Bioorganic & Medicinal Chemistry 23 (2015) 4277–4285

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc

Stereoselective synthesis and pharmacological evaluation of [4.3.3]propellan-8-amines as analogs of adamantanamines Héctor Torres-Gómez a, Kirstin Lehmkuhl a, Bastian Frehland a, Constantin Daniliuc b, Dirk Schepmann a, Christina Ehrhardt c,d, Bernhard Wünsch a,d,⇑ a

Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-Universität Münster, Corrensstraße 48, D-48149 Münster, Germany Organisch-Chemisches Institut der Westfälischen Wilhelms-Universität Münster, Corrensstraße 40, D-48149 Münster, Germany Institut für Molekulare Virologie, Zentrum für Molekularbiologie der Entzündung (ZMBE) der Westfälischen Wilhelms-Universität Münster, Von-Esmarch-Str. 56, D-48149 Münster, Germany d Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), University Münster, Germany b c

a r t i c l e

i n f o

Article history: Received 18 May 2015 Revised 11 June 2015 Accepted 12 June 2015 Available online 18 June 2015 Keywords: NMDA receptor PCP binding site Antiviral activity Amantadine Propellanamines Stereoselective synthesis

a b s t r a c t Amantadine (1) exerts its anti-Parkinson effects by inhibition of the NMDA associated cation channel and its antiviral activity by inhibition of the M2 protein channel of influenza A viruses. Herein the synthesis, NMDA receptor affinity and anti-influenza activity of analogous propellanamines 3 are reported. The key steps in the synthesis of the diastereomeric propellanamines syn-3 and anti-3 are diastereoselective reduction of the ketone 7 with L-Selectride to give anti-11, Mitsunobu inversion of the alcohol anti-13 into syn-13, and SN2 substitution of diastereomeric mesylates syn-14 and anti-14 with NaN3. The affinity of the propellanamines syn-3 and anti-3 to the PCP binding site of the NMDA receptor is similar to that of amantadine (Ki = 11 lM). However, both propellanamines syn-3 and anti-3 do not exhibit activity against influenza A viruses. Compared to amantadine (1), the structurally related propellanamines syn3 and anti-3 retain the NMDA antagonistic activity but loose the antiviral activity. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction The adamantane core is an interesting scaffold in Medicinal Chemistry, which has been called the ‘lipophilic bullet’1 due to its ability for providing the critical lipophilicity to bioactive molecules in order to enhance their pharmacokinetics.1–3 Exemplarily, introduction of the adamantane system into cannabinoid receptor subtype 2 (CB2) ligands leads to increased CB2 affinity and penetration into the central nervous system.4 The adamantanamines amantadine (1) and memantine (2) display anti-Parkinson, antiAlzheimer and antiviral activities.5,6 (Fig. 1) It has been shown that the anti-Parkinson and anti-Alzheimer effects of amantadine (1) and memantine (2) are due to their interaction with the phencyclidine (1-(1-phenylcyclohexyl)piperidine, PCP) binding site of the N-methyl-D-aspartate (NMDA) receptor.6–8 Both adamantanamines 1 and 2 belong to the class of open-channel blockers. They can interact with the PCP binding site within the channel pore and thus inhibit the flux of cations through the NMDA receptor associated ion channel only after activating and opening of the ion channel first. Due to this property they are called ⇑ Corresponding author. Tel.: +49 251 8333311; fax: +49 251 8332144. E-mail address: [email protected] (B. Wünsch). http://dx.doi.org/10.1016/j.bmc.2015.06.030 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

uncompetitive NMDA receptor antagonists.9–11 The affinity of amantadine (1) and memantine (2) towards the PCP binding site of the NMDA receptor is rather low. In our assay we found Ki values of 11 lM and 0.74 lM for amantadine and memantine, respectively, (see Table 1). However, the favorable off-rate kinetics at the NMDA receptor renders 1 and 2 valuable drugs for the treatment of neurodegenerative disorders. In addition to the blockade of the NMDA receptor, amantadine (1) exerts anti-influenza A virus activity by inhibition of the tetrameric M2 proton channel. The correct function of the M2 proton channel is vital for the viral replication. During the cell entry the influx of protons into the virus starts the uncoating and thus infection process.12,13 However, treatment with amantadine and derivatives results in the emergence of resistant virus variants and is not recommended for a general and uncontrolled use.14,15 Furthermore, various influenza virus strains already harbor different M2 gene mutations responsible for resistance to adamantine derivatives.16 Nonetheless, to characterize the mode of action of propellanamines 3, we tested its antiviral activity in comparison to amantadine within this study. Herein we wish to report on the synthesis and pharmacological evaluation of [4.3.3]propellanamines 3, which are regarded as analogs of the adamantanamines 1 and 2. Both, the adamantanamines

4278

H. Torres-Gómez et al. / Bioorg. Med. Chem. 23 (2015) 4277–4285

CH3 NH2 NH2 amantadine (1)

NH2 H3C memantine (2)

3

Figure 1. Comparison of prominent adamantanamines amantadine (1) and memantine (2) with the projected propellanamines 3.

1, 2 and the propellanamines 3 represent tricyclic, conformationally constrained alkanes bearing a primary amino moiety. The molecular formulas of memantine (2) and propellanamine 3 are identical (C12H21N). In contrast to the highly symmetric adamantanamines 1 and 2, two diastereomers of the propellanamines 3 exist, which are termed syn-3 and anti-3. In syn-3 the primary amino moiety is directed towards the larger cyclohexane ring, whereas it is oriented towards the smaller five-membered ring in anti-3. Both diastereomers syn-3 and anti-3 will be prepared diastereoselectively and their affinity towards the PCP binding site of the NMDA receptor and their antiviral activity will be evaluated. In Figures 2 and 3 size, shape, electrostatic surface potential and lipophilic surface map of protonated amantadine (1H+) and protonated propellanamine syn-3H+ are compared. Amantadine (1H+) has a spherical shape, whilst syn-3H+ is more elongated. As a consequence the propellanamine syn-3H+ has a larger volume (193 Å3) than amantadine (160 Å3). The electrostatic surface potentials of both primary amines 1H+ and syn-3H+ are very similar showing a positive region around the protonated amino moiety and a

negative potential along the lipophilic tricyclic alkane scaffold (Fig. 2). The total polar surface areas of both 1H+ and syn-3H+ are identical (27.64 Å2). The lipophilic surface maps are also very similar with a large lipophilic region along the bicyclic scaffold (Fig. 3). It is assumed that the similarity in the electrostatic surface potential and the lipophilic surface but the differences in the molecular shape and volume will result in similar but not identical drug-target molecular interactions. 2. Chemistry The synthesis of the key propellanedione 6 was achieved in two steps via a modified Weiss–Cook reaction as previously described.17 (Scheme 1) Briefly, cyclohexane-1,2-dione (4) was reacted with dimethyl 3-oxoglutarate (5) in a citrate phosphate buffer pH 5.6 resulting in a tetraester, which was hydrolyzed with HCl to afford the diketone 6 in 61% yield over two steps. The diketone 6 was crystallized from methanol affording colorless crystals suitable for X-crystal structure analysis. The X-ray crystal structure of 6 was recorded to prove the structure of this key precursor (Fig. 4). The chair conformation of the six-membered ring is clearly observed in the X-ray crystal structure of the diketone 6. The conjoining bond between C-1 and C-6 is slightly longer than a normal single covalent C–C bond (1.52 Å). The measured distance between the bridgehead C-atoms C1 and C6 is 1.572(2) Å confirming the existence of the conjoining bond in the [4.3.3]propellane 6. The C1/C6-bond length of 6 is in good agreement with the reported length of the conjoining bond in the crystal

Figure 2. Comparison of the electrostatic surface potentials of (a) protonated amantadine (1H+) and (b) protonated propellanamine syn-3H+. Blue: positive region; grey: neutral region; total polar surface areas (TPSA): 1H+: 27.64 Å2, syn-3H+: 27.64 Å2; Volumes: 1H+: 160 Å3, syn-3H+: 193 Å3; physicochemical parameters were calculated using www.molinspiration.com online services.

Figure 3. Comparison of the lipophilic surface maps of (a) protonated amantadine (1H+) and (b) protonated propellanamine syn-3H+. Green: lipophilic region; white: neutral region; purple: hydrophilic region; calculated using www.molinspiration.com online services.

4279

H. Torres-Gómez et al. / Bioorg. Med. Chem. 23 (2015) 4277–4285

O

O

+

O

O

H3CO

O

O

(a), (b)

O

6

O

O

O O

O

7

(c)

OCH3 5

4

O

O

O

(d)

8

(e)

X

(f)

9

(g)

10: X = NHBn 3: X = NH2

Scheme 1. Synthesis of a 1:1-mixture of diastereomeric propellanamines syn-3 and anti-3. Reagents and reaction conditions: (a) Citrate-phosphate buffer pH 5.6, MeOH 15% v/v, rt, 72 h. (b) HCl 6 M, 95 °C, 18 h, 61% (over two steps). (c) Ethylene glycol 0.9 equiv, p-TsOH, toluene, Dean–Stark apparatus, reflux, 10 h, 49%. (d) NH2NH2, KOH, diethylene glycol, 136 °C 2 h then 200 °C 6 h, 60%. (e) Acetone, p-TsOH, 60 °C, 2 h, 95%. (f) BnNH2, NaBH(OAc)3, HOAc, 1,2-dichloroethane, rt, 72 h, 99%, syn-10: anti-10 = 1:1. (g) HCO2NH4, Pd(OH)2/C, MeOH/EtOAc, reflux, 3 h, 75%, syn-3: anti-3 = 1:1.

structure of [12.3.3]propellan-16,19-dione (1.58 Å).20 The bonds between the bridgehead C-atoms and their adjacent C-atoms are slightly shorter (1.53–1.54 Å) and these lengths are in good accordance with the length of typical C–C single bonds. Moreover, the typical inverted pyramidal structure of around the bridgehead Catoms of the propellane is displayed. The monoketal 7 was previously prepared in 56% yield in a twostep procedure via a bis-ketal and subsequent transketalization.18 Herein, the monoketal was synthesized in a direct manner by portionwise addition of 0.9 equiv of ethylene glycol to the diketone 6. Thus the monoketal 7 was obtained in a single reaction step in 49% yield. The reduction of the remaining carbonyl moiety of the monoketal 7 to a methylene moiety was achieved by the Huang Minlon modification of the Wolff–Kishner reduction using H2NNH2 and KOH in boiling diethylene glycol (Scheme 1). Cleavage of the resulting ketal 8 with acetone and p-TsOH gave the monoketone 9 in 95% yield. The IR spectra of ketals 7 and 8 confirm the removal of the carbonyl moiety since the carbonyl band at 1750 cm1 disappeared in the IR spectrum of 8. Reductive amination of ketone 9 with benzylamine and NaBH(OAc)3 afforded a 1:1 mixture of the diastereoisomeric secondary amines syn-10 and anti-10. Hydrogenolytic removal of the benzyl moiety from the diastereoisomeric amines 10 with ammonium formate and Pd(OH)2/C yielded the diastereoisomeric primary amines syn-3 and anti-3 in the same 1:1 ratio. Neither the diastereomeric benzylamines 10 nor the diastereomeric primary amines 3 could be separated by flash chromatography. In order to evaluate the pharmacological properties of the pure diastereomeric primary propellanamines syn-3 and anti-3, a stereoselective synthesis of the diastereomers syn-3 and anti-3 was envisaged. For this purpose the monoketal 7 should be reduced diastereoselectively. The reduction of the ketone 7 under different conditions has been reported previously affording the diastereomeric alcohols 11 almost in the ratio 1:1.19 Herein the sterically demanding reducing agent L-Selectride (Li(secBu)3BH) was employed which produced the diastereomeric alcohols anti-11 and syn-11 in the ratio 85:15. anti-11 and syn-11 were separated by flash chromatography and isolated in 69% and 10% yield, respectively. (Scheme 2) The high diastereoselectivity during this reducing step was attributed to the dioxolane ring shielding the si-face of the carbonyl moiety of the ketone 7 in 8-position. Thus the re-face attack of the sterically demanding reducing agent L-Selectride was preferred resulting predominantly in the 8s-configured diastereomer anti-11 with anti-orientation of the OH moiety at the pseudochiral center. The configuration of the newly generated center of pseudochirality was determined by NOESY (nuclear Overhauser

enhancement spectroscopy) experiments. Whereas a cross peak between the anti-oriented proton in 11-position (tt at 4.53 ppm) and the protons 7-Hanti and 9-Hanti of the five-membered ring (d at 1.80 ppm) is observed for syn-11, the NOESY experiment performed with anti-11 does not show a cross peak between the syn-oriented proton in 11-position (tt at 4.49 ppm) and the antioriented protons of the five-membered ring. However, it shows a cross peak with the multiplet signal at 1.28 ppm belonging to the protons of the six-membered ring. This interaction is only possible when the proton in 11-position is oriented towards the six-membered ring of the propellane. In addition to the NOESY experiments, the alcohol syn-11 was crystallized from ethyl acetate to give crystals suitable for X-ray crystal structure analysis. The X-ray crystal structure confirmed unequivocally the syn-orientation of the OH-moiety in 11-position. (Fig. 5) Moreover the perpendicular orientation of the dioxolane ring towards the plane of the five-membered ring of the propellane scaffold is shown, which illustrates its shielding of the anti-face of the keto group in the opposite five-membered ring. As observed for the X-ray crystal structure of the diketone 6, the cyclohexane ring of the propellane system of syn-11 adopts a chair conformation. The length of the bond connecting the bridgehead carbon atoms C1 and C6 is 1.557(2) Å indicating that the conjoining bond of this propellane derivative is again slightly longer than a typical C–C single bond. The four bonds originating at C-1 and C-6, respectively, are oriented in only one hemisphere showing the distorted geometry around these sp3-hybridized C-atoms. Cleavage of the ketal of anti-11 and syn-11 with acetone and ptoluenesulfonic acid afforded the stereoisomerically pure hydroxyketones anti-12 and syn-12 in 98% and 97% yield, respectively, (Scheme 2). Spectroscopic data of the diastereomerically pure hydroxyketones anti-12 and syn-12 are in good agreement with the reported data for the mixtures of diastereomers.19–21 Wolff– Kishner reduction of the hydroxyketone anti-12 with H2NNH2 and KOH in diethylene glycol at 200 °C provided the alcohol anti13 in 60% yield. The diastereomerically pure alcohol anti-13 served as starting material for the stereoselective synthesis of diastereomerically pure primary amines syn-3 and anti-3 (Scheme 3). At first a Mitsunobu reaction was performed to convert the alcohol anti-13 in a one-step procedure into the azide syn-15. However, reaction of anti-13 with diphenyl phosphoryl azide, PPh3 and diisopropyl azodicarboxylate (DIAD) failed to produce the azide syn-15. Therefore, the mesylate anti-14 was prepared by reaction of anti13 with methanesulfonyl chloride. SN2 reaction of mesylate anti14 with NaN3 in DMF proceeded with inversion of configuration to provide the azide syn-15 as the only product. Since the azide

4280

H. Torres-Gómez et al. / Bioorg. Med. Chem. 23 (2015) 4277–4285 Table 1 Affinities of primary propellanamines 3 and reference compounds towards the PCP binding site of the NMDA receptor as well as their antiviral activity Compd

Ki ± SEM (lM) PCP binding site

Antiviral activitya (%)

3 syn-3 anti-3 Amantadine Memantine MK-801

10.5 11 ± 1.8 16 ± 2.9 11 ± 0.7 9.74 ± 0.074 0.003 ± 0.001

65 ± 11 116 ± 10 97 ± 10 3.9 ± 1.6 – –

a Replication of influenza A viruses in % of control (DMSO) in the presence of 5 lM test compound, mean value ± SD, n = 4.

Figure 4. XP diagram of the diketone 6 (30% thermal ellipsoids); selected bond lengths: C1–C2 1.531(2) Å; C1–C6 1.572(2) Å; C1–C9 1.539(2) Å; C6–C7 1.527(2); C6–C10 1.536(2); C6–C5 1.530(2).

O O

(a)

7

9

1

10

O

OH

7

H

6

+

O

H

anti -13

OH

(b)

9

O

H 11

7

syn-11

(b)

(c)

10

6

anti-11

OH

9 1

11

10 1

OH 11

7

H

O

H

6

anti-12

OH

syn-12

Scheme 2. Diastereoselective synthesis of alcohol anti-13. Reagents and reaction conditions. (a) L-Selectride, THF, 78 °C, 30 min, rt, 2 h, 85:15 mixture (1H NMR spectroscopy), anti-11 (69%), syn-11 (10%). (b) Acetone, p-TsOH, reflux, 2 h, anti-12 (98%), syn-12 (97%). (c) NH2NH2, KOH, diethylene glycol, 136 °C 2 h then 200 °C 6 h, 60%.

syn-15 is rather volatile and unstable at higher temperature, it was directly reduced with H2 and Pd/C to give the primary amine syn-3 in 65% yield over two steps. The diastereoisomeric amine anti-3 was obtained stereospecifically by double inversion of the configuration of alcohol anti-13 in a Mitsunobu reaction and subsequent SN2 reaction. The alcohol anti-13 was treated with benzoic acid in the presence of DIAD and Ph3P to obtain the benzoate syn-16 with inverted configuration in 89% yield (Scheme 3). Hydrolysis of the benzoate syn-16 with LiOH afforded the diastereomerically pure alcohol syn-13 in 98% yield. The synthesis of the primary amine anti-3 was achieved in analogy to the synthesis of syn-3. Reaction of the alcohol syn-13 with methanesulfonyl chloride afforded the mesylate syn-14 in 74% yield. Inversion of the configuration by SN2 reaction of syn14 with NaN3 in DMF led to the azide anti-15, which was reduced with H2 and Pd/C to obtain the primary amine anti-3 in 50% yield over two steps.

towards the PCP binding site of the NMDA receptor was determined in competitive receptor binding studies with the radioligand [3H]-(+)-MK-801. Pig brain membrane preparations were used as receptor material.22–24 The affinity data of the propellanamines 3 and reference compounds are summarized in Table 1. The propellanamine syn-3 (Ki = 11 lM) shows the same affinity at the PCP binding site of the NMDA receptor as amantadine (Ki = 11 lM) (Table 1). The recorded PCP affinity of amantadine in our assay is very similar to the reported affinity (Ki = 10 lM25). However, the propellanamine syn-3 is slightly more affine than anti-3 (Ki = 16 lM). The small difference in the PCP affinity of the diastereomeric propellanamines syn-3 and anti-3 is explained by the similar size of the bridges across the conjoining C-1/C-6 bond (3 vs 4 CH2 moieties). Since some ligands interacting with the PCP binding site of the NMDA receptor also bind at r1 and/or r2 receptors (e.g., phencyclidine itself, some benzomorphans),26–28 the affinity of propellanamines 3 towards r1 and r2 receptors was also recorded in receptor binding studies using radioligands.29–31 Up to a test compound concentration of 10 lM the diastereomeric propellanamines syn-3 and anti-3 did not interact with r1 and r2 receptors. The antiviral activity of the propellanamines 3 was investigated in the human lung epithelial cell line A549. The cells were infected with influenza A viruses and then the test compounds were added to the cell culture medium in a concentration of 5 lM. After 24 h, the amount of newly formed influenza A viruses in the supernatant was determined in the plaque assay using Madin-Darby canine kidney (MDCK) cells.32 Amantadine was able to reduce the amount of newly formed influenza A viruses to 3.9% of the control (DMSO). Although the 1:1-mixture of diastereomers 3 led to 65% of virus replication, this effect was not significant. A concentration of

3. Biological activity The affinity of the diastereomerically pure propellanamines syn-3 and anti-3 as well as the 1:1 mixture of diastereomers 3

Figure 5. XP diagram of hydroxyketal syn-11 (30% thermal ellipsoids). Selected bond lengths: C1–C2 1.539(2) Å; C1–C6 1.557(2) Å; C1–C9 1.543(2) Å; C6–C10 1.553(2) Å; C6–C5 1.533(2); C6–C7 1.533(2).

4281

H. Torres-Gómez et al. / Bioorg. Med. Chem. 23 (2015) 4277–4285

OMes (a)

anti-13

X

(b)

anti-14

(d)

(c)

syn-15: X = N3 syn-3: X = NH2

X OR

(e)

syn-16: R = Ph-C=O syn-13: R = H

(f)

OMes

syn-14

(g)

(h)

anti -15: X = N3 anti-3: X = NH2

Scheme 3. Stereoselective synthesis of diastereomerically pure primary amines syn-3 and anti-3. Reagents and reaction conditions. (a) MsCl, NEt3, DMAP, CH2Cl2, rt, overnight, 93%. (b) NaN3, DMF, 80 °C, 5 h. (c) H2, Pd/C 10%, 4 bar, 4.5 h, 65% (over two steps). (d) Benzoic acid, Ph3P, DIAD, 4 °C to rt, 3 h, 89%. (e) LiOH, THF:H2O, 70 °C, 10 h, 98%. (f) MsCl, NEt3, DMAP, CH2Cl2, rt, overnight, 74%. (g) NaN3, DMF, 80 °C, 4 h. (h) H2, Pd/C 10%, 4 bar, 4 h, 50% (over two steps).

5 lM of the pure diastereomers syn-3 and anti-3 could not reduce the replication of influenza A viruses. Unfortunately the propellanamines syn-3 and anti-3 don’t show any antiviral activity in comparison to the lead compound amantadine.

acid; (B) acetonitrile with 0:05% (v/v) trifluoroacetic acid: gradient elution: (A%): 0–4 min: 90%, 4–29 min: gradient from 90% to 0%, 29–31 min: 0%, 31–31:5 min: gradient from 0% to 90%, 31:5– 40 min: 90%.

4. Conclusion

5.2. Synthetic procedures

With respect to size, shape, electrostatic surface potential and lipophilic surface, the tricyclic propellanamines 3 are related to the adamantanamines amantadine (1) and memantine (2). The diastereomeric propellanamines syn-3 and anti-3 were synthesized stereoselectively. They interact with the PCP binding site of the NMDA receptor in the low micromolar range, which correlates with the PCP affinity of amantadine and memantine. In contrast to amantadine, both propellanamines syn-3 and anti-3 could not inhibit the replication of influenza A viruses. It can be concluded that the variation of the tricyclic scaffold leads to a separation of NMDA antagonistic and antiviral activity.

5.2.1. Spiro[[1,3]dioxolane-2,80 -[4.3.3]-propellan]-11-one (7) In a round bottom flask equipped with magnetic stirrer, Dean– Stark water separator and condenser in reflux position, the diketone 6 (12 g, 62 mmol) and p-toluenesulfonic acid (1.2 g, 6.3 mmol) were dissolved in dry toluene (100 mL) and ethylene glycol (3.3 g, 56 mmol) was added portionwise in 3 equal portions in intervals of 2.5 h while heating under reflux during 10 h. After cooling, the reaction mixture was washed with NaOH 1 M (3  20 mL) and brine (3  15 mL). Then it was dried (Na2SO4), filtered, the filtrate was concentrated in vacuo and the residue was purified by fc (8 cm, cyclohexane:ethyl acetate = 7:3, 80 mL, Rf = 0.46). Colorless solid, mp 48–49 °C, yield 7.3 g (49%). C14H20O3 (236.31). Exact mass (APCI): m/z = 237.1524 (calcd 237.1485 for C14H21O3 [M+H]+). FT-IR (ATR, film): m (cm1) = 2927 (aliphatic m C–H), 1735 (m C@O). 1H NMR (400 MHz, CDCl3): d (ppm) = 1.29–1.39 (m, 4H, 30 -CH2, 40 -CH2), 1.42–1.49 (m, 2H, 20 -CH2, 50 -CH2), 1.56–1.62 (m, 2H, 20 -H2, 50 CH2), 1.88 (d, J = 14.7 Hz, 2H, 70 -CH2, 90 -CH2), 2.11(d, J = 14.7 Hz, 2H, 70 -CH2, 90 -CH2), 2.19 (d, J = 19.3 Hz, 2H, 100 -CH2, 120 -CH2), 2.30 (d, J = 19.3 Hz, 2H, 100 -CH2, 120 -CH2), 3.77–3.84 (m, 4H, OCH2CH2O). 13C NMR (100 MHz, CDCl3): d (ppm) = 20.1 (2C, C-30 , C-40 ), 30.2 (2C, C-20 , C-50 ), 45.5 (2C, C-70 , C-90 ), 46.9 (2C, C-10 , C-60 ), 48.1 (2C, C-100 , C-120 ), 62.9 (1C, C-4 dioxolane), 63.0 (1C, C-5 dioxolane), 115.7 (1C, C-2 spiroketal), 218.1 (1C, C@O).

5. Experimental 5.1. Chemistry, general methods Unless otherwise mentioned, THF was dried with sodium/benzophenone and was freshly distilled before use. Thin layer chromatography (tlc): Silica gel 60 F254 plates (Merck). Preparative thin layer chromatography (ptlc): Silica gel 60 F254, plates (Merck) 20  20 cm, layer thickness 2 mm Flash chromatography (fc): Silica gel 60, 40–64 lm (Merck); parentheses include: diameter of the column, length of column, fraction size, eluent, Rf value. Melting point: Melting point apparatus SMP 3 (Stuart Scientific), uncorrected. IR: IR spectrophotometer IRAffinity with MIRacle 10 accessory FT-ATR-IR (Shimadzu). 1H NMR (400 MHz), 13C NMR (100 MHz): Mercury plus 400 spectrometer (Varian); d in ppm relative to tetramethylsilane; coupling constants are given with 0:5 Hz resolution. Where necessary, the assignment of the signals in the 1H NMR and 13C NMR spectra was performed using 1H–1H COSY, 1H–13C HSQC NMR spectra, the stereochemistry was assigned using NOESY NMR spectra. MS: EI = electron impact, ESI = electrospray ionization: MicroTof (Bruker Daltronics, Bremen), calibration with sodium formate clusters before measurement. HPLC method for determination of the product purity: Merck Hitachi Equipment; UV detector: L-7400; autosampler: L7200; pump: L-7100; degasser: L-7614; Method: column: LiChrospherÒ 60 RP-select B (5 mm), 250–4 mm cartridge; flow rate: 1:00 mL min1; injection volume: 5:0 mL; detection at l = 210 nm; solvents: (A) water with 0:05% (v/v) trifluoroacetic

5.2.2. Spiro-[[1,3]dioxolane-2,80 -[4.3.3]-propellane] (8) A solution of the monoketal 7 (5.0 g, 21.1 mmol), KOH (5.9 g, 0.10 mol, 5 equiv), and hydrazine monohydrate (10.6 g, 0.21 mol, 10 equiv) in diethylene glycol (15 mL) was heated to 136 °C during 2 h and then heated to reflux (200 °C) in a DeanStark apparatus for 6 h. The mixture was poured into cold water and washed with Et2O. The aqueous layer was acidified with 6 M HCl and extracted with CH2Cl2 (3  20 mL). The combined organic layers were washed with brine (20 mL), dried (Na2SO4), concentrated in vacuo and the residue was purified by fc (5 cm, cyclohexane:ethyl acetate = 9:1, 50 mL, Rf = 0.48). Pale yellow oil, yield 2.5 g (60%). C14H22O2 (222.3). FT-IR (ATR, film): m (cm1) = 2924 (aliphatic m C–H). 1H NMR (600 MHz, toluene-d8): d (ppm) = 1.24–1.39 (m, 8H, 20 -CH2, 30 -CH2, 40 -CH2, 50 -CH2), 1.50–1.58 (m, 4H, 100 -CH2, 120 -CH2), 1.71–1.75 (m, 2H, 110 -CH2), 1.93 (d, J = 14.0 Hz, 2H,

4282

H. Torres-Gómez et al. / Bioorg. Med. Chem. 23 (2015) 4277–4285

70 -H, 90 -H), 2.00 (d, J = 14.0 Hz, 2H, 70 -H, 90 -H), 3.47–3.49 (m, 4H, OCH2CH2O). 5.2.3. [4.3.3]Propellan-8-one (9) Dioxolane 8 (2.5 g, 11.2 mmol) and p-toluenesulfonic acid monohydrate (0.2 g, 1.1 mmol) were dissolved in acetone (25 mL) and heated to 60 °C during 2 h. The solvent was removed in vacuo and the residue was purified by fc (3 cm, cyclohexane:ethyl acetate = 9:1, 20 mL, Rf = 0.35). Colorless solid, mp 115–117 °C, yield 1.9 g (95%). C12H18O (178.3). Exact mass (APCI): m/z = 179.1441 (calcd 179.1430 for C12H19O [M+H]+). FT-IR (ATR, film): m (cm1) = 2924 (aliphatic m C–H), 1735 (m C@O). 1H NMR (600 MHz, CDCl3): d (ppm) = 1.33–1.37 (m, 2H, 2-Hax, 5-Heq), 1.39–1.44 (m, 2H, 3-H, 4-H), 1.45–1.53 (m, 4H, 2-Heq, 3-H, 4-H, 5-Hax), 1.58–1.63 (m, 2H, 10-Hsyn, 12-Hsyn), 1.78–1.82 (m, 2H, 10Hanti, 12-Hanti), 1.82–1.88 (m, 2H, 11-CH2), 2.16 (d, J = 19.3 Hz, 2H, 7-H, 9-H), 2.24 (d, J = 19.3 Hz, 2H, 7-H, 9-H). 13C NMR (150 MHz, CDCl3): d (ppm) = 20.5 (1C, C-11), 21.6 (2C, C-3, C-4), 31.5 (2C, C-2, C-5), 36.1 (2C, C-10, C-12), 47.9 (2C, C-1, C-6), 49.7 (2C, C-7, C-9), 219.9 (1C, C@Oketone). 5.2.4. syn- and anti-N-Benzyl-[4.3.3]propellan-8-amine (10) Under N2, NaBH(OAc)3 (3.6 g, 16.80 mmol) was added to a solution of ketone 9 (1.0 g, 5.61 mmol), benzylamine (0.92 mL, 8.41 mmol) and acetic acid (0.32 mL, 5.61 mmol) in 1,2-dichloroethane (15 mL, dried over molecular sieves 4 Å). The mixture was stirred at rt for 72 h. Then NaOH (1 M) was added (pH 8– 10), the mixture was extracted with CH2Cl2 (3) and the combined organic layers were washed with brine (1), dried (Na2SO4), filtered, the filtrate was concentrated in vacuo and the residue was purified by fc (3 cm, cyclohexane:ethyl acetate:methanol = 8:1:1 20 mL, Rf = 0.30) to obtain a mixture of diastereoisomeric amines syn-10 and anti-10 as a pale yellow oil, yield 1.5 g (99%). C19H27N (269.4). MS (ESI): m/z = 270 [M+H]+. Exact mass (APCI): m/z = 270.2243 (calcd 270.2216 for C19H28N [M+H]+). FT-IR (ATR, film): m (cm1) = 3350 (N–H st) 2924 (aliphatic m C–H). 1H NMR (400 MHz, CDCl3): d (ppm) = 1.26–1.50 (m, 9H, 2-CH2, 3-CH2, 4CH2, 5-CH2, 10(12)-CH2(1H)), 1.52–1.70 (m, 6H, 7-CH2(1H), 9CH2(1H), 10(12)-CH2(3H), 11-CH2(1H)), 1.71–1.81 (m, 1H, 11CH2(1H)), 1.87 (dd, J = 13.1/8.0 Hz, 2  0.5H, 7-H, 9-H), 1.94 (dd, J = 13.2/8.4 Hz, 2  0.5H, 7-H, 9-H), 3.29–3.38 (m, 2  0.5H, 8-H), 3.77 (m, 2H, NCH2Ph), 7.23–7.28 (m, 1H, 4-HPh), 7.30–7.38 (m, 4H, 2-HPh, 3-HPh, 4-HPh, 5-HPh). A signal for the NH proton is not seen in the spectrum. 13C NMR (100 MHz, CDCl3): d (ppm) = 20.9, 21.2 (2C, C-3, C-4), 21.3 (1C, C-11), 32.2, 32.8 (2C, C-2, C-5), 37.8 (2C, C-10, C-12), 44.8, 45.3 (2C, C-7, C-9) 50.1, 50.6 (2C, C-1, C-6), 52.5 (1C, NCH2Ph), 56.8, 55.9 (2  0.5C, C-8), 127.3 (1C, C-4phenyl), 128.4 (2C, C-3phenyl, C-5phenyl), 128.7 (2C, C-2phenyl, C-6phenyl), 129.2 (1C, C-1phenyl). Ratio syn-10: anti-10 = 1:1. Purity (HPLC): 91.7% (tR = 17.40 min). 5.2.5. syn- and anti-[4.3.3]Propellan-8-amine (3) Pd(OH)2/C (20%, 0.15 g) was added to a solution of the N-benzylpropellanamines syn-10/anti-10 (ratio 1:1, 1.5 g, 5.57 mmol) and ammonium formate (1.4 g, 22.28 mmol) in methanol (50 mL). The mixture was heated to reflux for 3 h. After evaporation of the solvent in vacuo, ethyl acetate (30 mL) was added to the residue. The mixture was washed with NaOH (1M, 10 mL) and brine (10 mL), filtered, the filtrate was concentrated in vacuo and the residue was purified by Kugelrohr distillation (bp 170 °C, 2 mbar). Colorless solid, mp 82–84 °C, yield 0.75 g (75%). C12H21N (179.3). MS (ESI): m/z = 180 [M+H]+. Exact mass (APCI): m/z = 180.1787 (calcd 180.1747 for C12H22N [M+H]+). FT-IR (ATR, film): m (cm1) = 2920 (aliphatic m C–H), 1570 (d N–H2). 1H NMR (600 MHz, CDCl3): d (ppm) = 1.29–1.31 (m, 2H, 2-CH2(1H), 5CH2(1H)) 1.37–1.52 (m, 8H, 2-CH2(1H), 3-CH2, 4-CH2, 5-CH2(1H),

7-CH2(1H), 9-CH2(1H)), 1.54–1.63 (m, 4H, 10-CH2, 12-CH2) 1.64– 1.70 (m, 1H, 11-CH2(1H)), 1.71–1.78 (m, 1H, 11-CH2(1H)), 1.85 (dd, J = 13.7/7.9 Hz, 2  0.5H, 7-H, 9-H), 1.89 (dd, J = 13.2/8.2 Hz, 2  0.5, 7-H, 9-H), 2.19 (s, 2H, NH2), 3.46–3.52 (m, 2  0.5 H, 8H). Signals for the protons of the NH2 group are not seen in the spectrum. 13C NMR (150 MHz, CDCl3): d (ppm) = 20.4, 20.7 (2C, C-3, C-4), 21.22 (1C, C-11), 31.9, 32.6 (2C, C-2, C-5), 37.7, 39.2 (2C, C-10, C-12), 47.9, 48.7 (2C, C-7, C-9), 50.0, 50.3, (2C, C-1, C6), 50.9 (1C, C-8). Ratio syn-3: anti-3 = 1:1. 5.2.6. 110 -syn-Spiro[[1,3]-dioxolane-2,80 -[4.3.3]propellan]-110 -ol (syn-11) and 11’-anti-Spiro[[1,3]-dioxolane-2,80 -[4.3.3]propellan]110 -ol (anti-11) Under N2 monoketal 7 (7.3 g, 30.9 mmol) was dissolved in THF (80 mL) and cooled to 78 °C. A solution of L-Selectride in THF (1 M, 37.1 mL, 44.5 mmol) was added dropwise. After stirring for 30 min at 78 °C, the cooling bath was removed and the mixture was stirred at rt for 2 h. A NaOH solution (1 M, 20 mL) was added and the mixture was stirred vigorously for 20 min. The mixture was extracted with EtOAc (3) and the combined EtOAc layers were washed with brine (1). Then it was dried (Na2SO4), filtered and the filtrate was concentrated in vacuo to obtain 8.4 g of a mixture of the diastereomeric alcohols syn-11 and anti-11 in the ratio 85: 15. This mixture was separated and purified by fc (8 cm, cyclohexane:ethyl acetate = 9:1 to 6:4, 80 mL). syn-11 (Rf = 0.29 (cyclohexane:ethyl acetate = 7:3)): Pale yellow solid, mp 77–80 °C, yield 0.74 g (10%). C14H22O3 (238.3). Exact mass (APCI): m/z = 239.1877 (calcd 239.1647 for C14H22O3 [M+H]+). FT-IR (ATR, film): m (cm1) = 3491 (m OH), 2927 (aliphatic m C–H). 1H NMR (400 MHz, CDCl3): d (ppm) = 1.39–1.49 (m, 4H, 30 CH2, 40 -CH2), 1.51–1.60 (m, 4H, 20 -CH2, 50 -CH2), 1.68 (dd, J = 14.2,/4.0 Hz, 2H, 100 -Hsyn, 120 -Hsyn), 1.80 (d, J = 14.4 Hz, 2H, 70 Hanti, 90 -Hanti), 1.96 (d, J = 14.4 Hz, 2H, 7’-Hsyn, 9’-Hsyn), 2.16 (dd, J = 14.1/8.0 Hz, 2H, 100 -Hanti, 120 -Hanti), 3.80–3.86 (m, 4H, OCH2CH2O), 4.53 (tt, J = 8.1, 4.0 Hz, 1H, 110 -H). A signal for the OH proton is not seen in the spectrum. 13C NMR (50 MHz, CDCl3): d (ppm) = 21.6 (2C, C-30 , C-40 ), 32.0 (2C, C-20 , C-50 ), 47.1 (2C, C-100 , C-120 ), 48.8 (2C, C-70 , C-90 ), 49.6 (2C, C-.10 , C-60 ), 64.00 (1C, C-4), 64.1 (1C, C-5), 72.3 (1C, C-110 ), 117.3 (1C, C-80 ). Crystals suitable for X-ray diffraction were obtained by slow evaporation of EtOAc. anti-11 (Rf = 0.22 (cyclohexane:ethyl acetate = 7:3)): Colorless solid, mp 48–50 °C, yield 5.1 g (69%). C14H22O3 (238.3). Exact mass (APCI): m/z = 239.1596 (calcd 239.1647 for C14H22O3 [M+H]+). FTIR (ATR, film): m (cm1) = 3491 (m OH), 2927 (aliphatic m C–H). 1H NMR (400 MHz, CDCl3): d (ppm) = 1.32–1.43 (m, 8H, 20 -CH2, 30 CH2, 40 -CH2, 50 -CH2,), 1.76 (dd, J = 14.1/4.7 Hz, 2H, 100 -Hanti, 120 Hanti), 2.00 (d, J = 14.4 Hz, 2H, 70 -H, 90 -H), 2.09 (d, J = 14.4 Hz, 2H, 70 -H, 90 -H), 2.11 (dd, J = 14.1/8.3 Hz, 2H, 100 -Hsyn, 120 -Hsyn), 3.80– 3.86 (m, 4H, OCH2CH2O), 4.49 (tt, J = 8.3/4.7 Hz, 1H, 80 -H). A signal for the OH proton is not seen in the spectrum. 13C NMR (100 MHz, CDCl3): d (ppm) = 21.3 (2C, C-30 , C-40 ), 32.1 (2C, C-20 , C-50 ), 47.3 (2C, C-100 , C-120 ), 48.8 (2C, C-70 , C-90 ), 49.8 (2C, C-10 , C-60 ), 64.0 (1C, C4), 64.07 (1C, C-5), 72.5 (1C, C-110 ), 117.5 (1C, C-80 ). 5.2.7. 11-anti-11-Hydroxy-[4.3.3]propellan-8-one (anti-12) The monoketal anti-11 (5.1 g, 21.4 mmol) and p-toluenesulfonic acid monohydrate (0.4 g, 2.1 mmol) were dissolved in acetone (50 mL) and the solution was heated to 60 °C during 2 h. The solvent was removed in vacuo and the residue was purified by fc (3 cm, cyclohexane:ethyl acetate = 7:3, 50 mL, Rf = 0.33). Colorless solid, mp 121–123 °C, yield 4.1 g (98%). C12H18O2 (194.1). Exact mass (APCI): m/z = 195.1385 (calcd 195.1380 for C12H19O2 [M+H]+). FT-IR (ATR, film): m (cm1) = 3329 (m O–H), 2924 (aliphatic m C–H), 1734 (m C@O). 1H NMR (400 MHz, CDCl3): d (ppm) = 1.33– 1.46 (m, 8H, 2-CH2, 3-CH2, 4-CH2, 5-CH2), 1.66 (dd,

H. Torres-Gómez et al. / Bioorg. Med. Chem. 23 (2015) 4277–4285

4283

J = 14.3/4.1 Hz, 2H, 10-Hanti, 12-Hanti), 1.80 (s, 1H, OH), 2.26 (d, J = 19.6 Hz, 2H, 7-Hanti, 9-Hanti), 2.28 (dd, J = 14.3/8.2 Hz, 2H, 10Hsyn, 12-Hsyn), 2.45 (d, J = 19.6 Hz, 2H, 7-Hsyn, 9-Hsyn), 4.61 (tt, J = 8.2/4.1 Hz, 1H, 11-H). 13C NMR (100 MHz, CDCl3): d (ppm) = 21.4 (2C, C-3, C-4), 32.2 (2C, C-2, C-5), 46.9 (2C, C-10, C12), 48.0 (2C, C-1, C-6), 50.1 (2C, C-7, C-9), 71.9 (1C, C-11), 219.7 (1C, C@O).

8H, 2-CH2, 3-CH2, 4-CH2, 5-CH2), 1.62–1.82 (m, 6H, 10-CH2, 11CH2, 12-CH2), 1.89 (dd, J = 14.4/5.1 Hz, 2H, 7-Hanti, 9-Hanti), 2.18 (dd, J = 14.4/8.4 Hz, 2H, 7-Hsyn, 9-Hsyn), 2.97 (s, 3H, CH3), 5.19 (tt, J = 8.5/5.1 Hz, 1H, 8-H). 13C NMR (151 MHz, CDCl3): d (ppm) = 20.9 (1C, C-11), 21.3 (2C, C-3, C-4), 31.8 (2C, C-2, C-5), 36.9 (2C, C-10, C-12), 38.4 (1C, CH3), 44.5 (2C, C-7, C-9), 50.8 (2C, C-1, C-6), 83.1 (1C, C-8).

5.2.8. 11-syn-11-Hydroxy-[4.3.3]propellan-8-one (syn-12) syn-11 (0.90 g, 3.8 mmol) and p-toluenesulfonic acid monohydrate (0.07 g, 0.38 mmol) were dissolved in acetone (10 mL) and heated to 60 °C during 2 h. The solvent was removed in vacuo and the residue was purified by fc (3 cm, cyclohexane:ethyl acetate = 7:3, 20 mL, Rf = 0.33). Colorless solid, mp 102–105 °C, yield 0.70 g (97%). C12H18O2 (194.1). Exact mass (APCI): m/z = 195.1383 (calcd 195.1380 for C12H19O2 [M+H]+). FT-IR (ATR, film): m (cm1) = 3329 (m O–H), 2931 (aliphatic m C–H), 1735 (m C@O). 1H NMR (400 MHz, CDCl3): d (ppm) = 1.38–1.48 (m, 4H, 3-CH2, 4CH2), 1.51–1.61 (m, 2H, 2-CH2, 5-CH2), 1.72–1.78 (m, 2H, 2-CH2, 5-CH2), 1.88 (dd, J = 14.5/3.6 Hz, 2H, 10-Hsyn, 12-Hsyn), 2.08 (dd, J = 14.5/7.9 Hz, 2H, 10-Hanti, 12-Hanti), 2.10 (d, J = 19.6, Hz, 2H, 7Hanti, 9-Hanti), 2.25 (d, J = 19.6, Hz, 2H, 7-Hsyn, 9-Hsyn), 4.58 (tt, J = 7.9/3.6 Hz, 1H, 11-H). A signal for the OH proton is not seen in the spectrum. 13C NMR (100 MHz, CDCl3): d (ppm) = 21.7 (2C, C3, C-4), 32.1 (2C, C-2, C-5), 46.9 (2C, C-10, C-12), 47.8 (2C, C-1, C6), 49.9 (2C, C-7, C-9), 71.9 (1C, C-11), 219.3 (1C, C@Oketone).

5.2.11. 8-syn-8-Azido-[4.3.3]propellane (syn-15) NaN3 (38 mg, 0.57 mmol) was added to a solution of anti-14 (0.1 g, 0.38 mmol) in DMF (5 mL) and the resulting mixture was heated at 80 °C for 4 h. Then cold water (10 mL) was added, the mixture was extracted with Et2O (3) and the combined organic layers were washed with brine (1), dried (Na2SO4) and filtered. The solvent was removed with a gentle N2 flow to yield the azide syn-15 as a colorless oil (Rf = 0.23, EtOAc:MeOH = 8:2), yield 77 mg (97%). The crude product was used in the next step without further purification. C12H19N3 (205.3). FT-IR (ATR, film): m (cm1) = 2924 (aliphatic m C–H), 2091 (s, m N@N@N). 1H NMR (400 MHz, CDCl3): d (ppm) = 1.40–1.52 (m, 8H, 2-CH2, 3-CH2, 4CH2, 5-CH2), 1.59–1.67 (m, 6H, 10-CH2, 11-CH2, 12-CH2), 1.71 (dd, J = 13.8/6.0 Hz, 2H, 7-Hsyn, 9-Hsyn), 1.94 (dd, J = 13.8/8.3 Hz, 2H, 7-Hanti, 9-Hanti), 4.07 (tt, J = 8.3/5.9 Hz, 1H, 8-H). 13C NMR (101 MHz, CDCl3): d (ppm) = 21.1 (1C, C-11), 21.6 (2C, C-3, C-4), 32.0 (2C, C-2, C-5), 38.0 (2C, C-10, C-12), 43.8 (2C, C-7, C-9), 50.5 (2C, C-1, C-6), 60.4 (1C, C-8).

5.2.9. 8-anti-[4.3.3]Propellan-8-ol (anti-13) A solution of the hydroxyketone anti-12 (0.80 g, 4.1 mmol), KOH (1.16 g, 2.0 mol, 5 equiv) and hydrazine monohydrate (2.06 g, 41.2 mol, 10 equiv) in diethylene glycol (10 mL) was heated to 136 °C during 2 h and then to reflux (200 °C) in a DeanStark apparatus for 6 h. The mixture was poured into cold water and washed with Et2O. The aqueous layer was acidified with 6 M HCl and extracted with CH2Cl2 (3  10 mL). The combined organic layers were washed with brine (10 mL), dried (Na2SO4), concentrated in vacuo and the residue was purified by fc (3 cm, cyclohexane:ethyl acetate = 7:3, 20 mL, Rf = 0.44). Colorless solid, mp 97–99 °C, yield 0.63 g (85%). C12H20O (180.3). Exact mass (APCI): m/z = 179.1452 (calcd 179.1430 for C12H19O [M+H]+). FTIR (ATR, film): m (cm1) = 3282 (m OH), 2924 (aliphatic m C–H). 1H NMR (600 MHz, CDCl3): d (ppm) = 1.25–1.42 (m, 8H, 2-CH2, 3CH2, 4-CH2, 5-CH2), 1.57 (dd, J = 13.6/5.7 Hz, 2H, 7-Hsyn, 9-Hsyn), 1.59–1.64 (m, 1H, 11-Hsyn), 1.66–1.74 (m, 4H, 10-CH2, 12-CH2), 1.75–1.84 (m, 1H, 11-Hanti), 2.05 (dd, J = 13.6/8.1 Hz, 2H, 7-Hanti, 9-Hanti), 4.45 (tt, J = 8.1/5.7, 1H, 8-H). A signal for the proton of the OH group is not observed. 13C NMR (150 MHz, CDCl3): d (ppm) = 21.2 (1C, C-11), 21.3 (2C, C-3, C-4), 32.5 (2C, C-2, C-5), 37.5 (2C, C-10, C-12), 47.9 (2C, C-7, C-9), 50.9 (2C, C-1, C-6), 72.8 (1C, C-8). 5.2.10. (8-anti-[4.3.3]Propellan-8-yl) methanesulfonate (anti14) Under N2, methanesulfonyl chloride (0.16 mL, 1.5 mmol) was added dropwise to a solution of the alcohol anti-13 (0.17 g, 0.7 mmol), Et3N (0.33 mL, 1.8 mmol) and DMAP (17 mg, 0.07 mmol) in CH2Cl2 (15 mL). The mixture was stirred overnight at rt. Then water (5 mL) was added and the mixture was extracted with CH2Cl2 (3). The combined organic layers were washed with brine (1), dried (Na2SO4), filtered, the filtrate was concentrated in vacuo and the residue was purified by fc (3 cm, cyclohexane: Et2O = 7.3, Rf = 0.67) to yield the mesylate anti-14 as a pale yellow oil, yield 0.23 g (93%). C13H22O3S (258.4). Exact mass (APCI): m/z = 258.1486 (calcd 258.1290 for C13H22O3S). FTIR (ATR, film): m (cm1) = 2924 (aliphatic m C–H), 1346 (m C– SO2–CH3). 1H NMR (600 MHz, CDCl3): d (ppm) = 1.24–1.42 (m,

5.2.12. syn-[4.3.3]Propellan-8-amine (syn-3) Pd/C 10% (10 mg, 0.1 equiv) was added to a solution of azide syn-15 (77 mg, 0.38 mmol) in MeOH (15 mL). The mixture was shaken under H2 (4 bar) at rt for 4.5 h. Then the reaction mixture was filtered over Celite(R) and the filtrate was concentrated in vacuo to afford the primary amine syn-3 as a colorless solid, mp 215 °C (dec), yield 45 mg (67% over 2 steps). C12H21N (179.3). Exact mass (APCI): m/z = 180.1746 (calcd 180.1747 for C12H21N). FT-IR (ATR, film): m (cm1) = 3387 (m NH2), 2924 (aliphatic m C–H). 1H NMR (400 MHz, CD3OD): d (ppm) = 1.44–1.70 (m, 16H, 2-CH2, 3-CH2, 4-CH2, 5-CH2, 10-CH2, 11-CH2, 12-CH2, 7-Hsyn, 9-Hsyn), 1.89 (dd, J = 13.4/8.1 Hz, 2H, 7-Hanti, 9-Hanti), 3.52 (quint, J = 8.2 Hz, 1H, 8-H). Signals for the protons of the NH2 moiety are not seen in the spectrum. 13C NMR (101 MHz, CD3OD); d (ppm) = 21.3 (1C, C-11), 23.1 (2C, C-3, C-4), 33.6 (2C, C-2, C-5), 40.4 (2C, C-10, C-12), 46.8 (2C, C-7, C-9), 50.7 (2Cm C-1, C-6), 51.3 (1C, C-8). 5.2.13. (8-syn-[4.3.3]Propellan-8-yl) benzoate (syn-16) Under N2, DIAD (0.34 g, 1.7 mmol) was added dropwise to an ice-cooled solution of alcohol anti-13 (0.12 g, 0.67 mmol), PPh3 (0.44 g, 1.7 mmol) and benzoic acid (0.12 g, 2.6 mmol) in dry THF (10 mL). The reaction mixture was stirred without cooling for 3 h. The solvent was removed in vacuo and the residue was purified by fc (3 cm, cyclohexane: Et2O = 8:2, Rf = 0.83). Pale yellow oil, yield 0.17 g (89%). C19H24O2 (284.4). Exact mass (APCI): m/z = 285.1840 (calcd 285.1849 for C19H25O2 [M+H]+). FT-IR (ATR, film): m (cm1) = 2924 (aliphatic m C–H), 1712 (m C@O). 1H NMR (400 MHz, CDCl3): d (ppm) = 1.41–1.78 (m, 14H, 2-CH2, 3-CH2, 4CH2, 5-CH2, 10-CH2, 11-CH2, 12-CH2), 1.87 (dd, J = 14.5/3.9 Hz, 2H, 7-Hsyn, 9-Hsyn), 2.18 (dd, J = 14.5, 8.0 Hz, 2H, 7-Hanti, 9-Hanti),), 5.48 (tt, J = 8.0/3.9 Hz, 1H. 8-H), 7.42–7.47 (m, 2H, 3-HPh, 5-HPh), 7.52–7.57 (m, 1H, 4-HPh), 8.02–8.06 (m, 2H, 2-HPh, 6-HPh). 13C NMR (101 MHz, CDCl3): d (ppm) = 21.2 (1C, C-11), 21.8 (2C, C-3, C-4), 32.0 (2C, C-2, C-5), 37.4 (2C, C-10, C-12), 44.6 (2C, C-7, C-9), 50.6 (2C, C-1, C-6), 76.3 (1C, C-8), 128.4 (2C, C-2Ph, C-6Ph), 129.6 (2C, C-3Ph, C-5Ph), 131.0 (1C, C-1Ph), 132.8 (1C, C-4Ph), 166.5 (1C, C@O).

4284

H. Torres-Gómez et al. / Bioorg. Med. Chem. 23 (2015) 4277–4285

5.2.14. 8-syn-[4.3.3]Propellan-8-ol (syn-13) LiOH (71 mg, 3.0 mmol) was added to a solution of ester syn-16 (0.17 g, 0.6 mmol) in THF:H2O (1:1, 15 mL). The mixture was heated to 70 °C for 10 h. The mixture was acidified (pH 6) with a solution 1 M HCl and extracted with EtOAc (3). The combined organic layers were washed with brine, dried (Na2SO4), filtered, the filtrate was concentrated in vacuo and the residue was purified by fc (3 cm, cyclohexane:Et2O = 8:2, Rf = 0.54). Colorless solid, mp 89.2–91 °C, yield 102 mg (98%). C12H20O (180.3). Exact mass (APCI): m/z = 180.1553 (calcd 180.1514 for C12H20O). FT-IR (ATR, film): m (cm1) = 3282 (m OH), 2924 (aliphatic m C–H). 1H NMR (600 MHz, CDCl3): d (ppm) = 1.35–1.61 (m, 14H, 2-CH2, 3-CH2, 4CH2, 5-CH2, 10-CH2, 11-CH2, 12-CH2), 1.64 (dd, J = 13.8/4.9 Hz, 2H, 7-Hsyn, 9-Hsyn), 1.98 (dd, J = 13.7/7.7 Hz, 2H, 7-Hanti, 9-Hanti), 4.46 (tt, J = 7.7/4.9 Hz, 1H, H-8). A signal for the proton of the OH group is not observed. 13C NMR (151 MHz, CDCl3): d (ppm) = 21.3 (1C, C-11), 21.6 (2C, C-3, C-4), 32.4 (2C, C-2, C-5), 38.3 (2C, C-10, C-12), 47.8 (2C, C-7, C-9), 50.5 (2C, C-1, C-6), 72.5 (C-8). 5.2.15. (8-syn-[4.3.3]Propellan-8-yl) methanesulfonate (syn-14) Under N2, methanesulfonyl chloride (0.16 g, 1.40 mmol) was added dropwise to a solution of the alcohol syn-13 (0.1 g, 0.55 mmol), Et3N (0.17 g, 1.68 mmol) and DMAP (7 mg, 0.05 mmol) in CH2Cl2 (10 mL). The mixture was stirred overnight at rt. Then water (5 mL) was added and the mixture was extracted with CH2Cl2 (3). The combined organic layers were washed with brine (1), dried (Na2SO4), filtered, the filtrate was concentrated in vacuo and the residue was purified by fc (3 cm, cyclohexane:Et2O = 8:2, Rf = 0.54) to yield the mesylate syn-14 as pale yellow oil, yield 0.11 g (74%). C13H22O3S (258.4). Exact mass (APCI): m/z = 258.1486 (calcd 258.1290 for C13H22O3S). FT-IR (ATR, film): m (cm1) = 2924 (aliphatic m C–H), 1346 (m C–SO2–CH3). 1H NMR (600 MHz, CDCl3): d (ppm) = 1.40–1.50 (m, 8H, 2-CH2, 3-CH2, 4-CH2, 5-CH2), 1.61–1.70 (m, 6H, 10-CH2, 11-CH2, 12-CH2), 1.97 (dd, J = 14.2/4.3 Hz, 2H, 7-Hsyn, 9-Hsyn), 2.12 (dd, J = 14.4/8.1 Hz, 2H, 7-Hanti, 9-Hanti), 2.98 (s, 3H, CH3), 5.22 (tt, J = 8.1/4.0 Hz, 1H, 8-H). 13C NMR (151 MHz, CDCl3): d (ppm) = 21.1 (1C, C-11), 21.2 (2C, C-3, C-4), 31.6 (2C, C-2, C-5), 37.2 (2C, C-10, C-12), 38.3 (1C, CH3), 44.6 (2C, C-7, C-9), 50.3 (2C, C-1, C-6), 83.1 (1C, C-8). 5.2.16. 8-anti-8-Azido-[4.3.3]propellane (anti-15) NaN3 (38 mg, 0.58 mmol) was added to a solution of syn-14 (0.11 g, 0.43 mmol) in DMF (5 mL) and the resulting mixture was heated at 80 °C for 4 h. Then cold water (10 mL) was added and the mixture was extracted with Et2O (3). The combined organic layers were washed with brine (1), dried (Na2SO4), filtered and the solvent was removed with a gentle N2 flow to yield the azide anti-15 as a colorless oil (Rf = 0.26, EtOAc:MeOH = 8:2), yield 80 mg (92%). The crude product anti-15 was used in the next step without further purification. C12H19N3 (205.3). FT-IR (ATR, film): m (cm1) = 2924 (aliphatic m C–H), 2090 (s, m N@N@N). 1H NMR (400 MHz, CDCl3): d (ppm) = 1.25–1.44 (m, 8H, 2-CH2, 3-CH2, 4-CH2, 5-CH2), 1.60–1.82 (m, 8H, 10-CH2, 11-CH2, 12-CH2, 7-Hanti, 9-Hanti), 2.01 (dd, J = 14.0/8.7 Hz, 2H, 7-Hsyn, 9-Hsyn), 4.03 (tt, J = 8.7/6.7 Hz, 1H, 8-H). 13C NMR (101 MHz, CDCl3): d (ppm) = 21.1 (1C, C-11), 21.2 (2C, C3, C-4), 31.9 (2C, C-2, C-5), 37.1 (2C, C-10, C-12), 44.0 (2C, C-7, C-9), 50.9 (2C, C-1, C-6), 60.8 (1C, C-8). 5.2.17. anti-[4.3.3]Propellan-8-amine (anti-3) Pd/C (10%. 10 mg) was added to a solution of azide anti-15 (80 mg, 0.39 mmol) in MeOH (15 mL). The mixture was shaken under H2 (4 bar) at rt for 4 h. Then the reaction mixture was filtered over Celite(R) and the filtrate was concentrated in vacuo to afford the amine anti-3 as a colorless solid, mp 230 °C (dec), yield

38 mg (50% over 2 steps). C12H21N (179.3). Exact mass (APCI): m/z = 180.1784 (calcd 180.1747 for C12H21N). FT-IR (ATR, film): m (cm1) = 3375 (m NH2), 2924 (aliphatic m C–H). 1H NMR (400 MHz, CD3OD): d (ppm) = 1.36–1.51 (m, 8H, 2-CH2, 3-CH2, 4-CH2, 5CH2), 1.68–1.86 (m, 8H, 10-CH2, 11-CH2, 12-CH2, 7-Hanti, 9-Hanti), 2.05 (dd, J = 13.7/8.6 Hz, 2H, 7-Hsyn, 9-Hsyn), 3.70 (quint, J = 8.6 Hz, 1H, 8-H). Signals for the protons of the NH2 moiety are not seen in the spectrum. 13C NMR (101 MHz, CD3OD): d (ppm) = 21.7 (1C, C-11), 22.1 (2C, C-3, C-4), 32.7 (2C, C-2, C-5), 38.5 (2C, C-10, C-12), 44.7 (2C, C-7, C-9), 51.3 (2C, C-1, C-6), 52.0 (1C, C-8). 5.3. X-ray crystal structures 5.3.1. X-Ray diffraction Data sets were collected with a Nonius KappaCCD diffractometer. Programs used: data collection, COLLECT (R. W. W. Hooft, Bruker AXS, 2008, Delft, The Netherlands); data reduction DenzoSMN;33 absorption correction, Denzo;34 structure solution SHELXS-97;35 structure refinement SHELXL-9736 and graphics, XP (BrukerAXS, 2000). R-values are given for observed reflections, and wR2 values are given for all reflections. CCDC-1057196 (6) and CCDC-1057197 (syn-11) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_ request/cif. 5.3.2. X-ray crystal structure analysis of 6 Recrystallization from methanol. Formula C12H16O2, M = 192.25, colorless crystal, 0.35  0.20  0.10 mm, a = 8.6986(3), b = 10.9284(6), c = 10.8111(3) Å, b = 96.185(3)°, V = 1021.7(1) Å3, qcalc = 1.250 g cm3, l = 0.664 mm1, empirical absorption correction (0.800 6 T 6 0.936), Z = 4, monoclinic, space group P21/n (No. 14), k = 1.54178 Å, T = 223(2) K, x and u scans, 11841 reflections collected (±h, ±k, ±l), [(sinh)/k] = 0.60 Å1, 1730 independent (Rint = 0.032) and 1685 observed reflections [I > 2r(I)], 127 refined parameters, R = 0.043, wR2 = 0.116, max. (min.) residual electron density 0.20 (0.12) e.Å3, hydrogen atoms were calculated and refined as riding atoms. CCDC-1057196. 5.3.3. X-ray crystal structure analysis of syn-11 Formula C14H22O3, M = 238.32, colorless crystal, 0.37  0.23  0.15 mm, a = 7.5624(1), b = 8.1560(1), c = 11.2544(1) Å, a = 69.946(1), b = 88.252(1), c = 74.759(1)°, V = 627.8(1) Å3, qcalc = 1.261 g cm-3, l = 0.696 mm1, empirical absorption correction (0.782 6 T 6 0.902), Z = 2, triclinic, space group P21/n (No. 2), k = 1.54178 Å, T = 223(2) K, x and u scans, 7672 reflections collected (±h, ±k, ±l), [(sinh)/k] = 0.60 Å1, 2130 independent (Rint = 0.032) and 2048 observed reflections [I > 2r(I)], 158 refined parameters, R = 0.042, wR2 = 0.113, max. (min.) residual electron density 0.22 (0.17) e Å3, the hydrogen at O1 atom was refined freely; others were calculated and refined as riding atoms. CCDC-1057197. 5.4. Receptor binding studies 5.4.1. Affinity towards the PCP binding site of the NMDA receptor22–24 The test was performed with the radioligand [3H]-(+)-MK-801 (22.0 Ci/mmol; Perkin Elmer). The thawed membrane preparation (about 100 lg of the protein) was incubated with various concentrations of test compounds, 2 nM [3H]-(+)-MK-801, and TRIS/EDTAbuffer (5 mM/1 mM, pH 7.5) in a total volume of 200 lL for 150 min at rt. The incubation was terminated by rapid filtration through the presoaked filtermats using a cell harvester. After

H. Torres-Gómez et al. / Bioorg. Med. Chem. 23 (2015) 4277–4285

washing each well five times with 300 lL of water, the filtermats were dried at 95 °C. Subsequently, the solid scintillator was placed on the filtermat and melted at 95 °C. After 5 min, the solid scintillator was allowed to solidify at rt. The bound radioactivity trapped on the filters was counted in the scintillation analyzer. The nonspecific binding was determined with 10 lM unlabeled (+)-MK801. The Kd-value of (+)-MK-801 is 2.26 nM. 5.4.2. Affinity towards r1 and r2 receptors The affinity towards r1 and r2 receptors was recorded according to Ref. 29–31 5.5. Antiviral activity32 The human lung epithelial cell line A549 was grown in Dulbecco modified Eagle medium (DMEM) and MDCK (MadinDarby canine kidney) cells were grown in minimal essential medium (MEM), respectively. All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS). The highly pathogenic avian influenza virus strain A/FPV/Bratislava/79 (H7N7) was taken from the virus strain collection of the Institute of Virology, Giessen, Germany. For infection A549 cells were washed in PBS and incubated with 0.001 MOI (multiplicities of infection) of the influenza virus A/FPV/Bratislava/79 (H7N7) diluted in PBS/BA (0.2% BSA, 1 mM MgCl2, 0.9 mM CaCl2, 100 U/mL penicillin and 0.1 mg/mL streptomycin) for 30 min at 37 °C. The inoculum was aspirated and cells were incubated with DMEM/BA (0.2% BSA, 1 mM MgCl2, 0.9 mM CaCl2, 100 U/mL penicillin and 0.1 mg/mL streptomycin). The test compounds [amantadine (Sigma), the 1:1-mixture of diastereomers 3, and the propellanamines syn-3 and anti-3] or solvent control (DMSO) were added along with the DMEM/BA at a final concentration of 5 lM. Supernatants were collected 24 h post infection and were used to assess the number of infectious particles (plaque titers) in the samples by standard plaque assays. Briefly, MDCK cells grown to 90% confluence in 6-well dishes were washed with PBS and infected with serial dilutions of the supernatants in PBS/BA for 30 min at 37 °C. The inoculum was aspirated and cells were incubated with MEM/BA (0.2% BSA, 1 mM MgCl2, 0.9 mM CaCl2, 100 U/mL penicillin and 0.1 mg/mL streptomycin) supplemented with 0.6% Agar (Oxoid), 0.3%DEAE-Dextran (Pharmacia Biotech) and 1.5% NaHCO3 at 37 °C, 5% CO2 for 2 days. Virus plaques were visualized by staining with neutral red. Acknowledgement We wish to thank the NRW Graduate School of Chemistry for a scholarship, which is funded by the Government of the State Nordrhein-Westfalen and the Westfälische Wilhelms-Universität Münster, Germany. We thank Anmari Christersson-Wiegers for excellent technical assistance.

4285

Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2015.06.030. References and notes 1. Wanka, L.; Iqbal, K.; Schreiner, P. R. Chem. Rev. 2013, 113, 3516. 2. Gerzon, K.; Krumalns, E. V.; Brindle, R.; Marshall, F. J.; Root, M. A. J. Med. Chem. 1963, 6, 760. 3. Rapala, R. T.; Kraay, R. J.; Gerzon, K. J. J. Med. Chem. 1965, 8, 580. 4. Nettekoven, M.; Fingerle, J.; Grether, U.; Grüner, S.; Kimbara, A.; Püllmann, B.; Rogers-Evans, M.; Röver, S.; Schuler, F.; Schulz-Gasch, T.; Ullmer, C. Bioorg. Med. Chem. Lett. 2013, 23, 1177. 5. Davies, W. L.; Grunert, R. R.; Haff, R. F.; McGahen, J. W.; Neumayer, E. M.; Paulschok, M.; Watts, J. C.; Wood, T. R.; Hermann, E. C.; Hoffmann, C. E. Science 1964, 144, 862. 6. Schwab, R. S.; England, A. C., Jr.; Poskanzer, D. C.; Young, R. R. J. Am. Med. Assoc. 1969, 208, 1168. 7. Sonkusare, S. K.; Kaul, C. L.; Ramarao, P. Pharmacol. Res. 2005, 51, 1. 8. Lipton, S. A. Nat. Rev. Drug Discovery 2006, 5, 160. 9. Kornhuber, J.; Schoppmeyer, K.; Riederer, P. Neurosci. Lett. 1993, 163, 129. 10. Chen, H. S.; Pellegrini, H. W.; Aggarwal, S. K.; Lei, S. Z.; Warach, S.; Jensen, F. E.; Lipton, S. A. J. Neurosci. 1992, 12, 4427. 11. Weller, M.; Finiels-Marlier, F.; Paul, S. M. Brain Res. 1993, 613, 143. 12. Pielak, R. M.; Chou, J. J. Biochim. Biophys. Acta 2011, 1808, 522. 13. Schnell, J. R.; Chou, J. J. Nature 2008, 451, 591. 14. Fleming, D. M. Int. J. Clin. Pract. 2001, 55, 189. 15. Hayden, F. G.; Hay, A. J. Curr. Top. Microbiol. Immunol. 1992, 176, 119. 16. Dong, G.; Peng, C.; Luo, J.; Wang, C.; Han, L.; Wu, B.; Ji, G.; He, H. PLoS One 2015, 10, e0119115. 17. Torres-Gómez, H.; Lehmkuhl, K.; Schepmann, D.; Wünsch, B. Eur. J. Med. Chem. 2013, 70, 78. 18. Natrajan, A.; Ferrara, J. D.; Hayz, J. D.; Khot, M.; Collonel, J.; Youngs, W. J.; Sukeni, C. N. J. Org. Chem. 1990, 55, 2164. 19. Senegör, L.; Ashkenazi, P.; Ginsburg, D. Tetrahedron 1984, 40, 5271. 20. Ginsburg, D.; Ashkenazi, P.; Kapo, M. Tetrahedron 1986, 42, 2555. 21. Bhanumati, S.; Ashkenazi, P.; Migdal, S.; Ginsburg, D. Helv. Chim. Acta 1986, 66, 2703. 22. Banerjee, A.; Schepmann, D.; Köhler, J.; Würthwein, E.-U.; Wünsch, B. Bioorg. Med. Chem. 2010, 18, 7855. 23. Köhler, J.; Bergander, K.; Fabian, J.; Schepmann, D.; Wünsch, B. J. Med. Chem. 2012, 55, 8953. 24. Wirt, U.; Schepmann, D.; Wünsch, B. Eur. J. Org. Chem. 2007, 462. 25. Kornhuber, J.; Quack, G.; Danysz, K.; Jellinger, K.; Danielczyk, W.; Gsell, W.; Riederer, P. Neuropharmacology 1995, 34, 713. 26. Quirion, R.; Chicheportiche, R.; Contreras, C. P.; Johnson, K. M.; Lodge, D.; Tam, S. W.; Woods, J. H.; Zukin, S. R. Trends Neurosci. 1987, 10, 444. 27. Caroll, F. I.; Abraham, P.; Parham, K.; Bai, X.; Zhang, X.; Brine, G. A.; Mascarella, S. W.; Martin, R. R.; May, E. L.; Sauss, C.; Di Paolo, L.; Wallace, P.; Walker, J. M.; Bowen, W. D. J. Med. Chem. 1992, 35, 2812. 28. May, E. L.; Aceto, M. D.; Bowman, E. R.; Bentley, C.; Martin, H. R.; Harris, L. S.; Medzihradsky, F.; Mattson, M. V.; Jacobson, A. E. J. Med. Chem. 1994, 37, 3408. 29. Meyer, C.; Neue, B.; Schepmann, D.; Yanagisawa, S.; Yamaguchi, J.; Würthwein, E.-U.; Itami, K.; Wünsch, B. Bioorg. Med. Chem. 2013, 21, 1844. 30. Miyata, K.; Schepmann, D.; Wünsch, B. Eur. J. Med. Chem. 2014, 83, 709. 31. Hasebein, P.; Frehland, B.; Lehmkuhl, K.; Fröhlich, R.; Schepmann, D.; Wünsch, B. Org. Biomol. Chem. 2014, 12, 5407. 32. Ehrhardt, C.; Dudek, S. E.; Holzberg, M.; Urban, S.; Hrincius, E. R.; Haasbach, E.; Seyer, R.; Lapuse, J.; Planz, O.; Ludwig, S. Plos One 2013, 8, e63657. 33. Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307. 34. Otwinowski, Z.; Borek, D.; Majewski, W.; Minor, W. Acta Crystallogr. A 2003, 59, 228. 35. Sheldrick, G. M. Acta Crystallogr. A 1990, 46, 467. 36. Sheldrick, G. M. Acta Crystallogr. A 2008, 64, 112.