Cover Page
The handle http://hdl.handle.net/1887/123227 holds various files of this Leiden University dissertation.
Author: Maurits, E.
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Oligopeptide-masked
doxorubicins
for
in
situ
immunoproteasome-mediated activation
IntroductionProteasome inhibitors are used in the clinic to kill rapidly proliferating cells that express a high content of misfolded proteins (for instance, cancerous cells characteristic for multiple myeloma or acute myeloid leukemia).[1] The currently clinically applied proteasome inhibitor (PI) carfilzomib (1) does not completely halt the proliferation, and as well drug resistance has emerged over the years.[2]
To overcome PI resistance a conjugate that combines apoptotic inducers with proteasome targeting scaffolds was developed. The design of drug release through the action of proteasome catalytic sites was inspired by anti-body drug conjugates (ADCs) in which the cytotoxic payload (e.g. 2, doxorubicin) is released through the action of another protease, namely, cathepsin B.[3] In ADCs, the oligopeptide portion connecting the antibody with the drug functions as a cleavable linker, with the antibody driving selectivity towards tumor tissue. In the here-presented proteasome targeting-drug conjugates, selectivity is hypothesized to be achieved through targeting of immunoproteasome expressing cells. For this purpose oligopeptide sequences found in earlier reported immunoproteasome selective fluorogenic substrates (3, LU-FS05i; 4, LU-FS01i) and inhibitors (5[4]; 6, LU-035i[5,6]) were selected.
Figure 1. Structures of clinically used PIs, doxorubicin and previously developed immunoproteasome selective fluorogenic
substrates and inhibitors.
The research described in Chapter 2 revealed that fluorogenic substrates can be designed that are highly selectively processed by proteasomes (and not by other proteases), and that also may be selectively processed by individual proteasome subunits. In general, healthy tissue contains mostly constitutive proteasome core particles (cCP), while certain cancerous blood cells almost exclusively express immunoproteasome core particles (iCP).[7] Recently reported iCP inhibitors have been developed with the aim to take advantage of this altered iCP/cCP ratio with the ultimate goal to arrive at more effective and less toxic clinical drugs.[8] Based on these results as well as related studies[8,9] pointing towards
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proteasome substrate-doxorubicin conjugates. As a toxic payload, doxorubicin (2) was chosen. Doxorubicin is a clinically used chemotherapeutic agent with high toxicity towards all mammalian cells.[10] The free amine within 2 is pivotal for its induction of apoptosis through DNA base pair
intercalation.[11] Acylation of this amine delivers non-toxic and inactive compounds.[12] Direct acylation
with the oligopeptide inherent to LU-FS05i (3) would yield conjugate 7 (Figure 2A). p-Aminobenzyl alcohol in turn is a commonly used spacer in anti-body drug conjugates that undergoes self immolative cleavage upon liberation of its amine.[13] Implementing this linker in the prodrug design provides conjugates 8 and 9 as β5i and β1i proteasome subunit targeting conjugates respectively.
Figure 2. Proteasome-targeting conjugates subject of studies described in this Chapter. A) Substrate-based conjugates targeting
β1i and β5i proteasome subunits. B) Sulfonyl-based cleavable proteasome inhibitors. C) Epoxyketone (EK) warhead based cleavable FRET-pair.
Next to substrate-based constructs 7-9, covalent inhibitor-based conjugates 10-14 were also envisioned (Figure 2B) as putative proteasome-targeting prodrug conjugates. Proteasome inhibition would obviate conjugate cleavage by the catalytic site, unless the inhibition mechanism would result in release of the toxic agent. Recently it was shown that oligopeptides featuring a methyl aryl sulfonate, such as 5, are potent, covalent and irreversible proteasome inhibitors that release, upon reacting within the proteasome active sites, the phenolate group (coumarin). Thus, inhibition is accompanied with release of a payload, and it was hypothesized that the phenolate moiety could be replaced by a doxorubicin conjugate, connected in an appropriate manner to the sulfonate.[4,14,15] Substitution of the coumarin
p-43
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aminobenzyloxycarbonyl (PABC) linker was used for construct 10,[17]whereas use of its tetrafluorinated
analogue (shown to be a better leaving group when compared to the tetrahdyro derivative) let to the design of conjugate 11.[18] Dubiella et al. have shown that a fluorinated phenol increases the potency of
the sulfonate ester by a 1000-fold compared to the non-fluorinated congeners.[4] Therefore, fluorinated
analogues 12 and 13 were also envisioned as potential covalent oligopeptide-masked doxorubicins. In an alternative strategy, an epoxyketone (EK) warhead, as present in the clinically used PI, carfilzomib (1) as well as immunoproteasome-selective PIs (for instance, 6), was equiped with a potential proteasome cleavable linker. Figure 3 displays a putative mechanism, taking in account the molecular mechanism by means of which proteasome N-terminal threonines react with an unsubstituted epoxyketone, and how this might result in payload release. Route A displays the original thinking on how epoxyketones react within proteasome active sites[19], with the covalent adduct formed as a morpholine.
Route B depicts how the more recently suggested oxazepane would be formed.[20] In both routes, opening up the epoxide yields an alcohol that may displace the phenolate, leading after formation of the quinone and CO2 the payload, doxorubicin.
Figure 3. A) Proposed release of the payload based on morpholine formation. B) Proposed release mechanism following
proteasome inhibition in case an oxazepane is formed in the first step.[20]
To evaluate whether the hydroxyl group that is freed after an attack by the proteasomal β subunit is capable of initiating elimination of the cleavable linker, a fluorophore/quencher FRET (fluorescence resonance energy transfer) pair was utilized (14, Figure 2). The fluorophore is attached to one side of the EK, while the cleavable linker and quencher are attached to the other side. Elimination of the cleavable linker will release the quencher, with (enhanced) fluorescence as the result.[21]
Results
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yield compound 21. Then, the primary alcohol was reacted with nitrophenyl chloroformate to obtain activated carbonate 22, which was subsequently transformed into conjugate 8 upon reaction with doxorubicin. Conjugate 9 was made in a similar fashion as conjugate 7 (Figure 2A).
Scheme 1. Structures and synthesis of conjugates 6 and 7. Reagents and conditions: a) SOCl2, MeOH, 0°C; b) TFA, DCM, quant.
over two steps; c) Fmoc-Trp(Boc)-OH, HCTU, DiPEA, DCM; d) NaN3, DMF, 50°C, 50% over two steps; e) Fmoc-D-Ala-OH, HCTU,
DiPEA, DCM, 51%; f) NaN3, DMF, 50°C, 76%; g) 3-methyl-1H-indene-2-carboxylic acid, HCTU, DiPEA, DCM, 64%; h) LiOH, THF,
H2O; i) HOSu-OH, DCC, THF, 57% over two steps; j) TFA, DCM, 37%; k) 2, DiPEA, DMF, 13%; l)
4-(((tert-butyldimethylsilyl)oxy)methyl)aniline, DMAP, Et3N, DCM; m) TFA, DCM, 74% over two steps; n) Nitrophenyl chloroformate, Pyr,
THF, quant.; o) 2, DiPEA, DMF, 30%.
Cell death assay of toxic proteasome substrates
As the next objective, the cytotoxicity of conjugates 7-9 was investigated. In order to test their susceptibility to proteolytic processing a LC-MS based assay was conducted (Figure 4A). Raji cell lysate (a cell line derived from tissue from a patient suffering from Burkitt′s lymphoma) was incubated with the conjugates as well as with LLVY-AMC, a commonly used β5 fluorogenic substrate.[22,23] After incubation
for 16 h the lysate was extracted with EtOAc, and the extract was compared with the non-lysate containing samples by LC-MS analysis. Hydrolysis of LLVY-AMC, 7 and 8 was observed while conjugate 9 did not show significant decomposition. As the proteasomes binding sites differ for each of the catalytic subunits, the β5i subunit could hypothetically tolerate the rigid anthracycline moiety of doxorubicin as present in 8 whereas the β1i subunit would not do so with respect to construct 9.
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Figure 4. Biological evaluation of conjugates 7, 8 and 9. A) Substrate hydrolysis as measured by LC-MS after exposure of 10 µM
conjugate to Raji cell lysate for 16 h. LLVY-AMC is used as a control compound. B) Cell survival of AMO-1 cells following treatment with conjugate 7 compared to doxorubicin. C) Cell survival of AMO-1 cells following treatment with conjugate 8, with and without pre-incubation of the β5i proteasome subunit, compared to doxorubicin. D) Cell survival of HEK cells following treatment with conjugate 8 compared to doxorubicin. E) Cell survival of AMO-1 cells following treatment with conjugate 9, with and without pre-incubation of the β1i proteasome subunit, compared to doxorubicin. Conditions: cell survival was measured by the Alamar Blue mitochondrial dye conversion assay after 3 days of incubation in a 5% CO2 humidified incubator. β1i and β5i
inhibition was obtained by pre-incubation with 1 µM LU-001i or LU-035i, respectively. SD: N=3, n=3
To further investigate this, human embryonic kidney (HEK) cells that do not express iCPs were tested on cell viability (Figure 4D).[24] Conjugate 8 was still able to induce apoptosis, again suggesting that other
proteases liberate doxorubicin from the construct. Finally, the cytotoxicity of conjugate 9 was evaluated (Figure 4E). Additional experiments were performed to determine cell survival with and without β1i pre-inhibition. This time, no significant decrease in cell viability was detected and even at elevated concentrations the cells were still viable. These results are in agreement with the slow hydrolysis, as inferred by LC-MS experiments (Figure 4A), and also confirm that N-acylated doxorubicins are much less toxic than the unmodified anthracycline itself.
Synthesis of sulfonyl-based inhibitors
The synthesis of peptide sulfonyl esters 30a-d and doxorubicin conjugate 10 is depicted in Scheme 2 and commenced with the construction of the sulfonyl ester via a modified Dubiella procedure.[4,14]
Cyclohexyl-L-alaninol 23 was N-Cbz protected followed by substitution of the alcohol with thioacetic acid via the mesylate, yielding 25. The sulfide was readily oxidized into sulfonyl chloride 26 with NCS, and can be reacted with a (protected) self-immolative linker of choice to form 27a-d. The tetrafluorophenol linkers proved however prone to hydrolysis due to their electron withdrawing character, requiring anhydrous conditions for the synthesis of 27c-d. Deprotection of both the TBS and Cbz groups in 27a was achieved by stirring in HBr/AcOH. However, when using these conditions to deprotect 27b-d,
C e ll S u rv iv a l, % C e ll S u rv iv a l, % C e ll S u rv iv a l, % C e ll S u rv iv a l, %
A
B
C
D
E
LLVY-AMC 7 8 9 0 50 10046
3
degradation was observed. Therefore, the Cbz group was first removed under hydrogenating conditions to yield 28b-d as TBS protected linked warhead. Then, the oligopeptidic backbone was attached via an acyl azide coupling with the free amine of the warhead moiety. Standard acyl azide coupling[25] led to
hydrolysis of the sulfonyl ester, but performing the amide bond formation under anhydrous conditions gave better results. Subsequent deprotection and reaction of the primary alcohol in 30a with nitrophenyl chloroformate obtained the activated carbonate 31a, in a sequence of events that was based on the literature procedure for doxorubicin-carbonate formation in anti-body drug conjugate synthesis.[12] Upon
condensation of 31a with the amine in doxorubicin, conjugate 10 was obtained.
a b, c e f i CbzHN SCl O O + H2N OH CbzHN OH CbzHN SR 1 O O OTBS N O N H O H N N H O O S R1 O O OH O O N O N H O H N N H O O S R1 O O O O O NO2 10 O N O N H O H N N H O NH2 O H2N SR1 O O OTBS 23 24 25 26 27a-d 28a-d 30a-d 31a d CbzHN S O 29 j g, h R2 R2 R2 R2 a, R1 = -O- R2 = 4 x H b, R1 = -NH- R2 = 4 x H c, R1 = -O- R2 = 4 x F d, R1= -NH- R2= 4 x F
Scheme 2. Synthesis of conjugate 10. Reagents and conditions: a) CbzCl, NaHCO3, dioxane, H2O, quant.; b) MsCl, DiPEA, DCM; c)
thioacetic acid, K2CO3, DMF, 80% over two steps; d) NCS, MeCN, 0oC, quant.; e) 4-(((tert-butyldimethylsilyl)oxy)methyl)aniline or
4-(((tert-butyldimethylsilyl)oxy)methyl)phenol, Et3N, THF, 72%; f) Pd/C, MeOH, H2; g) (i) tBuONO, HCl, DMF, -35°C; (ii) 28, DiPEA,
-35°C to RT, 50% over two steps; h) TFA, DCM; i) nitrophenol chloroformate, pyridine, THF, quant.; j) 2, DiPEA, DMF, 16%
Figure 5. Inhibition profiles of compounds 30a-d, as determined in Raji cell lysate. Cell lysates were treated with the indicated
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Having sulfonyl esters/amides 30a-d in hand, their ability to inhibit proteasome activities was first assessed using a competitive ABPP assay.
As expected, LU-005i gave completed inhibition of the β5i subunit at 0.3 µM. In contrast, conjugates 30a,
b and d did not inhibit any of the proteasome active sites even at high concentrations. Compound 30c
inhibited β5i to some extend at 10 µM final concentration, but nowhere near as potent as the lead compound (LU-005i) and no further experiments were conducted with this series of compounds.
Synthesis of epoxyketone conjugates
The synthesis of the epoxyketone-based quenched fluorophore 14, necessitates the construction of epoxyketone 41 (Scheme 3) suitable for elongation at both the cyclohexylalanine amine site and the primary hydroxyl moiety. The synthesis of 41 commenced with one-carbon elongation of 2,3-dibromopropene followed by triflation to yield 33 (Scheme 3).[28] The triflate in 33 was displaced with trityl protected 4-hydroxybenzyl alcohol 35 to yield 36. Carbolithiation of bromide 36 was effected by treatment with tBuLi, followed by addition of Weinreb ketone 37[5] to furnish enone 38. Acidic work-up
was carefully controlled in order to keep the trityl intact. Luche reduction of enone 38 afforded allylic alcohol 39, which facilitated a diastereoselective epoxidation and subsequent oxidation to furnish epoxyketone 40. Deprotection of both Trt and Boc groups under acidic conditions yielded 41.
Scheme 3. Synthesis of 41. Reagents and conditions: a) Formaldehyde, SnCl2·2H2O, KI, H2O, 88%; b) Tf2O, Pyr, DCM; c) Trt-Cl, pyr,
quant. over 2 steps; d) NaH, THF, 69%; e) t-BuLi, THF, 55%; f) CeCl3·7 H2O, NaBH4, MeOH, 71%; g) VO(acac)2, t-BuOOH, DCM; h)
DMP, DCM, 30% over 2 steps; i) TFA, DCM, 86%.
The synthesis of conjugate 14 commenced with the preparation of oligopeptide 46 (Scheme 4).[5] The
carboxylic acid and amine of tyrosine were first protected to obtain 43. Subsequently, the phenolic alcohol was equipped with an alkyne by substitution with propargyl bromide and then N-Boc deprotected to furnish 44. Standard HCTU based peptide bond formation with D-Ala and then the methylindene moiety yielded capped dipeptide 46, which was next transformed into hydrazide 47. Formation of compound 50 was achieved by conjugation of 47 with H2N-Cha-EK 48 followed by a
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Scheme 4. Synthesis of FRET-pair PI 14. Reagents and conditions: a) SOCl2, MeOH, quant; b) Boc2O, NaHCO3, dioxane, 93%; c)
Propargyl bromide, K2CO3, DMF, 96%; d) TFA, DCM, 98%; e) Boc-D-Ala-OH, HCTU, DIPEA, DCM, quant.; f) TFA, DCM, quant.; g)
3-Methylindene-2-carboxylic acid, HCTU, DIPEA, DCM, 66%; h) N2H4·H2O, MeOH; i) (i) tBuONO, HCl, DMF, 35°C; (ii) 48, DiPEA,
-35°C-rt, 83% over 2 steps; j) 52, CuSO4, sodium ascorbate, DMF, 74%; k) (i) tBuONO, HCl, DMF, -35°C; (ii) 41, DiPEA, -35°C-rt,
31%; l) nitrophenol chloroformate, pyr, THF, 88%; m) 53, DIPEA, DMF, 63%; n) 55, CuSO4, sodium ascorbate, DMF, 36%. Biological evaluation of peptide epoxyketone conjugate 14
In a first study to establish proteasome inhibitory potency, compound 14 was compared to compound 50 and intermediates 51, 52 and 53 in a competitive ABPP experiment (Figure 6A). EDANS-modified LU-035i analogue 50 showed complete inhibition of the β5i subunit at 0.01 µM (in comparison, the IC50 of
LU-035i is 0.0083 µM)[5], which confirms that P2 modification is tolerated. A drastic decrease in potency as
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Figure 6. Biological evaluation of peptide epoxyketone conjugate 14. A) Inhibition profiles of compounds 50-54 and 14,
determined in Raji cell lysate. Cell lysates were treated with the distinct compounds at indicated concentrations for 1 h followed by labeling with ABP cocktail. The upper red band indicates the targeted β5i subunit. B) Incubation of FRET-pair 14 in Raji lysate to induce proteasomal cleavage. Relative fluorescence of positive control compound 50 = 5797 ± 117. Shown are background-corrected 490 nm fluorescent emissions resulting from excitation at 340 nm (n = 3).
Conclusion
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Experimental
Synthetic procedures
All reagents were of commercial grade and used as received unless stated otherwise. Solvents used in synthesis were dried and stored over 4Å molecular sieves, except MeOH and ACN which were stored over 3Å molecular sieves. Triethylamine (Et3N) and Di-isopropylethylamine (DiPEA) were stored over
KOH pellets.
Column chromatography was performed on silica gel 60 Å (40-63 µm, Macherey-Nagel). TLC analysis was
performed on Macherey-Nagel aluminium sheets (silica gel 60 F254).
TLC was used to monitor reactions with visualization by UV at wavelength 254 nm and by one of
following treatments: spraying with either cerium molybdate spray (25 g/L (NH4)6Mo7O24, 10 g/L
(NH4)4Ce(SO4)4·H2O in 10% H2SO4 water solution) or KMnO4 spray (20 g/L KMnO4 and 10 g/L K2CO3 in
water) followed by charring at c.a. 250°C. TLC-MS analysis was performed on a Advion ExpressionL EMS
in combination with a Plate Express interface (eluted with MeOH/H2O 90%/10% v/v + 0.1% formic acid,
flow rate 0.20 mL/min).
LC-MS analysis was performed on a Finnigan Surveyor HPLC system with a Nucleodur C18 Gravity 3um 50
x 4.60 mm column (detection at 200-600 nm) coupled to a Finnigan LCQ Advantage Max mass spectrometer with ESI or coupled to a Thermo LCQ Fleet Ion mass spectrometer with ESI. The Method used was 10→90% 13.5 min (0→0.5 min: 10% MeCN; 0.5→8.5 min: 10% to 90% MeCN; 8.5→ 11 min: 90% MeCN; 11→13.5 min: 10% MeCN).
High-resolution mass spectrometry (HRMS) was performed on a Thermo Scientific Q Exactive HF
Orbitrap mass spectrometer equipped with an electrospray ion source in positive-ion mode (source voltage 3.5 kV, sheath gas flow 10, capillary temperature 275°C) with resolution R = 240.000 at m/z 400 (mass range of 150-6000) correlated to an external calibration, or on a Waters Synapt G2-Si (TOF) equipped with an electrospray ion source in positive mode (source voltage 3.5 kV) and LeuEnk (m/z = 556.2771) as internal lock mass.
1
H and 13C NMR spectra were recorded on a Bruker AV-400 NMR, a Bruker DMX-400 NMR instrument
(400 and 101 MHz respectively), a Bruker AV-500 NMR instrument (500 and 126 MHz respectively), and Bruker AV-600 NMR instrument (600 and 151 MHz respectively). Chemical shifts (δ) are given in ppm relative to tetramethylsilane as internal standard or the residual signal of the deuterated solvent. Coupling constants (J) are given in Hz. All given 13C-APT spectra are proton decoupled. Assignment of
NMR spectra was based on 1H-COSY and 1H-13C-HSQC.
HPLC purification was performed on a Gilson HPLC system coupled to a Magerey-Nagel Nucleodur C18
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Cytotoxic substrate synthesis
H2N-Cha-OMe (16). Boc-Cha-OH (1.89 g, 7.00 mmol) was dissolved MeOH (24 mL) and cooled to 0°C. Thionyl chloride (0.71 mL, 9.8 mmol) was slowly added over 10 min, followed by stirring overnight at RT. The solvent was evaporated and co-evaporated with toluene (3x). The crude methyl ester was dissolved in DCM (3 mL) and TFA (3 mL) and was stirred for 30 min. Evaporation of the solvent and co-evaporation with toluene (3x) and CHCl3 (2x) yielded the title compound 16 (2.1 g, 7.0 mmol, quant.) 1H NMR (400 MHz, CDCl3) δ 8.22
(s, 2H), 4.13 (m, 1H), 3.81 (s, 3H), 1.82 (d, J = 6.4 Hz, 2H), 1.73 (d, J = 13.0 Hz, 3H), 1.66 (d, J = 15.4 Hz, 2H), 1.31 – 1.18 (m, 3H), 1.14 (q, J = 12.1 Hz, 1H), 0.93 (p, J = 11.5 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ
170.83, 53.54, 51.44, 38.14, 33.58, 33.02, 32.54, 26.36, 25.96, 25.73. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 6.29 (ESI-MS (m/z): 186.14 (M+H+)).
H2N-Trp(Boc)-Cha-OMe (17). H2N-Cha-OMe (2.1 g, 7 mmol), Fmoc-Trp(Boc)-OH
(4.42 g, 8.4 mmol) and HCTU (3.48 g, 8.4 mmol) were dissolved in DCM (70 mL), followed by addition of DiPEA (4.8 mL, 28 mmol). The reaction mixture was then stirred overnight at RT. The reaction mixture was concentrated in vacuo and dissolved in EtOAc and washed with 1 M HCl, saturated NaHCO3 (2x) and brine. The
organic layer was dried over MgSO4, filtered and concentrated in vacuo. The crude
protected dipeptide was dissolved in DMF (30 mL) and NaN3 (0.55 g, 8.4 mmol) was
added. The mixture was then stirred overnight at 50°C followed by evaporation of the solvent and co-evaporation with n-heptane (5x). The crude product was purified via flash column chromatography on silica gel (EtOAC in Pentane – 0% to 100% v/v) to yield the title compound (1.63 g, 3.5 mmol, 50%). 1H
NMR (400 MHz, CDCl3) δ 8.13 (d, J = 8.1 Hz, 1H), 7.99 (d, J = 3.1 Hz, 1H), 7.70 – 7.50 (m, 1H), 7.32 (t, J =
7.6 Hz, 1H), 7.25 – 7.14 (m, 1H), 4.54 (td, J = 8.7, 5.5 Hz, 1H), 4.04 (t, J = 6.8 Hz, 1H), 3.69 (s, 1H), 3.36 (dd, J = 14.7, 5.4 Hz, 1H), 2.95 (d, J = 1.1 Hz, 3H), 2.17 (d, J = 0.6 Hz, 4H), 1.79 – 1.54 (m, 10H), 1.37 – 1.01 (m, 2H), 0.98 – 0.72 (m, 1H).13C NMR (101 MHz, CDCl3) δ 149.7, 129.2, 128.4, 127.9, 125.4, 125.1, 124.9, 123.0, 120.1, 119.1, 115.5, 84.1, 77.5, 77.2, 76.8, 54.4, 52.5, 50.5, 39.8, 38.7, 36.7, 34.1, 33.4, 32.5, 31.6, 31.1, 29.2, 28.3, 26.4, 26.1, 26.0. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt
(min): 6.92 (ESI-MS (m/z): 472.20 (M+H+)).
H2N-D-Ala-Trp(Boc)-Cha-OMe (18). H2N-Trp(Boc)-Cha-OMe (1.63 g, 3.5
mmol), Fmoc-D-Ala-OH (1.30 g, 4.15 mmol) and HCTU (1.72 g, 4.15 mmol) were dissolved in DCM (35 mL), followed by addition of DiPEA (2.4 mL, 14 mmol). The reaction mixture was then stirred overnight at RT. The reaction mixture was concentrated in vacuo and dissolved in EtOAc and washed with 1 M HCl, saturated NaHCO3 (2x) and brine. The organic layer was dried over
MgSO4, filtered and concentrated in vacuo. The crude protected tripeptide
was dissolved in DMF (15 mL) and NaN3 (0.23 g, 4.2 mmol) was added. The mixture was then stirred
overnight at 50°C followed by evaporation of the solvent and co-evaporation with n-heptane (5x). The crude product was purified via flash column chromatography on silica gel (MeOH in DCM – 0% to 4% v/v) to yield the title compound (1.44 g, 2.66 mmol, 76%). 1H NMR (400 MHz, CDCl
3) δ 8.23 – 8.05 (m, 1H),
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39.9, 38.8, 34.1, 33.4, 32.5, 28.3, 27.4, 26.4, 26.2, 26.1, 0.1. LC-MS (linear gradient 10 → 90% MeCN/H2O,
0.1% TFA, 12.5 min): Rt (min): 6.94 (ESI-MS (m/z): 543.20 (M+H+)).
MophAc-D-Ala-Trp(Boc)-Cha-OMe (19). H2
N-D-Ala-Trp(Boc)-Cha-OMe (0.747 g, 1.38 mmol), 1-methyl-3H-idene-2-carboxylic acid (0.29 g, 1.66 mmol) and HCTU (0.69 g, 1.66 mmol) were dissolved in DCM (35 mL), followed by addition of DiPEA (0.93 mL, 5.5 mmol). The reaction mixture was then stirred overnight at RT. The reaction mixture was concentrated in vacuo and dissolved in EtOAc and washed with 1 M HCl, saturated NaHCO3
(2x) and brine. The organic layer was dried over MgSO4, filtered
and concentrated in vacuo. The crude product was purified via flash column chromatography on silica gel (MeOH in DCM – 0% to 1% v/v) to yield the title compound (0.625 g, 0.89 mmol, 64%). 1H NMR (400 MHz, CDCl3) δ 8.10 (d, J = 8.3 Hz, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.52 (s, 1H), 7.47 – 7.41 (m, 2H), 7.33 (tdd, J = 15.0, 7.4, 1.3 Hz, 3H), 7.22 (td, J = 7.4, 1.2 Hz, 1H), 7.06 – 6.98 (m, 1H), 6.54 (d, J = 7.9 Hz, 1H), 6.42 (d, J = 6.8 Hz, 1H), 4.76 (q, J = 7.0 Hz, 1H), 4.56 (t, J = 6.9 Hz, 1H), 4.49 (td, J = 8.4, 5.8 Hz, 1H), 3.62 (s, 3H), 3.57 (t, J = 2.9 Hz, 2H), 3.34 – 3.09 (m, 3H), 2.80 (s, 5H), 2.49 (t, J = 2.3 Hz, 3H), 1.65 (s, 9H), 1.62 – 1.49 (m, 3H), 1.48 – 1.33 (m, 10H), 1.27 – 0.95 (m, 2H), 0.93 – 0.64 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 173.3, 173.2, 172.9, 172.7, 172.1, 171.0, 170.5, 166.4, 166.1, 149.7, 148.5, 145.6, 145.4, 142.2, 142.2, 131.3, 130.9, 130.3, 127.7, 127.5, 126.9, 126.9, 124.7, 123.9, 122.8, 121.0, 120.9, 119.2, 119.0, 115.5, 115.4, 83.8, 77.5, 77.2, 76.8, 55.7, 53.9, 53.4, 52.3, 52.2, 51.7, 50.6, 50.4, 49.9, 49.3, 43.6, 39.7, 38.7, 38.3, 34.0, 33.5, 33.3, 33.2, 32.5, 28.3, 27.6, 26.4, 26.1, 26.0, 18.7, 18.4, 17.3, 12.6, 12.6, 12.5, 0.1. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 9.94 (ESI-MS (m/z): 699.38 (M+H+)).
OSu (20).
MophAc-D-Ala-Trp(Boc)-Cha-OMe (0.156 g, 0.22 mmol) was dissolved in THF (3 mL) and H2O (1 mL)
and LiOH (54 mg, 2.2 mmol) was added. The mixture was heated to 45°C and stirred overnight. The reaction mixture was concentrated in vacuo and dissolved in EtOAc and washed with H2O and brine. The
organic layer was dried over MgSO4, filtered and concentrated in
vacuo. The crude product was dissolved in DCM (4 mL) and DCC (91 mg, 0.44 mmol) and HOSu (50 mg, 0.44 mmol) were added. After stirring overnight the solvent was evaporated followed by flash column chromatography on silica gel (MeOH in DCM – 0% to 4% v/v) to yield the title compound (98 mg, 0.125 mmol, 57%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 9.57 (ESI-MS
(m/z): 782.07 (M+H+)).
Trp(Boc)-Cha-NH-BnOTBS (21).
MophAc-D-Ala-Trp(Boc)-Cha-OSu (98 mg, 0.125 mmol) was dissolved in DCM (3 mL) and H2N-Tol-OTBS (31 mg, 0.132 mmol), DMAP (3 mg, 0.022
mmol) and Et3N (50 µL, 0.36 mmol) were added. After stirring
overnight at RT the solvent was evaporated followed by flash column chromatography on silica gel (MeOH in DCM – 0% to 1% v/v) to yield the title compound (80 mg, 0.088 mmol, 70%). 1H NMR (400 MHz, CDCl
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3
1.57 – 1.49 (m, 1H), 1.35 (dd, J = 6.8, 4.3 Hz, 3H), 1.25 (s, 2H), 1.13 – 1.01 (m, 2H), 0.95 – 0.91 (m, 9H), 0.10 – 0.06 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 173.0, 170.7, 170.5, 170.2, 145.5, 145.3, 142.2, 137.4, 136.9, 136.8, 130.3, 129.0, 127.8, 127.6, 127.5, 126.9, 126.5, 126.5, 124.9, 124.3, 123.9, 123.8, 123.0, 121.0, 120.2, 119.8, 119.6, 118.9, 115.6, 115.5, 115.4, 115.3, 112.8, 83.9, 65.2, 64.8, 64.7, 54.3, 53.9, 52.3, 51.8, 49.5, 49.2, 48.3, 38.4, 38.1, 34.3, 34.0, 33.7, 33.3, 32.9, 32.5, 30.8, 29.8, 28.3, 28.3, 26.4, 26.2, 26.1, 26.1, 26.1, 26.0, 26.0, 24.7, 18.5, 17.8, 12.6, 12.5, -5.0, -5.1, -5.1. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 13.5 min): Rt (min): 12.66 (ESI-MS (m/z): 904.00 (M+H+)).
MophAc-D-Ala-Trp-Cha-NH-BnO-PNP (22).
MophAc-D-Ala-Trp(Boc)-Cha-NH-BnOTBS (36 mg, 0.04 mmol) was dissolved in DCM (1 mL) and TFA (1 mL) and stirred for 30 min at RT. Removal of the solvent followed by co-evaporation with toluene (3x) and CHCl3 (2x)
delivered the deprotected intermediate. p-Nitrophenol chloroformate (24 mg, 0.08 mmol), THF (4 mL) and pyridine (10 µL, 0.12 mmol) were added and stirred overnight at RT. The mixture was quenched with sat. aq. citric acid, extracted with EtOAc (3x) and washed with brine. After drying over MgSO4 and
filtration, the solvent was evaporated followed by flash column chromatography on silica gel (MeOH in DCM – 0% to 2% v/v) to yield the title compound (34 mg, 0.04 mmol, quant.). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 9.73 (ESI-MS (m/z): 855.00 (M+H+)).
Cha-NH-Doxo (7).
MophAc-D-Ala-Trp-Cha-OSu (14.4 mg, 18 µmol) and Doxo-NH2 (10.7 mg, 18
µmol) were dissolved in DMF (200 µL) in an Eppendorf tube. DiPEA (12.3 µL, 72 µmol) was added and the tube was shaken overnight at RT. The solvent was evaporated and subsequently co-evaporated with n-heptane (3x). The crude product was purified via flash column chromatography on silica gel (MeOH in DCM – 0% to 5% v/v) was followed by Prep-HPLC purification. After freezedrying this yielded the title compound (2.56 mg, 2.3 µmol, 13%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 8.80 (ESI-MS
(m/z): 1110.13 (M+H+)). HRMS: calculated for C61H78N5O15 1110.47064 [M+H]+; found 1110.47004.
MophAc-Ala-Trp-Cha-NH-BnO-Doxo (8).
MophAc-D-Ala-Trp-Cha-NH-BnO-PNP (3.74 mg, 4.4 µmol) and Doxo-NH2 (2.54 mg, 4.4 µmol) were dissolved in DMF
(75 µL) in an Eppendorf tube. DiPEA (3 µL, 18 µmol) was added and the tube was shaken overnight at RT. The solvent was evaporated and subsequently coevaporated with n-heptane (3x). The crude product was purified via flash column chromatography on silica gel (MeOH in DCM – 0% to 3% v/v) to yield the title compound (1.66 mg, 1.3 µmol, 30%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 8.64 (ESI-MS (m/z): 1281.20
(M+Na+)). HRMS: calculated for C
69H75N6O17 1259.51887 [M+H]+; found 1259.51812.
H2N-Nle-Cha-OMe (56). H2N-Cha-OMe (1.22 g, 4.05 mmol), Boc-Nle-OH (1.12 g, 4.86
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3
filtered and concentrated in vacuo. The crude product was purified via flash column chromatography on silica gel (MeOH in DCM – 0% to 2% v/v) and deprotected by redissolving in DCM (2 mL) and TFA (2 mL). The mixture was then stirred for 30 min at RT followed by evaporation of the solvent and co-evaporation with toluene (3x) and CHCl3 (3x). This yielded the title compound as a TFA salt (2.07 g, 4.86 mmol,
quant.). 1H NMR (500 MHz, CDCl
3) δ 10.21 (s, 2H), 7.36 (d, J = 6.9 Hz, 1H), 4.51 – 4.40 (m, 1H), 4.18 (t, J =
6.6 Hz, 1H), 3.72 (s, 3H), 2.87 (s, 2H), 2.36 (d, J = 0.8 Hz, 1H), 1.93 – 1.80 (m, 2H), 1.74 – 1.54 (m, 6H), 1.43 – 1.23 (m, 5H), 1.21 – 1.06 (m, 2H), 0.96 – 0.74 (m, 5H). 13C NMR (126 MHz, CDCl3) δ 172.77, 169.38, 53.96, 53.66, 52.57, 51.36, 38.87, 38.84, 33.89, 33.17, 32.40, 31.34, 30.00, 27.46, 26.84, 26.45, 26.30, 25.94, 25.77, 22.20, 22.07, 21.54, 13.52, 13.41. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA,
12.5 min): Rt (min): 9.07 (ESI-MS (m/z): 299.20 (M+H+)).
H2N-F2Pro-Nle-Cha-OMe (57). H2N-Nle-Cha-OMe (0.432 g, 1.04 mmol), Boc-F2Pro-OH (0.293 g, 1.25
mmol) and HCTU (0.52 g, 1.25 mmol) were dissolved in DCM (11 mL), followed by addition of DiPEA (0.66 mL, 3.8 mmol). The reaction mixture was then stirred overnight at RT. The reaction mixture was concentrated in vacuo and dissolved in EtOAc and washed with 1 M HCl, saturated NaHCO3 (2x) and brine.
The organic layer was dried over MgSO4, filtered and concentrated in vacuo.
The crude product was purified via flash column chromatography on silica gel (MeOH in DCM – 0% to 1% v/v) and deprotected by redissolving in DCM (2 mL) and TFA (2 mL). The mixture was then stirred for 30 min at RT followed by evaporation of the solvent and co-evaporation with toluene (3x) and CHCl3 (3x). This yielded the title compound as a TFA salt (0.39 g,
0.74 mmol, 71%). 1H NMR (400 MHz, CDCl
3) δ 7.30 – 7.04 (m, 1H), 4.56 (dq, J = 17.4, 8.7, 8.3 Hz, 3H), 3.97
– 3.78 (m, 2H), 3.72 (s, 3H), 2.82 (s, 1H), 2.65 (dq, J = 47.5, 11.5 Hz, 1H), 1.68 (dq, J = 16.2, 8.6 Hz, 5H), 1.38 – 1.26 (m, 5H), 1.25 – 1.11 (m, 2H), 0.96 – 0.78 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 173.0, 171.3,
169.8, 125.9, 121.2, 108.4, 81.6, 77.5, 77.4, 77.2, 76.8, 61.5, 53.1, 52.0, 50.0, 39.4, 38.5, 33.9, 33.2, 32.3, 32.0, 28.0, 27.0, 26.2, 25.9, 25.8, 22.3, 13.7. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5
min): Rt (min): 9.29 (ESI-MS (m/z): 432.13 (M+H+)).
H2N-Ala-F2Pro-Nle-Cha-OMe (58). H2N-F2Pro-Nle-Cha-OMe (0.392 g, 0.74
mmol), Boc-Ala-OH (0.167 g, 0.88 mmol) and HCTU (0.36 g, 0.88 mmol) were dissolved in DCM (10 mL), followed by addition of DiPEA (0.44 mL, 2.6 mmol). The reaction mixture was then stirred overnight at RT. The reaction mixture was concentrated in vacuo and dissolved in EtOAc and washed with 1 M HCl, saturated NaHCO3 (2x) and brine. The organic layer was dried
over MgSO4, filtered and concentrated in vacuo. The crude product was purified via flash column
chromatography on silica gel (MeOH in DCM – 0% to 2% v/v) and deprotected by redissolving in DCM (2 mL) and TFA (2 mL). The mixture was then stirred for 30 min at RT followed by evaporation of the solvent and co-evaporation with toluene (3x) and CHCl3 (3x). This yielded the title compound as a TFA salt (0.36
g, 0.59 mmol, 80%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 8.69
(ESI-MS (m/z): 503.27 (M+H+)).
N3Ac-Ala-F2Pro-Nle-Cha-OH (59). H2N-Ala-F2Pro-Nle-Cha-OMe (0.143 g, 0.23 mmol), N3Ac-OH (0.029 g,
0.29 mmol) and HCTU (0.12 g, 0.29 mmol) were dissolved in DCM (5 mL), followed by addition of DiPEA (0.14 mL, 0.8 mmol). The reaction mixture was then stirred overnight at RT. The reaction mixture was concentrated in vacuo and dissolved in EtOAc and washed with 1 M HCl, saturated NaHCO3 (2x) and
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ester was purified via flash column chromatography on silica gel (MeOH in DCM – 0% to 5% v/v) and saponificated using LiOH (7 mg, 0.28 mmol) in THF (2 mL) and H2O (2 mL). The mixture was heated
to 45°C and stirred overnight. The reaction mixture was concentrated in vacuo and dissolved in EtOAc and washed with H2O
and brine. The organic layer was dried over MgSO4, filtered and
purified using flash column chromatography on silica gel (MeOH in DCM – 0% to 10% v/v). This yielded the title compound (81 mg, 0.14 mmol, 62%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 6.95 (ESI-MS (m/z): 572.20 (M+H+)).
N3Ac-Ala-F2Pro-Nle-Cha-NH-Bn-OH (60). N3Ac-Ala-F2
Pro-Nle-Cha-OH (10 mg, 18 µmol), EEDQ (5.2 mg, 19 µmol) and p-nitrobenzyl alcohol (2.3 mg, 19 µmol) were dissolved in THF (200 µmol) in an Eppendorf tube. Subsequently, the tube was shaken overnight at RT. The solvent was evaporated and crude product was purified via column chromatography on silica gel (MeOH in DCM – 0% to 5% v/v) to yield the title compound (8.4 mg, 12 µmol, 69%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 7.21 (ESI-MS (m/z): 677.13 (M+H+)).
N3Ac-Ala-F2Pro-Nle-Cha-NH-Bn-O-PNP (61). N3Ac-Ala-F2
Pro-Nle-Cha-NH-Bn-OH (4.3 mg, 6.4 µmol), p-nitrophenol chloroformate (3.8 mg, 12.7 µmol) and pyridine (1.5 µL, 19 µmol) were dissolved in THF (200 µmol) in an Eppendorf tube. Subsequently, the tube was shaken overnight at RT. The solvent was evaporated and crude product was purified via column chromatography on silica gel (MeOH in DCM – 0% to 1% v/v) to yield the title compound (5.4 mg, 6.4 µmol, quant.). 1H NMR (500 MHz, MeOD) δ 7.98 (s, 2H), 7.90 (s, 2H), 7.86 (dd, J = 7.2, 2.0 Hz, 1H), 7.65 (dd, J = 8.7, 2.6 Hz, 1H), 7.59 (ddd, J = 8.7, 5.1, 2.1 Hz, 1H), 4.54 (ddd, J = 22.7, 10.6, 4.6 Hz, 1H), 4.32 (dp, J = 13.5, 6.5 Hz, 1H), 4.24 – 4.08 (m, 1H), 2.99 (s, 5H), 2.86 (d, J = 0.7 Hz, 6H), 2.64 – 2.51 (m, 1H), 2.47 – 2.41 (m, 3H), 1.99 (d, J = 12.4 Hz, 3H), 1.87 – 1.72 (m, 2H), 1.73 (s, 1H), 1.72 (d, J = 3.3 Hz, 1H), 1.67 (ddd, J = 14.0, 10.9, 4.9 Hz, 1H), 1.44 – 1.31 (m, 4H), 1.31 – 1.15 (m, 3H), 1.09 – 0.88 (m, 2H). 13C NMR (126 MHz, MeOD) δ 175.82, 174.87, 174.85, 173.55, 173.51, 173.37, 173.34, 173.19, 173.13, 172.50, 164.83, 163.20, 163.16, 155.27, 155.20, 155.18, 155.17, 143.35, 143.25, 127.55, 126.64, 126.57, 126.51, 117.42, 117.27, 117.20, 117.18, 117.10, 117.05, 113.70, 113.61, 113.59, 108.18, 108.03, 107.93, 60.43, 56.39, 54.85, 54.59, 53.45, 53.31, 53.15, 51.09, 49.23, 46.99, 39.97, 39.91, 38.04, 37.85, 37.65, 36.95, 35.66, 35.37, 35.11, 34.98, 34.85, 33.26, 33.00, 32.88, 32.45, 31.72, 31.64, 29.28, 29.06, 27.53, 27.44, 27.39, 27.23, 27.17, 23.40, 23.28, 22.41, 22.39, 21.96, 19.32, 18.54, 17.58, 16.99, 16.17, 15.50, 14.34, 14.25. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 8.94 (ESI-MS (m/z): 842.13 (M+H+)).
N3Ac-Ala-F2Pro-Nle-Cha-NH-Bn-O-Doxo (9). N3
Ac-Ala-F2Pro-Nle-Cha-NH-Bn-O-PNP (5.4 mg, 6.4 µmol)
and Doxo-NH2 (3.7 mg, 6.4 µmol) were dissolved in
DMF (100 µL) in an Eppendorf tube. DiPEA (3 µL, 18 µmol) was added and the tube was shaken overnight at RT. The solvent was evaporated and subsequently co-evaporated with n-heptane (3x). The crude product was purified via flash column chromatography on silica gel (MeOH in DCM – 0% to 3% v/v) to yield the title compound (1.62 mg, 1.3 µmol, 20%). LC-MS
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(linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 8.41 (ESI-MS (m/z): 1268.40
(M+Na+)). HRMS: calculated for C
60H77F2N10O18 1263.53799 [M+NH4]+; found 1263.53849.
Self-immolative linker synthesis
Scheme 4. Example synthesis of self-immolative linkers. Reagents and conditions: a) BH3, THF, 60°C; b) TBS-Cl or Trt-Cl, Et3N,
DMAP, DMF, 40°C
Procedure A[29]: A solution of BH
3·THF (1 M, 5 eq.) was carefully added to a stirred solution of the
commercially available carboxylic acid (1 eq.) in THF (0.25 M) under N2. The mixture was then refluxed
overnight. Subsquently, the mixture was quenched at 0°C with HCl (2 M) and extracted with EtOAc. The organic layers were combined, washed with brine, dried over MgSO4, filtered and concentrated. The
crude product was purified via flash column chromatography on silica gel (EtOAc in MeOH – 0% to 5% v/v) to yield the title compound as a white solid.
Procedure B – TBS/Trt protection: The primary alcohol (1 eq.) was dissolved in DMF (0.3 M) and DMAP (0.2 eq.) and Et3N (1.2 eq.) were added. TBS-Cl or Trt-Cl (1.1 eq.) was added slowly and the reaction
mixture was stirred overnight at 40°C. The reaction mixture was concentrated in vacuo and dissolved in EtOAc and washed with 1 M HCl, saturated NaHCO3 (2x) and brine. The organic layer was dried over
MgSO4, filtered and concentrated in vacuo. The crude product was purified via flash column
chromatography on silica gel (EtOAc in pentane – 0% to 4% v/v) to yield the title compound.
HO-Tol-OTrt (62). This compound was prepared by Trt protection according to
procedure B on a 5.0 mmol scale using 4-(hydroxymethyl)phenol. This yielded the title compound (1.7 g, 4.8 mmol, 93%). 1H NMR (400 MHz, CDCl3) δ 7.54 – 7.48 (m, 6H), 7.36 – 7.28 (m,
9H), 7.24 (d, 2H), 6.82 (d, J = 8.6 Hz, 2H), 4.08 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 155.01, 144.32,
128.88, 128.82, 128.07, 127.95, 127.11, 115.27, 100.12, 65.56. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 10.21 (ESI-MS (m/z): 367.16 (M+H+)).
H2N-Tol-OTBS (63). This compound was prepared by TBS protection according to procedure B on a 8.1 mmol scale using (4-aminophenyl)methanol. This yielded the title compound (1.7 g, 7.0 mmol, 86%). 1H NMR (300 MHz, CDCl3) δ 7.18 – 7.05 (m,
2H), 6.69 – 6.60 (m, 2H), 4.62 (d, J = 0.8 Hz, 2H), 3.59 (s, 2H), 0.92 (s, 9H), 0.07 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 145.5, 127.8, 115.1, 77.6, 77.2, 76.7, 65.1, 26.1, -5.0.
HO-F4Tol-OTBS (64). This compound was prepared by a reduction according to procedure A on a 2.6 mmol scale using 2,3,5,6-tetrafluoro-4-hydroxybenzoic acid, followed by TBS protection using procedure B. This yielded the title compound (0.57 g, 1.8 mmol, 76%). 1H NMR (300 MHz, CDCl3) δ 4.75 (m, 1H), 2.99 (dd, J = 2.6, 1.2 Hz,
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H2N-F4Tol-OTBS (65). This compound was prepared by a reduction according to procedure A on a 2.4 mmol scale using 4-amino-2,3,5,6-tetrafluorobenzoic acid, followed by TBS protection using procedure B. This yielded the title compound (0.35 g, 1.1 mmol, 43%). 1H NMR (400 MHz, CDCl
3) δ 4.70 (q, J = 1.7 Hz, 2H), 4.21 – 3.98 (m,
2H), 0.89 (d, J = 3.4 Hz, 9H), 0.09 (d, J = 4.8 Hz, 6H). s13C NMR (101 MHz, CDCl
3) δ 146.9, 144.4, 137.5,
126.7, 53.1, 38.0, 25.8, -5.4.
Proteasome inhibitor synthesis
Cbz-Chaninol (24). Cyclohexyl-L-alaninol HCl salt (1.2 g, 6.2 mmol) and Na2CO3 (1.3 g, 12
mmol) were dissolved in a mixture of H2O (10 mL) and dioxane (20 mL) and cooled to 0°C.
Cbz-Cl (1.4 mL, 9.3 mmol) was added dropwise and the reaction was stirred overnight at RT. The mixture was then concentrated until approximately 10 mL of aqueous solution remained and this was extracted with DCM (3x). The combined organic layers washed with sat. aq. NH4Cl, sat. aq. NaHCO3 and brine followed by drying over MgSO4, filtration and concentration. The crude
was purified by chromatography on silica gel (EtOAC in Pentane – 0% to 50% v/v) to yield the title compound as a viscous colorless oil (1.8 g, 6.2 mmol, quant.). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 9.12 (ESI-MS (m/z): 306.23 (M+H+)).
Cbz-Cha-[CH2S]-Ac (25). Cbz-Chaninol (1.8 g, 6.2 mmol) was dissolved in DCM (30 mL) and cooled to 0°C. Et3N (1.04 mL, 7.5 mmol) was added followed by slow addition of
MsCl (0.57 mL, 7.5 mmol) and subsequently stirring overnight at RT. The reaction was then quenched with sat. aq. NH4Cl (30 mL) and washed with H2O and brine followed by
drying over MgSO4, filtration and concentration. The crude was purified by chromatography on silica gel
(EtOAC in Pentane – 0% to 40% v/v) to yield the intermediate mesylate. Subsequently, thioacetic acid (0.53 mL, 7.44 mmol) and K2CO3 (0.51 g, 3.72 mmol) were dissolved in DMF (25 mL) and stirred for 10
min at RT. The mesylate in DMF (5 mL) was then added and the mixture was heated to 50°C while stirring overnight. After evaporation of the solvent the crude product was purified via flash column chromatography on silica gel (EtOAC in Pentane – 0% to 40% v/v) to yield the title compound as a smelly brown solid (1.73 g, 4.96 mmol, 80%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min):
Rt (min): 8.67 (ESI-MS (m/z): 350.00 (M+H+)).
Cbz-Cha-[CH2SO2]-Cl (26). MeCN (3 mL) was cooled to 0°C and n-chlorosuccinimide (0.82 g, 6.2 mmol) and 2 M aq. HCl (387 μL, 0.77 mmol) was added. After stirring for 15 min, Cbz-Cha-[CH2S]-Ac (0.542 g, 1.55 mmol) was added and reaction mixture was left to stir at
RT for another 15 min. DCM and brine were added and the mixture was extracted with DCM (2x), dried over MgSO4 and filtered. After evaporation of the solvent, the crude product was co-evaporated with
toluene (4x) and CHCl3 (1x) followed by drying at high vacuum. The obtained product (0.57 g, 1.55 mmol,
quant.) was used without further purification in the next step. 1H NMR (500 MHz, CDCl
3 ) δ 7.41 – 7.28
(m, 5H), 5.30 (s, 1H), 5.14 – 5.07 (m, 2H), 4.30 (d, J = 8.6 Hz, 1H), 4.14 (dd, J = 14.3, 6.3 Hz, 1H), 3.89 (dd, J = 14.3, 4.6 Hz, 1H), 1.84 – 1.49 (m, 6H), 1.47 – 1.04 (m, 3H), 1.04 – 0.68 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 177.97, 136.20, 128.66, 128.56, 128.36, 128.16, 127.87, 69.20, 67.12, 46.40, 41.02, 34.12, 33.59, 32.35, 29.66, 26.56, 26.38, 26.31, 26.19, 25.99. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1%
TFA, 12.5 min): Rt (min): 8.15 (ESI-MS (m/z): 334.00 (M+H+)).
Cbz-Cha-[CH2SO2]-O-Tol-OTrt (27a). Cbz-Cha-[CH2SO2]-Cl (102 mg, 0.273
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3
0.328 mmol) was added and the mixture was stirred for 15 min. Afterwards, Et3N (74 μL, 0.546 mmol)
was added and the mixture was stirred overnight at RT. Evaporation of the solvent and purification via flash column chromatography (EtOAc in pentane – 0% to 20% v/v) yielded the title compound (160 mg, 0.227 mmol, 83%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 10.81
(ESI-MS (m/z): 704.30 (M+H+)).
Cbz-Cha-[CH2SO2]-NH-Tol-OTBS (27b). Cbz-Cha-[CH2SO2]-Cl (129 mg, 0.38 mmol) was dissolved in MeCN
(2 mL) and cooled to 0°C. H2N-Tol-OTBS (100 mg, 0.41 mmol) was added and
the mixture was stirred for 15 min. Afterwards, Et3N (103 μL, 0.76 mmol)
was added and the mixture was stirred overnight at RT. Evaporation of the solvent and purification via flash column chromatography (EtOAc in pentane – 0% to 20% v/v) yielded the title compound (142 mg, 0.244 mmol, 65%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 9.61 (ESI-MS (m/z): 575.08 (M+H+)).
Cbz-Cha-[CH2SO2]-O-F4Tol-OTBS (27d). Cbz-Cha-[CH2SO2]-Cl (25 mg, 76
μmol) was dissolved in MeCN (1 mL) and cooled to 0°C. H2N-F4Tol-OTBS (29
mg, 92 μmol) was added and the mixture was stirred for 15 min. Afterwards, Et3N (20 μL, 150 μmol) was added and the mixture was stirred
for 2 h at 55°C while changing from colorless to dark purple. Evaporation of the solvent and purification via column chromatography (MeOH in DCM – 0% to 1% v/v) yielded the title compound (26 mg, 40 μmol, 25%). 1H NMR (500 MHz, CDCl3) δ 7.37 – 7.24 (m, 5H), 5.20 (d, J = 8.1 Hz, 3H), 5.09 (d, J = 3.2 Hz, 6H), 4.77 (d, J = 1.6 Hz, 2H), 4.43 (d, J = 7.5 Hz, 3H), 3.91 – 3.80 (m, 3H), 3.76 – 3.65 (m, 3H), 3.23 – 3.03 (m, 6H), 2.28 (t, J = 2.2 Hz, 6H), 1.82 (d, J = 6.4 Hz, 2H), 1.73 (d, J = 13.0 Hz, 3H), 1.66 (d, J = 15.4 Hz, 2H), 1.31 – 1.18 (m, 3H), 1.14 (q, J = 12.1 Hz, 1H), 0.93 (p, J = 11.5 Hz, 2H), 0.91 (s, 9H), 0.13 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 155.53, 136.24, 129.43, 129.07, 128.63, 128.19, 127.44, 67.13, 54.83, 53.25, 51.44, 49.42, 39.30, 38.12, 33.54, 33.07, 32.53, 29.85, 26.3, 25.9, 25.87, 25.71, -5.40. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 11.29 (ESI-MS (m/z): 642.00 (M+H+)).
Cbz-Cha-[CH2SO2]-NH-F4Tol-OTBS (27c). Cbz-Cha-[CH2SO2]-Cl (30 mg, 88
μmol) was dissolved in MeCN (1 mL) and cooled to 0°C. H2N-F4Tol-OTBS (30
mg, 97 μmol) was added and the mixture was stirred for 15 min. Afterwards, Et3N (24 μL, 176 μmol) was added and the mixture was stirred for 3 h at
55°C. Evaporation of the solvent and purification via column chromatography (MeOH in DCM – 0% to 1% v/v) yielded the title compound (26 mg, 40 μmol, 45%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 10.41 (ESI-MS (m/z): 647.00 (M+H+)).
H2N-Cha-[CH2SO2]-O-Tol-OH (28a). Cbz-Cha-[CH2SO2]-O-Tol-OTrt (160 mg, 0.227
mmol) was dissolved DCM (1 mL) and HBr in acetic acid (33%, 0.9 mL, 5.45 mmol) was added dropwise to the solution and stirred at RT for 45 min. After concentration in vacuo the residue was co-evaporated with toluene (3x) and CHCl3 (2x) to yield the crude
deprotected product as an HBr salt (25 mg, 0.050 mmol, 22%). The crude product was used without further purification in the next step. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min):
Rt (min): 5.33 (ESI-MS (m/z): 328.33 (M+H+)).
H2N-Cha-[CH2SO2]-NH-Tol-OTBS (28b). Cbz-Cha-[CH2SO2]-NH-Tol-OTBS (20 mg,
76 μmol) was dissolved in MeOH (2 mL) and flushed with N2. Pd/C (10%, 10 mg)
was added and the reaction mixture was viscously stirred under an H2 flow for
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2 h. The mixture was then filtered over Celite. The eluent was concentrated in vacuo and purified by flash column chromatography (MeOH in DCM – 0% to 1% v/v) to yield the title compound (32 mg, 72 μmol, 95%). 1H NMR (500 MHz, CDCl
3) δ 7.28 (d, J = 8.3 Hz, 2H), 7.24 – 7.19 (m, 2H), 4.70 (s, 2H), 3.53 –
3.46 (m, 1H), 3.09 (dd, J = 14.4, 2.3 Hz, 1H), 2.95 (dd, J = 14.4, 10.2 Hz, 1H), 1.71 – 1.62 (m, 5H), 1.61 (s, 2H), 1.33 – 1.22 (m, 4H), 1.20 – 1.07 (m, 2H), 0.94 (s, 9H), 0.91 – 0.80 (m, 1H), 0.10 (s, 6H). 13C NMR (126
MHz, CDCl3) δ 138.67, 135.99, 127.31, 121.38, 64.52, 57.17, 45.83, 45.21, 34.04, 33.69, 32.83, 26.48,
26.26, 26.15, 26.06, 18.53, -5.13. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt
(min): 6.60 (ESI-MS (m/z): 441.17 (M+H+)).
H2N-Cha-[CH2SO2]-O-F4Tol-OTBS (28c). Cbz-Cha-[CH2SO2]-O-F4Tol-OTBS (49
mg, 76 μmol) was dissolved in MeOH (2 mL) and flushed with N2.Pd/C (10%,
10 mg) was added and the reaction mixture was viscously stirred under an H2
flow for 2 h. The mixture was then filtered over Celite. The eluent was concentrated in vacuo and purified by flash column chromatography (MeOH in DCM – 0% to 1% v/v) to yield the title compound (20 mg, 40 μmol, 52%). 1H NMR (500 MHz, CDCl
3) δ 4.74 (s, 2H), 4.01 – 3.84 (m,
2H), 3.38 (s, 1H), 3.25 (d, J = 14.4 Hz, 1H), 1.65 (dd, J = 46.8, 10.6 Hz, 2H), 1.25 (s, 4H), 1.20 – 1.05 (m, 2H), 0.89 (d, J = 3.8 Hz, 9H), 0.10 (d, J = 7.9 Hz, 6H). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA,
12.5 min): Rt (min): 8.28 (ESI-MS (m/z): 508.07 (M+H+)).
H2N-Cha-[CH2SO2]-NH-F4Tol-OTBS (28d). Cbz-Cha-[CH2SO2]-NH-F4Tol-OTBS
(40 mg, 62 μmol) was dissolved in MeOH (2 mL) and flushed with N2. Pd/C
(10%, 10 mg) was added and the reaction mixture was viscously stirred under an H2 flow for 2 h. The mixture was then filtered over Celite. The
eluent was concentrated in vacuo and purified by flash column chromatography (MeOH in DCM – 0% to 1% v/v) to yield the title compound (19 mg, 37μmol, 60%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 7.67 (ESI-MS (m/z): 513.20
(M+H+)).
Morp-Ala-Tyr(OMe)-Cha-[CH2SO2]-O-Tol-OH (30a). Morp-Ala-Tyr(OMe)-Cha-NHNH2 (0.229 g, 0.56 mmol, made via
literature procedure [9]) was dissolved in DMF (20 mL) and
cooled to -30°C. tBuONO (148 μL, 1.23 mmol) and HCl (0.78 mL, 4 M solution in dioxane) were added and the mixture was stirred at -30°C for 4 h. H2N-Cha-[CH2SO2]-O-Tol-OH
(9.18 mg, 22 μmol) was added to the reaction mixture as a solution in DMF (5 mL) and DiPEA (0.97 mL,
5.6 mmol). The reaction mixture was allowed to slowly come to RT and was stirred overnight. Then, the solvent was removed under vacuo and the crude was dissolved in DCM and washed with H2O and brine.
Column chromatography (MeOH in DCM – 0% to 2% v/v) yielded the title compound (28 mg, 40 μmol, 7%).LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 5.64 (ESI-MS (m/z):
697.20(M+H+)).
Morp-Ala-Tyr(OMe)-Cha-[CH2SO2]-NH-Tol-OH (30b). Morp-Ala-Tyr(OMe)-Cha-NHNH2 (15.24 mg, 34 μmol, made via literature
procedure [9]) was dissolved in DMF (2 mL) and cooled to -30°C
under an N2 atmosphere. tBuONO (9 μL, 76 μmol) and HCl (72
μL, 4 M solution in dioxane) were added and the mixture was
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stirred at -30°C for 4 h. H2N-Cha-[CH2SO2]-NH-Tol-OTBS (9.18 mg, 22 μmol) was added to the reaction
mixture as a solution in DMF (100 μL) and DiPEA (60 μL, 340 μmol). The reaction mixture was allowed to slowly come to RT and was stirred overnight. Then, the solvent was concentrated in vacuo and purified by flash column chromatography (MeOH in DCM – 0% to 1% v/v) to yield the protected intermediate. This was dissolved in DCM (0.5 mL) and TFA (0.5 mL) and stirred at RT for 30 min. After concentration in vacuo the residue was co-evaporated with toluene (3x) and CHCl3 (2x), followed by flash column
chromatography (MeOH in DCM – 0% to 5% v/v) to yield the title compound (6.82 mg, 9.7 μmol, 28%). 1H NMR (500 MHz, CDCl3) δ 7.71 (dd, J = 5.7, 3.3 Hz, 1H), 7.53 (dd, J = 5.7, 3.3 Hz, 1H), 7.40 – 7.35 (m, 1H), 7.16 – 7.11 (m, 2H), 6.92 – 6.87 (m, 1H), 6.85 – 6.80 (m, 2H), 6.75 (d, J = 8.7 Hz, 1H), 4.49 – 4.43 (m, 2H), 4.41 (s, 2H), 4.36 (t, J = 7.0 Hz, 1H), 4.29 (dt, J = 8.4, 4.8 Hz, 1H), 4.22 (qd, J = 10.9, 5.9 Hz, 2H), 3.78 (s, 3H), 3.68 (t, J = 4.7 Hz, 2H), 3.38 (d, J = 3.5 Hz, 2H), 3.31 (dd, J = 14.9, 4.0 Hz, 1H), 3.17 (ddd, J = 15.0, 6.6, 5.1 Hz, 2H), 2.99 – 2.84 (m, 2H), 2.50 – 2.38 (m, 2H), 1.72 – 1.53 (m, 5H), 1.42 (q, J = 7.1 Hz, 3H), 1.37 – 1.23 (m, 4H), 1.18 – 1.02 (m, 3H), 0.96 – 0.69 (m, 2H). 13C NMR (126 MHz, CDCl 3) δ 173.23, 171.48, 171.43, 158.69, 136.82, 135.40, 135.03, 131.03, 130.45, 129.20, 129.14, 128.95, 128.71, 121.10, 120.49, 114.23, 74.25, 74.22, 68.30, 67.02, 61.56, 58.32, 58.26, 55.96, 55.38, 54.10, 53.89, 49.05, 43.74, 41.55, 38.87, 35.75, 34.12, 33.13, 32.93, 32.62, 30.50, 29.85, 29.07, 26.46, 26.42, 26.18, 23.88, 23.13, 16.61, 14.20, 11.11. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 5.52 (ESI-MS
(m/z): 702.33 (M+H+)).
Morp-Ala-Tyr(OMe)-Cha-[CH2SO2]-O-F4Tol-OH(30c). Morp-Ala-Tyr(OMe)-Cha-NHNH2 (8.79 mg, 17 μmol, made via
literature procedure [9]) was dissolved in DMF (2 mL) and
cooled to -30°C under an N2 atmosphere. tBuONO (4.5 μL,
37 μmol) and HCl (37 μL, 4 M solution in dioxane) were added and the mixture was stirred at -30°C for 4h. H2
N-Cha-[CH2SO2]-O-F4Tol-OTBS (9.18 mg, 22 μmol) was added to the reaction mixture as a solution in DMF
(100 μL) and DiPEA (29 μL, 170 μmol). The reaction mixture was allowed to slowly come to RT and was stirred overnight. Then, the solvent was concentrated in vacuo and purified by flash column chromatography (MeOH in DCM – 0% to 1% v/v) to yield the protected intermediate. This was dissolved in DCM (0.5 mL) and TFA (0.5 mL) and stirred at RT for 30 min. After concentration in vacuo the residue was co-evaporated with toluene (3x) and CHCl3 (2x), followed by flash column chromatography (MeOH in
DCM – 0% to 5% v/v) to yield the title compound (1.93 mg, 2.5 μmol, 15%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 5.63 (ESI-MS (m/z): 775.23 (M+H+)).
Morp-Ala-Tyr(OMe)-Cha-[CH2SO2]-NH-F4Tol-OH (30d). Morp-Ala-Tyr(OMe)-Cha-NHNH2 (8.79 mg, 17 μmol, made
via literature procedure [9]) was dissolved in DMF (2 mL)
and cooled to -30°C under an N2 atmosphere. tBuONO
(4.5 μL, 37 μmol) and HCl (37 μL, 4 M solution in dioxane) were added and the mixture was stirred at -30°C for 4 h. H2N-Cha-[CH2SO2]-NH-F4Tol-OTBS (9.18 mg, 22 μmol) was added to the reaction mixture as a solution in
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DCM – 0% to 5% v/v) to yield the title compound (2.94 mg, 3.8 μmol, 22%). 1H NMR (600 MHz, MeOD) δ
7.72 (dd, J = 5.7, 3.3 Hz, 2H), 7.63 (dd, J = 5.7, 3.3 Hz, 2H), 7.16 – 7.13 (m, 1H), 6.81 (d, J = 8.7 Hz, 1H), 4.68 (d, J = 1.5 Hz, 1H), 4.59 (s, 1H), 4.54 (dd, J = 7.7, 5.2 Hz, 1H), 4.47 (dd, J = 9.3, 5.7 Hz, 1H), 4.35 (q, J = 7.1 Hz, 1H), 4.26 – 4.18 (m, 3H), 3.74 (s, 2H), 3.69 (dd, J = 5.8, 3.7 Hz, 3H), 3.61 (s, 6H), 3.26 – 3.15 (m, 2H), 3.06 – 2.95 (m, 2H), 2.91 – 2.84 (m, 1H), 2.47 (t, J = 4.7 Hz, 2H), 1.72 – 1.58 (m, 3H), 1.43 (qdd, J = 7.4, 6.4, 5.0 Hz, 3H), 1.37 (dd, J = 7.0, 1.5 Hz, 3H), 1.37 – 1.32 (m, 4H), 1.32 – 1.25 (m, 4H), 0.95 (t, J = 7.5 Hz, 3H), 0.93 – 0.86 (m, 2H). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min):
5.58 (ESI-MS (m/z): 774.29 (M+H+)).
Morp-Ala-Tyr(OMe)-Cha-[CH2SO2]-O-Tol-OCO-PNP (31a). Morp-Ala-Tyr(OMe)-Cha-[CH2SO2]-O-Tol-OH (28
mg, 40 µmol), p-nitrophenol chloroformate (24 mg, 80 µmol) and pyridine (10 µL, 120 µmol) were dissolved in THF (1 mL) and stirred overnight at RT. The mixture was quenched with sat. aq. citric acid, extracted with EtOAc (3x) and washed with brine. After drying over MgSO4 and filtration, the solvent was evaporated followed
by flash column chromatography on silica gel (MeOH in DCM – 0% to 2% v/v) to yield the title compound (34 mg, 40 µmol, quant.) 1H NMR (400 MHz, CDCl3) δ 8.33 – 8.22 (m, 2H), 7.51 – 7.44 (m, 1H), 7.39 (dt, J = 9.0, 1.6 Hz, 1H), 7.35 – 7.29 (m, 1H), 7.23 – 7.16 (m, 1H), 7.13 – 7.00 (m, 1H), 6.88 – 6.72 (m, 1H), 5.29 (dd, J = 7.5, 4.8 Hz, 2H), 4.71 (d, J = 6.4 Hz, 1H), 4.60 (q, J = 6.9 Hz, 1H), 4.52 (q, J = 7.2 Hz, 1H), 4.28 (d, J = 6.9 Hz, 1H), 4.23 – 4.07 (m, 1H), 3.80 – 3.73 (m, 4H), 3.70 (s, 5H), 3.64 – 3.57 (m, 1H), 3.54 – 3.43 (m, 1H), 3.09 (d, J = 7.2 Hz, 1H), 3.06 – 2.87 (m, 1H), 2.63 – 2.40 (m, 2H), 1.98 – 1.91 (m, 2H), 1.70 (dt, J = 13.3, 3.8 Hz, 1H), 1.61 (q, J = 7.2 Hz, 2H), 1.37 – 1.25 (m, 9H), 1.20 – 1.05 (m, 2H), 0.93 – 0.75 (m, 1H). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 9.77 (ESI-MS (m/z): 855.00 (M+H+)).
Morp-Ala-Tyr(OMe)-Cha-[CH2SO2 ]-O-Tol-OCNH-Doxo (10).
Morp-Ala-Tyr(OMe)-Cha-[CH2SO2]-O-Tol-OCO-PNP (13.6 mg, 15.8
µmol) and Doxo-NH2 (9.2 mg, 15.8 µmol)
were dissolved in DMF (500 µL) in an Eppendorf tube. DiPEA (11 µL, 63 µmol) was added and the tube was shaken overnight at RT. The solvent was evaporated and subsequently co-evaporated with n-heptane (3x). The crude product was purified via flash column chromatography on silica gel (MeOH in DCM – 0% to 5% v/v) to yield the title compound (3.13 mg, 2.5 µmol, 16%). LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1%
TFA, 12.5 min): Rt (min): 6.30 (ESI-MS (m/z): 1266.00 (M+H+)). HRMS: calculated for C63H78N5O21S
1272.49100 [M+H]+; found 1272.49023.
Epoxyketone conjugate synthesis
Br-C(CH2CH2O-Tol-OTrt)=CH2 (36). A mixture of 2,3-dibromopropene (80 %wt, 1.2 mL, 10 mmol), formaldehyde (37 %wt, 10 mL, 134 mmol), tin(II) chloride dihydrate (2.26 g, 10 mmol) and KI (1.66 g, 10 mmol) in H2O (13
mL) was stirred overnight at RT. The reaction mixture was extracted with DCM (5x), and the combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The resulting alcohol was
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trifluoromethanesulfonic anhydride (2 mL, 11.9 mmol) were added and the reaction mixture was left to stir at -10°C under argon for 1 h. The DCM mixture was then washed with cooled H2O (1x) and the cooled
H2O was extracted with cooled DCM (2x). The combined organic layers were then washed with cooled
H2O, dried over MgSO4, filtered and concentrated at 20°C, leaving a 3-bromobut-3-en-1-yl
trifluoromethanesulfonate crude. Next, 33 (3.30 g, 9 mmol) was dissolved in anhydrous THF (104 mL) and the resulting solution was cooled to 0°C in an ice bath, after which sodium hydride (60 %wt, 0.3842 g, 9.6 mmol) was slowly added. The 3-bromobut-3-en-1-yl trifluoromethanesulfonate crude was then dissolved in anhydrous THF (8 mL) and added to the reaction mixture, which was left to stir for 16 h while allowing it to warm from 0°C to RT. The solvent was then evaporated, the residue was dissolved in EtOAc and the organic phase was washed with H2O (2x), 1 M NaOH (1x), H2O (1x) and brine (1x). Then
the organic layer was dried over MgSO4, filtered and concentrated, and the residue was purified by
column chromatography over silica gel (EtOAc in pentane – 2% v/v) to afford the title compound (3.09 g, 6.18 mmol, 69%). 1H NMR (400 MHz, CDCl3) δ 7.53 – 7.49 (m, 6H), 7.33 – 7.28 (m, 9H), 7.25 – 7.24 (m,
2H), 6.91 – 6.87 (m, 2H), 5.74 (d, J = 1.7 Hz, 1H), 5.54 (d, J = 1.7 Hz, 1H), 4.16 (t, J = 6.4 Hz, 2H), 4.09 (s, 2H), 2.92 – 2.84 (m, 2H). 13C NMR (101 MHz, CDCl
3) δ 157.90, 144.30, 131.71, 128.87, 128.63, 127.96,
127.11, 119.16, 114.60, 87.03, 65.63, 65.54, 41.31. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1%
TFA, 12.5 min): Rt (min): 11.23 (ESI-MS (m/z): 499.32 (M+H+)).
Boc-Cha-C(CH2CH2O-Tol-OTrt)=CH2 (38). To a mixture of 36 (8.96 g, 17.9 mmol) in anhydrous THF (60 mL) at -78°C was added tBuLi (16 mL, 27 mmol, from 1.7 M in pentane). After stirring for 15 min at -78°C, a solution of 37 (1.8861 g, 6.0 mmol) in Et2O (2 mL) was added
over 10 min. The reaction mixture was then stirred for 3 h under N2,
while not heating up beyond -70°C. After TLC analysis revealed completion of the reaction, the reaction was quenched by the addition of sat. aq. NH4Cl (50 mL) and the mixture was allowed to come to RT. Now
the reaction mixture was transferred to a separatory funnel and extracted with EtOAc (4x). The combined organic layers were washed with brine (1x), dried over Na2SO4, filtered and concentrated. The
crude product was purified by column chromatography over silica gel (EtOAc in pentane – 0% to 20% v/v) to afford the title compound (2.21 g, 3.28 mmol, 55%). 1H NMR (400 MHz, CDCl
3) δ 7.52 – 7.48 (m, 6H), 7.35 – 7.26 (m, 9H), 7.25 – 7.20 (m, 2H), 6.85 (d, J = 8.6 Hz, 2H), 6.22 (s, 1H), 6.04 (s, 1H), 5.28 – 4.94 (m, 2H), 4.09 – 4.00 (m, 4H), 2.92 – 2.78 (m, 1H), 2.78 – 2.62 (m, 1H), 1.97 (d, J = 12.4 Hz, 1H), 1.83 – 1.54 (m, 5H), 1.53 – 1.45 (m, 1H), 1.45 – 1.28 (m, 10H), 1.23 – 1.05 (m, 3H), 0.94 – 0.80 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 201.60, 157.95, 155.67, 144.30, 143.05, 131.41, 128.85, 128.60, 127.94, 127.10, 114.43, 86.98, 79.80, 66.06, 65.54, 52.23, 41.65, 34.35, 34.15, 32.44, 31.51, 28.47, 26.53, 26.36, 26.16. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 8.73 (ESI-MS (m/z): 674.23 (M+H+)).
Boc-Cha-OH-C(CH2CH2O-Tol-OTrt)=CH2 (39). To a solution of 38 (0.833 g, 1.24 mmol) in MeOH (17 mL) was added CeCl3·7 H2O (0.746 g, 2.00 mmol),
and the mixture was stirred at RT. Once the solution became clear, the mixture was cooled to 0°C in an ice bath and NaBH4 (0.062 g, 1.6 mmol)
was added in small portions over 10 min. After 1 h of stirring at 0°C, the reaction was quenched by adding a few drops of AcOH until pH = 5 and stirring continued for 10 min at 0°C. Then sat. aq. NaHCO3
was added until pH = 7 and the mixture was stirred for 5 min. The solvent was evaporated and the residue dissolved in a 1:1 H2O / EtOAc, which was then transferred to a separatory funnel. The aqueous
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(400 MHz, CDCl3) δ 7.68 – 7.40 (m, 5H), 7.39 – 7.15 (m, 12H), 6.88 (d, J = 8.6 Hz, 2H), 5.17 (s, 1H), 5.10 (s, 1H), 4.65 (d, J = 8.9 Hz, 1H), 4.21 (s, 1H), 4.13 (t, J = 6.8 Hz, 2H), 4.08 (s, 2H), 3.88 (t, J = 9.3 Hz, 1H), 2.72 (d, J = 3.3 Hz, 1H), 2.66 – 2.56 (m, 1H), 2.56 – 2.40 (m, 1H), 1.85 (d, J = 12.5 Hz, 1H), 1.77 – 1.52 (m, 5H), 1.43 (s, 9H), 1.39 – 1.27 (m, 2H), 1.25 – 1.08 (m, 3H), 1.01 – 0.72 (m, 2H). 13C NMR (101 MHz, CDCl 3) δ 157.91, 146.11, 144.30, 131.56, 128.86, 128.61, 127.95, 127.10, 114.86, 114.47, 113.16, 87.00, 79.55, 77.85, 67.47, 65.55, 51.19, 36.53, 34.57, 34.24, 32.42, 32.09, 28.54, 26.70, 26.58, 26.27. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 8.44 (ESI-MS (m/z): 676.27 (M+H+)).
Boc-Cha-EK-CH2CH2O-Tol-OTrt (40). To a solution of 39 (0.598 g, 0.88 mmol) in anhydrous DCM (8.8 mL) at 0°C was added vanadyl acetylacetonate (0.024 g, 0.09 mmol), followed by the addition of tBuOOH (0.5 mL, 2.75 mmol, 5.5 M in decane). The reaction mixture was stirred under argon at 0°C for 3 h, after which it was concentrated. The resulting residue was dissolved in EtOAc, and the organic layer was washed with sat. aq. NaHCO3 (2x), H2O (1x) and brine (1x),
and consequently dried over Na2SO4, filtered and concentrated. A solution of the crude product in DCM
(8.8 mL) was cooled to 0°C and Dess-Martin periodinane (0.563 g, 1.33 mmol) was added. The solution was left to stir overnight under argon while warming up to RT. Then the reaction mixture was quenched by adding sat. aq. NaHCO3 (8 mL) and the mixture was transferred to a separatory funnel. The aqueous
layer was extracted with DCM (3x), and the combined organic layers were washed with sat. aq. NaHCO3
(1x) and brine (1x), dried over Na2SO4, filtered and concentrated. The crude was purified by column
chromatography over silica gel (EtOAc in pentane – 5% to 20% v/v) to obtain the title compound (0.181 g, 0.26 mmol, 30%). 1H NMR (400 MHz, CDCl 3) δ 7.59 – 7.42 (m, 6H), 7.32 – 7.21 (m, 11H), 6.83 (d, J = 8.6 Hz, 2H), 4.81 (d, J = 8.7 Hz, 1H), 4.43 – 4.25 (m, 1H), 4.20 – 3.93 (m, 4H), 3.32 (d, J = 4.9 Hz, 1H), 3.00 (d, J = 4.9 Hz, 1H), 2.73 (dt, J = 12.4, 6.0 Hz, 1H), 2.01 (dt, J = 14.2, 5.2 Hz, 1H), 1.84 (d, J = 12.4 Hz, 1H), 1.72 – 1.44 (m, 5H), 1.44 – 1.27 (m, 10H), 1.25 – 0.97 (m, 4H), 0.96 – 0.78 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 209.10, 157.67, 155.81, 144.30, 131.55, 128.84, 128.57, 127.92, 127.08, 114.34, 87.00, 79.88, 65.55, 63.55, 60.07, 51.61, 51.16, 38.63, 34.34, 34.11, 31.86, 30.32, 28.44, 26.51, 26.32, 25.99. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 8.61 (ESI-MS (m/z): 691.53 (M+H+)).
H2N-Cha-EK-CH2CH2O-Tol-OH (41). A mixture of 40 (0.181 g, 0.26 mmol) in DCM (5.5 mL) was cooled to 0°C, and TFA (2.7 mL, 35.3 mmol) was added. The mixture was then stirred for 1 h under N2 while warming up to RT
followed by evaporation of the solvent and co-evaporation with toluene (3x) and CHCl3 (3x). Finally, the crude product was purified using column chromatography over silica gel
(MeOH in DCM – 4% to 10% v/v) to obtain the title compound (0.103 g, 0.22 mmol, 86%). 1H NMR (400
MHz, CDCl3) δ 7.31 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 5.27 (s, 2H), 4.27 – 3.86 (m, 4H), 3.18 (d, J =
3.9 Hz, 1H), 3.02 (d, J = 4.1 Hz, 1H), 2.88 – 2.81 (m, 1H), 2.00 – 1.81 (m, 1H), 1.75 – 1.46 (m, 7H), 1.35 – 1.01 (m, 4H), 0.90 – 0.66 (m, 2H). 13C NMR (101 MHz, CDCl
3) δ 204.80, 158.95, 130.89, 126.03, 114.70,
69.61, 63.32, 59.90, 52.21, 51.97, 37.60, 33.66, 33.34, 31.38, 30.17, 26.22, 25.77, 25.36. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 7.71 (ESI-MS (m/z): 345.80 (M+H+)).
H2N-Cha-OH (66). Inside of a 500 mL pressure vessel, L-phenylalanine (90.8 mmol, 15.0 g) and PtO2 (3.1 mmol, 0.70 g) were dissolved in MeOH (40 mL) and 2 M aq. HCl (4 mL). The
