Design and Synthesis of Non-peptide RGD Mimics for Evaluation of Their Utility as Anti-platelet Agents

Abstract

Arg-Gly-Asp (RGD) mimics were synthesized and their anti-platelet activity was evaluated. A concise method was developed for the synthesis of the target compounds from dehydroepiandrosterone and Wieland–Miescher and Hajos–Parrish ketones, which are suitable for readily available platform. Among the synthesized compounds, the perhydronaphthalene framework with a 3-(4-piperidinyl)propoxyl structure 3e possessed the highest anti-aggregative activity. The IC₅₀ values of 3e were 0.91 mM (ADP initiation) and 0.54 mM (collagen initiation).

Platelets play an important role in hemostasis; however, its aggregation triggers several serious diseases, including cardiovascular, cerebrovascular, and peripheral vascular diseases. Platelet aggregation process is occurred by the stimulation of various physiological activators such as ADP, collagen and thrombin.^1,2) Finally the stimulation derives the structural transformation of glycoprotein IIb/IIIa (integrin αIIbβ3) on the platelet surface. The activated GPIIb/IIIa receptor interacts with fibrinogen to induce platelet aggregation.^3–5) The interaction between GPIIb/IIIa and fibrinogen is mediated by the Arg-Gly-Asp (RGD) sequence in fibrinogen.^6–8) Additionally, the β-turn structure formed by the sequence enhances the interaction of each molecules.^9,10) Therefore, RGD mimics are valuable platelet aggregation antagonists, and many compounds have been developed.^11–14) Among them, tirofiban (1)¹⁵⁾ and eptifibatide (2),¹⁶⁾ were launched for clinical use (Fig. 1).

Fig. 1. RGD Mimics for Acute Platelet Diseases

To develop a more efficient approach to RGD mimics, we assumed that the shape and volume of the β-turn structure is equal to that of perhydro polycyclic hydrocarbon structures such as perhydro-phenanthrene,^17,18) -naphthalene, and -indene (Fig. 2). Therefore, these hydrocarbons, with acidic and basic moieties of the appropriate position and size, were hypothesized to be good candidate for antiplatelet agents. Moreover, such compounds should be biologically stable and show better oral bioavailability than the peptide ligands. Additionally, a method for stereoselective transformation of such compounds is available, based on established concepts in steroid chemistry, enabling detailed conformational analysis of the ligand docking site. We planned to develop a method for synthesis of the orally active antiplatelet agents 3a–e from the readily available dehydroepiandrosterone (4) and Wieland–Miescher (5) and Hajos–Parrish ketones (6). Herein, we report the synthesis of the target peptide mimic compounds and an evaluation of those antiplatelet activities.

Fig. 2. Synthetic Strategy for RGD Mimics 3a–e

Chemistry

First, for preparation of the perhydrophenanthrene compounds 3a and b, the common intermediate 11 was synthesized from dehydroepiandrosterone 4 (Chart 1). Acetalization of the ketone 4¹⁹⁾ and Oppenauer oxidation of the secondary alcohol accompanying isomerization of the olefin yielded an enone.²⁰⁾ The stereoselective 1,4-reduction of the enone under Birch conditions²¹⁾ quenching with solid NH₄Cl afforded 7,²²⁾ and then Horner–Wadsworth–Emmons olefination provided the α,β-unsaturated ester 8a. One-electron reduction of the α,β-unsaturated ester in 8a with magnesium metal²³⁾ gave a mixture of stereoisomers (β : α=1 : 1); however, Birch reduction of α,β-unsaturated carboxylic acid 8b²⁴⁾ gave the β-rich isomer 9 with excellent selectivity (β : α=>10 : 1). Methylation of carboxylic acid 9b and deacetalization of 9a afforded compound 10, which was converted to the intermediate 11 by ozonolysis of the enol acetate of 10, followed by methylation of the carboxylic acid provided.^25,26)

Chart 1

Reagents and conditions: (a) (CH₂OH)₂, CSA, benzene, reflux, 80%; (b) Al(OPrⁱ)₃, cyclohexanone, toluene, reflux, 97%; (c) Li, liq. NH₃, THF, −78°C, then NH₄Cl, 88%; (d) (EtO)₂P(O)CH₂CO₂Me, KOBu^t, DMF, 0°C–r.t., 97%; (e) KOH, EtOH/H₂O, 90°C; (f) Li, liq. NH₃, ^tBuOH, THF/dioxane, −78°C, β : α=>10 : 1; (g) MeI, NaH, DMF, r.t.; (h) 1.5 M HCl, THF, r.t., 91% in 4 steps; (i) isopropenyl acetate, conc. H₂SO₄, reflux, 75%; (j) O₃, CH₂Cl₂/AcOH, then Me₂S, AcOH/H₂O, −78°C to r.t.; (k) CH₂N₂, Et₂O, 0°C, 83% in 2 steps.

Side-chain elongation of intermediate 11 was carried out by Horner–Wadsworth–Emmons (HWE) olefination with diethyl cyanomethyl phosphonate to yield 12 (Chart 2). Hydrogenation of the α,β-unsaturated nitrile in 12 with Adams’ catalyst^27,28) followed by tert-butoxycarbonyl (Boc) protection afforded the carbamate 13. Hydrolysis of the primary ester and subsequent deprotection afforded a primary amine. Finally, the guanidine formation from the amine with N,N′-bis(Boc)-1H-pyrazole-1-carboxamidine and subsequent deprotection²⁹⁾ gave the target compound 3a.

Chart 2

Reagents and conditions: (a) (EtO)₂P(O)CH₂CN, KOBu^t, DMF, 0°C to r.t., 96%; (b) PtO₂, H₂, HCl/MeOH/CHCl₃, r.t.; (c) Boc₂O, Et₃N, CH₂Cl₂, 0°C to r.t., 95% in 2 steps; (d) 10% LiOH aq., Bu₄NHSO₄, MeOH/THF, 0°C to r.t., quant.; (e) TFA, CH₂Cl₂, 0°C to r.t.; (f) N,Nʹ-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine, pyridine, 0°C to r.t., quant. in 2 steps; (g) TFA, CH₂Cl₂, 0°C to r.t., 87%.

An alternative target compound, 3b, which is one carbon shorter at the side chain than compound 3a, was synthesized from intermediate 11, by the following procedure (Chart 3).

Wittig chain elongation of intermediate 11, using methoxymethyl triphenylphosphonium chloride followed by hydrolysis of the enolate, produced aldehyde 14. Condensation between aldehyde 14 and hydroxylamine,³⁰⁾ followed by hydrogenation³¹⁾ and protection of the amine produced compound 15, at 78% yield. The following transformation to the target compound 3b was achieved by the same procedure as the synthesis of compound 3a.

Chart 3

Reagents and conditions: (a) MeOCH₂PPh₃·Cl, LHMDS, THF, −78°C, 4 h, 73%; (b) p-TsOH·H₂O, acetone/H₂O; 0°C to r.t., 73%; (c) NH₂OH·HCl, Et₃N, EtOH, r.t.; (d) PtO₂, H₂, AcOH, r.t.; (e) Boc₂O, Et₃N, CH₂Cl₂, 0°C to r.t., 78% in 3 steps; (f) 10% LiOH aq., Bu₄NHSO₄, MeOH/THF, 0°C to r.t., quant.; (g) TFA, CH₂Cl₂, 0°C to r.t.; (h) N,Nʹ-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine, pyridine, 0°C to r.t., 71% in 2 steps; (i) TFA, CH₂Cl₂, 0°C to r.t., quant.

For regulation of the hydrophilicity of the test compounds, we synthesized bicyclic compounds such as perhydro-naphthalene and -indene from Wieland–Miescher (5) and Hajos–Parrish ketones (6) (Charts 4, 5).

At first, a keto-acetal 17 was produced from 5, via a previously reported procedure^32–34) (Chart 4). To introduce the carboxyl unit, a HWE reaction of 17 yielded the α,β-unsaturated ester 18a at 93% yield, as a mixture of geometric isomers (E : Z=1 : 1). Hydrogenation or one-electron reduction of 18a with magnesium metal afforded a mixture of epimers (β : α=1 : 1 to 8 : 1). In contrast, the Birch reduction of carboxylic acid 18b followed by deacetalization and esterification gave the desired β compound 19 in good yield. Side-chain elongation with diethyl cyanomethylphosphonate and potassium tert-butoxide gave 20 at 95% yield. The direct transformation of the cyanomethylidene moiety in 20 to an aminoethyl group, using Adams’ catalyst, resulted in a mixture of diastereomers (β : α=1 : 1). Stepwise transformation by hydrogenation of the olefin in 20 with 10% palladium on carbon (β : α=14 : 1, determined the by ¹H-NMR spectrum) and hydrogenation of the cyano group in 21 with Adams’ catalyst followed by amidylation of the amine gave the guanidino compound 22 at good yield and diastereoselectivity. After hydrolysis of the ester 22 with 10% LiOH aq., the carboxylic acid was recrystallized to give the diastereomerically pure β-compound 24. Finally, deprotection of the N-Boc guanidino compound 24 using 4 M HCl yielded the target compound 3c.

Chart 4

Reagents and conditions: (a) (EtO)₂P(O)CH₂CO₂Me, KOBu^t, DMF, r.t., 98%; (b) KOH, EtOH/H₂O, 90°C, 93%; (c) Li, liq. NH₃, THF, −78°C; (d) 1.5 M HCl, THF, r.t.; (e) MeI, K₂CO₃, acetone, r.t., 91% in 3 steps; (f) (EtO)₂P(O)CH₂CN, KOBu^t, DMF, 70°C, 95%; (g) 10% Pd/C, H₂, EtOH, 1.5 h, quant. (β : α=14 : 1); (h) PtO₂, H₂, 10% HCl–MeOH, r.t.; (i) N,Nʹ-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine, Et₃N, DMF, r.t., 83% in 2 steps; (j) 10% LiOH aq., MeOH–THF, 0°C to r.t., 75%; (k) recrystallization 13%; (l) 4 M HCl–AcOEt, 0°C to r.t., quant.

The indene core compound 3d was also synthesized from a Hajos–Parrish ketone (6),^35,36) following the same procedure as described for compound 3c (Chart 5).

Chart 5

Reagents and conditions: (a) Li, liq. NH₃, THF, −78°C, then NH₄Cl, 70%; (b) (EtO)₂P(O)CH₂CO₂Me, KOBu^t, DMF, r.t., 97%; (c) KOH, EtOH/H₂O, r.t., 71%; (d) Li, liq. NH₃, THF, −78°C, then NH₄Cl; (e) 1.5 M HCl, THF, r.t.; (f) MeI, K₂CO₃, acetone, r.t., 98% in 3 steps; (g) (EtO)₂P(O)CH₂CN, NaH, DMF, r.t., 93%; (h) 10% Pd/C, H₂, EtOH, r.t., 80%; (i) PtO₂, H₂, 10% HCl–MeOH; (j) N,Nʹ-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine, Et₃N, DMF, r.t., 13% in 2 steps; (k) 10% LiOH aq., MeOH–THF, r.t., 82%; (l) 4 M HCl–AcOEt, r.t., 56%.

We also synthesized a pyrrolidino compound, 3e, from the intermediate 19 (Chart 6). The reduction of ketone 19, which was synthesized in Chart 4, with NaBH₄ afforded the alcohol 29 (β : α=8.5 : 1, determined by the ¹H-NMR spectrum). Etherification of the secondary alcohol in 29 with N-Boc bromopropyl piperidine³⁷⁾ gave compound 30. Finally, deprotection of the N-Boc guanidino compound 30, using 4 M HCl, afforded the target compound 3e.

Thus, accessibility to test compounds such as 3e will facilitate evolution of the structural diversity of RGD mimic analogs.

Chart 6

Reagents and conditions: (a) NaBH₄, MeOH, 0°C, 83%; (b) tert-butyl 4-(3-bromopropyl)piperidine-1-carboxylate, NaH, DMF, r.t., 29%; (c) 4 M HCl–AcOEt, r.t., 75%.

Biological Activity

In a preliminary study, the tricyclic compounds 3a and b were only slightly soluble in a buffer solution and dimethyl sulfoxide (DMSO), preventing testing of their anti-platelet activity. Therefore, we investigated the anti-platelet activity of the soluble bicyclic compounds 3c–e, which have low-lipophilicity cores relative to 3a and b. Initially, the dependence of the anti-platelet activity of compound 3c on concentration was measured by initiation with both ADP (Fig. 3, left) and collagen (Fig. 3, right) in platelet-rich plasma (PRP). The extent of platelet aggregation was measured at 5 min after addition of ADP. Maximum aggregation of the control was set as 100%. A strong correlation was observed between the concentration and the antiplatelet activity of 3c, using each inducer.

Fig. 3. Concentration Dependence of Platelet Aggregation by Compound 3c, Initiated by ADP (Left) and Collagen (Right)

Next, the anti-aggregative activity of compounds 3c–e was investigated in ADP-induced platelet aggregation (Fig. 4). The aggregation ratio of the perhydronaphthalene 3c was much higher than that of the perhydroindane 3d (Fig. 4, left). Comparison of the activities of the perhydronaphthalenes 3c and e indicated that the 3-(4-piperidinyl)propoxyl moiety in 3e is more effective than the guanidino group in 3c. In addition, Arg-Gly-Asp-Ser (RGDS) showed extremely smooth and strong anti-platelet activity compared to 3e (Fig. 4, right). Antiplatelet activity of RGD mimics were evaluated from maximum aggregation value of each compound against that of control (Fig. 5). The maximum anti-aggregation ratio of 3e (60%) was much higher than those of compounds 3c (41%) and d (17%).

Fig. 4. Anti-platelet Activity of Compounds 3c–e and RGDS (1.0 mM) on ADP-Induced Platelet Aggregation

Anti-platelet activities were monitored during 5 min after addition of an inducer.

Fig. 5. Anti-platelet Activity of Compounds 3c–e and RGDS (1.0 mM) on ADP-Induced Platelet Aggregation

Data are expressed as the mean±standard error (S.E.) (n=6 for 3c–e, n=5 for RGDS). ** p<0.01 compared with RGDS.

Conclusion

We have studied the synthesis of RGD-mimic compounds and evaluated the anti-platelet activity of each compound. We have developed a concise method for synthesis of RGD mimics from readily available steroid and bicyclic ketones. Among the compounds synthesized, the perhydronaphthalene framework with the 3-(4-piperidinyl)propoxyl structure 3e possessed the highest anti-aggregative activity. Further analogue syntheses and those biological evaluation are under investigation.

Experimental

General Methods

All melting points were measured on a Yanagimoto micro melting point apparatus and are uncorrected. Optical rotations were obtained using a JASCO P-2200 polarimeter. Optical purities were determined on a JASCO HPLC (PU-2089 plus, MD-2015 plus) instrument equipped with Daicel Chemical chiral columns. IR spectra were recorded on a Shimadzu IRPrestige-21 spectrophotometer. ¹H- and ¹³C-NMR spectra were measured on a JEOL JNM-AL300 (300 MHz), JEOL JMN-AL400 (400 MHz), JEOL JNM-LA500 (500 MHz) or JNM-ECA500 (500 MHz) spectrometers with tetramethylsilane as an internal standard. J-Values are given in Hertz. Mass spectra were recorded on a JEOL JMS-DX302 or JEOL JMS 700 instruments with a direct inlet system. Elemental analysis was performed using a Yanaco MT-6 elemental analyzer. Column chromatography was carried out on silica gel [Kanto Chemical Co., Inc., Japan (Silica Gel 60N, Spherical, neutral 40–50 µm) and Merck Ltd. (Silica Gel 60F₂₅₄, 230–400 mesh)]. TLC was carried out on silica gel plates [Merck Ltd., Germany (TLC Silica gel 60F₂₅₄)] and visualized with UV254 light and/or staining with phosphomolybdic acid in EtOH. The purity of compounds 3a–e were measured by the internal standard method using 1,4-bis(trimethylsilyl)benzene.³⁸⁾

(3S,8R,9S,10R,13S,14S)-10,13-Dimethyl-1,2,3,4,7,8,9,10,11,12,13,14,15,16-tetradecahydrospiro[cyclopenta[a]phenanthrene-17,2′-[1,3]dioxolan]-3-ol

To a solution of 4 (25.0 g, 86.7 mmol) in benzene (420 mL) were added ethylene glycol (14.5 mL, d 1.11, 259 mmol) and camphorsulfonic acid (4.0 g, 17.2 mmol). The reaction mixture was heated under reflux using Dean-Stark apparatus for 3 h. The mixture was cooled to room temperature (r.t.) and neutralized using satd aqueous NaHCO₃ on an ice cooling bath. The mixture was extracted with EtOAc and the combined organic layer was washed with brine. The extract was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified using column chromatography (AcOEt–n-hexane=1 : 8) and acetal (23 g, 80%) was obtained as a white powder.

mp 166–168°C (AcOEt–n-hexane); [α]_D²⁷ −92.3 (c=0.48, CHCl₃); Anal. Calcd for C₂₁H₃₂O₃: C, 75.86; H, 9.70. Found: C, 75.82; H, 9.60.

(8R,9S,10R,13S,14S)-10,13-Dimethyl-1,6,7,8,9,10,11,12,13,14,15,16-dodecahydrospiro[cyclopenta[a]phenanthrene-17,2′-[1,3]dioxolan]-3(2H)-one

Under nitrogen atmosphere, Al(Oi-Pr)₃ (2.6 g, 12.7 mmol) and cyclohexanone (39.7 mL, d 0.95, 383 mmol) were added to a toluene (255 mL) solution of acetal (8.5 g, 25.5 mmol) at r.t. After the 2 h reflux, the reaction mixture was cooled to r.t. and added satd aqueous potassium sodium tartrate. The aqueous layer was extracted with AcOEt and the combined extract was dried over MgSO₄. The concentrated residue was purified by column chromatography (AcOEt–n-hexane=1 : 3) to give enone (8.2 g, 97%) as colorless crystals.

mp 148–149°C (AcOEt–n-hexane); [α]_D²⁷ +62.2 (c=0.49, CHCl₃); Anal. Calcd for C₂₁H₃₀O₃: C, 76.33; H, 9.15. Found: C, 76.17; H, 8.98.

(5S,8R,9S,10S,13S,14S)-10,13-Dimethyltetradecahydrospiro[cyclopenta[a]phenanthrene-17,2′-[1,3]dioxolan]-3(2H)-one (7)

Under a nitrogen atmosphere, a lithium wire (1.0 g, 144 mmol) was added to liquid ammonia (300 mL) at −78°C. To the reaction mixture was added the solution of enone (4.0 g, 12.1 mmol) in tetrahydrofuran (THF) (150 mL) and stirred for 30 min. After the substrate disappearance was monitored by TLC, solid NH₄Cl was added until the mixture turned white suspension. The mixture was allowed to r.t. and the liquid ammonia was evaporated through the night. The residue was suspended with CH₂Cl₂ and filtrated. The concentrated residue was purified by column chromatography (AcOEt–n-hexane=1 : 3) to give compound 7 (3.5 g, 88%) as white crystals.

mp 203–204°C (MeOH); [α]_D²⁸ +2.1 (c=0.47, CHCl₃); Anal. Calcd for C₂₁H₃₂O₃: C, 75.86; H, 9.70. Found: C, 75.96; H, 9.91.

Methyl 2-((5S,8R,9S,10S,13S,14S)-10,13-Dimethyltetradecahydrospiro[cyclopenta[a]phenanthrene-17,2′-[1,3]dioxolan]-3(2H)-ylidene)acetate (8a)

To a suspension of t-BuOK (5.3 g, 47.5 mmol) in anhydrous DMF (95 mL) was added methyl diethylphosphonoacetate (10.5 mL, d 1.15, 57.0 mmol) at r.t. and stirred for 1 h under a nitrogen atmosphere. The mixture was cooled to 0°C and added the solution of 7 (6.3 g, 19.0 mmol) in anhydrous DMF (95 mL) and stirred for 12.5 h. The reaction mixture was poured into water and extracted with Et₂O. The combined extracts were washed with brine, dried over MgSO₄ and concentrated under the reduced pressure. The residue was purified by column chromatography (AcOEt–n-hexane=1 : 6) to give compound 8a (7.1 g, 97%) as a white powder.

mp 199–201°C (MeOH); [α]_D²⁸ +68.8 (c=0.16, CHCl₃); IR (CHCl₃) cm⁻¹: 2945, 1705, 1643, 1204, 1165; ¹H-NMR (CDCl₃, 400 MHz) δ: 5.59 (t, 1H, J=1.6 Hz), 4.04–3.80 (m, 4H), 3.67 (s, 3H), 2.44–2.26 (m, 1H), 2.22–2.08 (m, 1H), 2.06–0.45 (m, 20H), 0.94 (s, 3H), 0.85 (s, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 167.0, 163.5, 119.3, 112.1, 65.2, 64.6, 54.1, 50.8, 50.4, 47.6, 46.0, 40.2, 36.2, 35.8, 34.3, 33.7, 32.3, 31.4, 30.8, 29.0, 22.8, 20.7, 14.6, 12.1; HR-MS (EI) Calcd for C₂₄H₃₆O₄ m/z [M⁺]: 388.2614. Found 388.2612; Anal. Calcd for C₂₄H₃₆O₄: C, 74.19; H, 9.34. Found: C, 74.04; H, 9.59.

Methyl 2-((3S,5S,8R,9S,10S,13S,14S)-10,13-Dimethyl-17-oxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)acetate (10)

To a suspension of 8a (7.1 g, 18.3 mmol) in 1 : 1 EtOH/H₂O (184 mL) was added KOH (10.3 g, 183 mmol) and the mixture was heated at 90°C for 1.5 h. The reaction mixture was concentrated and neutralized with dilute HCl on the ice bath. The mixture was extracted with CH₂Cl_2, and the combined organic layer was dried over MgSO₄. The filtrate was concentrated to obtain the crude α,β-unsaturated carboxylic acid 8b.

Under the nitrogen atmosphere, a lithium wire (1.3 g, 187 mmol) was added to liquid ammonia (30 mL) at −78°C. To the mixture was added the solution of the above crude α,β-unsaturated carboxylic acid 8b in 1,4-dioxane (95 mL), THF (95 mL) and t-BuOH (7.1 mL, d 0.78, 74.2 mmol). After 3 h stirring, MeOH was added until the mixture turned white suspension. The mixture was allowed to r.t. and the liquid ammonia was evaporated through the night. The residue was suspended with CH₂Cl₂ and filtrated. The solution was acidified using dilute HCl and the water layer was extracted by CH₂Cl₂. The combined organic layer was dried over MgSO₄ and the concentrated residue was evaporated to give the crude carboxylic acid 9b. The ratio of isomers was determined the benzyl esters of 9b by ¹H-NMR.

Under the nitrogen atmosphere, sodium hydride (NaH) (2.2 g, 60% oil suspension, 54.9 mmol) was added to the above crude carboxylic acid 9b in anhydrous DMF (183 mL) at r.t. After the reaction mixture was stirred for 1 h, methyl iodide (MeI) (8.0 mL, 54.9 mmol) was added and the reaction mixture was stirred overnight. The reaction was concentrated, diluted by Et₂O and washed with H₂O. The organic layer was dried over MgSO₄ and concentrated to give crude methyl ester.

To the above crude methyl ester in THF (183 mL) was added 1.5 N HCl (245 mL) using the dropping funnel at 0°C and the mixture was stirred for 4 h at r.t. The reaction mixture was neutralized with 10% NaOH and concentrated under reduced pressure. The residue was extracted with AcOEt and the organic layer was dried over MgSO_4. The concentrated residue was purified by column chromatography (AcOEt–n-hexane=1 : 7) to give compound 10 (5.8 g, 91%, 4 steps) as a white powder.

mp 110–115°C (AcOEt–n-hexane); [α]_D²⁸ +74.3 (c=0.50, CHCl₃); IR (CHCl₃) cm⁻¹: 3012, 2920, 2855, 1728, 1439; ¹H-NMR (CDCl₃, 400 MHz) δ: 3.64 (s, 3H), 2.41 (dd, 1H, J=19.0, 8.7 Hz), 2.18 (d, 2H, J=7.1 Hz), 1.91 (dt, 1H, J=19.0, 9.0 Hz), 1.97–1.86 (m, 1H), 1.86–0.60 (m, 20H), 0.84 (s, 3H), 0.77 (s, 3H); ¹³C-NMR (CDCl_3, 100 MHz) δ: 220.9, 173.2, 54.6, 51.5, 51.4, 47.8, 46.4, 41.8, 38.3, 36.0, 35.9, 35.24, 35.21, 35.1, 31.7, 31.0, 28.59, 28.57, 21.9, 20.4, 13.9, 12.4; HR-MS (EI) Calcd for C₂₂H₃₄O₃ m/z [M⁺]: 346.2508. Found 346.2511.

Methyl 2-((3S,5S,8R,9S,10S,13S,14S)-17-Acetoxy-10,13-dimethyl-2,3,4,5,6,7,8,9,10,11,12,13,14,15-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)acetate

To a solution of 10 (6.1 g, 17.6 mmol) in isopropenyl acetate (38.4 mL) was added the solution of conc. H₂SO₄ (0.1 mL) in isopropenyl acetate (5.0 mL) under a nitrogen atmosphere. After stirring for 5.5 h at 90°C, above H₂SO₄ in isopropenyl acetate (1.9 mL) was added and then the reaction mixture was stirred for 4.5 h at same temperature. The reaction mixture was diluted with Et₂O and neutralized with satd aqueous NaHCO₃ at 0°C. The mixture was extracted with Et₂O and the combined organic layer was dried over MgSO₄. The concentrated residue was purified by column chromatography (AcOEt–n-hexane=1 : 15) to give acetate (5.2 g, 75%) as a white solid.

IR (CHCl₃) cm⁻¹: 2920, 2855, 1732, 1229, 1202; ¹H-NMR (CDCl₃, 400 MHz) δ: 5.44 (dd, 1H, J=2.9, 1.5 Hz), 3.65 (s, 3H), 2.29–2.07 (m, 1H), 2.19 (d, 2H, J=7.1 Hz), 2.13 (s, 3H), 1.98–0.67 (m, 20H), 0.87 (s, 3H), 0.77 (s, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 173.2, 168.6, 159.5, 111.1, 55.1, 54.2, 51.4, 46.7, 44.9, 41.9, 38.1, 36.1, 35.3 (CH), 35.3 (CH₂), 33.7, 33.5, 31.2, 29.0, 28.7, 28.6, 21.3, 20.6, 15.7, 12.4; HR-MS (EI) Calcd for C₂₄H₃₆O₄ m/z [M⁺]: 388.2614. Found 388.2617.

Methyl (1S,2S,4aS,4bS,7S,8aS,10aR)-7-(2-Methoxy-2-oxoethyl)-2,4b-dimethyl-1-(2-oxoethyl)tetradecahydrophenanthrene-2-carboxylate (11)

To a solution of acetate (1.0 g, 2.6 mmol) and AcOH (2.1 mL) in CH₂Cl₂ (26 mL) was bubbled with ozone at −78°C for 3 h. The dissolved ozone gas was removed by Ar bubbling through the solution. Then Me₂S (3.0 mL, d 0.85, 41.0 mmol) was added to the mixture, and the resulting mixture was stirred at 0°C for 6.5 h. After addition of AcOH (24 mL) and H₂O (8 mL), the mixture was stirred at r.t. for 1 d. The reaction mixture was added H₂O and extracted with CH₂Cl₂. The organic layer was dried over MgSO₄ and concentrated in vacuo. The residue was diluted with Et₂O, and a solution of CH₂N₂ in Et₂O was added until the resulting solution turned to yellow. The mixture was warmed to r.t. and stirred overnight. The concentrated residue was purified by column chromatography (Et₂O–n-hexane=1 : 5) to give compound 11 (845 mg, 83% in 2 steps) as a white solid.

mp 68–70°C (AcOEt–n-hexane); IR (CHCl₃) cm⁻¹: 3026, 2949, 2855, 1721, 1435; [α]_D²⁷ −26.6 (c=0.36, CHCl₃); ¹H-NMR (CDCl₃, 400 MHz) δ: 9.68 (d, 1H, J=3.2 Hz), 3.654 (s, 3H), 3.646 (s, 3H), 2.42–2.31 (m, 1H), 2.27 (ddd, 1H, J=17.2, 7.2, 3.2 Hz), 2.19 (d, 2H, J=6.8 Hz), 2.10 (dd, 1H, J=17.2, 2.2 Hz), 1.88–0.67 (m, 18H), 1.11 (s, 3H), 0.72 (s, 3H). ¹³C-NMR (CDCl₃, 100 MHz) δ: 201.9, 178.1, 173.2, 53.3, 52.0, 51.4, 47.4, 46.7, 45.8, 41.8, 41.7, 38.1, 37.4, 36.7, 35.9, 35.2, 35.0, 31.9, 28.7, 28.5, 19.8, 15.6, 12.3; HR-MS (FAB) Calcd for C₂₃H₃₇O₅ m/z [MH⁺]: 393.2641. Found 393.2641.

Methyl (1S,2S,4aS,4bS,7S,8aS,10aR)-1-(3-Cyanoallyl)-7-(2-methoxy-2-oxoethyl)-2,4b-dimethyltetradecahydrophenanthrene-2-carboxylate (12)

To a suspension of KO^tBu (44.0 mg, 0.38 mmol) in DMF (640 µL) was added diethyl cyanomethylphosphonate (59.6 µL, d 1.13, 0.38 mmol) and the mixture was stirred at 0°C for 1 h under a nitrogen atmosphere. To the solution was added compound 11 (50.0 mg, 0.13 mmol) in anhydrous DMF (640 µL) and stirred at r.t. for 10 h. The reaction mixture was poured into H₂O and extracted with Et₂O. The combined extract was dried over MgSO₄ and concentrated. The residue was purified by column chromatography (Et₂O–n-hexane=1 : 3) to give compound 12 (50.5 mg, 96%) as white amorphous.

IR (CHCl₃) cm⁻¹: 2949, 2922, 2224, 1721, 1630, 1435, 1236; [α]_D²⁷ −28.2 (c=0.24, CHCl₃); ¹H-NMR (CDCl₃, 300 MHz, E : Z=1 : 1.8) δ: 6.70 (dt, 0.36H, J=16.5, 7.2 Hz), 6.49 (ddd, 0.64H, J=10.9, 6.8, 6.6 Hz), 5.28 (dt, 0.36H, J=16.5, 1.7 Hz), 5.21 (dt, 0.64H, J=10.9, 1.7 Hz), 3.670 (s, 1.1H), 3.665 (s, 1.9H), 3.663 (s, 1.9H), 3.661 (s, 1.1H), 2.51–2.30 (m, 0.64H), 2.26–2.09 (m, 1H), 2.20 (d, 2H, J=7.0 Hz), 2.08–1.95 (m, 0.36H), 1.91–0.73 (m, 19H), 1.15 (s, 1.9H), 1.10 (s, 1.1H), 0.74 (s, 1.9H), 0.73 (s, 1.1H); ¹³C-NMR (CDCl₃, 100 MHz, E : Z=1 : 5.5) δ: 178.2, 172.9, 155.7, 115.7, 98.2, 53.3, 51.7, 51.2, 47.4, 46.9, 45.6, 41.6, 38.0, 37.3, 37.2, 35.8, 35.0, 34.9, 34.3, 31.5, 28.6, 28.3, 19.6, 15.2, 12.2; HR-MS (FAB) Calcd for C₂₅H₃₈NO₄ m/z [MH⁺]: 416.2801. Found 416.2806.

Methyl (1S,2S,4aS,4bS,7S,8aS,10aR)-1-(4-((tert-Butoxycarbonyl)amino)butyl)-7-(2-methoxy-2-oxoethyl)-2,4b-dimethyltetradecahydrophenanthrene-2-carboxylate (13)

Under a hydrogen atmosphere (10 atm), the suspension of compound 12 (144 mg, 0.35 mmol), Pt₂O (27 mg) and 10% HCl–MeOH (9.0 mL) in CHCl₃ (6.0 mL) was stirred at for 11.5 h in an autoclave at r.t. The reaction mixture was filtered through a Celite pad and the filtrate was concentrated. The residue was dissolved in CH₂Cl₂ (4.0 mL) and added Et₃N (146 µL, d 0.73, 1.05 mmol) and Boc₂O (161 µL, d 0.95, 0.70 mmol). After 7 h stirring, the reaction mixture was washed with H₂O and dried over MgSO₄. The concentrated residue was purified by column chromatography (AcOEt–n-hexane=1 : 5) to give compound 13 (174 mg, 95%, 2 steps) as a pale yellow oil.

[α]_D²⁸ −16.7 (c=0.46, CHCl₃); IR (CHCl₃) cm⁻¹: 3453, 3024, 2928, 2857, 2359, 1715, 1506; ¹H-NMR (CDCl₃, 300 MHz) δ: 4.49 (br, 1H), 3.64 (s, 3H), 3.63 (s, 3H), 3.03 (dd, 2H, J=11.8, 6.3 Hz), 2.18 (d, 2H, J=6.9 Hz), 2.00–0.50 (m, 25H), 1.42 (s, 9H), 1.05 (s, 3H), 0.70 (s, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 179.2, 173.5, 155.9, 78.9, 53.3, 51.5, 51.3, 47.7, 47.5, 45.8, 41.7, 40.3, 38.0, 37.7, 36.9, 35.8, 35.1, 35.0, 31.8, 31.6, 30.5, 28.8, 28.4, 28.4, 27.6, 19.6, 15.1, 12.2; HR-MS (EI) Calcd for C₃₀H₅₁NO₆ m/z [M⁺]: 521.3716. Found 522.3797.

2-((2S,4aS,4bS,7S,8S,8aR,10aS)-8-(4-((tert-Butoxycarbonyl)amino)butyl)-7-(methoxycarbonyl)-4a,7-dimethyltetradecahydrophenanthren-2-yl)acetic Acid

To a solution of 13 (400 mg, 0.77 mmol) and Bu₄NHSO₄ (40 mg) in 1 : 1 MeOH/THF (15.4 mL) was added 10% LiOH (3.6 mL, 15.2 mmol) and stirred at r.t. for 3.5 h. The mixture was concentrated in vacuo and dilute with CH₂Cl₂. The mixture was neutralized with 10% KHSO₄ and the aqueous layer was extracted with CH₂Cl₂. The concentrated residue was purified by column chromatography (CH₂Cl₂–MeOH=10 : 1) to give a desired carboxylic acid (389 mg, quant.) as a white amorphous.

¹H-NMR (CDCl₃, 300 MHz) δ: 4.51 (br, 1H), 3.64 (s, 3H), 3.04 (dd, 2H, J=11.6, 6.2 Hz), 2.22 (d, 2H, J=6.9 Hz), 1.95–0.65 (m, 25H), 1.43 (s, 9H), 1.11 (s, 3H), 0.72 (s, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 179.0, 178.2, 155.7, 79.0, 53.4, 51.6, 47.9, 47.6, 46.0, 41.8, 40.4, 38.2, 37.9, 37.0, 36.0, 35.1, 35.0, 32.0, 31.8, 30.7, 29.0, 28.5, 28.5, 27.8, 19.8, 15.3, 12.4.

2-((2S,4aS,4bS,7S,8S,8aR,10aS)-8-(4-Guanidinobutyl)-7-(methoxycarbonyl)-4a,7-dimethyltetradecahydrophenanthren-2-yl)acetic Acid (3a)

To a solution of the above carboxylic acid (350 mg, 0.71 mmol) in CH₂Cl₂ (7.1 mL) was added trifluoroacetic acid (TFA) (5.5 mL, d 1.48, 71 mmol) at 0°C, and the mixture was stirred at r.t. for 4.5 h. The reaction mixture was concentrated, and then pyridine (7.1 mL) and N,N′-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (1.1 g, 3.55 mmol) was added to the residue. The mixture was stirred for 11 d and the reaction mixture was diluted with Et₂O. The mixture was neutralized with 10% KHSO₄ and the water layer was extracted by Et₂O. The organic layer was dried over MgSO₄ and concentrated in vacuo. The residue was purified by column chromatography (CH₂Cl₂–MeOH=20 : 1) to give a N-Boc guanidine derivative (462 mg, 2 steps quant.) as white amorphous. To the solution of above compound (50 mg, 0.08 mmol) in CH₂Cl₂ (769 µL) was added TFA (592 µL, d 1.48, 7.69 mmol) at 0°C and stirred at r.t. for 5 h. The reaction mixture was concentrated to give crude guanidine 3a (30 mg, 87%, 68% purity) as a colorless oil.

[α]_D²⁸ −5.75 (c=0.72, EtOH); IR (KBr) cm⁻¹: 3354, 3171, 2937, 2857, 1705, 1662, 1451, 1383, 1243, 1148, 1120; ¹H-NMR (CD₃OD, 50°C, 500 MHz) δ: 3.64 (s, 3H), 3.11 (t, 2H, J=7.0 Hz), 2.15 (d, 2H, J=7.0 Hz), 1.92 (ddd, 1H, J=12.5, 6.7, 3.4 Hz), 1.85–0.60 (m, 24 H), 1.10 (s, 3H), 0.77 (s, 3H); ¹³C-NMR (CD₃OD, 50°C, 100 MHz) δ: 180.5, 176.5, 158.5, 55.1, 52.1, 49.0, 47.5, 42.8, 42.4, 39.5, 39.2, 39.1, 38.3, 37.1, 36.5, 36.3, 33.4, 32.8, 30.5, 30.2, 29.6, 28.7, 20.9, 15.7, 12.7; HR-MS (FAB) Calcd for C₂₅H₄₄N₃O₄ m/z [MH⁺]: 450.3332. Found 450.3339.

Methyl (1S,2S,4aS,4bS,7S,8aS,10aR)-7-(2-Methoxy-2-oxoethyl)-1-(3-methoxyallyl)-2,4b-dimethyltetradecahydrophenanthrene-2-carboxylate

To a suspension of (methoxymethyl)triphenylphosphonium chloride (263 mg, 0.78 mmol) in THF (2.6 mL) was added LHMDS (765 µL, 0.765 mmol) and stirred at −78°C for 15 min under a nitrogen atmosphere. After the solution of 11 (100 mg, 0.255 mmol) in THF (2.6 mL) was added to the reaction mixture and stirred for 4.5 h. The reaction was quenched with satd aqueous NH₄Cl and extracted with Et₂O. The organic layer was washed with brine and dried over MgSO₄. The concentrated residue was purified by column chromatography (AcOEt–n-hexane=1 : 10) to give a desired enol ether (78.4 mg, 73%) as a colorless oil.

¹H-NMR (CDCl₃, 300 MHz, E : Z=1 : 1.2) δ: 6.16 (d, 0.46H, J=12.6 Hz), 5.74 (dt, 0.54H, J=6.2, 1.6 Hz), 4.64 (dt, 0.46H, J=12.6, 7.6 Hz), 4.28 (dd, 0.54H, J=13.6, 7.1 Hz), 3.66 (s, 3H), 3.631 (s, 1.4H), 3.625 (s, 1.6H), 3.55 (s, 1.6H), 3.47 (s, 1.4H), 2.20 (d, 2H, J=6.9 Hz), 2.14–0.52 (m, 21H), 1.11 (s, 1.6H), 1.10 (s, 1.4H), 0.73 (s, 3H); ¹³C-NMR (CDCl₃, 100 MHz, E : Z=1 : 1.2) δ: 179.1 (Z), 179.0 (E), 173.23 (Z), 173.21 (E), 147.0 (E), 145.0 (Z), 106.9 (Z), 102.8 (E), 59.4, 55.9, 53.5, 51.5 (Z), 51.4 (E), 48.0 (E), 47.5 (Z), 47.1, 45.9 (0.54), 45.8 (E), 41.8, 38.2, 37.9 (E), 37.7 (Z), 37.4 (0.54), 37.3 (E), 36.0, 35.21 (Z), 35.20 (E), 35.16 (Z), 35.12 (E), 31.8 (E), 31.5 (0.54), 29.2 (E), 28.93 (Z), 28.91 (E), 28.6 (Z), 25.8, 19.93 (E), 19.90 (Z), 15.4 (E), 15.3 (Z), 12.3; HR-MS (FAB) Calcd for C₂₅H₄₁O₅ m/z [MH⁺]: 421.2954. Found 421.2963.

Methyl (1S,2S,4aS,4bS,7S,8aS,10aR)-7-(2-Methoxy-2-oxoethyl)-2,4b-dimethyl-1-(3-oxopropyl)tetradecahydrophenanthrene-2-carboxylate (14)

To a solution of the enol ether (114 mg, 0.271 mmol) in acetone (9 mL)–H₂O (300 µL) was added TsOH·H₂O (262 mg, 1.35 mmol) at 0°C and stirred at r.t. for overnight. The reaction mixture was neutralized with satd aqueous NaHCO₃ and extracted with Et₂O. The combined organic layer was washed with brine and dried over MgSO₄. The concentrated residue was purified by column chromatography (AcOEt–n-hexane=1 : 5) to give compound 14 (80.0 mg, 73%) as a colorless oil.

IR (CHCl₃) cm⁻¹: 3516, 2949, 2922, 2727, 1732, 1711, 1385, 1238; ¹H-NMR (CDCl₃, 300 MHz) δ: 9.62 (t, 1H, J=1.4 Hz), 3.62 (s, 3H), 3.62 (s, 3H), 2.43 (td, 2H, J=7.8, 1.4 Hz), 2.16 (d, 2H, J=7.2 Hz), 1.82–0.73 (m, 21H), 1.08 (s, 3H), 0.70 (s, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 201.8, 178.6, 173.1, 53.3, 51.7, 51.3, 47.8, 46.7, 45.8, 45.0, 41.7, 38.1, 37.9, 37.2, 35.9, 35.1, 35.0, 31.9, 28.8, 28.5, 24.0, 19.7, 15.2, 12.3; HR-MS (EI) Calcd for C₂₄H₃₈O₅ m/z [M⁺]: 406.2719. Found 406.2717.

Methyl (1S,2S,4aS,4bS,7S,8aS,10aR)-1-(3-((tert-Butoxycarbonyl)amino)propyl)-7-(2-methoxy-2-oxoethyl)-2,4b-dimethyltetradecahydrophenanthrene-2-carboxylate (15)

To a solution of 14 (355 mg, 0.87 mmol) in EtOH (9 mL) was added Et₃N (487 µL, d 0.73, 3.49 mmol) and hydroxylamine hydrochloride (152 mg, 2.18 mmol) and stirred vigorously at r.t. for 2.5 h. The reaction mixture was diluted with Et₂O and washed with water and brine. The organic layer was dried over MgSO₄ and concentrated in vacuo to give crude oxime. Under a hydrogen atmosphere (30 atm), a suspension of the crude oxime (165 mg, 0.39 mmol), PtO₂ (16.5 mg) in AcOH (15 mL) was vigorously stirred at r.t. for 10 h in an autoclave. The reaction mixture was filtered through a Celite pad and concentrated. The residue was dissolved in CH₂Cl₂ (4.0 mL) and added Et₃N (163 µL, d 0.73, 1.17 mmol) and Boc₂O (179 µL, d 0.95, 0.78 mmol). After 2 h stirring, the reaction mixture was washed with H₂O and dried over MgSO₄. The concentrated residue was purified by column chromatography (AcOEt–n-hexane=1 : 5) to give compound 15 (155 mg, 78%) as a white amorphous.

[α]_D²⁷ +18.2 (c=0.51, CHCl₃); IR (CHCl₃) cm⁻¹: 3455, 2947, 2924, 1715, 1506; ¹H-NMR (CDCl₃, 500 MHz) δ: 4.66 (br, 1H), 3.66 (s, 3H), 3.66 (s, 3H), 2.99 (quintet, 2H, J=6.2 Hz), 2.20 (d, 2H, J=7.4 Hz), 1.88–0.73 (m, 23H), 1.43 (s, 9H), 1.08 (s, 3H), 0.73 (s, 3H); ¹³C-NMR (CDCl₃, 125 MHz) δ: 179.0, 173.3, 155.8, 78.6, 53.2, 51.4, 51.1, 47.7, 47.0, 45.7, 41.6, 40.7, 37.9, 37.7, 36.8, 35.7, 34.94, 34.89, 31.7, 30.6, 29.0, 28.7, 28.3, 19.5, 15.0, 12.1; HR-MS (FAB) Calcd for C₂₉H₅₀NO₆ m/z [MH⁺]: 508.3638. Found 508.3637.

2-((2S,4aS,4bS,7S,8S,8aR,10aS)-8-(3-((tert-Butoxycarbonyl)amino)propyl)-7-(methoxycarbonyl)-4a,7-dimethyltetradecahydrophenanthren-2-yl)acetic Acid

To a solution of 15 (126 mg, 0.25 mmol) and Bu₄NHSO₄ (13 mg) in 1 : 1 MeOH/THF (5.0 mL) was added 10% aqueous LiOH (1.2 mL, 5.0 mmol) and stirred at r.t. overnight. The reaction mixture was concentrated in vacuo and diluted with CH₂Cl₂. The solution was neutralized with 10% aqueous KHSO₄ at 0°C, and the aqueous layer was extracted with CH₂Cl₂. The concentrated residue was purified by column chromatography (CH₂Cl₂–MeOH=20 : 1) to give a desired carboxylic acid (122 mg, quant) as a white amorphous.

mp 176–180°C (AcOEt–n-hexane); [α]_D²⁷ +83.1 (c=0.045, CHCl₃); IR (CHCl₃) cm⁻¹: 3455, 2945, 2924, 2857, 1709, 1506; ¹H-NMR (CDCl₃, 400 MHz) δ: 4.56 (br, 1H), 3.66 (s, 3H), 3.00 (br, 2H), 2.22 (d, 2H, J=5.1 Hz), 1.87–0.68 (m, 23H), 1.43 (s, 9H), 1.08 (s, 3H), 0.73 (s, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 179.0, 178.1, 155.7, 79.0, 53.4, 51.7, 47.9, 47.2, 45.9, 41.8, 41.0, 38.2, 37.9, 37.1, 36.0, 35.1, 35.0, 32.0, 31.5, 30.9, 29.3, 28.9, 28.5, 19.8, 15.3, 12.4; HR-MS (FAB) Calcd for C₂₈H₄₈NO₆ m/z [MH⁺]: 494.3482. Found: 494.3477.

2-((2S,4aS,4bS,7S,8S,8aR,10aS)-8-(3-((Z)-2,3-Bis(tert-butoxycarbonyl)guanidino)-propyl)-7-(methoxycarbonyl)-4a,7-dimethyltetradecahydrophenanthren-2-yl)acetic Acid (16)

To a solution of the carboxylic acid (110 mg, 0.22 mmol) in CH₂Cl₂ (2.2 mL) was added TFA (1.7 mL, 22 mmol) at 0°C and the mixture was stirred at r.t. for 4.5 h. The reaction mixture was concentrated, and then pyridine (2.2 mL) and N,N′-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (140 mg, 0.44 mmol) was added to the residue. The solution was stirred for 6 d and the reaction mixture was diluted with Et₂O. The mixture was neutralized with 10% aqueous KHSO_4, and the aqueous layer was extracted by Et₂O. The organic layer was dried over MgSO₄ and concentrated in vacuo. The residue was purified by column chromatography (CH₂Cl₂– MeOH=20 : 1) to give compound 16 (101 mg, 71%) as white amorphous.

IR (CHCl₃) cm⁻¹: 3688, 3327, 2928, 1719, 1634, 1616, 1578; [α]_D²⁰ −13.8 (c=0.87, CHCl₃); ¹H-NMR (CDCl₃, 400 MHz) δ: 8.24 (t, 1H, J=4.4 Hz), 3.66 (s, 3H), 3.27 (m, 2H), 2.22 (d, 2H, J=7.1 Hz), 1.83–0.72 (m, 23H), 1.48 (s, 18H), 1.02 (s, 3H), 0.72 (s, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 178.8, 178.2, 163.3, 155.8, 153.0, 82.9, 79.1, 53.4, 51.7, 47.9, 47.2, 46.0, 41.8, 41.4, 38.1, 37.8, 37.1, 36.0, 35.1, 35.0, 32.0, 30.0, 29.4, 28.9, 28.5, 28.4, 28.1, 19.8, 15.3, 12.4; HR-MS (FAB) Calcd for C₃₄H₅₈N₃O₈ m/z [MH⁺]: 635.4146. Found 636.4224;

2-((2S,4aS,4bS,7S,8S,8aR,10aS)-8-(3-Guanidinopropyl)-7-(methoxycarbonyl)-4a,7-dimethyltetradecahydrophenanthren-2-yl)acetic Acid (3b)

To a solution of 16 (50 mg, 0.08 mmol) in CH₂Cl₂ (786 µL) was added TFA (606 µL, d 1.48, 7.86 mmol) at 0°C, and the mixture was stirred at r.t. for 5 h. The reaction mixture was concentrated to give a crude guanidine 3b (34 mg, quant, 69% purity) as a colorless oil.

IR (CHCl₃) cm⁻¹: 2952, 2924, 1710, 1678, 1199; [α]_D²⁷ −56.0 (c=0.03, EtOH); ¹H-NMR (CDCl₃, 400 MHz) δ: 3.65 (s, 3H), 3.07 (t, 2H, J=6.7 Hz), 2.17 (d, 2H, J=7.1 Hz), 1.94 (dd, 1H, J=12.5, 2.8 Hz), 1.84–0.60 (m, 22 H), 1.11 (s, 3H), 0.78 (s, 3H); ¹³C-NMR (CD₃OD, 100 MHz) δ: 180.4, 176.6, 158.3, 55.0, 52.3, 49.1, 48.6, 47.4, 42.75, 42.69, 39.4, 39.2, 38.4, 37.1, 36.5, 36.2, 33.3, 31.0, 30.3, 30.1, 29.5, 20.8, 16.0, 12.8; HR-MS (FAB) Calcd for C₂₄H₄₂N₃O₄ m/z [M+H⁺]: 436.3186. Found 436.3181.

Methyl 2-((4aS,8aS)-8a-Methylhexahydro-2H-spiro[naphthalene-1,2′-[1,3]dioxolan]-6(5H)-ylidene)acetate (18a)

Under a nitrogen atmosphere, to a suspension of t-BuOK (3.0 g, 27 mmol) in anhydrous DMF (22 mL) was added methyl diethylphosphonoacetate (4.9 mL, d 1.15, 27 mmol) at r.t. and stirred for 1.5 h. The mixture was cooled to 0°C and the solution of 17 (2.0 g, 8.9 mmol) in anhydrous DMF (23 mL) was added, and the resulting mixture was stirred for 12.5 h. The reaction mixture was poured into water and extracted with Et₂O. The combined extracts was washed with brine, dried over MgSO₄ and concentrated under the reduced pressure. The residue was purified by column chromatography (AcOEt–n-hexane=1 : 4) to give compound 18a (2.6 g, 98%) as a colorless oil.

IR (CHCl₃) cm⁻¹: 2949, 1707, 1165; [α]_D²⁶ +0.88 (c=0.90, EtOH); ¹H-NMR (CDCl₃, 400 MHz, E : Z=1 : 1) δ: 5.59 (1/2H, s), 5.58 (1/2H, s), 3.95-3.81 (4H, m), 3.77 (1/2H, d, J=14.4 Hz), 3.67 (3/2H, s), 3.66 (3/2H, s), 3.56 (1/2H, d, J=13.6 Hz), 2.43–1.21 (12H, m), 1.10 (3H, s); ¹³C-NMR (CDCl₃, 100 MHz, E : Z=1 : 1) δ: 167.2, 167.1, 163.0, 162.9, 112.7, 112.54, 112.50, 112.4, 65.1, 65.0, 64.93, 64.87, 50.67, 50.66, 42.8, 42.3, 42.2, 42.0, 39.6, 33.1, 31.6, 31.4, 31.0, 30.5, 30.4, 27.9, 27.7, 24.8, 22.80, 22.77, 13.5, 13.3; HR-MS (EI) Calcd for C₁₆H₂₄O₄ m/z [M⁺]: 280.1675. Found 280.1672.

2-((4aS,8aS)-8a-Methylhexahydro-2H-spiro[naphthalene-1,2′-[1,3]dioxolan]-6(5H)-ylidene)acetic Acid (18b)

To a suspension of 18a (2.5 g, 8.6 mmol) in 1 : 1 EtOH/H₂O (86 mL) was added KOH (4.8 g, 86 mmol), and the mixture was heated at 90°C for 0.5 h. The reaction mixture was concentrated and neutralized with 3 M HCl on an ice bath. The mixture was extracted with CH₂Cl_2, and the combined organic layer was dried over MgSO₄. The concentrated residue was purified by column chromatography (CH₂Cl₂–MeOH=20 : 1) to give α,β-unsaturated carboxylic acid 18b (2.1 g, 93%) as a white amorphous.

IR (CHCl₃) cm⁻¹: 2933, 1685, 1631; [α]_D²⁶ −12.7 (c=0.06, EtOH); ¹H-NMR (CDCl₃, 400 MHz, E : Z=1 : 1) δ: 11.8 (1H, br s), 5.61 (1/2H, s), 5.60 (1/2H, s), 3.98–3.82 (4H, m), 3.77 (1/2H, d, J=14.8 Hz), 3.55 (1/2H, d, J=13.2 Hz), 2.35 (1/2H, dt, J=4.8, 13.6 Hz), 2.35–2.10 (m, 1H), 2.05–1.90 (m, 1H), 1.87–1.45 (m, 15/2H), 1.41–1.17 (m, 2H), 1.10 (s, 3/2H), 1.09 (s, 3/2H); ¹³C-NMR (CDCl₃, 100 MHz, E : Z=1 : 1) δ: 172.3, 172.2, 165.8, 165.6, 112.8, 112.6 (2C), 112.5, 65.14, 65.08, 65.0, 64.9, 42.9, 42.3, 42.2, 42.1, 39.9, 33.3, 31.6 (2C), 31.1, 30.5, 30.4, 27.9, 27.7, 25.1, 22.80, 22.77, 13.5, 13.4; HR-MS (EI) Calcd for C₁₅H₂₂O₄ m/z [M⁺]: 266.1518. Found 266.1517.

Methyl 2-((2S,4aS,8aS)-4a-Methyl-5-oxodecahydronaphthalen-2-yl)acetate (19)

Under a nitrogen atmosphere, a lithium wire (0.5 g, 68 mmol) was added to liquid ammonia (96 mL) at −78°C. To this mixture was added a solution of 18b (1.8 g, 6.8 mmol) in THF (45 mL), and the resulting mixture was stirred for 3 h. After the substrate disappearance was monitoring by TLC, solid NH₄Cl was added until the mixture turned white suspension. The mixture was allowed to r.t. and the liquid ammonia was evaporated through the night. The residue was suspended with CH₂Cl₂ and filtrated. To the concentrated residue in THF (68 mL) was added 1.5 M HCl (0.11 L) using a dropping funnel at 0°C, and the mixture and stirred for 1 h at r.t. The reaction mixture was extracted with CH₂Cl₂ and dried over MgSO₄. To the concentrated residue was added MeI (6.9 mL, 68 mmol) and K₂CO₃ (1.9 g, 14 mmol), and then the mixture was stirred at r.t. overnight. The reaction was concentrated, diluted with Et₂O and washed with H₂O. The organic layer was dried over MgSO₄ and concentrated. The crude material was purified by column chromatography (AcOEt–n-hexane=1 : 2) to give compound 19 (1.5 g, 91%, in 3 steps) as a colorless oil.

IR (CHCl₃) cm⁻¹: 3010, 2933, 2862, 1730, 1699; [α]_D²⁰ −7.9 (c=0.15, EtOH); ¹H-NMR (CDCl₃, 300 MHz) δ: 3.67 (3H, s), 2.66–2.63 (1H, m), 2.28–2.17 (3H, m), 2.08–1.96 (1H, m), 1.84–1.41 (9H, m), 1.26–1.12 (2H, m), 1.09 (3H, s); ¹³C-NMR (CDCl₃, 100 MHz) δ: 215.9, 173.1, 51.3, 47.8, 45.2, 41.3, 37.3, 34.6, 33.8, 32.1, 27.7, 27.3, 26.0, 15.5; HR-MS (EI) Calcd for C₁₄H₂₂O₃ m/z [M⁺]: 238.1569. Found 238.1562.

Methyl 2-((2S,4aS,8aS)-5-(Cyanomethylene)-4a-methyldecahydronaphthalen-2-yl)acetate (20)

To a suspension of KO^tBu (3.6 g, 32 mmol) in DMF (30 mL) was added diethyl cyanomethylphosphonate (5.0 mL, d 1.13, 32 mmol) under a nitrogen atmosphere, and the mixture was stirred at 0°C for 1 h. To the solution was added 19 (2.5 g, 11 mmol) in anhydrous DMF (25 mL) and the resulting mixture was stirred at 90°C for 1 h. The reaction mixture was added H₂O and extracted with Et₂O. The combined extracts was dried over MgSO₄ and concentrated. The residue was purified by column chromatography (AcOEt–n-hexane=1 : 5) to give compound 20 (2.6 g, 95%) as a colorless oil.

IR (CHCl₃) cm⁻¹: 2983, 2929, 2216, 1730; [α]_D²⁵ −71.9 (c=0.46, EtOH); ¹H-NMR (CDCl₃, 400 MHz) δ: 5.03 (1H, d, J=1.6 Hz), 3.67 (3H, s), 2.94–2.78 (1H, m), 2.42–2.30 (1H, m), 2.29–2.10 (2H, m), 1.98–1.67 (3H, m), 1.66–1.57 (1H, m), 1.51–1.03 (8H, m), 1.01 (3H, s); ¹³C-NMR (CDCl₃, 100 MHz) δ: 175.9, 173.0, 117.7, 90.2, 51.3, 45.2, 41.2, 40.6, 35.5, 34.58, 34.55, 30.1, 28.2, 28.0, 27.0, 16.9; HR-MS (EI) Calcd for C₁₆H₂₃NO₂ m/z [M⁺]: 261.1729. Found 261.1725.

Methyl 2-((2S,4aS,5R,8aS)-5-(Cyanomethyl)-4a-methyldecahydronaphthalen-2-yl)acetate (21)

Under a hydrogen balloon atmosphere, a suspension of compound 20 (12 mg, 0.046 mmol), 10% Pd/C (2.0 mg) in EtOH (1.4 mL) was stirred at r.t. for 1.5 h. The reaction mixture was filtered through a Celite pad and concentrated. The residue was purified by column chromatography (AcOEt–n-hexane=1 : 5) to give compound 21 (12 mg, quant, β : α=14 : 1) as a colorless oil.

IR (CHCl₃) cm⁻¹: 2927, 2858, 2247, 1730; [α]_D²³ +100 (c=0.004, EtOH); ¹H-NMR (CDCl₃, 400 MHz) δ: 3.66 (3H, s), 2.63–2.35 (1H, m), 2.21 (2H, d, J=7.2 Hz), 2.00 (1H, dd, J=16.4, 10.4 Hz), 1.90–1.71 (3H, m), 1.67–1.56 (2H, m), 1.53–0.96 (10H, m), 0.72 (3H, s); ¹³C-NMR (CDCl₃, 100 MHz) δ: 173.3, 120.1, 51.4, 45.7, 45.6, 41.5, 38.1, 36.3, 35.0, 34.8, 28.5, 28.2, 27.8, 25.9, 18.3, 10.9; HR-MS (EI) Calcd for C₁₆H₂₅NO₂ m/z [M⁺]: 263.1885. Found 263.1881.

Methyl 2-((2S,4aS,5R,8aS)-5-(2-((Z)-2,3-Bis(tert-butoxycarbonyl)guanidino)ethyl)-4a-methyldecahydronaphthalen-2-yl)acetate (22)

Under a hydrogen atmosphere (balloon), a suspension of compound 21 (0.11 g, 0.45 mmol) and PtO₂ (20 mg) in 10% HCl–MeOH (12 mL) was stirred at r.t. for 140 min. The reaction mixture was filtered through a Celite pad and concentrated. The residue was dissolved in DMF (3.9 mL), and Et₃N (0.16 mL, d 0.73, 1.2 mmol) and N,N′-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (0.15 g, 0.47 mmol) was added. After 1 h stirring, the reaction mixture was added H₂O, extracted with Et₂O, and dried over MgSO₄. The concentrated residue was purified by column chromatography (AcOEt–n-hexane=1 : 5) to give compound 22 (114 mg, 83%, 2 steps) as a colorless amorphous.

IR (CHCl₃) cm⁻¹: 2981, 2926, 2856, 1720, 1633; [α]_D²⁶ +2.2 (c=0.09, EtOH); ¹H-NMR (CDCl₃, 400 MHz) δ: 11.5 (1H, br s), 8.25 (1H, br s), 3.66 (3H, s), 3.62–3.22 (2H, m), 2.20 (2H, d, J=6.8 Hz), 1.99–1.38 (10H, m), 1.26 (9H, s), 1.22–0.70 (13H, m), 0.53 (3H, s); ¹³C-NMR (CDCl₃, 100 MHz) δ: 173.4, 163.6, 156.0, 153.3, 82.9, 79.1, 51.3, 46.3, 45.9, 41.7, 40.2, 37.9, 36.3, 35.2, 29.6, 28.9, 28.8, 28.4, 28.3, 28.0, 27.7, 26.3, 11.1; HR-MS (FAB) Calcd for C₂₇H₄₇N₃O₆ m/z [M⁺]: 510.3543. Found 510.3543.

2-((2S,4aS,5R,8aS)-5-(2-((Z)-2,3-Bis(tert-butoxycarbonyl)guanidino)ethyl)-4a-methyldecahydronaphthalen-2-yl)acetic Acid (24)

To a solution of 22 (1.5 g, 3.1 mmol, β : α=14 : 1) in MeOH–THF (62 mL, 1 : 1) was added 10% aqueous LiOH (15 mL, 0.2 M) and stirred at r.t. for 4 h. The mixture was concentrated in vacuo and diluted with CH₂Cl₂. The solution was neutralized with dil HCl and aqueous layer was extracted with CH₂Cl₂ and dried over MgSO₄. The concentrated residue was purified by column chromatography (CH₂Cl₂– MeOH=10 : 1) to give compound 23 (1.2 g, 75%, β : α=14 : 1) as a white solid. This product was recrystallized from Et₂O to give β-isomer 24 as a white powder (0.20 g, 13%).

mp 108–112°C (AcOEt–n-hexane); IR (CHCl₃) cm⁻¹: 3332, 2926, 1720, 1641, 1394; [α]_D²¹ −6.6 (c=0.09, EtOH); ¹H-NMR (CDCl₃, 400 MHz) δ: 8.24 (1H, brs), 3.49–3.38 (1H, m), 3.33–3.24 (1H, m), 2.24 (2H, d, J=6.8 Hz), 1.89–1.58 (8H, m), 1.50 (18H, s), 1.43–0.92 (11H, m), 0.69 (3H, s); ¹³C-NMR (CDCl₃, 100 MHz) δ: 178.7, 163.5, 156.0, 153.7, 83.0, 79.2, 46.3, 41.7, 40.3, 37.9, 36.3, 36.1, 35.1, 28.89, 28.85, 28.4, 28.3, 28.0, 27.6, 26.3, 11.1; HR-MS (FAB) Calcd for C₂₆H₄₆N₃O₆ m/z [MH⁺]: 496.3387. Found 496.3384.

2-((2S,4aS,5R,8aS)-5-(2-Guanidinoethyl)-4a-methyldecahydronaphthalen-2-yl)acetic Acid (3c)

To a solution of 24 (39 mg, 0.080 mmol) was added HCl–AcOEt (0.80 mL, 0.4 M) at 0°C and stirred at r.t. for 31 h. The mixture was concentrated in vacuo to give compound 3c (24 mg, quant, 70% purity) as a white amorphous.

IR (CHCl₃) cm⁻¹: 3435, 3417, 1633, 1261, 1095, 1022, 802; [α]_D²³ +22.7 (c=0.22, EtOH); ¹H-NMR (CDCl₃, 300 MHz) δ: 7.48–7.35 (1H, m), 3.24–2.82 (2H, m), 2.10 (2H, d, J=7.2 Hz), 1.90–0.83 (20H, m), 0.66 (3H, s); ¹³C-NMR (CDCl₃, 125 MHz) δ: 173.6, 156.6, 53.5, 52.3, 49.4, 37.5, 35.9, 34.7, 28.5, 28.3, 27.8, 27.6, 26.9, 25.9, 11.0, 0.05; HR-MS (FAB) Calcd for C₁₆H₃₀N₃O₂ m/z [MH⁺]: 296.2338. Found 296.2335.

(3aS,7aS)-7a-Methylhexahydrospiro[indene-1,2′-[1,3]dioxolan]-5(4H)-one (25)

Under a nitrogen atmosphere, a lithium wire (0.16 g, 23.0 mmol) was added to liquid ammonia (30 mL) at −78°C. To the reaction mixture was added the solution of acetal (1.5 g, 7.2 mmol) in THF (18 mL), and the resulting mixture was stirred for 100 min. After the substrate disappearance was monitored by TLC, solid NH₄Cl was added until the mixture turned white suspension. The mixture was allowed to r.t. and the liquid ammonia was evaporated through the night. The residue was suspended with CH₂Cl₂ and filtrated. The concentrated residue was purified by column chromatography (AcOEt–n-hexane=1 : 4) to give compound 25 (1.0 g, 70%) as a colorless oil.

IR (CHCl₃) cm⁻¹: 2956, 1705; [α]_D²² +58.8 (c=0.17, EtOH); ¹H-NMR (CDCl₃, 400 MHz) δ: 3.97–3.87 (4H, m), 2.52–2.19 (5H, m), 2.02–1.78 (4H, m), 1.72–1.66 (1H, m), 1.42–1.30 (1H, m), 1.16 (3H, s); ¹³C-NMR (CDCl₃, 100 MHz) δ: 213.0, 119.4, 65.2, 64.4, 44.3, 44.1, 42.2, 37.1, 32.9, 29.4, 25.7, 18.4; HR-MS (EI) Calcd for C₁₂H₁₈O₃ m/z [M⁺]: 210.1256. Found 210.1257.

Methyl 2-((3aS,7aS)-7a-Methylhexahydrospiro[indene-1,2′-[1,3]dioxolan]-5(4H)-ylidene)acetate

To a suspension of KO^tBu (0.11 g, 1.1 mmol) in DMF (0.75 mL) was added methyl 2-(diethoxyphosphoryl)acetate (0.19 mL, d 1.15, 1.1 mmol), and the mixture was stirred at 0°C for 10 min under a nitrogen atmosphere. To the mixture was added compound 25 (74 mg, 0.35 mmol) in anhydrous DMF (1.0 mL) and the resulting mixture was stirred at r.t. for 30 h. The reaction mixture was added H₂O and extracted with Et₂O. The combined extract was dried over MgSO₄ and concentrated. The residue was purified by column chromatography (AcOEt–n-hexane=1 : 4) to give α,β-unsaturated ester (90 mg, 97%) as a colorless oil.

IR (CHCl₃) cm⁻¹: 3012, 2949, 2879, 1708, 1649; [α]_D²¹ +65.5 (c=0.07, EtOH); ¹H-NMR (CDCl₃, 400 MHz, E : Z=1 : 1) δ: 5.76 (1/2H, s), 5.64 (1/2H, s), 3.95–3.81 (4H, m), 3.69 (3/2H, s), 3.68 (3/2H, s), 3.62–3.50 (1H, m), 2.47–1.19 (10H, m), 1.06 (3/2H, s), 1.05 (3/2H, s); ¹³C-NMR (CDCl₃, 100 MHz, E : Z=1 : 1) δ: 167.0, 166.7, 161.7, 161.6, 120.0 (2C), 115.0, 114.6, 65.2 (2C), 64.2, 64.1, 50.7 (2C), 44.7, 44.6, 43.9, 43.8, 37.2, 33.1, 32.7, 32.6, 31.3, 29.8, 28.7, 25.1, 23.9, 23.5, 17.6, 16.9; HR-MS (EI) Calcd for C₁₅H₂₂O₄ m/z [M⁺]: 266.1518. Found 266.1516.

2-((3aS,7aS)-7a-Methylhexahydrospiro[indene-1,2′-[1,3]dioxolan]-5(4H)-ylidene)acetic Acid (26)

To a solution of α,β-unsaturated ester (57 mg, 0.21 mmol) in 1 : 1 EtOH/H₂O (2.0 mL) was added powdered KOH (0.12 g, 2.1 mmol) and the mixture was heated at 90°C for 15 min. The reaction mixture was concentrated and neutralized with 10% HCl on an ice bath. The mixture was extracted with CH₂Cl₂ and the combined organic layer was dried over MgSO₄. The concentrated residue was purified by column chromatography (AcOEt–n-hexane=1 : 3) to give compound 26 (38 mg, 71%) as a white amorphous.

IR (CHCl₃) cm⁻¹: 3016, 2949, 2879, 1687; [α]_D²⁵ +100 (c=0.02, EtOH); ¹H-NMR (CDCl₃, 400 MHz, E : Z=1 : 1) δ: 5.78 (1/2H, s), 5.67 (1/2H, s), 3.95–3.79 (4H, m), 3.55 (1H, d, J=16.0 Hz), 2.49–1.10 (11H, m), 1.06 (3/2H, s), 1.05 (3/2H, s); ¹³C-NMR (CDCl₃, 100 MHz, E : Z=1 : 1) δ: 165.6, 165.5, 164.8, 164.6, 120.1, 120.1, 114.8, 114.3, 65.3, 64.3, 64.2, 44.7, 44.7, 44.1, 44.0, 37.6, 33.5, 32.8, 32.7, 31.3, 29.8, 29.1, 25.4, 24.1, 23.7, 17.9, 17.0; HR-MS (EI) Calcd for C₁₄H₂₀O₄ m/z [M⁺]: 252.1362. Found 252.1356.

Methyl 2-((3aS,5S,7aS)-7a-Methyl-1-oxooctahydro-1H-inden-5-yl)acetate (27)

Under a nitrogen atmosphere, a lithium wire (0.74 g, 0.10 mol) was added to liquid ammonia (150 mL) at −78°C. To the reaction mixture was added the solution of 26 (2.7 g, 11 mmol) in THF (100 mL), and the mixture was stirred for 25 min at r.t. After the substrate disappearance was monitored by TLC, solid NH₄Cl was added until the mixture turned white suspension. The mixture was allowed to r.t. and the liquid ammonia was evaporated through the night. The residue was suspended with CH₂Cl₂ and filtrated. To the concentrated residue in THF (100 mL) was added 1.5 M HCl (40 mL) using a dropping funnel at 0°C, and the mixture and stirred for 1.5 h at r.t. The reaction mixture was extracted with CH₂Cl₂ and dried over MgSO₄. To the concentrated residue was dissolved in acetone (100 mL), and MeI (11 mL, 0.10 mol) and K₂CO₃ (6.0 g, 43 mmol) were added, and then the mixture was stirred for overnight. The reaction was concentrated, diluted with Et₂O and washed with H₂O. The organic layer was dried over MgSO₄ and concentrated. The crude material was purified by column chromatography (AcOEt–n-hexane=1 : 2) to give compound 27 (2.4 g, 98%, 3 steps) as a colorless oil.

IR (CHCl₃) cm⁻¹: 2953, 2929, 2358, 2339, 1730; [α]_D²⁰ +35.6 (c=0.03, EtOH); ¹H-NMR (CDCl₃, 400 MHz) δ: 3.68 (3H, s), 2.54–2.38 (1H, m), 2.32–1.80 (7H, m), 1.75–1.56 (2H, m), 1.50–1.35 (2H, m), 1.29–1.10 (2H, m), 1.07 (3H, s); ¹³C-NMR (CDCl₃, 100 MHz) δ: 222.2, 172.9, 51.2, 46.6, 42.3, 41.0, 35.9, 30.9, 29.0, 27.7, 27.3, 22.9, 18.8; HR-MS (EI) Calcd for C₁₃H₂₀O₃ m/z [M⁺]: 224.1412. Found 224.1412.

Methyl 2-((3aS,5S,7aS)-1-(Cyanomethylene)-7a-methyloctahydro-1H-inden-5-yl)acetate

To a suspension of NaH (26 mg, 60% oil suspension, 0.66 mmol) in DMF (0.50 mL) was added diethyl cyanomethylphosphonate (0.10 mL, d 1.13, 0.66 mmol) and stirred at 0°C for 1 h under a nitrogen atmosphere. To the solution was added compound 11 (550 mg, 0.22 mmol) in anhydrous DMF (0.51 mL) and stirred at r.t. for 11 h. The reaction mixture was added H₂O and extracted with Et₂O. The combined extract was dried over MgSO₄ and concentrated. The residue was purified by column chromatography (Et₂O–n-hexane=1 : 4) to give the desired α,β-unsaturated nitrile (50 mg, 93%) as a colorless oil.

IR (CHCl₃) cm⁻¹: 3018, 2954, 2927, 2216, 1730; [α]_D²⁶ +11.7 (c=0.29, EtOH); ¹H-NMR (CDCl₃, 400 MHz) δ: 5.10 (1H, t, J=2.4 Hz), 3.65 (3H, s), 2.90–2.69 (1H, m), 2.57–2.46 (1H, m), 2.21 (2H, d, J=6.8 Hz), 2.07–1.15 (10H, m), 1.07 (3H, s); ¹³C-NMR (CDCl₃, 100 MHz) δ: 182.1, 172.9, 117.3, 89.2, 51.3, 44.9, 44.8, 41.0, 33.0, 30.9, 30.2, 28.8, 28.1, 26.0, 21.8; HR-MS (EI) Calcd for C₁₅H₂₁NO₂ m/z [M⁺]: 247.1572. Found 247.1572.

Methyl 2-((1R,3aS,5S,7aS)-1-(Cyanomethyl)-7a-methyloctahydro-1H-inden-5-yl)acetate (28)

Under a hydrogen balloon atmosphere, a suspension of the α,β-unsaturated nitrile (23 mg, 0.090 mmol), 10% Pd/C (2.3 mg) in EtOH (3.0 mL) was stirred at r.t. for 2 h. The reaction mixture was filtered through a Celite pad and concentrated. The residue was purified by column chromatography (AcOEt–n-hexane=1 : 5) to give nitrile 28 (18.6 mg, 80%, β: α=10 : 1) as a colorless oil.

IR (CHCl₃) cm⁻¹: 2953, 2926, 1730; [α]_D²⁶ −29.0 (c=0.06, EtOH); ¹H-NMR (CDCl₃, 400 MHz) δ: 3.67 (3H, s), 2.40–2.25 (1H, m), 2.20 (2H, d, J=5.6 Hz), 2.17–1.03 (14H, m), 1.02 (30/11H, s), 0.95 (3/11, s); ¹³C-NMR (CDCl₃, 100 MHz) δ: 173.3, 119.8, 51.4, 47.9, 45.6, 41.6, 41.0, 31.0, 29.0, 27.9, 27.6, 26.8, 25.5, 22.6, 17.1; HR-MS (EI) Calcd for C₁₅H₂₃NO₂ m/z [M⁺]: 249.1729. Found 249.1727.

Methyl 2-((1R,3aS,5S,7aS)-1-(2-((E)-2,3-Bis(tert-butoxycarbonyl)guanidino)ethyl)-7a-methyloctahydro-1H-inden-5-yl)acetate

Under a hydrogen atmosphere (balloon), a suspension of nitrile 28 (29 mg, 0.11 mmol) and PtO₂ (3 mg) in 10% HCl–MeOH (2.7 mL) was stirred at r.t. for 2 h. The reaction mixture was filtered through a Celite pad and concentrated. The residue was dissolved in DMF (1.1 mL), and Et₃N (45 µL, d 0.73, 0.33 mmol) and N,N′-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (40 mg, 0.13 mmol) were added. After 1 h stirring, the reaction mixture was added H₂O, extracted with Et₂O, and dried over MgSO₄. The concentrated residue was purified by column chromatography (AcOEt–n-hexane=1 : 5) to give N,N′-bisBoc guanidine (11 mg, 13%) as a colorless amorphous.

IR (CHCl₃) cm⁻¹: 2927, 1716, 1635; [α]_D²¹ +15.0 (c=0.02, EtOH); ¹H-NMR (CDCl₃, 400 MHz, 10 : 1 diastereo mixture) δ: 11.5 (1H, s), 8.27 (1H, s), 3.66 (3H, s), 3.43–3.33 (2H, m), 2.25 (2/11H, d, J=7.6 Hz), 2.18 (20/11H, d, J=7.2 Hz), 2.14–1.78 (2H, m), 1.77–1.54 (5H, m), 1.51 (9H, s), 1.49 (9H, s), 1.47–0.98 (8H, m), 0.94 (30/11H, s), 0.86 (3/11H, s); ¹³C-NMR (CDCl₃, 100 MHz) δ: 173.6, 163.6, 156.0, 153.3, 83.0, 79.2, 51.4, 49.6, 45.7, 41.8, 41.1, 40.7, 31.3, 29.3, 29.2, 28.30, 28.26, 28.1, 27.7, 27.1, 26.1, 22.7; HR-MS (FAB) Calcd for C₂₆H₄₆N₃O₆ m/z [MH⁺]: 496.3386. Found 496.3390.

2-((1R,3aS,5S,7aS)-1-(2-((E)-2,3-Bis(tert-butoxycarbonyl)guanidino)ethyl)-7a-methyloctahydro-1H-inden-5-yl)acetic Acid

To a solution of the N,N′-bisBoc guanidine (36 mg, 0.070 mmol) in MeOH (0.70 mL) and THF (0.70 mL) was added 10% aqueous LiOH (0.70 mL, 0.1 M), and the mixture was heated at r.t. for 4 h. The reaction mixture was concentrated and neutralized with dil HCl on an ice bath. The mixture was extracted with CH₂Cl₂, and the combined organic layer was dried over MgSO₄. The concentrated residue was purified by column chromatography (AcOEt–n-hexane=1 : 1) to give the desired carboxylic acid (28 mg, 82%) as a white amorphous.

IR (CHCl₃) cm⁻¹: 2981, 2927, 2872, 1714, 1633; ¹H-NMR (CDCl₃, 400 MHz) δ: 11.5 (1H, s), 8.27 (1H, s), 3.53–3.33 (2H, m), 2.18 (2H, d, J=7.6 Hz), 2.14–1.54 (7H, m), 1.53 (9H, s), 1.49 (9H, s), 1.47–0.98 (9H, m), 0.94 (3H, s); ¹³C-NMR (CDCl₃, 100 MHz) δ: 156.0, 153.3, 83.0, 79.3, 53.4, 49.6, 45.6, 41.1, 40.7, 31.2, 31.0, 29.1, 28.3, 28.2, 28.1, 27.7, 27.1, 26.1, 22.7; HR-MS (FAB) Calcd for C₂₅H₄₄N₃O₆ m/z [MH⁺]: 481.3230. Found 482.3233.

2-((1R,3aS,5S,7aS)-1-(2-Guanidinoethyl)-7a-methyloctahydro-1H-inden-5-yl)acetic Acid (3d)

To a solution of the carboxylic acid (24 mg, 0.050 mmol) was added 4 M HCl–AcOEt (0.5 mL) at 0°C, and the mixture was stirred at r.t. for 6.5 h. The mixture was concentrated in vacuo to give compound 3d (7.8 mg, 56, 75% purity) as a pale yellow oil.

IR (CHCl₃) cm⁻¹: 3152, 2956, 2928, 2868, 1725, 1678, 1642, 1615, 1252, 1148; [α]_D²⁰ −5.0 (c=0.12, EtOH); ¹H-NMR (DMSO-d₆, 300 MHz) δ: 11.95 (5H, br s), 8.07 (1H, br s), 7.15 (3H, brs), 3.24–2.97 (2H, m), 2.08 (2H, d, J=9.2 Hz), 1.08–0.97 (15H, m), 0.92 (3H, s); ¹³C-NMR (DMSO-d₆, 100 MHz) δ: 173.7, 171.9, 156.7, 48.5, 45.4, 41.4, 30.7, 28.6, 28.4, 27.7, 26.9, 26.6, 25.6, 22.6, 21.0; HR-MS (FAB) Calcd for C₁₅H₂₈N₃O₂ m/z [MH⁺]: 282.2181. Found 282.2184.

Methyl 2-((2S,4aS,5S,8aS)-5-Hydroxy-4a-methyldecahydronaphthalen-2-yl)acetate (29)

To a solution of 19 (10 mg, 0.040 mmol) in MeOH (0.2 mL) was added NaBH₄ at 0°C, and the mixture was stirred at r.t. for 5 min. The reaction mixture was concentrated and then purified by column chromatography (AcOEt–n-hexane=1 : 4) to give compound 29 (8.0 mg, 83%, β : α=8.5 : 1) as a colorless oil.

IR (CHCl₃) cm⁻¹: 2927, 2858, 1730; [α]_D²⁵ −13.0 (c=0.26, EtOH); ¹H-NMR (CDCl₃, 400 MHz) δ: 3.67 (3H, s), 3.23 (1H, dd, J=12.0, 4.0 Hz), 2.22 (2H, d, J=4.0 Hz), 2.60–0.90 (15H, m), 0.79 (3H, s); ¹³C-NMR (CDCl₃, 100 MHz) δ: 173.5, 79.5, 51.4, 43.5, 41.7, 38.7, 36.9, 35.2, 34.2, 30.4, 28.1, 27.8, 24.3, 9.8; HR-MS (EI) Calcd for C₁₄H₂₄O₃ m/z [M⁺]: 240.1725. Found 240.1724.

2-((2S,4aS,5S,8aS)-5-(3-(1-(tert-Butoxycarbonyl)piperidin-4-yl)propoxy)-4a-methyldecahydronaphthalen-2-yl)acetic Acid (30)

To a solution of 29 (22 mg, 0.090 mmol, β : α=8.5 : 1) in anhydrous DMF (1.0 mL) was added NaH (5.5 mg, 0.23 mmol, 60% oil suspension), and the mixture was stirred at r.t. for 1 h under a nitrogen atmosphere. The mixture was added tert-butyl 4-(3-bromopropyl)piperidine-1-carboxylate (50 mg, 0.090 mmol) and stirred for 30 h. Then the solution was neutralized with satd aqueous NH₄Cl and extracted with Et₂O. The extract was washed with brine and dried over Na₂SO₄. The concentrated residue was purified by column chromatography (AcOEt–n-hexane=1 : 3) to give compound 30 (12 mg, 29%) as a colorless oil and 29 (12 mg, 53%) was recovered.

IR (CHCl₃) cm⁻¹: 3444, 2929, 2358, 1722, 1678; [α]_D²⁸ +3.0 (c=0.82, CHCl₃); ¹H-NMR (CDCl₃, 500 MHz) δ: 4.05 (2H, t, J=6.5 Hz), 3.23 (1H, dd, J=4.0 Hz), 2.68 (2H, t, J=12.5 Hz), 2.20 (2H, d, J=6.0 Hz), 1.93–1.47 (11H, m), 1.45 (9H, s), 1.43–0.84 (15H, m), 0.80 (3H, s); ¹³C-NMR (CDCl₃, 100 MHz) δ: 173.1, 154.9, 79.5, 79.2, 64.3, 43.7, 43.5, 42.0, 38.7, 36.9, 35.6, 35.3, 34.2, 32.7, 32.1, 30.4, 28.4, 28.0, 27.8, 25.8, 24.3, 24.0, 10.9, 9.8; HR-MS (EI) Calcd for C₂₆H₄₅NO₅ m/z [M⁺]: 451.3298. Found 451.3295.

2-((2S,4aS,5S,8aS)-4a-Methyl-5-(3-(piperidin-4-yl)propoxy)decahydronaphthalen-2-yl)acetic Acid (3e)

To a solution of 30 (6.2 mg, 0.010 mmol) was added 4 M HCl–AcOEt (0.1 mL) at 0°C, and the mixture was stirred at r.t. for 5 h. The reaction mixture was concentrated in vacuo to give compound 3e (3.6 mg, 75, 72% purity) as a white amorphous.

IR (CHCl₃) cm⁻¹: 3685, 2928, 2857, 1722, 1601, 1453; [α]_D²⁸ +2.4 (c=0.45, EtOH); ¹H-NMR (CD₃OD, 400 MHz) δ: 4.21–4.02 (2H, m), 3.37 (2H, d, J=12.4 Hz), 3.15 (1H, dd, J=4.8, 11.2 Hz), 2.96 (2H, t, J=12.4 Hz), 2.21 (2H, d, J=7.2 Hz), 2.12–0.90 (25H, m), 0.90 (1H, s), 0.79 (2H, s); ¹³C-NMR (CD₃OD, 100 MHz) δ: 174.8, 80.2, 65.3, 45.3 (2C), 45.2, 42.8, 40.0, 38.4, 36.8, 35.5, 34.5, 33.4, 31.3, 30.0 (2C), 29.19, 29.16, 26.7, 25.6, 10.3; HR-MS (FAB) Calcd for C₂₁H₃₇NO₃ m/z [MH⁺]: 352.2852. Found 352.2857.

Platelet Aggregation Assay

Rabbit platelet rich-plasma (PRP) was obtained from fresh rabbit blood collected into a one-tenth volume of 3.8% sodium citrate by centrifugation at 1000 rpm (50×g) for 20 min at r.t. Platelet aggregometry followed the methods of Born³⁹⁾ and O’Brien.⁴⁰⁾ Rabbit PRP (175 µL) was added to 10 mM CaCl₂ (25 µL) and the mixture was preincubated at 37°C for 1 min. The mixture was added to each sample in 0.02 M Tris–HCl buffer (pH 7.4) containing 0.15 M NaCl (25 µL) and incubated at 37°C for 5 min. A total of 25 µL of ADP (final concentration: 1–2 µM) or collagen (final concentration: 1 µg/mL) was then added and incubated at 37°C for 5–7 min. Platelet aggregation was measured by the turbidimetric method using HEMA TRACER 704 from MC MEDICAL Inc. (Tokyo, Japan).

Statistical Analysis

Statistical analyses were performed by using Microsoft Excel 2007 software (Microsoft Co., Redmond, WA, U.S.A.). The statistical significance of differences was determined according to the student’s t-test. p<0.05 was considered to be statistically significant.

Acknowledgments

This work was supported by JSPS KAKENHI Grant-in-Aid for Encouragement of Young Scientists (B) 19790099. We are grateful to N. Eguchi, T. Koseki, and S. Yamada at the Analytical Center of Meiji Pharmaceutical University for performing microanalysis and mass spectrometry.

Conflict of Interest

The authors declare no conflict of interest.

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