Design and Synthesis of Mycophenolic Acid Analogues for Osteosarcoma Cancer Treatment

Abstract

Mycophenolic acid (MPA), a natural compound, was modified to new MPA analogues via the classical method of silylation and esterification. Their cytotoxicity was evaluated in vitro on four osteosarcoma cancer cell lines (MNNG/HOS, U2OS, 143B, and SaOS-2) and human normal cells (hFOB 1.19). The most potent silicon-containing compound 2d (R¹ = TPS, R² = H) exhibited good cytotoxic activity against all osteosarcoma cancer cell lines with IC₅₀ values ranging from 0.64 to 2.27 μM and showing low cytotoxicity against normal cells. Further investigations revealed that compound 2d (R¹ = TPS, R² = H) displayed significant inhibition of IMPDH2 with K_iapp 1.8 μM. Furthermore, molecular modeling studies were performed to investigate the binding affinity of 2d (R¹ = TPS, R² = H) which can effectively bind to critical amino acids of three proteins (vascular endothelial growth factor receptor 2; VEGFR-2, cyclin-dependent kinase 2; CDK2, inosine-5′-monophosphate dehydrogenase; IMPDH) involved in cancer therapy. This finding suggests that triphenylsilyl-MPA (TPS-MPA) analogue could serve as a promising starting point for developing new anticancer drugs for osteosarcoma.

Introduction

Osteosarcoma (OS) is a primary malignant bone tumor, and 70% of patients are children and adolescents. Although osteosarcoma is a rare malignancy that accounts for 2–5 patients per million people or 5–6% of all childhood tumors but shows a high predisposition to metastasize distributed from the original site to other organ sites, making it more difficult to cure which is the main cause of cancer-related death.^1,2 Nowadays, the treatment for osteosarcoma consists of chemotherapy and surgical resection.³ Chemotherapy is an integral approach to cancer therapy.⁴ Effective treatment of cancers needs accurate delivery of enough intracellular doses of chemo-drugs to kill the cancer cells and have not spread to normal cells.⁵ Recently, the mycophenolate analogues have shown selective cytotoxicity in cancer cells and were less toxic to normal cells in comparison to the lead compound, MPA 1, and a standard drug, ellipticine.⁶

Mycophenolic acid (MPA) was isolated from Penicillium in 1896 by Italian physician Bartolomeo Gosio.^7,8 It comprises a phthalide in which an aromatic ring is substituted with hydroxy, methyl, methoxy, and a six-carbon chain with a double bond in the E configuration and the free carboxylic acid.⁹ MPA acts as an inhibitor of inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo synthesis of guanine nucleotide that causes the inhibition of lymphocyte activity in humans.¹⁰⁻¹² Therefore, this compound is displayed as an immunosuppressive drug in transplantation to protect against acute and chronic transplant rejection.^13,14 Almost all research has focused on MPA and its derivatives for inhibitory immunosuppressive properties, while IMPDH is overexpressed in many types of cancer compared to normal cells. Inhibition of IMPDH can suppress the proliferation of and induce apoptosis in cancer cells.¹⁵ Therefore, MPA and derivatives might function as anticancer agents.¹⁶⁻¹⁸ Recently, rekindled interest has focused on synthesizing novel mycophenolic acid derivatives regarding their anticancer efficacy.

Typically, silicon-containing analogues are considered valuable structural moieties in pharmaceuticals and natural bioactive molecules because silicon-containing compounds are more lipophilic than their carbon counterparts.¹⁹ For instance, karenitecin²⁰ and silatecan²¹ are used clinically to inhibit topoisomerase I with potent anticancer activity. The organosilicon drug cisobitan can significantly inhibit prostate cancer.²² Silicon-containing andrographolide compounds have shown better anticancer activity in human cancer cells than parent andrographolide (Figure 1).^23,24 Given the rich applications of anticancer activity, our design concept was built on previous reports that replacing the silicon moieties would generate new analogues with potential anticancer activity and show good lipophilicity properties.

Figure 1.

Biologically active silicon analogues.

We then focused on exploring new MPA compounds with enhanced anticancer activity. Batovska et al.²⁵ have synthesized a series of hydroxamic acid-MPA derivatives and studied the anticancer activity of these compounds. One of the synthesized compounds bearing lauroyloxy group at C-7 showed potent inhibitory activities against HDACs, which had been proven to exert their effective anticancer activity with an IC₅₀ value of 0.5 μM. In our previous work (2021),⁶ we designed a series of propargylamine mycophenolate derivatives that showed selective inhibition of neuroblastoma (SH-SY5Y) cancer cell lines and were nontoxic to normal cells. Inspired by these works, we designed and synthesized a series of mycophenolic acid derivatives by classical method silylation and esterification and evaluated all synthetic analogues for cytotoxic activity against four OS cancer cells (Figure 2).

Figure 2.

Mycophenolic acid (MPA) 1, and derivatives.

Results and Discussion

Chemistry

The preparation of silyl-MPAs 2 and 3 is outlined in Scheme 1. Silylation of MPA 1 with silyl chloride (R-Cl; R = TBS, TBDPS, TIPS) in DMF afforded derivatives 2a–2c (R¹R² = TBS, TBDPS, TIPS) in 59–98% yields followed by the selective desilylation reaction of 2a–2c (R¹R² = TBS, TBDPS, TIPS) with HCOOH:H₂O (9:1) in THF at 0 °C gave compounds 3a–3c (R¹ = H, R² = TBS, TBDPS, TIPS) in 39–75% yields. Next, triphenylsilyl chloride (TPS) and triphenyl chloride (TrCl) were used as bulky protecting groups. MPA 1 was synthesized using Et₃N as the base in DMF, affording compounds 2d (R¹ = TPS, R² = H) and 2e (R¹ = Tr, R² = H) in yields of 48 and 30%, respectively.

Scheme 1. Synthesis of Silyl and Trityl Analogues of MPA 2 and 3^a.

Reaction conditions (a) R-Cl (8–10 equiv), imidazole (8–10 equiv) 80 °C, DMF, 24 h. (b) HCOOH:H₂O (9:1), THF, 0 °C-rt. (c) R-Cl (3.0 equiv), Et₃N (3.0 equiv), rt, DMF, 24 h, TBS = tert-butyldimethylsilyl, TBDPS = tert-butyldiphenylsilyl, TIPS = triisopropylsilyl, TPS = triphenylsilyl and Tr = triphenyl.

The next series of silyl ester analogues of MPA 4 (R¹ = TBS, TBDPS, TIPS, R² = H) are shown (Scheme 2). The reaction carried out in pyridine and silyl chloride (R¹ = TBS, TBDPS, TIPS) gave the silyl-protected analogues 4b (R¹ = TBDPS, R² = H) and 4c (R¹ = TIPS, R² = H) in 66 and 55% yield, respectively while 4a (R¹ = TBS, R² = H) was not stable.

Scheme 2. Synthesis of Silyl Ester Analogues of MPA 4^a.

Reaction conditions: MPA 1 (100 mg, 0.3121 mmol), pyridine 0.5 mL, R¹-Cl (0.4682 mmol). TBS = tert-butyldimethylsilyl, TBDPS = tert-butyldiphenylsilyl, TIPS = triisopropylsilyl.

Further, the MPA 1 was converted to the phthalimide ester analogue of MPA 6a in low yield due to the phenol group in MPA 1 not being protected, as shown in Scheme 3.

Scheme 3. Synthesis of Phthalimide Ester of MPA 6a^a.

Reaction conditions: MPA 1 (0.3121 mmol), DMAP 0.03125 mmol), EDCI (0.3125 mmol), N-hydroxyphthalimide (0.4688 mmol) in DCM 2.0 mL. DMAP = 4-dimethylaminopyridine, EDCI = 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide.

Through this method, we can obtain the esterification products in high yield when changing the substrate. TBDPS-protected phenol of MPA 3b (R¹ = H, R² = TBDPS) was utilized as a starting material for the synthesis of new mycophenolate analogues 5a–5c by reactions with different alcohol compounds, namely, N-hydroxyphthalimide, 2-(4-fluorophenyl)ethanol and 2-(4-methoxyphenyl)ethanol affording 5a–5c in 65–98% yields respectively (Scheme 4).

Scheme 4. Synthesis of Mycophenolate Analogues 5a–5c.

Reaction conditions: tert-butyldiphenylsilyl ether-MPA product (3b) (0.090 mmol), DMAP (0.0090 mmol), EDCI (0.3125 mmol), R¹–OH (0.135 mmol) in DCM 1.0 mL. DMAP = 4-dimethylaminopyridine, EDCI = 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide.

For the derivatives of the unprotected phenol, desilylation of compounds 5b and 5c using TBAF in MeOH, furnishing 6b and 6c in a yield of 50 and 99% respectively (Scheme 5).

Scheme 5. Synthesis of Mycophenolic Acid Derivatives 6b and 6c^a.

Reaction conditions: 5b and 5c (50 mg), TBAF (0.02793 mmol) in MeOH 1.0 mL at 0 °C for 30 min. TBAF = tetrabutylammonium fluoride.

Cytotoxic Studies

Cell Viability Using MTT Assay

The cell viability and survival of the cells will be evaluated using a microculture tetrazolium (MTT) assay. MNNG/HOS, U2OS, 143B, and SaOS-2 human osteosarcoma cell lines, and human normal osteoblastic cell lines (hFOB 1.19) were obtained from the ATCC. Osteosarcoma cells at 5 × 10³ cells will be seeded in each well of a 96-well plate and allowed to attach at 37 °C in a 5% CO₂ incubator overnight. On the following day, the cells will be treated with mycophenolic acid derivatives for 72 h. After the treatment period, culture media will be removed and replaced with 100 μL of freshly prepared 0.5 mg/mL MTT (A2231, AppliChem, Germany) in a culture medium and incubated at 37 °C for 2 h. After incubation, the reagent will be removed. Formazan crystals will be then dissolved in 100 μL of DMSO with agitation on a plate shaker. The optical density (OD) will be measured using a microplate reader at 570 nm wavelength. Survival rate of cell (%) = (OD of experiment group/OD of the control group) × 100%. The studies were performed in triplicate.

Measurement of IMPDH Activity in Osteosarcoma Cells

The inhibitory assay of human IMPDH1 and IMPDH2 (R&D Systems, USA) was performed by varying concentrations of the candidate IMPDH inhibitors and MPA (0.001 to 100 μM) with fixed concentrations of IMP (100 μM for IMPDH1 and 250 μM for IMPDH2) and NAD⁺ (200 μM for IMPDH1 and 500 μM for IMPDH2). The assays were performed in assay buffers under the following conditions: IMPDH1; 50 mM Tris, 300 mM NaCl, pH8.0; and IMPDH2; 50 mM Tris, 300 mM NaCl, 1 mM EDTA, and 1 mM DTT, pH 8.0. First, 25 μL of varying concentrations of candidate IMPDH inhibitors and MPA were loaded into a 96-well UV plate (Greiner Bio-one, Germany) followed by 25 μL of 40 μg/mL of the rhIMPDH enzyme (1 μg). For reaction control, 25 μL of assay buffer was loaded instead of the inhibitor. For the substrate blank, 50 μL of assay buffer was loaded. The reactions were started by adding 50 μL of 2× substrate mixture (IMP and NAD⁺). Kinetic analysis of IMPDH enzyme activity was performed by the measurement of NADH production at a wavelength of 340 nm every 1 min for 5 min. The enzyme velocity (μM/min) was calculated from absorbance using the molar extinction coefficient of the absorbing molecule in Beer’s law equation (6220 M^–1 cm^–1).

The apparent tight-binding inhibition constants (K_i,app) were calculated from enzyme velocity following Morrison’s eq 2 to assess the strength of enzyme/inhibitor binding using the Dynafit program.²⁶⁻²⁸

1

In eq 1, v₀ is the initial velocity in the absence of the inhibitor; [E] is the enzyme concentration; and [I] is the inhibitor concentration.

MPA 1 and mycophenolic acid analogues 2–6 were screened to inhibit the growth of four osteosarcoma cell lines and the IC₅₀ values were listed in Table 1. The results showed that MPA 1 exhibited cytotoxicity on all OS cell lines with IC₅₀ ranging from 0.64 to 4.00 μM. Modification by silylation of MPA 1 to give silyl-protected analogues of MPA (R¹R² = TBS; 2a) and (R¹R² = TBDPS; 2b) displayed high activity against all OS cell lines with IC₅₀ values ranging from 1.36 to 5.43 and 1.18–5.85 μM, respectively. The comparison of the results shows that the activity of (R¹R² = TBS; 2a) and (R¹R² = TBDPS; 2b) was better than analog (R¹R² = TIPS; 2c) with a bulky group (IC₅₀ 6.82–63.85 μM) in terms of in vitro cytotoxic activity against all OS cell lines (Table 1, entries 2–4). In MNNG cells, compound 2d (R¹ = TPS, R² = H) with a triphenyl silyl substitution and nonsubstitution of phenol showed great cytotoxicity of 0.64 μM, which is similar to MPA 1 (0.64 μM). Another triphenyl group in compound 2e (R¹ = Tr, R² = H) also displayed remarkable cytotoxicity against MNNG (IC₅₀ = 0.68 μM) and U2OS cells (0.98 μM) respectively. Compounds 2d (R¹ = TPS, R² = H) and 2e (R¹ = Tr, R² = H) exhibited good inhibition against all OS cell lines with IC₅₀ values ranging from 0.64 to 2.27 and 0.68–5.13 μM. The triphenyl group may interact well with the target molecule, enhancing binding and biological activity. Modification of MPA 1 by introducing TBDPS and TIPS group 4b (R¹ = TBDPS, R² = H) and 4c (R¹ = TIPS, R² = H at R¹ position led to the appearance of cytotoxicity against U2OS with an IC₅₀ value of 1.85 and 0.92 μM respectively (Table 1, entries 10 and 11). Remarkably, the free phenol of MPA derivatives 2d (R¹ = TPS, R² = H), 2e (R¹ = Tr, R² = H), 4b (R¹ = TBDPS, R² = H) and 4c (R¹ = TIPS, R² = H) with various R¹ (TPS-, Tr-, TBDPS-, TIPS-) of carboxylic acid analogues showed enhancement in cytotoxic activity. It should be noted that the inhibitory activities against all OS cell lines were somewhat positively improved by introducing the free phenol group. Meaningfully, the inhibitory activities of free carboxylic acid (3a–3c; R¹ = H, R² = TBS, TBDPS, TIPS) against all OS cell lines were relatively better than (2a–2c; R¹R² = TBS, TBDPS, TIPS), especially 3a (R¹ = H, R² = TBS), which had a TBS-group substituent placed at the R² position shown as the most attractive molecule with an IC₅₀ value of 0.86 μM in MNNG cell line. Compounds 3b (R¹ = H, R² = TBDPS) and 3c (R¹ = H, R² = TIPS) having a TBDPS- and TIPS- substituent at the R² position were found to be active against MNNK cancer cells with IC₅₀ 1.15 and 2.68 μM respectively (Table 1, entries 8 and 9).

Table 1. In Vitro Cytotoxicity, of MPA 1, and Derivatives 2–6.

entry	compd.	cytotoxic activity IC50 (μM)a
		143B	MNNG	U2OS	SaOS
1	MPA 1	1.55 ± 0.49	0.64 ± 0.08	0.68 ± 0.04	4.00 ± 0.28
2	2a	4.12 ± 0.28	1.36 ± 0.76	3.80 ± 0.88	5.43 ± 2.87
3	2b	5.85 ± 0.54	1.18 ± 0.74	2.52 ± 0.96	5.80 ± 1.15
4	2c	63.15 ± 3.20	6.82 ± 3.51	24.43 ± 0.04	17.38 ± 9.03
5	2d	1.65 ± 0.26	0.64 ± 0.15	1.01 ± 0.40	2.27 ± 0.68
6	2e	5.13 ± 1.10	0.68 ± 0.11	0.98 ± 0.035	4.83 ± 0.50
7	3a	3.90 ± 0.76	0.86 ± 0.20	2.31 ± 1.26	4.55 ± 2.60
8	3b	4.74 ± 0.76	1.15 ± 0.35	2.96 ± 2.32	4.58 ± 1.05
9	3c	6.31 ± 0.40	2.68 ± 1.02	3.75 ± 1.06	5.44 ± 0.82
10	4b	6.80 ± 1.41	13.00 ± 4.24	1.85 ± 0.07	1.85 ± 0.35
11	4c	35.00 ± 9.90	5.70 ± 0.71	0.92 ± 0.00	5.50 ± 1.51
12	5a	31.00 ± 1.41	19.50 ± 3.54	10.25 ± 3.89	12.00 ± 0.00
13	5b	ND	ND	ND	ND
14	5c	14.50 ± 4.95	43.50 ± 2.12	10.25 ± 1.06	39.5 ± 0.71
15	6a	4.85 ± 1.48	ND	1.38 ± 0.60	6.90 ± 0.00
16	6b	2.00 ± 0.14	ND	7.00 ± 2.12	5.75 ± 1.06
17	6c	2.60 ± 0.28	38.00 ± 11.31	7.35 ± 1.63	9.00 ± 1.41

^aIC₅₀ values (drug concentration causing 50% growth inhibition) in μM, ND = Not determined.

On the contrary, the esterification products 5a–5c with TBDPS substitution at the R² position on the phenol resulted in lower activities with IC₅₀ more than 10 μM toward all OS cancer cells. Compound 5b, having a 4-fluorophenethylester, performed less efficiently than the rest of the phthalimide ester 5a and 4-methoxyphenethylester 5c. Due to the free phenol of MPA 1 being a key position in the cytotoxic activity. Attempts to enhance the cytotoxicity resulted in the removal of the TBDPS group at the R² position. Analogues 6a–6c based on ester analogues 5a–5c returned more promising levels of cytotoxicity. Compounds 6a–6c showed IC₅₀ values in the range of 1.38–9.00 μM in three OS cell lines and no cytotoxic effects on the MNNG cell line (Table 1, entries 15–17). Among the three compounds tested (6a–6c), phthalimide ester 6a exhibited remarkable cytotoxicity in U2OS cell lines showing an IC₅₀ of 1.38 μM and better than 5a. Therefore, the analogues that were obtained after deprotection of R² at the phenol showed better activity profiles than all of the corresponding protected analogues.

Among all compounds examined for cytotoxicity in Table 1, compound 2d (R¹ = TPS, R² = H) was the most potent against all four OS cancer cell lines. Silicon incorporation can be used to increase the biological activity of the target molecule.²⁹ Therefore, we studied the effect of silicon 2d (R¹ = TPS, R² = H) and carbon 2e (R¹ = Tr, R² = H) on the cytotoxic activity. In the cases of triphenyl silyl analogue 2d (R¹ = TPS, R² = H) was approximately 2 to 3-fold more potent than its corresponding carbon compound 2e (R¹ = Tr, R² = H) by inhibiting two specific OS cancer cell lines (143B and SaOS). Also, the lactone function, phenol, and triphenyl group in compound 2d (R¹ = TPS, R² = H) can engage in various interactions with biomolecular targets, including hydrogen bonding, π–π stacking, and dipole interactions (Figure 3). The study suggests that the incorporation of silicon (Si) into structure 2d (R¹ = TPS, R² = H) led to an increase in its cytotoxic activity.

Figure 3.

Most active compound of MPA derivative 2d.

Regarding the safety of 2d (R¹ = TPS, R² = H) toward the normal cell line, compound 2d (R¹ = TPS, R² = H) showed low cytotoxicity to human normal osteoblast cells (hFOB 1.19) at the tested concentrations, introducing that this compound is much less cytotoxic to normal cells as compared to OS cancer cells (Table 2). Therefore, compound 2d (R¹ = TPS, R² = H) led to significant development in cytotoxicity against the OS cancer cell line and nontoxic on the normal cell line (hFOB 1.19) compared with MPA 1.

Table 2. Cytotoxic Effect of Mycophenolic Acid Analogues in Osteoblast Cell Lines hFOB 1.19.

compounds	cell viability of hFOB 1.19a ± SD (%)
	0.1 μM	1.0 μM	10.0 μM	100.0 μM
control	100
MPA 1	74.3 ± 4.24	20.14 ± 1.41	7.13 ± 0.67	3.38 ± 3.40
2d	93.91 ± 1.01	91.95 ± 1.78	84.5 ± 2.95	73.34 ± 1.39

^aCell viability (%): the cell viability in control was taken as 100%, all data were expressed as mean ± SD (n = 3). hFOB 1.19 = human fetal osteoblastic cell line.

Inosine monophosphate dehydrogenase 2 (IMPDH2) is an enzyme that plays an important role in the biosynthesis of guanine nucleotides, which are essential for DNA and RNA synthesis, and related to cell differentiation, tumor transformation, and rapidly increasing tumor cells.^30,31 Cancer cells often require elevated levels of nucleotides to support their uncontrolled growth and division.^31,32 In some cases, overexpression or increased activity of IMPDH2 has been observed in cancers such as the fatal brain tumor glioblastoma, acute myelogenous leukemia (AML), and chronic myelogenous leukemia (CML).^33,34 From these results, it might be possible to cure some cancers by blocking the IMPDH-mediated de novo pathway of guanine nucleotide production.³⁵ Therefore, a renewed determination is being made on the anticancer potential of IMPDH inhibitors. MPA 1 is an inhibitor of IMPDH and is a known anticancer and immunosuppressive agent. Table 3 illustrates that MPA 1 has promising activity against both IMPDH 1 and IMPDH 2 enzymes with K_i,app values of 0.136 and 0.046 μM, respectively. In our study, MPA 1 was further modified to explore new and selective inhibitors of IMPDH2. However, we found that compound 2d (R¹ = TPS, R² = H) did not exhibit potent inhibition of either IMPDH1 or IMPDH2 compared to that of MPA 1.

Table 3. Selected Inhibitors of IMPDH.

compounds	Ki,app (μM)a
	IMPDH1	IMPDH2
MPA 1	0.136	0.046
2d	NO	1.8

^aThe apparent tight-binding inhibition constants (K_i,app) in μM, NO = No inhibition observed.

Computational Docking

Molecular docking calculations for model compounds were employed to illustrate the factors resolving the cytotoxic activity of the studied MPA derivatives. MPA analogues were explored for the first time on cytotoxicity to osteosarcoma cancer cells, confirming the important role played by the molecular docking method in understanding the interaction mode of MPA derivatives.

Vascular endothelial growth factor (VEGF) is a highly specific vascular endothelial growth factor overexpressed in tumor cells which release higher levels of VEGF compared to normal cells.^36,37 The VEGFR-2 subtype is the most critical regulator of the angiogenesis process and plays an important role in the dissolution, migration, and proliferation of endothelial cancer cells. Targeting VEGFR-2 specifically can be an effective way to inhibit the angiogenic process in osteosarcoma and potentially slow down tumor growth and metastasis.³⁸ Therefore, the anti-VEGFR-2 subtype is an essential form of suppressing OS tumor growth and metastasis.

Molecular docking was employed to explain the promising VEGFR-2 inhibitory activity of the mycophenolic acid analogues by finding their binding mode and their interaction with the critical amino acids (Glu885, Cys919, and Asp1046) in the active site of the VEGFR-2. In our work, we selected (PDB ID: 4ASD) as the protein data bank for VEGFR-2 to screen three compounds (MPA 1, 2d, and 5c) in molecular docking studies.³⁹

MPA 1 displayed the binding energy values of −6.60 kcal/mol with VEGFR-2 and was involved in strong hydrogen bonding with the key amino acids Cys919 and Asp1046 via the carbonyl of lactone, phenol, and carboxylic acid at a distance of 2.05, 2.24, and 1.78 Å respectively because these three functional groups highly boosted cytotoxic efficiency (Figure 4). Compound 2d (R¹ = TPS, R² = H) exhibits the best experimental cytotoxic activity against all four OS cancer cell lines with IC₅₀ ranging from 0.64 to 2.27 μM and showed the highest predicted binding affinity with a docking score of–9.11 kcal/mol. Also, the hybridization of the triphenyl silyl group with the MPA scaffold 2d (R¹ = TPS, R² = H) binds in the same favorable hydrogen bond and with the backbone of Asp1046 via the oxygen atom of phenol (distance: 2.05 Å) as shown in Figure 5. The triphenyl silyl substitution of carboxylic acid increases the hydrophobic interaction with the key amino acid Glu885 and Asp1046 enhancing the binding interaction, as can be shown in its docking score (−9.11 kcal/mol) when compared to the MPA 1 (−6.60 kcal/mol) (Table 4). Due to the low cytotoxic activity of all four OS cancer cell lines in this series, compound 5c was selected to study molecular docking interactions in the active site of VEGFR-2. Docking results of compound 5c showed poor binding affinity and did not bind at the key amino acids of the active site which may be because of steric clashes with the VEGFR-2 binding site (Table 4). Accordingly, the obtained results confirmed that the molecular docking was consistent with the cytotoxic activities of the tested compounds.

Figure 4.

Proposed binding mode of MPA 1 in the active site of VEGFR-2 (PDB code: 4ASD).

Figure 5.

Proposed binding mode of compound 2d in the active site of VEGFR-2 (PDB code: 4ASD).

Table 4. Molecular Docking Analysis of VEGFR-2 with MPA 1 and Mycophenolic Acid Analogues 2d and 5c^a.

compounds	binding energy (kcal/mol)	intermolecular hydrogen bonding			intermolecular hydrophobic contacts
		amino acids interaction	distance (Å)	interaction groups
MPA 1	–6.60	Asp1046	1.78	CO of lactone	Val916, Lys868, Val899, Cys1045
		Asp1046	2.24	OH of phenol
		Cys919	2.05	OH of COOH
2d	–9.11	Asp1046	2.05	OH of phenol	Asp1046, Ala881, Glu885, Leu1049, Cys1045, Lys868, Val916
5c	–6.81				Asp1046, Leu1049, Arg1027, Val898, Leu1019

^aThe binding energies were evaluated using AutoDock.

The cyclin-dependent kinases (CDKs) play an essential role in cell cycle progression and are validated as cancer therapy targets. CDK2 is commonly overexpressed in human tumors and is necessary for the proliferation of tumor cells.^40,41 Although rare, osteosarcoma is an aggressive cancer that often metastasizes to other organs. In addition, the research studies evidenced that the cyclin-dependent kinase (CDK) inhibitor proved to induce the apoptosis of several osteosarcoma cell lines.⁴² The molecular docking study was employed to understand the binding of the most potent compound 2d (R¹ = TPS, R² = H) from the Protein Data Bank with PDB code 2a4l to study their interactions with the key amino acids in the active site of the CDK2 enzyme to explain the structure–activity relationship (SARs) (Table 5).⁴³

Table 5. Molecular Docking Analysis of CDK2 with Mycophenolic Acid Analogue 2d^a.

compounds	binding energy (kcal/mol)	intermolecular hydrogen bonding			intermolecular hydrophobic contacts
		amino acids interaction	distance (Å)	interaction groups
2d	–8.63	Leu83	2.15	OH of phenol	Val18, Leu134, Asp86
		Lys89	1.84	CO of ester

^aThe binding energies were evaluated using AutoDock.

As shown in the docking mode of compound 2d (R¹ = TPS, R² = H) with CDK2, the phenol and ester of 2d (R¹ = TPS, R² = H) have the potential to form two hydrogen bonds with the essential Leu83 and Lys89 residues. Moreover, the triphenylsilyl group and aromatic phenol are extended in the pocket to form a hydrophobic interaction with the surrounding Asp86 and His84 amino acids (Figure 6). To better understand the SARs study shows that the free phenol group attached to MPA can form favorable interactions in the active site of CDK2.

Figure 6.

Proposed binding mode of compound 2d in the active site of CDK2 (PDB code: 2a4l).

To provide a basis for identifying IMPDH inhibitors as anticancer agents, molecular docking was conducted to show the potential binding mode of compound 2d (R¹ = TPS, R² = H) with the IMPDH enzymes (PDB: 1JR1).^44,45 This compound could interact with the key amino acid residues in the active site of the enzyme, including hydrogen-bonding interactions between the carbonyl group of the lactone ring and THR333. Similarly, the phenol group on the compound 2d (R¹ = TPS, R² = H), as a hydrogen donor, also interacted with THR333. The oxygen atom of lactone forms a hydrogen bonding interaction with the backbone NH moiety of GLY326 that is necessary for efficient inhibitors. At the same time, the carbonyl group of ester (COOSiPh₃) forms a hydrogen bonding interaction with Ser276 (Table 6 and Figure 7).

Table 6. Molecular Docking Analysis of IMPDH with Mycophenolic Acid Analogue 2d^a.

compounds	binding energy (kcal/mol)	intermolecular hydrogen bonding			intermolecular hydrophobic contacts
		amino acids interaction	distance (Å)	interaction groups
2d	–8.02	THR333	2.62	OH of phenol	SER275, PHE282
		THR333	2.01	CO of lactone
		GLY326	2.09	O of lactone
		SER276	1.75	CO of ester

^aThe binding energies were evaluated using AutoDock.

Figure 7.

Proposed binding mode of compound 2d in the active site of IMPDH (PDB code: 1JR1).

Conclusions

In this work, a new series of mycophenolic acid analogues 2–6 were synthesized by using convenient reactions to enhance the cytotoxic activity of MPA. All synthetic analogues were tested for cytotoxicity against four OS cancer cell lines. The triphenyl silyl analog 2d (R¹ = TPS, R² = H) showed potent cytotoxicity and selectivity to OS cancer cells than MPA 1 and displayed the strongest IC₅₀ values ranging from 0.64 to 2.27. The triphenyl analogue 2e (R¹ = Tr, R² = H) also showed high cytotoxicity (IC₅₀ = 0.68–5.13 μM). The triphenyl group and free phenol group in MPA play a significant impact on cytotoxicity. The molecular docking represents the poses of triphenyl silyl-MPA 2d (R¹ = TPS, R² = H) on three cancer proteins (VEGFR-2, CDK2, and IMPDH). Computational studies revealed that compound 2d (R¹ = TPS, R² = H) possesses a high affinity for VEGFR-2 (−9.11 kcal/mol), CDK2 (−8.63 kcal/mol), and IMPDH (−8.02 kcal/mol). Our results showed the triphenyl silyl of MPA plays a very significant role in cytotoxic activity against cancer cell lines. Our synthetic MPA analogues might serve as a starting point of building blocks for future structure modification against OS cancer cells.

Experimental Section

General Information

All chemicals and mycophenolic acid as a starting material (CAS NO. 24280-93-1) were purchased from commercial sources and used without further purification. Proton NMR spectra were recorded by using a BRUKER AVANC (400 MHz) spectrometer. All spectra were recorded in the CDCl₃ solvent, chemical shifts are reported as δ values in parts per million (ppm) relative to tetramethylsilane (δ 0.00), and CDCl₃ (δ 7.26) was used as an internal standard. Carbon NMR spectra were recorded on a BRUKER AVANC (100 MHz) spectrometer. All spectra were recorded in CDCl₃ solvent and chemical shifts are reported as δ values in parts per million (ppm) relative to CDCl₃ (δ 77.0) as an internal standard. High-resolution mass spectrometry (HRMS) data were recorded at Naresuan University. Analytical thin-layer chromatography (TLC) was conducted on precoated TLC plates; silica gel 60F-254 [E. Merck, Darmstadt, Germany]. Silica gel columns for open-column chromatography utilized silica gel 60 PF254 [E. Merck, Darmstadt, Germany]. Melting points were measured using a melting point apparatus (Griffin) and are uncorrected.

General Procedure for the Preparation of Silyl Ether-MPA Analogues (2a–2c)

MPA 1 (100 mg, 0.3121 mmol), silyl chloride (R-Cl; R = TBS, TBDPS, TIPS) (1.8726 mmol), and imidazole (170 mg, 2.4968 mmol) were dissolved in dry DMF (2 mL), and the reaction mixture was stirred at room temperature for 24 h. After TLC showed the reaction completed, the mixture was quenched with saturated NH₄Cl solution, then extracted with EtOAc, and washed with brine. The combined organic layers were dried over anhydrous Na₂SO₄ and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography.

Synthesis of tert-Butyldimethylsilyl (E)-6-(4-((tert-Butyldimethylsilyl)oxy)-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (2a)

MPA 1 (100 mg, 0.3121 mmol), tert-butyldimethylsilyl chloride (282 mg, 1.8726 mmol), and imidazole (170 mg, 2.4968 mmol) were dissolved in dry DMF (2 mL), and the reaction mixture was stirred at room temperature for 24 h. The crude product 2a was purified by column chromatography (6:1 n-hexane:EtOAc) to afford compound 2a (112.5 mg) as a white solid in 66% yield.

2a: A white solid in 66% yield; Mp: 76.6–80.6 °C; R_f = 0.79 (2:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 5.18 (1H, t, J = 6.4 Hz), 5.05 (2H, brs), 3.73 (3H, s), 3.37 (2H, d, J = 6.4 Hz), 2.40–2.33 (2H, m), 2.30–2.22 (2H, m), 2.14 (3H, s), 1.75 (3H, s), 1.02 (9H, s), 0.88 (9H, s), 0.23 (6H, s), 0.18 (6H, s); ¹³C NMR (100 MHz, CDCl₃): δ 173.8, 169.2, 163.2, 151.4, 146.0, 135.2, 133.8, 127.7, 125.0, 123.3, 117.9, 111.6, 67.6, 60.7, 34.7, 34.6, 32.2, 26.4, 26.0, 25.6, 25.5, 23.6, 23.4, 18.7, 17.5, 16.4, 11.4, −3.6, −4.9 ppm; HRMS (m/z): calcd for C₂₉H₄₈O₆Si₂Na [M + Na]⁺ 571.2887, found: 571.2886.

Synthesis of tert-Butyldiphenylsilyl (E)-6-(4-((tert-Butyldiphenylsilyl)oxy)-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (2b)

MPA 1 (100 mg, 0.3121 mmol), tert-butyldiphenylsilyl chloride (481 μL, 1.8726 mmol), and imidazole (170 mg, 2.4968 mmol) were dissolved in dry DMF (2 mL), and the reaction mixture was stirred at room temperature for 24 h. The crude product 2b was purified by column chromatography (6:1 n-hexane:EtOAc) to afford compound 2b (245 mg) as a white oil in 98% yield.

2b: A white oil in 98% yield; R_f = 0.74 (2:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 7.69 (4H, d, J = 6.8 Hz), 7.64 (4H, d, J = 6.8 Hz), 7.50–7.28 (12H, m), 5.10–4.95 (3H, m), 3.59 (3H, s), 3.16 (2H, d, J = 5.6 Hz), 2.50–2.40 (2H, m), 2.23 (2H, t, J = 8.4 Hz), 2.11 (3H, s), 1.48 (3H, s), 1.09 (9H, s), 1.07 (9H, s) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 172.4, 168.3, 163.1, 151.5, 145.9, 135.2, 134.9, 134.7, 133.6, 133.5, 131.8, 129.9, 129.4, 129.3, 127.6, 127.2, 123.4, 117.8, 111.1, 67.3, 60.5, 34.6, 34.3, 26.8, 26.4, 24.2, 20.3, 19.0, 16.2, 11.2 ppm; HRMS (m/z): calcd for C₄₉H₅₆O₆Si₂Na [M + Na]⁺ 819.3513, found: 819.3513.

Synthesis of Triisopropylsilyl (E)-6-(6-Methoxy-7-methyl-3-oxo-4-((triisopropylsilyl)oxy)-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (2c)

MPA 1 (100 mg, 0.3121 mmol), triisopropylsilyl chloride (400 μL, 1.8726 mmol), and imidazole (170 mg, 2.4968 mmol) were dissolved in dry DMF (2 mL), and the reaction mixture was stirred at room temperature for 24 h. The crude product 2c was purified by column chromatography (5:1 n-hexane:EtOAc) to afford compound 2c (117.4 mg) as a white solid in 59% yield.

2c: A white solid in 59% yield; Mp: 54–58 °C; R_f = 0.68 (2:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 5.16 (1H, t, J = 4.8 Hz), 5.06 (2H, s), 3.72 (3H, s), 3.39 (2H, d, J = 6.0 Hz), 2.43–2.38 (2H, m), 2.37–2.26 (2H, m), 2.14 (3H, s), 1.77 (3H, s), 1.56–1.48 (3H, m), 1.27–1.20 (3H, m), 1.24–1.00 (36H, m) ppm; ¹³C NMR (100 MHz, CDCl₃): δ173.3, 169.4, 163.1, 152.7, 145.8, 133.8, 127.3, 123.7, 117.3, 111.0, 67.6, 60.6, 34.5, 23.6, 18.0, 17.6, 16.4, 14.3, 11.3, 11.0 ppm; HRMS (m/z): calcd for C₃₅H₆₀O₆Si₂Na [M + Na]⁺ 655.3826, found 655.3826.

General Procedure for the Preparation of Triphenyl Ether-MPA Analogues (2d–2e)

MPA 1 (200 mg, 0.6240 mmol), R-Cl (R = Tr, TPS) (1.872 mmol), and Et₃N (263 μL, 1.872 mmol) were dissolved in dry DMF (2 mL), and the reaction mixture was stirred at room temperature for 24 h. After TLC showed the reaction was completed, the mixture was quenched with saturated NH₄Cl solution, then extracted with EtOAc, and washed with brine. The combined organic layers were dried over anhydrous Na₂SO₄ and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography.

Synthesis of Triphenylsilyl (E)-6-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (2d)

MPA 1 (200 mg, 0.6240 mmol), triphenylsilyl chloride (552 mg, 1.872 mmol), and Et₃N (263 μL, 1.872 mmol) were dissolved in dry DMF (2 mL), and the reaction mixture was stirred at room temperature for 24 h. The crude product 2e was purified by column chromatography (3:1 n-hexane:EtOAc) to afford compound 2e (175 mg) as a white solid in 48% yield.

2d: A white solid in 48% yield; Mp: 114–120 °C; R_f = 0.53 (1:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 7.61 (6H, d, J = 6.8 Hz), 7.42–7.37 (4H, m), 7.33 (2H, s), 7.31 (3H, s), 7.28 (1H, s), 4.98 (1H, t, J = 5.2 Hz), 4.87 (2H, s), 3.67 (3H, s), 3.17 (2H, d, J = 6.0 Hz), 2.31 (2H, t, J = 7.2 Hz), 2.17 (2H, t, J = 8.4 Hz), 2.09 (3H, s), 1.54 (3H, s) ppm; ¹³C NMR (100 MHz, CDCl₃): δ178.6, 168.4, 162.9, 151.2, 145.4, 135.7, 134.9, 133.5, 133.2, 130.0, 129.9, 127.8, 127.7, 127.4, 123.7, 118.3, 111.8, 67.4, 60.7, 33.9, 32.5, 23.7, 16.1, 11.3 ppm; HRMS (m/z): calcd for C₃₅H₃₄O₆SiNa [M + Na]⁺ 601.2022, found: 601.2022.

Synthesis of Trityl (E)-6-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (2e)

MPA 1 (200 mg, 0.6240 mmol), trityl chloride (528 mg, 1.872 mmol), and Et₃N (263 μL, 1.872 mmol) were dissolved in dry DMF (2 mL), and the reaction mixture was stirred at room temperature for 24 h. The crude product 2d was purified by column chromatography (5:1 n-hexane:EtOAc) to afford compound 2d (103 mg) as a white solid in 30% yield.

2e: A white solid in 30% yield; Mp: 92–98 °C; R_f = 0.15 (5:1 n-hexane:EtOAc) ¹H NMR (400 MHz, CDCl₃): δ 7.59 (1H, s), 7.28–7.23 (6H, m), 7.23–7.12 (9H, m), 5.20 (1H, t, J = 6.8 Hz), 5.12 (2H, s), 3.65 (3H, s), 3.31 (2H, d, J = 6.8 Hz), 2.43–2.38 (2H, m), 2.24 (2H, t, J = 8.0 Hz), 2.06 (3H, s), 1.72 (3H, s) ppm; ¹³C NMR (100 MHz, CDCl₃): δ172.9, 171.0, 163.7, 153.7, 146.9, 144.1, 143.4, 134.3, 128.4, 128.0, 128.0, 127.9, 127.7, 127.3, 127.2, 122.6, 116.8, 106.4, 89.8, 82.0, 70.1, 61.0, 34.3, 22.6, 16.3, 11.6 ppm; HRMS (m/z): calcd for C₃₆H₃₄O₆Na [M + Na]⁺ 585.2253, found: 585.2252.

General Procedure for the Preparation of Silyl Ether-MPA Analogues (3a–3c)

MPA 1, silyl chloride (R-Cl; R = TBS, TBDPS, TIPS) (10 equiv), and imidazole (10 equiv) were dissolved in dry DMF (5 mL), and the reaction mixture was stirred at rt-80 °C for 24 h. After TLC showed that the reaction was completed, the mixture was quenched with saturated NH₄Cl solution. Then, extracted with EtOAc and washed with brine, the combined organic layers were dried over anhydrous Na₂SO_4, and the solvent was removed by a rotary evaporator. The crude product 2 was dissolved in THF (5 mL) and then was added HCOOH:H₂O (9:1) (5 mL) at 0 °C to the mixture and allowed to stir at room temperature for a further 2 h. After TLC indicated that the reaction was complete, the reaction mixture was diluted with EtOAc (10 mL) and quenched with cold saturated NaHCO₃, extracted with EtOAc and washed with brine, then dried over with Na₂SO₄ anhydrous, and concentrated in vacuo to obtain crude product 3. The crude product was purified by column chromatography (n-hexane:EtOAc).

Synthesis of (E)-6-(4-((tert-Butyldimethylsilyl)oxy)-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoic acid (3a)

MPA 1 (100 mg, 0.3121 mmol), tert-butyldimethylsilyl chloride (470 mg, 3.121 mmol), imidazole (212 mg, 3.121 mmol) were dissolved in dry DMF (5 mL), and the reaction mixture was stirred at 80 °C for 24 h to obtain crude product 2a. The crude product was dissolved in THF (5 mL) and then was added HCOOH:H₂O (9:1) (5 mL) at 0 °C to the mixture and allowed to stir at room temperature for a further 2 h. The crude product was purified by column chromatography (3:1 n-hexane:EtOAc) to afford compound 3a (52.9 mg) as a white solid in 39% yield in two steps.

3a: A white solid in 39% yield; Mp: 122–124 °C, R_f = 0.61 (1:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 7.29 (1H, s), 5.20 (1H, t, J = 5.6 Hz), 5.07 (2H, s), 3.74 (3H, s), 3.38 (2H, d, J = 6.0 Hz), 2.41 (2H, t, J = 6.4 Hz), 2.30 (2H, t, J = 7.6 Hz), 2.15 (3H, s), 1.76 (3H, s), 1.02 (9H, s), 0.23 (6H, s) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 179.2, 169.2, 163.2, 151.7, 146.1, 138.4, 127.6, 123.9, 117.9, 111.6, 67.7, 60.7, 34.1, 32.7, 26.0, 23.7, 18.7, 16.3, 11.4, −3.6 ppm; HRMS (m/z): calcd for C₂₃H₃₄O₆SiNa [M + Na]⁺ 457.2022, found: 457.2019.

Synthesis of (E)-6-(4-((tert-Butyldimethylsilyl)oxy)-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoic acid (3b)

MPA 1 (500 mg, 1.5625 mmol), tert-butyldiphenylsilyl chloride (2.4 mL, 9.375 mmol), and imidazole (638 mg, 9.375 mmol) were dissolved in dry DMF (10 mL), and the reaction mixture was stirred at room temperature for 24 h to obtain crude product 2b. The crude product was dissolved in THF (10 mL) and then HCOOH:H₂O (9:1) (10 mL) at 0 °C was added to the mixture and allowed to stir at room temperature for a further 2 h. The crude product was purified by column chromatography (3:1 n-hexane:EtOAc) to afford compound 3b (0.6573 g) as a sticky white oil in 75% yield in two steps.

3b: A yellow oil in 75% yield; R_f = 0.34 (2:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 7.68 (4H, d J = 6.8 Hz), 7.40–7.28 (6H, m), 5.00–4.92 (4H, m), 3.60 (3H, s), 3.15 (2H, d, J = 5.6 Hz), 2.27 (2H, t, J = 7.2 Hz), 2.15 (2H, t, J = 8.4 Hz), 2.10 (3H, s), 1.46 (3H, s), 1.08 (9H, s) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 178.6, 168.4, 163.1, 151.5, 145.9, 134.9, 133.7, 129.4, 127.3, 123.5, 117.8, 111.2, 67.4, 60.5, 34.3, 29.6, 26.4, 24.2, 20.3, 16.0, 11.2 ppm; HRMS (m/z): calcd for C₃₃H₃₈O₆SiNa [M + Na]⁺ 581.2335, found: 581.2335.

Synthesis of (E)-6-(6-Methoxy-7-methyl-3-oxo-4-((triisopropylsilyl)oxy)-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoic acid (3c)

MPA 1 (100 mg, 0.3121 mmol), triisopropylsilyl chloride (668 μL, 3.121 mmol), and imidazole (209 mg, 3.121 mmol) were dissolved in dry DMF (5 mL), and the reaction mixture was stirred at 80 °C for 24 h to obtain crude product 2c. The crude product was dissolved in THF (5 mL) and then was added HCOOH:H₂O (9:1) (5 mL) at 0 °C to the mixture and allowed to stir at room temperature for a further 6 h. The crude product was purified by column chromatography (3:1 n-hexane:EtOAc) to afford compound 3c (82.3 mg) as a white solid in 55% yield in two steps.

3c: A yellow solid in 55% yield; Mp: 110–114 °C, R_f = 0.24 (3:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 7.27 (1H, s), 5.16 (1H, t, J = 6.0 Hz), 5.06 (2H, s), 3.72 (3H, s), 3.38 (2H, d, J = 6.0 Hz), 2.41 (2H, t, J = 7.2 Hz), 2.31 (2H, t, J = 7.6 Hz), 2.13 (3H, s), 1.77 (3H, s), 1.60–1.40 (3H, m), 1.09 (18H, s) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 179.4, 169.5, 163.2, 152.7, 146.0, 133.4, 127.3, 124.4, 117.4, 111.1, 67.7, 60.7, 34.1, 32.7, 23.7, 18.1, 16.3, 14.3, 11.4 ppm; HRMS (m/z): calcd for C₂₆H₄₀O₆SiNa [M + Na]⁺ 499.2492, found: 499.2486.

General Procedure for the Preparation of Silyl Ether-MPA Analogues (4a–4c)

To a rapidly stirring solution of MPA, 1 (100 mg, 0.3121 mmol) in 0.5 mL of pyridine was added silyl chloride (R-Cl; R = TBS, TBDPS, TIPS) (0.4682 mmol). The reaction mixture was stirred at room temperature for 0.5 h. After TLC showed the reaction completed, the mixture was diluted with EtOAc 5 mL, quenched with saturated NH₄Cl solution, and then extracted with EtOAc. The mixture was quenched with CuSO₄·5H₂O to remove the pyridine, extracted with EtOAc, and washed with brine. The combined organic layers were dried over anhydrous Na₂SO₄, and the solvent was removed by rotary evaporation and purified by column chromatography.

Synthesis of tert-Butyldiphenylsilyl (E)-6-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (4b)

To a rapidly stirring solution of MPA 1 (100 mg, 0.3121 mmol) in 0.5 mL of pyridine was added tert-butyldiphenylsilyl chloride (120 μL, 0.4682 mmol). The reaction mixture was stirred at room temperature for 0.5 h. The crude product was purified by column chromatography (5:1 n-hexane:EtOAc) to afford compound 4b (112.5 mg) as a white solid in 64% yield.

4b: A white solid in 64% yield; Mp: 64–68 °C, R_f = 0.15 (5:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 7.67 (1H, s), 7.67–7.63 (4H, m), 7.42–7.30 (6H, m), 5.28 (1H, t, J = 7.2 Hz), 5.19 (2H, s), 3.73 (3H, s), 3.40 (2H, d, J = 6.8 Hz), 2.55 (2H, t, J = 6.8 Hz), 2.36 (2H, t, J = 8.0 Hz), 2.14 (3H, s), 1.82 (3H, s), 1.07 (9H, s) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 172.9, 172.5, 163.7, 153.6, 144.0, 135.2, 134.2, 131.9, 129.9, 127.6, 122.5, 122.1, 116.7, 106.3, 70.0, 60.9, 34.7, 29.6, 26.8, 22.6, 19.1, 16.2, 11.5 ppm; HRMS (m/z): calcd for C₃₃H₃₈O₆SiNa [M + Na]⁺ 581.2335, found: 581.2335.

Synthesis of Triisopropylsilyl (E)-6-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (4c)

To a rapidly stirring solution of MPA 1 (100 mg, 0.3121 mmol) in 0.5 mL of pyridine was added triisopropylsilyl chloride (100 μL, 0.4682 mmol). The reaction mixture was stirred at room temperature for 0.5 h. The crude product was purified by column chromatography (4:1 n-hexane:EtOAc) to afford compound 4c (81.6 mg) as a white solid in 55% yield.

4c: A white solid in 55% yield; Mp: 48–50 °C, R_f = 0.31 (3:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 7.66 (1H, s), 5.24 (1H, t, J = 6.8 Hz), 5.19 (2H, s), 3.76 (3H, s), 3.38 (2H, d, J = 6.8 Hz), 2.43 (2H, t, J = 6.8 Hz), 2.32 (2H, t, J = 8.0 Hz), 2.14 (3H, s), 1.81 (3H, s), 1.04 (3H, m), 1.05–1.00 (18H, m) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 173.4, 172.9, 163.7, 153.7, 144.0, 134.4, 122.3, 122.2, 116.7, 106.4, 70.0, 61.0, 34.8, 34.5, 22.6, 17.7, 16.3, 11.9 ppm; HRMS (m/z): calcd for C₂₆H₄₀O₆SiNa [M + Na]⁺ 499.2492, found: 499.2489.

General Procedure for the Preparation of Mycophenolate Analogues (5a–5c) and (6b–6c)

To a solution of tert-butyldiphenylsilyl ether-MPA product (3b) (50 mg, 0.090 mmol) in 1.0 mL of DCM were added DMAP (1.0 mg, 0.0090 mmol) and EDCI (60 mg, 0.3125 mmol) and stirred for 10 min, then alcohol (0.135 mmol) was added to the reaction mixture and allowed to stir at room temperature. After TLC indicated that the reaction was complete, the reaction mixture was diluted with DCM 1.0 mL, quenched with H₂O, extracted with DCM, and washed with brine, the combined organic layers were dried over anhydrous Na₂SO₄ and the solvent was removed by rotary evaporation and purified by column chromatography to obtain product 5.

To a stirred solution of compound 5 (50 mg) in MeOH 1.0 mL and TBAF (0.02793 mmol) was added at 0 °C and stirred for 30 min. After TLC showed complete conversion of the reaction, the reaction mixture was diluted with 3.0 mL of DCM and quenched with cold saturated NH₄Cl. The reaction mixture was extracted with DCM and washed with brine, followed by drying over Na₂SO₄ anhydrous, and concentrated in vacuo to obtain crude product and purified by column chromatography to afford compound product 6.

Synthesis of 1,3-Dioxoisoindolin-2-yl (E)-6-(4-((tert-Butyldiphenylsilyl)oxy)-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (5a)

N-Hydroxyphthalimide (22 mg, 0.135 mmol) was added to the reaction mixture and allowed to stir at room temperature for a further 5 h. The crude product was purified by column chromatography (3:1 n-hexane:EtOAc) to afford compound 5a (61.7 mg) as a sticky white oil in 98% yield.

5a: A stricky yellow oil in 98% yield; R_f = 0.30 (3:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 8.90–7.75 (4H, m), 7.70 (4H, d, J = 6.8 Hz), 7.40–7.30 (6H, m), 5.00 (1H, t, J = 6.8 Hz), 4.97 (2H, s), 3.63 (3H, s), 3.18 (2H, d, J = 6.0 Hz), 2.63 (2H, t, J = 7.6 Hz), 2.35 (2H, t, J = 8.0 Hz), 2.12 (3H, s), 1.52 (3H, s), 1.10 (9H, s) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 169.0, 168.2, 163.1, 161.7, 151.4, 145.9, 134.8, 134.6, 133.4, 132.2, 129.3, 128.7, 127.2, 126.8, 124.6, 123.7, 117.7, 111.0, 67.2, 60.4, 33.5, 29.4, 26.3, 24.1, 20.2, 15.8, 11.1 ppm; HRMS (m/z): calcd for C₄₁H₄₁NO₈SiNa [M + Na]⁺ 726.2499, found: 726.2490.

Synthesis of 4-Fluorophenethyl (E)-6-(4-((tert-Butyldiphenylsilyl)oxy)-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (5b)

4-Fluorophenethyl alcohol (17 μL, 0.135 mmol) was added to the reaction mixture and allowed to stir at room temperature for a further 5 h. The crude product was purified by column chromatography (5:1 n-hexane:EtOAc) to afford compound 5b (51.2 mg) as a colorless oil in 84% yield.

5b: A colorless oil in 84% yield; R_f = 0.25 (5:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 7.66 (4H, brs), 7.40–7.25 (6H, m), 7.10 (2H, brs), 6.94 (2H, t, J = 8.4 Hz), 4.95 (1H, brs), 4.88 (2H, s), 4.16 (2H, t, J = 6.4 Hz), 3.60 (3H, s), 3.15 (2H, brs), 2.80 (2H, t, J = 7.2 Hz), 2.26 (2H, t, J = 7.2 Hz), 2.15 (2H, t, J = 6.8 Hz), 2.08 (3H, s), 1.47 (3H, s), 1.07 (9H, s) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 173.0, 168.2, 163.0, 151.4, 134.8, 133.5, 130.1, 129.2, 127.2, 123.5, 117.7, 115.2, 115.0, 111.0, 67.2, 64.5, 60.4, 34.0, 32.7, 26.4, 24.1, 20.2, 16.0, 11.1 ppm; HRMS (m/z): calcd for C₄₁H₄₅FO₆SiNa [M + Na]⁺ 703.2867, found: 703.2866.

Synthesis of 4-Methoxyphenethyl (E)-6-(4-((tert-Butyldiphenylsilyl)oxy)-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (5c)

A mixture of tert-butyldiphenylsilyl ether-MPA (3b) (100 mg, 0.2793 mmol) in 2.0 mL of DCM, DMAP (3.4 mg, 0.02793 mmol), and EDCI (54 mg, 0.2793 mmol) was added and stirred for 10 min, and then, 4-methoxyphenethyl alcohol (64 mg, 0.4190 mmol) was added to the reaction mixture and allowed to stir at room temperature further 24 h. The crude product was purified by column chromatography (5:1 n-hexane:EtOAc) to afford compound 5c (122.7 mg) as a colorless oil in 65% yield.

5c: A colorless oil in 65% yield; R_f = 0.47 (4:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 7.69 (4H, d, J = 9.6 Hz), 7.40–7.24 (6H, m), 7.05 (2H, d, J = 8.4 Hz), 6.80 (2H, t, J = 8.4 Hz), 4.96 (1H, t, J = 6.8 Hz), 4.87 (2H, s), 4.16 (2H, t, J = 7.2 Hz), 3.75 (3H, s), 3.60 (3H, s), 3.16 (2H, d, J = 6.0 Hz), 2.74 (2H, t, J = 7.2 Hz), 2.29 (2H, t, J = 7.2 Hz), 2.16 (2H, t, J = 6.8 Hz), 2.07 (3H, s), 1.48 (3H, s), 1.08 (9H, s) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 173.1, 168.2, 163.0, 158.1, 151.4, 145.8, 134.8, 133.5, 133.4, 129.6, 129.3, 127.2, 127.1, 123.5, 117.7, 113.7, 111.0, 67.2, 64.8, 60.4, 55.1, 34.1, 32.7, 26.4, 24.1, 20.2, 16.0, 11.1 ppm; HRMS (m/z): calcd for C₄₂H₄₈O₇SiNa [M + Na]⁺ 715.3067, found: 715.3067.

Synthesis of 4-Fluorophenethyl (E)-6-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (6b)

A mixture of tert-butyldiphenylsilyl ether-MPA (3b) (100 mg, 0.2793 mmol) in DCM 2.0 mL, DMAP (3.4 mg, 0.2793 mmol) and EDCI (54 mg, 0.2793 mmol) was added and stirred for 10 min, then 4-fluorophenethyl alcohol (35 μL, 0.2793 mmol) was added to the reaction mixture and allowed to stir at room temperature further 24 h. To a solution of crude product 5b in MeOH 1.0 mL and TBAF (0.02793 mmol) was added at 0 °C and stirred for 30 min to obtain the crude product 5b. The crude product 5b was dissolved in MeOH 1.0 mL and TBAF (73 mg, 0.2793 mmol) was added at 0 °C and continued stirring for 30 min to obtain the crude product 6b. The crude product 6b was purified by column chromatography (3:1 n-hexane:EtOAc) to afford compound 6b (62.4 mg) as a yellow oil in 50% yield.

6b: A yellow oil in 50% yield; R_f = 0.24 (3:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 7.66 (1H, brs), 7.12 (2H, q, J = 8.4, 4.8 Hz), 6.96 (2H, t, J = 11.6 Hz), 5.23 (1H, t, J = 7.6 Hz), 5.13 (2H, s), 4.18 (2H, t, J = 7.2 Hz), 3.75 (3H, s), 3.37 (2H, d, J = 7.2 Hz), 2.80 (2H, t, J = 6.8 Hz), 2.38 (2H, t, J = 7.2 Hz), 2.27 (2H, t, J = 6.8 Hz), 2.12 (3H, s), 1.78 (3H, s) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 173.1, 172.8, 163.5, 162.8, 160.3, 153.5, 143.9, 133.9, 133.4, 130.2, 122.6, 122.0, 116.6, 115.2, 115.0, 106.2, 69.9, 64.6, 60.9, 34.4, 34.2, 32.9, 22.5, 16.0, 11.4 ppm; HRMS (m/z): calcd for C₂₅H₂₇FO₆Na [M + Na]⁺ 465.1689, found: 465.1688.

Synthesis of 4-Methoxyphenethyl (E)-6-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (6c)

The product 5b (50 mg, 0.072 mmol) was dissolved in 1.0 mL of MeOH, and TBAF (19 mg, 0.072 mmol) was added at 0 °C and continued stirring for 30 min to obtain the crude product of 6b. The crude product 6b was purified by column chromatography (3:1 n-hexane:EtOAc) to afford compound 6b (48 mg) as a yellow oil in 99% yield.

6c: A yellow oil in >99% yield; R_f = 0.44 (3:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 7.69 (1H, bs), 7.07 (2H, d, J = 8.4 Hz), 6.82 (2H, d, J = 8.4 Hz), 5.22 (1H, t, J = 7.6 Hz), 5.11 (2H, s), 4.16 (2H, t, J = 7.2 Hz), 3.78 (3H, s), 3.75 (3H, s), 3.37 (2H, d, J = 6.8 Hz), 2.75 (2H, t, J = 7.2 Hz), 2.42–2.35 (2H, m), 2.32–2.25 (2H, m), 2.11 (3H, s) 1.78 (3H, s) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 173.2, 172.9, 163.6, 158.2, 153.6, 144.0, 135.2, 134.1, 129.8, 125.0, 122.6, 122.1, 116.6, 113.9, 106.3, 70.0, 65.0, 61.0, 55.2, 34.6, 34.2, 33.0, 32.2, 26.4, 23.4, 16.1, 11.5 ppm; HRMS (m/z): calcd for C₂₆H₃₀O₇Na [M + Na]⁺ 477.1889, found: 477.1886.

Synthesis of 1,3-Dioxoisoindolin-2-yl (E)-6-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (6a)

To a solution of MPA 1 (100 mg, 0.3125 mmol) in DCM 2 mL, DMAP (3.8 mg, 0.03125 mmol) and EDCI (60 mg, 0.3125 mmol) were added and stirred for 10 min, then N-hydroxyphthalimide (76 mg, 0.4688 mmol) was added to the mixture and allowed to stir at room temperature for a further 5 h. After TLC indicated that the reaction was complete, the reaction mixture was diluted with DCM 3 mL and quenched with H₂O, extracted with DCM and washed with brine, then dried over with Na₂SO₄ anhydrous, and concentrated in vacuo to obtain crude product 6a. The crude product was purified by column chromatography (4:1 n-hexane:EtOAc) to afford compound 6a (46.6 mg) as a yellow solid in 32% yield.

6a: A yellow solid in 32% yield; Mp: 108–110 °C, R_f = 0.59 (1:1 n-hexane:EtOAc); ¹H NMR (400 MHz, CDCl₃): δ 8.90–7.75 (4H, m), 5.33 (1H, t, J = 6.8 Hz), 5.18 (2H, s), 3.76 (3H, s), 3.40 (2H, d, J = 6.8 Hz), 2.74 (2H, t, J = 7.6 Hz), 2.44 (2H, t, J = 8.0 Hz), 2.13 (3H, s), 1.84 (3H, s) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 172.8, 169.1, 163.6, 167.8, 153.5, 144.0, 134.6, 132.8, 128.8, 123.8, 123.7, 121.8, 116.6, 106.3, 69.9, 60.9, 33.9, 29.6, 22.5, 16.0, 11.4 ppm; HRMS (m/z): calcd for C₂₅H₂₃NO₈Na [M + Na]⁺ 488.1321, found: 488.1317.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsbiomedchemau.4c00079.

• ¹H and ¹³C NMR spectra (PDF)

Author Contributions

CRediT: Patamawadee Silalai data curation, investigation, methodology, writing - original draft; Pimpisa Teeyakasem investigation; Dumnoensun Pruksakorn investigation; Rungnapha Saeeng conceptualization, project administration, writing - review & editing.

The authors declare no competing financial interest.