Discovery of 8-Cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8- dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7x) as a Potent Inhibitor of Cyclin- Dependent Kinase 4 (CDK4) and AMPK-related Kinase 5 (ARK5)

Abstract

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 multi kinase 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 30–100nM. In vitro kinase profiling revealed that 7x is a multi-kinase 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.

INTRODUCTION

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 human tumors exhibit deregulation of the CDK4/6-cyclin D-RB pathway.^1,9,10 For example, CDK4/6 is hyperactivated in a number of human cancers 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 CDK4^R24C 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 non-toxic 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 non-selective 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 towards 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 human tumor 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 Based on 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 evalueated 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-RB hyperactivity.

Chemistry

To generate an ATP-competitive kinase inhibitor library, we used pyrido[2,3-d]pyrimidines (7) as the back bone since 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)^a.

^aReagents and conditions (i) X-NH₂, Et₃N, THF, rt, 1–3 h, 80–95%; (ii) LiAlH₄, THF, −10 ^oC - 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 ^oC, 6 h, 62–73%; (v) m-CPBA, CH₂Cl₂, rt, 3 h, 87–94%; (vi) Z-H, Toluene, 100 ^oC, 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–j. The ester group in compounds 2a–j was then reduced with lithium aluminum hydride (LiAlH₄) to obtain the corresponding alcohols 3a–j, which when further oxidized with manganese dioxide (MnO₂), yielded the corresponding pyrimidine carbaldehyde 4a–j. The aldehyde 4a–j were converted to pyridopyrimidines 5a–s 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–s. 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 C-2 position with substituted aryl/heteroaryl amines, it was oxidized to methyl sulfoxides 6a–s 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–j 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 ⁰C 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 (5bj) from 2-methylsulfanyl-7-oxo-7,8-dihydro[2,3-d]pyrimidine-6-carbonitrile (5a)^a.

^aReagents and conditions: (i) X-I, NaH, DMF, 50 ^oC, 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)^a.

^aReagents and conditions: (i) AC₂O, 120 ^oC, 3 h, 64%; (ii) 4-CF₃PhCOCl, NaH, DMF, rt, 3 h, 62%.

The active methylene compounds, arylmethanesulfonylacetic acids (10a–c), 4-chlorophenylsulfonylacetic acid (13) and N-arylmalonamic acids (16a–b) were prepared as shown in Schemes 4, 5 and 6 as per the reported procedures.^49–51

Scheme 4. Synthesis of Arylmethanesulfonyl acetic acids^a.

^aReagents 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^a.

^aReagents 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^a.

^aReagents and conditions: (i) ClCOCH₂CO₂Me, Et₃N, DCM, rt, 3 h, 90%; (ii) 10% NaOH, HCl, rt, 1 h, 78–80%.

Arylmethyl bromides (8a–c) were treated with thioglycolic acid in the presence of sodium hydroxide to produce corresponding arylmethylsulfanyl acetic acids (9a–c). De-protection of tert-butyldimethylsilyl ether (TBDMS) group in 9c with tetrabutylammonium fluoride (TBAF) yielded 9d.^49a The oxidation of 9a–b and 9d with 30% H₂O₂ in acetic acid yielded the corresponding arylmethylsulfonyl acetic acids (10a–c) (Scheme 4).

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.

The reaction of aromatic amines (14a–b) with methyl-3-chloro-3-oxopropionate in the presence of triethylamine generated 3-anilino-3-oxopropionic acid methyl esters (15a–b), which, when subjected to hydrolysis, resulted in the formation of 3-anilino-3-oxopropionic acids (16a–b) (Scheme 6).

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^a.

^aReagents and conditions: (i) N-methylpiperazine, acetonitrile, 100 ⁰C, 5 h, 70%; (ii) N-methylpiperazine, tetra-n-butyl ammonium iodide, DMSO, 80 ⁰C, overnight, 65%; (iii) 10% Pd/C, NH₂NH₂.H₂O, rt, 4 h, 75%.

Structure Activity Relationships (SARs)

It is evident from Table 1 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 Table 1 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) 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 pyridylpiperazine (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.

Compd.	Z	IC50 (μM)
		K562	DU145
7a		100	75
7b		30	30
7c		75	5
7d		15	15
7e		15	30
7f		5	0.5
7g		75	25
7h		15	15
7i		2	5
7j		15	15
7k		2.5	5
7l		75	75
7m		0.5	3
7n		5	30
7o		30	15
7p		5	5
7q		5	5
7r		1	1
7s		0.25	3
7t		5	15
7u		5	15
7v		5	15
7w		30	100
7x		0.05	0.025
PD0332991 (Palbociclib)	-	2	7.5

Table 4.

In vitro cytotoxicity of 7aq and 7ar compounds.

Compd.	R	IC50 (μM)
		K562	DU145
7aq		0.75	5
7ar		0.5	1
7x	H	0.05	0.025

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.

Compd.	X	IC50 (μM)
		K562	DU145
7y	H	30	30
7z		75	75
7aa		0.75	1.5
7ab		5	15
7ac		2.5	2.5
7ad		0.75	0.75
7ae		0.75	1.5
7af		2.5	2.5
7ag		1.5	50
7x		0.05	0.025

Table 3.

In vitro cytotoxicity of pyrido[2,3-d]pyrimidines (7ah – 7ap) with variables at C-6- position.

Compd.	Y	IC50 (μM)
		K562	DU145
7ah		0.75	2.5
7ai		5	5
7aj		30	75
7ak		5	15
7al		10	75
7am		2	2
7an		2	5
7ao		5	30
7ap		15	30
7x	CN	0.05	0.025

Biological Results and Discussion

In vitro anti-tumor activity of compound 7x

We next tested the cyototoxic activity of the most active compound (7x) against a panel of human tumor 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 Supplemental 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 over-expression 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 the supplemental Table 2. Interestingly, this study revealed that 7x is a multi-kinase inhibitor, with the highest inhibitory activity against CDK4, CDK6, ARK5, FGFR1, PDGFRs and PI3K-δ, all of which are intimately associated with the growth, survival and metastasis in human tumor 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 nM, 50–100 nM, 100–150 nM 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	>10,000
FYN	11.09	>10,000
FMS	10.00	>10,000
PDGFRs	26.00	>10,000
FGFR1	26.00	>10,000
ABL	53.32	>10,000
PI3K-δ	144	>10,000

Molecular Modeling of 7x

Docking prediction of 7x to CDK6 suggest different binding than PD-0332991

In order to explain the difference in inhibitor potency between 7x and PD0332991, binding of 7x to the kinase domain of CDK6 was predicted by molecular docking and energy minimization based upon the X-ray co-crystal 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 co-crystal 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 co-crystal 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-PD0332991 (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 stick. (B and 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 A that may be involved in Van der Waals interactions that aid in the stabilization of the small molecule. It is worth to emphasize 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 supplemental). Results of these docking studies show a great variability of binding between kinases and since 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 human breast 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 (5RM ATP, 10Rci γ^32P-ATP, 10 mM MgCl₂ and 1Rg recombinant RB substrate) and incubated at 30 ^oC for 20 min. The reactions were terminated by the addition of 2X Laemmli sample buffer and heated at 95 ^oC 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 non-phosphorylated 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.

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.

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 supplemental 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 non-phosphorylated 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. 100% 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 sub-cutaneous 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 communication, we describe the synthesis of pyrido[2,3-d]pyrimidine analogs that induce apoptotic death of a wide variety of human tumor 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 introductory section, 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 human tumors. However, clinical trials with PD-0332991 suggest that reduction of tumor burden in human tumors that have increased levels 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 multi-targeted 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 human cancers that over-express 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 A. Yields were of purified product and were not optimized. Melting points were determined using an Electro thermal Mel-Temp 3.0 micro melting 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 to m/z 1000. The purity of the final compounds was determined by HPLC and is 95% or higher unless specified otherwise. Zorbax Exlipse XDB C18 (150 × 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 16⁵¹ 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.

4-Amino-2-methylsulfanyl-pyrimidine-5-carboxylic acid ethyl ester (2a)

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 ⁰C; 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.

4-Methylamino-2-methylsulfanyl-pyrimidine-5-carboxylic acid ethyl ester (2b)

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 ⁰C; 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.

4-Ethylamino-2-methylsulfanyl-pyrimidine-5-carboxylic acid ethyl ester (2c)

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.

2-Methylsulfanyl-4-propylmino-pyrimidine-5-carboxylic acid ethyl ester (2d)

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.

4-Isopropylamino-2-methylsulfanyl-pyrimidine-5-carboxylic acid ethyl ester (2e)

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.

4-Butylamino-2-methylsulfanyl-pyrimidine-5-carboxylic acid ethyl ester (2f)

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.

2-Methylsulfanyl-4-pentylamino-pyrimidine-5-carboxylic acid ethyl ester (2g)

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.

4-Cyclopropylamino-2-methylsulfanyl-pyrimidine-5-carboxylic acid ethyl ester (2h)

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.

4-Cyclopentylamino-2-methylsulfanyl-pyrimidine-5-carboxylic acid ethyl ester (2i)

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.

4-Cyclohexylamino-2-methylsulfanyl-pyrimidine-5-carboxylic acid ethyl ester (2j)

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 drop wise to the cooled LiAlH₄ solution while keeping the reaction temperature bellow −10 ⁰C. 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.

(4-Amino-2-methylsulfanyl-pyridine-5-yl)-methanol (3a)

Starting from 2a, 85% of 3a was obtained according to method described for the synthesis of 3. mp 157–159 ⁰C; 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.

(4-Methylamino-2-methylsulfanyl-pyridine-5-yl)-methanol (3b)

Starting from 2b, 80% of 3b was obtained according to method described for the synthesis of 3. mp 145–146 ⁰C; 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.

(4-Ethylamino-2-methylsulfanyl-pyridine-5-yl)-methanol (3c)

Starting from 2c, 84% of 3c was obtained according to method described for the synthesis of 3. mp 155–157 ⁰C; 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.

(2-Methylsulfanyl-4-propylamino-pyridine-5-yl)-methanol (3d)

Starting from 2d, 83% of 3d was obtained according to method described for the synthesis of 3. mp 120–121; 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.

(4-Isopropylamino-2-methylsulfanyl-pyridine-5-yl)-methanol (3e)

Starting from 2e, 85% of 3e was obtained according to method described for the synthesis of 3. mp 127–129 ⁰C; 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.

(4-Butylamino-2-methylsulfanyl-pyridine-5-yl)-methanol (3f)

Starting from 2f, 83% of 3f was obtained according to method described for the synthesis of 3. mp 105–107 ⁰C; 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.

(2-Methylsulfanyl-4-pentylamino-pyridine-5-yl)-methanol (3g)

Starting from 2g, 86% of 3g was obtained according to method described for the synthesis of 3. mp 110–112 ⁰C; 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.

(4-Cyclopropylmino-2-methylsulfanyl-pyridine-5-yl)-methanol (3h)

Starting from 2h, 82% of 3h was obtained according to method described for the synthesis of 3. mp 135–137 ⁰C; 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.

(4-Cyclopentylamino-2-methylsulfanyl-pyridine-5-yl)-methanol (3i)

Starting from 2i, 84% of 3i was obtained according to method described for the synthesis of 3. mp 150–151 ⁰C; 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.

(4-Cyclohexylamino-2-methylsulfanyl-pyridine-5-yl)-methanol (3j)

Starting from 2j, 84% of 3j was obtained according to method described for the synthesis of 3. mp 175–177 ⁰C; 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 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.

4-Amino-2-methylsulfanyl-pyrimidine-5-carbaldehyde (4a)

Starting from 3a, 72% of 4a was obtained according to the procedure described for the synthesis of 4. mp 186–188 ⁰C; 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.

4-Methylamino-2-methylsulfanyl-pyrimidine-5-carbaldehyde (4b)

Starting from 3b, 75% of 4b was obtained according to the procedure described for the synthesis of 4. mp 98–99 ⁰C; 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.

4-Ethylamino-2-methylsulfanyl-pyrimidine-5-carbaldehyde (4c)

Starting from 3c, 72% of 4c was obtained according to the procedure described for the synthesis of 4. mp 60–61 ⁰C; 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.

2-Methylsulfanyl-4-propylmino-pyrimidine-5-carbaldehyde (4d)

Starting from 3d, 70% of 4d was obtained according to the procedure described for the synthesis of 4. mp 50–51 ⁰C; 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.

4-Isopropylamino-2-methylsulfanyl-pyrimidine-5-carbaldehyde (4e)

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 ⁰C; 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.

4-Butylamino-2-methylsulfanyl-pyrimidine-5-carbaldehyde (4f)

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.

2-Methylsulfanyl-4-pentylmino-pyrimidine-5-carbaldehyde (4g)

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.

4-Cyclopropylamino-2-methylsulfanyl-pyrimidine-5-carbaldehyde (4h)

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 ⁰C; 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.

4-Cyclopentylamino-2-methylsulfanyl-pyrimidine-5-carbaldehyde (4i)

Starting from 3i, 87% of 4i was obtained as solid according to the procedure described for the synthesis of 4. mp 41–42 ⁰C; 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.

4-Cyclohexylamino-2-methylsulfanyl-pyrimidine-5-carbaldehyde (4j)

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)

Mixture of 4-alkylamino-2-methylsulfanyl-pyrimidine-5-carbaldehyde 4 (4.2 mmol), 1.2 equivalent of active methylene compound and 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, 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, water and dried over vacuo. The crude product was recrystallized in 2-propanol to get pure product (5).

2-Methylsulfanyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (5a)

Starting from 4a and cyanoacetic acid, 65% of 5a was obtained according to the method described for the synthesis of 5. mp 328–330 ⁰C; 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.

8-Methyl-2-methylsulfanyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (5b)

Starting from 4b and cyanoacetic acid, 67% of 5b was obtained according to the method described for the synthesis of 5. mp 292–294 ⁰C; 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.

8-Ethyl-2-methylsulfanyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (5c)

Starting from 4c and cyanoacetic acid, 70% of 5c was obtained according to the method described for the synthesis of 5. mp 244–245 ⁰C; 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.

2-Methylsulfanyl-7-oxo-8-propyl-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (5d)

Starting from 4d and cyanoacetic acid, 70% of 5d was obtained according to the method described for the synthesis of 5. mp 230–231 ⁰C; 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.

8-Isopropyl-2-methylsulfanyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (5e)

Starting from 4e and cyanoacetic acid, 70% of 5e was obtained according to the method described for the synthesis of 5. mp 200–202 ⁰C; 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.

8-Butyl-2-methylsulfanyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (5f)

Starting from 4f and cyanoacetic acid, 70% of 5f was obtained according to the method described for the synthesis of 5. mp 220–222 ⁰C; 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.

2-Methylsulfanyl-7-oxo-8-pentyl-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (5g)

Starting from 4g and cyanoacetic acid, 70% of 5g was obtained according to the method described for the synthesis of 5. mp 160–161 ⁰C; 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.

8-Cyclopropyl-2-methylsulfanyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (5h)

Starting from 4h and cyanoacetic acid, 71% of 5h was obtained according to the method described for the synthesis of 5. mp 158–159 ⁰C; 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.

8-Cyclopentyl-2-methylsulfanyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (5i)

Starting from 4i and cyanoacetic acid, 71% of 5i was obtained according to the method described for the synthesis of 5. mp 209–210 ⁰C; 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.

8-Cyclohexyl-2-methylsulfanyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (5j)

Starting from 4j and cyanoacetic acid, 71% of 5j was obtained according to the method described for the synthesis of 5. mp 239–240 ⁰C; 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.

8-Cyclopentyl-2-methylsulfanyl-6-nitro-8H-pyrido[2,3-d]pyrimidin-7-one (5k)

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 ⁰C; 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.

8-Cyclopentyl-6-methanesulfonyl-2-methylsulfanyl-8H-pyrido[2,3-d]pyrimidin-7-one (5l)

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 ⁰C; 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 C₁₄H₁₇N₃O₃S₂ m/z: 339.07.

6-Benzenesulfonyl-8-cyclopentyl-2-methylsulfanyl-8H-pyrido[2,3-d]pyrimidin-7-one (5m)

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 ⁰C; 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 C₁₉H₁₉N₃O₃S₂ m/z: 401.09.

6-(4-Chloro-benzenesulfonyl)-8-cyclopentyl-2-methylsulfanyl-8H-pyrido[2,3-d]pyrimidin-7-one (5n)

Starting from 4i and 4-chlorobenzene sulfonyl acetic acid 13, 70% of 5n was obtained according to the method described for the synthesis of 5. mp 222–223 ⁰C; 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 C₁₉H₁₈ClN₃O₃S₂ m/z: 435.05.

6-(4-Chloro-phenylmethanesulfonyl)-8-cyclopentyl-2-methylsulfanyl-8H-pyrido[2,3-d]pyrimidin-7-one (5o)

Starting from 4i and 4-chloro-phenylmethane sulfonyl acetic acid 10a, 67% of 5o was obtained according to the method described for the synthesis of 5. mp 250–252 ⁰C; ¹H NMR (300 MHz, CDCl₃), δ 8.68 (s, 1H), 8.57 (s, 1H), 7.42 (d, 2H), 7.25 (d, 2H), 5.84–5.93 (m, 1H), 4.65 (s, 2H), 2.59 (s, 3H), 2.21–2.29 (m, 2H), 1.99–2.09 (m, 2H), 1.82–1.89 (m, 2H), 1.64–1.72 (m, 2H). MS found (M+H)⁺ (m/z): 450.10, Calcd for C₂₀H₂₀ClN₃O₃S₂ m/z: 449.06.

8-Cyclopentyl-6-(3-hydroxy-4-methoxy-phenylmethanesulfonyl)-2-methylsulfanyl-8Hpyrido[ 2,3-d]pyrimidin-7-one (5p)

Starting from 4i and 4-hydroxy-3-methoxy-phenylmethane sulfonyl acetic acid 10c, 65% of 5p was obtained according to the method described for the synthesis of 5. mp 180–181 ⁰C; 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 C₂₁H₂₃N₃O₅S₂ m/z: 461.11.

8-Cyclopentyl-6-(4-methoxy-3-nitro-phenylmethanesulfonyl)-2-methylsulfanyl-8H-pyrido [2,3-d]pyrimidin-7-one (5q)

Starting from 4i and 4-methoxy-3-nitro-phenylmethane sulfonyl acetic acid 10b, 62% of 5q was obtained according to the method described for the synthesis of 5. mp 200–201 ⁰C; ¹H NMR (300 MHz, CDCl₃), δ 8.68 (s, 1H), 8.27 (s, 1H), 7.85 (s, 1H), 7.80–7.82 (m, 1H), 7.58–7.63 (m, 1H), 5.90–6.09 (m, 1H), 4.82 (s, 2H), 3.90 (s, 3H), 2.67 (s, 3H), 2.32–2.43 (m, 2H), 2.09–2.20 (m, 2H), 1.88–2.00 (m, 2H), 1.69–1.77 (m, 2H). MS found (M+H)⁺ (m/z): 491.10, Calcd for C₂₁H₂₂N₄O₆S₂ m/z: 490.10.

8-Cyclopentyl-2-methylsulfanyl-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carboxylicacid(4-chloro-phenyl)-amide (5r)

Starting from 4i and N-(4-chloro-phenyl)-malonamic acid 16a, 65% of 5r was obtained according to the method described for the synthesis of 5. mp 260–262 ⁰C; ¹H NMR (300 MHz, CDCl₃), δ 11.71 (bs, 1H), 8.86 (s, 1H), 8.82 (s, 1H), 7.68–7.78 (m, 2H), 7.33–7.39 (m, 2H), 6.04–6.10 (m, 1H), 2.65 (s, 3H), 2.30–2.42 (m, 2H), 2.10–2.20 (m, 2H), 1.89–2.00 (m, 2H), 1.79–1.81 (m, 2H). MS found (M+H)⁺ (m/z): 415.10, Calcd for C₂₀H₁₉ClN₄O₂S m/z: 414.09.

8-Cyclopentyl-2-methylsulfanyl-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carboxylicacid (4-fluoro-phenyl)-amide (5s)

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 ⁰C; 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-j) 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 ⁰C for 10 min until to get the clear solution, and then NaH (5 mmol) was added and stirred about 5 min. The reaction mixture was brought to room temperature and was added alkyl iodides (6.5 mmol). 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, organic layer was dried over Na₂SO₄ and concentrated to produce the corresponding methylsuloxide 6 and was purified with flash chromatography.

2-Methylsulfinyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (6a)

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.

2-Methylsulfinyl-8-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (6b)

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.

8-Ethyl-2-methylsulfinyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (6c)

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.

2-Methylsulfinyl-7-oxo-8-propyl-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (6d)

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.

8-Isopropyl-2-methylsulfinyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (6e)

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.

8-Butyl-2-methylsulfinyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (6f)

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.

2-Methylsulfinyl-7-oxo-8-pentyl-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (6g)

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.

8-Cyclopropyl-2-methylsulfinyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (6h)

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.

8-Cyclopentyl-2-methylsulfinyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (6i)

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.

8-Cyclohexyl-2-methylsulfinyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine-6-carbonitrile (6j)

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.

8-Cyclopentyl-2-methylsulfinyl-6-nitro-8H-pyrido[2,3-d]pyrimidin-7-one (6k)

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.

8-Cyclopentyl-2-methylsulfinyl-6-methanesulfonyl-8H-pyrido[2,3-d]pyrimidin-7-one (6l)

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 C₁₄H₁₇N₃O₄S₂ m/z: 355.07.

6-Benzenesulfonyl-8-cyclopentyl-2-methylsulfinyl-8H-pyrido[2,3-d]pyrimidin-7-one (6m)

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 C₁₉H₁₉N₃O₄S₂ m/z: 417.08.

6-(4-Chloro-benzenesulfonyl)-8-cyclopentyl-2-methylsulfinyl-8H-pyrido[2,3-d]pyrimidin-7-one (6n)

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 C₁₉H₁₈ClN₃O₄S₂ m/z: 451.04.

6-(4-Chloro-phenylmethanesulfonyl)-8-cyclopentyl-2-methylsulfinyl-8H-pyrido[2,3-d]pyrimidin-7-one (6o)

Starting from 5o and, 3-chloro-benzenecarboperoxoic acid 92% of 6o was obtained according to the method described for the synthesis of 6. ¹H NMR (300 MHz, CDCl₃), δ 8.70 (s, 1H), 8.56 (s, 1H), 7.43 (d, 2H), 7.24 (d, 2H), 5.83–5.91 (m, 1H), 4.67 (s, 2H), 2.96 (s, 3H), 2.20–2.28 (m, 2H), 1.97–2.10 (m, 2H), 1.81–1.88 (m, 2H), 1.65–1.73 (m, 2H). MS found (M+H)⁺ (m/z): 466.10, Calcd for C₂₀H₂₀ClN₃O₄S₂ m/z: 465.06.

8-Cyclopentyl-6-(3-hydroxy-4-methoxy-phenylmethanesulfonyl)-2-methylsulfinyl-8Hpyrido[ 2,3-d]pyrimidin-7-one (6p)

Starting from 5p and, 3-chloro-benzenecarboperoxoic acid 91% of 6p was obtained according to the method described for the synthesis of 6. ¹H NMR (300 MHz, CDCl₃), δ 8.50 (s, 1H), 8.14 (s, 1H), 6.51–6.72 (m, 3H), 5.74–5.91 (m, 1H), 4.58 (s, 2H), 3.67 (s, 3H), 2.97 (s, 3H), 2.13–2.27 (m, 2H), 1.88–2.02 (m, 2H), 1.66–1.80 (m, 2H), 1.49–1.60 (m, 2H). MS found (M+H)⁺ (m/z): 478.10, Calcd for C₂₁H₂₃N₃O₆S₂ m/z: 477.10.

8-Cyclopentyl-2-methylsulfinyl-6-(4-methoxy-3-nitro-phenylmethanesulfonyl)-2-methylsulfanyl-8H-pyrido[2,3-d]pyrimidin-7-one (6q)

Starting from 5q and, 3-chloro-benzene-carboperoxoic acid 93% of 6q was obtained according to the method described for the synthesis of 6. ¹H NMR (300 MHz, CDCl₃), δ 8.67 (s, 1H), 8.30 (s, 1H), 7.83 (s, 1H), 7.81–7.83 (m, 1H), 7.59–7.63 (m, 1H), 5.93–6.09 (m, 1H), 4.85 (s, 2H), 3.90 (s, 3H), 2.96 (s, 3H), 2.30–2.42 (m, 2H), 2.08–2.18 (m, 2H), 1.89–2.01 (m, 2H), 1.67–1.78 (m, 2H). MS found (M+H)⁺ (m/z): 507.10, Calcd for C₂₁H₂₂N₄O₇S₂ m/z: 506.09.

8-Cyclopentyl-2-methylsulfinyl-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carboxylicacid(4-chloro-phenyl)-amide (6r)

Starting from 5r and, 3-chloro-benzenecarboperoxoic acid 88% of 6r was obtained according to the method described for the synthesis of 6. ¹H NMR (300 MHz, CDCl₃), δ 11.68 (bs, 1H), 8.85 (s, 1H), 8.80 (s, 1H), 7.69–7.77 (m, 2H), 7.35–7.38 (m, 2H), 6.06 (bs, 1H), 2.95 (s, 3H), 2.33–2.43 (m, 2H), 2.13–2.21 (m, 2H), 1.92–2.00 (m, 2H), 1.77–1.80 (m, 2H). MS found (M+H)⁺ (m/z): 431.10, Calcd for C₂₀H₁₉ClN₄O₃S m/z: 430.09.

8-Cyclopentyl-2-methylsulfinyl-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carboxylicacid(4-fluoro-phenyl)-amide (6s)

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 ⁰C 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.

2-Benzylamino-8-cyclopentyl-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7a)

Starting from 6i and benzylamine, 51% of 7a was obtained according to the method described for the synthesis of 7. mp 211–212 ⁰C; 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.

2-(4-Chloro-phenylamino)-8-cyclopentyl-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7b)

Starting from 6i and 4-chlorophenylamine, 50% of 7b was obtained according to the method described for the synthesis of 7. mp 272–273 ⁰C; 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.

2-(4-Cyano-phenylamino)-8-cyclopentyl-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7c)

Starting from 6i and 4-aminobenzonitrile, 55% of 7c was obtained according to the method described for the synthesis of 7. mp 285–287 ⁰C; 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.

8-Cyclopentyl-2-(2-hydroxy-phenylamino)-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimi-dine-6-carbonitrile (7d)

Starting from 6i and 2-aminophenol, 42% of 7d was obtained according to the method described for the synthesis of 7. mp 303–305 ⁰C; ¹H NMR (CDCl₃, 300 MHz): δ 9.69 (bs, 1H), 8.76 (s, 1H), 8.52 (s, 1H), 7.42–7.45 (m, 1H), 7.03–7.08 (m, 1H), 6.89–6.90 (m, 1H), 6.79–6.84 (m, 1H), 5.58–5.59 (m, 1H), 3.37 (bs, 1H), 2.14–2.21 (m, 2H), 1.59–1.65 (m, 4H), 1.39–1.43 (M, 2H). MS found (M+H)⁺ (m/z): 348.20, Calcd for C₁₉H₁₇N₅O₂ m/z: 347.14.

8-Cyclopentyl-2-(2-methoxy-phenylamino)-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7e)

Starting from 6i and 2-methoxyphenylamine, 54% of 7e was obtained according to the method described for the synthesis of 7. mp 159–160 ⁰C; 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 C₂₀H₁₉N₅O₂ m/z: 361.15.

8-Cyclopentyl-2-(3-methoxy-phenylamino)-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7f)

Starting from 6i and 3-methoxyphenylamine, 50% of 7f was obtained according to the method described for the synthesis of 7. mp 179–180 ⁰C; ¹H NMR (DMSO-d₆, 300 MHz): δ 8.61 (s, 1H), 8.00 (s, 1H), 7.56 (bs, 1H), 7.35–7.36 (m, 1H), 7.17–7.19 (m, 1H), 7.05–7.07 (m, 1H), 6.73–6.76 (m, 1H), 5.88–5.94 (m, 1H), 3.85 (s, 3H), 2.24–2.33 (m, 2H), 2.05 (bs, 2H), 1.83–1.90 (m, 2H), 1.62–1.67 (m, 2H). MS found (M+H)⁺ (m/z): 362.20, Calcd for C₂₀H₁₉N₅O₂ m/z: 361.15.

8-Cyclopentyl-2-(4-methoxy-phenylamino)-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7g)

Starting from 6i and 4-methoxyphenylamine, 54% of 7g was obtained according to the method described for the synthesis of 7. mp 238–239 ⁰C; 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 C₂₀H₁₉N₅O₂ m/z: 361.15.

8-Cyclopentyl-2-(2,4-dimethoxy-phenylamino)-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7h)

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 ⁰C; 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 C₂₁H₂₁N₅O₃ m/z: 391.16.

8-Cyclopentyl-2-(3,4-dimethoxy-phenylamino)-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7i)

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 ⁰C; 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 C₂₁H₂₁N₅O₃ m/z: 391.16.

8-Cyclopentyl-2-(3,5-dimethoxy-phenylamino)-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7j)

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 ⁰C; 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 C₂₁H₂₁N₅O₃ m/z: 391.16.

8-Cyclopentyl-2-(3,4,5-trimethoxy-phenylamino)-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7k)

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 ⁰C; 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 C₂₂H₂₃N₅O₄ m/z: 421.18.

8-Cyclopentyl-2-(5-fluoro-2-methoxy-phenylamino)-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7l)

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 ⁰C; 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 C₂₀H₁₈FN₅O₂ m/z: 379.14.

8-Cyclopentyl-7-oxo-2-(pyridin-2-ylamino)-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7m)

Starting from 6i and 2-aminopyridine, 48% of 7m was obtained according to the method described for the synthesis of 7. mp 284–286 ⁰C; 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.

2-(4-Cyano-pyridin-2-ylamino)-8-cyclopentyl--7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7n)

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 ⁰C; 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.

8-Cyclopentyl-2-(1H-indole-4-ylamino)-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7o)

Starting from 6i and 4-aminoindole, 40% of 7o was obtained according to the method described for the synthesis of 7. mp 320–322 ⁰C; 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.

8-Cyclopentyl-2-(1H-indole-5-ylamino)-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7p)

Starting from 6i and 5-aminoindole, 42% of 7p was obtained according to the method described for the synthesis of 7. mp 192–193 ⁰C; 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.

8-Cyclopentyl-7-oxo-2-[quinolin-3-ylamino]-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7q)

Starting from 6i and 3-aminoquinoline, 40% of 7q was obtained according to the method described for the synthesis of 7. mp 212–213 ⁰C; ¹H NMR (DMSO-d₆, 300 MHz): δ 10.92 (bs, 1H), 8.07–9.08 (m, 1H), 8.88 (s, 1H), 8.69–8.70 (m, 1H), 8.62 (s, 1H), 7.99–8.09 (m, 1H), 7.81–7.85 (m, 1H), 7.58–7.68 (m, 2H), 5.85–5.87 (m, 1H), 2.10–2.28 (m, 2H), 1.85 (bs, 4H), 1.56 (bs, 2H). MS found (M+H)⁺ (m/z): 383.30, Calcd for C₂₂H₁₈N₆O m/z: 382.15.

8-Cyclopentyl-7-oxo-2-[quinolin-8-ylamino]-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7r)

Starting from 6i and 8-aminoquinoline, 40% of 7r was obtained according to the method described for the synthesis of 7. mp 222–224 ⁰C; ¹H NMR (DMSO-d₆, 300 MHz): δ 10.84 (bs, 1H), 8.91 (s, 1H), 8.79–8.81 (m, 1H), 8.62 (s, 1H), 8.39–8.41 (m, 1H), 8.18–8.21 (m, 1H), 7.94–8.02 (m, 2H), 7.50–7.54 (m, 1H), 5.87–5.89 (m, 1H), 2.20–2.28 (m, 2H), 1.83–1.87 (m, 4H), 1.57–1.59 (m, 2H). MS found (M+H)⁺ (m/z): 383.20, Calcd for C₂₂H₁₈N₆O m/z: 382.15.

8-Cyclopentyl-2-(2-methoxy-quinolin-6-ylamino)-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7s)

Starting from 6i and 6-amino-2-methoxy-quinoline, 40% of 7s was obtained according to the method described for the synthesis of 7. mp 223–224 ⁰C; ¹H NMR (DMSO-d₆, 300 MHz): δ 10.82 (bs, 1H), 8.64 (s, 1H), 8.10–8.11 (m, 1H), 8.01 (s, 1H), 7.90–7.96 (m, 1H), 7.86 (s, 1H), 7.68–6.72 (m, 1H), 6.95 (d, 1H), 5.86–5.98 (m, 1H), 4.09 (s, 3H), 2.29–2.36 (m, 2H), 2.01 (bs, 2H), 1.88–1.95 (m, 2H), 1.65 (bs, 2H). MS found (M+H)⁺ (m/z): 413.20, Calcd for C₂₃H₂₀N₆O₂ m/z: 412.16.

8-Cyclopentyl-2-(4-morpholin-4-yl-phenylamino)-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7t)

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 ⁰C; 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 C₂₃H₂₄N₆O₂ m/z: 416.20.

8-Cyclopentyl-2-(5-morpholin-4-yl-pyridin-2-ylamino)-7-oxo-7,8-dihydro-pyrido[2,3-d] pyrimidine-6-carbonitrile (7u)

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 ⁰C; 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 C₂₂H₂₃N₇O₂ m/z: 417.19.

8-Cyclopentyl-2-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-7-oxo-7,8-dihydro-pyrido[ 2,3-d]pyrimidine-6-carbonitrile (7v)

Starting from 6i and 5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamine 22, 50% of 7v was obtained according to the method described for the synthesis of 7. mp 295–297 ⁰C; ¹H NMR (DMSO-d₆, 300 MHz): δ 10.53 (bs, 1H), 8.82 (s, 1H), 8.56 (s, 1H), 8.08 (d, 1H), 7.77 (d, 1H), 7.49-7.45 (m, 1H), 5.79-5.73 (m, 1H), 3.19-3.17 (m, 4H), 2.55-2.52 (m, 4H), 2.32 (s, 3H), 2.19-2.16 (m, 2H), 1.84-1.75 (m, 4H), 1.58-1.55 (m, 2H). MS found (M+H)⁺ (m/z): 431.20, Calcd for C₂₃H₂₆N₈O m/z: 430.22.

8-Cyclopentyl-7-oxo-2-[4-(5-trifluoromethyl-pyridin-2-yl)-piperazin-1-yl]-7,8-dihydro-pyrido[ 2,3-d]pyrimidine-6-carbonitrile (7w)

Starting from 6i and 1-(5-trifluoromethyl-pyridin-2-yl)-piperazine, 40% of 7w was obtained according to the method described for the synthesis of 7. mp 259–260 ⁰C; ¹H NMR (CDCl₃, 300 MHz): δ 8.53 (s, 1H), 8.44 (s, 1H), 7.94 (s, 1H), 7.68–7.71 (m, 1H), 6.68–6.71 (m, 1H), 5.82–5.88 (m, 1H), 4.09–4.14 (m, 4H), 3.82 (bs, 4H), 2.27–2.38 (m, 2H), 2.02–2.09 (m, 2H), 1.82–1.93 (m, 2H), 1.70–1.76 (m, 2H). MS found (M+H)⁺ (m/z): 470.10, Calcd for C₂₃H₂₂F₃N₇O m/z: 469.18.

8-Cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7x)

Starting from 6i and 4-(4-methyl-piperazin-1-yl)-phenyl amine 21, 55% of 7x was obtained according to the method described for the synthesis of 7. mp 290–292 ⁰C; ¹H NMR (CDCl₃, 300 MHz): δ 8.55 (s, 1H), 7.79 (s, 1H), 7.40–7.45 (m, 2H), 6.91–6.99 (m, 2H), 5.83–5.84 (m, 1H), 3.23–3.27 (m, 4H), 2.63–2.66 (m, 4H), 2.43 (s, 3H), 2.21-2.30 (m, 2H), 1.85 (bs, 4H), 1.62 (bs, 2H). ¹³C NMR (CDCl₃, 300 MHz): δ 25.6, 28.0, 46.1, 49.2, 54.7, 55.0, 102.9, 105.6, 105.7, 115.3, 116.3, 123.0, 129.3, 143.4, 148.9, 157.0, 160.2. HPLC purity 99.63%, retention time 8.41 min. HRMS: m/z Calcd [M+H] 430.2355; found 430.2374. Anal. Calcd for C₂₄H₂₇N₇O: C 67.11, H 6.34, N 22.83. Found: C 67.00, H 6.27, N 22.77.

2-[4-(4-Methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7y)

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 ⁰C; 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.

8-Methyl-2-[4-(4-Methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d] pyrimidine-6-carbonitrile (7z)

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 ⁰C; 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.

8-Ethyl-2-[4-(4-Methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d] pyrimidine-6-carbonitrile (7aa)

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 ⁰C; 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.

2-[4-(4-Methyl-piperazin-1-yl)-phenylamino]-7-oxo-8-propyl-7,8-dihydro-pyrido[2,3-d] pyrimidine-6-carbonitrile (7ab)

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 ⁰C; 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.

8-Isopropyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d] pyrimidine-6-carbonitrile (7ac)

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 ⁰C; 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.

8-Butyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7ad)

Starting from 6f and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 60% of 7ad was obtained according to the method described for the synthesis of 7. mp 282–283 ⁰C; ¹H NMR (DMSO-d₆, 300 MHz): δ 8.58 (s, 1H), 8.00 (s, 1H), 7.64 (bs, 1H), 7.54-7.52 (m, 2H), 6.98-6.95 (m, 2H), 4.37-4.32 (m, 2H), 3.30-3.26 (m, 4H), 2.73-2.70 (m, 4H), 2.45 (s, 3H), 1.75-1.68 (m, 2H), 1.49–126 (m, 2H), 1.01-0.94 (t, 3H). MS found (M+H)⁺ (m/z): 418.30, Calcd for C₂₃H₂₇N₇O m/z: 417.23.

2-[4-(4-Methyl-piperazin-1-yl)-phenylamino]-7-oxo-8-pentyl-7,8-dihydro-pyrido[2,3-d] pyrimidine-6-carbonitrile (7ae)

Starting from 6g and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 60% of 7ae was obtained according to the method described for the synthesis of 7. mp 291–292 ⁰C; ¹H NMR (DMSO-d₆, 300 MHz): δ 10.48 (bs, 1H), 8.80 (s, 1H), 8.56 (s, 1H), 7.64-7.62 (m, 2H,), 6.95-6.92 (m, 2H), 4.18 (bs, 2H), 3.34 (bs, 4H), 3.14 (bs, 4H), 2.30 (s, 3H), 1.63 (bs, 2H), 1.33 (bs, 4H), 0.86 (bs, 3H). MS found (M+H)⁺ (m/z): 432.40, Calcd for C₂₄H₂₉N₇O m/z: 431.24.

8-Cyclopropyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7af)

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 ⁰C; 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.

8-Cyclohexyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d] pyrimidine-6-carbonitrile (7ag)

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 ⁰C; 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.

8-Cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-6-nitro-8H-pyrido[2,3-d]pyrimidin-7-one (7ah)

Starting from 6k and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 55% of 7ah was obtained according to the method described for the synthesis of 7. mp 220–222 ⁰C; ¹H NMR (DMSO-d₆, 300 MHz): δ 8.67 (s, 1H), 8.46 (s, 1H), 7.72 (bs, 1H), 7.47-7.44 (m, 2H,), 6.99-6.96 (m, 2H), 5.90(bs, 1H), 3.28-3.25 (bs, 4H), 2.68-2.64 (m, 4H), 2.41 (s, 3H), 2.32-2.28 (m, 2H), 2.07 (bs, 2H), 1.88 (bs, 2H), 1.63 (bs, 2H). MS found (M+H)⁺ (m/z): 450.20, Calcd for C₂₃H₂₇N₇O₃ m/z: 449.22.

8-Cyclopentyl-6-methylsulfonyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-8H-pyrido[2, 3-d]pyrimidin-7-one (7ai)

Starting from 6l and 4-(4-methyl-piperazin-1-yl)-phenyl amine 21, 52% of 7ai was obtained according to the method described for the synthesis of 7. mp 255–257 ⁰C; ¹H NMR (CDCl₃, 300 MHz): δ 8.56 (s, 1H), 8.35 (s, 1H), 7.35 (d, 2H), 6.90 (d, 2H), 5.75 (bs, 1H), 3.25 (s, 3H), 3.14–3.23 (m, 4H), 2.52–2.55 (m, 4H), 2.30 (s, 3H), 2.12–2.27 (m, 2H), 1.75–1.97 (m, 2H), 1.57–1.76 (m, 4H). MS found (M+H)⁺ (m/z): 483.20, Calcd for C₂₄H₃₀N₆O₃S m/z: 482.21.

6-Benzenesulfonyl-8-cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-8H-pyrido [2,3-d]pyrimidine-7-one (7aj)

Starting from 6m and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 51% of 7aj was obtained according to the method described for the synthesis of 7. mp 199–200 ⁰C; ¹H NMR (CDCl₃, 300 MHz): δ 8.65 (s, 1H), 8.58 (s, 1H), 8.09–8.13 (m, 2H), 7.55–7.63 (m, 3H), 7.37–7.46 (m, 1H), 7.13–7.24 (m, 1H), 6.80–6.96 (m, 2H), 5.82–5.84 (m, 1H), 3.22–3.32 (m, 4H), 2.63–2.71 (m, 4H), 2.46 (s, 3H), 2.18–2.33 (m, 2H), 1.45–1.76 (m, 6H). MS found (M+H)⁺ (m/z): 545.30, Calcd for C₂₉H₃₂N₆O₃S m/z: 544.23.

6-(4-Chloro-benzenesulfonyl)-8-cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-8H-pyrido[2,3-d]pyrimidine-7-one (7ak)

Starting from 6n and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 50% of 7ak was obtained according to the method described for the synthesis of 7. mp 220–221 ⁰C; ¹H NMR (CDCl₃, 300 MHz): δ 8.64 (s, 1H), 8.62 (s, 1H), 8.04–8.06 (m, 2H), 7.49–7.50 (m, 2H), 7.37–7.40 (m, 2H), 6.92–6.95 (m, 2H), 5.65–5.69 (m, 1H), 3.23–3.27 (m, 4H), 2.67–2.70 (m, 4H), 2.42 (s, 3H), 2.09–2.2.20 (m, 2H), 1.71–1.82 (m, 4H), 1.51–1.63 (m, 2H). MS found (M+H)⁺ (m/z): 579.30, Calcd for C₂₉H₃₁ClN₆O₃S m/z: 578.19.

6-(4-Chloro-phenylmethanesulfonyl)-8-cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-8H-pyrido[2,3-d]pyrimidine-7-one (7al)

Starting from 6o and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 60% of 7al was obtained according to the method described for the synthesis of 7. mp 200–202 ⁰C; ¹H NMR (DMSO-d₆, 300 MHz): δ 10.59 (bs, 1H), 8.98 (s, 1H), 8.41 (s, 1H), 8.09–8.11 (m, 1H), 7.77–7.81 (d, 2H), 7.48–7.53 (m, 1H), 7.33–7.42 (m, 2H), 7.20–7.31 (m, 2H), 5.75–7.91 (m, 1H), 4.83 (s, 2H), 3.24–3.29 (m, 4H), 2.68–2.72 (m, 4H), 2.31–2.44 (s, 3H), 2.37 (m, 2H), 2.11–2.20 (m, 2H), 1.55–2.05 (m, 4H). MS found (M+H)⁺ (m/z): 593.10, Calcd for C₃₀H₃₃ClN₆O₃S m/z: 592.20.

8-Cyclopentyl-6-(3-hydroxy-4-methoxy-phenylmethanesulfonyl)-2-[4-(4-methyl-piperzin-1-yl)-phenylamino]-8H-pyrido[2,3-d]pyrimidin-7-one (7am)

Starting from 6p and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 53% of 7am was obtained according to the method described for the synthesis of 7. mp 165–166 ⁰C; ¹H NMR (DMSO-d₆, 300 MHz): δ 10.35 (bs, 1H), 9.08 (s, 1H), 8.88 (s, 1H), 7.46–7.51 (m, 2H), 6.95-6.92 (m, 2H), 6.83 (d, 1H), 6.67–6.68 (m, 1H), 6.56–6.60 (m, 1H), 5.89 (bs, 1H), 4.61 (s, 2H), 3.71 (s, 3H), 2.48–2.50 (m, 4H), 2.42–2.45 (m, 4H), 2.41 (s, 3H), 2.23–2.25 (m, 2H), 1.83–1.97 (m, 4H), 1.60 (bs, 2H). MS found (M+H)⁺ (m/z): 605.30, Calcd for C₃₁H₃₆N₆O₅S m/z: 604.25.

8-Cyclopentyl-6-(4-methoxy-3-nitro-phenylmethanesulfonyl)-2-[4-(4-methyl-piperzin-1-yl)-phenylamino]-8H-pyrido[2,3-d]pyrimidin-7-one (7an)

Starting from 6q and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 45% of 7an was obtained according to the method described for the synthesis of 7. mp 260–262 ⁰C; ¹H NMR (CDCl₃, 300 MHz): δ 8.52 (s, 1H), 8.12 (s, 1H), 7.80–7.81 (m, 1H), 7.59–7.62 (m, 1H), 7.38–7.42 (m, 2H), 7.02–7.05 (m, 1H), 6.91–1.94 (m, 2H), 5.89 (bs, 1H), 4.81 (s, 2H), 3.93 (s, 3H), 3.23–2.35 (m, 4H), 2.64–2.66 (m, 4H), 2.41 (s, 3H), 1.93–2.05 (m, 6H), 1.64–1.76 (m, 2H). MS found (M+H)⁺ (m/z): 634.30, Calcd for C₃₁H₃₅N₇O₆S m/z: 633.24.

8-Cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carboxylicacid(4-chloro-phenyl)-amide (7ao)

Starting from 6r and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 55% of 7ao was obtained according to the method described for the synthesis of 7. mp 301–302 ⁰C; ¹H NMR (DMSO-d₆, 300 MHz): δ 11.79 (s, 1H), 10.23 (bs, 1H), 8.77 (s, 1H), 8.67 (s, 1H), 7.71–7.74 (m, 2H), 7.44–7.61 (m, 2H), 7.31–7.33 (m, 2H), 6.91–6.99 (m, 2H), 5.89–6.03 (m, 1H), 3.47–3.499 (m, 4H), 3.22–3.25 (m, 4H), 2.63 (s, 3H), 2.28–2.38 (m, 2H), 1.89–2.05 (m, 4H), 1.67 (bs 2H). MS found (M+H)⁺ (m/z): 558.30, Calcd for C₃₀H₃₂ClN₇O₂ m/z: 557.23.

8-Cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carboxylicacid(4-fluoro-phenyl)-amide (7ap)

Starting from 6s and 4-(4-methyl-piperazin-1-yl)-phenylamine 21, 55% of 7ap was obtained according to the method described for the synthesis of 7. mp 289–290 ⁰C; ¹H NMR (DMSO-d₆, 300 MHz): δ 11.63 (bs, 1H), 10.25 (bs, 1H), 9.01 (s, 1H), 8.77 (s, 1H), 7.76–7.77 (m, 2H), 7.43–7.52 (m, 2H), 7.19–7.29 (m, 2H), 6.92–6.95 (m, 2H), 5.88–5.97 (m, 1H), 3.08–3.11 (m, 4H), 2.50 (s, 3H), 2.45–2.48 (m, 4H), 2.20–2.30 (m, 2H), 1.81–1.96 (m, 4H), 1.46–1.60 (m, 2H). MS found (M+H)⁺ (m/z): 542.30, Calcd for C₃₀H₃₂FN₇O₂ m/z: 541.26.

N-(6-Cyano-8-cyclopentyl-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrim-idin-2-yl)-N-[4-(4-methyl-piperazin-1-yl)-phenyl]-acetamide (7aq)

The compound 7x (1 g, 2.3 mmol) was taken into acetic anhydride and stirred at 120 ⁰C temperature for 3 h. the reaction mixture was cooled to room temperature and filtered the crude product. The pure product 7aq was obtained by flash chromatography with 2% methanol in chloroform. Yield 64%; light orange solid, mp 223–224 ⁰C; 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 C₂₆H₂₉N₇O₂ m/z: 471.24.

N-(6-Cyano-8-cyclopentyl-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimi-din-2-yl)-N-[4-(4-methyl-piperazin-1-yl)-phenyl]-4-trifluoromethyl-benzamide (7ar)

The compound 7x (1 g, 2.3 mmol) was taken into DMF and was added NaH at room temperature. After 10 min 4-trifluoromethyl benzoyl chloride (0.58 g, 2.8 mmol) was added and continued the stirring 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 ⁰C; 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 C₃₂H₃₀F₃N₇O₂ m/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 ⁰C 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 post-plating 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

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) 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% acrylamide SDS-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 non-linear 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 post-plating. Both non-adherent 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 ⁰C. 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, 50mM NaF, 0.1 mM sodium orthovanadate, (pH 7.4) and protease inhibitors) and 100 Rg 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; cat#9307), Rb (Cell Signaling; catalog # 9309), AKT (Cell Signaling, catalog # 4691), phosphospecific AKT Ser473 (Cell Signaling; catalog # 9271) and PARP (BD Biosciences;cat # 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 bi-laterally 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 (QDx15), 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)xlongx0.5. 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-PD0332991 (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 stick. B and 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.

ABBREVIATIONS USED

CDK

cyclin-dependent kinase

MPF

M-phase promoting factor

ARK5

AMPK-related protein kinase 5

FGFR

fibroblast growth factor receptor

PDGFR

platelet-derived growth factor receptor

PI3K

Phosphatidylinositide 3-kinases

RB

retinoblastoma protein

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

PVDF

polyvinylidene difluoride

mTOR

mammalian target of rapamycin

FACS

Fluorescence activated cell sorting

PARP

poly(ADP-ribose)polymerase

DMEM

dulbecco’s modified eagle medium

RPMI

Roswell Park Memorial Institute

PBS

Phosphate buffered saline