Design, Synthesis and Biological Evaluation of Novel Rapamycin Benzothiazole Hybrids as mTOR Targeted Anti-cancer Agents

Abstract

The immunosuppressant drug rapamycin, was firstly identified as a mammalian target of rapamycin (mTOR) allosteric inhibitor, and its derivatives have been successfully developed as anti-cancer drugs. Therefore, finding rapamycin derivatives with better anti-cancer activity has been proved to be an effective way to discover new targeted anti-cancer drugs. In this paper, structure modification was performed at the C-43 position of rapamycin using bioisosterism and a hybrid approach: a series of novel rapamycin–benzothiazole hybrids 4a–e, 5a–c, and 9a, b have been designed, synthesized and evaluated for their anti-cancer activity against Caski, CNE-2, SGC-7901, PC-3, SK-NEP-1 and A-375 human cancer cell lines. Some of these compounds (4a–e, 9a, b) displayed good to excellent potency against the Caski and SK-NEP-1 cell line as compared with rapamycin. Compound 9b as the most active compound showed IC₅₀ values of 8.3 (Caski) and 9.6 μM (SK-NEP-1), respectively. In addition, research on the mechanism showed that 9b was able to cause G1 phase arrest and induce apoptosis in the Caski cell line. Most importantly, it significantly decreased the phosphorylation of S6 ribosomal protein, p70S6K1 and 4EBP1, which indicated that 9b inhibited the cancer cell growth by blocking the mTOR pathway and may have the potential to become a new mTOR inhibitor.

Rapamycin, a 31-member polyketide macrolide, was discovered as a fermentation product of the Streptomyces hygroscopicus AYB-944 and FC904, best known as a potent immunosuppressive agent later.^1–5) In recent years, rapamycin and its derivatives showed potent and broad anti-cancer properties in vitro and in vivo.^6–11) The two most prominent rapamycin derivatives, Temsirolimus (CCI-779)^12,13) and Everolimus (RAD001)^14,15) (Fig. 1) have been developed as mammalian target of rapamycin (mTOR) targeting anti-cancer drugs. Evidently, as all rapamycin derivatives currently approved (Temsirolimus, Everolimus) for cancer therapy or in clinical trials (Ridaforolimus) are C-43 substituted molecules, these results remind us that selective modification at the C-43 hydroxyl is an important asset.

Fig. 1. The Structure of the Rapamycin Derivatives

To the best of our knowledge, semi-synthesis of biologically active products through selective deoxygenation of available structures is an attractive strategy to gain novel active compounds.^16–19) Interestingly, a similar strategy had already been successfully applied in the synthesis of rapamycin derivative-zotarolimus,²⁰⁾ an anti-proliferative drug, which modified by replacement of hydroxyl with tetrazole at C-43 postion. Intrigued by the above observation, using a similar design as zotarolimus, we plan to replace the tetrazole moiety of zotarolimus with benzothiazole group by means of structure-based bioisosterism,^21,22) both of them with the same heterocyclic backbone. Additionally, benzothiazole has been an attractive pharmacological scaffold present in anti-cancer drugs, such as phosphatidyl inositol 3-kinase (PI3K) inhibitor Xl-147,^23,24) it is reasonable to combine benzothiazole with rapamycin nucleus to form a single molecular framework.^25,26) Through above mentioned molecular hybridization and bioisosterism process, it will be possible to obtain novel rapamycin derivatives with better anti-cancer activity with increasing bioavailability and better pharmacokinetics properties.

In this study, 8 new rapamycin derivatives (4a–e, 5a–c) were firstly synthesized by bonding benzothiazole moiety into the rapamycin nucleus at the C-43 position directly. The rapamycin derivatives of 9a and b were also prepared to explore whether the length of the spacer between rapamycin nucleus and benzothiazole moiety has an effect on their activities, in which the benzothiazole group was combined into the rapamycin nucleus through an oxyethylflexible linkage (Fig. 2).

Fig. 2. The Structure of the Targeted Compounds

Results and Discussion

Chemical Synthesis

The synthetic route of the target compounds 4a–e, 5a–c and 9a, b were illustrated in Chart 1. First of all, we decided to introduce the azido group selectively at C-43 of rapamycin by exploiting the superior reactivity of C-43 hydroxyl group with respect to the hydroxyl groups at C-28.²⁷⁾ Thus, the azido compound (2) was synthesized from rapamycin (1) via triflate activation of the C-43 hydroxyl group followed by treatment with sodium azide according to our reported procedure.²⁸⁾ Meanwhile, it should be pointed out that this method involves an S_N2 process and therefore gives rise to inversion of configuration at C-43. As already observed in our previous work,²⁸⁾ the stereochemistry of compound (2) had been determined by single crystal X-ray diffraction studies. Next, the azido compound (2) was transformed to C43-aminorapamycin (3) via Staudinger reduction, which subsequently reacted with side chains (12) to afford targeted compounds (4a–e, 5a–c) as white powder. On the side chain, benzothiazole-2-thiol (11a–e) was obtained by cyclization of the starting material polyhaloanilines bearing an ortho halogen atom in the presence of potassium O-ethyl xanthate at relatively mild temperatures (95–120°C).²⁹⁾ Next, benzothiazolium salts (12a–e, 13a–c) were obtained by alkylation of 11 with dimethylsulfate and diethylsulfate under reflux for 10 h, respectively.³⁰⁾

Chart 1

Reagents and conditions: (a) (CF₃SO₂)₂O, 2,6-lutidine, DCM, 0°C; (b) Acetone, NaN₃, 25°C; (c) Ph₃P, H₂O, THF 25°C; (d) CH₃CN, Et₃N, side chain 12; (e) 2-Bromoethyl trifluoromethanesulfonate, DIPEA, toluene, 60°C; (f) NaN₃, KI, DMF; (g) CH₃CH₂C(S)SK, DMF, 130°C; (h) Me₂SO₄, 110°C.

43-O-(2-Bromoethyl)rapamycin (6) was prepared with rapamycin and side chain trifluoromethanesulfonate, followed by treating with sodium azide to give 43-O-(2-azidoethyl)-rapamycin (7) according to literature.³¹⁾ The product (8) was obtained from (7) in a similar manner as described for the preparation of compound (3). The targeted compounds (9a, 9b) were prepared using the same procedure as synthesizing compounds (4a–e, 5a–c) by nucleophilic reaction involving compound (8), the side chains, and organic base as catalyst. The products obtained were purified by column chromatography on silica gel. The chemical structures of all the synthesized novel compounds were confirmed by IR, MS, high resolution (HR)-MS, ¹H-NMR and ¹³C-NMR.

Biological Activity

Anti-cancer Activity

The targeted rapamycin derivatives (4a–e, 5a–c and 9a, b) were firstly evaluated at the single concentration of 10 μM toward Caski, CNE-2, SGC-7901, PC-3, SK-NEP-1 and A-375 cancer cell lines (Table 1). The targeted derivatives containing N-methyl group on the thiazole ring (4a–e, 9a, b) displayed more potent activity than rapamycin against Caski and SK-NEP-1 cell lines. On the contrary, compounds (5a–c) with N-ethyl group on the thiazole ring showed lower activity in Caski and SK-NEP-1 cell lines as compared with 4a–e and 9a, b. The results implied that the different N-substituted groups may be related with their activity. Among 4a–e and 9a, b, the compounds 9a and b were superior to 4a–e on Caski and SK-NEP-1 cell lines. The results indicated that the introduction of oxyethyl between rapamycin nucleus and benzothiazole moiety has positive impact on their anti-cancer activities. Therefore, 9a and b, as most active compounds, were selected for further evaluation, and the results expressed as IC₅₀ were summarized in Table 2.

Table 1. Anti-tumor Activity of the Compounds against Caski, CNE-2, SGC-7901, PC-3, SK-NEP-1, A-375 Cell Lines in Concentration of 10 μM

Compd. No.	Growth inhibition rate (%)a)
	Caskib)	CNE-2b)	SGC-7901b)	PC-3b)	SK-NEP-1b)	A-375b)
4a	59.0	35.2	22.4	33.1	52.3	n.d.d)
4b	69.5	26.1	21.9	33.6	n.d.d)	26.7
4c	56.4	28.0	36.3	n.d.d)	51.8	23.1
4d	65.8	n.d.d)	25.2	31.4	47.9	38.2
4e	69.0	24.5	15.1	29.9	50.1	41.0
5a	32.1	23.9	39.5	n.d.d)	28.7	36.9
5b	31.9	33.2	46.4	43.2	33.4	28.3
5c	28.4	35.2	21.5	32.5	35.7	n.d.d)
9a	75.7	n.d.d)	42.3	48.1	66.2	36.2
9b	78.3	42.3	33.6	27.3	68.6	42.8
Rapamycinc)	40.1	46.0	25.9	25.7	42.5	31.0

a) Growth inhibition rate was determined by the MTT assay. Each experiment was run at least three times, and the results are presented as average values. b) Caski, cervical cancer cell line; SK-NEP-1, renal cell carcinoma; SGC-7901, human gastric carcinoma cell line; PC-3, human prostate cancer cell line; A375, human malignant melanoma cell line; CNE-2, nasopharyngeal carcinoma cell line. c) Used as a positive control. d) n.d.: not determined.

The data in Table 2 indicated that 9a and b displayed superior activity to rapamycin. Meanwhile, 9b as the most promising compound with IC₅₀ values 8.3 (Caski) and 9.6 μM (SK-NEP-1), was single digital times more active than that of rapamycin. More importantly, 9a and b were more potent against Caski and SK-NEP-1 cell lines than CNE-2, SGC-7901, PC-3, and A-375 cell lines. These results revealed that this series of compounds possessed selectivity for Caski and SK-NEP-1 cell lines.

Table 2. Anti-tumor Activity of the Selected Compounds 9a, b and Rapamycin in Vitro

Compd. No.	IC50a) µM, mean±S.D.
	Caski	SK-NEP-1	SGC-7901
9a	9.7±0.33	10.1±1.09	32.3±1.23
9b	8.3±0.61	9.6±0.96	23.6±1.26
Rapamycinb)	35.13±1.25	32.0±1.32	25.9±1.03

a) Data presented is the mean±S.D. value of three independent determinations. b) Used as positive control.

Cell Apoptosis Assays

The apoptosis of Caski cells induced by compound 9b were quantitatively assessed. As shown in Fig. 3, comparing with the control, compound 9b at 10 μM significantly induced the apoptosis in Caski cells, with increasing percentages of the apoptotic cells from 3.3 to 19.4%, also more active than that (5.6%) of rapamycin.

Cell Cycle Arrest

The effect of 9b on the cell cycle of Caski cells was studied by flow cytometry in propidium iodide (PI)-stained cells after treatment for 48 h. As shown in Fig. 4, 9b was able to significantly induce G1 phase cell cycle arrest at 5 μM, meanwhile, it induced G1 phase cell cycle arrest with a dose-dependent manner. In addition, the capability of 9b inducing G1 phase cell cycle arrest is stronger than parent rapamycin.

Fig. 3. Representative Dot Plots of PI and Annexin V Double Staining on Caski Cell Line Treated with Rapamycin and 9b for 48 h

(a) Control; (b) Rapamycin, 10 μM; (c) 9b, 10 μM.

Effect on mTOR Signaling Pathway

Rapamycin and its derivatives exert their anti-cancer activity through inhibiting the phosphorylation of mTOR, resulting in decreased phosphorylation of the mTOR downstream protein S6, P70S6K1 and 4EBP1. Therefore, we examined the effect of 9b on inhibition of phosphorylation of S6, p70S6K1 and 4EBP1 at different concentration. As showed in Fig. 5, 9b inhibited the phosphorylation of S6 ribosomal protein, p70S6K1 and 4EBP1 effectively at 0.01 μM. The suppressive effects of compound 9b on p-p70S6K1, p-S6 and p-4EBP1 were almost equal to rapamycin. The results implied that compound 9b inhibited the cancer cells growth through blocking the PI3K/AKT/mTOR pathway.

Fig. 4. A: Cell Cycle Analysis on Caski Cell Line Treated by the Compounds for 48 h; B: Quantitative Analysis of Cell Cycle Arrest

Fig. 5. Western Blot Analysis in Caski Cell Line Treated with Compounds for 24 h

Conclusion

In conclusion, we present the synthesis of rapamycin derivatives and their anti-cancer activity on Caski, CNE-2, SGC-7901, PC-3, SK-NEP-1 and A-375 human cancer cell lines. The pharmacological results showed that 9a and b displayed most potent inhibitory activity in all of compounds. Meanwhile, conclusions regarding the structure–activity relationships could be tentatively drawn as two aspects. (1) Significant improvements of the cell growth inhibitory capability were achieved when benzothioazole group were introduced to the C-43 position with ethyloxy linkage. When the ethyloxy linkage was replaced by C–N bonds (compounds 4a–e, 5a–c), the anti-cancer activity were decreased. (2) Compounds of the thiazole ring with N-methyl group were more active than those with N-ethyl group. Importantly, 9b could induce apoptosis and cause the G₁ phase arrest in Caski cells, and also decreased phosphorylation of S6 ribosomal protein, p70S6K1 and 4EBP1, suggesting it could block the PI3K/AKT/mTOR pathway and may become a promising mTOR inhibitor candidate for the treatment of cervical and renal cancer.

Experimental

Biology Experiment

Cell Culture

The different human cancer cell lines (Caski, CNE-2, SGC-7901, PC-3, SK-NEP-1 and A-375) were maintained in RPMI-1640 Medium (Gibco, U.S.A.) supplemented with 10% Fetal Bovine Serum (Gibco) and Gentamycin (80 U/mL) in a humidified incubator and 5% CO₂ atmosphere at 37°C.

Sulforhodamine B (SRB) Proliferation Assay

The anti-cancer activity of the synthesized compounds was evaluated against Caski, CNE-2, SGC-7901, PC-3, SK-NEP-1 and A-375 human cancer cell lines using SRB assay. The different human cancer cell lines were maintained in RPMI-1640 Medium (Gibco) supplemented with 10% Fetal Bovine Serum (Gibco) and Gentamycin (80 U/mL) in a humidified incubator and 5% CO₂ atmosphere at 37°C. The SRB assay is used for cell density determination, based on the measurement of cellular protein content. Briefly, the cells were seeded in a 96-well cell culture plate at a density of 10⁵ cells/well and incubated for 24 h at 37°C in a humidified 5% CO₂ incubator. The compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted with culture medium. Cells were treated with the compounds at different concentrations to achieve a final concentration of 1.0, 2.0, 5.0, 10.0, 25.0, 50.0 and 100.0 μM and incubated for 72 h at 37°C in a humidified 5% CO₂ incubator. After an incubation period, cell monolayers are fixed with 10% (w/v) trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1% (v/v) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for optical density (OD) determination at 510 nm using a microplate reader (Thermo Scientific, U.S.A.). The IC₅₀ values were calculated from the chart of percent cell viability against dose of compounds (μM) treated.

Flow Cytometry Analysis

Prepared Caski cells (10⁵/mL) were washed twice with cold phosphate buffered saline (PBS) and then re-suspended gently in 500 μL binding buffer. Thereafter, cells were stained in 5 μL Annexin V-fluorescein isothiocyanate (FITC) and shaked well. Finally, 5 μL propidium iodide (PI) was added to these cells and incubated for 10 min in a dark place, analyzed by BD ACCURI C6.

Cell Cycle Assay

The Caski cancer cells were harvested and washed with PBS and resuspended in 75% ethanol in PBS and kept at −20°C for at least 4 h. Cells were resuspended and incubated in 20 μL RNase A for 30 min at 37°C. Cells were resuspended and incubated in propidium iodidestaining solution for 30 min at room temperature and kept in the dark at 4°C, analyzed by BD ACCURI C6.

Western Blotting Analysis

Cells were lysed in 2% sodium dodecyl sulfate (SDS). To equate every sample, BCA kit was used to determine the protein concertration of the lysate. Equal amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes. The target protein-carrying membranes were blocked with 5% milk and incubated with the primary antibody (CST, U.S.A.) at 4°C overnight. The membranes were then washed in Tris-buffered saline with tween (TBST) buffer three times and incubated with the secondary antibody for 1 h. Wash the membrane as previous procedure. Bands were visualized by using the enhanced chemiluminescence Western blotting detection system (GE, U.S.A.). Densitometric analysis was performed under certain conditions that yielded a linear response.

Chemical Synthesis

Reagents and General Procedures

All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and were uncorrected. MS were taken in electrospray ionization (ESI) mode on Agilent 1100 LC-MS (Agilent, Palo Alto, CA, U.S.A.), [α]_D values are were recorded on Autopol I-Rudolph (U.S.A.). High resolution (HR)-MS were recorded on Agilent Technologies 6540 instrument. Nuclear magnetic resonance spectroscopy was performed using Bruker ARX-400, 400 MHz spectrometers (Bruker Bioscience, Billerica, MA, U.S.A.) with tetramethylsilane (TMS) as an internal standard. IR spectra (KBr disks) were recorded with a Bruker IFS 55 instrument (Bruker). Unless otherwise noted, all the materials were obtained from commercially available sources and were used without further purification.

Preparation of 43-Azidorapamycin (2)

To a mixture solution of rapamycin (2.6 g, 2.8 mmol) and 2,6-lutidine (1.2 g, 11.3 mmol) in dichloromethane (DCM) (30.0 mL) was added trifluoromethanesulfonic anhydride (2.4 g, 8.5 mmol) dropwise at 0°C. The mixture was stirred at 0°C for 2 h. Water was added to quench the reaction. The solution was extracted with dichloromethane; the organic layer was washed with saturated NaHCO₃ aqueous solution and brine, dried over Na₂SO₄, filtered, and evaporated. Sodium azide (0.18 g, 8.5 mmol) was added gradually to the solution of residue in acetone (60 mL). The reaction mixture was stirred vigorously at 25°C for 6 h. The reaction mixture was diluted with EtOAc and washed thoroughly with brine, dried over Na₂SO₄, filtered, and concentrated. Silica gel chromatography (PE/EtOAc=3 : 1) of the crude mixture afforded 2 (1.2 g, 45%). mp: 162.3–164.6°C; MS (ESI) m/z: 961. 5 (M+Na)⁺; [α]_D −32.6 (c=1.0, MeOH); ¹H-NMR (400 MHz, DMSO-d₆) δ: 6.46 (s, 1H), 6.43–6.33 (m, 1H), 6.27–6.19 (m, 1H), 6.17–6.07 (m, 2H), 5.49–5.42 (m, 1H), 5.27 (s, 1H), 5.08 (d, J=10.2 Hz, 1H), 5.03–4.95 (m, 1H), 4.93–4.86 (m, 1H), 4.28–4.07 (m, 2H), 3.88–3.62 (m, 2H), 3.60–3.52 (m, 1H), 3.51–3.36 (m, 2H), 3.28–3.22 (m, 1H), 3.21 (s, 3H), 3.15 (s, 3H), 3.07 (s, 3H), 2.86–2.76 (m, 1H), 2.74–2.65 (m, 1H), 2.44–2.31 (m, 2H), 2.33 (s, 3H), 2.26–2.12 (m, 1H), 2.10–2.07 (m, 2H), 2.06–1.97 (m, 1H), 1.95–1.78 (m, 2H), 1.74 (s, 3H), 1.62 (s, 3H), 1.60–1.02 (m, 9H), 0.98 (d, J=6.5 Hz, 3H), 0.88 (d, J=6.5 Hz, 3H), 0.82 (d, J=6.5 Hz, 3H), 0.78 (d, J=6.4 Hz, 3H), 0.73 (d, J=6.5 Hz, 3H).

Preparation of 43-Aminorapamycin (3)

A 100 mL round-bottomed flask was charged with compound 2 (1.2 g, 1.3 mmol) and tetrahydrofuran (THF) (60.0 mL). To this solution, triphenyl phosphine (1.3 g, 3.9 mmol) was added slowly, and the reaction mixture was stirred for 2 h at 60°C. Several drops of water (0.3 mL, 18.3 mmol) were added, and the resulting suspension was stirred for 6 h. The mixture was concentrated under reduced pressure. The crude mixture was purified by flash column chromatography (silica gel, dichloromethane/methanol, 200 : 1) to afford the desired compound 3 (0.8 g, 68%) as yellow solids. mp: 121.7–123.3°C; MS (ESI, m/z): 913.6(M+H)⁺; [α]_D −38.1 (c=1.1, MeOH); ¹H-NMR (400 MHz, DMSO-d₆) δ: 6.46 (s, 1H), 6.42–6.31 (m, 1H), 6.22–6.15 (m, 1H), 6.14–6.03 (m, 2H), 5.52–5.45 (m, 1H), 5.27 (s, 1H), 5.08–5.01 (m, 3H), 4.96–4.80 (m, 2H), 4.53–4.41 (m, 2H), 4.06–3.83 (m, 3H), 3.67–3.52 (m, 1H), 3.42–3.36 (m, 1H), 3.34–3.23 (m, 1H), 3.21 (s, 3H), 3.16 (s, 3H), 3.07 (s, 3H), 3.03–2.94 (m, 2H), 2.85–2.767 (m, 1H), 2.74–2.53 (m, 1H), 2.42–2.34 (m, 2H), 2.22–2.16 (m, 1H), 2.15–2.08 (m, 1H), 2.05–1.97 (m, 1H), 1.95–1.76 (m, 2H), 1.73 (s, 3H), 1.63 (s, 3H), 1.61–1.02 (m, 10H), 0.98 (d, J=6.5 Hz, 3H), 0.88 (d, J=6.5 Hz, 3H), 0.81 (d, J=6.4 Hz, 2H), 0.78 (d, J=6.5 Hz, 2H), 0.74 (d, J=6.4 Hz, 3H).

Preparation of 43-O-(2-Bromoethyl)rapamycin (6)

2-Bromoethyl trifluoromethanesulfonate (5.0 g, 19.5 mmol) was added to a solution of rapamycin (6.0 g, 6.6 mmol) and DIPEA (4.2 g, 32.8 mmol) in 100 mL of toluene at 25°C. The mixture was stirred at 60°C until the reaction completed. The reaction mixture was washed with a saturated NaHCO₃ solution, water and brine. The organic layer was dried and concentrated. The residue was purified by flash column chromatography (silica gel, petrol/EA, 5 : 1) to afford the desired compound 6 (5.0 g, 75%) as yellow solids. mp: 183.4–185.9°C; MS (ESI) m/z: 1042.5 (M+Na)⁺; [α]_D −12.6 (c=1.1, MeOH); ¹H-NMR (400 MHz, DMSO-d₆) δ: 6.45 (s, 1H), 6.42–6.32 (m, 1H), 6.22–6.17 (m, 1H), 6.16–6.05 (m, 2H), 5.51–5.45 (m, 1H), 5.26 (s, 1H), 5.08 (d, J=9.8 Hz, 1H), 4.95–4.82 (m, 2H), 4.56–4.43 (m, 2H), 4.07–3.82 (m, 3H), 3.66–3.51 (m, 1H), 3.43 (m, 3.6 Hz, 1H), 3.30–3.22 (m, 1H), 3.21 (s, 3H), 3.15 (s, 3H), 3.06 (s, 3H), 3.02–2.93 (m, 2H), 2.84–2.76 (m, 1H), 2.73–2.55 (m, 1H), 2.44–2.31 (m, 2H), 2.22 (m, 1H), 2.15–2.06 (m, 1H), 2.05–1.96 (m, 1H), 1.94–1.77 (m, 2H), 1.73 (s, 3H), 1.62 (s, 3H), 1.60–1.01 (m, 10H), 0.98 (d, J=6.5 Hz, 3H), 0.87 (d, J=6.5 Hz, 3H), 0.81 (d, J=6.3 Hz, 2H), 0.77 (d, J=6.5 Hz, 2H), 0.73 (d, J=6.4 Hz, 3H).

Preparation of 43-O-(2-Azidoethyl)rapamycin (7)

NaN₃ (4.0 g, 60.2 mmol) was added to a stirred solution of compound 6 (2.1 g, 2.1 mmol) and sodium iodide (0.3 g, 4.6 mmol) in N,N-dimethylformamide (DMF) (40 mL). The reaction mixture was heated to 60°C for 1.5 h. The mixture was poured into ice water, stirred for 1h and separated by filtration to give white compound 7 (1.0 g, 49%). mp: 171.3–173.6°C; MS (ESI, m/z): 1005.6 (M+Na)⁺; [α]_D −36.5 (c=1.0, MeOH); ¹H-NMR (400 MHz, DMSO-d₆) δ: 6.45 (s, 1H), 6.38 (m, 1H), 6.27–6.18 (m, 1H), 6.17–6.06 (m, 2H), 5.48–5.40 (m, 1H), 5.27 (s, 1H), 5.09 (d, J=10.4 Hz, 1H), 5.02–4.96 (m, 1H), 4.95–4.89 (m, 1H), 4.29–4.06 (m, 2H), 3.85–3.66 (m, 2H), 3.60 (m, 1H), 3.50–3.39 (m, 1H), 3.29–3.23 (m, 1H), 3.22 (s, 3H), 3.14 (s, 3H), 3.06 (s, 3H), 2.85–2.75 (m, 1H), 2.76–2.68 (m, 1H), 2.43–2.34 (m, 2H), 2.32 (s, 3H), 2.28–2.15 (m, 1H), 2.10 (m, 2H), 2.06–1.95 (m, 1H), 1.96–1.78 (m, 2H), 1.73 (s, 3H), 1.62 (s, 3H), 1.61–1.01 (m, 9H), 0.98 (d, J=6.5 Hz, 3H), 0.87 (d, J=6.5 Hz, 3H), 0.82 (d, J=6.3 Hz, 3H), 0.76 (d, J=6.4 Hz, 3H), 0.73 (d, J=6.5 Hz, 3H).

Preparation of 43-O-(2-Aminoethyl)rapamycin (8)

A 100 mL round-bottomed flask was charged with compound 7 (1.0 g, 1.0 mmol) and THF (20.0 mL). To this solution, triphenyl phosphine (1.0 g, 3.0 mmol) was added slowly, and the reaction mixture was stirred for 2 h at 60°C. Several drops of water (0.2 mL, 11.1 mmol) were added, and the resulting suspension was stirred for 6 h. The mixture was concentrated under reduced pressure. The crude mixture was purified by flash column chromatography (silica gel, dichloromethane/methanol, 100 : 1) to afford the desired compound 8 (0.7 g, 72%) as yellow solids. mp: 131.1–133.8°C; MS (ESI, m/z): 957.6 (M+H)⁺; [α]_D −12.9 (c=1.1, MeOH); ¹H-NMR (400 MHz, DMSO-d₆) δ: 6.46 (s, 1H), 6.40–6.36 (m, 1H), 6.26–6.17 (m, 1H), 6.16–6.05 (m, 2H), 5.47–5.41 (m, 1H), 5.27 (s, 1H), 5.09 (d, J=10.4 Hz, 1H), 5.04–4.99 (m, 3H), 4.93–4.86 (m, 1H), 4.28–4.05 (m, 2H), 3.84–3.67 (m, 3H), 3.60–3.52 (m, 2H), 3.51–3.38 (m, 2H), 3.28–3.24 (m, 1H), 3.21 (s, 3H), 3.15 (s, 3H), 3.07 (s, 1H), 2.86–2.76 (m, 1H), 2.74–2.66 (m, 1H), 2.42–2.33 (m, 2H), 2.33 (s, 3H), 2.25–2.13 (m, 1H), 2.11–2.09 (m, 2H), 2.05–1.94 (m, 1H), 1.92–1.78 (m, 2H), 1.73 (s, 3H), 1.63 (s, 3H), 1.62–1.01 (m, 8H), 0.98 (d, J=6.4 Hz, 3H), 0.88 (d, J=6.5 Hz, 3H), 0.83 (d, J=6.4 Hz, 3H), 0.77 (d, J=6.5 Hz, 3H), 0.75 (d, J=6.5 Hz, 3H).

General Procedure for the Preparation of Intermediates 11a–e

The polyhaloanilines (0.1 mol) and potassium O-ethyldithiocarbonate (250.0 mmol) were mixed in a 1000 mL round-bottomed flask with 400 mL of the DMF. The reaction mixture was stirred at 110–130°C for 3–8 h. The products 11a–e were acidified to pH 3–4 with CH₃COOH and isolated by filtration.²⁹⁾

General Procedure for the Preparation of Intermediates 12a–e and 13a–c

Intermediate 11a–e (50.0 mmol) was dissolved in dimethyl sulfate or dimethyl sulfate (50.0 mL), the reaction mixture was refluxed for 2–4 h. After cooling the product precipitated as solid and was isolated by filtration. The solid was washed with diethyl ether. Yields: 55–83%.³⁰⁾

General Procedure for the Preparation of Compounds 4a–e

The 43-aminorapamycin (0.2 g, 0.2 mmol) was dissolved in 10 mL of acetonitrile and then added dropwise to a solution of 10 mL of acetonitrile containing of Et₃N (0.2 mL) and side chains 12 (0.66 mmol) at 25°C, and the mixture was stirred for 1 h. The mixture was quenched with a saturated NaHCO₃ solution (100.0 mL) and diluted with dichloromethane. Two phases were separated and the organic phase was washed with brine, the organic phase was then collected and dried overnight over anhydrous Na₂SO₄. After filtering, the solvent was evaporated under reduced pressure. The crude mixture was purified by column chromatography (silica, 1–5% EtOAc/hexanes) to furnish the targeted compound 4a–e.

43-N-(6-Fluoro-3-methylbenzo[d]thiazol-2(3H)-ylidene)aminerapamycin (4a)

Yield: 36%. mp: 161–163°C; MS (ESI, m/z): 1078.6 (M+H)⁺; [α]_D −15.2 (c=1.1, MeOH); IR (KBr) cm⁻¹: 3439.6, 2930.3, 2858.7, 1719.6, 1646.4, 1580.7, 1561.9, 1452.7, 1381.6, 1195.5, 1092.8, 989.7; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.46 (dd, J=8.3, 1.9 Hz, 1H), 7.08 (t, J=7.9 Hz, 1H), 7.03 (d, J=4.5 Hz, 1H), 6.46 (s, 1H), 6.42 (m, 1H), 6.24 (d, 1H), 6.17 (d, J=13.7 Hz, 1H), 6.10 (m, 1H), 5.45 (dd, J=14.6, 9.7 Hz, 1H), 5.27 (s, 1H), 5.08 (d, J=10.1 Hz, 1H), 5.05–4.98 (m, 1H), 4.95 (d, J=5.2 Hz, 1H), 4.03 (m, 1H), 4.01 (d, J=4.2 Hz, 1H), 3.91 (d, J=4.7 Hz, 1H), 3.62 (d, J=12.1 Hz, 1H), 3.43 (m, 2H), 3.35 (s, 3H), 3.20 (s, 3H), 3.15 (s, 3H), 3.05 (s, 3H), 2.77 (d, J=16.1 Hz, 1H), 2.48–2.31 (m, 2H), 2.25–1.94 (m, 3H), 1.85 (m, 2H), 1.74 (s, 3H), 1.65 (s, 3H), 1.63–1.46 (m, 5H), 1.44–1.00 (m, 10H), 0.96 (d, J=6.4 Hz, 3H), 0.87 (d, J=6.5 Hz,), 0.82 (d, J=6.5 Hz, 3H), 0.79 (d, J=6.9 Hz, 3H), 0.73 (d, J=6.6 Hz, 3H); ¹³C-NMR (101 MHz, DMSO-d₆) δ: 211.16, 208.02, 199.54, 169.66, 167.50, 158.58, 156.23, 152.08, 139.81, 138.35, 137.82, 137.72, 132.78, 130.95, 127.52, 127.47, 125.70, 123.30, 110.36, 109.56, 99.51, 86.21, 82.83, 81.70, 76.19, 74.09, 66.70, 61.56, 57.49, 55.97, 55.89, 51.10, 45.70, 43.95, 35.72, 35.29, 33.58, 33.44, 33.34, 30.73, 30.66, 30.08, 28.03, 26.90, 26.73, 24.98, 22.12, 20.84, 16.06, 16.03, 15.14, 14.03, 13.71, 10.98; HR-MS (ESI): Calcd for C₅₉H₈₄FClN₃O₁₂S [M+Cl]⁻=1112.5760. Found=1112.5781.

43-N-(4-Fluoro-3-methylbenzo[d]thiazol-2(3H)-ylidene)aminerapamycin (4b)

Yield: 45%. mp: 153–155°C; MS (ESI, m/z): 1078.5 (M+H)⁺; [α]_D −11.9 (c=1.0, MeOH); IR (KBr) cm⁻¹: 3434.9, 2930.2, 2858.6, 2044.7, 1720.5, 1645.4, 1608.1, 1585.6, 1490.5, 1458.8, 1380.9, 1195.7, 1112.7, 990.6; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.31 (m, J=6.9 Hz, 1H), 7.11 (dd, J=12.1, 8.7 Hz, 1H), 7.01–6.90 (m, 1H), 6.46 (s, 1H), 6.41 (dd, J=14.2, 11.4 Hz, 1H), 6.23 (m, 1H), 6.17 (d, J=13.4 Hz, 1H), 6.11 (m, 1H), 5.46 (dd, J=14.6, 9.7 Hz, 1H), 5.26 (s, 1H), 5.09 (d, J=10.0 Hz, 1H), 5.05–4.98 (m, 1H), 4.95 (m, 1H), 4.08–3.97 (m, 2H), 3.93 (m, 1H), 3.66–3.59 (m, 1H), 3.29 (m, 3H), 3.20 (s, 3H), 3.18 (s, 3H), 3.15 (s, 3H), 3.05 (s, 3H), 2.76 (d, J=16.0 Hz, 1H), 2.41 (dd, J=24.6, 8.9 Hz, 1H), 2.29–1.94 (m, 3H), 1.85 (dd, J=21.1, 9.0 Hz, 2H), 1.75 (s, 3H), 1.64 (s, 3H), 1.62–0.99 (m, 16H), 0.96 (d, J=6.4 Hz, 3H), 0.87 (d, J=6.5 Hz, 3H), 0.83 (d, J=7.0 Hz, 3H), 0.80 (d, J=7.2 Hz, 3H), 0.73 (d, J=6.5 Hz, 3H); ¹³C-NMR (101 MHz, DMSO-d₆) δ: 211.08, 208.03, 199.49, 169.68, 167.48, 139.78, 138.35, 137.68, 132.78, 130.94, 128.67, 128.57, 127.53, 127.46, 125.60, 124.51, 124.49, 121.70, 119.04, 114.60, 99.51, 86.14, 82.81, 81.67, 76.20, 74.06, 66.70, 61.87, 57.46, 55.96, 55.91, 51.14, 45.70, 43.96, 35.71, 35.28, 33.72, 33.55, 33.45, 30.77, 30.58, 30.10, 27.99, 26.91, 26.74, 24.98, 22.11, 20.85, 16.07, 16.03, 15.19, 13.95, 13.79, 10.97; HR-MS (ESI): Calcd for C₅₉H₈₄FClN₃O₁₂S [M+Cl]⁻=1112.5760. Found=1112.5728.

43-N-(5-Chloro-3-methylbenzo[d]thiazol-2(3H)-ylidene)aminerapamycin (4c)

Yield: 31%. mp: 163–165°C; MS (ESI, m/z): 1094.5 (M+H)⁺; [α]_D −35.4 (c=1.1, MeOH); IR (KBr) cm⁻¹: 3439.6, 2930.3, 2858.7, 2041.1, 1719.6, 1646.4, 1588.6, 1481.0, 1452.7, 1381.6, 1195.5, 1092.7, 989.7; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.47 (d, J=8.2 Hz, 1H), 7.15 (s, 1H), 7.00 (d, J=8.2 Hz, 1H), 6.43 (s, 1H), 6.42 (dd, J=14.5, 11.1 Hz, 1H), 6.26–6.20 (m, 1H), 6.21–6.13 (m, 1H), 6.11 (dd, J=10.8, 5.5 Hz, 1H), 5.46 (dd, J=14.8, 9.6 Hz, 1H), 5.23 (s, 1H), 5.10 (d, J=10.2 Hz, 1H), 5.06–5.00 (m, 1H), 4.95 (d, J=5.2 Hz, 1H), 4.06–3.96 (m, 2H), 3.90 (d, J=4.8 Hz, 1H), 3.63 (d, J=10.3 Hz, 1H), 3.44 (dd, J=12.0, 2.1 Hz, 2H), 3.37 (s, 3H), 3.20 (s, 3H), 3.15 (s, 3H), 3.06 (s, 3H), 2.77 (dd, J=17.6, 2.0 Hz, 1H), 2.48–2.36 (m, 2H), 2.23–2.16 (m, 1H), 2.07–1.99 (m, 1H), 1.90–1.78 (m, 2H), 1.74 (s, 3H), 1.65 (s, 3H), 1.64–1.46 (m, 4H), 1.46–1.20 (m, 8H), 1.13–0.99 (m, 3H), 0.97 (d, J=6.4 Hz, 3H), 0.89 (d, J=6.5 Hz, 3H), 0.83 (d, J=6.4 Hz, 3H), 0.80 (d, J=6.7 Hz, 3H), 0.73 (d, J=6.6 Hz, 3H). ¹³C-NMR (126 MHz, DMSO-d₆) δ: 210.64, 207.50, 199.01, 169.15, 166.98, 151.65, 142.04, 139.30, 137.83, 137.19, 132.29, 131.04, 130.43, 127.00, 126.95, 125.19, 123.24, 120.33, 120.17, 108.78, 99.00, 85.71, 82.33, 81.18, 75.67, 73.59, 66.19, 61.23, 56.99, 55.46, 55.41, 50.59, 45.19, 43.44, 40.07, 39.64, 39.54, 39.20, 39.00, 35.21, 34.78, 33.06, 32.95, 32.81, 30.14, 30.10, 29.56, 27.52, 26.39, 26.22, 24.47, 21.61, 20.33, 15.54, 15.53, 14.66, 13.53, 13.20, 10.47; HR-MS (ESI): Calcd for C₅₉H₈₄Cl₂N₃O₁₂S [M+Cl]⁻=1128.5158. Found=1128.5131.

43-N-(3-Methylbenzo[d]thiazol-2(3H)-ylidene)aminerapamycin (4d)

Yield: 38%. mp: 159–162°C; MS (ESI, m/z): 1060.5 (M+H)⁺; [α]_D −19.8 (c=1.0, MeOH); IR (KBr) cm⁻¹: 3427.1, 2930.8, 2873.2, 2041.1, 1720.2, 1643.6, 1586.6, 1480.9, 1452.2, 1378.5, 1194.9, 1100.9, 989.9; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.46 (d, J=7.6 Hz, 1H), 7.24 (t, J=7.8 Hz, 1H), 7.04 (d, J=7.9 Hz, 1H), 6.97 (t, J=7.6 Hz, 1H), 6.47 (s, 1H), 6.45–6.35 (m, 1H), 6.25 (m, 1H), 6.23–6.16 (m, 1H), 6.13 (m, 1H), 5.46 (dd, J=14.7, 9.7 Hz, 1H), 5.27 (d, J=4.5 Hz, 1H), 5.09 (d, J=10.1 Hz, 1H), 5.05–5.00 (m, 1H), 4.96 (d, J=5.1 Hz, 1H), 4.02 (m, 2H), 3.92 (d, J=4.7 Hz, 1H), 3.63 (d, J=12.2 Hz, 1H), 3.52–3.39 (m, 2H), 3.37 (s, 3H), 3.31–3.24 (m, 3H), 3.21 (s, 3H), 3.15 (s, 3H), 3.06 (s, 3H), 2.78 (d, J=16.9 Hz, 1H), 2.48–2.34 (m, 2H), 2.26 (m, 3H), 2.14–2.01 (m, 2H), 1.92–1.80 (m, 2H), 1.75 (s, 3H), 1.66 (s, 3H), 1.63–1.21 (m, 10H), 0.96 (d, J=6.4 Hz, 3H), 0.88 (d, J=6.4 Hz, 3H), 0.83 (d, J=6.6 Hz, 3H), 0.80 (d, J=7.0 Hz, 3H), 0.73 (d, J=6.6 Hz, 3H); ¹³C-NMR (101 MHz, DMSO-d₆) δ: 211.18, 207.93, 199.54, 169.65, 167.50, 152.32, 141.17, 139.81, 138.35, 137.73, 132.79, 130.94, 127.52, 127.47, 126.82, 125.72, 122.61, 121.83, 121.14, 109.22, 103.51, 99.51, 86.23, 82.84, 81.73, 76.18, 74.10, 66.71, 61.59, 60.21, 57.51, 55.97, 55.87, 51.10, 45.70, 43.94, 35.73, 35.30, 33.61, 33.45, 33.38, 30.68, 30.43, 30.08, 28.07, 26.90, 26.73, 24.95, 22.13, 21.22, 16.04, 15.15, 14.55, 14.06, 13.69; HR-MS (ESI): Calcd for C₅₉H₈₅ClN₃O₁₂S [M+Cl]⁻=1094.5548. Found=1094.5499.

43-N-(6-Chloro-3-methylbenzo[d]thiazol-2(3H)-ylidene)aminerapamycin (4e)

Yield: 39%. mp: 160–162°C; MS (ESI, m/z): 1094.6 (M+H)⁺; [α]_D −22.1 (c=1.1, MeOH); IR (KBr) cm⁻¹: 3428.5, 2930.5, 2871.9, 2044.7, 1719.9, 1647.4, 1593.1, 1585.6, 1490.5, 1451.9, 1381.5, 1200.9, 1103.2.7, 989.7; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.61 (s, 1H), 7.27 (d, J=7.9 Hz, 1H), 7.04 (d, J=8.5 Hz, 1H), 6.44 (s, 1H), 6.43–6.38 (m, 1H), 6.22 (m, 1H), 6.20–6.14 (m, 1H), 6.13–6.07 (m, 1H), 5.46 (dd, J=15.3, 9.1 Hz, 1H), 5.24 (s, 1H), 5.10 (d, J=9.7 Hz, 1H), 5.09 (m, 1H), 5.06–5.00 (m, 1H), 4.96 (d, J=4.7 Hz, 1H), 4.06–3.96 (m, 2H), 3.90 (d, J=4.1 Hz, 1H), 3.68–3.59 (m, 1H), 3.50–3.40 (m, 2H), 3.36 (s, 3H), 3.21 (s, 3H), 3.16 (s, 3H), 3.06 (s, 3H), 2.77 (d, J=16.8 Hz, 1H), 2.47–2.29 (m, 2H), 2.19 (s, 1H), 2.15–1.97 (m, 2H), 1.93–1.78 (m, 2H), 1.75 (s, 3H), 1.66 (s, 3H), 1.64–1.00 (m, 14H), 0.97 (d, J=6.3 Hz, 3H), 0.89 (d, J=6.4 Hz, 3H), 0.83 (d, J=6.3 Hz, 3H), 0.80 (d, J=6.5 Hz, 3H), 0.73 (d, J=6.4 Hz, 3H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 210.64, 207.50, 199.01, 169.15, 166.98, 151.31, 139.72, 139.29, 137.83, 137.19, 132.27, 130.42, 127.00, 126.95, 126.09, 125.20, 124.26, 123.37, 121.77, 109.73, 98.99, 85.70, 82.32, 81.18, 75.68, 73.59, 66.19, 61.16, 56.99, 55.45, 55.40, 50.59, 45.19, 43.43, 35.20, 34.78, 33.07, 32.94, 32.80, 30.17, 30.10, 29.57, 27.52, 26.39, 26.22, 24.46, 21.61, 20.32, 15.54, 15.52, 14.65, 13.53, 13.19, 10.47; HR-MS (ESI): Calcd for C₅₉H₈₄Cl₂N₃O₁₂S [M+Cl]⁻=1128.5158. Found=1128.5199.

General Procedure for the Preparation of Compounds 5a–c

The foregoing method for the preparation of the 4a–e was applied to prepare the 5a–c except that side chains 13 was used instead of side chains 12.

43-N-(6-Fluoro-3-ethylbenzo[d]thiazol-2(3H)-ylidene)aminerapamycin (5a)

Yield: 33%. mp: 151–153°C; MS (ESI, m/z): 1091.6 (M+H)⁺; [α]_D −45.2 (c=1.1, MeOH); IR (KBr) cm⁻¹: 3428.7, 2930.1, 2872.4, 2043.4, 1715.8, 1645.4, 1592.1, 1583.6, 1491.4, 1451.6, 1382.3, 1200.6, 1103.3.7, 991.2; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.42 (dd, J=8.2, 1.9 Hz, 1H), 7.06 (t, J=8.0 Hz, 1H), 7.01 (d, J=4.5 Hz, 1H), 6.44 (s, 1H), 6.41 (m, 1H), 6.22 (d, 1H), 6.15 (d, J=13.6 Hz, 1H), 6.08 (m, 1H), 5.44 (dd, J=14.2, 9.6 Hz, 1H), 5.26 (s, 1H), 5.06 (d, J=10.2 Hz, 1H), 5.03–4.96 (m, 1H), 4.92 (d, J=5.2 Hz, 1H), 4.4–4.03 (m, 2H), 4.01 (d, J=4.6 Hz, 1H), 3.88 (d, J=4.8 Hz, 1H), 3.63 (d, J=12.2 Hz, 1H), 3.49–3.31 (m, 4H), 3.20 (s, 3H), 3.15 (s, 3H), 3.05 (s, 3H), 2.73 (d, J=16.0 Hz, 1H), 2.45–2.29 (m, 2H), 2.23–1.92 (m, 3H), 1.84 (m, 2H), 1.73 (s, 3H), 1.66 (s, 3H), 1.63–1.46 (m, 5H), 1.44–1.00 (m, 13H), 0.97 (d, J=6.4 Hz, 3H), 0.87 (d, J=6.5 Hz,), 0.82 (d, J=6.5 Hz, 3H), 0.79 (d, J=6.8 Hz, 3H), 0.74 (d, J=6.6 Hz, 3H); ¹³C-NMR (101 MHz, DMSO-d₆) δ: 211.14, 208.03, 199.54, 169.67, 167.51, 158.56, 156.22, 152.08, 139.82, 138.37, 137.81, 137.71, 132.78, 130.92, 127.52, 127.46, 125.71, 123.32, 110.37, 109.55, 99.52, 86.22, 82.82, 81.72, 76.17, 74.04, 66.73, 61.55, 57.48, 55.96, 55.89, 51.11, 45.72, 44.31, 43.92, 35.71, 35.27, 33.52, 33.41, 30.72, 30.67, 30.09, 28.02, 26.91, 26.73, 24.97, 22.13, 20.85, 16.10, 16.03, 15.15, 14.03, 13.71, 13.20, 10.98. HR-MS (ESI): Calcd for C₆₀H₈₆ClFN₃O₁₂S [M+Cl]⁻=1126.5610. Found=1126.5661.

43-N-(4-Fluoro-3-ethylbenzo[d]thiazol-2(3H)-ylidene)aminerapamycin (5b)

Yield: 42%. mp: 156–158°C; MS (ESI, m/z): 1091.7 (M+H)⁺; [α]_D −15.2 (c=1.0, MeOH); IR (KBr) cm⁻¹: 3427.8, 2930.2, 2871.5, 2044.1, 1716.8, 1645.7, 1592.6, 1583.0, 1491.0, 1451.6, 1382.6, 1202.2, 1103.7, 991.0; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.30 (m, 1H), 7.11 (dd, J=12.1, 8.5 Hz, 1H), 7.01–6.92 (m, 1H), 6.46 (s, 1H), 6.42 (m, 1H), 6.23–6.19 (m, 1H), 6.13 (d, J=13.4 Hz, 1H), 6.06 (m, 1H), 5.45 (dd, J=14.1, 9.8 Hz, 1H), 5.26 (s, 1H), 5.06 (d, J=10.2 Hz, 1H), 5.02–4.97 (m, 1H), 4.93 (d, J=5.4 Hz, 1H), 4.41–4.02 (m, 2H), 4.02 (d, J=4.7 Hz, 1H), 3.89 (d, J=4.9 Hz, 1H), 3.64 (d, J=12.4 Hz, 1H), 3.48–3.30 (m, 4H), 3.20 (s, 3H), 3.15 (s, 3H), 3.06 (s, 3H), 2.72 (d, J=16.2 Hz, 1H), 2.43–2.26 (m, 2H), 2.21–1.91 (m, 3H), 1.85 (m, 2H), 1.72 (s, 3H), 1.66(s, 3H), 1.62–1.48 (m, 6H), 1.44–1.02 (m, 12H), 0.98 (d, J=6.4 Hz, 3H), 0.86 (d, J=6.5 Hz,), 0.83 (d, J=6.4 Hz, 3H), 0.79 (d, J=6.6 Hz, 3H), 0.75 (d, J=6.6 Hz, 3H); ¹³C-NMR (101 MHz, DMSO-d₆) δ: 211.15, 208.04, 199.55, 169.66, 167.52, 158.57, 156.21, 152.06, 139.83, 138.36, 137.82, 137.70, 132.79, 130.91, 127.51, 127.44, 125.72, 123.31, 110.36, 109.56, 99.53, 86.21, 82.82, 81.73, 76.16, 74.06, 66.74, 61.56, 57.49, 55.97, 55.88, 51.12, 45.71, 44.32, 43.91, 35.72, 35.26, 33.53, 33.42, 30.73, 30.66, 30.08, 28.01, 26.92, 26.74, 24.96, 22.15, 20.86, 16.11 16.04, 15.16, 14.04, 13.70, 13.21, 10.48; HR-MS (ESI): Calcd for C₆₀H₈₆FClN₃O₁₂S [M+Cl]⁻=1126.5610. Found=1126.5582.

43-N-(5-Chloro-3-ethylbenzo[d]thiazol-2(3H)-ylidene)aminerapamycin (5c)

Yield: 42%. mp: 157–159°C; MS (ESI, m/z): 1108.6 (M+H)⁺; [α]_D −35.6 (c=1.1, MeOH); IR (KBr) cm⁻¹: 3428.9, 2931.2, 2872.0, 2043.8, 1716.2, 1645.3, 1592.5, 1583.2, 1491.8, 1451.9, 1382.4, 1202.7, 1103.1, 991.5; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.46 (d, J=8.2 Hz, 1H), 7.16 (s, 1H), 7.02 (d, J=8.2 Hz, 1H), 6.45 (s, 1H), 6.41–6.35 (m, 1H), 6.22–6.19 (m, 1H), 6.13 (d, J=13.4 Hz, 1H), 6.08–5.96 (m, 1H), 5.45 (dd, J=14.1, 9.8 Hz, 1H), 5.26 (s, 1H), 5.07 (d, J=10.2 Hz, 1H), 5.02–4.95 (m, 1H), 4.91 (d, J=5.3 Hz, 1H), 4.43–4.03 (m, 2H), 4.01 (d, J=4.7 Hz, 1H), 3.89 (d, J=4.6 Hz, 1H), 3.64 (d, J=12.0 Hz, 1H), 3.48–3.30 (m, 4H), 3.20 (s, 3H), 3.15 (s, 3H), 3.06 (s, 3H), 2.74 (d, J=16.0 Hz, 1H), 2.45–2.27 (m, 2H), 2.21–1.90 (m, 3H), 1.85 (m, 2H), 1.72 (s, 3H), 1.66 (s, 3H), 1.64–1.45 (m, 5H), 1.42–1.01 (m, 13H), 0.98 (d, J=6.4 Hz, 3H), 0.87 (d, J=6.5 Hz,), 0.82 (d, J=6.6 Hz, 3H), 0.79 (d, J=6.7 Hz, 3H), 0.75 (d, J=6.6 Hz, 3H); ¹³C-NMR (101 MHz, DMSO-d₆) δ: 211.13, 208.02, 199.54, 169.67, 167.52, 158.53, 156.21, 152.09, 139.83, 138.35, 137.81, 137.72, 132.76, 130.93, 127.51, 127.47, 125.72, 123.33, 110.36, 109.56, 99.51, 86.23, 82.83, 81.71, 76.18, 74.05, 66.74, 61.56, 57.49, 55.95, 55.86, 51.13, 45.73, 44.32, 43.91, 35.71, 35.27, 33.51, 33.41, 30.72, 30.66, 30.09, 28.03, 26.95, 26.73, 24.94, 22.12, 20.88, 16.11, 16.04, 15.15, 14.02, 13.72, 13.21, 10.96; HR-MS (ESI): Calcd for C₆₀H₈₅ClN₃O₁₂S [M−H]⁻=1106.5621. Found=1106.5661.

General Procedure for the Preparation of Compounds 9a and b

The foregoing method for the preparation of the 5a–c was applied to prepare the 9a and b except that compound 3 was used instead of 43-aminorapamycin.

43-O-(2-(3-Methylbenzo[d]thiazol-2(3H)-ylideneamino)ethyl)oxygenrapamycin (9a)

Yield: 39%. mp: 158–160°C; MS (ESI, m/z): 1104.5 (M+H)⁺; [α]_D −25.1 (c=1.1, MeOH); IR (KBr) cm⁻¹: 3428.6, 2933.5, 2872.7, 2045.1, 1717.5, 1646.5, 1591.0, 1582.9, 1492.1, 1452.0, 1384.8, 1203.6, 1102.5, 993.2; ¹H-NMR (400 MHz, DMSO-d₆) δ7.42 (d, J=7.7 Hz, 1H), 7.20 (t, J=7.9 Hz, 1H), 7.04 (d, J=7.9 Hz, 1H), 6.99 (t, J=7.7 Hz, 1H), 6.46 (s, 1H), 6.44–6.32 (m, 1H), 6.24–6.18 (m, 1H), 6.16 - 6.13 (m, 1H), 6.10–6.06 (m, 1H), 5.46–5.34 (m, 1H), 5.26 (s, 1H), 5.08 (d, J=10.2 Hz, 1H), 5.02–4.96 (m, 1H), 4.91 (d, J=5.6 Hz, 1H), 4.05–3.96 (m, 3H), 3.95 (d, J=4.6 Hz, 1H), 3.79–3.53 (m, 5H), 3.49–3.37 (m, 2H), 3.34 (s, 3H), 3.32 (s, 3H), 3.30–3.19 (m, 2H), 3.15 (s, 3H), 3.05 (s, 3H), 2.88–2.67 (m, 2H), 2.46–2.34 (m, 2H), 2.24–2.16 (m, 1H), 2.14–1.79 (m, 4H), 1.72 (s, 3H), 1.64 (s, 3H), 1.61–0.98 (m, 11H), 0.97 (d, J=6.4 Hz, 3H), 0.87 (d, J=6.5 Hz, 3H), 0.82 (d, J=6.6 Hz, 3H), 0.77 (d, J=6.6 Hz, 3H), 0.73 (d, J=6.6 Hz, 3H).¹³C-NMR (101 MHz, DMSO-d₆) δ: 211.04, 208.03, 199.45, 169.69, 167.48, 155.01, 148.33, 140.31, 140.27, 139.82, 138.34, 137.66, 132.84, 130.92, 127.51, 126.74, 125.51, 124.89, 123.77, 122.35, 110.52, 99.57, 86.04, 83.08, 82.95, 82.77, 76.21, 74.06, 69.95, 66.71, 57.71, 57.43, 55.96, 55.21, 51.24, 45.65, 43.99, 38.75, 36.62, 35.69, 35.32, 33.88, 32.75, 31.42, 30.53, 30.32, 29.46, 26.94, 26.71, 24.96, 22.19, 20.87, 16.04, 15.13, 13.92, 13.85, 10.96. HR-MS (ESI): Calcd for C₆₁H₈₈N₃O₁₃S [M−H]⁻=1102.6116. Found=1102.6158.

43-O-(2-(6-Chloro-3-methylbenzo[d]thiazol-2(3H)-ylideneamino)ethyl)oxygenrapamycin (9b)

Yield: 34%. mp: 160–162°C; MS (ESI, m/z): 1138.6 (M+H)⁺; [α]_D −35.3 (c=1.1, MeOH); IR (KBr) cm⁻¹: 3428.9, 2933.1, 2872.4, 2045.3, 1717.6, 1646.7, 1591.6, 1582.9, 1491.7, 1451.3, 1384.1, 1203.2, 1102.7, 993.6; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.66 (s, 1H), 7.29 (d, J=8.5 Hz, 1H), 7.08 (d, J=8.6 Hz, 1H), 6.47 (s, 1H), 6.45–6.34 (m, 1H), 6.23 (dd, J=10.1, 3.7 Hz, 1H), 6.17 (d, J=12.3 Hz, 1H), 6.14–6.06 (m, 1H), 5.46 (dd, J=14.6, 9.6 Hz, 1H), 5.29 (d, J=4.5 Hz, 1H), 5.09 (d, J=10.0 Hz, 1H), 5.01–4.96 (m, 1H), 4.94 (d, J=5.4 Hz, 1H), 4.05–3.98 (m, 1H), 3.94 (d, J=4.4 Hz, 1H), 3.79–3.57 (m, 2H), 3.49–3.38 (m, 1H), 3.34 (s, 1H), 3.33 (s, 1H), 3.30–3.19 (m, 2H), 3.15 (s, 1H), 3.05 (s, 1H), 2.86–2.67 (m, 1H), 2.45–2.32 (m, 1H), 2.20 (dt, J=16.1, 9.2 Hz, 1H), 2.14–1.79 (m, 3H), 1.74 (s, 1H), 1.63 (s, 1H), 1.61–0.99 (m, 9H), 0.97 (d, J=6.4 Hz, 1H), 0.87 (d, J=6.4 Hz, 1H), 0.82 (d, J=6.2 Hz, 1H), 0.77 (d, J=6.6 Hz, 1H), 0.73 (d, J=6.6 Hz, 1H). ¹³C-NMR (101 MHz, DMSO-d₆) δ: 211.03, 208.02, 199.44, 169.68, 167.49, 155.02, 148.32, 140.34, 140.28, 139.81, 138.33, 137.67, 132.83, 130.91, 127.50, 126.75, 125.50, 124.88, 123.75, 122.36, 110.50, 99.58, 86.05, 83.09, 82.93, 82.76, 76.22, 74.07, 69.94, 66.70, 57.70, 57.45, 55.95, 55.20, 51.21, 45.69, 43.98, 38.76, 36.64, 35.69, 35.30, 33.85, 32.77, 31.40, 30.54, 30.31, 29.45, 26.93, 26.71, 24.96, 22.15, 20.86, 16.04, 15.19, 13.95, 13.84, 10.96; HR-MS (ESI): Calcd for C₆₁H₈₇ClN₃O₁₃S [M−H]⁻=1172.5420. Found=1172.5466.

Acknowledgments

This study was supported by Grants from National Natural Science Foundation of China (81502935), and the Natural Science Foundation of Fujian province (2012J05014).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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