.84
(m, 4H), 3.55–3.52 (m, 4H), 2.22–2.06 (m, 2H), 1.63–1.29
(m, 6H). MS found (M + H)+ (m/z), 418.20; calcd for C22H23N7O2m/z, 417.19.
Starting
from 6a and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 45% of 7y was obtained according to the method
described for the synthesis of 7; mp >300 °C. 1H NMR (DMSO-d6, 300 MHz) δ
13.10 (bs, 1H), 9.03 (s, 1H), 8.82 (s, 1H), 7.85–7.82 (m, 2H),
7.55–7.53 (m, 2H), 3.88–3.86 (m, 4H), 2.97–2.95
(m, 4H), 2.47 (s, 3H). MS found (M + H)+ (m/z), 362.20; calcd for C19H19N7O m/z, 361.17.
Starting from 6b and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 55% of 7z was obtained according to the method
described for the synthesis of 7; mp 211–212 °C. 1H NMR (DMSO-d6, 300 MHz) δ
8.57 (s, 1H), 8.04 (s, 1H), 7.89 (bs, 1H), 7.58–7.54 (m, 2H),
7.12–7.09 (m, 2H), 3.30–3.24 (m, 4H), 2.65–2.61
(m, 4H), 2.65 (s, 3H), 2.44 (s, 3H). MS found (M + H)+ (m/z), 376.20; calcd for C20H21N7O m/z, 375.18.
Starting from 6c and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 60% of 7aa was obtained according to the
method described for the synthesis of 7; mp 262–264
°C. 1H NMR (DMSO-d6, 300
MHz) δ 8.58 (s, 1H), 8.01 (s, 1H), 7.71 (bs, 1H), 7.53–7.50
(m, 2H), 6.99–6.96 (m, 2H), 4.44 (q, 2H), 3.27–3.24
(m, 4H), 2.67–2.63 (m, 4H), 2.40 (s, 3H), 1.36 (t, 3H). MS
found (M + H)+ (m/z),
390.20; calcd for C21H23N7O m/z, 389.20.
Starting from 6d and 4-(4-methyl-piperzin-1-yl)-phenylamine 21, 52% of 7ab was obtained according to the
method described for the synthesis of 7; mp 291–293
°C. 1H NMR (CDCl3, 300 MHz) δ 8.56
(s, 1H), 8.07 (s, 1H), 7.51–7.57 (m, 2H), 6.93–6.99
(2H), 4.30 (t, 2H), 3.21–3.25 (m, 4H), 2.61–2.63 (m,
4H), 2.38 (s, 3H), 1.73–1.78 (m, 2H), 1.02 (t, 3H). MS found
(M + H)+ (m/z), 404.21;
calcd for C22H25N7O m/z, 403.21.
Starting from 6e and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 65% of 7ac was obtained according to the
method described for the synthesis of 7; mp 296–298
°C. 1H NMR (CDCl3, 300 MHz) δ 8.56
(s, 1H), 7.96 (s, 1H), 7.47–7.44 (m, 2H,), 6.99–6.96
(m, 2H), 5.78–5.71 (m, 1H), 3.27–3.24 (bs, 4H), 2.66–2.62
(m, 4H), 2.40 (s, 3H), 1.60–1.57 (m, 6H). MS found (M + H)+ (m/z), 404.30; calcd for
C22H25N7O m/z, 403.21.
Starting from 6h and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 55% of 7af was obtained according to the
method described for the synthesis of 7; mp 285–287
°C. 1H NMR (DMSO-d6, 300
MHz) δ 10.44 (bs, 1H), 8.74 (s, 1H), 8.50 (s, 1H), 7.84–7.82
(m, 2H,), 6.98–6.95 (m, 2H), 3.36–3.34 (bs, 4H), 3.15–3.12
(m, 4H), 2.91–2.85 (m, 1H), 2.29 (s, 3H), 1.25 (bs, 2H), 0.86
(bs, 2H). MS found (M + H)+ (m/z), 402.30; calcd for C22H23N7O m/z, 401.20.
Starting from 6j and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 52% of 7ag was obtained according to the
method described for the synthesis of 7; mp 279–281
°C. 1H NMR (CDCl3, 300 MHz) δ 8.53
(s, 1H), 7.94 (s, 1H), 7.45–7.66 (m, 2H), 6.95–6.98
(m, 2H), 5.43–5.47 (m, 1H), 3.22–3.26 (m, 4H), 2.62–2.65
(m, 4H), 2.39 (s, 3H), 1.88 (bs, 2H), 1.64 (bs, 4H), 1.33 (bs, 4H).
MS found (M + H)+ (m/z), 444.30; calcd for C25H29N7O m/z, 443.24.
The compound 7x (1 g, 2.3
mmol) was taken into acetic anhydride and stirred at 120 °C temperature
for 3 h. The reaction mixture was cooled to room temperature, and
the crude product was filtered. The pure product 7aq was
obtained by flash chromatography with 2% methanol in chloroform. Yield
64%; light orange solid, mp 223–224 °C. 1H
NMR (CDCl3, 300 MHz) δ 8.88 (s, 1H), 8.11 (s, 1H),
7.12–7.09 (m, 2H), 7.01–6.97 (m, 2H), 5.61–5.55
(m, 1H), 3.30–3.26 (m, 4H), 2.62–2.59 (m, 4H), 2.42
(s, 3H), 2.38 (s, 3H), 2.11–2.04 (m, 2H), 1.72–1.68
(m, 4H), 1.54–1.51 (m, 2H). MS found (M + H)+ (m/z), 472.30; calcd for C26H29N7O2m/z, 471.24.
The compound 7x (1 g, 2.3
mmol) was taken into DMF and NaH was added at room temperature. After
10 min, 4-trifluoromethyl benzoyl chloride (0.58 g, 2.8 mmol) was
added and stirring continued for about 1 h. The reaction mixture was
quenched with water and filter off the crude product, and it was purified
with column chromatography by using 1–2% methanol in dichloromethane
as eluents. Yield 62%; brown solid, mp 155–156 °C. 1H NMR (CDCl3, 300 MHz) δ 8.60 (s, 1H), 7.97
(s, 1H), 7.72 (d, 2H), 7.56 (d, 2H), 7.07–7.04 (m, 2H), 6.91–6.88
(m, 2H), 5.01–4.95 (m, 1H), 3.21–3.18 (m, 4H), 2.52–2.48
(m, 4H), 2.29 (s, 3H), 1.87–1.85 (m, 2H), 1.69–1.68
(m, 2H), 1.35–1.33 (m, 4H). MS found (M + H)+ (m/z), 602.30; calcd for C32H30F3N7O2m/z, 601.24.
Biology: Materials and
Methods
Cell lines were purchased
from ATCC and were maintained in DMEM or RPM1 (CellGro) supplemented
with 10% fetal bovine serum (Cellgeneration, CO) and 1 unit/mL penicillin–streptomycin
(Invitrogen) at 37 °C under humidified conditions.
Cytotoxicity
Assays
Cells were seeded at a cell density
of 1.5 × 103 cells/0.1 mL/well in a 96-well plate.
The compounds were added 24 h postplating at the indicated concentrations.
Cell counts were determined from duplicate wells 96 h post-treatment.
The total number of viable cells was determined using the Cell Titer
Blue assay (Promega, WI) in conjunction with the GloMax plate reader
(Promega, WI).
Kinase Assays and IC50 Determination
First,
10 ng of recombinant CDK4/cyclin D1 (Life Technologies PV4204) was
diluted in kinase buffer (20 mM Tris pH 7.5, 10 mM MgCl2, 0.01% NP-40, 2 mM DTT) and incubated with the indicated concentration
of inhibitor at room temperature for 30 min. Kinase reactions were
initiated by the addition of 1 μg (1.5 μM) of recombinant
Rb protein, 5 μM ATP, and 10 μCi γ-32P-ATP. The reactions were incubated at 30 °C for 20 min, terminated
by the addition of 2× Laemmli sample buffer, heated at 95 °C
for 3 min, resolved using 12% acrylamideSDS-PAGE, and subjected to
autoradiography. The autoradiograms were scanned, and the band corresponding
to the phosphorylated protein substrate was quantitated using a densitometer
(Bio-Rad). The densitometric values obtained were plotted as a function
of log drug concentration using Prism 4 Graphpad software and IC50 values determined by plotting sigmoidal nonlinear regression
curves with a variable slope.
Flow Cytometry
MCF-7 (human estrogen positive breast
carcinoma) and MDA-MB-231 (human triple negative breast carcinoma)
cells were plated onto 100 mm2 dishes at a cell density
of 1.0 × 106 cells/dish. All cells were treated with
increasing concentrations of the indicated compounds 24 h postplating.
Both nonadherent cells (floating) and adherent cells were harvested
24 h post-treatment, washed in phosphate buffered saline (PBS), and
fixed in ice cold 70% ethanol for at least 24 h. The fixed cells were
then washed with room temperature PBS and stained with propidium iodide
(50 mg/mL) in the presence of RNase A (0.5 mg) for 30 min at 37 °C.
The stained cells were then analyzed using a FACSCAN (BD Biosciences)
and the resulting data analyzed with cell cycle analysis software
(Modfit, BD).
Western Blot Analysis
Cells were
treated with increasing
concentrations of compound and harvested 24 h post-treatment. All
cell pellets were frozen on dry ice before lysis. Cells were lysed
in lysis buffer ((50 mM Tris-HCl, 0.1% Triton-X100, 250 mM NaCl, 5
mM EDTA, 50 mM NaF, 0.1 mM sodium orthovanadate (pH 7.4), and protease
inhibitors), and 100 μg of clarified lysates were resolved by
10%-SDS-polyacrylamide gel electrophoresis. The resolved proteins
were transferred onto nitrocellulose filter paper and hybridized with
the following antibodies: phosphospecific Rb (Cell Signaling; catalogue
no. 9307), Rb (Cell Signaling; catalogue no. 9309), AKT (Cell Signaling,
catalogue no. 4691), phosphospecific AKTSer473 (Cell Signaling; catalogue
no. 9271), and PARP (BD Biosciences; catalogue no. 556362). Lysates
used for Western blot analysis to detect cleaved PARP were obtained
as above except that the cells were lysed in 1% NP40/PBS lysis buffer
containing protease inhibitors. Following hybridization with primary
antibodies, the blots were washed, treated with secondary antibodies
conjugated to infrared dyes (IRDye 800 or IRDye 680) and analyzed
on an infrared scanning system (Odyssey, Li-Cor Biosciences, NE) according
to the manufacturer’s instructions.
Orthotopic Nude Mouse Assay
MDA-MB-231 triple negative
breast cancer cells (1 × 106) were injected bilaterally
in the mammary fat pads of 7–8 week old female athymic nude
mice (NCR nu/nu, Taconic, NY). Once the tumors grew to a volume of
approximately 100 mm3, they were placed into two treatment
groups (n = 6, with a total tumor number of 11).
The mice were treated daily for 15 days (QD × 15), a dose of
100 mg/kg (0.1 mL, intraperitoneally), or placebo (sterile PBS). Body
weights and tumor size were determined every other day. Tumor measurements
were used using a digital vernier caliper, and the volumes were determined
using the following calculation: (short2) × long ×
0.5. Experiments were performed under an approved IACUC protocol according
to federal and institutional guidelines and regulations.
Statistical
Analysis
Statistical analysis was performed
using a standard, unpaired, two-tailed Student’s t test. Data are graphed as mean ± SEM.
Model of 7x Binding to CDK6
Small molecule 7x binding
was predicted by docking and energy minimization
using the X-ray crystal structure of CDK6–Vcyclin–PD-0332991
(2EUF) as a reference. Representations of the superimposition of X-ray
crystal structure (CDK6/PD-0332991) and predicted lowest energy binding
(CDK6/7x) were prepared using PyMOL (Figure 2). Figure 2A, ribbon representation
of CDK6 (green) bound to PD-0332991 (red) and 7x (cyan).
Small molecules are shown as sticks. Figure 2B,C, closeup view showing proximal residues of CDK6 to 7x (blue) and PD-0332991 (pink), respectively. Hydrogen bonds are shown
as a dotted back lines.
Authors: John P Leonard; Ann S LaCasce; Mitchell R Smith; Ariela Noy; Lucian R Chirieac; Scott J Rodig; Jian Q Yu; Shankar Vallabhajosula; Heiko Schoder; Patricia English; Donna S Neuberg; Peter Martin; Michael M Millenson; Scott A Ely; Rachel Courtney; Naveed Shaik; Keith D Wilner; Sophia Randolph; Annick D Van den Abbeele; Selina Y Chen-Kiang; Jeffrey T Yap; Geoffrey I Shapiro Journal: Blood Date: 2012-03-01 Impact factor: 22.113
Authors: M V Ramana Reddy; Muralidhar Reddy Mallireddigari; Venkat R Pallela; Padmavathi Venkatapuram; Rengasamy Boominathan; Stanley C Bell; E Premkumar Reddy Journal: Bioorg Med Chem Date: 2005-03-01 Impact factor: 3.641
Authors: E Verdaguer; E G Jordà; A M Canudas; A Jiménez; F X Sureda; V Rimbau; D Pubill; E Escubedo; J Camarasa; M Pallàs; A Camins Journal: Neuroscience Date: 2003 Impact factor: 3.590
Authors: Guoxin Zhu; Scott E Conner; Xun Zhou; Chuan Shih; Tiechao Li; Bryan D Anderson; Harold B Brooks; Robert Morris Campbell; Eileen Considine; Jack A Dempsey; Margaret M Faul; Cathy Ogg; Bharvin Patel; Richard M Schultz; Charles D Spencer; Beverly Teicher; Scott A Watkins Journal: J Med Chem Date: 2003-05-22 Impact factor: 7.446
Authors: T Wölfel; M Hauer; J Schneider; M Serrano; C Wölfel; E Klehmann-Hieb; E De Plaen; T Hankeln; K H Meyer zum Büschenfelde; D Beach Journal: Science Date: 1995-09-01 Impact factor: 47.728
Authors: Raghupathi Neelarapu; Jordany R Maignan; Cynthia L Lichorowic; Andrii Monastyrskyi; Tina S Mutka; Alexis N LaCrue; Lynn D Blake; Debora Casandra; Sherwin Mashkouri; Jeremy N Burrows; Paul A Willis; Dennis E Kyle; Roman Manetsch Journal: J Med Chem Date: 2018-02-09 Impact factor: 7.446
Authors: M V Ramana Reddy; Balireddy Akula; Shashidhar Jatiani; Rodrigo Vasquez-Del Carpio; Vinay K Billa; Muralidhar R Mallireddigari; Stephen C Cosenza; D R C Venkata Subbaiah; E Vijaya Bharathi; Venkat R Pallela; Poornima Ramkumar; Rinku Jain; Aneel K Aggarwal; E Premkumar Reddy Journal: Bioorg Med Chem Date: 2015-12-01 Impact factor: 3.641
Authors: T Narita; A Inagaki; T Kobayashi; Y Kuroda; T Fukushima; M Nezu; S Fuchida; H Sakai; N Sekiguchi; I Sugiura; Y Maeda; H Takamatsu; N Tsukamoto; D Maruyama; Y Kubota; M Kojima; K Sunami; T Ono; M Ri; K Tobinai; S Iida Journal: Blood Cancer J Date: 2015-02-27 Impact factor: 11.037
Authors: S K A Divakar; M V Ramana Reddy; S C Cosenza; S J Baker; D Perumal; A C Antonelli; J Brody; B Akula; S Parekh; E Premkumar Reddy Journal: Leukemia Date: 2015-07-15 Impact factor: 11.528
Chart 1
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Scheme 5
Scheme 6
Scheme 7
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7