Strained bi-functionalized trans-cyclooctenes
20240294457 · 2024-09-05
Assignee
Inventors
- Floris Petrus Johannes Theodorus Rutjes (Wijchen, NL)
- Luuk Maartense (Nijmegen, NL)
- Daniel Blanco-Ania (Utrecht, NL)
Cpc classification
C07C69/757
CHEMISTRY; METALLURGY
C07H15/252
CHEMISTRY; METALLURGY
C07C2602/24
CHEMISTRY; METALLURGY
C07C2602/32
CHEMISTRY; METALLURGY
C07C35/23
CHEMISTRY; METALLURGY
C07C271/34
CHEMISTRY; METALLURGY
A61K47/542
HUMAN NECESSITIES
International classification
C07C69/74
CHEMISTRY; METALLURGY
C07C69/757
CHEMISTRY; METALLURGY
C07C271/34
CHEMISTRY; METALLURGY
C07C35/23
CHEMISTRY; METALLURGY
Abstract
The present invention relates to novel bi-functionalized trans-cyclooctenes of formula (1). It also relates to conjugates, compositions, medical uses of such compounds, and methods for producing such compounds as well as conjugates of such compounds.
Claims
1. A compound of general formula (I), or a salt thereof: ##STR00045## wherein one of X.sup.1 and X.sup.2 is R.sup.a or Z.sup.1C(?Z.sup.2)R.sup.b, and the other is H; Y is chosen from C(?Z.sup.3)R.sup.c or CH.sub.2R.sup.a; Z.sup.1, Z.sup.2, and Z.sup.3 are in each instance chosen independently from O, S or NQ.sup.N; R.sup.a, R.sup.b, R.sup.c, and R.sup.d are in each instance chosen independently from OPr, SPr, NQ.sup.N1Q.sup.N2, a leaving group, a pharmaceutically active substance, or a targeting moiety; Pr is in each instance chosen independently from H, a linear, branched, or cyclic C1-6 alkyl or acyl, or a protecting group, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, alkoxy, C(?O)O(CH.sub.2).sub.0-4H, or haloalkoxy; Q, Q.sup.N, Q.sup.N1, and Q.sup.N2 are in each instance chosen independently from H or a linear, branched or cyclic C1-6 alkyl, wherein alkyl is optionally unsaturated and optionally substituted with halogen, alkoxy, or haloalkoxy; and m is 0, 1, 2, 3 or 4.
2. The compound according to claim 1, wherein m is 0.
3. The compound according to claim 1, wherein X.sup.2 is R.sup.a or OC(?O)R.sup.b, and X.sup.1 is H.
4. The compound according to claim 1, wherein Y is C(?O)R.sup.c or CH.sub.2R.sup.d.
5. The compound according to claim 1, wherein the protecting group is acetyl (Ac), benzoyl (Bz), benzyl (Bn), ?-methoxyethoxymethyl (MEM), dimethoxytrityl, bis-(4-methoxyphenyl)phenylmethyl (DMT), methoxymethyl (MOM), methoxytrityl (MMT), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Tr), silyls such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS), or ethoxyethyl (EE).
6. The compound according to claim 1, wherein the leaving group is a halogen, or wherein the leaving group is ##STR00046##
7. The compound according to claim 1, wherein the compound is of general formula (V-OO), (V-HO), (V-OH) or (V-HH): ##STR00047##
8. The compound according to claim 1, wherein the compound is of general formula (III-a), (III-c), or (III-e): ##STR00048##
9. The compound according to claim 1, wherein the compound is of general formula (II-a) or (VI-a) or an enantiomer thereof: ##STR00049##
10. The compound according to claim 1, wherein the compound is of general formula (VII-O) or (VII-OH) or an enantiomer thereof, wherein R.sup.b, R.sup.C and R.sup.d are in each instance chosen independently from OH or a leaving group: ##STR00050##
11. (canceled)
12. A method for producing a conjugate, the method comprising the steps of: i) providing a compound as defined in claim 1; ii) providing a second compound; iii) reacting the compound provided in step i) with the second compound to form a conjugate; and optionally iv) isolating the conjugate obtained in step iii).
13. A composition comprising a compound as defined in claim 1, and a pharmaceutically acceptable excipient.
14.-15. (canceled)
16. The method according to claim 12, wherein the second compound is a protein.
17. The method according to claim 12, wherein the second compound is an antibody.
18. The composition according to claim 13, wherein the composition comprises a tetrazine.
19. The composition according to claim 16, wherein the composition comprises a dipyridyl tetrazine.
Description
LEGENDS TO THE FIGURES
[0151]
[0152]
EXAMPLES
Example 1Synthesis of Compounds and Intermediates
[0153] ##STR00021##
Synthesis of ethyl (1R,4Z,8S,9r)-bicyclo[6.1.0]non-4-ene-9-carboxylate (Z-01) and ethyl (1R,4Z,8S,9s)-bicyclo[6.1.0]non-4-ene-9-carboxylate (Z-02)
[0154] A 1-I flask was charged with 1,5-cyclooctadiene (466 g, 530 mL, 6 equiv, 4.31 mol) and copper(II) acetylacetonate (14.1 g, 0.075 equiv, 53.9 mmol). The mixture was stirred and purged with argon for 15 minutes. Next, the temperature was elevated to 90? C. (ext) followed by the addition of ethyl diazoacetate (100.00 g, 90.9 mL, 82.0% Wt, 1 equiv, 719 mmol) over ca. 3 hours with a piston pump (flow rate of 0.555 ml/min). A TLC (10% EtOAc in heptane) showed complete consumption of the ethyl diazoacetate 30 minutes after the addition was finished. The mixture was cooled to 40? C. and the flask was placed in a distillation setup with the collection flask place in LN.sub.2. The 1,5-COD was distilled off from the reaction mixture under reduced pressure at 40? C. To the distillation residue was added ammonium hydroxide (25 g, 28 mL, 25% Wt, 0.25 equiv, 180 mmol) in brine (250 ml). The aqueous layers were back extracted with diethyl ether (2?500 ml). Subsequently, the organic layer was washed with ammonium hydroxide (10 g, 11 mL, 25% Wt, 0.10 equiv, 71.9 mmol) in brine (250 ml), an aqueous sat. Na.sub.4EDTA solution (250 ml), dried over MgSO.sub.4 and concentrated to afford the crude product as a yellow oil in 200 g yield with a significant 1,5-COD content. Z-01: .sup.1H NMR [400 MHz, ? (ppm), CDCl.sub.3]: 5.68-5.59 (m, 2H), 4.10 (q, J=7.2 Hz, 2H), 2.35-2.25 (m, 2H), 2.24-2.14 (m, 2H), 2.13-2.02 (m, 2H), 1.61-1.53 (m, 2H), 1.53-1.42 (m, 2H), 1.25 (t, J=7.2 Hz, 3H), 1.18 (t, J=4.5 Hz, 1H). RF (heptane/EtOAc 24:1): 0.20. Z-02: .sup.1H NMR [400 MHz, ? (ppm), CDCl.sub.3]: 5.64-5.55 (m, 2H), 4.10 (q, J=7.1 Hz, 2H), 2.55-2.44 (m, 2H), 2.24-2.14 (m, 2H), 2.09-1.98 (m, 2H), 1.86-1.76 (m, 2H), 1.69 (t, J=8.8 Hz, 1H), 1.41-1.32 (m, 2H), 1.25 (t, J=7.1 Hz, 3H). RF (heptane/EtOAc 24:1): 0.30.
##STR00022##
Synthesis of ethyl (1R,8S,9R, Z)-bicyclo[6.1.0]non-4-ene-9-carboxylate (Z-01
[0155] Ethyl (Z)-bicyclo[6.1.0]non-4-ene-9-carboxylate (6.0 g, 1 equiv, 31 mmol) was loaded into a dropping funnel. A dry 100-ml reaction vial was purged with argon for 15 minutes and charged with a 1M KOtBu (4.5 g, 40 mL, 1.00 molar, 1.30 equiv, 40 mmol) solution in THF and dry ethanol (1.6 g, 2.0 mL, 1.1 equiv, 34 mmol). The substrate solution was drop wise added to the stirred ethanol-KOtBu solution at r.T. under argon atmosphere. After full conversion was observed on TLC, the reaction was quenched with ethanol (4 ml) and a large portion of THF (ca. 20 ml) was removed via distillation. After ethanol (40 ml) was added, the reaction mixture was refluxed overnight. EtOAc (200 ml), brine (150 ml) and 1M aqueous HCl (45 ml) were added to the reaction mixture. After the organic layer was collected, the aqueous layer was back extracted with EtOAc (100 ml). The combined organic layers were then washed with 1M aqueous NaOH (150 ml) followed by brine (150 ml). After the collected organic layer was dried over MgSO.sub.4, the mixture was concentrated to afford the crude product in 5.2 g (87% of theory) yield. The mixture was purified via distillation under reduced pressure at 150? C. Z-01: .sup.1H NMR [400 MHz, ? (ppm), CDCl.sub.3]: 5.68-5.59 (m, 2H), 4.10 (q, J=7.2 Hz, 2H), 2.35-2.25 (m, 2H), 2.24-2.14 (m, 2H), 2.13-2.02 (m, 2H), 1.61-1.53 (m, 2H), 1.53-1.42 (m, 2H), 1.25 (t, J=7.2 Hz, 3H), 1.18 (t, J=4.5 Hz, 1H). RF (heptane/EtOAc 24:1): 0.20.
##STR00023##
Synthesis of ethyl (1S,8R,9R)-4-acetoxy-5-iodobicyclo[6.1.0]nonane-9-carboxylate (Z-03
[0156] A suspension of ethyl (Z)-bicyclo[6.1.0]non-4-ene-9-carboxylate (20.4 g, 1 equiv, 105 mmol) and sodium acetate (25.8 g, 3.0 equiv, 315 mmol) in acetic acid (252 g, 240 mL, 40 equiv, 4.20 mol) was stirred and cooled with a water bath until the temperature stabilized to ambient temperature. Next, NIS (28.4 g, 1.2 equiv, 126 mmol) was added and the mixture was stirred at ambient temperature for 3 hours. After full conversion was observed on TLC, brine (50 ml) and heptane (200 ml) were added to the reaction mixture. The organic layer was collected and washed with brine (250 ml), a mixture of sat. aqueous sodium thiosulfate solution (50 ml) and brine (200 ml) followed by sat. aqueous sodium bicarbonate solution (200 ml). The aqueous layers were back-extracted in similar order with two additional portions of heptane (2?200). The combined organic layers were dried over MgSO.sub.4 and concentrated to afford the crude product in 39 g. The crude was purified over silica (2?120 g) with gradient of 25% to 75% EtOAc in heptane to afford the purified product as white crystals in 33.2 g (83% of theory) yield. .sup.1H NMR (400 MHz, CDCl.sub.3) ? 4.86 (ddd, J=7.7, 6.6, 3.5 Hz, 1H), 4.75 (td, J=7.8, 5.3 Hz, 1H), 4.08 (q, J=7.2 Hz, 2H), 2.24-2.17 (m, 2H), 2.15-2.08 (m, 1H), 2.07 (s, 3H), 2.05-1.96 (m, 2H), 1.84-1.70 (m, 1H), 1.54-1.46 (m, 1H), 1.46-1.38 (m, 1H), 1.34-1.25 (m, 2H), 1.23 (t, J=7.1 Hz, 3H), 1.15 (t, J=4.3 Hz, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3) ? 173.66, 169.75, 73.30, 60.44, 37.53, 33.71, 33.22, 26.19, 26.19, 26.04, 25.71, 23.61, 21.49, 14.29. RF (heptane/EtOAc 3:1) 0.14.
##STR00024##
Synthesis of ethyl (1S,5R,8R,9S, Z)-5-acetoxybicyclo[6.1.0]non-3-ene-9-carboxylate (Z-04
[0157] To a stirred solution of ethyl 4-acetoxy-5-iodobicyclo[6.1.0]nonane-9-carboxylate (33.2 g, 1 equiv, 87.3 mmol) in toluene (241 g, 279 mL, 30 equiv, 2.62 mol) was added DBU (39.9 g, 39.5 mL, 3.0 equiv, 262 mmol). The mixture was stirred at 100? C. (ext.) overnight. The reaction mixture was cooled with an ice bath and filtered to remove solids. The filter residue was washed with toluene (2?50 ml). The filtrate was washed with brine (2?250 ml) and the aqueous layers were back extracted with toluene (100 ml). The combined organic layers were dried over MgSO.sub.4 and concentrated to afford a yellow oil as the crude product in 25 g. The crude was purified over silica (2?120 g) with a gradient of 10 to 20% EtOAc in heptane to afford the purified product as white crystals in 19.6 g (89% of theory) yield. .sup.1H NMR (500 MHz, CDCl.sub.3) ? 5.81 (dddd, J=11.3, 9.2, 6.9, 2.0 Hz, 1H), 5.62 (dd, J=11.1, 5.3 Hz, 1H), 5.32 (dd, J=10.6, 5.2 Hz, 1H), 4.10 (q, J=7.2 Hz, 2H), 2.44-2.34 (m, 2H), 2.05 (s, 3H), 1.91-1.85 (m, 1H), 1.79-1.63 (m, 3H), 1.44-1.28 (m, 3H), 1.25 (t, J=7.1 Hz, 3H). .sup.13C NMR (126 MHz, CDCl.sub.3) ? 173.50, 170.19, 133.93, 129.18, 75.30, 60.37, 36.46, 31.22, 28.67, 26.92, 26.43, 25.42, 21.31, 14.25. RF (heptane/EtOAc 4:1) 0.26.
##STR00025##
Synthesis of ethyl (1S,5R,8R,9S, E)-5-acetoxybicyclo[6.1.0]non-3-ene-9-carboxylate (E-01a. E-01b
[0158] A custom-made long-necked flask was charged with an aqueous solution of silver nitrate (7.00 g, 2.97 equiv, 41.2 H mmol) in water (100 ml). Next, a solution of ethyl (1R,5S,8S,9R, Z)-5-acetoxybicyclo[6.1.0]non-3-ene-9-carboxylate (3.50 g, 92.5 mL, 150H mmolar, 1 equiv, 13.9 mmol) and methyl benzoate (3.84 g, 3.56 mL, 2.03 equiv, 28.2 mmol) in deoxygenated heptane (160 ml) and MTBE (40 ml) HE-b was loaded into a UV irradiation setup (as described in Blanco-Ania et al., ChemPhotoChem 2018, 2(10), 898-905.). The continuous process ran overnight for 16 hours. Next, the biphasic reaction mixture was loaded into a separation funnel and the aqueous layer was collected. The organic layer was washed with water (100 ml). The combined aqueous layers were washed with heptane (100 ml). Subsequently, 25% aqueous ammonium hydroxide (25 ml) was added to the aqueous layer before extracting it with EtOAc (100 ml). The combined organic layers were dried over MgSO.sub.4 and concentrated to afford a diastereomeric mixture with a ratio of 7:13 (E-01a:E-01b) of two products as clear oil in 1.6 g yield. (E-01a) .sup.1H NMR (500 MHz, CDCl.sub.3) ? 6.11 (ddd, J=16.8, 10.9, 5.9 Hz, 0.35H), 5.55 (dd, J=16.9, 3.4 Hz, 0.35H), 5.15 (q, J=3.1 Hz, 0.35H), 4.14-4.05 (m, 0.70H), 2.67 (dt, J=12.6, 6.0 Hz, 0.35H), 2.51-2.45 (m, 0.35H), 2.25-2.20 (m, 0.35H), 2.08 (m, 0.35H), 2.05 (q, 0.70H), 2.00-1.95 (m, 0.35H), 1.80-1.75 (m, 0.35H), 1.60-1.55 (m, 0.70H), 1.55-1.45 (m, 0.35H), 1.24 (dt, J=10.5, 7.1 Hz, 1.05H). .sup.13C NMR (126 MHz, CDCl.sub.3) ? 174.95, 170.04, 131.77, 129.56, 70.78, 60.57, 37.26, 31.37, 29.29, 26.75, 21.17, 20.62, 20.62, 14.29. RF (EtOAc/heptane 1:1) 0.54. (E-01b) .sup.1H NMR (500 MHz, CDCl.sub.3) ? 5.91 (ddd, J=16.0, 9.1, 6.4 Hz, 0.65H), 5.67 (ddd, J=16.6, 9.7, 1.5 Hz, 0.65H), 5.01 (td, J=9.8, 5.3 Hz, 0.65H), 4.14-4.05 (m, 1.30H), 2.86 (dtt, J=14.7, 9.2, 1.1 Hz, 0.65H), 2.40-2.35 (m, 0.65H), 2.38-2.30 (m, 0.65H), 2.17 (m, 0.65H), 1.55-1.45 (m, 0.65H), 1.40 (t, J=5.6 Hz, 0.65H), 1.25 (dt, J=10.5, 7.1 Hz, 3H), 1.25-1.20 (m, 0.65H), 0.77 (dt, J=15.4, 11.4 Hz, 0.65H). .sup.13C NMR (126 MHz, CDCl.sub.3) ? 174.28, 170.62, 136.17, 131.20, 77.46, 60.47, 37.67, 36.47, 34.61, 30.23, 29.06, 28.47, 21.25, 14.26. RF (EtOAc/heptane 1:1) 0.54.
##STR00026##
Synthesis of ethyl (1S,5R,8R,9S, E)-5-hydroxybicyclo[6.1.0]non-3-ene-9-H carboxylate (E-02a. E-02b
[0159] A solution of ethyl (1R,5S,8S,9R, E)-5-acetoxybicyclo[6.1.0]non-3-ene-9-carboxylate (2.00 g, 1 equiv, 7.93 mmol) and potassium carbonate (2.19 g, 2.0 equiv, 15.9 mmol) in ethanol (15.7 g, 19.9 mL, 43 equiv, 341 mmol) was stirred at ambient temperature overnight. The flask was shielded from light with aluminium foil. After completion was observed with TLC, acetic acid (2.00 g, 1.91 mL, 4.2 equiv, 33.3 mmol) in a 1:1 water-brine mixture (50 ml) was added to quench the reaction. After EtOAc (25 ml) was added and separated from the aqueous layer, the organic layer was washed with aqueous sat. NaHCO.sub.3 (25 ml), brine (25 ml). All aqueous layers were back-extracted with EtOAc (2?25 ml). The combined organic layers were dried over MgSO.sub.4 and concentrated to afford the crude product as a yellow oil in 1.8 g yield. The diastereomers were separated over silica (80 g, 10% acetone in toluene) to afford E-02a in 470 mg yield and E-02b in 980 mg yield. (E-02a) .sup.1H NMR (500 MHz, CDCl.sub.3) ? 6.26 (ddd, J=16.8, 10.9, 5.9 Hz, 1H), 5.55 (ddd, J=16.8, 3.1, 0.9 Hz, 1H), 4.36 (q, J=3.0 Hz, 1H), 4.08 (qd, J=7.1, 2.1 Hz, 2H), 2.66 (dt, J=12.5, 6.0 Hz, 1H), 2.49-2.42 (m, 1H), 2.25 (dd, J=16.1, 11.9 Hz, 1H), 2.00-1.91 (m, 1H), 1.87 (q, J=8.0 Hz, 1H), 1.79-1.74 (m, 1H), 1.68 (t, J=13.1 Hz, 1H), 1.55-1.48 (m, 2H), 1.46-1.40 (m, 1H), 1.24 (t, J=7.2 Hz, 3H). .sup.13C NMR (126 MHz, CDCl.sub.3) ? 175.25, 133.55, 130.42, 68.52, 60.49, 39.75, 31.73, 29.13, 27.05, 20.57, 19.31, 14.27. RF (EtOAc/heptane 1:1) 0.37 (E-02b) .sup.1H NMR (500 MHz, CDCl.sub.3) ? 5.76 (ddd, J=15.8, 8.9, 6.5 Hz, 1H), 5.62 (ddd, J=16.5, 9.4, 1.4 Hz, 1H), 4.08 (dd, J=9.8, 5.2 Hz, 1H), 4.05 (q, J=7.1 Hz, 2H), 2.81 (dtt, J=14.8, 9.3, 1.2 Hz, 1H), 2.34-2.29 (m, 1H), 2.28-2.23 (m, 1H), 2.16-2.08 (m, 1H), 2.10-2.05 (m, 1H), 1.43 (dddd, J=13.1, 11.4, 9.7, 0.8 Hz, 1H), 1.32-1.29 (m, 1H), 1.20 (t, J=7.1 Hz, 3H), 1.18-1.13 (m, 1H), 0.68 (dt, J=15.4, 11.3 Hz, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) ? 174.53, 140.34, 129.39, 76.10, 60.44, 38.02, 37.75, 30.33, 29.31, 28.63, 28.50, 14.23. RF (EtOAc/heptane 1:1) 0.27.
##STR00027##
Ethyl (1R,4Z,8S,9s)-bicyclo[6.1.0]Non-4-Ene-9-carboxylate (Z-02
[0160] Z-02 was separated from Z-01 by subjecting a mixture of Z-01 and Z-02 to silica gel column chromatography.
Ethyl (1S,4R,5R,8R,9S)-4-acetoxy-5-iodobicyclo[6.1.0]nonane-9-carboxylate (Z-021
[0161] ##STR00028##
[0162] Ethyl (1R,8S,9s, Z)-bicyclo[6.1.0]non-4-ene-9-carboxylate (Z-02, 501 mg, 2.6 mmol) and NIS (696 mg, 3.1 mmol) were dissolved in acetic acid (3.0 mL) under an inert atmosphere. To the solution was added 3.0 mL of a saturated NaOAc in AcOH solution. The reaction mixture was stirred for 72 h. after which it was diluted with ethyl acetate (20 mL), water (15 mL), brine (15 mL) and 10% aqueous Na.sub.2S.sub.2O.sub.3 solution (10 mL). The organic phase was separated from the aqueous layers and the aqueous layers were extracted with ethyl acetate (5?40 mL). The combined organic layers were dried with MgSO.sub.4, concentrated in vacuo and purified with silica gel column chromatography (0%.fwdarw.20% EtOAc in n-heptane) to afford Z-021 (800 mg, 82%) as a yellow dense liquid. TLC (EtOAc/n-heptane, 3:7 v/v): R.sub.f=0.55. .sup.1H NMR (500 MHz, CDCl.sub.3) ? 4.95 (ddd, J=7.8, 6.8, 3.2 Hz, 1H), 4.76 (ddd, J=9.3, 7.8, 3.8 Hz, 1H), 4.14 (q, J=7.1 Hz, 2H), 2.40-2.23 (m, 2H), 2.22-2.08 (m, 6H), 1.91-1.64 (m, 4H), 1.42 (dtd, J=10.9, 8.7, 4.0 Hz, 1H), 1.35-1.21 (m, 4H). .sup.13C NMR (126 MHz, CDCl.sub.3) ? 171.8, 169.8, 74.0, 60.2, 38.2, 33.9, 33.9, 23.6, 23.5, 21.7, 21.3, 21.3, 18.7, 14.5.
Ethyl (1R,5R,8S,9S, Z)-5-acetoxybicyclo[6.1.0]Non-3-Ene-9-carboxylate(Z-022
[0163] ##STR00029##
[0164] Ethyl (1S,4R,5R,8R,9S)-4-acetoxy-5-iodobicyclo[6.1.0]nonane-9-carboxylate (Z-021 800 mg, 2.1 mmol) was dissolved in dry toluene (10 mL). To the solution was added DBU (1 mL, 6.3 mmol) and the solution was heated to 100? C. The reaction was stirred for a total of 2 days at 100? C. The reaction was cooled to room temperature, washed with water (10 mL), 1M HCl (10 mL) and brine (10 mL). Subsequently the combined aqueous layers were extracted with toluene (10 mL) and the combined organic layers were dried with MgSO.sub.4, concentrated in vacuo and purified with silica gel column chromatography (0%.fwdarw.30% EtOAc in n-heptane) to afford Z-022 (254 mg, 48%) as a colorless oil. TLC (EtOAc/n-heptane, 3:7 v/v): R.sub.f=0.66. .sup.1H NMR (500 MHz, CDCl.sub.3) ? 5.82 (dddd, J=11.1, 9.8, 7.0, 2.1 Hz, 1H), 5.57 (ddd, J=10.9, 5.2, 1.2 Hz, 1H), 5.48-5.39 (m, 1H), 4.12 (q, J=7.1 Hz, 2H), 2.60 (dddd, J=13.7, 12.7, 9.8, 1.3 Hz, 1H), 2.29-2.14 (m, 1H), 2.11-2.02 (m, 4H), 1.98-1.90 (m, 2H), 1.76-1.64 (m, 2H), 1.45 (dtd, J=12.2, 8.7, 3.3 Hz, 1H), 1.34-1.22 (m, 4H). .sup.13C NMR (126 MHz, CDCl.sub.3) ? 172.0, 170.2, 134.0, 130.2, 75.6, 60.1, 36.6, 28.5, 23.3, 22.2, 21.5, 20.6, 14.5. HRMS (m/z): [M+Na]+ calcd. for C.sub.14H.sub.20O.sub.4Na: 275.1259, found 275.1243.
Ethyl (1R,8S,9S, E)-5-acetoxybicyclo[6.1.0]Non-3-Ene-9-carboxylate (E-021a/E-021b
[0165] ##STR00030##
[0166] A custom-made long-necked flask was charged with an aqueous solution of silver nitrate (2.69 g, 15.85 mmol) in water (3 ml). Next, a solution of ethyl (1R,5R,8S,9S, Z)-5-acetoxybicyclo[6.1.0]non-3-ene-9-carboxylate (Z-022, 2.00 g, 40 mmolar, 7.93 mmol) and methyl benzoate (400 ?L, 3.17 mmol) in deoxygenated heptane (20 ml) was loaded into a UV irradiation setup (as described in Blanco-Ania et al., ChemPhotoChem 2018, 2 (10), 898-905.). The continuous process ran for 43 h. The extraction vial was disconnected from the setup and residual solution in the photoreactor was collected by flushing the remaining system with heptane (150 ml). Next, the biphasic reaction mixture was loaded into a separation funnel and additional water (150 ml) was added before starting the extraction. After phase separation, the organic layer was washed with a solution of silver nitrate (1.5 g) in water (100 ml). The combined aqueous layers were then back extracted with heptane (100 mL) to remove residual starting material from the aqueous layers. Next, ammonium hydroxide (5.6 mL, 25% Wt, 35.67 mmol) was added to the aqueous layer to decomplex the product from the silver ions. The turbid aqueous solution was extracted with ethyl acetate (3?200 ml). The combined organic layers were washed with water (100 ml), dried with MgSO.sub.4 and concentrated to afford a diastereomeric mixture with a ratio of 1:1 (E-021a:E-021b) of two products as clear oil (610 mg, 31%). HRMS (m/z): [M+Na]+ calcd. for C.sub.14H.sub.20O.sub.4Na: 275.1259, found 275.1241.
E-021a (Axial): TLC (EtOAc/n-heptane, 1:1 v/v): R.sub.f=0.73. .sup.1H NMR (500 MHz, CDCl.sub.3) ? 5.86-5.80 (m, 2H), 5.06-5.00 (m, 1H), 4.16-4.09 (m, 2H), 2.68-2.60 (m, 1H), 2.51 (ddd, J=11.7, 9.2, 1.8 Hz, 1H), 2.48-2.43 (m, 1H), 2.07 (s, 3H), 1.81 (t, J=9.0 Hz, 1H), 1.74-1.71 (m, 2H), 1.62-1.59 (m, 1H), 1.58-1.54 (m, 1H), 1.29-1.25 (m, 3H), 1.25-1.22 (m, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) ? 171.7, 170.7, 134.0, 131.3, 69.9, 60.1, 38.8, 26.1, 26.0, 24.2, 22.0, 21.4, 17.4, 14.5.
E-021b (Equatorial): TLC (EtOAc/n-heptane, 1:1 v/v): R.sub.f=0.73. .sup.1H NMR (500 MHz, CDCl.sub.3) ? 6.32 (dddd, J=16.8, 8.4, 6.8, 0.8 Hz, 1H), 5.62 (ddd, J=16.8, 9.7, 1.5 Hz, 1H), 5.15-5.07 (m, 1H), 4.16-4.09 (m, 2H), 2.79-2.71 (m, 1H), 2.34 (ddd, J=14.5, 6.7, 2.1 Hz, 1H), 2.23 (dt, J=12.0, 6.2 Hz, 1H), 2.10-2.08 (m, 1H), 2.05 (s, 3H), 1.90-1.84 (m, 1H), 1.73 (d, J=1.4 Hz, 1H), 1.50-1.41 (m, 2H), 1.27 (t, J=0.9 Hz, 3H), 1.15-1.07 (m, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) ? 171.7, 170.7, 135.3, 134.3, 77.9, 60.1, 35.0, 34.8, 26.1, 25.4, 24.0, 24.0, 21.4, 14.5.
Synthesis of ethyl (1S,5R,8R,9S, E)-5-(((perfluorophenoxy)carbonyl)oxy)bicyclo[6.1.0]non-3-ene-9-carboxylate (E-03a
[0167] ##STR00031##
[0168] To a stirred solution of ethyl (1R,5S,8S,9R, E)-5-hydroxybicyclo[6.1.0]non-3-ene-9-carboxylate (E-02a, 38.0 mg, 1 equiv, 181 ?mol) and DIPEA (70.1 mg, 94.4 ?L, 3.0 equiv, 542 ?mol) in DCM (1.30 g, 988 ?L, 85 equiv, 15.4 mmol) was added DPFPC (196 mg, 2.75 equiv, 497 ?mol) at 0? C. After the addition was complete, the mixture was covered from light and allowed to warm up to ambient temperature. After completion, water (1 ml) was added and subsequently the mixture was neutralized with acidic acid. The organic layer was washed with water (2 ml). The collected organic layer was dried over MgSO.sub.4 and concentrated to afford crude product in 190 mg yield. Purification over silica (gradient of 10 to 500/EtOAc in heptane, 4 g silica) afforded an isolated yield of 24 mg (320/of theory). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 6.27 (ddd, J=16.9, 10.9, 5.9 Hz, 1H), 5.57 (dd, J=16.9, 3.3 Hz, 1H), 5.17 (d, J=3.1 Hz, 1H), 4.13 (q, J=7.2 Hz, 2H), 2.76 (dt, J=12.8, 6.3 Hz, 1H), 2.57-2.47 (m, 1H), 2.34-2.19 (m, 2H), 2.08 (s, 1H), 1.88 (t, J=13.9 Hz, 1H), 1.64-1.54 (m, 2H), 1.53-1.46 (m, 1H), 1.26 (t, J=7.1 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) ? 174.70, 150.31, 142.46, 141.01, 140.11, 139.12, 138.49, 136.65, 133.26, 127.54, 77.10, 60.66, 37.31, 31.25, 29.41, 26.42, 20.47, 14.19. .sup.19F NMR (377 MHz, CDCl.sub.3) ? ?153.22 (d, J=17.0 Hz, 2F), ?157.50 (t, J=21.7 Hz, 1F), ?162.04 (dd, J=21.8, 17.0 Hz, 2F). RF (EtOAc/heptane 1:1) 0.55.
##STR00032##
Synthesis of ethyl (1S,5R,8R,9S, E)-5-(((perfluorophenoxy)carbonyl)oxy)bicyclo[6.1.0]non-3-ene-9-carboxylate (E-03b
[0169] To a stirred solution of ethyl (1R,5S,8S,9R, E)-5-hydroxybicyclo[6.1.0]non-3-ene-9-carboxylate (E-02b, 52.0 mg, 1 equiv, 247 ?mol) and DIPEA (95.9 mg, 129 ?L, 3.0 equiv, 742 ?mol) in DCM (1.79 g, 1.35 mL, 85 equiv, 21.0 mmol) was added DPFPC (268 mg, 2.75 equiv, 680 ?mol) at 0? C. After the addition was complete, the mixture was covered from light and allowed to warm up to ambient temperature. After completion, water (1 ml) was added and subsequently the mixture was neutralized with acidic acid. The organic layer was washed with water (2 ml). The collected organic layer was dried over MgSO.sub.4 and concentrated to afford the crude product in 270 mg yield. Purification over silica (gradient of 10 to 50% EtOAc in heptane, 4 g silica) afforded an isolated yield of 45 mg (43% of theory). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 6.01 (ddd, J=16.0, 9.0, 6.5 Hz, 1H), 5.79 (ddd, J=16.6, 9.6, 1.4 Hz, 1H), 5.03 (td, J=9.9, 5.3 Hz, 1H), 4.10 (q, J=7.1 Hz, 2H), 2.99-2.86 (m, 1H), 2.49-2.31 (m, 3H), 2.26-2.18 (m, 1H), 1.70 (dt, J=12.9, 10.7 Hz, 1H), 1.39 (t, J=5.6 Hz, 1H), 1.30-1.19 (m, 1H), 1.25 (t, J=7.1 Hz, 3H), 0.78 (dt, J=15.1, 11.3 Hz, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3) ? 174.18, 150.62, 142.57, 140.97, 140.14, 139.16, 138.45, 136.41, 134.56, 132.97, 83.57, 60.63, 37.50, 34.32, 30.06, 28.79, 28.72, 28.49, 14.22. .sup.19F NMR (377 MHz, CDCl.sub.3) ? ?153.16 (d, J=16.9 Hz), ?157.66 (t, J=21.7 Hz), ?162.15 (dd, J=21.7, 17.0 Hz). RF (EtOAc/heptane 1:1) 0.57.
##STR00033##
COOEt-Exo-Axial-TCO-AMC (E-022a
[0170] 7-Amino-4-methylcoumarin (121 mg, 0.69 mmol) was dissolved in dry toluene (15 mL) and DIPEA (442 ?L, 2.54 mmol) and triphosgene (205 mg, 0.69 mmol) were added. The reaction mixture was stirred for 1 h. at 120? C. and allowed to cool down to rt. COOEt-R-TCO-OH (E-02a, 116 mg, 0.52 mmol) was dissolved in dry DCM (10 mL) and DMAP (202 mg, 1.66 mmol) was added. The cooled down 7-amino-4-methylcoumarin solution was added dropwise to the TCO mixture in DCM on ice. The reaction mixture was stirred for 18 h. in the dark. The reaction mixture was concentrated and suspended in EtOAc (80 mL). 1 M aqueous HCl (40 mL) was added and the aqueous phase was separated. The organic layer was washed brine (30 mL). The combined aqueous layers were back extracted with EtOAc (2?20 mL). The combined organic layers were dried with MgSO.sub.4, concentrated in vacuo and purified with silica gel column chromatography (5%.fwdarw.50% EtOAc in n-pentane) to afford E-022a (142.3 mg, 63%) as an off-white solid. TLC (EtOAc/n-pentane, 1:1 v/v): R.sub.f=0.54. .sup.1H NMR (500 MHz, CDCl.sub.3) ? 7.55 (d, J=8.6 Hz, 1H), 7.48-7.43 (m, 2H), 7.15 (s, 1H), 6.25-6.16 (m, 1H), 5.63 (dd, J=16.9, 3.3 Hz, 1H), 5.23 (d, J=3.2 Hz, 1H), 4.17-4.11 (m, 2H), 2.72 (dt, J=12.5, 6.0 Hz, 1H), 2.55-2.48 (m, 1H), 2.43 (d, J=1.3 Hz, 3H), 2.30-2.14 (m, 2H), 2.04 (q, J=6.5 Hz, 1H), 1.86 (t, J=13.6 Hz, 1H), 1.60 (q, J=8.7 Hz, 3H), 1.50 (dd, J=9.1, 6.4 Hz, 1H), 1.30 (t, J=7.1 Hz, 3H). .sup.13C NMR (126 MHz, CDCl.sub.3) ? 175.0, 161.2, 154.5, 152.4, 152.1, 141.6, 132.4, 129.4, 125.5, 115.6, 114.5, 113.3, 106.0, 72.2, 60.7, 37.6, 31.5, 29.5, 26.8, 21.1, 20.8, 18.7, 14.4. HRMS (m/z): [M+Na]+ calcd. for C.sub.23H.sub.25N.sub.1O.sub.6Na: 434.1579, found 434.1575.
##STR00034##
COOEt-Exo-Axial-TCO-Glycine-OMe (E-023a
[0171] To a stirred H solution of ethyl(1R,5S,8S,9R, E)-5-hydroxybicyclo[6.1.0]non-3-ene-9-carboxylate (E-02a, 46 mg, 219 ?mol) in dry acetonitrile (3 mL) at 0? C., was added DPFPC (172 mg, 438 ?mol), DIPEA (191 ?L, 1.09 mmol), and DMAP (2.7 mg, 21.9 ?mol). After the addition was complete, the mixture was shielded from light and allowed to warm up to ambient temperature overnight. After completion, the reaction mixture was diluted with diethyl ether (10 mL) and washed with water (2?5 mL). The organic layer was dried with MgSO.sub.4 and concentrated in vacuo. Without further purification, the product was dissolved in dry acetonitrile (2.5 mL) and to this a solution of glycine methyl ester hydrochloride (55 mg, 438 ?mol) and DIPEA (95.4 ?L, 547 ?mol) in dry acetonitrile (2.5 mL) was added. The reaction was shielded from light. Upon completion, the reaction was diluted with EtOAc (10 mL) and extracted with water (2?5 mL). The organic layers were dried with MgSO.sub.4, concentrated in vacuo and purified with silica gel column chromatography (5% acetone in toluene) to afford E-023a (19 mg, 27%) as a slightly yellow oil. TLC (acetone/toluene, 1:9 v/v): R.sub.f=0.21. .sup.1H NMR (500 MHz, CDCl.sub.3) ? 6.13 (ddd, J=16.9, 11.0, 5.9, 1H), 5.56 (dd, J=16.9, 3.3, 1H), 5.22 (t, J=5.6, 1H), 5.08 (q, J=3.1, 1H), 4.11 (qd, J=7.2, 2.6, 2H), 3.97 (dd, J=5.6, 3.1, 2H), 3.76 (s, 3H), 2.67 (dt, J=9.8, 6.0, 1H), 2.46 (t, J=11.9, 1 H), 2.24-2.17 (m, 1H), 2.10 (ddd, J=15.2, 6.7, 2.8, 1H), 2.01-1.89 (m, 1H), 1.76 (t, J=13.8, 1H), 1.65-1.50 (m, 2H), 1.48-1.41 (m, 1H), 1.26 (t, J=7.1, 3H). .sup.13C NMR (126 MHz, CDCl.sub.3) ? 175.1, 170.6, 155.5, 131.8, 129.9, 71.8, 60.7, 52.5, 42.7, 31.6, 29.4, 27.1, 20.6, 14.4. HRMS (m/z): [M+Na]+ calcd. for C.sub.16H.sub.23NO.sub.6Na: 348.1423, found 348.1413.
##STR00035##
COOEt-Exo-Axial-TCO-Sarcosine-OMe (E-024a
[0172] To a stirred solution of ethyl(1R,5S,8S,9R, E)-5-hydroxybicyclo[6.1.0]non-3-ene-9-carboxylate (E-02a, 50.3 mg, 239 ?mol) in dry acetonitrile (3 mL) at 0? C., was added DPFPC (191 mg, 485 ?mol), DIPEA (208 ?L, 1.2 mmol), and DMAP (3.6 mg, 29 ?mol). After the addition was complete, the mixture was shielded from light and allowed to warm up to ambient temperature overnight. After completion, the reaction mixture was diluted with diethyl ether (10 mL) and washed with water (2?5 mL). The organic layer was dried with MgSO.sub.4 and concentrated in vacuo. Without further purification, the product was dissolved in dry acetonitrile (2.5 mL) and to this a solution of sarcosine methyl ester hydrochloride (66.4 mg, 476 ?mol) and DIPEA (104 ?L, 595 ?mol) in dry acetonitrile (2.5 mL) was added. The reaction was shielded from light. Upon completion, the reaction mixture was diluted with EtOAc (10 mL) and it was washed with water (2?5 mL). The organic layer was dried with MgSO.sub.4, concentrated in vacuo and purified with silica gel column chromatography (5% acetone in toluene) to afford E-024a as a colorless oil (31 mg, 31%). TLC (acetone/toluene, 1:9 v/v) R.sub.f=0.32. .sup.1H NMR (500 MHz, CDCl.sub.3) rotamer 1: ? 6.12 (ddd, J=16.8, 10.9, 5.9, 1H), 5.56 (td, J-16.7, 3.3, 1H), 5.11 (dd, J=5.9, 3.0, 1H), 4.11 (dtd, J=10.1, 7.1, 2.8, 2H), 4.06-3.95 (m, 2H), 3.73 (s, 3H), 3.03 (s, 3H), 2.67 (tt, J=12.4, 6.2, 1H), 2.52-2.42 (m, 1H) 2.22 (dd, J=16.2, 11.8, 1H), 2.15-2.10 (m, 1H), 2.10-2.02 (m, 1H), 1.81-1.68 (m, 1H), 1.55 (td, J=11.3, 10.4, 5.8, 2H), 1.45 (ddd, J=15.0, 9.9, 6.1, 1H), 1.26 (td, J=7.1, 2.1, 3H), rotamer 2: ? 5.99 (ddd, J=16.8, 10.9, 5.9, 1H), 5.56 (td, J=16.7, 3.3, 1H), 5.11 (dd, J=5.9, 3.0, 1H), 4.11 (dtd, J=10.1, 7.1, 2.8, 2H), 4.06-3.95 (m, 2H), 3.77 (s, 3H), 2.99 (s, 3H), 2.67 (tt, J=12.4, 6.2, 1H), 2.52-2.42 (m, 1H) 2.22 (dd, J=16.2, 11.8, 1H), 2.15-2.10 (m, 1H), 2.10-2.02 (m, 1H), 1.81-1.68 (m, 1H), 1.55 (td, J=11.3, 10.4, 5.8, 2H), 1.45 (ddd, J=15.0, 9.9, 6.1, 1H), 1.26 (td, J=7.1, 2.1, 3H).sup.13C NMR (126 MHz, CDCl.sub.3) rotamer 1: ? 175.1, 170.1, 155.9, 131.5, 130.1, 72.3, 60.7, 52.2, 50.6, 37.6, 35.3, 31.6, 29.4, 26.9, 20.7, 14.4, rotamer 2: ? 175.2, 170.2, 155.2, 131.6, 129.9, 72.2, 60.7, 52.3, 50.7, 37.6, 36.2, 31.6, 29.4, 26.9, 20.6, 14.43. HRMS (m/z): [M+Na]+ calcd. for C.sub.17H.sub.25NO.sub.6Na: 362.1579, found 362.1598.
##STR00036##
COOEt-Exo-Equatorial-TCO-AMC (E-022b
[0173] 7-Amino-4-methylcoumarin (15 mg, 86 ?mol) was dissolved in dry toluene (2 mL) and DIPEA (55 ?L, 317 ?mol) and triphosgene (25 mg, 86 ?mol) were added. The reaction mixture was stirred for 1 h. at 120? C. and allowed to cool down to rt. COOEt-NR-TCO-OH (E-02b, 14 mg, 67 ?mol) was dissolved in dry DCM (5 mL) and DMAP (26 mg, 214 ?mol) was added. The cooled down 7-amino-4-methylcoumarin solution was added dropwise to the TCO mixture in DCM on ice. The reaction mixture was stirred for 18 h. in the dark. The reaction mixture was concentrated in vacuo and purified with silica gel column chromatography (10%.fwdarw.60% EtOAc in n-pentane) to afford E-022b (14 mg, 42%) as an off-white solid. TLC (EtOAc/n-pentane, 1:1 v/v): R.sub.f=0.40. .sup.1H NMR (500 MHz, DMSO) ? 7.69 (d, J=8.7 Hz, 1H), 7.54 (d, J=2.1 Hz, 1H), 7.41 (dd, J=8.7, 2.1 Hz, 1H), 6.23 (d, J=1.4 Hz, 1H), 6.12-6.03 (m, 1H), 5.67 (ddd, J=16.6, 9.7, 1.3 Hz, 1H), 5.02 (td, J=9.9, 5.1 Hz, 1H), 4.01 (q, J=7.0 Hz, 2H), 2.83 (dt, J=14.4, 9.3 Hz, 1H), 2.38 (d, J=1.2 Hz, 3H), 2.32-2.24 (m, 2H), 2.20 (dt, J=12.6, 6.2 Hz, 1H), 2.08-2.00 (m, 1H), 1.56-1.47 (m, 2H), 1.17 (t, J=7.1 Hz, 3H), 1.14-1.07 (m, 1H), 0.94 (q, J=12.1 Hz, 1H). .sup.13C NMR (126 MHz, DMSO) ? 173.5, 160.0, 153.8, 153.2, 152.7, 142.8, 135.6, 131.8, 114.3, 114.2, 111.8, 104.4, 77.8, 59.9, 37.1, 34.6, 28.5, 28.0, 17.9, 14.1. HRMS (m/z): [M+Na]+ calcd. for C.sub.23H.sub.25N.sub.1O.sub.6Na: 434.1579, found 434.1578.
##STR00037##
COOEt-Exo-Equatorial-TCO-Glycine-OMe (E-023b)
[0174] To a H stirred solution of DPFPC (356 mg, 904 ?mol) and DMAP (276 mg, 2.26 mmol) in dry DMF (1 mL) was added a solution of ethyl(1R,5S,8S,9R, E)-5-hydroxybicyclo[6.1.0]non-3-ene-9-carboxylate (E-02b, 95 mg, 452 ?mol) and DIPEA (393 uL, 2.26 mmol) in DMF (1 mL). After the addition was complete, the mixture was covered from light. After overnight stirring, half of the reaction (1 mL) was transferred into a new, flame dried flask. Glycine methyl ester hydrochloride (30.2 mg, 339 ?mol) was added to this solution. After 2.5 h, 2 additional equivalents of glycine methyl ester hydrochloride and 2.5 additional equivalents of DIPEA were added. When the reaction was complete, the reaction mixture was diluted with DCM and washed with 5% NaHCO.sub.3 (5 mL), aqueous sat. NH.sub.4Cl (5 ml) and brine (5 mL). The organic layer was dried with MgSO.sub.4 and concentrated in vacuo and purified with silica gel column chromatography (30% EtOAc in heptane) to afford E-023b (20.2 mg, 42%). TLC (EtOAc/n-heptane, 1:1 v/v) R.sub.f=0.33. .sup.1H NMR (500 MHz, CDCl.sub.3) ? 5.91 (ddd, J=16.0, 9.2, 6.5 Hz, 1H), 5.67 (ddd, J=16.5, 9.6, 1.5 Hz, 1H), 5.15 (s, 1H), 4.95 (td, J=9.3, 8.7, 4.9, 1H), 4.09 (q, J=7.1, 2H), 3.97 (dd, J=5.6, 2.9, 2H), 3.76 (s, 3H), 2.91-2.81 (m, 1H), 2.40-2.28 (m, 2H), 2.22 (dt, J=12.8, 6.3, 1H), 2.16 (q, J=7.7, 1H), 1.55-1.45 (m, 1H), 1.36 (t, J=5.6, 1H), 1.26 (t, J=7.1, 3H), 1.22-1.17 (m, 1H), 0.82-0.72 (m, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) ? 174.5, 170.6, 156.0, 136.5, 131.1, 81.1, 60.6, 52.4, 42.6, 37.8, 35.0, 30.3, 29.2, 28.6, 26.5, 14.4. HRMS (m/z): [M+Na]+ calcd. for C.sub.16H.sub.23NO.sub.6Na: 348.1423, found 348.1417.
##STR00038##
COOEt-Exo-Equatorial-TCO-Sarcosine-OMe (E-024b
[0175] To a stirred solution of ethyl(1R,5S,8S,9R, E)-5-hydroxybicyclo[6.1.0]non-3-ene-9-carboxylate (E-02b, 37 mg, 177 ?mol) in dry acetonitrile (1.5 mL) at 0? C., was added DPFPC (140 mg, 355 ?mol), DIPEA (77.2 ?L, 443 ?mol), and DMAP (2.17 mg, 18 ?mol). After the addition was complete, the mixture was shielded from light and allowed to warm up to ambient temperature overnight. After completion, the reaction mixture was diluted with diethyl ether (5 mL) and washed with water (2?5 mL). The organic layer was dried with MgSO.sub.4 and concentrated in vacuo. Without further purification, the product was dissolved in dry acetonitrile (2.5 mL) and to this a solution of sarcosine methyl ester hydrochloride (49.5 mg, 355 ?mol) and DIPEA (77.2 ?L, 444 ?mol) in dry acetonitrile (2.5 mL) was added. The reaction was shielded from light. Upon completion, the reaction mixture was diluted with EtOAc (10 mL) and washed with water (2?5 mL). The organic layer was dried with MgSO.sub.4, concentrated in vacuo and purified with silica gel column chromatography (2%.fwdarw.5% acetone in toluene) yielding E-024b (15.1 mg, 25%) as an oil. TLC (acetone/toluene, 1:9 v/v) R.sub.f=0.34. .sup.1H NMR (500 MHz, CDCl.sub.3) rotamer 1: ? 5.96-5.86 (m, 1H), 5.72 (ddd, J=16.5, 9.6, 1.4, 1H), 4.96 (td, J=9.8, 5.3, 1H), 4.09 (q, J=7.3, 2H), 4.05-3.91 (m, 2H), 3.75 (s, 3H), 2.97 (s, 3H), 2.86 (dq, J=17.8, 9.1, 1H), 2.42-2.29 (m, 2H), 2.28-2.20 (m, 1H), 2.20-2.11 (m, 1H), 1.58-1.38 (m, 1H), 1.38-1.32 (m, 1H), 1.24 (t, J=7.2, 3H), 1.22-1.12 (m, 1H), 0.85-0.72 (m, 1H), rotamer 2: ? 5.96-5.86 (m, 1H), 5.63 (ddd, J=16.5, 9.7, 1.4, 1H), 4.96 (td, J=9.8, 5.3, 1H), 4.09 (q, J=7.3, 2H), 4.05-3.91 (m, 2H), 3.73 (s, 3H), 2.98 (s, 3H), 2.86 (dq, J=17.8, 9.1, 1H), 2.42-2.29 (m, 2H), 2.28-2.20 (m, 1H), 2.20-2.11 (m, 1H), 1.58-1.38 (m, 1H), 1.38-1.32 (m, 1H), 1.24 (t, J=7.2, 3H), 1.22-1.12 (m, 1H), 0.85-0.72 (m, 1H). .sup.13C NMR (126 MHz, CDCl.sub.3) rotamer 1: ? 174.5, 170.2, 155.8, 136.8, 131.6, 78.8, 60.6, 52.2, 50.6, 37.8, 35.3, 35.0, 30.3 29.3, 29.2, 28.6, 14.4, rotamer 2: ? 174.5, 170.2, 156.6, 136.6, 131.6, 79.0, 60.6, 52.2, 50.6, 37.8, 35.9, 35.0, 30.3 29.3, 29.2, 28.6, 14.4. HRMS (m/z): [M+Na]+ calcd. for C.sub.17H.sub.25NO.sub.6Na: 362.1579, found 362.1565.
##STR00039##
COOEt-Exo-Axial-TCO-PNP (E-025a
[0176] COOEt-R-TCO-OH (E-02a, 99 mg, 471 ?mol) was dissolved in dry DCM (10 mL) and pyridine (95 ?L, 1.18 mmol) was added. A solution of 4-nitrophenyl chloroformate (85 mg, 424 ?mol) in dry DCM (3 mL) was added. The reaction mixture was stirred for 4 h. before it was quenched with aqueous sat. NH.sub.4Cl (10 ml). The phases were separated and the aqueous phase was extracted with DCM (2?15 mL) and the combined organic layers were dried with MgSO.sub.4, concentrated in vacuo and purified with silica gel column chromatography (0%.fwdarw.10% EtOAc in n-pentane) to afford the Intermediate carbonate (65 mg, 70%) as an inseparable mixture of the product and starting 4-nitrophenyl chloroformate. TLC (EtOAc/n-pentane, 1:9 v/v): R.sub.f=0.27. .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.30-8.24 (m, 2H), 7.41-7.35 (m, 2H), 6.27 (ddd, J=16.9, 10.9, 5.9 Hz, 1H), 5.59 (dd, J=17.0, 3.3 Hz, 1H), 5.18 (q, J=3.0 Hz, 1H), 4.12 (qd, J=7.2, 1.6 Hz, 2H), 2.74 (dt, J=12.7, 6.2 Hz, 1H), 2.56-2.46 (m, 1H), 2.33-2.19 (m, 2H), 2.09-2.00 (m, 1H), 1.88 (t, J=13.8 Hz, 1H), 1.63-1.55 (m, 2H), 1.53-1.47 (m, 1H), 1.27 (t, J=7.1 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) ? 174.9, 155.6, 150.1, 146.0, 133.3, 128.2, 125.4, 121.8, 75.9, 60.8, 37.5, 31.4, 29.5, 26.6, 20.7, 14.4.
HRMS (m/z): [M+Na]+ calcd. for C.sub.19H.sub.21N.sub.1O.sub.7Na: 398.1215, found 398.1228. The Intermediate carbonate (6.5 mg, 17 ?mol) was dissolved in dry DMF (2 mL) and triethylamine (2.6 ?L, 19 ?mol) and Doxorubicin hydrochloride (9.0 mg, 16 ?mol) were added. The reaction mixture was stirred in the dark overnight. LCMS indicated still a significant amount of starting material so the temperature was raised to 40? C. and the reaction was stirred for another night. The reaction mixture was concentrated in vacuo and the crude product was purified using reversed-phase preparative HPLC (0.fwdarw.100% MeCN (0.1% formic acid) in MiliQ (0.1% formic acid)) and lyophilized to afford E-025a (3.8 mg, 30%). TLC (DCM/MeOH, 9:1 v/v): R.sub.f=0.07. .sup.1H NMR (500 MHz, CDCl.sub.3) ? 14.02 (s, 1H), 13.29 (s, 1H), 8.07 (d, J=7.7 Hz, 1H), 7.81 (td, J=8.1, 2.1 Hz, 1H), 7.44-7.39 (m, 1H), 6.16-5.99 (m, 1H), 5.58-5.48 (m, 2H), 5.37-5.30 (m, 1H), 5.18-5.11 (m, 1H), 5.03-4.97 (m, 1H), 4.61-4.49 (m, 1H), 4.21-4.14 (m, 2H), 4.11 (s, 3H), 4.09-4.03 (m, 1H), 3.92-3.83 (m, 1H), 3.73-3.66 (m, 1H), 3.35-3.25 (m, 1H), 3.11-3.03 (m, 1H), 2.47-2.32 (m, 2H), 2.09-2.02 (m, 2H), 1.93-1.86 (m, 2H), 1.73 (d, 7H), 1.28-1.26 (m, 6H). .sup.13C NMR (126 MHz, CDCl.sub.3) ? 214.0, 187.3, 186.9, 175.1, 161.2, 156.3, 155.8, 154.9, 135.9, 135.7, 133.7, 131.5, 129.9, 129.7, 121.0, 120.0, 118.6, 111.7, 111.6, 100.8, 71.4, 69.7, 67.4, 65.6, 60.6, 56.8, 50.9, 47.0, 46.0, 37.5, 35.8, 34.9, 32.0, 31.0, 29.8, 26.9, 22.8, 21.0, 17.0, 14.2. HRMS (m/z): [M+Na]+ calcd. for C.sub.40H.sub.45N.sub.1O.sub.15Na: 802.2686, found 802.2710.
##STR00040##
Synthesis of (1S,5R,8R,9S, E)-5-hydroxybicyclo[6.1.0]non-3-ene-9-carboxylic acid (E-04a
[0177] To a stirred solution of E-02a (500 mg, 1 equiv, 2.40 mmol) in THF (8 mL) and water (4 ml) was added LiOH (172 mg, 3.0 equiv, 7.20 mmol). The mixture was stirred at 50? C. and covered with aluminum foil. After completion was seen on TLC, EtOAc (5 ml) and 1M aqueous HCl (5 ml) were added to the mixture. The organic layer was washed with brine (5 ml) and both aqueous layers were back-extracted with EtOAc (2?5 ml). The combined organic layers were dried over MgSO.sub.4 and concentrated to afford the crude product in 376 mg (87% of theory) yield. .sup.1H NMR (400 MHz, D.sub.2O) ? 6.18 (ddd, J=16.9, 10.8, 5.8 Hz, 1H), 5.63 (dd, J=17.1, 3.4 Hz, 1H), 4.35 (q, J=3.0 Hz, 1H), 2.66 (dt, J=12.8, 6.4 Hz, 1H), 2.43 (t, J=11.8 Hz, 1H), 2.14 (dd, J=15.3, 11.4 Hz, 1H), 1.90-1.74 (m, 3H), 1.44 (t, J=5.9 Hz, 1H), 1.38-1.30 (m, 1H), 1.28-1.21 (m, 1H). .sup.13C NMR (101 MHz, D.sub.2O) ? 186.83, 134.80, 133.14, 70.48, 41.13, 32.10, 31.07, 27.68, 25.40, 21.35. RF (methanol/dichloromethane 1-9) 0.23.
##STR00041##
Synthesis of (1S,5R,8R,9S, E)-5-hydroxybicyclo[6.1.0]non-3-ene-9-carboxylic acid (E-04b
[0178] To a stirred solution of E-02b (500 mg, 1 equiv, 2.40 mmol) in THF (8 mL) and water (4 ml was added LiOH (172 mg, 3.0 equiv, 7.20 mmol). The mixture was stirred at 50? C. and covered with aluminum foil. After completion was seen on TLC, EtOAc (5 ml) and 1M aqueous HCl (5 ml) were added to the mixture. The organic layer was washed with brine (5 ml) and both aqueous layers were back-extracted with EtOAc (2?5 ml). The combined organic layers were dried over MgSO.sub.4 and concentrated to afford the crude product in 370 mg (85% of theory) yield. .sup.1H NMR (400 MHz, D.sub.2O) ? 5.93 (ddd, J=15.9, 9.0, 6.4 Hz, 1H), 5.63 (ddd, J=16.5, 9.5, 1.4 Hz, 1H), 4.09 (td, J=9.7, 5.3 Hz, 1H), 2.86-2.69 (m, 1H), 2.30-2.17 (m, 2H), 2.06 (ddd, J=12.8, 6.9, 5.4 Hz, 1H), 2.00-1.86 (m, 1H), 1.39 (dt, J=12.9, 10.6 Hz, 1H), 1.18 (t, J=5.7 Hz, 1H), 0.95 (ddt, J=11.3, 8.9, 4.9 Hz, 1H), 0.70 (dt, J=15.5, 11.2 Hz, 1H). .sup.13C NMR (101 MHz, D.sub.2O) ? 184.19, 138.61, 131.78, 75.51, 37.57, 36.76, 31.87, 29.93, 28.18, 28.14. RF (methanol/dichloromethane 1:9) 0.20.
##STR00042##
Synthesis of (1R,4R,8S,9S, E)-9-(hydroxymethyl)bicyclo[6.1.0]non-5-en-4-ol (E-05a
[0179] To a suspension of lithium aluminum hydride (14.2 mg, 2.4 equiv, 0.38 mmol) in dry diethyl ether (750 ?l), was added a solution of E-02a (40 mg, 2 equiv, 0.16 mmol) in dry diethyl ether (750 ?l) at 0? C. The mixture was stirred at ambient temperature for one hour. After completion was seen on L, an aqueous solution of 1.0 M HCl (800 ?l) was dropwise added to reaction mixture. Extra diethyl ether (5 ml) and aqueous 1.0 M HCl solution (5 ml) were added to the reaction mixture. The organic layer was collected and washed with brine (5 ml). The aqueous layers were back extracted with diethyl ether (2?5 ml). The combined organic layers were dried over MgSO.sub.4 and concentrated to afford the desired product in 26 mg yield. The crude was purified over silica (8 g) with 10% MeOH in DCM to afford a clear oil in 20 mg (62% of theory) yield. .sup.1H NMR (400 MHz, CDCl.sub.3) ? 6.27 (ddd, J=16.7, 10.8, 5.9 Hz, 1H), 5.54 (dd, J=16.8, 3.2 Hz, 1H), 4.35 (q, J=3.1 Hz, 1H), 3.52-3.43 (m, 2H), 2.65 (dt, J=12.8, 6.5 Hz, 1H), 2.40 (t, J=11.6 Hz, 1H), 2.25 (dd, J=16.3, 11.8 Hz, 1H), 1.99-1.88 (m, 1H), 1.88-1.76 (m, 1H), 1.77-1.66 (m, 1H), 1.04 (p, J=6.4 Hz, 1H), 0.81 (dd, J=14.7, 7.1 Hz, 1H), 0.65 (td, J=8.8, 6.2 Hz, 1H). .sup.13C (NMR 101 MHz, CDCl.sub.3) ? 132.92, 131.28, 68.68, 67.72, 39.90, 29.70, 25.98, 22.41, 22.40, 19.88. RF (methanol/dichloromethane 1:9) 0.22.
Example 3Stability Test
[0180] An NMR tube was charged with a solution of E-04a (41 mg) in deuterated PBS buffer (500 ?l). The NMR sample was placed in a water bath at 37? C. while being covered from light by aluminum foil. The sample showed no changes in the .sup.1H-NMR spectrum after 7 days.
Example 4Kinetics Study of Click Reaction
[0181] The reaction rate in a TCO-Tz click reaction of E-02a was measured under similar conditions as described in Versteegen et al., Angewandte Chemie International Edition 2013, 52 (52), 14112-14116. The second order reaction constant of the reaction between 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine and E-02a was determined under second order conditions in MeCN at 20? C. by UV spectroscopy. A cuvette was filled with MeCN (2.8 mL) and equilibrated at 20? C. A stock solution of tetrazine in DMSO (100 ?l) was added to the cuvette. The absorption of the tetrazine moiety was measured at 540 nm. Next, the cuvette was removed from the apparatus to add a stock solution of E-02a in DMSO (100 ?l) and briefly mixed the solution by shaking the cuvette before placing it back into the apparatus. The absorption at 540 nm was measured for 15 minutes. From this absorption at 540 nm, the concentration of tetrazine was calculated using a molar absorption coefficient of ?=430 M.sup.?1 cm.sup.?1. The second order rate constant k2 was obtained from the slope of a plot of (1/c?1/c0) versus time. The calculated reaction rates at a concentration of 10 ?M and 50 ?M were respectively 249 M.sup.?1s.sup.?1 and 437 M.sup.?1s.sup.?1.
Example 5Kinetics Study of Release Reaction
[0182] The .sup.19F NMR (377 MHz) spectrum of an NMR-tube filled with a solution of E-03a (5 mg, 1.0 equiv, 11.9 ?mol) in CDCl.sub.3 (500 ?l) was measured. Next, a solution of 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine (3.1 mg, 1.1 equiv, 13 ?mol) in CDCl.sub.3 (100 ?l) was added to the NMR tube. The tube was vigorously shaken and placed back in the NMR apparatus. The sample was measured within two minutes after the addition of the second compound. Every 48 seconds a measurement (number of scans: 4, relaxation delay: 5.8 see, range: ?147.0 to ?178.5 ppm) was taken of the sample. After 160 minutes the measurements were stopped. Almost complete release of PFP was seen after 20 minutes.
Further investigation of the E-03a click-to-release reaction at lower temperatures showed that the release was instantaneous upon tautomerization from the click product. .sup.19F-NMR kinetic study of the click reaction of 40 mM with dipyridyl tetrazine was performed at controlled temperatures. At ?20? C. the click conjugate of the compound of the invention and dipyridyl tetrazine could be observed and did not show any formation of pentafluorophenol. Only after elevating the temperature to +10? C., the pentafluorophenyl carbonate was released from the conjugated product.
Release of payload from compounds of the invention was not only found to be fast, but also complete. Versteegen et al. (Angewandte Chemie International Edition 2018, 57 (33), 10494-10499 DOI: 10.1002/ange.201800402) tested various payloads on the allylic position of TCO compounds known from the prior art and observed only a maximum of 60% release under conditions that were comparable to those used in our .sup.19F-NMR studies. The fastest payload release observed with their compounds afforded less than 20% release over 250 minutes. Compounds of the present invention showed near complete release within 20 minutes.
Example 6Kinetics Study of Fluorophore Release
[0183] A procedure adapted from prior art was followed (Fan, X. et al., Optimized tetrazine derivatives for rapid bioorthogonal decaging in living cells. Angew. Chem. 2016, 128 (45), 14252-14256.)
##STR00043##
Stock solutions (all in DMSO) [0184] Stock A (R): E-022a (10 mmolar) [0185] Stock A (NR): E-022b (10 mmolar) [0186] Stock B (DM): 3,6-Dimethyl-1,2,4,5-tetrazine (DM) (20 mmolar) [0187] Stock B (BP): 3,6-Di-2-pyridyl-1,2,4,5-tetrazine (BP) (20 mmolar) [0188] Stock PC: 7-Amino-4-methylcoumarin (10 mmolar)
2.5 ?L of the tetrazine stock (DM or BP) was mixed with 395.5 ?L of PBS (pH=7.2). 2 ?L of the TCO stock (A (R) or A (NR)) was added to this. The reaction mixtures were incubated at 37? C. under mixing (300 rpm). Subsequently, the fluorescence (F1) was measured (ex: 380 nm, em. 450 nm) at certain time points after the addition of the TCO by aliquoting 30 ?L in a 384 wells plate (Greiner, black non-binding flat bottom) and directly measuring with a Tecan Spark plate-reader. As control experiment the maximum amount of released AMC was measured (F2, Stock PC without tetrazine or TCO). Negative control experiments revealed no significant fluorescent signal for either exclusively the tetrazine or the TCO's. The release efficiency (%) was calculated: F1/F2?100%. All experiments were executed in triplo and the data was processed using GraphPad Prism (version 9.0). The K.sub.elim. values were determined by first-order exponential one phase decay approximations. E-022a with DM displayed a K.sub.elim. value of 8.15*10.sup.?5S.sup.?1. For BP this value was 2.96*10.sup.?6S.sup.?1. As for example 5, release was found to be slower for the E-022b analogue.
Example 7Kinetics Study of Doxorubicin Release
[0189] ##STR00044##
Stock solutions (all in DMSO) [0190] Stock E-025a: E-025a (10 mmolar) [0191] Stock Ttz: 3,6-dimethyl-1,2,4,5-tetrazine (XA, 20 mmolar)
Procedure: 100 ?L of the TCO-Dox stock was added to 900 ?L of PBS (pH=7.2) and HPLC analysis was done which only indicated starting material (t=13.08 min). Hereafter the reaction was incubated at 37? C. under mixing (300 rpm) and 500 ?L of the Ttz stock was added. At certain time points after the addition of the Ttz the reaction mixture was analyzed by aliquoting 200 ?L and analysis by HPLC/LCMS. Directly after the addition of the tetrazine (t=6.11 min) almost instant release of doxorubicin (t=7.57 min) and formation of the elimination product (t=7.19 min) was observed.