Design, synthesis and biological evaluation of novel pyrimidine derivatives as bone anabolic agents promoting osteogenesis via the BMP2/SMAD1 signaling pathway

Sumit K Rastogi; Sonu Khanka; Santosh Kumar; Amardeep Lakra; Rajat Rathur; Kriti Sharma; Amol Chhatrapati Bisen; Rabi Sankar Bhatta; Ravindra Kumar; Divya Singh; Arun K Sinha

doi:10.1039/d3md00500c

. 2024 Jan 4;15(2):677–694. doi: 10.1039/d3md00500c

Design, synthesis and biological evaluation of novel pyrimidine derivatives as bone anabolic agents promoting osteogenesis via the BMP2/SMAD1 signaling pathway^†

Sumit K Rastogi ^a,^c,^‡, Sonu Khanka ^b,^c,^‡, Santosh Kumar ^a,^c, Amardeep Lakra ^b, Rajat Rathur ^b,^c, Kriti Sharma ^b,^c, Amol Chhatrapati Bisen ^c,^d, Rabi Sankar Bhatta ^c,^d, Ravindra Kumar ^a,^c,^✉, Divya Singh ^b,^c,^✉, Arun K Sinha ^a,^c,^✉

PMCID: PMC10880903 PMID: 38389884

Abstract

Anti-resorptive inhibitors such as bisphosphonates are widely used but they have limited efficacy and serious side effects. Though subcutaneous injection of teriparatide [PTH (1–34)] is an effective anabolic therapy, long-term repeated subcutaneous administration is not recommended. Henceforth, orally bio-available small-molecule-based novel therapeutics are unmet medical needs to improve the treatment. In this study, we designed, synthesized, and carried out a biological evaluation of 31 pyrimidine derivatives as potent bone anabolic agents. A series of in vitro experiments confirmed N-(5-bromo-4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)hexanamide (18a) as the most efficacious anabolic agent at 1 pM. It promoted osteogenesis by upregulating the expression of osteogenic genes (RUNX2 and type 1 col) via activation of the BMP2/SMAD1 signaling pathway. In vitro osteogenic potential was further validated using an in vivo fracture defect model where compound 18a promoted the bone formation rate at 5 mg kg⁻¹. We also established the structure–activity relationship and pharmacokinetic studies of 18a.

Out of thirty-one pyrimidine derivatives, compound 18a was identified as the most efficacious orally bioavailable bone anabolic agent (1 pM in vitro and 5 mg kg⁻¹in vivo). It promoted osteogenesis by upregulating osteogenic gene expression via activation of the BMP2/SMAD1 signaling pathway.

Introduction

Bone is mineralized connective tissue that undergoes constant remodeling through the coordinated actions of bone cells, including bone resorption by osteoclasts and bone formation by osteoblasts.¹ Bone remodeling is an incredibly complex process through which old bone is replaced by new bone and it is essential for calcium balance, fracture repair, and skeletal adaptability. However, an imbalance between bone resorption and creation (bone remodeling) leads to osteoporosis, characterized by a decrease in bone mineral density, bone mass, and bone strength that can raise the chances of fractures.² The most prevalent osteoporotic fractures include hip, forearm, proximal humerus, and vertebral compression fractures.³ Around 9 million osteoporotic or fragility fractures occur each year globally.⁴ In 2017, it was estimated that 200 million people are at high fracture risk and this estimate will increase 2-fold by 2040.⁵ According to estimates, 1 in 3 women and 1 in 5 men over the age of 50 will sustain an osteoporosis fracture during the remaining years of their life. A bone fracture heals in a complex manner, requiring interactions between heterotypic cells at the fracture site and involving an anabolic period of expanding tissue volume and mineralization, followed by remodeling at the defect site to restore the original structure.⁶ There is a necessity to cure the fractured bone in order to restore normal anatomy and avoid the possibility of an atypical posture. Halting the osteoclast activity and instigating the osteoblast function are the two crucial approaches for ameliorating atypical bone remodeling. Current therapeutics used to treat osteoporosis and related fractures are either antiresorptive or osteoanabolic agents. Anti-resorptive agents, including bisphosphonates (alendronic acid, ibandronic acid, risedronic acid, and zoledronic acid), and anti-RANKL antibodies, calcitonin, estrogen, selective estrogen receptor modulators (SERMs), etc. suppress bone resorption but they have shown serious side effects such as hyperplasia, carcinoma, constipation, diarrhea, and GI irritation. Currently, teriparatide hPTH (1–34), abaloparatide (PTHrp), and romosozumab are FDA-approved drugs available in bone anabolic treatments administered parenterally but these are also associated with serious side effects.⁷ The anabolic strategy is superior and preferable since it encourages bone formation, including the restoration of lost bone. In addition, oral medication has a higher rate of treatment compliance than parenteral medications. Thus, an unmet medical need for orally active bone anabolic agents attracted researchers to develop small molecules^8–11 including phyto- and natural molecules¹² which became an ideal choice for modern chemical biology and drug discovery. Almost 84% of small molecule drugs approved by the US FDA have at least one nitrogen atom in them, and 59% drugs contain some sort of nitrogen heterocycles. Heterocyclics containing pyrimidine moieties are of great significance because they constitute an important class of natural and non-natural products. Moreover, this privileged pyrimidine scaffold is well documented in the literature for a wide range of biological activities including antioxidant, anti-inflammatory, and anti-analgesic activities.¹³ As a result, strategic structural modification of the pyrimidine scaffold allowed varying substitution at 2, 4, 5, or 6 positions of its aromatic ring for generating mono-, di-, tri-, and tetra-substituted classes of highly functionalized pyrimidine derivatives to achieve selectivity with greater affinity for the biological targets. Some pyrimidine derivatives such as thienopyrimidine bisphosphonate (ThPBP),¹⁴ thienopyrimidines,¹⁵ pyrazolopyrimidine-2,4-dione sulfonamides,¹⁶ 2-cyano-pyrimidines,¹⁷ tetrahydropyrazolopyrimidines,¹⁸ 7-(aminoalkyl)pyrazolo[1,5-a]pyrimidine,¹⁹etc. have been reported as anti-resorptive agents for bone health. 2-Amino-4-(3,4,5-trimethoxyphenyl)pyrimidine-5-carboxamide was reported as an anabolic agent via a calcium-sensing receptor (CaSR) antagonist for the treatment of osteoporosis with an IC₅₀ of 190 nM.²⁰ Similarly, 3,4,5-tri-methoxyphenyl amide conjugates of flavanones were also reported for bone anabolic properties with 1 pM.¹⁰

Given the importance of poly-substituted pyrimidines, we became interested to develop novel tri- and tetra-substituted pyrimidine derivatives. In addition, polyphenolic compounds, such as gallic acid, or their derivatives are natural antioxidants, explored for many activities. We began our study with 2,4,5-tri-methoxyphenyl substituted pyrimidine, derived from natural asaronaldehyde (commonly known as 2,4,5-tri-methoxylated benzaldehyde), one of the key secondary metabolites found in Acorus calamus.²¹ The inception of this basic scaffold is inspired by the above reports^10,20 and our ongoing interest in the development of bioactive molecules.^22–24 To the best of our knowledge, the effects of bioactive asaronaldehyde in the core structure of 2-aminopyrimidine have not been reported for osteoanabolic properties.

Chemistry

There are several basic approaches towards the construction of a pyrimidine core, we have utilized chalcones (Scheme 1A) as a simple and economical synthon.²⁵ A series of unsymmetrical pyrimidines were synthesized by the cyclization of the respective chalcone (3) with guanidine hydrochloride (4) in the presence of NaOH in ethanol under reflux conditions for 24–48 hours and the obtained product upon crystallization provides pyrimidine derivatives 5a–5h (51–69% yields, Scheme 1A). At first, 2,4,5-tri-methoxy group (in ring A; electron-rich environment) and 4-halo group (in ring B) containing pyrimidines were synthesized with 51–58% yields (Scheme 1B, 5a–5c). These compounds were screened for ALP activity. Furthermore, to generate the SAR, 4-methoxy, 4-methyl (electron-rich in ring B), and 4-bromobiphenyl pyrimidine derivatives were synthesized instead of halogens and obtained the corresponding products with 46–69% yields (Scheme 1B, 5d, 5e, and 5h). Next, the 3,4,5-tri-methoxy group was investigated instead of the 2,4,5-tri-methoxy group in ring A, which gave the corresponding desired product 5f with a 51% yield. The 4-methoxy group (less electron-rich) (in ring A) and 4-Cl (in ring B) containing pyrimidine derivative was synthesized in 65% yield (entry 5g). In the ALP assay, both compounds (5a and 5b) were found to be active at 100 pM (Fig. 1). It was necessary to check the toxicity of compounds and therefore the cell viability for 5a and 5b was measured. Compound 5a was found to be relatively nontoxic (see Fig. 2) over 5b. Therefore, 5a was selected as a hit compound for further tuning of the structure and for better activity.

In the subsequent study, a series of amine group containing amides (8) were synthesized by the reaction of 5a with chloroacetyl chloride in the presence of Et₃N in toluene at reflux temperature resulting in the corresponding chloroacetyl amide (5′, 61% yield), followed by treatment with secondary amines in DMF at room temperature for 3 h (Scheme 1B, path (iii)). Wide substrate scopes with secondary amines such as 3-methyl piperidine, 4-methyl piperidine, morpholine, ethyl piperidine-4-carboxylate, and piperidin-4-ylmethanol were examined which gave the corresponding products in 70–95% yields (Scheme 1B, 8, 9, 10, 11, and 16). Similarly, 4-methyl, 4-phenyl, 4-propyl piperazine, and 2-(piperazin-1-yl)ethan-1-ol gave the desired products in 53–73% yields (Scheme 1B, 12–15). However, none of these compounds were found to be active in the ALP assay.

In order to investigate the impact of the 2-amino group on the activity, a series of different acyclic amides (7a, 7b, 7d, 7e, 7f, and 7g) were synthesized by the reaction of 5a with the corresponding acyl chlorides in the presence of Et₃N in 1,4-dioxane at reflux temperature for 3 hours. Butyryl chloride, hexanoyl chloride, heptanoyl chloride, octanoyl chloride, and nonyl chloride underwent amide formation smoothly from 5a and 5b, and the corresponding desired products were obtained in 67–70% yields (Scheme 1B, 7a–7g). Notably, 7b showed activity at 100 pM and 1 pM in the ALP assay (Fig. 1). For curiosity, equipotent molecule (ALP study) 5b was also converted into hexanoyl amide (active chain length) 7c (70% yield) under the same reaction conditions and it was found to be inactive.

Next, we intended to incorporate halogens at the C-5 position in the previously obtained ALP active compound 7b (Scheme 1B (i)). Therefore, halogenation of 5a and 5b was carried out, which were subjected to acylation under standard conditions, as shown in Scheme 1C. Thus, compounds 17a and 17b were obtained in 73% yields by the reaction of 5a with NBS and NCS, respectively, which were further reacted with hexanoyl chloride in the presence of Et₃N in 1,4-dioxane to obtain 18a and 18b in 87% and 80% yields. Similarly, 17c was also synthesized in 78% yield by the reaction of 5b with NCS at room temperature, which upon acylation with hexanoyl chloride gave 17c in 80% yield (Scheme 1C (ii)). Notably, all these compounds were tested for ALP activity, in which compounds 17c, 18a, and 18c were found to be active. Pleasingly, compound 18a was found to be active at a lower concentration i.e. 1 pM in ALP, MTT, and mineralization assays (Fig. 1, 2, and 3, respectively). To validate the in vitro experiments, 18a was selected as a potent molecule for an in vivo study.

Primary screening of compounds using the ALP activity assay

The primarily osteogenic potential of synthetic compounds was assessed by ALP activity on calvarial osteoblast cells according to a previously published protocol.²⁶ ALP is a crucial primary osteoblast differentiation marker located at the outer surface of osteoblast cells and plays an essential role in skeletal mineralization.²⁷ Briefly, osteoblast cells were treated with different concentrations of compounds ranging from 1 pM to 1 μM for 48 h. Medicarpin (Med), a pterocarpan that stimulated osteoblast differentiation at 100 pM, was taken as a positive control.²⁸ In series, among 31 compounds, 6 compounds namely 5a, 5b, 7b, 17c, 18a, and 18c significantly increased the ALP activity compared to the control cells (with no treatment). Compounds 5a, 5b, 17c, and 18c increased the activity significantly at concentrations ranging from 100 pM to 10 nM. 7b increased the activity from 1 pM to 10 nM and compound 18a stimulated the ALP activity from 1 pM to 1 μM (Fig. 1). These results demonstrated that these six compounds act as effective osteoblast differentiating agents. Screening data of inactive compounds are available in the ESI† (Fig. S1).

Active compounds enhanced the osteoblast cell viability

The cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed to check the toxic effect of active compounds on osteoblast cells based on published protocols.²⁹ In osteoblast cells, no toxicity was observed with the treatment of the above six active compounds compared to the control cells. Compound 7b enhanced cell proliferation significantly at doses of 1 pM (∼22%) and 100 pM (∼25%), 5a and 17c promoted proliferation at 100 pM (∼30% and ∼29%) and 18c increased it at 1 pM (∼18%). However, compound 18a increased osteoblast cell proliferation significantly at 1 pM (∼32%), 100 pM (∼31%) and 10 nM (∼23%) concentrations (Fig. 2).

Compounds promoted mineralized nodule formation in bone marrow mesenchymal cells

In order to find out the most effective osteogenic agent, compounds active in ALP activity and cell viability were further assessed for their mineralization potential in bone marrow cells.³⁰ MSCs have the ability to self-renew and differentiate into a variety of cell lineages such as osteoblasts, adipocytes, and chondrocytes.³¹ To ascertain the mineralization efficacy, in the present study, active compounds were evaluated for mineral nodule formation. Bone marrow cells were treated with the two lowest active doses of compounds (1 pM and 100 pM). The cells were supplemented with osteogenic differentiation media (media containing dexamethasone) for 21 days. Treatment of compounds was refreshed every 48 h for 21 days. The cells were fixed and calcium mineralized nodules were stained with Alizarin red S, a calcium chelator, and extracted using a 10% cetylpyridinium chloride method (CPC) (Fig. 3A).

Quantification of Alizarin-stained nodules showed that compounds 5a, 7b, 18a, 17c, and 18c significantly increased mineral nodule formation in vitro compared to the control cells. Data revealed that 5a, 17c, and 18c were the most effective at 100 pM dose (∼23%, ∼26% and ∼27% respectively), 7b increased mineralization at 1 pM (∼24%) and 100 pM (∼25%) and 18a promoted mineralization at 1 pM (∼40%) and 100 pM (∼39%) (Fig. 3B), which confirmed the osteogenic potential of these compounds. To identify the lead compound, we considered the lowest active in vitro dose for osteogenesis. Among the six compounds, 18a at the lowest concentration (1 pM) increased osteoblast differentiation, cell viability, and mineral nodule formation. Based on these results, 18a was selected as a lead compound for further experiments.

Compound 18a increased the osteogenic gene expression

After examining the mineralizing ability, we evaluated the potential of lead compound 18a in the expression of osteogenic genes (RUNX2 and type 1 col). Calvarial osteoblast cells were treated with compound 18a at concentrations of 1 pM and 100 pM, RNA was isolated and cDNA was synthesized. The expression of genes was assessed using the quantitative real-time polymerase chain reaction (qPCR).³² GAPDH was taken as a positive control. RUNX2 is a crucial osteogenic transcription factor, plays an important role in the early stage of osteogenic differentiation, and also induces the proliferation of osteoblast progenitors. RUNX2 stimulates osteogenesis by up-regulating the expression of ALP and type 1 col. Collagen 1 is the most abundant collagenous bone matrix protein synthesized by active osteoblasts and closely linked to extracellular matrix mineralization in osteoblasts.³³ Compound 18a increased the expression of RUNX2 and type 1 col at both 1 pM and 100 pM concentrations, however more significantly at 1 pM (Fig. 3C).

Compound 18a promoted osteogenesis via upregulating the BMP2/SMAD1 signaling pathway

Further, to gain an insight into the mechanism by which 18a stimulates osteoblastogenesis, its role in the activation of BMP2/SMAD1 was evaluated through western blotting. The bone morphogenetic protein 2 (BMP2)/SMAD signaling pathway is an important regulatory route that regulates osteoblast differentiation, bone mineralization, and osteogenic potential which are highly associated with skeletal tissue repair and regeneration.³⁴ BMP2 phosphorylates SMAD1 and translocates it into the nucleus, where it activates signaling pathways that increase the expression of RUNX2, a key transcription factor for osteoblast differentiation as it regulates and induces the expression of osteoblast marker genes such as osteocalcin and collagen type I alpha 1 (Col1α1).³⁵ Treatment of compound 18a led to significantly enhanced phosphorylation of SMAD1 as a result of the binding of the BMP2 ligand to its receptor. This resulted in downstream stimulation of osteogenic genes like RUNX-2 and type 1 col (Fig. 4A and B). Thus, 18a exerts its bone-forming effect by stimulating the BMP2/SMAD1 signaling pathway.

Pharmacokinetic profile for 18a

In vitro pharmacokinetic profile

The in vitro experimental data of 18a suggested that the analyte could have mild susceptibility toward gastric and intestinal pH conditions. In contrast, it has a good stability profile under physiological pH and in vitro plasma conditions. The log percent remaining vs. time was plotted to determine the percent remaining of the unchanged compound (Fig. 5A). It was observed that >50% of the unchanged analyte was found in gastric fluids (SGF) and intestinal fluids (SIF), whereas >85% of the unchanged analyte was found in physiological fluids (SPF) and plasma.

In vivo pharmacokinetic profile

To identify a candidate's druggability, pharmacokinetic assessment is crucial.³⁶ Therefore, the in vivo pharmacokinetics of 18a was studied. The plasma samples were processed and run in an LC-MS/MS system. The plasma–concentration curve plot of 18a shows the good pharmacokinetic profile of the candidate (Fig. 5B). After the oral administration of 18a (5 mg kg⁻¹), the maximum concentration of 18a (C_max) in the blood reached 830 ± 53.54 pg mL⁻¹ within 0.83 ± 0.24 h (T_max), and declined to half of its value (t_1/2) in 12.45 ± 1.04 h, for which the area under the curve (AUC_0–∞) was found to be 6180.06 ± 304.92 h pg mL⁻¹ (Table 1).

In vivo pharmacokinetic parameters for 18a.

Parameters	Unit	18a
C _max	pg mL⁻¹	830 ± 53.54
T _max	h	0.83 ± 0.24
AUC	h pg mL⁻¹	6180.06 ± 304.92
t _1/2	h	12.45 ± 1.04
Cl	L h⁻¹ kg⁻¹	117.84 ± 4.03
V _d	L kg⁻¹	2113.99 ± 165.89
MRT	h	18.11 ± 1.13

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Moreover, its apparent mean residence time (MRT), volume of distribution (V_d), and total clearance (Cl) were about 18.11 ± 1.13 h, 2113 ± 165.89 L kg⁻¹, and 117.84 ± 4.03 L h⁻¹ kg⁻¹, indicating good oral exposure of the compound in correlation to its in vivo efficacy outcomes. The compound has significant oral bioavailability, as evidenced by the fact that its C_max of 18a (830 pg mL⁻¹ or 1398.9 pM) at 5 mg kg⁻¹ (the maximum active dose) was a log scale higher than its in vitro osteogenic EC_max ∼ 100 pM. The overall pharmacokinetic profile of the analyte is promising and thus could be identified as the lead for future developments.

In vivo assessment of18a as a bone healing and osteogenic agent through micro-computed tomography (μCT) and bone histomorphometry

After confirming the in vitro bone-forming potential of 18a, we next evaluated its osteogenic effect in an animal model. To accomplish this aim, we tested compound 18a for its bone regenerative potential in a femur drill hole defect model which depicts a scheme fracture that heals through intramembranous ossification where bone regeneration is promoted by eliminating endochondral ossification.³⁷ Additionally, a drill-hole defect in the femur diaphysis is an easy and highly reproducible model to use because it does not require a metal fixation device.³⁸ Oral treatment of the compound at 1, 5, and 10 mg kg⁻¹ body weight doses was started the next day post-fracture and continued for 12 days. Human parathyroid hormone (1–34) (PTH) was taken as a positive control. It is a standard osteogenic medication and plays a crucial role in fracture healing.³⁹ μCT was performed according to a previously published protocol. Three-dimensional (3D) representative μCT images showed that in comparison with the control group, compound 18a enhanced the fracture healing and increased the callus formation at the fracture site (Fig. 6A). The μCT quantitative data depict that 18a at 5 mg and 10 mg kg⁻¹ doses significantly increased BV/TV and Tb.Th which was comparable to PTH. However, Tb.N increased more significantly at 5 mg kg⁻¹ dose, although less than the positive reference control i.e. PTH (Fig. 6B). Increased μCT parameters at the fracture site indicated increased callus formation and bone regeneration due to the increased thickness of trabecularized spicules and conversion of woven bone into the lamellar bone.⁴⁰ A further calcein labeling experiment showed that compound 18a increased calcein intensity compared to the control group (Fig. 6C). The calcein labeling quantitative data depict that 18a at 5 mg kg⁻¹ dose more significantly increased calcein binding at the fracture site, which was comparable to PTH (Fig. 6D). The fluorochrome calcein intensity was correlated with new bone formation at the callus region, therefore, these data depicted that compound 18a at 5 mg kg⁻¹ dose enhanced bone regeneration and new bone formation.

18a enhanced the expression of osteogenic genes (RUNX2, BMP2, and type 1 col) at the site of bone regeneration

To determine the osteogenic gene expression at the bone regeneration site, RNA was isolated from bone tissue from the fracture site and the expression of osteogenic markers like RUNX2, BMP2, and type 1 col was evaluated.⁴¹18a at 5 mg and 10 mg kg⁻¹ doses significantly increased the expression of osteogenic marker genes compared to the control group, which was more significant at 5 mg kg⁻¹ (Fig. 7A). The data depicted that bone healing in 18a treated groups was due to the recruitment of osteoprogenitors at the fracture region.

Compound 18a restores the bone morphological pattern at the fracture site

To ascertain the effect of 18a on the bone morphology, sections were prepared using the bone area from the fracture site and stained with H&E. The histological structure of the callus around the fracture area depicted that in comparison with the control group, 18a at 1, 5 and 10 mg kg⁻¹ doses accelerated the bone tissue formation with greater extracellular matrix deposition. These results were similar to PTH (Fig. 7B).

Structure–activity relationship

We began the biological investigation with 5a having 2,4,5-trimethoxyphenyl and 4-bromophenyl groups at 4th and 6th positions, respectively, in the pyrimidine ring. The selection of the 2,4,5-trimethoxyphenyl group was based on some previous reports with polyhydroxy/methoxy arenes^10,20 and our own interest.^21–23 Keeping the 4-bromophenyl group on the other side of the pyrimidine ring was based on two hypotheses. From the chemistry point of view, it provides a reactive handle to tune the structure by coupling reactions. However, an example of a Suzuki coupled product like 5h did not show ALP activity. In addition, there are several reports where halogens, particularly, chloro, bromo, and fluoro substituents, in small molecules a play very critical role in biological activity and improving bioavailability; thus, they are present in several drug molecules.^42,43 Halogen-containing compounds also induce osteogenic differentiation of human mesenchymal stem cells.⁴⁴ Thus, compounds 5a–5c having a 4-Br/Cl/F phenyl ring were synthesized in the first set and 5a and 5b were found to be ALP active (initial hits). To understand the electronic and substituent effects on the activity, a structure–activity relationship was established with a total of 31 compounds (Scheme 1). The proposed SAR and selection of the hit molecule were based on in vitro activity including ALP, MTT, and mineralization studies. The initial hit molecule, 5a (having a 2,4,5-tri-methoxy group in ring A and a 4-bromo group in ring B), indicates that an electron-rich environment in ring A and a relatively electron-poor environment in ring B are required for the activity. With this thought, we maintained the electronic environment in ring A with a 3,4,5-tri-methoxy group instead of the 2,4,5-tri-methoxy group and the 4-Br group in ring B remained intact (5avs.5f), and the ALP outcomes suggested that 5f was not found to be active. Keeping the 2,4,5-tri-methoxy group intact, electron-releasing groups (4-OMe, 4-Me) were introduced in place of 4-Br in ring B, and the in vitro outcomes showed no activity (5d and 5e). This clearly indicates that the particular 2,4,5-tri-methoxy group in ring A and 4-Br in ring B are crucial for activity. Amide plays a very crucial role in activity, and pyrimidine carboxamide was also reported with better efficacy in osteoblast activity.²⁰ Next, we sought to evaluate the activity of corresponding amides from the initial hit molecule, 5a having free primary amine. Among different possible amides, a long aliphatic chain was chosen in order to increase the lipophilicity and efficacy²⁰ of the initial hit. Therefore, different amides were synthesized from long-chain acid chloride (n = 4–9 carbons) and among them, the hexanoyl chain containing amide 7b was found to be more active (Scheme 1B). Further, bromine/chlorine was introduced at the reactive 5th position of the previous active compound 7b/7c, and compound 18a, having a bromo group at the 5th position of pyrimidine and 4-bromo at the B-ring, was found to be more efficacious at a lower dose (1 pM) (Scheme 1C). Thus, 18a was chosen as a lead compound for the in vivo study (Fig. 8).

Conclusions

In conclusion, a straightforward synthetic strategy has been developed for highly substituted pyrimidine derivatives (overall 31 compounds). Structure–activity relationship studies beginning with tri-substituted pyrimidine derivatives led to the identification of two compounds 5a and 5b possessing a free amino-group at the 2-position of the pyrimidine ring as potent bone anabolic agents. Further structural optimization of the 2-amino pyrimidine basic core led to 18a as the most potent derivative, which upon evaluation of their osteoanabolic efficacy in vitro (ALP, cell viability and mineralization nodule formation at 1 pM) and in vivo showed bone formation and regeneration in a drill hole injury model in BALB/c mice at 5 mg kg⁻¹, which was comparable to PTH. This compound was orally bioavailable which promoted osteogenesis via upregulating the BMP2/SMAD1 signaling pathway. As an outcome, 18a could serve as a lead molecule in the development of a new class of osteoanabolic and fracture healing medications. However, the identification of the exact molecular target of the hit compound 18a is needed using RNA sequencing studies.

Chemistry

Materials and methods

All materials and reagents were purchased from commercial suppliers from Sigma Aldrich or Alfa-Aesar or Spectrochem and used without further purification. All the glass apparatus was oven-dried prior to use. Silica gel (mesh size 100–200) was used for column chromatography and TLC was performed on Merck-pre-coated silica gel 60-F254 and aluminum oxide 60-F254 plates. Solvents from Thermo-Fisher were used for the column chromatography. The melting point was recorded with a COMPLAB melting point apparatus. All the synthesized compounds were fully characterized by ¹H, ¹³C, ESI-MS, and ESI-HRMS analysis. ¹H spectra were recorded at 300/400/500 MHz, and ¹³C spectra were recorded at 100/125 MHz. CDCl₃ and DMSO-d₆ were used as solvents for NMR recording, and tetramethyl silane as the internal standard. Chemical shifts were reported in parts per million (ppm) downfield from the solvent reference, and coupling constants (J) were measured in Hz. ESI-MS spectra were obtained on an LCQ Advantage Ion Trap mass spectrometer (Finnigan Thermo Fisher Scientific) and high-resolution mass spectra (ESI-HRMS) were recorded on an Agilent 6520 ESI-QTOP mass spectrometer.

Experimental procedure and characterization of the compounds (5a–5h)

In a 50 mL round bottom flask, the corresponding chalcone (1 mmol), guanidine (2.5 mmol), and NaOH (5 mmol) were refluxed in 10 mL ethanol for 24–48 h. The reaction progress was monitored by TLC (30% ethyl acetate in hexane). After the completion of the reaction, ethanol was evaporated using a rotavapor. The obtained crude was washed with hot water (3 × 15 mL) and crystallized in MeOH and dried under vacuum resulting in pure products (5a–5h) obtained with 51% to 69% yields.

5a. 4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

The general procedure was followed for the synthesis of 5a by the reaction of (E)-1-(4-bromophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (377 mg, 1 mmol) and guanidinium hydrochloride (142.5 mg, 1.5 mmol) in the presence of NaOH (200 mg, 5 mmol) in 10 mL ethanol at reflux for 24 h. Pure compound 5a (225 mg, yield: 54%) was obtained as an off-white solid after crystallization in MeOH. M.P. 168 °C; HPLC purity: 98.7%; ¹H NMR (400 MHz, CDCl₃): δ 7.91 (d, J = 8.6 Hz, 2H), 7.71 (s, 1H), 7.63 (d, J = 8.6 Hz, 2H), 7.59 (s, 1H), 6.63 (s, 1H), 5.33 (s, 2H), 3.99 (s, 3H), 3.96 (s, 3H) 3.92 (s, 3H).¹³C{¹H} NMR (100 MHz, CDCl₃): δ 164.4, 164.0, 163.2, 153.1, 151.6, 143.5, 136.9, 131.9, 128.8, 124.8, 118.1, 113.4, 108.5, 97.8, 56.8, 56.4, 56.1; HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₁₉H₁₉BrN₃O₃: 416.0610, found: 416.0606.

5b. 4-(4-Chlorophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

The general procedure was followed for the synthesis of 5b by the reaction of (E)-1-(4-chlorophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (332 mg, 1 mmol) and guanidinium hydrochloride (142.5 mg, 1.5 mmol) with NaOH (200 mg, 5 mmol) in 10 mL ethanol at reflux for 24 h. Pure compound 5b (215 mg, yield: 58%) was obtained as an off-white solid after crystallization in MeOH. M.P. 171 °C; HPLC purity: 96.1%; ¹H NMR (400 MHz, CDCl₃): δ 7.97 (d, J = 8.6 Hz, 2H), 7.73 (s, 1H), 7.62 (s, 1H), 7.44 (d, J = 8.6 Hz, 2H), 6.61 (s, 1H), 5.22 (s, 2H), 3.96 (s, 3H), 3.93 (s, 3H), 3.90 (s, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 164.4, 163.9, 163.3, 153.3, 151.5, 143.6, 136.6, 136.3, 128.9, 128.5, 118.3, 113.3, 108.4, 97.8, 56.8, 56.4, 56.1. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₁₉H₁₉ClN₃O₃: 372.1115, found: 372.1130.

5c. 4-(4-Fluorophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

The general procedure was followed for the synthesis of 5c by the reaction of (E)-1-(4-fluorophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (316 mg, 1 mmol) and guanidinium hydrochloride (142.5 mg, 1.5 mmol) with NaOH (200 mg, 5 mmol) in 10 mL ethanol at reflux for 24 h. Pure compound 5c (181 mg, yield: 51%) was obtained as an off-white solid after crystallization in MeOH. M.P. 171 °C; HPLC purity >95%; ¹H NMR (400 MHz, DMSO-d₆): δ 8.12 (m, 2H), 7.63 (s, 1H), 7.58 (s, 1H), 7.34 (dd, J = 8.8, 8.9 Hz, 2H), 6.81 (s, 1H), 6.61 (s, 2H), 3.91 (s, 3H), 3.89 (s, 3H), 3.77 (s, 3H). ¹³C{¹H} NMR (100 MHz, DMSO-d₆): δ 165.0, 164.1, 163.9 (d, J¹ = 245.9 Hz), 162.7, 153.7, 151.9, 143.2, 134.7, 129.5 (d, J³ = 8.6 Hz) 118.1, 116.1 (d, J² = 21.4 Hz), 114.2, 106.3, 99.0, 57.0, 56.7, 56.3. ¹⁹F NMR (375 MHz, DMSO-d₆): δ −111.3. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₁₉H₁₉FN₃O₃: 356.1410, found: 356.1399.

5d. 4-(4-Methoxyphenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

The general procedure was followed for the synthesis of 5d by the reaction of (E)-1-(4-methoxyphenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (328 mg, 1 mmol) and guanidinium hydrochloride (142.5 mg, 1.5 mmol) with NaOH (200 mg, 5 mmol) in 10 mL ethanol at reflux for 24 h. Pure compound 5d (212 mg, yield: 58%) was obtained as a white solid after crystallization in MeOH. M.P. 205 °C; HPLC purity: 94.2%; ¹H NMR (400 MHz, DMSO-d₆): δ 7.63 (d, J = 8.8 Hz, 2H), 6.88 (s, 1H), 6.83 (d, J = 8.9 Hz, 2H), 6.70 (s, 1H), 5.34 (d, J = 4.5 Hz, 2H), 5.10 (d, J = 4.4 Hz, 1H), 3.82 (s, 3H), 3.79 (s, 3H), 3.73 (s, 3H), 3.64 (s, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 164.1, 163.7, 163.6, 161.5, 153.6, 151.7, 143.1, 131.3, 130.5, 128.7, 118.3, 114.5, 114.2, 113.6, 105.9, 99.0, 57.0, 56.7, 56.3, 55.8. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₀H₂₂N₃O₄: 368.1610, found: 368.1614.

5e. 4-(p-Tolyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

The general procedure was followed for the synthesis of 5e by the reaction of (E)-1-(p-tolyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (312 mg, 1 mmol) and guanidinium hydrochloride (142.5 mg, 1.5 mmol) with NaOH (200 mg, 5 mmol) in 10 mL ethanol at reflux for 24 h. Pure compound 5e (242 mg, yield: 69%) was obtained as an off-white solid after crystallization in MeOH. M.P. 140 °C; HPLC purity: 99.1%; ¹H NMR (400 MHz, CDCl₃): δ 7.93 (d, J = 8.2 Hz, 2H), 7.72 (s, 1H), 7.60 (s, 1H), 7.28 (d, J = 8.0 Hz, 2H), 6.60 (s, 1H), 5.25 (s, 2H), 3.95 (s, 3H), 3.93 (s, 3H), 3.88 (s, 3H), 2.41 (s, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 165.2, 164.0, 163.4, 153.1, 151.3, 143.5, 140.4, 135.4, 129.4, 127.1, 118.7, 113.3, 108.4, 98.0, 56.8, 56.4, 56.1, 21.4. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₀H₂₂N₃O₃: 352.1661, found: 352.1660.

5f. 4-(4-Bromophenyl)-6-(3,4,5-trimethoxyphenyl)pyrimidin-2-amine

The general procedure was followed for the synthesis of 5f by the reaction of (E)-1-(4-bromophenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (376 mg, 1 mmol) and guanidinium hydrochloride (142.5 mg, 1.5 mmol) with NaOH (200 mg, 5 mmol) in 10 mL ethanol. The reaction time was 20 h at reflux and pure compound 5f (212 mg, yield: 51%) was obtained as an off-white solid after crystallization in MeOH. M.P. 172 °C; HPLC purity: >94%; ¹H NMR (500 MHz, CDCl₃): δ 7.94 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 7.34 (s, 1H), 7.29 (s, 2H), 5.20 (s, 2H), 3.97 (s, 6H), 3.92 (s, 3H). ¹³C{¹H} NMR (100 MHz, CDCl3): δ 166.1, 165.0, 163.5, 153.5, 140.6, 136.6, 133.0, 132.0, 128.7, 125.1, 104.6, 103.7, 61.0, 56.4. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₁₉H₁₉BrN₃O₃: 416.0610, found: 416.0609.

5g. 4-(4-Chlorophenyl)-6-(4-methoxyphenyl)pyrimidin-2-amine

The general procedure was followed for the synthesis of 5g by the reaction of (E)-1-(4-chlorophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (272 mg, 1 mmol) and guanidinium hydrochloride (142.5 mg, 1.5 mmol) with NaOH (200 mg, 5 mmol) in 10 mL ethanol at reflux for 16 h. Pure compound 5g (202 mg, yield: 65%) was obtained as a white solid after crystallization in MeOH. M.P. 161 °C; HPLC purity: >94%; ¹H NMR (400 MHz, DMSO-d₆): δ 8.02 (d, J = 8.9 Hz, 2H), 7.98 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.6 Hz, 2H), 7.35 (s, 1H), 6.99 (d, J = 8.9 Hz, 2H), 5.29 (s, 2H), 3.86 (s, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 165.9, 164.7, 163.6, 161.8, 136.5, 136.3, 129.9, 129.0, 128.7, 128.4, 114.2, 103.2, 55.4. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₁₇H₁₅ClN₃O: 312.0903, found: 312.0899.

5h. 4-(4-Bromo-[1,1′-biphenyl]-4-yl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

The general procedure was followed for the synthesis of 5h by the reaction of (E)-1-(4′-bromo-[1,1′-biphenyl]-4-yl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (453 mg, 1 mmol) and guanidinium hydrochloride (142.5 mg, 1.5 mmol) with NaOH (200 mg, 5 mmol) in 10 mL ethanol at reflux for 36 h. Pure compound 5h (256 mg, yield: 52%) was obtained as a pale yellowish solid after crystallization in MeOH. M.P. 212 °C; HPLC purity: >94%; ¹H NMR (400 MHz, DMSO-d₆): δ 7.81 (d, J = 8.4 Hz, 2H), 7.63 (s, 3H), 7.59 (d, J = 8.5 Hz, 2H), 6.89 (s, 1H), 6.72 (s, 1H), 5.47 (s, 2H), 5.40 (d, J = 4.54 Hz, 1H), 5.31 (d, J = 4.52 Hz, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.64 (s, 3H). ¹³C{¹H} NMR (100 MHz, DMSO-d₆): δ 155.2, 154.2, 147.9, 144.4, 137.0, 133.7, 132.1, 131.1, 130.8, 130.6, 125.8, 118.1, 103.7, 101.5, 61.9, 61.5, 61.2. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₅H₂₃BrN₃O₃: 492.0923, found: 492.0922.

Experimental procedure and characterization of the compounds (7a–7g)

A mixture of 5a/5b (4-(4-bromo/chloro-phenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine, 1 mmol), acyl chloride (3 mmol), and Et₃N (3 mmol) was refluxed in 5 mL 1,4-dioxane for 3 h. The reaction progress was monitored by TLC (20% ethyl acetate in hexane). After completion of the reaction, the mixture was diluted with 10 mL water and extracted with (3 × 25 mL) EtOAc. Then, the organic layer was combined, dried over anhydrous Na₂SO₄, and concentrated using a rotavapor. The crude product was purified by column chromatography using 10–20% ethyl acetate in hexane as an eluent resulting in pure products (7a–7g) obtained in 66–70% yields.

7a. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)butyramide

The general procedure was followed for the synthesis of 7a by the reaction of 5a (416 mg, 1 mmol), butyryl chloride (310 μL, 3 mmol), and Et₃N (417 μL, 3 mmol) in 5 mL 1,4-dioxane at reflux for 3 h. The crude product was purified by column chromatography using 15% EtOAc in hexane and pure compound 7a (340 mg, yield: 70%) was obtained as a white solid. M.P. 180 °C; HPLC purity: 100%; ¹H NMR (400 MHz, CDCl₃): δ 8.17 (s, 1H), 8.08 (s, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.78 (s, 1H), 7.63 (d, J = 8.6 Hz, 2H), 6.62 (s, 1H), 3.98 (s, 3H), 3.95 (s, 3H), 3.95 (s, 3H), 2.97 (t, J = 7.3 Hz, 2H), 1.88–1.78 (m, 2H), 1.07–1.02 (t, J = 7.4 Hz, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 164.1, 163.9, 157.4, 153.9, 152.3, 143.6, 136.2, 132.1, 128.8, 125.4, 117.0, 113.2, 111.6, 97.5, 56.7, 56.4, 56.2, 39.4, 18.4, 14.0. HRMS (ESI⁺): m/z: [M + 2 + H]⁺ calculated for C₂₃H₂₅BrN₃O₄: 488.1028, found: 488.1009.

7b. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)hexanamide

The general procedure was followed for the synthesis of 7b by the reaction of 5a (416 mg, 1 mmol), hexanoyl chloride (417 μL, 3 mmol), and Et₃N (417 μL, 3 mmol) in 5 mL 1,4-dioxane at reflux for 3 h. The crude product was purified by column chromatography using 15% EtOAc in hexane and pure compound 7b (355 mg, yield: 69%) was obtained as a white solid. M.P. 193 °C; HPLC purity: 98.2%; ¹H NMR (400 MHz, CDCl₃): δ 8.19 (s, 1H), 8.07 (s, 1H), 7.99 (d, J = 8.6 Hz, 2H), 7.80 (s, 1H), 7.66 (d, J = 8.6 Hz, 2H), 6.64 (s, 1H), 4.00 (s, 3H), 3.97 (s, 6H), 3.01 (t, J = 7.0 Hz 2H), 1.86–1.78 (m, 2H), 1.45–1.38 (m, 4H), 0.93 (t, J = 7.1 Hz, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 164.3, 163.9, 157.4, 153.9, 152.3, 143.6, 136.2, 132.1, 128.8, 125.4, 117.1, 113.2, 111.6, 97.5, 56.7, 56.4, 56.2, 37.5, 31.6 24.6, 22.6, 14.0. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₅H₂₉BrN₃O₄: 514.1341, found: 514.1337.

7c. N-(4-(4-Chlorophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)hexanamide

The general procedure was followed for the synthesis of 7c by the reaction of 5b (371 mg, 1 mmol), hexanoyl chloride (417 μL, 3 mmol), and Et₃N (417 μL, 3 mmol) in 5 mL 1,4-dioxane at reflux for 3 h. The crude product was purified by column chromatography using 17% EtOAc in hexane and pure compound 7c (328 mg, yield: 70%) was obtained as a white solid. M.P. 157 °C; HPLC purity: 100%; ¹H NMR (400 MHz, CDCl₃): δ 8.17 (s, 1H), 8.05 (s, 1H), 8.04 (d, J = 6.8 Hz, 2H), 7.77 (s, 1H), 7.48 (d, J = 8.7 Hz, 2H), 6.62 (s, 1H), 3.98 (s, 3H), 3.95 (s, 3H), 3.95 (s, 3H), 2.99 (t, J = 7.2 Hz, 2H), 1.84–1.76 (m, 2H), 1.43–1.35 (m, 4H), 0.91 (t, J = 7.1 Hz, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 164.2, 163.8, 157.4, 153.9, 152.3, 143.7, 137.0, 135.7, 129.1, 128.5, 117.1, 113.3, 111.6, 97.5, 56.7, 56.4, 56.2, 37.5, 31.6, 24.6, 22.6, 14.0. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₅H₂₉ClN₃O₄: 470.1846, found: 470.1849.

7d. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)heptanamide

The general procedure was followed for the synthesis of 7d by the reaction of 5a (416 mg, 1 mmol), heptanoyl chloride (464 μL, 3 mmol), and Et₃N (417 μL, 3 mmol) in 5 mL 1,4-dioxane at reflux for 3 h. The crude product was purified by column chromatography using 18% EtOAc in hexane and pure compound 7d (354 mg, yield: 67%) was obtained as a white solid. M.P. 116 °C; HPLC purity; 100%; ¹H NMR, (400 MHz, CDCl₃): δ 8.17 (s, 1H), 8.04 (s, 1H), 7.97 (d, J = 8.6 Hz, 2H), 7.77 (s, 1H), 7.64 (d, J = 8.6 Hz, 2H), 6.62 (s, 1H), 3.98 (s, 3H), 3.95 (s, 3H), 3.95 (s, 3H), 2.99 (t, J = 7.2 Hz, 2H), 1.83–1.75 (m, 2H), 1.46–1.39 (m, 2H), 1.34–1.30 (m, 4H), 0.88 (t, J = 7.1 Hz, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 164.2, 163.9, 157.4, 153.9, 152.3, 143.7, 136.2, 132.1, 128.8, 125.4, 118.0, 117.1, 113.3, 111.6, 97.6, 56.7, 56.4, 56.2, 37.5, 31.7, 29.1, 24.9, 22.6, 14.0. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₆H₃₁BrN₃O₄: 528.1498, found: 528.1495.

7e. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-3,5,5-trimethylhexanamide

The general procedure was followed for the synthesis of 7e by the reaction of 5a (416 mg, 1 mmol), 5,5-dimethylhexanoyl chloride (570 μL, 3 mmol), and Et₃N (417 μL, 3 mmol) in 5 mL 1,4-dioxane at reflux for 3 h. The crude product was purified by column chromatography using 15% EtOAc in hexane and pure compound 7e (367 mg, yield: 66%) was obtained as a white solid. M.P. 136 °C; HPLC purity: 100%; ¹H NMR (400 MHz, CDCl₃): δ 8.17 (s, 1H), 7.98 (d, J = 8.7 Hz, 3H), 7.77 (s, 1H), 7.64 (d, J = 8.7 Hz, 2H), 6.62 (s, 1H), 3.98 (s, 3H), 3.96 (s, 3H), 3.95 (s, 3H), 2.94–2.84 (m, 2H), 2.31–2.24 (m, 1H), 1.40–1.36 (m, 1H), 1.21–1.16 (m, 1H), 1.08 (d, J = 6.6 Hz, 3H), 0.92 (s, 9H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 164.2, 163.9, 157.4, 153.9, 152.3, 143.6, 136.2, 132.1, 128.8, 125.4, 117.1, 113.3, 111.7, 97.5, 56.7, 56.5, 56.2, 51.0, 46.9, 31.2, 30.1, 26.7, 22.9. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₈H₃₅BrN₃O₄: 556.1811, found: 556.1802.

7f. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)octanamide

The general procedure was followed for the synthesis of 7f by the reaction of 5a (416 mg, 1 mmol), octanoyl chloride (511 μL, 3 mmol), and Et₃N (417 μL, 3 mmol) in 5 mL 1,4-dioxane at reflux for 3 h. The crude product was purified by column chromatography using 18% EtOAc in hexane and pure compound 7f (363 mg, yield: 67%) was obtained as a white solid. M.P. 142 °C; HPLC purity: 100%; ¹H NMR (400 MHz, CDCl₃): δ 8.17 (s, 1H), 8.08 (s, 1H), 7.96 (d, J = 8.2 Hz, 2H), 7.77 (s, 1H), 7.63 (d, J = 8.2 Hz, 2H), 6.62 (s, 1H), 3.98 (s, 3H), 3.95 (s, 3H), 3.94 (s, 3H), 2.98 (t, J = 7.2 Hz, 2H), 1.80–1.77 (m, 2H), 1.41–1.28 (m, 8H), 0.87 (t, J = 6.1 Hz, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 164.2, 163.9, 157.4, 153.9, 152.3, 143.6, 136.2, 132.1, 128.8, 125.4, 117.0, 113.2, 111.6, 97.4, 56.7, 56.4, 56.2, 37.5, 31.7, 29.4, 29.2, 25.0, 22.6, 14.1. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₇H₃₃BrN₃O₄: 542.1654, found: 542.1647.

7g. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)nonanamide

The general procedure was followed for the synthesis of 7g by the reaction of 5a (416 mg, 1 mmol), nonanoyl chloride (540 μL, 3 mmol), and Et₃N (417 μL, 3 mmol) in 5 mL 1,4-dioxane at reflux for 3 h. The crude product was purified by column chromatography using 20% EtOAc in hexane and pure compound 7g (372 mg, yield: 67%) was obtained as a white solid. M.P. 131 °C; HPLC purity: 99.2%; ¹H NMR (400 MHz, CDCl₃): δ 8.17 (s, 1H), 8.02 (s, 1H), 7.97 (d, J = 8.6 Hz, 2H), 7.77 (s, 1H), 7.64 (d, J = 8.6 Hz, 2H), 6.62 (s, 1H), 3.98 (s, 3H), 3.95 (s, 6H), 2.99 (t, J = 7.2 Hz, 2H), 1.83–1.75 (m, 2H), 1.46–1.38 (m, 2H), 1.31–1.26 (m, 8H), 0.87 (t, J = 7.2 Hz, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 164.2, 163.9, 157.4, 153.9, 152.3, 143.6, 136.2, 132.1, 128.8, 125.4, 117.1, 113.2, 111.6, 97.5, 56.7, 56.4, 56.2, 37.5, 31.9, 29.5, 29.2, 24.9, 22.7, 14.1. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₈H₃₅BrN₃O₄: 556.1811, found: 556.1798.

Experimental procedure and characterization of the compounds (5′ and 8–16)

The compound (4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-chloroacetamide was synthesized by the reaction of 5a (416 mg, 1 mmol) with chloro acetyl chloride (416 μL, 3 mmol) and Et₃N (417 μL, 3 mmol) in 10 mL toluene at reflux for 3 h. The reaction progress was monitored by thin-layer chromatography in DCM : EtOAc. After completion of the reaction, the mixture was diluted with (20 mL) water and extracted with EtOAc (3 × 25 mL). The organic layer was combined and dried over sodium sulfate. Then, the organic layer was evaporated using a rotavapor, and the crude product was purified by column chromatography using 12% EtOAc in hexane as an eluent. Pure product (4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-chloroacetamide (5′) was obtained (150 mg, 61%) as a yellowish solid. Compound 8 was also found to be inactive in the ALP assay (see Fig. S1,† compound 5′) M.P. 183 °C; HPLC purity: >95%; ¹H NMR (400 MHz, CDCl₃): δ 8.54 (s, 1H), 8.24 (s 1H), 7.97 (d, J = 8.6 Hz, 2H), 7.80 (s, 1H), 7.65 (d, J = 8.7 Hz, 2H), 6.62 (s, 1H), 4.71 (s, 2H), 3.99 (s, 3H) 3.96 (s, 6H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 164.4, 164.2, 156.6, 154.0, 152.5, 143.7, 135.8, 132.2, 128.8, 125.7, 116.6, 113.1, 112.4, 97.4, 56.6, 56.4, 56.2, 44.2. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₁H₂₀BrClN₃O₄: 492.0325, found: 492.0318. Then, compound 5′ (492 mg, 1 mmol) and secondary amine (2 mmol) were stirred in 3 mL DMF at room temperature for 2–3 h. The reaction progress was monitored by thin-layer chromatography. After completion of the reaction, the mixture was diluted with water (20 mL) and extracted with EtOAc (3 × 25 mL). The organic layer was combined and washed with water (3 × 20 mL) to remove the excess amount of amines and dried over sodium sulfate. The combined organic layer was evaporated using a rotavapor, and the crude product (thick liquid) was saturated with diethyl ether and further washed with diethyl ether (3 × 5 mL) to remove impurities. Thus, pure products were obtained with 53–95% yields.

8. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-(3-methylpiperidin-1-yl)acetamide

The general procedure was followed for the synthesis of 8 by the reaction of N-(4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-chloroacetamide (492 mg, 1 mmol) and 3-methyl piperidine (234 μL, 2 mmol) in DMF at rt for 3 h. Pure compound 8 (395 mg, yield: 70%) was obtained as a yellow thick liquid. HPLC purity: 100%; ¹H NMR (400 MHz, CDCl₃): δ 9.91 (s, 1H), 8.24 (s, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.98 (s, 1H), 7.64 (d, J = 8.5 Hz, 2H), 6.61 (s, 1H), 3.99 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H), 2.96 (s, 2H), 2.88 (s, 3H), 2.03–1.94 (m, 1H), 1.84–174 (m, 6H), 0.93 (d, J = 6.4 Hz, 2H). ¹³C{¹H} NMR (100 MHz CDCl₃): δ 169.2, 164.2, 164.1, 157.1, 153.9, 152.2, 143.7, 136.3, 132.0, 128.9, 125.3, 117.3, 113.4, 112.1, 97.5, 63.2, 62.2, 56.7, 56.3, 56.1, 54.5, 32.1, 31.3, 25.5, 19.4. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₇H₃₂BrN₄O₄: 555.1607, found: 555.1625.

9. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-(4-methylpiperidin-1-yl)acetamide

The general procedure was followed for the synthesis of 9 by the reaction of N-(4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-chloroacetamide (492 mg, 1 mmol) and 4-methyl piperidine (236 μL, 2 mmol) in DMF at rt for 3 h. Pure compound 9 (536 mg, yield: 95%) was obtained as a yellow thick liquid. HPLC purity: 100%; ¹H NMR (400 MHz, CDCl₃): δ 9.90 (s, 1H), 8.23 (s, 1H), 8.03 (d, J = 8.6 Hz, 2H), 7.98 (s, 1H), 7.64 (d, J = 8.6 Hz, 2H), 6.61 (s, 1H), 3.99 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H), 3.24 (s, 2H), 2.97 (d, J = 10.64 Hz, 2H), 2.33–2.29 (m, 2H), 1.70 (d, J = 10.1 Hz, 2H), 1.42–1.26 (m, 3H), 0.98 (d, J = 5.68 Hz, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 169.0, 164.2, 164.1, 157.1, 153.9, 152.2, 143.7, 136.3, 132.0, 128.9, 125.3, 117.2, 113.4, 112.1, 97.5, 62.9, 56.7, 56.3, 56.1, 54.4, 34.4, 30.1, 21.8. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₇H₃₂BrN₄O₄: 555.1607, found: 555.1628.

10. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-morpholinoacetamide

The general procedure was followed for the synthesis of 10 by the reaction of N-(4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-chloroacetamide (492 mg, 1 mmol) and morpholine (173 μL, 2 mmol) in DMF at rt for 3 h. Pure compound 10 (472 mg, yield: 87%) was obtained as a yellow solid. M.P. 190 °C; HPLC purity: 96%; ¹H NMR (400 MHz, CDCl₃): δ 9.66 (s, 1H), 8.24 (s, 1H), 8.02 (d, J = 8.5 Hz, 2H), 7.94 (s, 1H), 7.65 (d, J = 8.5 Hz, 2H), 6.61 (s, 1H), 3.99 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H), 3.86–3.84 (m, 4H), 3.35 (s, 2H), 2.74 (s, 4H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 164.3, 164.1, 157.0, 154.0, 152.3, 143.7, 136.1, 132.0, 128.9, 125.4, 117.1, 113.4, 112.3, 97.4, 66.8, 62.9, 56.7, 56.4, 56.2, 53.7. HRMS (ESI⁺): m/z: [M + 2]⁺ calculated for C₂₅H₂₇BrN₄O₅: 544.1243, found: 544.1264.

11. Ethyl 1-(2-((4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)amino)-2-oxoethyl)piperidine-4-carboxylate

The general procedure was followed for the synthesis of 11 by the reaction of N-(4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-chloroacetamide (492 mg, 1 mmol) and ethyl piperidine-4-carboxylate (308 μL, 2 mmol) in DMF at rt for 3 h. Pure compound 11 (410 mg, yield: 70%) was obtained as a yellow thick liquid. HPLC purity: 99.5%; ¹H NMR (500 MHz, CDCl₃): δ 9.76 (s, 1H), 8.24 (s, 1H), 8.03 (d, J = 8.6 Hz, 2H), 7.98 (s, 1H), 7.65 (d, J = 8.6 Hz, 2H), 6.61 (s, 1H), 419–4.14 (m, 2H), 3.99 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H), 3.32 (s, 2H), 2.99 (d, J = 11.5 Hz, 2H), 2.35 (t, J = 8.8 Hz, 3H), 2.04–1.90 (m, 4H), 1.29–1.26 (m, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 174.8, 168.6, 164.21, 164.15, 157.1, 153.9, 152.2, 143.7, 136.2, 132.1, 128.9, 125.3, 117.2, 113.4, 112.2, 97.5, 63.0, 60.5, 56.7, 56.4, 56.1, 53.4, 40.4, 29.7, 28.4, 14.2. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₉H₃₄BrN₄O₆: 613.1661, found: 616.1658.

12. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-(4-methylpiperazin-1-yl)acetamide

The general procedure was followed for the synthesis of 12 by the reaction of N-(4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-chloroacetamide (492 mg, 1 mmol) and N-methyl piperazine (222 μL, 2 mmol) in DMF at rt for 3 h. Pure compound 12 (406 mg, yield: 73%) was obtained as a yellow solid. M.P. 210 °C; HPLC purity: 100%; ¹H NMR (400 MHz, CDCl₃): δ 9.76 (s, 1H), 8.24 (s, 1H), 8.03 (d, J = 8.6 Hz, 2H), 7.98 (s, 1H), 7.65 (d, J = 8.6 Hz, 2H), 6.62 (s, 1H), 3.99 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H), 3.27 (s, 2H), 2.71–2.56 (m, 8H), 2.34 (s, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 168.5, 164.2, 164.1, 157.1, 154.0, 152.2, 143.7, 136.3, 132.1, 128.9, 125.3, 117.2, 113.5, 112.2, 97.5, 62.6, 56.7, 56.3, 56.2, 55.2, 53.5, 46.0. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₆H₃₁BrN₅O₄: 556.1559, found: 556.1571.

13. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-(4-phenylpiperazin-1-yl)acetamide

The general procedure was followed for the synthesis of 13 by the reaction of N-(4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-chloroacetamide (492 mg, 1 mmol) and N-phenyl piperazine (222 μL, 2 mmol) in DMF at rt for 3 h. Pure compound 13 (352 mg, yield: 57%) was obtained as a yellow solid. M.P. 203 °C; HPLC purity: 98.6%; ¹H NMR (400 MHz, CDCl₃): δ 9.77 (s, 1H), 8.25 (s, 1H), 8.02 (d, J = 8.6 Hz, 2H), 7.97 (s, 1H), 7.64 (d, J = 8.6 Hz, 2H), 7.31–7.27 (m, 2H), 6.96 (d, J = 7.8 Hz, 2H), 6.91–6.88 (m, 1H), 6.61 (s, 1H), 3.98 (s, 3H), 3.97 (s, 3H), 3.95 (s, 3H), 3.34–3.32 (m, 6H), 2.86–2.84 (m, 4H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 168.3, 164.3, 164.1, 157.0, 154.0, 152.3, 151.1, 143.7, 136.2, 132.1, 129.2, 128.9, 125.4, 120.1, 117.2, 116.2, 113.4, 112.2, 97.5, 62.7, 56.7, 56.4, 56.2, 53.6, 49.4. HRMS (ESI⁺): m/z: [M + 2 + H]⁺ calculated for C₃₁H₃₃BrN₅O₄: 620.1713, found: 620.1684.

14. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-(4-propylpiperazin-1-yl)acetamide

The general procedure was followed for the synthesis of 14 by the reaction of N-(4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-chloroacetamide (492 mg, 1 mmol) and N-propyl piperazine (263 μL, 2 mmol) in DMF at rt for 3 h. Pure compound 14 (310 mg, yield: 53%) was obtained as a yellow thick liquid. HPLC purity: 100%; ¹H NMR (400 MHz, CDCl₃): δ 9.77 (s, 1H), 8.24 (s, 1H), 8.03 (d, J = 6.8 Hz, 2H), 7.98 (s, 1H), 7.65 (d, J = 6.8 Hz, 2H), 6.61 (s, 1H), 3.99 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H), 3.27 (s, 2H), 2.72–2.59 (m, 8H), 2.37–2.35 (m, 2H), 1.55–1.51 (m, 2H), 0.92 (t, J = 5.9 Hz, 3H). ¹³C{¹H} NMR, (100 MHz, CDCl₃): δ 168.6, 164.2, 164.1, 162.5, 157.1, 154.0, 152.2, 143.7, 136.3, 132.1, 128.9, 125.3, 117.2, 113.4, 112.2, 97.5, 62.7, 60.6, 56.7, 56.3, 56.2, 53.6, 53.3, 36.5, 20.0, 11.9. HRMS (ESI⁺): m/z: [M + 2 + H]⁺ calculated for C₂₈H₃₅BrN₄O₄: 586.1872, found: 586.1741.

15. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-(4-(2-hydroxyethyl)piperazin-1-yl)acetamide

The general procedure was followed for the synthesis of 15 by the reaction of N-(4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-chloroacetamide (492 mg, 1 mmol) and N-ethanol piperazine (244 μL, 2 mmol) in DMF at rt for 3 h. Pure compound 15 (376 mg, yield: 64%) was obtained as a yellow solid. M.P. 163 °C. HPLC purity; >94%; ¹H NMR (500 MHz, CDCl₃): δ 9.70 (s, 1H), 8.23 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.97 (s, 1H), 7.65 (d, J = 8.4 Hz, 2H), 6.62 (s, 1H), 3.99 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H), 3.64 (t, J = 5.3 Hz, 2H), 3.29 (s, 2H), 2.72–2.67 (m, 8H), 2.61 (t, J = 5.3 Hz, 2H). ¹³C{¹H} NMR, (100 MHz, CDCl₃): δ 164.3, 164.1, 157.1, 154.0, 152.3, 143.7, 136.2, 132.1, 128.9, 125.4, 117.2, 113.5, 112.2, 97.5, 62.6, 59.2, 57.8, 56.7, 56.4, 56.2, 53.6, 52.9. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₇H₃₃BrN₅O₅: 586.1665, found: 586.1741.

16. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-(4-(hydroxymethyl)piperidin-1-yl)acetamide

The general procedure was followed for the synthesis of 16 by the reaction of N-(4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-chloroacetamide (492 mg, 1 mmol) and piperidin-4-ylmethanol (235.6 μL, 2 mmol) in DMF at rt for 3 h. Pure compound 16 (399 mg, yield: 70%) was obtained as a yellow solid. M.P. 163 °C; HPLC purity: 100%; ¹H NMR (500 MHz, CDCl₃): δ 9.81 (s, 1H), 8.23 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.97 (s, 1H), 7.64 (d, J = 8.5 Hz, 2H), 6.61 (s, 1H), 3.99 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H), 3.56 (d, J = 6.2 Hz, 2H), 3.25 (s, 1H), 3.03 (d, J = 11.4 Hz, 2H), 2.96 (s, 2H), 2.88 (s, 2H), 2.34–2.29 (m, 2H), 1.81 (d, J = 11.7 Hz, 2H), 1.47–1.44 (m, 1H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 168.9, 164.2, 164.1, 157.1, 153.9, 152.2, 143.7, 136.3, 132.1, 128.9, 125.3, 117.2, 113.5, 112.2, 97.5, 67.6, 63.0, 56.7, 56.4, 56.1, 54.0, 37.9, 36.5, 28.9. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₇H₃₂BrN₄O₅: 571.1556, found: 571.1549.

Experimental procedure and characterization of the compounds (17a–17c)

5a/5b/5c (1 mmol) was dissolved in 10 mL MeCN and N-halosuccinimide (1 mmol) was added in three portions at 0 °C or rt for 2–3 h. The reaction progress was monitored by TLC (30% ethyl acetate in hexane). After the completion of the reaction, the mixture was diluted with water (10 mL) and extracted with EtOAc (3 × 25 mL). The organic layer was combined and dried over sodium sulfate. Then the organic layer was evaporated using a rotavapor. The obtained crude was dried and purified by column chromatography using 20–28% EtOAc in hexane as an eluent resulting in pure products obtained with 71% to 78% yields.

17. 5-Bromo-4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

The general procedure was followed for the synthesis of 17a by the reaction of 5a (416 mg, 1 mmol) and NBS (195 mg, 1.1 mmol) in 10 mL MeCN in an ice bath for 2 h. The crude product was purified by column chromatography using 20% EtOAc in hexane as an eluent and pure compound 17a (361 mg, yield: 73%) was obtained as a yellowish solid. M.P. 136 °C; HPLC purity: 98.8%; ¹H NMR (400 MHz, CDCl₃): δ 7.60 (s, 4H), 6.84 (s, 1H), 6.60 (s, 1H), 5.18 (s, 2H), 3.95 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 167.3, 165.4, 161.2, 150.9, 143.2, 137.5, 131.2, 130.8, 124.0, 119.4, 112.8, 108.4, 97.4, 56.5, 56.2. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₁₉H₁₈Br₂N₃O₃: 493.9715, found: 493.9705.

17b. 4-(4-Bromophenyl)-5-chloro-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

The general procedure was followed for the synthesis of 17b by the reaction of 5a (416 mg, 1 mmol) and NCS (146 mg, 1.1 mmol) in 10 mL MeCN at room temperature for 3 h. The crude product was purified by column chromatography using 25% EtOAc in hexane and pure compound 17b (328 mg, yield: 73%) was obtained as a yellowish solid. M.P. 196 °C; HPLC purity: 100%; ¹H NMR (400 MHz, CDCl₃): δ 7.68 (d, J = 8.2 Hz, 2H), 7.62 (d, J = 7.2 Hz, 2H), 6.89 (s, 1H), 6.62 (s, 1H), 5.29 (s, 2H), 3.97 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 165.4, 163.3, 160.6, 151.3, 151.0, 143.3, 136.0, 131.3, 131.0, 124.2, 118.2, 117.8, 112.9, 97.4, 56.5, 56.2. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₁₉H₁₈BrClN₃O₃: 450.0220, found 450.0216.

17c. 5-Chloro-4-(4-chlorophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

The general procedure was followed for the synthesis of 17c by the reaction of 5b (372 mg, 1 mmol) and NCS (146 mg, 1.1 mmol) in 10 mL MeCN at room temperature for 3 h. The crude product was purified by column chromatography using 25% EtOAc in hexane and pure compound 17c (317 mg, yield: 78%) was obtained as an off-white solid. M.P. 177 °C; HPLC purity: 100%; ¹H NMR (400 MHz, CDCl₃): δ 7.73 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.6 Hz, 2H), 6.87 (s, 1H), 6.61 (s, 1H), 5.33 (s, 2H), 3.95 (s, 3H), 3.87 (s, 3H), 3.83 (s, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 165.3, 163.3, 160.6, 151.3, 151.0, 143.2, 135.8, 135.7, 130.7, 128.3, 118.2, 117.8, 112.9, 97.4, 56.5, 56.2. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₁₉H₁₈Cl₂N₃O₃: 406.0725, found 406.0725.

Experimental procedure and characterization of the compounds (18a–18c)

The compounds 17a or 17b or 17c (1 mmol), hexanoyl chloride (3 mmol), and Et₃N (3 mmol) were taken in 10 mL 1,4-dioxane and refluxed for 3 h. The reaction progress was monitored by thin-layer chromatography. After completion of the reaction, the mixture was diluted with water (20 mL) and extracted with EtOAc (3 × 25 mL). The organic layer was combined and dried over sodium sulfate. Then, the organic layer was evaporated using a rotavapor and the crude product was purified by column chromatography using 28–30% EtOAc in hexane as an eluent resulting in pure products obtained with 80% to 87% yields.

18a. N-(5-Bromo-4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)hexanamide

The general procedure was followed for the synthesis of 18a by the reaction of 17a (495 mg, 1 mmol), hexanoyl chloride (417 μL, 3 mmol), and Et₃N (417 μL, 3 mmol) in 1,4-dioxane at reflux for 3 h. The crude product was purified by column chromatography using 30% EtOAc in hexane. Pure compound 18a (516 mg, yield: 87%) was obtained as a white solid. M.P. 161 °C; HPLC purity: 98.6%; ¹H NMR (400 MHz, CDCl₃): δ 7.97 (s, 1H), 7.71 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 8.6 Hz, 2H), 6.87 (s, 1H), 6.60 (s, 1H), 3.96 (s, 3H), 3.88 (s, 3H), 3.85 (s, 3H), 2.79 (t, J = 7.5 Hz, 2H), 1.75–1.70 (m, 2H), 1.33–1.30 (m, 4H), 0.87 (t, J = 7.1 Hz, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 173.5, 167.6, 165.3, 155.4, 151.4, 151.1, 143.2, 136.8, 131.3, 131.2, 124.6, 118.7, 113.5, 113.1, 97.2, 56.6, 56.3, 56.2, 37.3, 31.5, 24.6, 22.4, 13.9. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₅H₂₈Br₂N₃O₄: 592.0446, found 592.0439.

18b. N-(4-(4-Bromophenyl)5-chloro-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)hexanamide

The general procedure was followed for the synthesis of 18b by the reaction of 17b (450 mg, 1 mmol), hexanoyl chloride (417 μL, 3 mmol), and Et₃N (417 μL, 3 mmol) in 1,4-dioxane at reflux for 3 h. The crude product was purified by column chromatography using 28% EtOAc in hexane. Pure compound 18b (438 mg, yield: 80%) was obtained as a white solid. M.P. 109 °C; HPLC purity: 98.6%; ¹H NMR (400 MHz, CDCl₃): δ 8.02 (s, 1H), 7.78 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 8.6 Hz, 2H), 6.91 (s, 1H), 6.62 (s, 1H), 3.97 (s, 3H), 3.88 (s, 3H), 3.85 (s, 3H), 2.80 (t, J = 7.5 Hz, 2H), 1.76–1.69 (m, 2H), 1.35–130 (m, 4H), 0.87 (t, J = 7.1 Hz, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 165.7, 163.1, 154.7, 151.5, 143.3, 135.3, 131.4, 131.3, 124.9, 122.9, 117.0, 113.3, 97.2, 56.6, 56.4, 56.2, 37.3, 31.5, 24.6, 22.5, 13.9. HRMS (ESI⁺): m/z: [M + 2 + H]⁺ calculated for C₂₅H₂₈BrClN₃O₄: 550.0951, found 550.0929.

18c. N-(4-(4-Chlorophenyl)5-chloro-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)hexanamide

The general procedure was followed for the synthesis of 18c by the reaction of 17c (450 mg, 1 mmol), hexanoyl chloride (406 μL, 3 mmol), and Et₃N (417 μL, 3 mmol) in 1,4-dioxane at reflux for 3 h. The crude product was purified by column chromatography using 28% EtOAc in hexane. Pure compound 18c (403 mg, yield: 80%) was obtained as a white solid. M.P. 116 °C; HPLC purity: 100%; ¹H NMR (400 MHz, CDCl₃): δ 7.99 (s, 1H), 7.85 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 8.7 Hz, 2H), 6.91 (s, 1H), 6.62 (s, 1H), 3.97 (s, 3H), 3.88 (s, 3H), 3.85 (s, 3H), 2.80 (t, J = 7.5 Hz, 2H), 1.76–1.69 (m, 2H), 1.35–1.31 (m, 4H), 0.88 (t, J = 7.2 Hz, 3H). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 165.7, 163.0, 154.6, 151.5, 143.3, 136.5, 134.9, 131.1, 128.4, 122.9, 117.0, 113.3, 97.2, 56.6, 56.4, 56.2, 37.3, 31.5, 24.6, 22.5, 13.9. HRMS (ESI⁺): m/z: [M + H]⁺ calculated for C₂₅H₂₈Cl₂N₃O₄: 504.1457, found 504.1449.

Biology

General materials and methods

Reagents and chemicals

Cell culture media α-MEM and supplements like non-essential amino acids, antibiotics and sodium bicarbonate were purchased from Sigma-Aldrich (St. Louis, MO). FBS was procured from Gibco (Waltham, Massachusetts, USA). p-Nitrophenylphosphate (PNPP) was purchased from MP Biomedical (USA). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and calcein were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human PTH (1–34) was procured from Calbiochem (Darmstadt, USA). An Immobilon western chemiluminescent HRP substrate kit was procured from Millipore (WBKLS0500) USA. Primary antibodies for western blot analysis were obtained from cell signaling technologies, affinity, abcam, and biorbyt.

In vitro study

Mouse calvarial osteoblast cell isolation and culture

For osteoblast cell culture, the calvariae were isolated from 1–2 days old neonatal pups of BALB/c mice. The calvaria was isolated surgically from the skull region of each pup. Adherent muscle tissues were removed from the calvariae. After cleaning with PBS, calvariae were chopped into small pieces. An enzyme cocktail was prepared by combining dispase (0.1%) and collagenase type II (0.1%) for the digestion process. Chopped calvariae underwent five repeated digestions (15–20 min each) with the enzyme cocktail at 37 °C to release the cells. The enzyme supernatant was discarded from the 1st digestion and cells were collected from the 2nd to 5th digestion in FBS. The enzyme-containing cells were centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and the cell pellet was resuspended and cultured in α-MEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified incubator at 37 °C temperature in 5% CO₂ and 95% air. Cells were used for further experiments after attaining 80% to 90% confluency.²⁶

Mouse bone marrow cell isolation and culture

For the mineralization assay, the long bones of 6 to 8 weeks old BALB/c mice were surgically removed, adherent flesh around the legs was removed to clean the bones and bone marrow cells were flushed out in 10% α-MEM complete media and centrifuged at 1500 rpm. Cell pellets were resuspended and cultured in mineralization media.³⁰

Cell differentiation assay (alkaline phosphatase activity)

Primary osteoblast cells were seeded in a 96-well plate at a density of 2 × 10³ cells per well for 24 h. When the cells were adhered and attained the required confluency, they were treated with pyrimidine derivatives at 1 pM to 1 μM concentration with osteoblast differentiation media for the next 48 h. Next, the media were decanted from each well, rinsed with PBS, and kept at −80 °C or −20 °C overnight. For the ALP determination, cells were placed at 37 °C for rupturing of cells to expose ectoenzyme (alkaline phosphatase). Then, 100 μL p-nitrophenyl phosphate (PNPP) substrate was added to each well and ALP activity was measured by spectrophotometry at 405 nm.²⁶

Cell viability assay

To check the effect of test compounds on cell viability, osteoblast cells at a density of 2 × 10³ were seeded in 96-well plates. After 24 h, cells were treated with different concentrations (from 1 pM to 1 μM) of compounds in 5% differentiation media (α-MEM with 5% FBS, 10 mM β-glycerophosphate, 50 μg mL⁻¹ ascorbic acid, and 1% penicillin/streptomycin). After 48 h of incubation, 20 μL of MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well and incubated for 4 h at 37 °C. 100 μL dimethyl sulfoxide (DMSO) was added to each well to dissolve formazan crystals and absorbance was taken at 570 nm using a spectrophotometer (Spectra MaxM2).²⁹

Mineral nodule formation assay (mineralization)

For the mineralization assay, bone marrow cells were isolated from the long bones of BALB/c mice and seeded in a 12-well plate at a density of 2 × 10⁶ cells per well. The cells were supplemented with 10% FBS, 1% antibiotic solution, 100 nM dexamethasone, 50 μg mL⁻¹ ascorbic acid, and 10 mM β-glycerophosphate. Treatment of active compounds with respective concentrations was given and media were changed every 48 h for 21 days. After 21 days, the cells were washed with PBS and fixed with 4% paraformaldehyde, and counterstained with Alizarin red (4.2 pH) for 30–40 min. After staining, the cells were washed under tap water 2–3 times and images were captured under an EVOS FL bright-field microscope. Moreover, mineral nodules were quantified according to the cetyl pyridinium chloride method. In this method, 10% CPC (500 μL) was poured into each well and kept at room temperature for 1 h. Notably, the colored solution was transferred to 96 well plates and quantified spectrophotometrically at 595 nm.⁴⁵

Quantitative PCR

In order to assess the real-time expression of osteogenic genes, total RNA was isolated from the cultured osteoblast cells using TRIzol based on the manufacturer‘s protocol. Furthermore, RNA was quantified and 1 μg of total RNA was reverse transcribed to cDNA using a cDNA reverse transcription kit (Thermo Fisher). Real-time PCR was performed for transcript level expression of osteogenic marker genes such as runt-related transcription factor 2 (RUNX2), bone morphogenetic proteins, and type 1 collagen (type 1 col) on a Quant Studio 3 (Applied Biosystem Foster City, California) real-time PCR machine using SYBR Green chemistry.³² Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control (Scheme S1†).

Western blot analysis

Osteoblast cells were seeded in a 6-well plate at a density of 10⁶ cells per well. After 24 h, the cells were treated with the most active compound 18a at the two lowest active concentrations: 1 pM and 100 pM. After 48 h of treatment, the cells were washed with chilled 1× PBS and total protein was extracted using cell lytic solution containing a 1× protease inhibitor and 1× phosphatase inhibitor. A BCA kit (Thermo Scientific) was used to quantify protein. 30 μg protein was used and separated on SDS polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Blocking was done with 5% BSA for 1–2 h, followed by overnight incubation with primary antibodies such as BMP2 (1 : 1000) (AF5163), SMAD1 (1 : 1000) (9512S), PSMAD1 (1 : 1000) (9511S), RUNX2 (1 : 1000) (AF5186), type I col (1 : 1000) (E8I9Z), and β-actin (1 : 30 000) (A3854) at 4 °C. The horseradish peroxidase-conjugated secondary antibody was used to identify the expression of the protein. A chemiluminescence kit was used for signal detection on a ChemiDoc XRS+ system based on the manufacturer's instruction. Densitometry was done using ImageJ software.⁴⁷

In vivo study

Fracture study using BALB/c mice

All animal care and methodologies were approved by the Institutional Animal Ethical Committee (IAEC) at CDRI, Lucknow. The in vivo research work was conducted in compliance with the approval of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India with approval number IAEC/2020/52/Renew-1/Dated 04/01/2021.

A fracture study was carried out in 8 to 9 weeks-old female BALB/c mice (body weight 24 ± 5 gm). All the animals were acclimatized for a week before the experiments. Mice were kept at constant room temperature (22–24 °C), with light and dark cycles (12 h) and fresh air in the rooms.

Drill hole defect at the femur mid-diaphysis

To assess the bone healing and regeneration efficiency, fifty adult BALB/c mice were randomly divided into five groups: control, 18a (1 mg, 5 mg, and 10 mg kg⁻¹ BW) and PTH used as a reference standard (20 μg kg⁻¹ per day body weight) for the drill hole defect. The animals were anesthetized with a mixture of ketamine (45 mg kg⁻¹ body weight) and xylazine (5 mg kg⁻¹ body weight) via the intraperitoneal route. A straight small incision in the mid-diaphysis of the femur was made and muscles were removed to expose the femoral bone area. A drill-hole defect (0.6 mm) was done in the mid-diaphysis of the femur using a small electric drill machine. During defect drilling, saline was continuously irrigated to remove small-sized debris. Muscles were rearranged and the skin was stitched with a nylon thread. Melonex was given to animals as a painkiller after the surgery. Treatment of the compound was given orally in a 0.25% CMC (carboxymethyl cellulose) suspension from the next day of surgery and continued for 12 days. However, PTH was given subcutaneously 4 days in a week. At the end of the experiment, animals were euthanized and femur bones having drill-hole defects were isolated, fixed in neutral formaldehyde (4%, pH 7.0) for 2 days, and transferred to 70% isopropanol for micro-CT scanning and histomorphometry.⁴⁶

Micro-computed tomography

High-resolution micro-computed tomography (SkyScan scanner, 1076; Aartselaar, Belgium) was utilized to analyze the efficacy of compound 18a at the drill hole injury site based on microarchitectural changes. Muscles and soft connective tissues were removed without disturbing the callus area and MicroCT scanning was performed using an X-ray source at 70 kV and 142 mA with an aluminum filter (0.5 mm) and 9 μm per pixel resolution. Nrecon software was used to accomplish the reconstruction of scanned images, which was based on a modified Feldkamp approach, supported by network-distributed reconstruction running on computers. The cortical bone injury (regenerated callus) area was analyzed using CTan software by drawing a cylindrical positioned generated region of interest (ROI) around the entire drill hole area (6 mm diameter). The micro-architectural parameters such as bone volume/tissue volume (BV/TV) and trabecular thickness (Tb.Th) were quantified using Batman software.^37,47

Bone dynamic histomorphometry at the fracture site

Bone regeneration at the drill hole injury site was assessed by a single fluorochrome (calcein) labeling experiment. Calcein (20 mg kg⁻¹) was dissolved in normal saline and administered intraperitoneally to each animal before 24 hours of the autopsy to measure the new bone generation. After an autopsy, undecalcified bones (mid-diaphysis, drill-hole region) were embedded in an acrylic material and 50 μm sections were cut using an Isomet bone cutter. Calcein-labeled images were captured using a Leica confocal microscope (Leica SP8TCS) with 485 nm (excitation wavelength) and an analyzing wavelength of 510 nm (emission wavelength). New bone formation and the regeneration quantity were confirmed by the calcein binding intensity at the drill hole injury site. New bone formation (intensity of the fluorescence signal) was measured by creating a z-stack image of the drill hole injury site by using image analyzing software.⁴⁹

Histological analysis

Femur bones with the drill hole injury site were collected and cleaned by removing soft tissues, skin, and connective tissues. For histological analysis, the bone tissue within the injury site was fixed in 4% paraformaldehyde for 48 h and transferred to 70% isopropanol for another 48 h. The bones were then decalcified using a decalcifying buffer (Sigma-Aldrich) and subsequently embedded in paraffin wax. Sections of 5 μm size were cut transversely using a microtome (Leica microsystem, model number RM2165). After deparaffinization, the sections were processed in different grades of isopropanol, stained with hematoxylin and eosin (H&E) (Sigma-Aldrich, St. Louis, MO), and mounted using DPX. Histological images were taken under a bright field microscope (EVOS auto FL) for analysis of bone regeneration at the drill hole injury site.

qPCR using bone samples

The area surrounding the drill hole was dissected with a 1 mm margin from the femoral bone. To check the mRNA expression in the callus region of the femur bone, three femur samples from each group were used. Samples were frozen in liquid nitrogen and crushed into a fine powder using a mortar and pestle. Total RNA was isolated using TRIzol (Qiagen), while 1 μg of total RNA was used to synthesize cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems TM, Thermo Fisher Scientific, USA). SYBR Green (Applied Biosystems, MA, USA) was used to assess the changes in the expression of osteogenic genes such as RUNX2, BMP2, and type 1 col in a Quant Studio 3 cycler (Thermo Fisher, MA, USA). GAPDH was used as the standard internal control.⁴¹

Pharmacokinetic analysis

Analysis of the in vitro pharmacokinetics of the compound was performed to assess the p. o. stability in different virtual physiological microenvironments. The in vitro stability was assessed at pH 1.2, pH 6.8, and pH 7.4 in female BALB/c mice plasma using simulated solution systems mimicking SGF, SIF, and SPF. The solutions were formulated as per the USP guidelines.⁴⁸ The compound was exposed to the different simulated systems and aliquots were collected at multiple time points from zero to 60 min. All the experiments were performed in triplicate. The samples were extracted with acetonitrile as a quenching solvent, vortexed, centrifuged at 12 000g for 15 min and injected into RP-UHPLC for the quantification of the percent compound remaining against the function of time.

Similarly, the in vivo studies for 18a are planned and executed using female BALB/c mice (n = 6, three per group) to analyze the pharmacokinetic profile. The animals were treated with an oral dose of 5 mg kg⁻¹. The blood samples were collected at multiple time points from 0.83–24 h into heparinized microcentrifuge tubes. The samples were processed by centrifuging at 6800g for 15 min at 4 °C, the supernatant was collected and RBC residues were discarded. The plasma samples were stored at −80 °C until final sample preparation.^49,50 The bioanalysis was done using a hyphenated Shimadzu LC-MS/MS 8050 system assembled with a Shimadzu Nexera series UFLC. A Waters Symmetry column (150 × 4.6 mm 2, 5 μM) was used against 0.1% formic acid in deionized water and acetonitrile as an organic modifier (10 : 90%, v/v).

The compound was optimized in positive ESI-MS polarity and the analyte's MRM ion pair for data quantification was selected to be m/z 594.13 → 495.8 (Q1 → Q3). The pharmacokinetic parameters based on experimental data were derived using Phoenix WinNonlin 7.0 (Pharsight) software by Certara Inc.

Abbreviations

SAR: Structure–activity relationship
SERMs: Selective estrogen receptor modulators
PTH: Parathyroid hormone
FDA: Food and drug administration
PBS: Phosphate buffer saline
FBS: Fetal bovine serum
α-MEM: Alpha-minimum essential media
CO₂: Carbon dioxide
rpm: Rotation per minute
pM: Picomolar
μM: Micromolar
μL: Microliter
nM: Nanometer
mM: Millimolar
μg: Microgram
ALP: (Alkaline phosphatase)
MTT: (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
DMSO: Dimethyl sulfoxide
CPC: Cetyl pyridinium chloride
cDNA: Complementary DNA
PCR: Polymerase chain reaction
BMP2: Bone morphogenetic protein 2
ALP: Alkaline phosphatase
RUNX2: Runt-related transcription factor 2
Type1 col: Collagen type I
GAPDH: Glyceraldehyde-3-phosphate dehydrogenase
pNPP: p-Nitrophenyl phosphate
BCA: Bicinchoninic acid
SDS: Sodium dodecyl-sulfate
BSA: Bovine serum albumin
μCT: Micro-computed tomography
ROI: Region of interest
BV/TV: Bone volume/tissue volume
Tb.Th: Trabecular thickness
Tb.N: Trabecular number
H&E: Hematoxylin and eosin
RP-UHPLC: Reverse-phase ultra high-performance liquid chromatography
ESI: Electrospray ionization; source
t _1/2: Half-time
C _max: Maximum concentration
V _d: Volume of distribution
AUC: Area under the curve
MRT: Mean residence time
IAEC: Institutional Animal Ethics Committee
LC-MS/MS: Liquid-chromatography tandem mass spectrometry

Author contributions

R. K. and A. K. S. conceptualized the study. D. S. planned for the biological activities. S. K. R. and S. K. performed the chemistry and biology experiments respectively and wrote the manuscript, contributing equally to this work. S. K., A. L., R. R., and K. S. supported the work. A. C. B. performed the pharmacokinetic study under the guidance of R. S. B.

Conflicts of interest

The authors declare no conflicts of interest.

Supplementary Material

MD-015-D3MD00500C-s001

MD-015-D3MD00500C-s001.pdf^{(8.6MB, pdf)}

Acknowledgments

The authors acknowledged the CSIR-FBR project MLP-2028 for the funding. All the authors are thankful to the Director of CSIR-CDRI, for providing the necessary research facilities. S. K. R. and S. K., are thankful to the CSIR for the fellowship. S. K., R. R., K. S., and A. C. B. are thankful to UGC, DBT, DST, and ICMR for the fellowship, respectively. The authors are thankful to Ayus Dubey for the support in pharmacokinetic study and also thankful to in-house Sophisticated Analytical Instrumentation Facility & Research (SAIF & R) for providing the required valuable spectral data. This manuscript has CDRI Communication no. 10723.

^†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3md00500c

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Associated Data

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Supplementary Materials

MD-015-D3MD00500C-s001

MD-015-D3MD00500C-s001.pdf^{(8.6MB, pdf)}

[cit1] Robling A. G. et al., Biomechanical and molecular regulation of bone remodeling. Annu. Rev. Biomed. Eng. 2006;8:455–498. doi: 10.1146/annurev.bioeng.8.061505.095721. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Design, synthesis and biological evaluation of novel pyrimidine derivatives as bone anabolic agents promoting osteogenesis via the BMP2/SMAD1 signaling pathway†

Sumit K Rastogi

Sonu Khanka

Santosh Kumar

Amardeep Lakra

Rajat Rathur

Kriti Sharma

Amol Chhatrapati Bisen

Rabi Sankar Bhatta

Ravindra Kumar

Divya Singh

Arun K Sinha

Abstract

Introduction

Chemistry

Primary screening of compounds using the ALP activity assay

Active compounds enhanced the osteoblast cell viability

Compounds promoted mineralized nodule formation in bone marrow mesenchymal cells

Compound 18a increased the osteogenic gene expression

Compound 18a promoted osteogenesis via upregulating the BMP2/SMAD1 signaling pathway

Pharmacokinetic profile for 18a

In vitro pharmacokinetic profile

Fig. 5. [A] In vitro pharmacokinetic profile of 18a in different physiological microenvironments. [B] In vivo pharmacokinetic profile of 18a. The inset image in [B] signifies the enlarge pk profile from 0 to 6 h.

In vivo pharmacokinetic profile

In vivo pharmacokinetic parameters for 18a.

In vivo assessment of18a as a bone healing and osteogenic agent through micro-computed tomography (μCT) and bone histomorphometry

18a enhanced the expression of osteogenic genes (RUNX2, BMP2, and type 1 col) at the site of bone regeneration

Compound 18a restores the bone morphological pattern at the fracture site

Structure–activity relationship

Fig. 8. Structure–activity relationship around hit molecule 18a.

Conclusions

Chemistry

Materials and methods

Experimental procedure and characterization of the compounds (5a–5h)

5a. 4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

5b. 4-(4-Chlorophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

5c. 4-(4-Fluorophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

5d. 4-(4-Methoxyphenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

5e. 4-(p-Tolyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

5f. 4-(4-Bromophenyl)-6-(3,4,5-trimethoxyphenyl)pyrimidin-2-amine

5g. 4-(4-Chlorophenyl)-6-(4-methoxyphenyl)pyrimidin-2-amine

5h. 4-(4-Bromo-[1,1′-biphenyl]-4-yl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

Experimental procedure and characterization of the compounds (7a–7g)

7a. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)butyramide

7b. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)hexanamide

7c. N-(4-(4-Chlorophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)hexanamide

7d. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)heptanamide

7e. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-3,5,5-trimethylhexanamide

7f. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)octanamide

7g. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)nonanamide

Experimental procedure and characterization of the compounds (5′ and 8–16)

8. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-(3-methylpiperidin-1-yl)acetamide

9. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-(4-methylpiperidin-1-yl)acetamide

10. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-morpholinoacetamide

11. Ethyl 1-(2-((4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)amino)-2-oxoethyl)piperidine-4-carboxylate

12. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-(4-methylpiperazin-1-yl)acetamide

13. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-(4-phenylpiperazin-1-yl)acetamide

14. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-(4-propylpiperazin-1-yl)acetamide

15. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-(4-(2-hydroxyethyl)piperazin-1-yl)acetamide

16. N-(4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)-2-(4-(hydroxymethyl)piperidin-1-yl)acetamide

Experimental procedure and characterization of the compounds (17a–17c)

17. 5-Bromo-4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

17b. 4-(4-Bromophenyl)-5-chloro-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

17c. 5-Chloro-4-(4-chlorophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine

Experimental procedure and characterization of the compounds (18a–18c)

18a. N-(5-Bromo-4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)hexanamide

18b. N-(4-(4-Bromophenyl)5-chloro-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)hexanamide

18c. N-(4-(4-Chlorophenyl)5-chloro-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-yl)hexanamide

Biology

General materials and methods

Reagents and chemicals

In vitro study

Mouse calvarial osteoblast cell isolation and culture

Mouse bone marrow cell isolation and culture

Cell differentiation assay (alkaline phosphatase activity)

Cell viability assay

Mineral nodule formation assay (mineralization)

Quantitative PCR

Western blot analysis

Design, synthesis and biological evaluation of novel pyrimidine derivatives as bone anabolic agents promoting osteogenesis via the BMP2/SMAD1 signaling pathway^†