An Inexpensive and Scalable Synthesis of Shld

Article abstract of DOI:10.1021/acs.joc.8b00698

A synthesis of the important FKBP ligand Shld is reported. The synthesis avoids stoichiometric use of expensive and chiral reagents, maintains enantioselectivity and provides a high overall yield (39%). The main features in the method are enantioselective alkylation for preparation of the phenyl acetic acid moiety (building block A), catalytic enantioselective reduction for obtaining the chiral diaryl-1-propanol (building block C), and direct acylation of S-pipecolic tartrate rather than use of expensive Fmoc-pipecolic acid. The assembly of the building blocks A-C is reversed in comparison to previous synthesis, which also eliminates the need for protective groups.

Full text of DOI:10.1021/acs.joc.8b00698

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Article
An inexpensive and scalable synthesis of Shld
Frederik Præstholm Jørgensen, and Mikael Bols
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00698 • Publication Date (Web): 11 May 2018
Downloaded from http://pubs.acs.org on May 12, 2018
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An inexpensive and scalable synthesis of Shld
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Frederik Præstholm Jørgensen and Mikael Bols*
Department of chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen Ø,
Denmark
bols@chem.ku.dk
Graphical abstract (TOC)
Resolution
OMe
with tartaric acid
MeO
OMe
This work:
C
Enantioselective
alkylation
Shld
Shld
A+B
Corey-Bakshi-
39%
N
O
Shibata reduction
O
O
Shld
O
Previous:
(±)-A
OMe
OMe
N
+
C B
<16%
O
Abstract
A synthesis of the important FKBPꢀligand Shld (or Shieldꢀ1) is reported. The synthesis avoids
stoichiometric use of expensive and chiral reagents, maintains enantioselectivity and provides a
high overall yield (39%). The main features in the method are enantioselective alkylation for
preparation of the phenyl acetic acid moiety (building block A), catalytic enantioselective reduction
for obtaining the chiral diarylꢀ1ꢀpropanol (building block C) and direct acylation of Sꢀpipecolic
tartrate rather than use of expensive Fmocꢀpipecolic acid. The assembly of the building blocks A, B,
and C is reversed in comparison to previous synthesis, which also eliminates the need for protective
groups.
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Introduction
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Shld (or Shield1)1 is a synthetic simplification2,3 of the immunosuppressive drugs Rapamycin
and FK506 (Figure 1) that similarly to the parent molecules has affinity for FK binding proteins, but
differs by having specificity for a mutated binding site. More specifically, Shld binds to FKBP12
with the point mutation F36V with a 1000 fold selectivity over the wild type.4 Shld is used to
stabilize destabilized protein domains attached to a protein of interest – when Shld binds the
unstable protein domain it is stabilized, and the proteinꢀconjugate is not degraded. Shld can thus be
used to promote the production of a protein of interest in a genetically engineered cell and has been
used in mammalian cells,5 parasites6 and plants.7
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Figure 1. Structural similarities of Rapamycin and Shld. In Shld the building blocks from which it is
logically assembled have been colored. The similar fragments of Rapamycin are highlighted.
In connection with a project, where gram quantities of Shld were required, for use on plants it
was necessary to review the methods of Shld synthesis and evaluate their usefulness. While no
specific Shld synthesis has previously been published, synthesis of the building blocks8,9 and of
analogues10 such as the corresponding acid (the Nꢀmorpholinoethyl substituent being replaced with
carboxymethyl) has been reported (though not always with yields being given).3 Shld has
reportedly been made in a ‘similar fashion’ which was as follows: 1) The molecule was assembled
from building blocks A, B and C (Figure 1) with the ester being prepared first and subsequently the
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amide.3,11,12 2) Building block C was made by an aldol condensation with the stereocenter being
created by chiral reduction with excess (+)ꢀBꢀchlorodiisopinocamphenylborane ((+)ꢀDIPꢀCl).13 3)
Building block B was obtained from commercial but expensive FmocꢀLꢀpipecolic acid3 or Lꢀ
pipecolic acid methyl ester.14 4) Building block A was made from synthesis of the racemic acid
followed by resolution14 or attachment to the chiral BC fragment followed by chromatographic
separation of diastereomers.3
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Reviewing these methods it appeared that Shld synthesis could be made cheaper and more
efficient if the building blocks were assembled from A to C by a SchottenꢀBaumann like coupling
of acid A to pipecolic acid (B) followed by esterꢀcoupling of AꢀB to C. This would eliminate
protection steps. Secondly, DLꢀpipecolic acid is inexpensive and resolution with tartrate is possible
so this might be the source of B. Thirdly, replacing stoichiometric use of costly chiral reagent such
as (+)ꢀDIPꢀCl would also make the synthesis better suited for large scale use. Finally, the synthesis
of racemic A and the resulting loss of half of this comparatively expensive building block should be
replaced with an enantioselective synthesis. We here report a synthesis with the above changes and
modifications so that A and C are now made enantioselectively with reagents that are catalytic or
readily recycled. The overall yield of the synthesis is now 39%, where the previous synthesis was
below 16%.
Results and discussion
It was recently reported that phenylacetic acids could be αꢀalkylated with high enantiomeric
excess when chiral Kogaꢀbase 1 was used as a stoichiometric complexing agent.15 This appeared an
ideal methodology to improve the synthesis of building block A even if Kogaꢀbase is used
stoichiometrically, as it can readily be recovered from the reaction mixture by extraction.
Alkylation of 2 with 1.05 equivalent 1, 4 equivalents of butyl lithium and 4 equivalents of ethyl
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iodide gave 81% of 3 with 91% e.e as determined by HPLC. This is similar to what was observed
with mono methoxy substituted aryl acetic acids, which gave yields of 71ꢀ87% with an e.e. of 89ꢀ
93%.15 This acid could be converted to the acid chloride 4 in quantitative yield by heating to 40o C
with thionyl chloride in dichloromethane (Scheme 1).
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Scheme 1. Synthesis of AꢀB conjugate
Moldavi et al. described the resolution of pipecolic acid (5) using tartrate.16 We used this
method to obtain 6 from racemic 5 by mixing and dissolving with stoichiometric amounts of (ꢀ)ꢀ
tartaric acid in hot methanol allowing the salt to crystallize. In this manner we obtained 82% yield
of a levarotatory salt 6 with 84% e.e. as determined from rotation and after a recrystallization from
waterꢀacetone a 44% yield of a product with 96% e.e (Scheme 1). Functionalizing the pipecolic acid
moiety of the salt 6 proved a difficult task due to the zwitterionic character of the pipecolic acid and
the resulting poor solubility in organic solvents. Previously this problem has been dealt with by
converting the salt 6 into either the Fmoc protected amine17 or the pipecolic methyl ester18. In both
cases the zwitterionic character disappears and further functionalization is possible. Here we report
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a method in which protection or modification of the salt 6 can be avoided as direct acylation of 6
with 4 was performed by refluxing 22 h in 1,4ꢀdioxane with solid NaHCO3 present. This gave 66%
yield of 7, when the acid chloride 4 was used in excess. When the less expensive tartrate 6 was used
in excess 70% of 7 was obtained. The advantage of this comparatively slow procedure is that since
the tartrate is only slightly soluble, selective acylation of the amine occurs and no significant
acylation of tartaric acid is seen.
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Scheme 2. Synthesis of buildingꢀblock C
Synthesis of building block C was performed as shown in Scheme 2. Aldol condensation
between 8 and 9 to adduct 10 and subsequent selective reduction was performed essentially as
previously described13,14 giving 85% and 68% yields, respectively. CoreyꢀBakshiꢀShibata reduction
of 11 using boraneꢀdimethylsulfide and 10 mol% methyl oxazaborolidene catalyst gave 98% of the
Sꢀalcohol 12 with 97% e.e. The optical purity was determined by HPLC. Finally, alkylation of the
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phenol in 12 using K2CO3 and Nꢀ(2ꢀchloroethyl) morpholine gave 13 in quantitative yield (Scheme
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2).
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The coupling of 7 and 13 to give Shld (Scheme 3) was a reaction potentially prone to give
epimerization in the pipecol residue due to the risk of formation of oxazolidinone 14 during the
coupling step.19 This problem is proportional with propensity of the substituent on nitrogen to
deliver neighboring group assistance during the peptide formation and is therefore higher when that
substituent is an amide (as in 7) rather than a carbamate such as is normally employed in peptide
couplings. A number of different coupling conditions were attempted in order to minimize
epimerization (Table 1). Coupling 7 with 13 using of 1ꢀ(mesitylenesulfonyl)ꢀ3ꢀnitroꢀ1,2,4ꢀtriazole
(MSNT) and Nꢀmethylimidazole gave a high yield of Shld, but as a 1:1 mixture of diastereomers
(entry 1). Coupling with HATU or PyBOP gave a slow and lowꢀyielding reaction and considerable
(2:1) epimerization (entries 2ꢀ3). Inspired by the work by Holt et al.20 we also tried Yamaguchi
lactonization conditions (entry 4), but this gave low yield and complete epimerization.
Scheme 3. Conjugation of AB and C to Shld
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The high degree of epimerization in these reactions could very well be caused by the use of the
relatively strong Hünig’s base (DIPEA, EtNiPr2). An attempt to convert 7 to the acid chloride and
couple that to 13 in the presence of base failed to give any product (entry 5).
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We then tried a series of carbodiimide coupling reactions (Table 1). Dicyclohexyl carbodiimide
(DCC) with catalytic 4ꢀdimethylaminopyridine (DMAP, entry 6), with Nꢀhydroxybenzotriazole
(HOBt) and collidine (entry 7) or with hydroxysuccinimide (HOSu, entry 8) all gave meagre yields
and considerable epimerization. In the reaction with HOSu, activated OꢀSuccinimide ester was
isolated in 82% yield, which could be reacted with compound 13 (alkohol) in the precence of 2
equiv. DMAP and triethylamine giving Shld. Counterintuitively the best results was achieved from
DCC with excess DMAP, which afforded 57% yield of Shld and neglible epimerization (entry 9).
It is likely that with sufficient quantity of DMAP, neighboring group effect from the amide and the
accompanying epimerization via 14, can no longer compete with the faster esterification. With a
change of DCC to diisopropyl carbodimide (DIC) the yield was increased to 69% and epimerization
was still low (entry 10). Repeated in a larger scale 70% yield of Shld was obtained (Scheme 3).
Table 1. Results of a variety of ester couplings of 7 and 13 to form Shld.
Entrya
Reagent (equiv.)
MSNT (1.1)
HATU (1.5)
Additive (equiv.)
Nꢀmethyl imidazole (3)
DIPEA (2)
Time (h) Yield 2S/2R ratio
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2
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96
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90%
28%
24%
20%
0%
1:1
1:0.55
1:0.62
1:1
PyBOP (1.5)
DIPEA (2)
DIPEA (2)
DIPEA
DMAP (0.1)
2,4,6ꢀCl3C6H2COCl (1.2)
Thionyl chloride (5)
DCC (1.1)
ꢀ
ꢀ
3
81%
15%
8%
57%
69%
1:0.23
1:1.35
1:0.4
1:0.03
1:0.02
DCC (1.1)
DCC (1.1)
DCC (1.1)
DIC (1.1)
HOBt (1.1) + collidine (2)
HOSu (2)
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3
DMAP (2)
DMAP (2)
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3
a) All reaction were started at 0oC and left to run at room temperature in dichloromethane, except entry
7 which was run similarly in DMF. The diastereomeric ratio was determined by NMR integration of
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selected isolated peaks from Shld and compared to the corresponding peak in the epimerized
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product.
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This completes the Shld synthesis, and the overall yield can be evaluated. Since it is a
convergent synthesis it can be calculated from 2, from (±)ꢀ5 or from 8/9, which gives respectively
39% (from 2, 81% x 99% x 70% x 70%), 10% (from (±)ꢀ5, ½ x 44% x 66% x 70%) or 39% (from
8/9, 85% x 68% x 98% x 99% x 70%). Since 2 is by far the most expensive building block for
Shld21 the yield based on 2, 39%, is the most meaningful. The most related synthesis in the
literature (where yields are recorded) use alkylation of 2 to racemic 3, resolution, coupling to
methyl pipecolate, hydrolysis and coupling to dimeric C gave 7% overall yield of Shld related
dimer (from 2, 81% x 21% x 73% x 59%),14 which makes the present method a improvement based
on use of 2. Our use of (±)ꢀ5 is much less efficient (10% overall yield), and (±)ꢀ5 is admittedly a
comparatively expensive building block. However all previous synthesis have used (-)-5, methyl Lꢀ
pipecolate or Fmoc Lꢀpipecolate in lieu of (±)-5 which have prices that presently are 3ꢀ400 times
more expensive. So on the use of pipecolic acid our syntheses is also more costꢀeffective. Finally,
this synthetic approach is convenient by having only three chromatographic purifications.
Compounds 3, 13 and Shld were purified by flash column chromatography, 12 was used crude
while the remaining compounds were purified by crystallization.
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By NMR Shld is observed to have a full set of duplicated peaks in a 1:3 ratio, indicating that the
compound consists of two stable conformations most likely due to rotamer formation around the
tertiary amide. To confirm this, a VT (variable temperature) NMR experiment was performed in
tetrachloroethaneꢀd2. Upon heating the sample the duplicated peaks merged confirming the
existence of rotamers (see Supporting Information). Only small amounts of diastereoisomer (<3%)
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is observed by NMR, while LCMS analysis only gave one UV/Vis trace with a mass corresponding
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to Shld (see Supporting Information).
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Experimental
General information.
Air and water sensitive reactions were carried out under nitrogen (balloon technique). All
commercial available chemicals and solvents were used as received. 1H NMR spectra was measured
on a Bruker instrument with cryoꢀprobe at 500 MHz. 13C NMR was measured on the same
instrument but at 126 MHz. NMR solvents used were CDCl3 (referenced to δH = 7.26 ppm and δC =
77.16), D2O (referenced to δH = 4.79 ppm) and DMSOꢀd6 (referenced to δH = 2.50 ppm and δC =
39.52). Coupling constants (J) was given in Hertz (Hz). CDCl3 was passed through activated Al2O3
(basic) prior to use. Anhydrous solvents were collected from an IT (Innovative Technology)
installation of the model PSꢀMDꢀ05. Thinꢀlayer chromatography (TLC) was performed on
precoated (silica 60) aluminum plates with fluorescence indicator. Flash column chromatography
was carried out on silica (SiO2) with particle size of 40ꢀ63 µm from ROTH. Chiral HPLC (High
Performance Liquid Chromatography) was performed on a Shimadzu Prominence LCꢀ20AD with
SPDꢀ20A UV/Vis detector and a chiral Kromasil 5ꢀAmyCoat 4.6 x 250 mm column. Optical
rotation was measured on an Anton Paar MCP 300 polarimeter with a 50 x 5 mm cuvette. UPLCꢀ
MS (Ultra Performance Liquid Chromatography ꢀ Mass Spectrometry) was performed on a Dionex
UltiMate 3000 RS with an AcclaimTM RSLC 120 C18 column (2.2 µm, 120 Å, 2.1 x 100 mm)
connected to a Bruker micrOTOF QꢀIII mass spectrometer. High resolution mass spectrometry
(HRꢀMS) was carried out on a FTꢀICR spectrometer using either matrix assisted laser desorption
ionization (MALDI) with dithranol as matrix or electrospray ionization (ESI+) with methanol + 1%
TFA. Melting points are uncorrected.
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N1,N3-bis-(R)-1-phenyl-2-(piperidin-1-yl)ethyl)propane-1,3-diamine (1)22
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To a stirred solution of (R)ꢀstyrene oxide (19.0 mL, 166 mmol) in abs. EtOH (100 mL),
piperidine (19.3 mL, 195 mmol) was added, and the solution was heated to reflux and left at this
temperature for 5 hours. The reaction mixture was cooled to ambient temperature, and concentrated
in vacuo. The residue was dissolved in toluene (25 mL) and concentrated in vacuo (azeotropic
distillation). This distillation was performed two additional times (2 x 25 mL). Methyl tertꢀbutyl
ether (20 mL) and Et3N (85 mL, 609 mmol) was added to the solids, and the suspension was stirred
for 10 min, allowing the solids to dissolve. The contents of the reaction vessel were cooled to ‒10
ºC, and then methanesulfonyl chloride (15.4 mL, 199 mmol) was added dropwise over 1 hour. The
reaction mixture was left stirring for 30 min at ‒10 ºC, before addition of diaminopropane (7.0 mL,
83.9 mmol) and water (60 mL). The reaction mixture was allowed to warm to ambient temperature
and left stirring at this temperature for 30 hours. The phases were separated and the organic phase
washed with sat. aq. NH4Cl followed by a wash with brine. The organic phase was dried with
NaSO4, filtered and concentrated in vacuo. The residual oil was dissolved in iꢀPrOH (80 mL) and
water was added slowly until a suspended precipitation persists. The suspension was stored in the
freezer overnight and the resulting crystals was collected and washed with cold iꢀPrOH/water (1:1).
The crystals were dried in a desiccator under vacuum over P2O5 for 3 days, yielding 1 as colorless
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1
crystals (22.0 g, 59%). [ꢀ]ꢂꢁꢃ − 113 (c = 1.1, CHCl3). H NMR (500 MHz, CDCl3): δ 7.36 – 7.33
(m, 4H), 7.32 – 7.28 (m, 4H), 7.24 – 7.20 (m, 2H), 3.72 (dd, J = 11.0, 3.4 Hz, 2H), 2.59 – 2.15 (m,
18H), 1.71 – 1.62 (m, 2H), 1.61 – 1.35 (m, 12H) ppm. 13C NMR (126 MHz, CDCl3): δ 143.5, 128.4,
127.5, 127.1, 66.7, 60.4, 54.8, 46.5, 30.7, 26.3, 24.7 ppm. HRMS (MALDI/FTICRꢀTOF) m/z: [M +
+
H]+ Calcd for C29H45N4 449.3639; Found 449.3650.
(S)-2-(3,4,5-trimethoxyphenyl)butanoic acid (3)15
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To a stirring solution of 2,3,4ꢀtrimethoxyphenyl acetic acid (2, 1.50 g, 6.63 mmol) and 1 (3.15
g, 7.03 mmol) in anhydrous THF (10 mL) at 0 ºC was added dropwise nꢀBuLi (2.5 M in hexane,
10.6 mL, 26.5 mmol). After addition the reaction mixture was left stirring at 0 ºC for 15 min, then
cooled to ꢀ78 ºC and left at this temperature for 5 min before adding ethyl iodide (2.1 mL, 26.1
mmol). The reaction mixture was left at ꢀ78 ºC for 2.5 hours, then quenched with MeOH (2 mL)
and allowed to heat to ambient temperature. The resulting pale yellow solution was partitioned
between 1 M aq. HCl (75 mL) and EtOAc (50 mL). The phases were separated and the aquous
phase was extracted 3 times with EtOAc (3 x 50 mL). The combined organic phases were washed
with 10% aq. Na2S2O3 followed by a wash with brine, dried over MgSO4, filtered and concentrated
in vacuo. The crude residue was purified by flash column chromatography (40% EtOAc in toluene
+ 1% formic acid) to yield 3 as colorless crystalline solid (1.36 g, 81%, 91% ee). [ꢀ]ꢂꢁꢃ + 54.1 (c =
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1.05, MeOH). Mp 80 – 82 °C. H NMR (500 MHz, CDCl3): δ 6.53 (s, 2H), 3.85 (s, 6H), 3.83 (s,
3H), 3.38 (t, J = 7.7 Hz, 1H), 2.15 – 2.02 (m, 1H), 1.86 – 1.74 (dp, J = 13.6, 7.4 Hz 1H), 0.93 (t, J =
7.4 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3): δ 179.3, 153.4, 137.5, 134.1, 105.2, 61.0, 56.3,
+
53.5, 26.6, 12.3 ppm. HRMS (ESI/FTICRꢀTOF) m/z: [M + H]+ Calcd for C13H19O5 255.1227;
Found 255.1234. Ee: 91% (determined by chiral HPLC (heptane (0.1% TFA)/iꢀPrOH, 0.9:0.03),
flow rate: 0.93 mL/min)
(S)-2-carboxypiperidin-1-ium (2S,3S)-3-carboxy-2,3-dihydroxypropanoate (6) 16
To a boiling solution of (±)ꢀpiperidineꢀ2ꢀcarboxylic acid (5, 10.01 g, 77.4 mmol) in methanol
(150 mL) was added Dꢀ(‒)ꢀtartaric acid (11.7 g, 78.0 mmol). The solution was heated for 5 min, and
then filtered hot. To the cooled solution was added one grain of 6, and the solution was left at 5 ºC
overnight. The resulting crystals was filtered and washed with cold methanol to yield 6 as colorless
crystals (8.82 g, 82%). [ꢀ]ꢂꢁꢃ −17.2 (c = 2.0, CHCl3). The colorless crystals were transfered to a
flask equipped with a condenser. H2O/acetone (12 mL, 1:2) was added and the suspension was
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heated to 60 ºC. After 20 min the crystals were dissolved and acetone (25 mL) was added dropwise
over 1.5 hours from an addition funnel on top of the condenser. Once addition was complete the
suspension was cooled to ambient temperature, filtered, and washed with 80% wet acetone to yield
6 as a white crystalline solid (4.8 g, 44%). [ꢀ]ꢂꢁꢃ −19.7 (c = 1.6, CHCl3). 1H NMR (500 MHz,
D2O): δ 4.69 (s, 2H), 3.70 (dd, J = 11.6, 3.4 Hz, 1H), 3.47 – 3.38 (m, 1H), 3.06 – 2.97 (m, 1H), 2.28
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– 2.19 (m, 1H), 1.93 – 1.81 (m, 2H), 1.74 – 1.53 (m, 3H) ppm. C NMR (126 MHz, D2O, DMSOꢀ
d6 lock tube): δ 174.1, 172.7, 71.2, 57.4, 42.8, 25.3, 20.7, 20.5 ppm.
(S)-2-(3,4,5-trimethoxyphenyl)butanoyl chloride (4)
To a solution of 3 (1.89 g, 7.41 mmol) in anhydrous CH2Cl2 (15 mL) was added thionyl
chloride (5.0 mL, 69 mmol) and the reaction mixture was heated to reflux point and stirred at this
temperature for 2 hours. The volatiles were removed in vacuo to yield 4 as yellow oil (2.05 g,
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quantitative). The crude oil was used without further purification. H NMR (500 MHz, CDCl3): δ
6.47 (s, 2H), 3.87 (s, 6H), 3.85 (s, 3H), 3.79 (t, J = 7.6 1H), 2.24 – 2.13 (m, 1H), 1.90 – 1.79 (m,
1H), 0.96 (t, J = 7.4 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3): δ 175.1, 153.7, 138.1, 131.4,
105.5, 65.5, 61.0, 56.3, 26.9, 11.9 ppm. HRMS (ESI/FTICRꢀTOF) m/z: [M + H]+ Calcd for
+
C13H18ClO4 273.0888; Found 273.0891.
(S)-1-((S)-2-(3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylic acid (7)
To a refluxing suspension of 6 (3.90 g, 14.0 mmol) and NaHCO3 (5.00 g, 59.0 mmol) in
anhydrous 1,4ꢀdioxane (200 mL) was added a solution of 4 (2.05 g, 7.41 mmol) in anhydrous 1,4ꢀ
dioxane (5 ml). The reaction mixture was refluxed for 22 hours, then cooled to ambient temperature
and partitioned between 1 M aq. HCl (100 mL) and EtOAc (100 mL). The phases were separated
and the aqueous phase extracted two additional times with 100 mL EtOAc. The combined organic
phases were washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The crude
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white solid was dissolved in 20 mL boiling MeOH and refrigerated overnight. The resulting crystals
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was filtered and washed with cold MeOH to yield 7 as a white crystalline solid (1.89 g, 70%).
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[ꢀ]ꢂꢁꢃ + 7.3 (c = 0.85, DMSO). Mp 176 – 178 °C (lit.14: 173.5 – 174 °C). H NMR (500 MHz,
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CDCl3, mixture of rotamers 0.25:1, A:B): δ 6.44 (s, 0.4H, A), 6.42 (s, 1.6H, B), 5.38 – 5.34 (m,
0.8H, B), 4.69 (d, J = 5.6 Hz, 0.2H, A), 4.65 (d, J = 13.6 Hz, 0.2H, A) 3.85 – 3.83 (br s, 1.8H, A),
3.82 (s, 2.4H, B), 3.81 (s, 4.8H, B), 3.78 – 3.75 (m, 0.8H, B), 3.58 (t, J = 7.1 Hz, 0.8H, B), 3.36 (t, J
= 7.1 Hz, 0.2H, A), 2.87 – 2.80 (m, 0.8H, B), 2.65 (td, J = 13.6, 2.2 Hz, 0.2H, A), 2.29 – 2.23 (m,
0.8H, B), 2.15 – 2.04 (m, 1H, A + B), 2.00 (d, J = 14.2 Hz, 0.2H, A), 1.78 – 1.56 (m, 3.8H, A + B),
1.51 – 1.33 (m, 1.8H, A + B), 1.26 – 1.22 (m, 0.2H, A), 1.07 – 0.98 (m, 0.2H, A), 0.92 (t, J = 7.3
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Hz, 2.4H, B), 0.87 (t, J = 7.3 Hz, 0.6H, A) ppm. C NMR (126 MHz, CDCl3, mixture of rotamers
1:4, A:B): δ 176.3 (A), 175.8 (B), 173.7 (B), 172.5 (A), 153.7 (A), 153.4 (B), 137.1 (A), 136.8 (B),
136.1 (A), 135.3 (B), 104.9 (B), 104.7 (A), 61.0 (A), 61.0 (B), 56.4 (A), 56.2 (B), 55.6 (A), 52.3
(B), 51.4 (A), 51.3 (B), 43.7 (B), 39.8 (A), 28.5 (A), 28.4 (B), 26.7 (A), 26.5 (B), 25.2 (B), 24.6
(A), 21.0 (A), 20.9 (B), 12.7 (B), 12.6 (A) ppm. HRMS (MALDI/FTICRꢀTOF) m/z: [M + H]+
+
Calcd for C19H28NO6 366.1911; Found 366.1908.
(E)-3-(3,4-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (10)13
To a solution of 3,4ꢀdimethoxyphenylbenzaldehyde (14.35 g, 86.4 mmol) and 3ꢀ
hydroxyacetophenone (11.56 g, 84.9 mmol) in EtOH (200 mL) at 0 °C was added a solution of
KOH (19.5 g, 348 mmol) in H2O (100 mL) and the reaction mixture was stirred overnight. The
reaction mixture was acidified with conc. HCl and then partitioned between 1 M aq. HCl (100 mL)
and EtOAc/Et2O (100 mL, 1:3) and the phases were separated. The aquous phase was extracted
with additional EtOAc/Et2O (100 mL 1:3). The combined organic phases were washed with brine,
dried over MgSO4, filtered and concentrated in vacuo. The crude product was dissolved in Et2O (40
mL) and put in the freezer overnight. The resulting solid was collected affording 10 as a yellow
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solid (21.18 g, 86%). Mp 140 – 142 °C (lit.23: 140 – 142 °C). H NMR (500 MHz, CDCl3): δ 7.76
(d, J = 15.6 Hz, 1H), 7.66 (dd, J = 2.5, 1.5 Hz, 1H), 7.55 (ddd, J = 7.7, 1.5, 0.9 Hz, 1H), 7.36 (d, J =
15.6 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.22 (s, 1H), 7.20 (dd, J = 8.3, 1.8 Hz, 1H), 7.12 (d, J = 1.8
Hz, 1H), 7.12 (ddd, J = 7.7, 2.5, 0.9 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 3.92 (s, 3H), 3.91 (s, 3H)
ppm. 13C NMR (126 MHz, CDCl3): δ 191.2, 156.8, 151.7, 149.3, 145.9, 139.8, 129.9, 127.8, 123.5,
120.8, 120.5, 120.0, 115.4, 111.3, 110.4, 56.11, 56.09 ppm.
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3-(3,4-dimethoxyphenyl)-1-(3-hydroxyphenyl)propan-1-one (11)14
To a suspension of Pd/C (0.6 g, 10 wt%, 0.56 mmol) and NH4HCO3 (1.99 g, 31.5 mmol) in
MeOH (10 mL) was added a solution of 10 (6.01 g, 21.1 mmol) in MeOH (150 mL). The resulting
black suspension was heated to reflux point and left at this temperature for 3 hours. After cooling to
ambient temperature, the reaction mixture was filtered through a pad of celite and the filtrate was
concentrated in vacuo until the volume was reduced to approximately 40 mL. The solution was
stored in the freezer overnight, and the formed precipitate was filtered off and washed with cold
MeOH to yield 11 as an offꢀwhite solid (4.1 g, 68%). Mp 119 – 121 °C (lit.23: 116 – 118 °C). H
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NMR (500 MHz, CDCl3): δ 7.52 – 7.49 (m, 2H), 7.32 (t, J = 8.1 Hz, 1H), 7.08 – 7.05 (m, 1H), 6.81
– 6.75 (m, 3H), 5.91 (br s, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.26 (t, J = 7.6 Hz, 2H), 3.01 (t, J = 7.6
Hz, 2H) ppm. 13C NMR (126 MHz, CDCl3): δ 199.8, 156.4, 149.0, 147.5, 138.4, 133.9, 130.1,
120.8, 120.6, 120.3, 114.7, 112.0, 111.5, 56.1, 56.0, 41.0, 30.0 ppm. HRMS (MALDI/FTICRꢀTOF)
m/z: [M ꢀ e]+ Calcd for C17H18O4+● 286.1200; Found 286.1198.
(R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenol (12)24
To a solution of (S)ꢀ1ꢀmethylꢀ3,3ꢀdiphenyltetrahydroꢀ1H,3Hꢀpyrrolo[1,2ꢀc][1,3,2]oxazaborole
(145 mg, 0.523 mmol) in anhydrous THF (20 mL) was added BH3SMe2 (2 M in THF, 0.26 mL,
0.520 mmol). To the resulting mixture separate solutions of 11 (1.50 g, 5.24 mmol) in anhydrous
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THF (17 mL) and BH3 SMe2 (2M in THF, 4.0 mL, 8.0 mmol) were added simultaneously over a
periode of 1.5 hours via syringe pumps. After the addition was complete the reaction mixture was
left stirring 30 min before slowly adding MeOH (2 mL). The solution was partitioned between 0.5
M aq. HCl (30 mL) and EtOAc (40 mL) and the organic phase was washed with water followed by
brine. The organic phase was dried over MgSO4, filtered and concentrated in vacuo. The crude
product was purified by flash column chromatography (30% EtOAc in CH2Cl2) to yield 12 as an
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oily solid (1.48 g, 98%). [ꢀ]ꢂꢁꢃ + 2.2 (c = 2.1, DMSO). H NMR (500 MHz, CDCl3): δ 7.16 (t, J =
7.8 Hz, 1H), 6.87 – 6.85 (m, 1H), 6.83 – 6.81 (m, 1H), 6.76 – 6.73 (m, 2H), 6.70 – 6.66 (m, 2H),
6.46 (s, 1H), 4.60 (dd, J = 7.7, 5.4 Hz, 1H), 3.82 (s, 3H), 3.81 (s, 3H), 2.69 – 2.54 (m, 2H), 2.10 –
1.91 (m, 2H) ppm. 13C NMR (126 MHz, CDCl3): δ 156.3, 148.9, 147.3, 146.3, 134.4, 129.8, 120.4,
118.3, 114.9, 112.9, 112.0, 111.5, 74.0, 56.04, 55.95, 40.4, 31.7 ppm. HRMS (MALDI/FTICRꢀ
TOF) m/z: [M ꢀ e]+ Calcd for C17H20O4+● 288.1356; Found 288.1354.
(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-morpholinoethoxy)phenyl)propan-1-ol (13)
To a suspension of 12 (1.50 g, 5.20 mmol) and K2CO3 (2.90 g, 21.0 mmol) in anhydrous DMF
(30 mL) was added Nꢀ(2ꢀchloroethyl)morpholine hydrochloride (1.02 g, 5.46 mmol) and the
resulting mixture was heated to 90 ºC for 3 hours before it was concentrated in vacuo and purified
by flash column chromatography (40% acetone in toluene + 1% Et3N) to yield 13 as an offꢀwhite
oil (2.08 g, 99%). [ꢀ]ꢂꢁꢃ − 0.4 (c = 2.0, MeOH). 1H NMR (500 MHz, CDCl3): δ 7.26 – 7.23 (m,
1H), 6.94 – 6.91 (m, 2H), 6.84 – 6.81 (m, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.73 (dd, J = 8.0, 2.0 Hz,
1H), 6.71 (d, J = 2.0 Hz, 1H), 4.68 – 4.64 (m, 1H), 4.12 (t, J = 5.7 Hz, 2H), 3.86 (s, 3H), 3.86 (s,
3H), 3.75 – 3.72 (m, 4H), 2.80 (t, J = 5.7 Hz, 2H), 2.74 – 2.60 (m, 2H), 2.60 – 2.55 (m, 4H), 2.14 –
1.96 (m, 2H), 1.87 (d, J = 2.9 Hz, 1H) ppm. 13C NMR (126 MHz, CDCl3): δ 159.1, 149.0, 147.4,
146.5, 134.5, 129.7, 120.3, 118.6, 113.8, 112.3, 111.9, 111.4, 73.9, 67.1, 66.0, 57.8, 56.1, 56.0,
54.3, 40.8, 31.8 ppm. HRMS (MALDI/FTICRꢀTOF) m/z: [M + H]+ Calcd for C23H32NO5
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402.2275; Found 402.2285. Ee: 97% (determined by chiral HPLC (heptane (0.1% Et2NH)/iꢀPrOH,
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0.7:0.13), flow rate: 0.83 mL/min)
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(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-morpholinoethoxy)phenyl)propyl (S)-1-((S)-2-(3,4,5-
trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (Shld)
To a solution of 13 (708 mg, 1.76 mmol), 7 (698 mg, 1.91 mmol) and 4ꢀ
(dimethylamino)pyridine (431 mg, 3.53 mmol) in anhydrous CH2Cl2 (10 mL) at 0 ºC was added
N,N’ꢀdiisopropylcarbodiimide (0.3 mL, 1.94 mmol). The reaction mixture was allowed to heat to
ambient temperature over a periode of 1 hour and left an additional 2 hours at this temperature. The
reaction mixture was washed with water (5 x 100 mL) and brine (100 mL) before the organic phase
was concentrated in vacuo. The crude product was purified by flash column chromatography (20%
acetone in toluene + 1% Et3N) to yield Shld as an offꢀwhite oil (920 mg, 70%). [ꢀ]ꢂꢁꢃ + 23.7 (c =
0.9, MeOH). 1H NMR (500 MHz, CDCl3, mixture of rotamers 0.3:1, A:B): δ 7.31 – 7.27 (m, 0.23H,
A), 7.18 – 7.13 (m, 0.77H, B), 6.95 – 6.92 (m, 0.23H, A), 6.89 – 6.83 (m, 0.46H, A), 6.79 – 6.76
(m, 2.54H, A + B), 6.70 – 6.67 (m, 0.23H, A), 6.66 – 6.63 (m, 1.77H, A + B), 6.62 – 6.58 (m,
0.77H, B), 6.41 (s, 0.46H, A), 6.40 (s, 1.54H, B), 5.80 (dd, J = 7.5, 6.2 Hz, 0.23H, A), 5.60 (dd, J =
8.1, 5.5 Hz, 0.77H, B), 5.48 – 5.45 (m, 0.77H, B), 4.65 – 4.58 (m, 0.46H, A), 4.13 – 4.08 (m, 2H, A
+ B), 3.86 (s, 0.69H, A), 3.85 (s, 0.69H, A), 3.85 (s, 2.31H, B), 3.85 (s, 2.31H, B), 3.84 – 3.83 (m,
2.08H, A), 3.80 – 3.78 (m, 3.08H, B), 3.74 – 3.71 (m, 4H, A + B), 3.69 (s, 4.62H, B), 3.57 (dd, J =
8.0, 6.4 Hz, 0.77H, B), 3.31 – 3.28 (m, 0.23H, A), 2.86 – 2.81 (m, 0.77H), 2.81 – 2.77 (m, 2H, A +
B), 2.59 – 2.56 (m, 4H, A + B), 2.55 – 2.51 (m, 1.23H, A + B), 2.49 – 2.42 (m, 0.77H, B), 2.33 –
2.27 (m, 0.77H, B), 2.27 – 2.22 (m, 0.23H, A) 2.12 – 2.03 (m, 2.23H, A + B), 1.97 – 1.89 (m,
0.77H, B), 1.74 – 1.63 (m, 3H, A + B), 1.61 – 1.53 (m, 1H, A + B), 1.48 – 1.37 (m, 1H, A + B),
1.33 – 1.28 (m, 1H, A + B), 1.11 – 1.03 (m, 0.23H, A), 0.90 (t, J = 7.3 Hz, 2.31H, B), 0.87 (t, J =
7.3 Hz, 0.69H, A) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:3, A:B): δ 172.7 (B),
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172.5 (A), 170.72 (B), 170.65 (A), 159.1 (A), 158.9 (B), 153.6 (A), 153.3 (B), 149.1 (A), 149.0 (B),
147.6 (A), 147.5 (B), 142.0 (B), 141.5 (A), 137.1 (A), 136.8 (B), 136.2 (A), 135.5 (B), 133.6 (B),
133.4 (A), 129.9 (A), 129.7 (B), 120.3 (B), 120.2 (A), 119.2 (A), 118.6 (B), 114.3 (A), 114.0 (B),
113.3 (A), 112.9 (B), 111.9 (B), 111.8 (A), 111.5 (A), 111.4 (B), 105.1 (B), 104.7 (A), 76.9 (A),
76.1 (B), 67.08 (B), 67.06 (A), 66.0 (A), 65.8 (B), 61.0 (A), 60.9 (B), 57.84 (B), 57.81 (A), 56.4
(A), 56.1 (B), 56.08 (A), 56.07 (B), 56.00 (A), 55.98 (B), 55.90 (A), 54.27 (A), 54.26 (B), 52.2 (B),
51.4 (A), 51.0 (B), 43.5 (B), 39.8 (A), 38.4 (B), 38.1 (A), 31.6 (A), 31.4 (B), 28.6 (A), 28.5 (B),
27.0 (B), 26.7 (A), 25.5 (B), 24.6 (A), 21.1 (B), 20.9 (A), 12.8 (A), 12.7 (B) ppm. HRMS
(MALDI/FTICRꢀTOF) m/z: [M + H]+ Calcd for C42H57N2O10+ 749.4008; Found 749.3997.
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Supporting information
Supporting information containing NMR spectra of all compounds, VT NMR data for Shld
together with chromatograms of Shld, 13 and 3 is available.
Acknowledgement
We thank the Danish national research council for production and technology and the
University of Copenhagen for financial support.
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References and notes
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1 MaynardꢀSmith, L. A.; Chen, L.ꢀC.; Banaszynski, L. A.; Ooi, A. G. L.; Wandless, T. J. A Directed Approach for
Engineering Conditional Protein Stability Using Biologically Silent Small Molecules. J. Biol. Chem. 2007, 282,
24866ꢀ24872.
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2 Clackson, T.; Yang, W.; Rozamus, L. W.; Hatada, M.; Amara, J. F.; Rollins, C. T.; Stevenson, L. F.; Magari, S. R.;
Wood, S. A.; Courage, N. L.; Lu, X.; Cerasoli, F.; Gilman, M.; Holt, D. A. Redesigning an FKBP–ligand interface to
generate chemical dimerizers with novel specificity. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 10437–10442.
3 Yang, W.; Rozamus, L. W.; Narula, S.; Rollins, C. T.; Yuan, R.; Andrade, L. J.; Ram, M. K.; Phillips, T. B.; van
Schravenkijk, M. R.; Dalgarno, D.; Clackson, T.; Holt, D. A. Investigating ProteinꢀLigand Interactions with a Mutant
FKBP Possessing a Designed Specificity Pocket. J. Med. Chem. 2000, 43, 1135–1142.
4 Lampson, M. A.; Kapoor, T. M. Targeting Protein Stability with a Small Molecule. Cell 2006, 126, 827ꢀ829.
5 Banaszynski, L.A.; Chen, L.C.; MaynardꢀSmith, L.A.; Ooi, A.G.; Wandless, T.J. A Rapid, Reversible, and Tunable
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21 Prices are presently: 2: ~ 6 $/g, (±)ꢀ5: ~ 2.3 $/g, 8: ~ 1 $/g, 9: ~ 0.1 $/g
22 Frizzle, M. J.; Caille, S.; Marshall, T. L.; McRae, K.; Nadeau, K.; Guo, G.; Wu, S.; Martinelli, M. J.; Moniz, G. A.
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and 1,3ꢀdiarylpropane analogs as antiꢀinflammatory agents. Bioorg. Med. Chem. Lett. 2017, 27, 1547ꢀ1550.
24 Besong, G.; Jarowicki, K.; Kocienski, P. J.; Sliwinski, E.; Boyle, F. T. Synthesis of (S)ꢀ(−)ꢀNꢀacetylcolchinol using
intramolecular biaryl oxidative coupling. Org. Biomol. Chem. 2006, 4, 2193ꢀ2207.
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