LETTER RESEARCH
This foam was slurried in isopropanol to deliver material with purity .98%. Mel-
ting point 188–191 uC; 1H-NMR (400 MHz, DMSO-d6) d 8.09 (dd, J 5 1.0, 4.9 Hz,
1 H), 7.71 (dd, J 5 1.1, 3.6 Hz, 1 H), 7.38 (d, J 5 8.6Hz, 2 H), 7.32 (dd, J 5 3.9,
4.9 Hz, 1 H), 6.89 (d, J 5 9.0 Hz, 2 H), 6.36 (s, 1 H), 4.08 (br. d, J 5 9.8 Hz, 1 H), 3.93
(d, J 5 10.8 Hz, 1 H), 3.72–3.61 (m, 2 H), 3.56 (dd, J 5 2.6, 10.9 Hz, 1 H), 3.51–3.39
(m, 2 H), 3.20 (t, J 5 11.6 Hz, 1 H), 3.14–2.99 (m, 2 H), 2.89 (d, J 5 11.5 Hz, 1 H),
2.44–2.18 (m, 3 H), 1.96 (dt, J 5 2.6, 11.0 Hz, 1 H), 1.73–1.56 (m, 4 H), 0.92 (d,
J 5 6.3 Hz, 3 H); 13C-NMR (101 MHz, DMSO-d6) d 148.5, 134.1 (2), 133.4, 129.5,
128.4, 127.3(2), 126.1(q, J 5 289Hz), 114.2(2), 72.8(q, J 5 28 Hz), 72.3,66.6, 54.8,
51.8, 50.9, 48.0, 46.6, 45.7, 41.2, 22.7, 14.5; 19F-NMR (377 MHz, DMSO-d6)
d 279.95. HRMS (m/z): [M1H]1 calculated for C23H31F3N3O4S2, 533.1708;
found, 533.1706.
Synthesis of AMG-3969. A 2-l Erlenmeyer flask was charged with 2-piperazinone
(36.5g,364 mmol), sodiumcarbonate(116g,1.10mol),600 mldioxane,and150 ml
water at room temperature. To this was slowly added benzyl chloroformate (62.1 g,
364 mmol) over 20min. After the addition was complete, the mixture was stirred
for 2 h, diluted with water and extracted with 2 l of ethyl acetate. The combined
organic extractswere dried(MgSO4), filteredand concentratedtogivea white solid.
To this solid was added 500ml CH2Cl2, triethylamine (92.6 g, 128 ml, 911mmol),
4-dimethylaminopyridine (4.45 g, 36.4 mmol), and di-tert-butyl dicarbonate (119 g,
546 mmol). After 1h at room temperature, the mixture was diluted with water and
the organics were separated, dried (MgSO4), filtered and concentrated to give a
brown oil. To this oil was added 100 ml CH2Cl2 followed by 1 l hexane. The resulting
white solid was collected by filtration to give 4-benzyl 1-tert-butyl 2-oxo-1,4-
piperazinedicarboxylate (101 g, 81%). 1H-NMR (400 MHz, CDCl3) d 7.45–7.29
(m, 5 H), 5.15 (s, 2 H), 4.24 (s, 2 H), 3.88–3.74 (m, 2 H), 3.74–3.59 (m, 2 H), 1.54 (s, 9 H).
A 150-ml round-bottomed flask charged with a solution of 4-benzyl1-tert-butyl
2-oxo-1,4-piperazinedicarboxylate (1.41 g, 4.22 mmol) in 5 ml THF was cooled to
0uC. 1-propynylmagnesium bromide (0.5 M in THF, 20.0 ml, 10.0 mmol) was added
dropwise over 15 min. The mixture was stirred at 0 uC for 2 h and then saturated
aqueous NH4Cl (40ml) was added. The aqueous phase was extracted with ethyl
acetate (200 ml, then 23100ml). The organic extracts were dried (Na2SO4), filtered
and concentrated. The crude product was purified by silica gel chromatography
(0–50%ethylacetateinhexanes)toaffordbenzyl(2-((tert-butoxycarbonyl)amino)
ethyl)(2-oxo-3-pentyn-1-yl)carbamate (1.55 g, 99%) as a clear oil. 1H-NMR (400 MHz,
CDCl3) d 7.39–7.27 (m, 5 H), 5.13 (d, J 5 19.4 Hz, 2 H), 4.97 (d, J 5 3.9 Hz, 1 H),
4.18 (s, 1 H), 4.16–4.08 (m, 1 H), 3.44 (td, J 5 5.8, 11.7 Hz, 2 H), 3.32–3.16 (m, 2 H),
2.00 (d, J 5 13.3 Hz, 3 H), 1.42 (d, J 5 4.7 Hz, 9 H).
METHODS
Preparation of recombinant GK and GKRP. RecombinanthumanGK (hepatic),
native or with an N-terminal AviTag (avi), was expressed in Escherichia coli and
purified and formulated in 50 mM Tris, pH 7.5, 150mM NaCl and 20 mM glucose.
A 1:1 ratio of 5-bromomethylfluorescein (Invitrogen) and GK was prepared and
the reaction mixture incubated overnight at 4 uC in the dark. Unreacted dye was
removed by dialysis into formulation buffer (25 mM Tris, pH7.2, 150mM NaCl,
10% glycerol and 2 mM dithiothreitol (DTT)). Human native GKRP (full-length
or 20–625 truncation) or with a C-terminal AviTag was expressed in a baculovirus
system. Purified avi-GK or GKRP-avi were incubated overnight with recombinant
biotin ligase in the presence of D-biotin, ATP and MgCl2. Labelled proteins were
isolated from reaction components by chromatography on a Superdex 200 size-
exclusion column into 50 mM HEPES, 150mM NaCl and 5 mM DTT. Untagged
native proteins were isolated from their purification tags using caspase 3.
GK–GKRP LC–MS/MS assay. Direct substrate conversion by GK was measured
by a liquid chromatography tandem mass spectrometry (LC–MS/MS) assay that
monitoredmass shift of universally labelled13C-glucose-6-phosphate (2269R79 m/z).
The final reaction mixture included 300 mM fructose-6-phosphate, 5 mM 13C-
glucose (Sigma), 240 mM ATP, 50 mM Tris, pH 7.5, 4 mM MgCl2, 8% dimethyl-
sulphoxide (DMSO), 6.8 mM DTT, 0.001% Brij-35, 0.01% BSA, 6 nM GK, and
either a range (0.05–1,000 nM) of GKRP concentrations, or 28 nM GKRP plus a
range (0.009–20 mM) of AMG-1694 concentrations. The reaction was incubated
for 30 min at room temperature, stopped with acetonitrile, injected into Biobasic
AX column LC–MS/MS and then analyte peak area (counts) was determined.
EC50 values were calculated using GraphPad Prism software.
GK–GKRP binding assay. GKRP-biotin was incubated with AMG-1694 for 20 min
at 5 nM GKRP-biotin, 1 mM S6P, 20 mM Tris, pH 7.5, 0.05% BSA and 1 mM DTT.
Afterwards, 1 nM fluorescein-GK and a 1:1,000 dilution of AlphaScreen anti-
fluorescein (FITC) acceptor beads and streptavidin donor beads (PerkinElmer)
were added followed by an additional 4 h incubation. Using an EnVision Instru-
ment(PerkinElmer), GK–GKRPbinding was detected andIC50 valueswerecalcu-
lated using GraphPad Prism software.
Synthesis of AMG-1694. Unless otherwise noted, all reagents were commercially
available and used asreceived. 1H-, 13C-and19F-NMR data arereportedas follows:
chemical shift (p.p.m., d units) from an internal standard, multiplicity (s 5 singlet,
d 5doublet, t 5triplet, q 5 quartet, m5 multiplet, and br 5broad), coupling con-
stant (Hz), and integration.
A 2-l round-bottomedflaskwascharged with (S)-3-methylmorpholine(10.10 g,
100 mmol), HATU (39.7 g, 104 mmol), (R)-1,4-bis(tert-butoxycarbonyl)piperazine-
2-carboxylic acid (30.0 g, 91 mmol) and 100 ml dimethylformamide. To this was
added Hu¨nig’s base (20.3 ml, 114 mmol). After stirring at room temperature for
1 h, the mixture was diluted with water (1 l) and extracted with 750 ml of ether.
The organic layer was separated and washed with water (4 3 500 ml), saturated
aqueous NaHCO3 (250 ml) and brine (250 ml). The organic extracts were dried
(MgSO4), filtered and concentrated to give (2R)-2-(((3S)-3-methyl-4-morpholinyl)-
carbonyl)-1,4-piperazinedicarboxylate (33.8 g, 82% yield) as a white solid.
A 1-l round-bottomed flask was charged with (2R)-2-(((3S)-3-methyl-4-morpholinyl)
carbonyl)-1,4-piperazinedicarboxylate (33.8g, 82.0 mmol), and 100ml tetrahy-
drofuran (THF). To this was added borane (1 M in THF, 327ml, 327mmol).
The mixture was warmed to 50 uC for 2 h and then cooled to 0 uC and slowly
quenched with 100ml methanol. The mixture was concentrated and diluted with
200ml ethyl acetate.
To this residue wasadded 100ml 4 NHClin dioxane. The mixturewasheated at
70 uC for 2.5 h and the resulting white precipitate was collected by filtration to
provide the amine HCl salt. This solid was suspended in 200ml CH2Cl2 and
triethylamine (57.0 ml, 408mmol). After cooling to 0 uC, 2-thiophenesulphonyl
chloride (14.9 g, 82.0 mmol) was added. The mixture wasallowed to warm to room
temperature and stirred for 1 h. Water (200 ml) was then added and the mixture
was extracted with ethyl acetate. The combined organics were dried (MgSO4),
filtered and concentrated to give an oil. Purification via silica gel chromatography
(0–10% methanol in CH2Cl2) gave (3S)-3-methyl-4-(((2S)-4-(2-thiophenylsulphonyl)-
2-piperazinyl)methyl)morpholine (20.2g, 71% yield) as a white solid.
A 3-l round-bottomed flask was charged with 2-((tert-butoxycarbonyl)amino)
ethyl)(2-oxo-3-pentyn-1-yl)carbamate (82.2 g, 219 mmol) and 300 ml CH2Cl2. After
cooling to 210 uC, TFA (169 ml, 2,195 mmol) was added and the resulting dark
solution was stirred at room temperature for 15 min. Sodium triacetoxyborohy-
dride (186 g, 878 mmol) was added portion-wise over 10 min. After 2 h, the mix-
ture was concentrated, diluted with ethyl acetate (1 l), and neutralized with 5 N
NaOH. The layers were separated and the organic extracts were washed with
brine, dried (MgSO4), filtered and concentrated. The resulting orange oil was
purified via silica gel chromatography (0–4.5% methanol in CH2Cl2) to give
benzyl 3-(1-propyn-1-yl)-1-piperazinecarboxylate (43.7 g, 77%) as a brown foam.
1H-NMR (400 MHz, CDCl3) d 7.43–7.30 (m, 5 H), 5.28–5.04 (m, 2 H), 4.05–3.61
(m, 5 H), 3.37 (br s, 1 H), 3.03 (br s, 1 H), 1.79 (br s, 3 H) (one exchangeable proton
was not observed).
A 150-ml reactionvessel was charged withbenzyl 3-(prop-1-yn-1-yl)piperazine-
1-carboxylate (2.88 g, 11.2 mmol), 2-(4-bromophenyl)-1,1,1,3,3,3-hexafluoro-2-
propanol26 (4.36 g, 13.5 mmol), dicyclohexyl(29,69-diisopropoxy-[1,19-biphenyl]-2-yl)
phosphine(RuPhos)(0.530g, 1.14 mmol), chloro(2-dicyclohexylphosphino-29,69-
di-i-propoxy-1,19-biphenyl)[2-(2-aminoethylphenyl)]palladium(II), methyl-tert-
butylether adduct (RuPhos first generation precatalyst) (0.417g, 0.572mmol),
sodiumtert-butoxide (2.73g, 28.4 mmol), and toluene (35 ml). Argon was bubbled
through the solution for 10 min. The vessel was sealed and heated at 100 uC for
1.5 h. After the reaction was allowed to cool to room temperature, water (100 ml)
was added. The aqueous phase was extracted with ethyl acetate (33 100ml) and
the combined organic layers were washed with brine (150 ml), dried (Na2SO4),
filtered and concentrated. The crude product was purified by silica gel chromato-
graphy (0–50% ethyl acetate in hexanes) to afford benzyl 3-(1-propyn-1-yl)-4-(4-
(2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl)phenyl)-1-piperazinecarboxylate
(3.84 g, 69%) as a yellow solid. 1H-NMR (400 MHz, CDCl3) d 7.59 (d, J 5 8.6 Hz,
2 H), 7.44–7.29 (m, 5 H), 6.99 (d, J 5 9.0 Hz, 2 H), 5.32–5.08 (m, 2 H), 4.48–4.17
(m, 3 H), 3.50–3.02 (m, 5 H), 1.69 (br s, 3 H).
A 1-l pressure vessel was charged with (S)-3-methyl-4-(((S)-4-(thiophen-2-
ylsulphonyl)piperazin-2-yl)methyl)morpholine (10.50g, 30.4mmol), 100 ml tolu-
ene, (R)-2-(4-bromophenyl)-1,1,1-trifluoropropan-2-ol (ref. 25; 9.81g, 36.5mmol)
and sodium 2-methylpropan-2-olate (7.30 g, 76mmol). Oxygen-free nitrogen gas
wasbubbledthroughthe solutionfor3 min. RuPhosprecatalyst(1.11 g,1.52mmol)
and RuPhos (0.709 g, 1.52 mmol) were then added. The vessel was sealed and heated
at 60 uC for 12 h. The mixture was diluted with water and extracted with ethyl acetate;
A 500-ml round-bottomed flask was charged with benzyl 3-(1-propyn-1-yl)-4-
the combined organics were dried (MgSO4), filtered, concentrated and purified by (4-(2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl)phenyl)-1-piperazinecarboxylate
silica gel chromatography (0–70% ethyl acetate in hexanes) to deliver (R)-1,1, (3.13 g, 6.25 mmol) and TFA (40 ml). Trifluoromethanesulphonic acid (1.25 ml,
1-trifluoro-2-(4-((S)-2-(((S)-3-methylmorpholino)methyl)-4-(thiophen-2-ylsulphonyl)- 14.1 mmol) was added dropwise at room temperature. After 5 min, additional tri-
piperazin-1-yl)phenyl)propan-2-ol (AMG-1694) (8.15 g, 50% yield) as a tan foam. fluoromethanesulphonic acid (0.45 ml, 5.1 mmol) was added. After an additional
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