Structure-Based Design of High-Affinity Macrocyclic FKBP51 Inhibitors

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Structure-Based Design of High-Aﬃnity Macrocyclic FKBP51

Inhibitors

Michael Bauder, Christian Meyners, Patrick L. Purder, Stephanie Merz, Wisely Oki Sugiarto,

Andreas M. Voll, Tim Heymann, and Felix Hausch *

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ABSTRACT: The FK506-binding protein 51 (FKBP51) emerged

as a key player in several diseases like stress-related disorders,

chronic pain, and obesity. Linear analogues of FK506 called SAFit

were shown to be highly selective for FKBP51 over its closest

homologue FKBP52, allowing the proof-of-concept studies in

animal models. Here, we designed and synthesized the ﬁrst

macrocyclic FKBP51-selective ligands to stabilize the active

conformation. All macrocycles retained full FKBP51 aﬃnity and

selectivity over FKBP52 and the incorporation of polar

functionalities further enhanced aﬃnity. Six high-resolution crystal

structures of macrocyclic inhibitors in complex with FKBP51

conﬁrmed the desired selectivity-enabling binding mode. Our

results show that macrocyclization is a viable strategy to target the

shallow FKBP51 binding site selectively.

thereby increasing bioavailability.^39,41,42However, the design

of appropriate macrocycles is still far from trivial. Quite

recently, macrocyclization strategies were crucial in the

discovery of potent and orally bioavailable cyclophilin

inhibitors.^43,44We here set out to investigate the suitability

of linear FKBP51 ligands to macrocyclization and report on

the discovery of two series of highly potent and selective

macrocyclic FKBP51 inhibitors.

INTRODUCTION

■

The FK506-binding protein 51 (FKBP51, encoded by the

FKBP5 gene) is a peptidyl-prolyl isomerase (PPIase) that

belongs to the immunophilin family and is best known for the

modulation of the glucocorticoid receptor activity^1,2and its

participation as a cochaperone in the Hsp90 protein folding

machinery.^3,4In numerous studies, FKBP51 was associated

with stress-related mental disorders,⁵⁻¹⁰obesity-induced

diabetes,¹¹⁻¹⁵and several forms of chronic pain,¹⁶⁻¹⁸

collectively suggesting FKBP51 as a new drug target.^3,19

The recent discovery of the ﬁrst FKBP51-selective ligands

(SAFit1 and SAFit2) provided powerful tools to study the role

of FKBP51 in cellular and animal models.^{13,16,17,20−27}A newly

discovered transient binding pocket is the key to selectivity for

FKBP51 over the highly homologous FKBP52.^20,28−31Never-

theless, SAFit1 and SAFit2 are far from becoming drug

candidates due to their high molecular weight and the

associated poor physicochemical properties.³²Additionally,

the binding site of FKBP51 is shallow and the creation of low

molecular mass inhibitors with high ligand eﬃciency and

suﬃcient potency is extremely challenging.^28,33−36

RESULTS AND DISCUSSION

■

Crystallographic Analysis and Design of the Macro-

cyclization Strategy. We used existing 3D information on

high-aﬃnity inhibitors in a complex with the FK1 domain of

FKBP51 to identify promising attachment points for the design

of macrocyclic SAFit analogues. The crystal structures of

known, selective FKBP51 inhibitors served as templates for our

structure-based rigidiﬁcation strategy.^{20,28,31,33,34}SAFit ana-

logues contain two aryl rings (A and B, Figure 1A), which are

consistently in close proximity when bound to FKBP51, as

illustrated for the FKBP51-SAFit2 complex (PDB 6TXX,³¹

Figure 1B). Structural analysis suggested the meta- or para-

Macrocycles are thought to possess improved drug-relevant

properties, which are especially important for diﬃcult drug

targets requiring ligands beyond the rule-of-ﬁve space.³⁵⁻⁴²

Preorganizing the bioactive conformation in a ring structure is

thought to improve aﬃnity by reducing the entropic loss upon

binding.³⁹Moreover, macrocyclizations can improve cell

permeability and provide enhanced proteolytic stability,

Received: December 19, 2020

Published: March 5, 2021

American Chemical Society

https://dx.doi.org/10.1021/acs.jmedchem.0c02195

J. Med. Chem. 2021, 64, 3320−3349

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tion, Scheme S1).^45,46Methylation of the remaining hydroxy

groups in 18 aﬀorded 19 in a good yield of 93%. After

substantial exploration of several alkylation conditions, we

referred to a direct racemic alkylation protocol, where the

carboxylic ester 19 is converted to the corresponding enolate

with LiHMDS, alkylated with 3-bromocyclohexene, and the

methyl ester 20 is afterward hydrolyzed in a mixture of

aqueous LiOH and THF to generate carboxylic acid 21.

Conversion to the corresponding acyl chloride with SOCl₂and

subsequent Schotten−Baumann reaction with L-pipecolic acid

and aqueous KOH in 1,4-dioxane provided the corresponding

amide as a mixture of four diastereomers (not shown).

Fortunately, the acyl chloride-activation/amidation sequence

provides a good yield of 71% with an additional 28% of

recovered 21 after hydrolysis of the Schotten−Baumann

reaction. Pd/C-catalyzed hydrogenation aﬀected alkene

reduction and simultaneous deprotection of the benzyl ether

to aﬀord 22b and desired 22a as a 1:1 mixture of diastereomers

that could be separated by silica gel chromatography (the

assignment of diastereomers 22a/22b to the desired (S)

conﬁguration is described in the Supporting Information).

Alkylation to install the alkene for later RCM followed by ester

cleavage aﬀorded the carboxylic acids 23−26 in very good

yields (81−96%) over two steps. After substantial optimization

to minimize epimerization,⁴⁷esteriﬁcation with alcohol 14 was

achieved with EDC and an excess of 4-pyrrolidinopyridine,

which aﬀorded 59−64% of the desired esters 27−30 with low

amounts of epimerization (<5%). Two key ﬁndings substan-

tially contributed to the optimized conditions: (a) the excess

use of 4-pyrrolidinopyridine, a more reactive analogue to

standard DMAP, and (b) the use of toluene as a nonpolar

solvent, probably due to decreased stabilization of the polar

intermediates that lead to epimerization.

Figure 1. (A) Chemical structure of the selective FKBP51 ligands

SAFit1 and SAFit2 and the macrocyclization strategy. (B) SAFit2 (red

sticks) in complex with FKBP51 (PDB 6TXX); key interactions are

highlighted (black dotted lines; distances in Å), and the macro-

cyclization strategy is outlined. Lys121 has been omitted for clarity.

Synthesis of Macrocyclic FKBP51 Ligands. The

precursors for the para- (9−12) and the meta-series (27−

30) were cyclized via ring-closing metathesis to give the

macrocycles (31−38, Scheme 3). The ring-closing metatheses

always aﬀorded mixtures of E/Z-alkene isomers, except for 35

and the longest linker of each series (34 and 38), where only

the E-alkenes were observed. Fortunately, we were able to

separate all E/Z-alkene mixtures and hence were able to test

the inﬂuence of the alkene geometry on FKBP51 binding

aﬃnity. Hydrogenation of the alkenes aﬀorded the saturated

macrocycles (39−46).

The shorter variants of the alkene macrocycles (31-(E) and

35-(E)) were subjected to an OsO₄dihydroxylation⁴⁸to

introduce additional functionalities into the macrocyclic frame.

Fortunately, we were able to separate the resulting pair of

isomers for the meta-series (64a/64b), but not for the para-

series (63). Furthermore, the alkenes 31-(E), 35-(E), 36-(E),

and 37-(E) were oxidized under Wacker conditions with

palladium acetate and 1,4-benzoquinone⁴⁹to obtain the

corresponding ketones (59, 60, 61a/61b, and 62). Interest-

ingly, the oxidation of the short-chain alkenes (31-(E) and 35-

(E)) resulted in ketones 59 and 60 as the sole products of two

possible regioisomers. Oxidation of the medium-length linker

(36-(E)) provided major and minor product regioisomers

(61a and 61b), whereas oxidation of the longest linker (37-

(E)) resulted in a single isolable product isomer (62).

position of ring A and the meta-position of ring B as possible

attachment points for linkers and modeling led us to postulate

that macrocyclization within this structural framework would

lead to macrocyclic ligands that could maintain all the binding

interactions observed for the existing acyclic analogues.

Synthesis of para- and meta-Aryl-Substituted Cycli-

zation Precursors. Since the precise requirements for the

cyclizing linkers were unknown, we implemented a macro-

cyclization strategy that allows various linkers to be

incorporated. The synthesis toward the cyclization precursors

for para-aryl-linked macrocycles starts with commercially

available phenol 1 (Scheme 1). After TBPDPS protection of

the para hydroxyl group, the carboxylic acid 2 was preactivated

to introduce the chiral auxiliary. The key step in this sequence

is the introduction of the cyclohexene ring via α-alkylation of

the enolate derived through deprotonation of 4. Hydro-

genation of the alkene followed by hydrolysis aﬀords the chiral

TBDPS-protected intermediate 6 in 33% yield over six steps.

Amide coupling with amine 15 and TBAF-mediated

desilylation provides the phenol 8, which is further derivatized

to the cyclization precursors (9 - 12) through alkylation with

the corresponding allyl-containing building blocks.

The synthesis of the cyclization precursors for meta-aryl-

linked macrocycles turned out to be far more challenging

(Scheme 2) since ﬁve steps were necessary to prepare the

hydroxy group in the meta-position of the aryl ring accessible

for later derivatizations. 18 was synthesized over ﬁve steps in

51% yield from commercially available 3,4,5-trimethoxyphenyl-

acetic acid following known procedures (Supporting Informa-

The bis-O-allyl intermediates bearing an alkyl O-allyl moiety

(11, 12, 29, and 30) oﬀered the potential for an additional

approach toward cyclization. Global deallylation of these

intermediates with Wilkinson catalyst yielded the diols (47−

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a

Scheme 1. Synthesis of para-Aryl-Substituted Cyclization Precursors

a

Reagents and conditions: a) TBDPSCl, imidazole, DCM/DMF, rt; then K₂CO₃, THF/H₂O, rt, 93%; b) C₆F₅OH, EDC, 4-DMAP, DCM, rt, 92%;

c) (S)-4-phenyloxazolidin-2-one, n-BuLi, THF, 0 °C - rt, 81%; d) 3-bromocyclohexene, LiHMDS, THF, −78 °C - rt, 50%; e) Pd/C, H₂, CH/EA,

rt, 99%; f) LiOH, H₂O₂, THF/H₂O, rt, 95%; g) 15, HATU, DIPEA, DCM/DMF, rt, 88%; h) TBAF, THF, 0 °C - rt, 95%; (i) Bromide or tosylate,

K₂CO₃, MeCN, reﬂux, 92−98%; j) Allyl bromide, K₂CO₃, acetone, reﬂux, 96%; k) (S)-Fmoc-Pip−OH, DCC, 4-DMAP, DCM, 0 °C - rt; then 4-

methylpiperidine, DCM, rt, 67% (2 steps).

50), which were transformed into the corresponding

monoiodides (51−54). Subsequent ring-closure was achieved

through an intramolecular S_N2 reaction⁵⁰under high dilution

to yield the smallest macrocycles of the para- (55 and 56) and

meta-series (57 and 58). Taken together, we were able to

synthesize 33 macrocycles of various ring sizes and linker

compositions to test their binding to FKBPs.

Structure-Aﬃnity Relationship (SAR) of Macrocyclic

FKBP51 Ligands. All macrocyclic compounds were tested in

a competitive ﬂuorescence polarization assay^51,52for binding

toward FKBP51 as well as the homologues FKBP12,

FKBP12.6, and FKBP52 (Table 1 and Table 2). To compare

the binding aﬃnities of our constrained, macrocyclic ligands

with a ﬂexible analogue, we use the acyclic SAFit analogue 65

(structure shown in Table 1), which has the same number and

type of nonhydrogen atoms, hydrogen bond donors, and

acceptors and the same functional groups as macrocycles 55

and 57 and thereby served as a reference compound also for

the larger macrocycles. Gratifyingly, almost all compounds

bound to FKBP51 with a K_d< 150 nM, similar to the

unfunctionalized, linear reference compound 65. Interestingly,

there appears to be a lower limit for the unfunctionalized

macrocycles regarding FKBP51 binding aﬃnity.

However, the introduction of polar groups increased aﬃnity

in several cases (e.g., 59, 60, 63, or 64a/64b). Gratifyingly, all

compounds displayed a striking selectivity for FKBP51 over

FKBP52, suggesting binding to a Phe67^outconformation that

was shown to be highly unfavorable for FKBP52.²⁰However,

the linker functionalization seemed to restore weak binding to

FKBP52 for several macrocycles (e.g., 61a, 63, and 64a).

Moreover, a slight preference for FKBP51 over FKBP12 and

FKBP12.6 was observed, similar to previous ﬁndings for acyclic

SAFit analogues.^{20,28,33,34,52}This preference seems to be

somewhat stronger for the para-linked series than for the

meta-linked series.

The ﬁnding that the SAFit analogues are extremely tolerant

to a variety of macrocyclic constraints was unexpected. Even

the shortest linkers such as in 55 or 57, which were designed to

challenge the binding mode of the acyclic SAFit compounds,

neither enhanced nor compromised binding.

Cocrystal Structures and Binding Thermodynamics.

To understand the molecular binding mode of our macrocyclic

FKBP51 inhibitors and to evaluate the role of the linker, we

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a

Scheme 2. Synthesis of meta-Aryl-Substituted Cyclization Precursors

a

Reagents and conditions: (a) MeI, K₂CO₃, DMF, rt, 93%; (b) 3-bromocyclohexene, LiHMDS, THF, −78 °C−rt, 94%; (c) LiOH, THF/H₂O, rt,

91%; (d) SOCl₂, DCM, reﬂux; then L-Pipecolic acid, KOH aq, 1,4-dioxane, 0 °C−rt, 71% + 28% of recovered substrate 21; (e) Pd/C, H₂, THF/

MeOH, rt, 44% for 22b, 41% for 22a, separation of isomers by ﬂash column chromatography; (f) bromide or tosylate, K₂CO₃, MeCN, reﬂux, 87−

99%; (g) LiOH, THF/MeOH/H₂O, rt, 90−97%; (h) 14, EDC, 4-(pyrrolidin-1-yl)pyridine, toluene, 0 °C−rt, 59−64%.

solved cocrystal structures of six macrocycles in complex with

the FK506-binding domain of FKBP51, covering a wide range

of linker lengths and functionalization (Figure 2 and

Supporting Information, Figure S7).⁵³Both the 18-membered

macrocycle 55, which has the shortest linker of the para-series,

and macrocyclic diol 64a show binding modes consistent with

those of linear FKBP51-selective ligands (PDB 4TW6,²⁰

4TW7,²⁰5DIT,²⁸5DIU,³⁴5DIV,³⁴6SAF,³³and 6TXX³¹),

including the conformational rearrangement of Phe67, which

underlies the strong selectivity vs the highly homologous

FKBP52 (Figure 2). The crystal structure of 64a also reveals

that both linker hydroxy groups are solvent exposed and do not

contact the protein surface. Gratifyingly, the conformations of

the linkers in all cocrystal structures were well-deﬁned

(Supporting Information, Figures S1 −S6), although neither

of them directly contacts FKBP51.

A superposition of the cocrystallized macrocycles 35-(E)

and 64a with cocrystallized SAFit2 (Figure 3) and other SAFit

analogues (Supporting Information, Figure S9) revealed

almost identical binding modes of the pipecolate core and

the cyclohexyl ring. Superposed 35-(E) shows a signiﬁcant

rotation of ring B (21.1°) due to the rigid E-conﬁguration in

the linker, which in turn aﬀects the position of the ring C-

bearing arm (Figure 3A). The highly potent macrocycle 64a

shows an almost perfect overlay with SAFit2, only the A ring

shows a minimal outward shift of 0.3 Å.

dihydroxylated macrocycle 64a by isothermal titration

calorimetry (ITC) and compared it to the acyclic ligand

SAFit1.²⁰The ITC data closely matched the aﬃnity constants

obtained by ﬂuorescence polarization and revealed that

binding of both compounds is enthalpically driven (Table 3

and Supporting Information, Figure S10).

CONCLUSION

■

Macrocyclization is thought to work in part by reducing the

conformational ﬂexibility and in turn the entropic penalty of

small molecules for adopting the active conformation. Since we

did not observe an aﬃnity enhancement for any of the

unfunctionalized linkers, irrespective of linker size or linker

rigidity, we conclude that the conformation of SAFit analogues

previously observed in the crystal structures is already

energetically favorable, e.g., by a hydrophobic collapse of the

two A and B rings.⁵⁴In this circumstance, no energy gain can

be expected by additional preorganization, as also conﬁrmed

by the results of the ITC experiments.

Remarkably, the introduction of polar modiﬁcations in the

linker enhanced aﬃnity in all cases. The macrocyclic linker,

while not contacting the protein directly, thus seems to provide

an eﬃcient framework for the positioning of hydrophilic

groups, as has been suggested as a recurring principle for

macrocyclic natural products.³⁸

Macrocyclization restricts conformational freedom and may

thus reduce the entropic penalty upon binding in a single

active conformation. To explore the thermodynamic con-

sequences of macrocyclization in more detail, we analyzed

EXPERIMENTAL SECTION

■

General Information. Air and water-sensitive reactions were

performed under argon atmosphere with commercially available dry

solvents. All commercially available chemicals and solvents were used

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a

Scheme 3. Synthetic Pathways Towards Macrocyclic FKBP51 Inhibitors

a

Reagents and conditions: (a) Grubbs second generation catalyst (C₄₆H₆₅Cl₂N₂PRu), DCM, reﬂux, 33−78%; (b) Pd/C, H₂, MeOH, rt, 37−45%

(for 39 and 40); (c) RhCl(PPh₃)₃, H₂, toluene, or MeOH/THF, rt, 66−87%, (for 41−46); (d) RhCl(PPh₃)₃, DABCO, EtOH/H₂O, reﬂux, 16−

47%; (e) I₂, PPh₃, imidazole, toluene or DCM, rt, 38−85%; (f) K₂CO₃, MeCN, reﬂux, 26−81%; (g) Pd(OAc)₂, p-BQ, HBF₄, MeCN/H₂O, rt, 33−

67%; (h) OsO₄, NMO, H₂O/acetone, 0 °C−rt, 26−40%.

as received. Solutions of n-butyllithium were titrated prior to use in

toluene using 2-propanol with 1,10-phenanthroline as an indicator

(red end point of titration; average of two determinations used).

When indicated, solvents or reagents were degassed by sparging with

argon. Chromatographic separations were performed by manual ﬂash

chromatography on silica (SiO₂) from Macherey−Nagel (particle size

0.04−0.063 mm). Reverse-phase puriﬁcations were performed either

with a Beckman System Gold 126 NMP programmable solvent

module ﬁtted with a Phenomenex Jupiter 4 μ Proteo 90 Å column

(250 mm × 10 mm) and a Beckman System Gold 166 programmable

detector module or a Interchim puriFlash 5.250 system ﬁtted with a

Uptisphere Strategy C18-HQ 5 μm column (250 mm × 21.2 mm).

Eluents were 0.1% TFA in water (eluent A) and 0.1% TFA in

acetonitrile (eluent B). Thin-layer chromatography (TLC) was

performed on precoated aluminum plates with a ﬂuorescence

indicator from Merck (silica gel 60 F₂₅₄). ¹H NMR spectra, ¹³C

NMR spectra, 2D HSQC, HMBC, COSY, and NOESY were obtained

from the NMR Department of the Technical University of Darmstadt,

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Table 1. Binding Aﬃnities of Macrocyclic FKBP51 Ligands (para-Linked Series) Determined by a Competitive Fluorescence

a

Polarization Assay

a

b

Values are given as mean weighted standard deviation from at least two independent measurements. No binding can be observed within the

c

range of solubility. Tested as a diastereomeric mixture (dr = 34:66).

on a Bruker DRX 500 spectrometer at room temperature, unless

stated otherwise. Proton chemical shifts are expressed as parts per

million (ppm, δ scale) and are referenced to residual solvent (¹H:

CDCl₃, δ = 7.26; C₆D₆, δ = 7.16; DMSO-d₆, δ = 2.50; THF-d₈, δ =

3.58; MeOD, δ = 3.31. ¹³C: CDCl₃, δ = 77.16; C₆D₆, δ = 128.06;

DMSO-d₆, δ = 39.52; THF-d₈, δ = 67.57; MeOD, δ = 49.00).

Coupling constants (J) were given in hertz (Hz). LC−MS (liquid

chromatography−mass spectrometry) measurements were performed

either on an LC−MS system with a Beckmann Coulter System Gold

126 solvent module, Beckmann Coulter System Gold 508

autosampler, Beckman Coulter System 166 detector connected to a

Thermo Finnigan LCQ Deca XP Plus or on an Agilent 1260 Inﬁnity

II System consisting of a 1260 Inﬁnity II ﬂexible pump, a vialsampler,

a multicolumn thermostat, and a diode array detector connected to a

6125B MSD single quadrupole detector. Eluents were 0.1% formic

acid in water (eluent A) and 0.1% formic acid in acetonitrile (eluent

B). High-resolution mass spectra (HRMS) were obtained by the Mass

Spectrometry Department of the Technical University of Darmstadt

using a Bruker Daltonics Impact II mass spectrometer (quadrupole

time-of-ﬂight). Analytical HPLC analyses were performed on a

Dionex P580 pump, Dionex ASI-100 automated sample injector ﬁtted

with a Phenomenex Kinetex 5 μm C18 100 Å column (250 mm × 4.6

mm), and a Dionex UCD 340U photodiode array detector (eluent A,

0.1% TFA in water; eluent B, 0.1% TFA in acetonitrile) or an Agilent

1260 Inﬁnity II System consisting of a 1260 Inﬁnity II ﬂexible pump, a

vialsampler, and a multicolumn thermostat ﬁtted with a Poroshell 120

3 mm × 150 mm, 2.7 μm EC-C18 column or a Poroshell 120 50 mm

× 2.1 mm, 1.9 μm EC-C18, a diode array detector, and a 6125B MSD

single quadrupole detector (eluent A, 0.1% formic acid in water;

eluent B, 0.1% formic acid in acetonitrile). Detailed measurement

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Table 2. Binding Aﬃnities of Macrocyclic FKBP51 Ligands (meta-Linked Series) Determined by a Competitive Fluorescence

a

Polarization Assay

a

b

Values are given as mean weighted standard deviation from at least two independent measurements. No binding can be observed within the

range of solubility.

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Figure 2. X-ray structures of the FK506-binding domain of FKBP51 (gray surface) in complex with the macrocyclic ligands 55 (A, PDB 7AWX,

green sticks) and 64a (B, PDB 7B9Y, blue sticks). Lys121 has been omitted for clarity. Hydrogen bonds to Ile87 and Tyr113 (yellow) are indicated

as black dotted lines (distances in Å). Phe67 (yellow) has been displaced by the cyclohexyl moiety upon binding of the ligands. Novel linker

moieties are highlighted in bright green and dark blue, respectively. Linker oxygens are highlighted in red.

Figure 3. Crystal structures of the FK506-binding domain of FKBP51 (gray surface) in complex with the macrocyclic ligands 35-(E) (A, PDB

7B9Z, yellow sticks) and 64a (B, PDB 7B9Y, blue sticks) superposed on the cocrystal structure of SAFit2 (PDB 6TXX, red sticks). Lys121 has

been omitted for clarity. Novel linker moieties and rings A, B and C are highlighted in orange and dark blue, respectively. Linker oxygens are

highlighted in red.

1

alkene bonds was determined by H−¹H-homonuclear decoupling of

Table 3. Thermodynamic Parameters for Binding of

the two sets of vicinal methylene protons, providing informative

coupling constants between the double bond protons for at least one

of the two isomers. Compound 13 was prepared as previously

Ligands SAFit1 and 64a to FKBP51FK1, Obtained by ITC

a

at 25 °C

described.²⁰18 was synthesized over ﬁve steps from commercially

available 3,4,5-trimethoxyphenylacetic acid.^45,46A dedicated reaction

scheme as well as analytical data can be found in the Supporting

Information. Diﬀraction data have been collected on BL14.1 at the

BESSY II electron storage ring operated by the Helmholtz-Zentrum in

Berlin (Germany),⁵⁵the beamline PX at the Swiss Light Source (SLS)

in Villigen (Switzerland),⁵⁶and the beamline P13 operated by EMBL

Hamburg at the PETRA III storage ring (DESY) in Hamburg

(Germany).⁵⁷A detailed description of the crystallization procedure,

the structure solution, the reﬁnement, as well as the reﬁnement data

are shown in the Supporting Information. The purity of all tested

compounds was ≥95% based on reverse phase HPLC (λ = 220 nm).

General Experimental Procedures. General Procedure A

(Alkylation with Bromides or Tosylates). To a stirred solution of

the substrate and K₂CO₃in the solvent was added the corresponding

bromide or tosylate, and the reaction mixture was heated at reﬂux

until completion of the reaction. The resulting suspension was ﬁltered

through Celite and washed with EA (200 mL), and the solvent was

removed under reduced pressure. The crude product was puriﬁed by

ﬂash column chromatography.

ΔG

ΔH

−TΔS

ligand

K_d[nM]

[kcal/mol]

SAFit1

64a

9.8 3.6

6.9 5.6

−10.9 0.2

−11.1 0.1

−11.2 0.3

−11.3 0.4

−0.24 0.29

−0.10 0.10

a

Mean

weighted standard deviation from three independent

measurements is reported.

parameters (e.g., used gradient) were speciﬁed in the analytical data of

the compounds. The pipecolic acid structural motive led to the

presence of rotamers in the ¹H and ¹³C NMR spectra of all linear and

most of the macrocyclic compounds of this work. Due to this, the

proton and carbon NMR spectra became fairly complex. When only

two rotamers were present, we tried to distinguish between the major

(indicated with B) and the minor rotamer (indicated with A) where

possible, even though some signals overlapped. This applied to

compounds 7−12, 15, 22a, 22b, 23−26, 33-(E), 35-(E), 36-(Z), 36-

(E), 37-(E), 38-(E), 41, 43−48, 51, 52, 61a, 61b, and 62. For

compounds 42, 58, and 64a the portion of the minor rotamer was too

small (≤1:10, minor/major), and we have therefore only reported the

major rotamer. If multiple rotamers were present, a detailed

assignment was not possible, and we indicated signals originating

from the major rotamer (alone or together with overlapping rotamer

signals) with an A. This applied to compounds 27−30, 49, 50, 53,

and 54. Compounds 20 and 21 were mixtures of four diastereomers

(stereocenter conﬁgurations: RR, RS, SR, SS), consisting of two pairs

of enantiomers. Here the NMR signals were normalized to the major

diastereomer, and all signals were reported. The geometry of the

General Procedure B (Ring-Closing Metathesis (RCM)). A stirred

solution of the substrate in DCM was degassed by sparging with

argon for 15 min. Then Grubbs second generation catalyst and 1,4-

benzoquinone (if indicated) were added and the mixture was heated

at reﬂux. After completion of the reaction the mixture was either (a)

ﬁltered through silica and the solvent removed under reduced

pressure or (b) quenched by the addition of tris(hydroxymethyl)-

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phosphine solution (1 M in iPrOH) and washed with brine. The

organic phase was dried over MgSO₄, ﬁltered, and concentrated under

reduced pressure. The crude product was puriﬁed by ﬂash column

chromatography or semipreparative HPLC.

General Procedure C (Nucleophilic Substitution with Iodine). To

a stirred solution of the substrate, PPh₃, and base in anhydrous

solvent, iodine was added. The mixture was stirred at room

temperature until completion of the reaction. The reaction mixture

was diluted with DCM or Et₂O (25 mL) and washed with aqueous

Na₂S₂O₃(10 mL). The organic phase was washed with 1 M HCl

solution and brine (10 mL each), dried over MgSO₄, and

concentrated under reduced pressure. The crude product was puriﬁed

by ﬂash column chromatography or semipreparative HPLC.

General Procedure D (Intramolecular Nucleophilic Substitution).

To a stirred solution of the substrate in MeCN was added K₂CO₃, and

the mixture was degassed by sparging with argon for 15 min. The

resulting mixture was then heated at reﬂux until completion of the

reaction. After cooling to room temperature, the suspension was

ﬁltered through Celite and washed with EA (100 mL), and the solvent

was removed under reduced pressure. The crude product was puriﬁed

by ﬂash column chromatography or semipreparative HPLC.

layer was then washed with 1 M HCl and brine (50 mL each), dried

over MgSO₄, ﬁltered, and concentrated under reduced pressure to

give 51.2 g crude product of the bis-protected compound as a brown

oil. The crude was dissolved in THF/H₂O (v/v = 2:1, 150 mL),

K₂CO₃(13.03 g, 94.2 mmol, 2.00 equiv) was added, and the reaction

mixture was stirred at room temperature for 16 h. 0.1 M HCl was

added to adjust a neutral pH, and the reaction mixture was extracted

with Et₂O (3 × 100 mL). The combined organic phases were washed

with brine, dried over MgSO₄, ﬁltered, and concentrated under

reduced pressure. The crude product was puriﬁed by ﬂash column

chromatography (CH/EA = 5:1) to yield 2 (19.81 g, 93%) as a white

solid over two steps. TLC (CH/EA = 3:1 + 1% HCOOH, v/v): R_f=

0.31. ¹H NMR (500 MHz, CDCl₃): δ 7.78−7.64 (m, 4H), 7.40−7.29

(m, 6H), 6.35 (s, 2H), 3.51 (s, 2H), 3.43 (s, 6H), 1.11 (s, 9H). ¹³C

NMR (126 MHz, CDCl₃): δ 178.0, 151.0, 135.3, 134.8, 133.8, 129.2,

127.1, 125.6, 106.4, 55.4, 41.3, 26.9, 20.3. LC−MS (m/z): (ESI⁺)

calculated for C₂₆H₃₁O₅Si [M + H]⁺: 451.19, found 451.13. HPLC

(5−95% sSolvent B, 1.0 mL/min, 19 min): t_R= 13.65 min, purity

(254 nm) = 99%.

Perﬂuorophenyl 2-(4-((tert-butyldiphenylsilyl)oxy)-3,5-

dimethoxyphenyl)acetate (3). To a stirred solution of 2 (19.81

g, 44.0 mmol, 1.00 equiv) and 2,3,4,5,6-pentaﬂuorophenol (8.90 g,

48.4 mmol, 1.10 equiv) in DCM (220 mL, 0.2 M) were added EDC

hydrochloride (9.27 g, 48.4 mmol, 1.10 equiv) and DMAP (1.61 g,

13.2 mmol, 0.30 equiv), and the reaction mixture was stirred at room

temperature overnight. The reaction mixture was washed with 1 M

HCl and brine (50 mL each), dried over MgSO₄, ﬁltered, and

concentrated under reduced pressure. The crude product was puriﬁed

by ﬂash column chromatography (CH/EA = 20:1) to yield 3 (24.90

General Procedure E (Double Bond Hydrogenation). A stirred

solution of the substrate in the solvent was degassed by sparging with

́

Argon for 10 min. After the addition of Wilkinsons catalyst

[RhCl(PPh₃)₃] the solution was sparged with hydrogen for 15 min

and was then stirred under a hydrogen atmosphere at room

temperature. When the reaction was deemed complete by LC-MS

analysis, the solvent was removed under reduced pressure. The crude

product was puriﬁed by ﬂash column chromatography or semi-

preparative HPLC.

1

g, 92%) as a colorless oil. TLC (CH/EA = 9:1, v/v): R_f= 0.51. H

General Procedure F (Ester Hydrolysis). To a stirred solution of

the substrate in H₂O/MeOH/THF (v/v/v = 1:1:2) was added

lithium hydroxide. The reaction mixture was stirred at room

temperature until completion of the reaction and subsequently

extracted with Et₂O (2 × 50 mL). The organic layer was removed,

and the aqueous layer was acidiﬁed with 1 M HCl solution (pH = 1−

2). After extraction with Et₂O (3 × 50 mL), the organic phase was

washed with brine, dried over MgSO₄, and concentrated under

reduced pressure. The crude product was puriﬁed by ﬂash column

chromatography.

General Procedure G (Esteriﬁcation with Alcohol 14). To a

stirred solution of the substrate, 14 and 4-(1-pyrrolidinyl)pyridine in

dry toluene at 0 °C was added EDC. After stirring for 1 h at 0 °C, the

reaction mixture was allowed to reach ambient temperature and left

stirring for the indicated time. During this time, the reaction was

controlled via LC-MS and HPLC to monitor product formation and

epimerization. Then the reaction mixture was acidiﬁed with 1 M HCl

solution and extracted with DCM (3 × 30 mL). The organic phase

was washed with brine, dried over MgSO₄and concentrated under

reduced pressure. The crude product was puriﬁed by ﬂash column

chromatography.

General Procedure H (Wacker Oxidation of Internal Alkenes).

Palladium acetate (Pd(OAc)₂) and 1,4-benzoquinone were charged in

a ﬂask under air. A mixture of MeCN and water (v/v = 9:1) was

added, followed by the addition of aqueous HBF₄. After the addition

of the corresponding substrate, the mixture was stirred at room

temperature until completion of the reaction. The reaction mixture

was diluted with brine (20 mL) and extracted with DCM (3 × 30

mL). The combined organic phases were dried over MgSO₄and

concentrated under reduced pressure. The crude product was puriﬁed

by ﬂash column chromatography or semipreparative HPLC.

2-(4-((tert-Butyldiphenylsilyl)oxy)-3,5-dimethoxyphenyl)-

acetic Acid (2). 3,5-Dimethoxy-4-hydroxyphenylacetic acid (10.00 g,

47.1 mmol, 1.00 equiv) and imidazole (12.83 g, 188.5 mmol, 4.00

equiv) were dissolved in a mixture of DCM/DMF (v/v = 2:1, 150

mL). TBDPSCl (38.87 g, 141.4 mmol, 3.00 equiv) is added dropwise

and the reaction mixture was stirred at room temperature for 20 h.

The reaction was quenched by the addition of water (50 mL) and

stirred for 5 min. The layers were separated, and the organic phase

was washed with water for another three times (100 mL). The organic

NMR (300 MHz, CDCl₃): δ 7.86−7.60 (m, 4H), 7.49−7.29 (m, 6H),

6.43 (s, 2H), 3.83 (s, 2H), 3.47 (s, 6H), 1.13 (s, 9H). Note: The

carbon NMR signals of the pentaﬂuorophenyl ring are broad and

diﬀuse with low intensity. ¹³C NMR (75 MHz, CDCl₃): δ 167.7,

151.3, 135.3, 134.6, 134.1, 129.2, 127.1, 124.4, 106.1, 55.4, 40.5, 26.9,

20.3. HPLC (50−95% Solvent B, 1.0 mL/min, 19 min): t_R= 13.73

min, purity (254 nm) = 99%.

(S )-3-(2-( 4-(( t er t-Butyldiph en yl silyl) oxy)-3, 5-

dimethoxyphenyl)acetyl)-4-phenyloxazo-lidin-2-one (4). (S)-

4-Phenyloxazolidin-2-one (6.72 g, 40.8 mmol, 1.01 equiv) was

dissolved in dry THF (350 mL, c = 0.1 M) and cooled in an ice

bath to 0 °C. n-Butyllithium (2.3 M in hexanes, 17.72 mL, 40.8 mmol,

1.01 equiv) was added dropwise, and the reaction mixture was stirred

for 1 h at 0 °C. Afterward, 3 (24.90 g, 40.4 mmol, 1.00 equiv)

dissolved in dry THF (50 mL) was added to the above solution and

stirred for 3 h at 0 °C and then overnight at room temperature. The

reaction mixture was quenched by the addition of saturated NH₄Cl

solution, and the aqueous phase was extracted by Et₂O (3 × 50 mL).

The combined organic phases were dried over MgSO₄, ﬁltered, and

concentrated under reduced pressure. The crude product was puriﬁed

by ﬂash column chromatography (CH/EA = 8:1) to yield 4 (19.58 g,

81%) as a white solid. TLC (CH/EA = 3:1, v/v): R_f= 0.49. ¹H NMR

(300 MHz, CDCl₃): δ 7.77−7.61 (m, 4H), 7.41−7.25 (m, 9H),

7.15−7.08 (m, 2H), 6.31 (s, 2H), 5.40 (dd, J = 4.2, 8.8 Hz, 1H), 4.65

(t, J = 8.8 Hz, 1H), 4.30−4.12 (m, 2H), 4.05 (d, J = 14.4 Hz, 1H),

3.36 (s, 6H), 1.09 (s, 9H). ¹³C NMR (75 MHz, CDCl₃): δ 170.8,

153.6, 150.8, 138.8, 135.3, 134.8, 133.5, 129.2, 129.2, 128.7, 127.1,

126.0, 125.5, 106.6, 69.9, 57.9, 55.3, 41.7, 26.9, 20.3. LC−MS (m/z):

(ESI⁺) calculated for C₃₅H₃₈NO₆Si [M + H]⁺: 596.25, found 596.11.

HPLC (50−95% solvent B, 1.0 mL/min, 19 min): t_R= 12.52 min,

purity (254 nm) = 98%.

(S)-3-((S)-2-(4-((tert-Butyldiphenylsilyl)oxy)-3,5-dimethoxy-

phenyl)-2-cyclohexylacetyl)-4-phenyloxazolidin-2-one (5). To

a stirred solution of 4 (19.58 g, 32.9 mmol, 1.0 equiv) in dry THF

(500 mL) was added LiHMDS (1 M in THF, 39.4 mL, 1.20 equiv) at

0 °C, and the reaction mixture was stirred at this temperature for 1 h.

The mixture was then cooled to −78 °C and 3-bromocyclohex-1-ene

(11.14 g, 65.7 mmol, 2.00 equiv) was added dropwise. The reaction

mixture was stirred at −78 °C for 5 h and was then allowed to warm

to room temperature overnight. Then the reaction mixture was

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quenched by the addition of saturated NH₄Cl solution (100 mL), and

the aqueous phase was extracted by Et₂O (3 × 40 mL). The

combined organic phases were washed with brine, dried over MgSO₄,

ﬁltered, and concentrated under reduced pressure. The crude product

was puriﬁed by ﬂash column chromatography (CH/EA = 10:1) to

yield S1 (11.12 g, 50%) as white solid and a mixture of diastereomers

(analytics shown in the Supporting Information). Subsequently, a

stirred solution of S1 (11.09 g, 16.41 mmol, 1.00 equiv) in a mixture

of CH/EA/MeOH (v/v/v = 2:1:1, 400 mL) was degassed by sparging

with argon for 15 min. After addition of Pd/C (10 wt %, 1.74 g, 1.64

mmol, 0.10 equiv), the solution was sparged with hydrogen for 15

min and was then stirred under a hydrogen atmosphere at room

temperature overnight. The dark suspension was ﬁltered through

Celite, and the solvent was removed under reduced pressure to yield 5

(11.10 g, 99%) as white solid without further puriﬁcation. TLC (CH/

EA = 9:1, v/v): R_f= 0.29. ¹H NMR (500 MHz, CDCl₃): δ 7.76−7.60

(m, 4H), 7.43−7.26 (m, 11H), 6.45 (s, 2H), 5.33 (dd, J = 3.4, 8.7 Hz,

1H), 4.69 (d, J = 10.6 Hz, 1H), 4.54 (t, J = 8.8 Hz, 1H), 4.19 (dd, J =

3.5, 8.8 Hz, 1H), 3.42 (s, 6H), 1.95−1.80 (m, 1H), 1.66−1.44 (m,

4H), 1.39−1.28 (m, 1H), 1.20−1.04 (m, 12H), 0.96−0.83 (m, 1H),

0.79−0.59 (m, 1H). ¹³C NMR (126 MHz, CDCl₃): δ 174.1, 153.6,

150.9, 139.6, 135.4, 134.6, 133.7, 130.0, 129.2, 129.1, 128.8, 127.0,

126.0, 106.1, 69.5, 58.3, 55.4, 54.5, 42.0, 31.7, 30.2, 26.9, 26.5, 26.0,

25.9, 20.2. LC−MS (m/z): (ESI⁺) calculated for C₄₁H₄₈NO₆Si [M +

H]⁺: 678.33, found 678.29. HPLC (80−95% solvent B, 1.5 mL/min,

20 min): t_R= 13.33 min, purity (220 nm) = 99%.

6.29 (d, J = 1.0 Hz, 1.15H, B), 6.24 (d, J = 1.1 Hz, 0.87H, A), 6.14−

5.98 (m, 0.94H, A+B), 5.82 (t, J = 6.9 Hz, 0.42H, A), 5.63 (dd, J =

5.5, 8.0 Hz, 0.56H, B), 5.49−5.43 (m, 1.10H, B), 5.43−5.38 (m,

0.47H, A), 5.35−5.25 (m, 0.99H, A+B), 4.73−4.67 (m, 0.42H, A),

4.60−4.47 (m, 2.51H, A+B), 3.88 (d, J = 1.5 Hz, 3.00H, A+B), 3.86

(d, J = 2.2 Hz, 3.88H, A+B), 3.44 (s, 2.71H, A+B), 3.29 (s, 3.34H, A

+B), 3.27 (d, J = 10.0 Hz, 0.83H, A+B), 2.93 (d, J = 9.4 Hz, 0.43H,

A), 2.79 (m, 0.54H, B), 2.67−2.41 (m, 2.48H, A+B), 2.34−2.23 (m,

1.04H, A+B), 2.18−2.09 (m, 0.43H, A), 2.06−1.95 (m, 2.11H, A+B),

1.94−1.84 (m, 1.71H, A+B), 1.75−1.50 (m, 6.79H, A+B), 1.47−1.22

(m, 6.04H, A+B), 1.18−1.13 (m, 5.74H, A+B), 1.13−1.09 (m, 6.60H,

A+B), 1.04−0.77 (m, 6.03H, A+B), 0.73−0.62 (m, 1.13H, A+B),

0.61−0.51 (m, 0.46H, A). ¹³C NMR (126 MHz, CDCl₃, mixture of

rotamers 4:5, A:B): δ 172.62 (B), 172.18 (A), 170.76 (A), 170.57

(B), 158.95 (A), 158.62 (B), 151.32 (A), 150.94 (B), 149.12 (A),

148.96 (B), 147.63 (A), 147.39 (B), 141.94 (B), 141.61 (A), 135.46

(A), 135.44 (A), 135.34 (B), 134.56 (B), 134.52 (B), 134.26 (A),

134.22 (A), 133.78 (A), 133.46 (A), 133.44 (B), 133.37 (A),

133.32(B), 133.17 (B), 131.40 (A), 130.42 (B), 129.79 (A), 129.67

(B), 129.14 (B), 129.03 (A), 129.00 (A), 126.97 (A+B), 120.41 (B),

120.24 (A), 119.24 (A), 118.84 (B), 117.85 (A), 117.64 (B), 114.40

(A), 114.13 (B), 113.31 (A), 113.08 (B), 111.98 (B), 111.88 (A),

111.57 (A), 111.51 (B), 105.66 (B), 105.02 (A), 76.78 (A), 75.74

(B), 68.91 (A), 68.86 (B), 56.08 (A), 56.05 (B), 55.99 (A), 55.96

(B), 55.86 (A), 55.76 (B), 55.54 (A+B), 55.27 (B), 55.21 (A), 52.20

(B), 43.65 (B), 41.36 (B), 40.91 (A), 39.50 (A), 38.14 (B), 37.98

(A), 33.31 (A), 33.03 (A), 31.57 (B), 31.16 (A), 30.63 (B), 30.49

(B), 26.96 (A), 26.88 (A+B), 26.77 (A+B), 26.64 (B), 26.63 (A),

26.42 (A), 26.39 (B), 26.35 (B), 26.26 (A), 25.62 (B), 24.51 (A),

21.10 (B), 20.90 (A), 20.16 (A+B). LC-MS (m/z): (ESI⁺) calculated

for C₅₈H₇₂NO₉Si [M + H]⁺: 954.50, found 954.28. HPLC (90−95%

solvent B, 1.5 mL/min, 20 min): t_R= 10.91 min, purity (220 nm) ≥

99%.

(S)-(R)-1-(3-(Allyloxy)phenyl)-3-(3,4-dimethoxyphenyl)-

p r o p y l 1 - ( ( S ) - 2 - C y c l o h e x y l - 2 - ( 4 - h y d r o x y - 3 , 5 -

dimethoxyphenyl)acetyl)piperidine-2-carboxylate (8). To a

stirred solution of 7 (2.86 g, 3.00 mmol, 1.00 equiv) in THF (50

mL, c = 0.06 M) at 0 °C was added TBAF (1 M in THF, 3.00 mL,

3.00 mmol, 1.00 equiv), and the reaction was allowed to warm to

room temperature and stirred overnight. The reaction was quenched

by the addition of H₂O (20 mL) and extracted with Et₂O (3 × 10

mL). The combined organic phases were washed with brine, dried

over MgSO₄, ﬁltered, and concentrated under reduced pressure. The

crude product was puriﬁed by ﬂash column chromatography (CH/EA

= 4:1) to aﬀord 8 (2.04 g, 95%) as light pink solid. TLC (CH/EA =

2:1, v/v): R_f= 0.58. ¹H NMR (500 MHz, CDCl₃, mixture of rotamers

0.37:1, A/B): δ 7.28 (t, J = 7.9 Hz, 0.28H, A), 7.10 (t, J = 7.9 Hz,

0.73H, B), 6.97−6.91 (m, 0.59H, A+B), 6.89−6.85 (m, 0.3H, A),

6.82−6.74 (m, 1.81H, A+B), 6.73−6.67 (m, 1.06H, A+B), 6.66−6.59

(m, 1.81H, A+B), 6.50−6.41 (m, 2.83H, A+B), 6.10−5.96 (m, 0.95H,

A+B), 5.81 (dd, J = 6.3, 7.7 Hz, 0.27H, A), 5.58 (dd, J = 5.6, 8.0 Hz,

0.74H, B), 5.48−5.34 (m, 2.70H, A+B), 5.31−5.23 (m, 1.01H, A+B),

4.71 (d, J = 5.6 Hz, 0.27H, A), 4.61−4.45 (m, 2.40H, A+B), 3.96−

3.89 (m, 0.77H, B), 3.88−3.81 (m, 8.16H, A+B), 3.71 (s, 4.52H, A

+B), 3.34 (d, J = 9.8 Hz, 0.72H, B), 2.95 (d, J = 9.6 Hz, 0.27H, A),

2.79 (td, J = 3.0, 13.3 Hz, 0.72H, B), 2.65−2.51 (m, 0.87H, A+B),

2.50−2.34 (m, 1.50H, A+B), 2.32−2.23 (m, 1.03H, A+B), 2.15−2.01

(m, 2.04H, A+B), 2.01−1.92 (m, 0.76H, A+B), 1.92−1.79 (m, 1.86H,

A+B), 1.73−1.49 (m, 6.52H, A+B), 1.48−1.04 (m, 8.43H, A+B),

1.04−0.68 (m, 3.22H, A+B), 0.67−0.52 (m, 0.58H, B). ¹³C NMR

(126 MHz, CDCl₃, mixture of rotamers 2:5, A:B): δ 172.67 (B),

172.51 (A), 170.77 (A), 170.70 (B), 158.98 (A), 158.62 (B), 149.12

(A), 149.00 (B), 147.64 (A), 147.45 (B), 147.25 (A), 147.00 (B),

141.89 (B), 141.62 (A), 133.76 (B), 133.74 (B), 133.45 (A), 133.38

(B), 133.15 (A), 129.89 (A), 129.58 (B), 129.04 (A+B), 120.40 (B),

120.25 (A), 119.32 (A), 118.69 (B), 117.91 (A), 117.70 (B), 114.34

(A), 114.05 (B), 113.45 (A), 113.21 (B), 112.05 (B), 111.89 (A),

111.58 (A), 111.49 (B), 105.70 (B), 105.14 (A), 76.90 (A), 75.81

(B), 68.94 (A), 68.86 (B), 56.66 (A), 56.37 (B), 56.07 (B), 56.01

(B), 55.90 (A), 55.71 (A), 55.05 (A+B), 52.17 (B), 43.69 (B), 41.45

(S)-2-(4-((tert-butyldiphenylsilyl)oxy)-3,5-dimethoxyphen-

yl)-2-cyclohexylacetic acid (6). To a stirred solution of 5 (11.21 g,

16.54 mmol, 1.00 equiv) in THF/H₂O (v/v = 8:5, 130 mL) at 0 °C

under air is added lithium hydroxide (792 mg, 33.07 mmol, 2.00

equiv) and H₂O₂(30% w/w in water, 8.45 mL, 82.68 mmol, 5.00

equiv). After stirring overnight at room temperature, the reaction

mixture was quenched by the addition of 1.5 M Na₂SO₃solution (50

mL) and extracted with Et₂O (3 × 20 mL). The aqueous phase was

acidiﬁed with 1 M HCl (pH < 2) and extracted with Et₂O (3 × 30

mL). The combined organic phases were washed with brine, dried

over MgSO₄, ﬁltered, and concentrated under reduced pressure. The

crude product was puriﬁed by ﬂash column chromatography (CH/EA

= 8:1 + 1% HCOOH) to aﬀord 6 (8.36 g, 95%) as white solid. TLC

1

(CH/EA = 5:1 + 1% HCOOH, v/v/v): R_f= 0.40. H NMR (500

MHz, CDCl₃): δ 7.71−7.65 (m, 4H), 7.36−7.30 (m, 2H), 7.30−7.25

(m, 4H), 6.36 (s, 2H), 3.41 (s, 6H), 3.02 (d, J = 10.5 Hz, 1H), 1.89−

1.80 (m, 2H), 1.76−1.70 (m, 1H), 1.68−1.58 (m, 2H), 1.33−1.24

(m, 2H), 1.17−1.08 (m, 11H), 1.06−0.96 (m, 1H), 0.73−0.58 (m,

1H). ¹³C NMR (126 MHz, CDCl₃): δ 179.6, 151.0, 135.4, 134.6,

133.9, 129.8, 129.1, 127.1, 105.6, 58.8, 55.4, 41.1, 32.1, 30.3, 26.9,

26.5, 26.1, 20.2. HRMS (m/z): (ESI⁺) calculated for C₃₂H₄₁O₅Si [M

+ H]⁺: 533.2718, found 533.2718. HPLC (70−100% solvent B, 0.8

mL/min, 2 min): t_R= 1.81 min, purity (220 nm) = 98%.

(S)-(R)-1-(3-(Allyloxy)phenyl)-3-(3,4-dimethoxyphenyl)-

propyl-1-((S)-2-(4-((tert-butyldiphenyl-silyl)oxy)-3,5-dime-

thoxyphenyl)-2-cyclohexylacetyl)piperidine-2-carboxylate

(7). To a stirred solution of 6 (1.82 g, 3.42 mmol, 1.00 equiv) in a

mixture of DCM/DMF (v/v = 1:1, 100 mL) at room temperature

were added HATU (1.37 g, 3.59 mmol, 1.05 equiv) and DIPEA (1.33

g, 10.25 mmol, 3.00 equiv) and stirred for 30 min, before 15 (1.65 g,

3.76 mmol, 1.10 equiv) was added. After stirring overnight, additional

portions of HATU (0.20 equiv), DIPEA (1.00 equiv) and 15 (0.10

equiv) were added and the reaction was stirred for another 4 h. Then

the reaction mixture was diluted with DCM (50 mL), washed with 1

M HCl and H₂O (50 mL each). The organic phase was washed with

brine, dried over MgSO₄, ﬁltered, and concentrated under reduced

pressure. The crude product was puriﬁed by ﬂash column

chromatography (CH/EA = 10:1) to yield 7 (2.86 g, 88%) as

1

colorless resin. TLC (CH/EA = 5:1, v/v): R_f= 0.37. H NMR (500

MHz, CDCl₃, mixture of rotamers 0.76:1, A/B): δ 7.75−7.68 (m,

2.92H, A+B), 7.68−7.65 (m, 1.15H, A+B), 7.40−7.21 (m, 6.92H, A

+B), 7.16 (t, J = 7.9 Hz, 0.57H, A), 6.98−6.87 (m, 1.33H, A+B),

6.84−6.75 (m, 2.21H, A+B), 6.74−6.70 (m, 0.44H, A), 6.69−6.67

(m, 0.44H, A), 6.67−6.63 (m, 1.16H, B), 6.57−6.53 (m, 0.56H, A),

3329

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Journal of Medicinal Chemistry

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Article

(B), 41.27 (A), 39.54 (A), 38.15 (B), 37.93 (A), 33.14 (A), 32.97

(B), 31.59 (A), 31.14 (B), 30.78 (B), 30.60 (A), 26.94 (B), 26.93

(A), 26.73 (B), 26.59 (A), 26.36 (B), 26.35 (A), 26.32 (B), 26.23

(A), 25.64 (B), 24.49 (A), 21.07 (B), 20.82 (A). HRMS (m/z):

(ESI⁺) calculated for C₄₂H₅₄NO₉[M + H]⁺: 716.37931, found

3.86−3.76 (m, 8.39H, A+B), 3.67 (s, 4.55H, A+B), 3.36 (d, J = 9.7

Hz, 0.72H, B), 2.95 (d, J = 9.6 Hz, 0.28H, A), 2.78 (td, J = 2.9, 13.3

Hz, 0.71H, B), 2.61−2.32 (m, 4.61H, A+B), 2.30−2.22 (m, 1.08H, A

+B), 2.12−2.00 (m, 2.17H, A+B), 1.98−1.75 (m, 2.67H, A+B),

1.70−1.48 (m, 6.32H, A+B), 1.45−1.03 (m, 7.64H, A+B), 1.01−0.80

(m, 1.32H, A+B), 0.78−0.68 (m, 0.76H, B), 0.66−0.52 (m, 0.57H,

A). ¹³C NMR (126 MHz, CDCl₃, mixture of rotamers 2:5, A/B): δ

172.34 (B), 172.20 (A), 170.57 (A), 170.48 (B), 158.85 (A), 158.47

(B), 153.53 (A), 153.27 (B), 149.00 (A), 148.87 (B), 147.52 (A),

147.31 (B), 141.79 (B), 141.50 (A), 136.08 (A), 136.04 (B), 135.02

(A+B), 134.18 (A), 133.51 (B), 133.47 (B), 133.31 (A), 133.27 (B),

133.04 (A), 129.69 (A), 129.47 (B), 120.30 (B), 120.15 (A), 119.16

(A), 118.56 (B), 117.72 (A), 117.50 (B), 116.28 (A), 116.17 (B),

114.23 (A), 113.88 (B), 113.28 (A), 113.17 (B), 111.89 (B), 111.79

(A), 111.49 (A), 111.39 (B), 105.98 (B), 105.49 (A), 76.70 (A),

75.57 (B), 72.37 (A), 72.30 (B), 68.79 (A), 68.71 (B), 56.35 (B),

56.08 (A+B), 55.95 (A), 55.91 (B), 55.87 (A), 55.84 (B), 55.78 (A),

54.98 (B), 52.02 (A+B), 43.63 (B), 41.40 (B), 41.05 (A), 39.43 (A),

37.93 (B), 37.80 (A), 34.55 (A), 34.52 (B), 33.00 (A), 32.77 (B),

31.45 (A), 30.99 (B), 30.63 (B), 30.46 (A), 26.74 (B), 26.71 (A),

26.59 (B), 26.45 (A), 26.22 (A+B), 26.18 (B), 26.10 (A), 25.55 (B),

24.35 (A), 20.94 (B), 20.69 (A). HRMS (m/z): (ESI⁺) calculated for

C₄₆H₆₀NO₉[M + H]⁺: 770.42626, found 770.42659. HPLC (70−

95% solvent B, 1.5 mL/min, 20 min): t_R= 11.52 min, purity (220

nm) = 99%.

(S)-(R)-1-(3-(Allyloxy)phenyl)-3-(3,4-dimethoxyphenyl)-

propyl 1-((S)-2-(4-(2-(Allyloxy)ethoxy)-3,5-dimethoxyphenyl)-

2-cyclohexylacetyl)piperidine-2-carboxylate (11). The substrate

8 (500 mg, 0.7 mmol, 1.00 equiv) was applied to general procedure A

with K₂CO₃(290 mg, 2.1 mmol, 3.00 equiv) and 16 (718 mg, 2.8

mmol, 4.00 equiv) in MeCN (25 mL). 11 (550 mg, 98%) was

obtained after puriﬁcation by ﬂash column chromatography (CH/EA

= 4:1) as light yellow resin. TLC (CH/EA = 4:1, v/v): R_f= 0.44. ¹H

NMR (500 MHz, CDCl₃, mixture of rotamers 0.44:1, A/B): δ 7.29 (t,

J = 7.9 Hz, 0.33H, A), 7.10 (t, J = 7.9 Hz, 0.70H, B), 6.97−6.91 (m,

0.64H, A+B), 6.90−6.85 (m, 0.32H, A), 6.81−6.74 (m, 1.79H, A+B),

6.71−6.67 (m, 1.03H, B), 6.66−6.61 (m, 1.80H, A+B), 6.48 (s,

1.41H, A+B), 6.46−6.43 (m, 0.75H, A+B), 6.41 (s, 0.63H, A), 6.09−

5.99 (m, 0.97H, A+B), 5.97−5.86 (m, 0.93H, A+B), 5.81 (dd, J = 6.2,

7.7 Hz, 0.31H, A), 5.56 (dd, J = 5.5, 8.2 Hz, 0.71H, B), 5.49−5.44 (m,

0.69H, B), 5.44−5.36 (m, 1.05H, A+B), 5.31−5.22 (m, 2.27H, A+B),

5.18−5.12 (m, 0.98H, A+B), 4.70 (d, J = 5.7 Hz, 0.29H, A), 4.59−

4.47 (m, 2.42H, A+B), 4.16−4.12 (m, 0.64H, A), 4.10−4.02 (m,

3.47H, A+B), 3.97−3.90 (m, 0.71H, B), 3.87−3.79 (m, 8.42H, A+B),

3.77−3.73 (m, 0.68H, A), 3.72−3.67 (m, 5.73H, A+B), 3.37 (d, J =

9.8 Hz, 0.70H, B), 2.97 (d, J = 9.6 Hz, 0.30H, A), 2.80 (td, J = 2.9,

13.3 Hz, 0.70H, B), 2.65−2.51 (m, 0.94H, A+B), 2.50−2.35 (m,

1.54H, A+B), 2.32−2.24 (m, 1.07H, A+B), 2.15−2.00 (m, 1.77H, A

+B), 1.99−1.78 (m, 2.58H, A+B), 1.74−1.50 (m, 6.80H, A+B),

1.47−1.05 (m, 6.92H, A+B), 1.04−0.85 (m, 1.18H, A+B), 0.79−0.69

(m, 0.75H, A+B), 0.67−0.53 (m, 0.65H, A+B). ¹³C NMR (126 MHz,

CDCl₃, mixture of rotamers 1:2, A:B): δ 172.50 (B), 172.33 (A),

170.74 (A), 170.62 (B), 158.99 (A), 158.62 (B), 153.57 (A), 153.31

(B), 149.14 (A), 149.01 (B), 147.66 (A), 147.45 (B), 141.95 (B),

141.62 (A), 136.19 (A), 135.19 (B), 135.13 (A), 134.39 (A), 133.70

(B), 133.67 (B), 133.40 (B), 133.17 (A), 129.84 (A), 129.63 (B),

120.43 (B), 120.27 (A), 119.31 (A), 118.69 (B), 117.93 (A), 117.71

(B), 116.85 (A), 116.79 (B), 114.38 (A), 114.03 (B), 113.46 (A),

113.31 (B), 112.01 (B), 111.90 (A), 111.59 (A), 111.51 (B), 106.11

(B), 105.59 (A), 76.91 (A), 75.74 (B), 72.33 (A), 72.27 (B), 69.61

(A), 69.55 (B), 68.96 (A), 68.89 (B), 56.52 (B), 56.26 (A+B), 56.11

(A), 56.08 (B), 56.02 (A), 56.00 (B), 55.94 (A), 55.15 (B), 52.18 (A

+B), 43.77 (B), 41.55 (B), 41.21 (A), 39.58 (A), 38.12 (B), 37.96

(A), 33.16 (A), 32.94 (B), 31.61 (A), 31.15 (B), 30.81 (B), 30.63

(A), 26.92 (B), 26.88 (A), 26.74 (B), 26.60 (A), 26.37 (A+B), 26.32

(B), 25.70 (B), 24.50 (A), 21.11 (B), 20.84 (A). HRMS (m/z):

(ESI⁺) calculated for C₄₇H₆₂NO₁₀[M + H]⁺: 800.43682, found

800.43719. HPLC (70−100% solvent B, 1.5 mL/min, 20 min): t_R=

10.08 min, purity (220 nm) = 95%.

716.37915. HPLC (50−95% solvent B, 1.5 mL/min, 20 min): t_R

13.49 min, purity (220 nm) = 99%.

=

(S)-(R)-1-(3-(Allyloxy)phenyl)-3-(3,4-dimethoxyphenyl)-

propyl 1-((S)-2-(4-(Allyloxy)-3,5-dimethoxy-phenyl)-2-

cyclohexylacetyl)piperidine-2-carboxylate (9). The substrate 8

(400 mg, 0.56 mmol, 1.00 equiv) was applied to general procedure A

with K₂CO₃(116 mg, 0.84 mmol, 1.50 equiv) and allyl bromide (76

mg, 0.62 mmol, 1.10 equiv) in MeCN (20 mL). Additional portions

of K₂CO₃(1.00 equiv) and allyl bromide (2 × 2.00 equiv) were added

until completion of the reaction. 9 (411 mg, 97%) was obtained as a

light brown solid after puriﬁcation by ﬂash column chromatography

(CH/EA = 4:1). TLC (CH/EA = 3:1, v/v): R_f= 0.26. ¹H NMR (500

MHz, CDCl₃, mixture of rotamers 0.38:1, A/B): δ 7.26−7.23 (m,

0.27H, A), 7.06 (t, J = 7.9 Hz, 0.71H, B), 6.93−6.88 (m, 0.58H, A

+B), 6.85−6.82 (m, 0.28H, A), 6.77−6.70 (m, 1.78H, A+B), 6.68−

6.64 (m, 1.03H, B), 6.64−6.58 (m, 1.80H, A+B), 6.47 (s, 1.47H, A

+B), 6.42−6.38 (m, 1.32H, A+B), 6.08−5.94 (m, 1.93H, A+B), 5.78

(dd, J = 6.2, 7.7 Hz, 0.28H, A), 5.54 (dd, J = 5.6, 8.1 Hz, 0.72H, B),

5.46−5.42 (m, 0.72H, B), 5.40−5.32 (m, 1.02H, A+B), 5.27−5.19

(m, 2.28H, A+B), 5.14−5.05 (m, 1.01H, A+B), 4.70−4.66 (m, 0.27H,

A), 4.56−4.44 (m, 3.02H, A+B), 4.42−4.38 (m, 1.47H, A+B), 3.96−

3.89 (m, 0.77H, B), 3.83−3.76 (m, 8.23H, A+B), 3.66 (s, 4.49H, A

+B), 3.35 (d, J = 9.8 Hz, 0.72H, B), 2.95 (d, J = 9.6 Hz, 0.27H, A),

2.77 (td, J = 2.9, 13.4 Hz, 0.71H, B), 2.61−2.49 (m, 0.86H, A+B),

2.47−2.31 (m, 1.49H, A+B), 2.29−2.21 (m, 1.05H, A+B), 2.12−1.99

(m, 1.76H, A+B), 1.96−1.75 (m, 2.66H, A+B), 1.70−1.46 (m, 6.38H,

A+B), 1.44−1.02 (m, 8.83H, A+B), 1.01−0.67 (m, 3.33H, A+B),

0.65−0.52 (m, 0.59H, B). ¹³C NMR (126 MHz, CDCl₃, mixture of

rotamers 2:5, A/B): δ 172.30 (B), 172.15 (A), 170.51 (A), 170.44

(B), 158.81 (A), 158.43 (B), 153.49 (A), 153.19 (B), 148.96 (A),

148.83 (B), 147.48 (A), 147.26 (B), 141.73 (B), 141.46 (A), 135.73

(B), 135.59 (A), 134.65 (B), 134.52 (A), 134.22 (A), 133.53 (B),

133.47 (B), 133.27 (A), 133.22 (B), 133.00 (A), 129.65 (A), 129.43

(B), 120.25 (B), 120.11 (A), 119.11 (A), 118.53 (B), 117.66 (A),

117.44 (B), 117.36 (A), 117.12 (B), 114.18 (A), 113.85 (B), 113.23

(A), 113.12 (B), 111.86 (B), 111.76 (A), 111.46 (A), 111.36 (B),

105.88 (B), 105.38 (A), 76.66 (A), 75.53 (B), 73.98 (A+B), 68.74

(A), 68.66 (B), 56.30 (B), 56.03 (B), 55.90 (A), 55.87 (B), 55.82

(A), 55.79 (B), 55.75 (A), 54.93 (B), 51.99 (A), 43.59 (B), 41.36

(B), 41.00 (A), 39.39 (A), 37.88 (B), 37.75 (A), 32.96 (A), 32.72

(B), 31.41 (A), 30.95 (B), 30.58 (B), 30.41 (A), 26.69 (B), 26.64

(A), 26.54 (B), 26.41 (A), 26.18 (A+B), 26.13 (B), 26.06 (A), 25.50

(B), 24.30 (A), 20.90 (B), 20.64 (A). HRMS (m/z): (ESI⁺)

calculated for C₄₅H₅₈NO₉[M + H]⁺: 756.41061, found 756.40996.

HPLC (60−90% solvent B, 1.5 mL/min, 20 min): t_R= 15.34 min,

purity (220 nm) = 99%.

(S)-(R)-1-(3-(Allyloxy)phenyl)-3-(3,4-dimethoxyphenyl)-

propyl 1-((S)-2-(4-(But-3-en-1-yloxy)-3,5-dimethoxyphenyl)-2-

cyclohexylacetyl)piperidine-2-carboxylate (10). The substrate 8

(300 mg, 0.42 mmol, 1.00 equiv) was applied to general procedure A

with K₂CO₃(174 mg, 1.26 mmol, 3.00 equiv) and 4-bromo-1-butene

(146 mg, 1.05 mmol, 2.50 equiv) in MeCN (10 mL). Additional

portions of K₂CO₃(2.00 equiv) and 4-bromo-1-butene (3 × 3.00

equiv) were added until completion of the reaction. 10 (307 mg,

95%) was obtained as yellow resin after puriﬁcation by ﬂash column

chromatography (CH/EA = 5:1). TLC (CH/EA = 3:1, v/v): R_f=

0.41. ¹H NMR (500 MHz, CDCl₃, mixture of rotamers 0.38:1, A/B):

δ 7.29−7.24 (m, 0.35H, A), 7.07 (t, J = 7.9 Hz, 0.72H, B), 6.94−6.89

(m, 0.59H, A+B), 6.87−6.83 (m, 0.3H, A), 6.79−6.71 (m, 1.81H, A

+B), 6.69−6.58 (m, 2.86H, A+B), 6.47 (s, 1.49H, A+B), 6.45−6.38

(m, 1.34H, A+B), 6.06−5.96 (m, 0.96H, A+B), 5.93−5.76 (m, 1.26H,

A+B), 5.54 (dd, J = 5.6, 8.1 Hz, 0.73H, B), 5.47−5.43 (m, 0.72H, B),

5.41−5.33 (m, 1.00H, A+B), 5.28−5.20 (m, 1.07H, A+B), 5.13−5.07

(m, 0.29H, A), 5.07−4.95 (m, 1.72H, A+B), 4.69 (d, J = 5.7 Hz,

0.27H, A), 4.57−4.44 (m, 2.40H, A+B), 4.01−3.89 (m, 2.96H, A+B),

3330

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Article

(S)-(R)-1-(3-(Allyloxy)phenyl)-3-(3,4-dimethoxyphenyl)-

propyl 1-((S)-2-(4-(3-(Allyloxy)propoxy)-3,5-dimethoxyphen-

yl)-2-cyclohexylacetyl)piperidine-2-carboxylate (12). The sub-

strate 8 (550 mg, 0.77 mmol, 1.00 equiv) was applied to general

procedure A with K₂CO₃(213 mg, 1.54 mmol, 2.50 equiv) and 17

(625 mg, 2.31 mmol, 3.00 equiv) in MeCN (20 mL). Additional

portions of K₂CO₃(1.00 equiv) and 17 (1.00 equiv) were added until

completion of the reaction. 12 (576 mg, 92%) was obtained after

puriﬁcation by ﬂash column chromatography (CH/EA = 4:1) as

(2.24 g, 6.83 mmol, 1.00 equiv) and (S)-1-(((9H-ﬂuoren-9-

yl)methoxy)-carbonyl)piperidine-2-carboxylic acid (2.64 g, 7.52

mmol, 1.10 equiv) in DCM (40 mL) at 0 °C were added DMAP

(167 mg, 1.37 mmol, 0.20 equiv) and DCC (1.55 g, 7.52 mmol, 1.10

equiv). The reaction mixture was stirred at 0 °C for 30 min and then

overnight at room temperature. The resulting precipitate was ﬁltered

oﬀ and washed with DCM (3 × 20 mL), and the solvent was removed

under reduced pressure. The crude product was puriﬁed by ﬂash

column chromatography (CH/EA = 5:1) to aﬀord the Fmoc-

protected amine (4.31 g, 6.52 mmol, 95%) as a light yellow resin.

Subsequently the Fmoc-protected amine (4.31 g, 6.52 mmol, 1.00

equiv) was dissolved in a mixture of 4-methylpiperidine/DCM (v/v =

1:20, 63 mL), and the mixture was stirred overnight at room

temperature. The solvent was removed under reduced pressure and

the crude product directly subjected to ﬂash column chromatography

(CH/EA = 2:1 + 1% NEt₃) to aﬀord 15 (2.11 g, 70%) as a light

1

colorless resin. TLC (CH/EA = 3:1, v/v): R_f= 0.26. H NMR (500

MHz, CDCl₃, mixture of rotamers 0.42:1, A/B): δ 7.30−7.26 (m,

0.25H, A), 7.11−7.06 (m, 0.69H, B), 6.95−6.90 (m, 0.62H, B),

6.88−6.85 (m, 0.31H, A), 6.80−6.73 (m, 1.77H, A+B), 6.70−6.66

(m, 1.04H, A+B), 6.66−6.60 (m, 1.80H, A+B), 6.47 (s, 1.43H, B),

6.46−6.42 (m, 0.75H, A+B), 6.41−6.39 (m, 0.63H, A), 6.08−5.98

(m, 0.96H, A+B), 5.95−5.83 (m, 0.95H, A+B), 5.80 (t, J = 6.9 Hz,

0.31H, A), 5.58−5.53 (m, 0.71H, B), 5.45 (d, J = 5.5 Hz, 0.70H, B),

5.43−5.35 (m, 1.04H, A+B), 5.30−5.20 (m, 2.05H, A+B), 5.17−5.10

(m, 1.01H, A+B), 4.72−4.68 (m, 0.30H, A), 4.58−4.46 (m, 2.42H, A

+B), 4.07−4.02 (m, 0.70H, B), 4.01−3.90 (m, 4.42H, A+B), 3.86−

3.77 (m, 8.55H, A+B), 3.70−3.63 (m, 5.14H, A+B), 3.63−3.58 (m,

1.51H), A+B, 3.36 (d, J = 9.7 Hz, 0.70H, B), 2.96 (d, J = 9.5 Hz,

0.3H, A), 2.83−2.75 (m, 0.69H, B), 2.64−2.50 (m, 0.96H, A+B),

2.49−2.34 (m, 1.46H, A+B), 2.31−2.23 (m, 1.06H, A+B), 2.13−2.04

(m, 1.58H, A+B), 2.01−1.90 (m, 2.71H, A+B), 1.90−1.78 (m, 2.00H,

A+B), 1.72−1.49 (m, 6.50H, A+B), 1.46−1.25 (m, 3.15H, A+B),

1.20−1.06 (m, 2.98H, A+B), 1.03−0.82 (m, 1.21H, A+B), 0.79−0.68

(m, 0.69H, A+B), 0.66−0.52 (m, 0.57H, A+B). ¹³C NMR (126 MHz,

CDCl₃, mixture of rotamers 2:5, A/B): δ 172.47 (B), 172.31 (A),

170.71 (A), 170.59 (B), 158.95 (A), 158.58 (B), 153.62 (A), 153.36

(B), 149.10 (A), 148.97 (B), 147.62 (A), 147.41 (B), 141.89 (B),

141.59 (A), 136.24 (A), 135.23 (B), 135.19 (A), 134.23 (A), 133.63

(B), 133.50 (B), 133.42 (A), 133.37 (B), 133.13 (A), 129.80 (A),

129.58 (B), 120.39 (B), 120.23 (A), 119.27 (A), 118.67 (B), 117.87

(A), 117.64 (B), 116.68 (A), 116.60 (B), 114.33 (A), 114.00 (B),

113.42 (A), 113.25 (B), 111.98 (B), 111.87 (A), 111.56 (A), 111.47

(B), 106.06 (B), 105.55 (A), 76.83 (A), 75.69 (B), 72.01 (A), 71.96

(B), 70.56 (A), 70.48 (B), 68.91 (A), 68.84 (B), 67.55 (B), 67.49

(A), 56.43 (B), 56.16 (B), 56.06 (A), 56.03 (B), 55.98 (A), 55.95

(B), 55.90 (A), 55.12 (B), 52.14 (B), 43.72 (B), 41.50 (B), 41.18

(A), 39.54 (A), 38.04 (B), 37.91 (A), 33.12 (A), 32.90 (B), 31.56

(A), 31.10 (B), 30.76 (B), 30.69 (A+B), 30.58 (A), 26.87 (B), 26.83

(A), 26.70 (B), 26.56 (A), 26.33 (A+B), 26.29 (B), 26.21 (A), 25.65

(B), 24.44 (A), 21.06 (B), 20.80 (A). HRMS (m/z): (ESI⁺)

calculated for C₄₈H₆₄NO₁₀[M + H]⁺: 814.45247, found 814.45247.

HPLC (70−100% solvent B, 1.5 mL/min, 20 min): t_R= 11.28 min,

purity (220 nm) = 99%.

(R)-1-(3-(Allyloxy)phenyl)-3-(3,4-dimethoxyphenyl)propan-

1-ol (14). To a stirred solution of 13 (2.00 g, 6.94 mmol, 1.00 equiv,

synthesized as previously described²⁰) and K₂CO₃(1.44 g, 10.40

mmol, 1.50 equiv) in acetone was added allyl bromide (1.68 g, 13.88

mmol, 2.00 equiv), and the reaction mixture was heated at reﬂux for 2

days. After the ﬁrst day, another portion of allyl bromide (0.50 equiv)

was added to the reaction mixture. The suspension was ﬁltered

through Celite, and the solvent was removed under reduced pressure.

The crude product was puriﬁed by ﬂash column chromatography

(CH/EA = 3:1) to aﬀord 14 (2.18 g, 96%) as cloudy, yellow oil. TLC

(CH/EA = 2:1, v/v): R_f= 0.40. ¹H NMR (500 MHz, CDCl₃): δ 7.25

(t, J = 7.8 Hz, 1H), 6.96−6.89 (m, 2H), 6.86−6.81 (m, 1H), 6.78 (d,

J = 8.0 Hz, 1H), 6.75−6.68 (m, 2H), 6.12−5.98 (m, 1H), 5.46−5.37

(m, 1H), 5.33−5.23 (m, 1H), 4.65 (dd, J = 5.2, 7.9 Hz, 1H), 4.53 (dt,

J = 1.5, 5.3 Hz, 2H), 3.85 (s, 3H), 3.85 (s, 3H), 2.80−2.48 (m, 2H),

2.18−1.90 (m, 2H). ¹³C NMR (126 MHz, CDCl₃): δ 158.9, 148.9,

147.3, 146.5, 134.5, 133.3, 129.6, 120.3, 118.5, 117.7, 113.8, 112.4,

111.9, 111.4, 73.8, 68.9, 56.0, 55.9, 40.7, 31.7. HRMS (m/z): (ESI⁺)

calculated for C₂₀H₂₄NaO₄[M + Na]⁺: 351.15668, found 351.15644.

HPLC (5−95% solvent B, 1.5 mL/min, 20 min): t_R= 14.25 min,

purity (220 nm) = 99%.

1

yellow oil. TLC (DCM/MeOH = 20:1, v/v): R_f= 0.24. H NMR

(500 MHz, DMSO-d₆, T = 353 K, mixture of rotamers 0.3:1, A/B): δ

7.43−7.18 (m, 1H, A+B), 7.00−6.87 (m, 4.03H, A+B), 6.82 (d, J =

2.0 Hz, 0.72H, B), 6.78 (d, J = 2.0 Hz, 0.26H, A), 6.76−6.68 (m,

0.99H, A+B), 6.19−5.99 (m, 0.86H, A+B), 5.84−5.73 (m, 0.98H, A

+B), 5.54−5.38 (m, 0.94H, A+B), 5.35−5.18 (m, 0.94H, A+B),

4.71−4.55 (m, 2.02H, A+B), 3.81 (s, 2.04H, B), 3.80−3.73 (m,

3.70H, A+B), 3.48 (t, J = 4.9 Hz, 0.25H, A), 3.39 (dd, J = 3.3, 9.3 Hz,

0.69H, B), 3.03−2.88 (m, 1.88H, A+B), 2.74−2.49 (m, 3.05H, A+B),

2.48−2.38 (m, 0.25H, A), 2.31−2.18 (m, 1.03H, A+B), 2.17−2.05

(m, 1.05H, A+B), 2.00−1.82 (m, 1.12H, A+B), 1.83−1.63 (m, 1.11H,

A+B), 1.62−1.35 (m, 3.99H, A+B), 1.29−1.20 (m, 0.21H, A+B),

1.01−0.80 (m, 0.28H, A). ¹³C NMR (126 MHz, DMSO-d₆, T = 353

K, mixture of rotamers 1:3, A/B): δ 172.22 (B), 172.14 (A), 158.42

(B), 158.39 (A), 149.20 (A+B), 147.58 (A+B), 142.20 (B), 142.08

(A), 133.83 (B), 133.74 (A+B), 133.69 (A), 129.36 (B), 129.29 (A),

120.25 (B), 120.20 (A), 118.67 (A), 118.51 (B), 117.09 (B), 116.99

(A), 114.01 (A+B), 113.18 (B), 113.11 (A), 113.07 (A), 112.96 (A

+B), 112.87 (B), 74.64 (B), 74.54 (A), 68.41 (B), 68.39 (A), 60.10

(A), 58.21 (B), 55.96 (A+B), 55.82 (B), 55.79 (A), 47.20 (A), 45.02

(B), 37.41 (B), 37.35 (A), 30.68 (B), 30.66 (A), 28.89 (B), 28.22

(A), 25.62 (B), 24.87 (A), 23.59 (B), 21.53 (A). HRMS (m/z):

(ESI⁺) calculated for C₂₆H₃₄NO₅[M + H]⁺: 440.24315, found

440.24365. HPLC (40−70% solvent B, 1.5 mL/min, 20 min): t_R

6.47 min, purity (220 nm) = 96%.

=

2-(Allyloxy)ethyl 4-Methylbenzenesulfonate (16). To a

stirred solution of 2-(allyloxy)ethanol (3.00 g, 29.37 mmol, 1.00

equiv) and pyridine (3.49 g, 44.06 mmol, 1.50 equiv) in dry DCM

(50 mL) at 0 °C was added p-toluenesulfonyl chloride (6.72 g, 35.25

mmol, 1.20 equiv). The reaction mixture was allowed to reach

ambient temperature and left stirring overnight. Then additional

portions of pyridine (0.50 equiv) and p-toluenesulfonyl chloride (0.50

equiv) were added, and the reaction was stirred for another night. The

reaction mixture was diluted with DCM (50 mL), washed with 1 M

HCl solution (100 mL), 4% aqueous NaHCO₃, water, and brine (50

mL each). The organic phase was dried over MgSO₄and

concentrated under reduced pressure. The crude product was puriﬁed

by ﬂash column chromatography (CH/EA = 8:1) to yield 16 (4.77 g,

1

63%) as red liquid. TLC (CH/EA = 5:1, v/v): R_f= 0.45. H NMR

(500 MHz, CDCl₃): δ 7.77−7.73 (m, 2H), 7.33−7.28 (m, 2H), 5.77

(ddt, J = 5.5, 10.4, 17.3 Hz, 1H), 5.20−5.15 (m, 1H), 5.12−5.08 (m,

1H), 4.15−4.11 (m, 2H), 3.91−3.87 (m, 2H), 3.60−3.55 (m, 2H),

2.39 (s, 3H). ¹³C NMR (126 MHz, CDCl₃): δ 144.7, 134.1, 132.9,

129.7, 127.8, 117.0, 71.9, 69.3, 67.3, 21.4. LC−MS (m/z): (ESI⁺)

calculated for C₁₂H₁₇O₄S [M + H]⁺: 257.08, found 257.05.

3-(Allyloxy)propyl 4-Methylbenzenesulfonate (17). To a

stirred solution of 1,3-propanediol (4.00 g, 52.56 mmol, 1.00 equiv)

in dry THF (50 mL) at 0 °C was added sodium hydride (2.32 g,

57.82 g, 1.10 equiv) in portions. After stirring for 30 min at 0 °C, allyl

bromide (9.54 g, 78.84 mmol, 1.50 equiv) was added to the gray

suspension and the resulting yellow suspension was allowed to reach

ambient temperature and was left stirring overnight. The reaction

mixture was diluted with Et₂O (50 mL) and washed with 10%

aqueous K₂CO₃and brine (25 mL each). The organic phase was dried

(S)-(R)-1-(3-(Allyloxy)phenyl)-3-(3,4-dimethoxyphenyl)-

propyl Piperidine-2-carboxylate (15). To a stirred solution of 14

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over MgSO₄and concentrated under reduced pressure (75 mbar, 40

°C; product is volatile). The crude product was puriﬁed by ﬂash

column chromatography (CH/EA = 2:1) to yield S2 (2.94 g, 48%,

analytics shown in the Supporting Information) as a light yellow

liquid. To a stirred solution of S2 (2.94 g, 25.34 mmol, 1.00 equiv)

and pyridine (4.01 g, 50.67 mmol, 2.00 equiv) in dry DCM (50 mL)

at 0 °C was added p-toluenesulfonyl chloride (9.66 g, 50.67 mmol,

2.00 equiv). The reaction mixture was allowed to reach ambient

temperature and left stirring overnight. The reaction mixture was

diluted with DCM (50 mL), washed with 1 M HCl solution (100

mL), 4% aqueous NaHCO₃, water, and brine (50 mL each). The

organic phase was dried over MgSO₄and concentrated under reduced

pressure. The crude product was puriﬁed by ﬂash column

chromatography (CH/EA = 8:1 → 6:1) to yield 17 (5.06 g, 74%)

as a light yellow liquid. TLC (CH/EA = 4:1, v/v): R_f= 0.46. ¹H NMR

(500 MHz, CDCl₃): δ 7.80−7.75 (m, 2H), 7.35−7.30 (m, 2H), 5.80

(ddt, J = 5.6, 10.4, 17.2 Hz, 1H), 5.18 (dq, J = 1.7, 17.3 Hz, 1H), 5.12

(dq, J = 1.4, 10.4 Hz, 1H), 4.13 (t, J = 6.2 Hz, 2H), 3.85 (dt, J = 1.4,

5.5 Hz, 2H), 3.43 (t, J = 6.0 Hz, 2H), 2.43 (s, 3H), 1.90 (p, J = 6.1

Hz, 2H). ¹³C NMR (126 MHz, CDCl₃): δ 144.8, 134.7, 133.2, 129.9,

128.0, 117.0, 72.0, 67.8, 65.6, 29.5, 21.7. LC−MS (m/z): (ESI⁺)

calculated for C₁₃H₁₉O₄S [M + H]⁺: 271.10, found 271.08.

by ﬂash column chromatography (CH/EA = 4:1 → 3:1) to yield 18

(5.13 g, 81%) as a yellow oil. TLC (CH/EA = 3:1, v/v): R_f= 0.22. ¹H

NMR (500 MHz, CDCl₃): δ 7.43−7.32 (m, 5H), 6.54 (d, J = 1.9 Hz,

1H), 6.50 (d, J = 1.9 Hz, 1H), 5.49−5.36 (m, 2H), 5.07 (s, 2H), 3.68

(s, 3H), 3.49 (s, 2H). ¹³C NMR (126 MHz, CDCl₃): δ 172.4, 146.3,

144.2, 136.4, 132.0, 128.8, 128.6, 128.1, 125.8, 110.2, 105.8, 71.5,

52.2, 41.1. LC−MS (m/z): (ESI⁺) calculated for C₁₆H₁₇O₅[M + H]⁺:

289.11, found 289.16.

Methyl 2-(3-(benzyloxy)-4,5-dimethoxyphenyl)acetate (19).

To a stirred solution of 18 (5.13 g, 17.8 mmol, 1.00 equiv) in

degassed DMF (60 mL, 0.3 M) were added K₂CO₃(7.37 g, 53.3

mmol, 3.00 equiv) and methyl iodide (10.10 g, 71.1 mmol, 4.00

equiv), and the reaction mixture was stirred at room temperature

overnight. The resulting red solution was diluted with water (100 mL)

and then extracted with Et₂O (4 × 50 mL). The combined organic

phases were washed with brine, dried over MgSO₄, ﬁltered, and

concentrated under reduced pressure. The crude product was puriﬁed

by ﬂash column chromatography (CH/EA = 6:1 → 3:1) to yield 19

(5.25 g, 93%) as a light yellow oil. TLC (CH/EA = 3:1, v/v): R_f=

0.42. ¹H NMR (500 MHz, CDCl₃): δ 7.46−7.43 (m, 2H), 7.39−7.36

(m, 2H), 7.33−7.29 (m, 1H), 6.55 (d, J = 1.9 Hz, 1H), 6.51 (d, J =

1.9 Hz, 1H), 5.12 (s, 2H), 3.86 (s, 6H), 3.68 (s, 3H), 3.52 (s, 2H).

¹³C NMR (126 MHz, CDCl₃): δ 172.0, 153.6, 152.6, 138.1, 137.3,

Methyl 2-(3-(benzyloxy)-4,5-dihydroxyphenyl)acetate (18).

A solution of 2-(3,4,5-trimethoxyphenyl)acetic acid (60.00 g, 265.2

mmol, 1.00 equiv) and acetic acid (160 mL) in concentrated

hydrobromic acid (48% w/w aq, 200 mL) was heated at reﬂux until

the starting material was no longer detected by TLC. A saturated

solution of sodium sulfate (100 mL) was added to the reaction

mixture and was then extracted with EA (8 × 100 mL). The organic

phase was dried over MgSO₄and concentrated under reduced

pressure. The crude product S3 (analytics shown in the Supporting

Information) was ﬁltered through a plug of silica and concentrated

under reduced pressure. Subsequently, crude S3 was dissolved in

MeOH (300 mL), and a catalytic amount of H₂SO₄(0.26 g, 2.7

mmol, 0.01 equiv) was added and the reaction mixture was stirred at

room temperature for 14 h. Then ice water (300 mL) was added to

the reaction mixture, and the layers were separated. The aqueous

phase was extracted with Et₂O (3 × 150 mL) and the combined

organic phases were washed with brine, dried over MgSO₄, ﬁltered,

and concentrated under reduced pressure to yield S4 (38.22 g, 73%,

analytics shown in the Supporting Information) as dark-red solid

without further puriﬁcation. A solution of S4 (12.40 g, 62.6 mmol,

1.00 equiv), p-toluenesulfonic acid monohydrate (1.19 g, 6.3 mmol,

0.10 equiv), and 2,2-dimethoxypropane (32.59 g, 312.9 mmol, 5.00

equiv) in toluene (1250 mL) was heated at reﬂux for 2 h. Then

additional portions of p-toluenesulfonic acid monohydrate (0.05

equiv) and 2,2-dimethoxypropane (1.00 equiv) were added, and the

reaction was heated at reﬂux for another hour. Then the reaction

mixture was diluted with Et₂O (500 mL), washed with saturated aq

NaHCO₃solution and brine, dried over MgSO₄, ﬁltered, and

concentrated under reduced pressure. The crude product was puriﬁed

by ﬂash column chromatography (CH/EA = 8:1 → 2:1) to yield S5

(14.21 g, 95%) as brown oil. Subsequently, to a stirred solution of S5

(31.20 g, 131.0 mmol, 1.00 equiv) in acetone (525 mL, 0.25 M) were

added K₂CO₃(21.72 g, 157.2 mmol, 1.20 equiv) and benzyl bromide

(24.64 g, 144.1 mmol, 1.10 equiv), and the reaction mixture was

heated at reﬂux overnight. The resulting suspension was ﬁltered

through Celite and washed with EA (500 mL), and the solvent was

removed under reduced pressure. The crude product was puriﬁed by

ﬂash column chromatography (CH/EA = 20:1) to yield S6 (42.16 g,

98%, analytics shown in the Supporting Information) as a light yellow

solid. To a stirred solution of methyl 2-(7-(benzyloxy)-2,2-

dimethylbenzo[d][1,3]dioxol-5-yl)acetate S6 (7.23 g, 22.0 mmol,

1.00 equiv) in MeCN/H₂O (70 mL, v/v = 1:1) was added

triﬂuoroacetic acid (170 mL), and the reaction mixture was stirred

at room temperature for 4 h. The reaction mixture was quenched by

the addition of saturated aq NaHCO₃solution. The aqueous phase

was extracted with EA (3 × 150 mL) and the combined organic phase

subsequently washed with brine, dried over MgSO₄, ﬁltered, and

concentrated under reduced pressure. The crude product was puriﬁed

129.5, 128.6, 128.0, 127.5, 108.8, 107.0, 71.3, 61.0, 56.3, 52.2, 41.5.

HRMS (m/z): (ESI⁺) calculated for C₁₈H₂₁O₅[M + H]⁺: 317.13835,

found 317.13795.

Methyl 2-(3-(benzyloxy)-4,5-dimethoxyphenyl)-2-(cyclo-

hex-2-en-1-yl)acetate (20). To a stirred solution of 19 (4.85 g,

15.3 mmol, 1.0 equiv) in dry THF (50 mL) was added LiHMDS (1

M in THF, 16.9 mL, 1.10 equiv) dropwise at −78 °C, and the

reaction mixture was stirred at this temperature for 20 min. Then 3-

bromocyclohex-1-ene (5.20 g, 30.7 mmol, 2.00 equiv) was added

dropwise. The reaction mixture was allowed to warm to −40 °C over

a period of 3 h (TLC indicated full conversion) before the reaction

mixture was quenched by the addition of saturated NH₄Cl solution

(50 mL). The aqueous phase was extracted with Et₂O (3 × 50 mL),

and the combined organic phases were washed with brine, dried over

MgSO₄, ﬁltered, and concentrated under reduced pressure. The crude

product was puriﬁed by ﬂash column chromatography (CH/EA =

10:1) to yield 20 (5.72 g, 94%) as a light yellow oil and a mixture of

1

diastereomers. TLC (CH/EA = 7:1, v/v): R_f= 0.38. H NMR (500

MHz, CDCl₃, mixture of diastereomers): δ 7.47−7.41 (m, 3.07H),

7.38−7.33 (m, 3.00H), 7.32−7.27 (m, 1.40H), 6.62 (d, J = 1.9 Hz,

0.53H), 6.58−6.57 (m, 1.44H), 6.54−6.52 (m, 1.00H), 5.80−5.74

(m, 1.14H), 5.67−5.61 (m, 0.48H), 5.60−5.53 (m, 1.10H), 5.18−

5.10 (m, 2.19H), 3.91−3.81 (m, 9.92H), 3.67−3.62 (m, 4.53H),

3.21−3.14 (m, 1.59H), 2.82−2.66 (m, 1.67H), 2.01−1.93 (m,

3.12H), 1.90−1.83 (m, 0.54H), 1.79−1.53 (m, 3.17H), 1.47−1.37

(m, 1.46H), 1.36−1.27 (m, 1.82H), 1.04−0.94 (m, 1.17H), 0.91−

0.82 (m, 0.41H). ¹³C NMR (126 MHz, CDCl₃, mixture of

diastereomers): δ 174.04, 173.96, 153.45, 153.42, 152.26, 152.19,

138.19, 137.20, 133.08, 132.91, 129.35, 128.95, 128.91, 128.57,

127.95, 127.90, 127.58, 127.53, 108.22, 107.94, 106.25, 105.89, 71.20,

71.16, 60.96, 57.62, 57.54, 56.27, 51.98, 38.82, 38.67, 28.04, 26.34,

25.38, 25.28, 21.25, 20.80. HRMS (m/z): (ESI⁺) calculated for

C₂₄H₂₉O₅[M + H]⁺: 397.20095, found 397.20114. HPLC (55−65%

solvent B, 1.5 mL/min, 20 min): t_R,1= 13.39 min, t_R,2= 13.62 min (no

baseline separation of peaks achieved), purity (220 nm) = 99%.

2-(3-(Benzyloxy)-4,5-dimethoxyphenyl)-2-(cyclohex-2-en-1-

yl)acetic Acid (21). To a stirred solution of 20 (1147 mg, 2.90

mmol, 1.00 equiv) in THF/H₂O (v/v = 1:1, 50 mL) under air at

room temperature was added lithium hydroxide (348 mg, 14.50

mmol, 5.00 equiv), and the mixture was heated at reﬂux overnight.

The reaction mixture was extracted with Et₂O (3 × 30 mL), the

organic layer removed, and the aqueous layer subsequently acidiﬁed

with 1 M HCl solution (pH = 1−2). After extraction with Et₂O (3 ×

30 mL), the organic phase was washed with brine, dried over MgSO₄,

and concentrated under reduced pressure. The crude product was

puriﬁed by ﬂash column chromatography (CH/EA = 6:1 + 0.2%

HCOOH) to aﬀord 21 (1.01 g, 91%) as a colorless resin and mixture

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of diastereomers. TLC (DCM/MeOH/HCOOH = 80:1:1, v/v/v): R_f

= 0.29. ¹H NMR (500 MHz, CDCl₃, mixture of diastereomers):

7.45−7.40 (m, 2H), 7.38−7.31 (m, 2H), 7.30−7.25 (m, 1H), 6.67−

6.65 (m, 0.5H), 6.62−6.58 (m, 1H), 6.57−6.54 (m, 0.5H), 5.83−5.77

(m, 0.5H), 5.70−5.63 (m, 1H), 5.18−5.10 (m, 2.5H), 3.89−3.83 (m,

6H), 3.19 (t, J = 11.3 Hz, 1H), 2.81−2.67 (m, 1H), 2.02−1.93 (m,

2.5H), 1.81−1.73 (m, 0.5H), 1.63−1.55 (m, 1H), 1.45−1.32 (m,

1.5H), 1.03−0.95 (m, 0.5H). ¹³C NMR (126 MHz, CDCl₃, mixture

of diastereomers): δ 179.52, 179.40, 153.51, 153.47, 152.33, 152.26,

138.42, 138.39, 137.07, 132.46, 132.30, 129.63, 129.23, 128.61,

128.57, 127.96, 127.64, 127.59, 108.40, 108.12, 106.44, 106.10, 71.27,

71.23, 60.99, 57.67, 57.62, 56.29, 38.45, 38.29, 28.01, 26.22, 25.35,

25.25, 21.19, 20.76. HRMS (m/z): (ESI⁺) calculated for C₂₃H₂₇O₅

[M + H]⁺: 383.18530, found 383.18513. HPLC (50−60% solvent B,

1.5 mL/min, 20 min): t_R= 9.39 min, purity (220 nm) = 99% (no peak

separation achieved).

20.96 (A), 20.86 (B). HRMS (m/z): (ESI⁺) calculated for

C₂₂H₃₂NO₆[M + H]⁺: 406.22241, found 406.22231. HPLC (30−

50% solvent B, 1.5 mL/min, 20 min): t_R= 13.42 min, purity (220

nm) = 99%. Data for 22b: TLC (DCM/MeOH/HCOOH = 80:1:1,

v/v/v; two runs): R_f= 0.38. ¹H NMR (500 MHz, CDCl₃, mixture of

rotamers 0.12:1, A/B): δ 6.51−6.47 (m, 0.84H, A+B), 6.47−6.43 (m,

0.86H, A+B), 6.42−6.39 (m, 0.34H, A), 5.48−5.43 (m, 0.82H, B),

4.94−4.90 (m, 0.10H, A), 4.53 (d, J = 13.4 Hz, 0.08H, A), 4.01−3.94

(m, 0.86H, B), 3.87−3.84 (m, 2.56H, A+B), 3.84−3.77 (m, 3.43H, A

+B), 3.36 (d, J = 10.0 Hz, 0.84H, B), 3.23 (td, J = 3.1, 13.1 Hz,

0.99H, A+B), 2.80−2.72 (m, 0.10H, A), 2.30−2.14 (m, 1.03H, A+B),

2.14−2.01 (m, 1.04H, A+B), 1.89 (d, J = 12.7 Hz, 1.00H, A+B),

1.74−1.42 (m, 6.60H, A+B), 1.42−1.20 (m, 4.08H, A+B), 1.19−0.93

(m, 3.33H, A+B), 0.93−0.79 (m, 1.25H, A+B), 0.79−0.65 (m,

1.05H). ¹³C NMR (126 MHz, CDCl₃, mixture of rotamers 1:7, A:B):

δ 176.28 (B), 176.20 (A), 173.87 (B), 172.98 (A), 152.68 (B),

149.17 (B), 134.87 (B), 134.61 (B), 133.67 (A), 109.65 (A), 108.95

(B), 104.22 (A), 103.90 (B), 61.02 (B), 60.85 (A), 56.10 (B), 56.03

(A), 55.34 (A), 54.78 (B), 53.53 (A), 52.32 (B), 43.76 (B), 41.35

(B), 41.08 (A), 39.97 (A), 32.73 (A), 32.61 (B), 30.80 (B), 29.78

(A), 28.10 (A), 26.66 (B), 26.27 (B), 26.21 (B), 25.04 (B), 24.97

(A), 21.00 (A), 20.86 (B). HRMS (m/z): (ESI⁺) calculated for

C₂₂H₃₂NO₆[M + H]⁺: 406.22241, found 406.22269. HPLC (30−

50% solvent B, 1.5 mL/min, 20 min): t_R= 11.78 min, purity (220

nm) = 99%.

(S)-1-((S)-2-(3-(Allyloxy)-4,5-dimethoxyphenyl)-2-

cyclohexylacetyl)piperidine-2-carboxylic Acid (23). The sub-

strate 22a (350 mg, 0.86 mmol, 1.00 equiv) was applied to general

procedure A with K₂CO₃(358 mg, 2.59 mmol, 3.00 equiv) and allyl

bromide (313 mg, 2.59 mmol, 3.00 equiv) in MeCN (20 mL). An

additional portion of allyl bromide (0.50 equiv) was added until

completion of the reaction. The product S8 (390 mg, 93%, analytics

shown in the Supporting Information) was obtained after puriﬁcation

by ﬂash column chromatography (CH/EA = 6:1) as a light yellow oil.

Subsequently, S8 (390 mg, 0.80 mmol, 1.00 equiv) was applied to

general procedure F with LiOH (78 mg, 3.20 mmol, 4.00 equiv) in

H₂O/MeOH/THF (12 mL). 23 (322 mg, 90%) was obtained after

puriﬁcation by ﬂash column chromatography (CH/EA = 3:1 + 0.2%

HCOOH) as a white solid. TLC (CH/EA = 2:1 + 1% HCOOH, v/v/

v): R_f= 0.34. ¹H NMR (500 MHz, CDCl₃, mixture of rotamers

0.36:1, A/B): δ 8.31 (br s, 0.79H, A+B), 6.47−6.40 (m, 2.12H, A+B),

6.07−5.95 (m, 0.99H, A+B), 5.39−5.33 (m, 1.03H, A+B), 5.29 (dd, J

= 2.4, 6.1 Hz, 0.74H, B), 5.25−5.19 (m, 1.04H, A+B), 4.75 (d, J = 5.6

Hz, 0.26H, A), 4.63−4.49 (m, 2.41H, A+B), 3.90−3.73 (m, 7.19H, A

+B), 3.32 (d, J = 9.6 Hz, 0.74H, B), 3.09 (d, J = 9.7 Hz, 0.26H, A),

2.94−2.85 (m, 0.73H, B), 2.67−2.59 (m, 0.26H, A), 2.26−2.19 (m,

0.73H, B), 2.11−1.99 (m, 1.48H, A+B), 1.93−1.79 (m, 1.06H, A+B),

1.72−1.52 (m, 6.16H, A+B), 1.51−1.24 (m, 3.90H, A+B), 1.21−0.97

(m, 3.11H, A+B), 0.96−0.85 (m, 0.82H, A+B), 0.83−0.64 (m, 1.38H,

A+B). ¹³C NMR (126 MHz, CDCl₃, mixture of rotamers 3:1, A/B): δ

175.84 (A), 175.71 (B), 173.68 (B), 172.26 (A), 153.62 (A), 153.28

(B), 152.13 (A), 151.94 (B), 137.59 (A), 137.55 (B), 134.09 (A),

133.57 (B), 133.39 (A), 132.98 (B), 117.68 (A), 117.53 (B), 108.10

(B), 107.58 (A), 106.01 (B), 105.33 (A), 70.04 (B), 69.96 (A), 60.96

(A), 60.88 (B), 56.41 (A), 56.26 (B), 55.73 (A), 55.39 (B), 52.44

(B), 43.77 (B), 41.29 (B), 41.20 (A), 39.65 (A), 32.93 (B), 30.80

(B), 30.68 (A), 26.88 (A), 26.66 (B), 26.55 (B), 26.33 (A+B), 26.22

(A), 25.37 (B), 24.50 (A), 20.98 (A), 20.89 (B). HRMS (m/z):

(ESI⁺) calculated for C₂₅H₃₆NO₆[M + H]⁺: 446.25371, found

(S)-1-((S)-2-Cyclohexyl-2-(3-hydroxy-4,5-dimethoxyphenyl)-

acetyl)piperidine-2-carboxylic Acid (22a) and (S)-1-((R)-2-

Cyclohexyl-2-(3-hydroxy-4,5-dimethoxyphenyl)acetyl)-

piperidine-2-carboxylic Acid (22b). To a stirred solution of 21

(3.11 g, 8.14 mmol mmol, 1.00 equiv) in DCM (125 mL) at 0 °C was

added DMF (6 μL, 0,08 mmol, 0.01 equiv) and thionyl chloride (0.89

mL, 12.21 mmol, 1.50 equiv), and the reaction mixture was stirred at

0 °C overnight. Then additional portions of DMF (0.05 equiv) and

thionyl chloride (0.20 equiv) were added, and the reaction was stirred

for one more hour. The volatiles were removed under reduced

pressure to yield the crude acid chloride as a yellow oil, which was

used in the following step without further puriﬁcation. (S)-piperidine-

2-carboxylic acid (1168 mg, 8.95 mmol, 1.10 equiv) was dissolved in 1

M KOH aqueous solution (18 mL, 17.9 mmol, 2.20 equiv) and

cooled to 0 °C. In a separate ﬂask, the crude acid chloride was

dissolved in 1,4-dioxane (25 mL) and was added to the above

aqueous solution at 0 °C. After stirring for 1 h at 0 °C, the reaction

mixture was allowed to reach ambient temperature and was left

stirring overnight. Then the reaction mixture was quenched and

acidiﬁed with 1 M HCl solution (pH = 1−2), extracted with EA (3 ×

30 mL), dried over MgSO_4,and concentrated under reduced pressure.

The crude product was puriﬁed by ﬂash column chromatography

(CH/EA = 5:1 + 1% HCOOH) to aﬀord product S7 (2858 mg, 71%,

white solid, mixture of diastereomers, analytics shown in the

Supporting Information) and recovered starting material 21 (885

mg, 28%). Subsequently, a stirred solution of S7 (6.15 g, 12.46 mmol,

1.00 equiv) in THF/MeOH (v/v = 1:1, 300 mL) was degassed by

sparging with argon for 15 min. After addition of Pd/C (10 wt %,

1330 mg, 1.25 mmol, 0.10 equiv), the solution was sparged with

hydrogen for 15 min and was then stirred under a hydrogen

atmosphere at room temperature overnight. The dark suspension was

ﬁltered through Celite, and the solvent was removed under reduced

pressure. The crude product was puriﬁed by ﬂash column

chromatography (DCM/MeOH/HCOOH = 90:1:1) to aﬀord the

desired product 22a (2089 mg, 41%) and the corresponding

diastereomer 22b (2210 mg, 44%) as white solids. A detailed

description of the assignment of diastereomers 22a and 22b to the

desired (S) conﬁguration can be found in the Supporting Information.

Data for 22a: TLC (DCM/MeOH/HCOOH = 80:1:1, v/v/v; two

1

runs): R_f= 0.36. H NMR (500 MHz, CDCl₃, mixture of rotamers

0.32:1, A/B): δ 6.47−6.34 (m, 1.96H, A+B), 4.78 (d, J = 5.5 Hz,

0.17H, A), 4.58 (d, J = 13.7 Hz, 0.13H, A), 3.95−3.66 (m, 6.90H, A

+B), 3.30 (d, J = 9.6 Hz, 0.76H, B), 3.10 (d, J = 9.7 Hz, 0.24H, A),

2.97−2.85 (m, 0.70H, B), 2.64 (td, J = 2.8, 13.4 Hz, 0.22H, A), 2.29−

2.17 (m, 0.75H, B), 2.12−1.97 (m, 1.30H, A+B), 1.92−1.76 (m,

1.22H, A+B), 1.75−1.50 (m, 6.15H, A+B), 1.50−1.00 (m, 7.50H, A

+B), 0.98−0.63 (m, 2.32H, A+B). ¹³C NMR (126 MHz, CDCl₃,

mixture of rotamers 3:1, A:B): δ 175.65 (A), 175.52 (B), 173.75 (B),

172.53 (A), 152.84 (A), 152.60 (B), 149.30 (A), 148.99 (B), 134.73

(A), 134.64 (B), 134.60 (A), 133.53 (B), 109.29 (B), 108.72 (A),

104.27 (B), 103.57 (A), 61.04 (A), 60.96 (B), 56.13 (A), 56.02 (B),

55.79 (A), 55.44 (A), 55.22 (B), 53.53 (A), 52.52 (B), 43.79 (B),

41.12 (B), 39.72 (A), 32.89 (B), 32.85 (A), 30.77 (B), 30.65 (A),

26.92 (A), 26.64 (B), 26.30 (B), 26.22 (A), 25.34 (B), 24.49 (A),

446.25383. HPLC (40−60% solvent B, 1.5 mL/min, 20 min): t_R

13.45 min, purity (220 nm) = 97%.

=

(S)-1-((S)-2-(3-(But-3-en-1-yloxy)-4,5-dimethoxyphenyl)-2-

cyclohexylacetyl)piperidine-2-carboxylic Acid (24). The sub-

strate 22a (400 mg, 0.99 mmol, 1.00 equiv) was applied to general

procedure A with K₂CO₃(409 mg, 2.96 mmol, 3.00 equiv) and 4-

bromo-1-butene (532 mg, 3.94 mmol, 4.00 equiv) in MeCN (20 mL).

Additional portions of K₂CO₃(2 × 2.00 equiv) and 4-bromo-1-

butene (2.00 + 3.00 equiv) were added until completion of the

reaction. S9 (443 mg, 87%) was obtained as a light yellow resin after

puriﬁcation by ﬂash column chromatography (CH/EA = 7:1,

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analytics shown in the Supporting Information). Subsequently, the

substrate S9 (360 mg, 0.70 mmol, 1.00 equiv) was applied to general

procedure F with LiOH (67 mg, 2.80 mmol, 4.00 equiv) in H₂O/

MeOH/THF (16 mL). 24 (298 mg, 93%) was obtained as a white

solid after puriﬁcation by ﬂash column chromatography (CH/EA =

4:1 + 0.5% HCOOH). TLC (CH/EA = 2:1 + 1% HCOOH, v/v): R_f

obtained as a colorless resin after puriﬁcation by ﬂash column

chromatography (CH/EA = 5:1). Subsequently, the substrate S11

(694 mg, 1.15 mmol, 1.00 equiv) was applied to general procedure F

with LiOH (110 mg, 4.60 mmol, 4.00 equiv) in H₂O/MeOH/THF

(20 mL). 26 (563 mg, 97%) was obtained as white solid after

puriﬁcation by ﬂash column chromatography (CH/EA = 4:1 + 0.5%

HCOOH). TLC (CH/EA = 2:1 + 1% HCOOH, v/v): R_f= 0.35. ¹H

NMR (500 MHz, CDCl₃, mixture of rotamers 0.33:1, A/B): δ 8.73

(br s, 0.81H, A+B), 6.49−6.40 (m, 2.01H, A+B), 5.92−5.83 (m,

1.04H, A+B), 5.30 (dd, J = 2.3, 6.0 Hz, 0.74H, B), 5.27−5.21 (m,

1.09H, A+B), 5.16−5.11 (m, 1.04H, A+B), 4.77−4.74 (m, 0.23H, A),

4.61−4.55 (m, 0.23H, A), 4.09−4.02 (m, 2.00H, A+B), 4.00−3.94

(m, 2.45H, A+B), 3.87−3.73 (m, 7.33H, A+B), 3.64−3.58 (m, 2.14H,

A+B), 3.32 (d, J = 9.4 Hz, 0.72H, B), 3.11 (d, J = 9.7 Hz, 0.24H, A),

2.88−2.81 (m, 0.70H, B), 2.63 (td, J = 2.6, 13.3 Hz, 0.23H, A), 2.24−

2.19 (m, 0.71H, B), 2.08−1.99 (m, 3.12H, A+B), 1.91−1.80 (m,

1.27H, A+B), 1.68−1.54 (m, 6.02H, A+B), 1.46−1.25 (m, 3.49H, A

+B), 1.22−1.01 (m, 2.50H, A+B), 0.94−0.86 (m, 0.80H, B), 0.81−

0.67 (m, 1.19H, A+B). ¹³C NMR (126 MHz, CDCl₃, mixture of

rotamers 1:3, A/B): δ 175.12 (A), 174.90 (B), 173.31 (B), 172.30

(A), 153.48 (A), 153.12 (B), 152.65 (A), 152.25 (B), 137.43 (B),

134.85 (A), 134.67 (B), 134.22 (A), 133.02 (B), 117.28 (B), 116.95

(A), 107.84 (B), 107.04 (A), 106.02 (B), 105.21 (A), 71.97 (A),

71.93 (B), 66.92 (B), 66.27 (A), 66.21 (B), 60.88 (A), 60.83 (B),

56.37 (A), 56.22 (B), 55.74 (A), 55.60 (A), 55.40 (B), 52.30 (B),

43.71 (B), 41.14 (B), 39.62 (A), 32.97 (B), 32.89 (A), 30.79 (B),

30.66 (A), 29.83 (A), 29.68 (B), 26.84 (A), 26.64 (B), 26.54 (A),

26.33 (B), 26.20 (A), 25.38 (B), 24.50 (A), 20.94 (A), 20.88 (B).

HRMS (m/z): (ESI⁺) calculated for C₂₈H₄₂NO₇[M + H]⁺:

504.29558, found 504.29563. HPLC (50−100% solvent B, 0.8 mL/

min, 10.50 min): t_R= 4.95 min, purity (220 nm) = 91%.

1

= 0.37. H NMR (500 MHz, CDCl₃, mixture of rotamers 0.36:1, A/

B): δ 8.52 (br s, 0.81H, A+B), 6.51−6.37 (m, 2.16H, A+B), 5.97−

5.83 (m, 0.99H, A+B), 5.33−5.27 (m, 0.73H, B), 5.18−5.03 (m,

2.08H, A+B), 4.76 (d, J = 5.7 Hz, 0.26H, A), 4.63−4.55 (m, 0.26H,

A), 4.01 (q, J = 6.6 Hz, 2.21H, A+B), 3.92−3.84 (m, 0.77H, B),

3.83−3.72 (m, 6.23H, A+B), 3.32 (d, J = 9.6 Hz, 0.76H, B), 3.10 (d, J

= 9.7 Hz, 0.26H, A), 2.95−2.86 (m, 0.71H, B), 2.68−2.60 (m, 0.28H,

A), 2.57−2.47 (m, 2.15H, A+B), 2.26−2.19 (m, 0.76H, B), 2.12−

1.99 (m, 1.46H, A+B), 1.93−1.79 (m, 1.09H, A+B), 1.73−1.52 (m,

6.21H, A+B), 1.50−0.98 (m, 7.17H, A+B), 0.96−0.85 (m, 0.87H, B),

0.83−0.65 (m, 1.38H, A+B). ¹³C NMR (126 MHz, CDCl₃, mixture of

rotamers 2:5, A/B): δ 175.74 (B), 173.60 (B), 172.31 (A), 153.51

(A), 153.20 (B), 152.58 (A), 152.26 (B), 137.54 (A), 137.45 (B),

134.72 (B), 134.55 (A), 134.14 (A), 133.01 (B), 117.22 (A), 117.08

(B), 107.67 (B), 107.07 (A), 105.95 (B), 105.40 (A), 68.57 (A),

68.46 (B), 60.96 (A), 60.88 (B), 56.43 (A), 56.27 (B), 55.77 (A),

55.67 (A), 55.38 (B), 52.41 (B), 43.75 (B), 41.29 (B), 41.20 (A),

39.65 (A), 33.92 (B), 32.93 (B), 30.80 (B), 30.68 (A), 26.86 (A),

26.64 (B), 26.59 (B), 26.37 (A), 26.32 (B), 26.21 (A), 25.38 (B),

24.51 (A), 20.97 (A), 20.91 (B). HRMS (m/z): (ESI⁺) calculated for

C₂₆H₃₈NO₆[M + H]⁺: 460.26936, found 460.26956. HPLC (40−

60% solvent B, 1.5 mL/min, 20 min): t_R= 16.20 min, purity (220

nm) = 95%.

(S)-1-((S)-2-(3-(2-(Allyloxy)ethoxy)-4,5-dimethoxyphenyl)-2-

cyclohexylacetyl)piperidine-2-carboxylic Acid (25). The sub-

strate 22a (502 mg, 1.24 mmol, 1.00 equiv) was applied to general

procedure A with K₂CO₃(684 mg, 4.95 mmol, 4.00 equiv) and 16

(1587 mg, 6.19 mmol, 5.00 equiv) in MeCN (20 mL). S10 (635 mg,

94%) was obtained as a light yellow oil after puriﬁcation by ﬂash

column chromatography (CH/EA = 5:1, analytics shown in the

Supporting Information). Subsequently, the substrate S10 (635 mg,

1.11 mmol, 1.00 equiv) was applied to general procedure F with

LiOH (106 mg, 4.43 mmol, 4.00 equiv) in H₂O/MeOH/THF (20

mL). 25 (511 mg, 94%) was obtained as a white solid after

puriﬁcation by ﬂash column chromatography (CH/EA = 5:1 + 0.5%

HCOOH). TLC (CH/EA = 2:1 + 1% HCOOH, v/v): R_f= 0.34. ¹H

NMR (500 MHz, CDCl₃, mixture of rotamers 0.34:1, A/B): δ 9.33

(br s, 0.90H, A+B), 6.48−6.43 (m, 2.11H, A+B), 5.96−5.85 (m,

1.06H, A+B), 5.33−5.24 (m, 1.89H, A+B), 5.20−5.14 (m, 1.07H, A

+B), 4.75 (d, J = 5.6 Hz, 0.26H, A), 4.62−4.56 (m, 0.26H, A), 4.17−

4.05 (m, 4.11H, A+B), 3.88−3.74 (m, 10.02H, A+B), 3.32 (d, J = 9.5

Hz, 0.75H, B), 3.10 (d, J = 9.6 Hz, 0.24H, A), 2.91−2.82 (m, 0.75H,

B), 2.67−2.60 (m, 0.25H, A), 2.27−2.19 (m, 0.77H, B), 2.11−2.00

(m, 1.33H, A+B), 1.90−1.81 (m, 0.96H, A+B), 1.70−1.56 (m, 5.93H,

(S)-(R)-1-(3-(Allyloxy)phenyl)-3-(3,4-dimethoxyphenyl)-

propyl 1-((S)-2-(3-(allyloxy)-4,5-dimethoxy-phenyl)-2-

cyclohexylacetyl)piperidine-2-carboxylate (27). The substrate

23 (246 mg, 0.55 mmol, 1.00 equiv) was applied to general procedure

G with 14 (181 mg, 0.55 mmol, 1.00 equiv), 4-(1-pyrrolidinyl)-

pyridine (334 mg, 2.21 mmol, 4.00 equiv), and EDC (119 mg, 0.61

mmol, 1.10 equiv) in dry toluene (40 mL). After stirring at room

temperature for 3 h, 27 (267 mg, 64%) was obtained after puriﬁcation

by ﬂash column chromatography (CH/EA = 4:1 + 1% MeOH) as

1

light yellow resin. TLC (CH/EA = 3:1, v/v): R_f= 0.35. H NMR

(500 MHz, CDCl₃, mixture of rotamers; major rotamer indicated with

an A): δ 7.29 (t, J = 7.9 Hz, 0.31H), 7.21−7.17 (m, 0.12H), 7.11 (t, J

= 7.9 Hz, 0.58H, A), 6.97−6.90 (m, 0.66H, A), 6.90−6.85 (m,

0.33H), 6.81−6.74 (m, 2.03H, A), 6.71−6.67 (m, 0.93H, A), 6.67−

6.58 (m, 1.80H, A), 6.53−6.48 (m, 1.42H, A), 6.46−6.40 (m, 1.21H,

A), 6.09−5.93 (m, 1.95H, A), 5.82−5.78 (m, 0.29H), 5.58 (dd, J =

5.6, 8.1 Hz, 0.70H, A), 5.48−5.44 (m, 0.59H, A), 5.44−5.34 (m,

1.87H, A), 5.33−5.17 (m, 2.38H, A), 4.73−4.67 (m, 0.34H), 4.60−

4.45 (m, 4.48H, A), 3.97−3.90 (m, 0.78H, A), 3.88−3.75 (m, 10.90H,

A), 3.69 (s, 1.79H, A), 3.38−3.32 (m, 0.70H, A), 3.09 (d, J = 9.5 Hz,

0.04H), 3.01−2.91 (m, 0.37H), 2.80 (td, J = 2.9, 13.3 Hz, 0.58H, A),

2.65−2.50 (m, 1.01H, A), 2.50−2.34 (m, 1.42H, A), 2.32−2.23 (m,

1.05H, A), 2.15−1.79 (m, 4.32H, A), 1.72−1.51 (m, 6.67H, A),

1.47−1.05 (m, 6.68H, A), 1.04−0.84 (m, 1.19H, A), 0.80−0.68 (m,

0.81H, A), 0.65−0.50 (m, 0.55H). ¹³C NMR (126 MHz, CDCl₃,

mixture of rotamers; major rotamer indicated with an A): δ 172.81,

172.41 (A), 172.31, 170.76, 170.66 (A), 170.52, 158.98, 158.60 (A),

153.61, 153.37 (A), 153.27, 152.12, 152.03 (A), 151.98, 149.12,

148.99 (A), 147.63, 147.43 (A), 142.31, 141.87 (A), 141.62, 137.68,

137.62 (A), 134.19, 133.64 (A), 133.59 (A), 133.56 (A), 133.49,

133.44, 133.37 (A), 133.16, 129.86, 129.66 (A), 120.41 (A), 120.34,

120.26, 119.30, 118.69 (A), 118.61, 117.96, 117.73 (A), 117.67,

117.49 (A), 114.35, 114.02 (A), 113.49, 113.36 (A), 112.89, 111.97

(A), 111.91, 111.85, 111.54, 111.47 (A), 108.21 (A), 107.75, 106.10,

106.03 (A), 105.47, 75.74 (A), 70.05 (A), 68.96, 68.88 (A), 60.98,

60.87 (A), 56.47, 56.25, 56.19 (A), 56.10, 56.06 (A), 56.01, 55.97

(A), 55.91, 55.86, 55.15, 55.08 (A), 52.32, 52.18 (A), 43.77 (A),

41.58, 41.51 (A), 41.24, 39.57, 38.26, 38.08 (A), 38.01, 37.96, 33.13,

32.91 (A), 31.64, 31.60, 31.34, 31.14 (A), 30.84, 30.79 (A), 30.61,

A+B), 1.47−1.02 (m, 7.05H, A+B), 0.95−0.67 (m, 2.01H, A+B). ¹³

C

NMR (126 MHz, CDCl₃, mixture of rotamers 1:3, A/B): δ 175.58

(A), 175.28 (B), 173.53 (B), 172.29 (A), 153.54 (A), 153.24 (B),

152.57 (A), 152.20 (B), 137.70 (A), 137.68 (B), 134.67 (A), 134.63

(B), 134.18 (A), 132.98 (B), 117.35 (B), 117.29 (A), 108.37 (B),

107.57 (A), 106.19 (B), 105.56 (A), 72.38 (B), 69.04 (A), 68.94 (B),

68.74 (B), 68.63 (A), 60.95 (A), 60.88 (B), 56.42 (A), 56.26 (B),

55.75 (A), 55.60 (A), 55.40 (B), 52.39 (B), 43.74 (B), 41.22 (B),

39.64 (A), 32.96 (B), 32.91 (A), 30.80 (B), 30.67 (A), 26.88 (A),

26.65 (B), 26.54 (B), 26.33 (B), 26.22 (A), 25.36 (B), 24.51 (A),

20.97 (A), 20.88 (B). HRMS (m/z): (ESI⁺) calculated for

C₂₇H₄₀NO₇[M + H]⁺: 490.27993, found 490.28008. HPLC (50−

60% solvent B, 1.5 mL/min, 20 min): t_R= 8.17 min, purity (220 nm)

= 94%.

(S)-1-((S)-2-(3-(3-(Allyloxy)propoxy)-4,5-dimethoxyphenyl)-

2-cyclohexylacetyl)piperidine-2-carboxylic Acid (26). The

substrate 22a (474 mg, 1.17 mmol, 1.00 equiv) was applied to

general procedure A with K₂CO₃(647 mg, 4.68 mmol, 4.00 equiv)

and 17 (1582 mg, 5.85 mmol, 5.00 equiv) in MeCN (20 mL). S11

(694 mg, 99%, analytics shown in the Supporting Information) was

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26.94 (A), 26.73 (A), 26.59, 26.37 (A), 26.31 (A), 26.23, 25.71 (A),

24.49, 21.13 (A), 21.07, 20.85. HRMS (m/z): (ESI⁺) calculated for

C₄₅H₅₈NO₉[M + H]⁺: 756.41061, found 756.41056. HPLC (75−

80% solvent B, 1.5 mL/min, 20 min): t_R= 9.25 min, purity (220 nm)

= 98%.

0.72H, A), 5.48−5.44 (m, 0.62H, A), 5.44−5.35 (m, 1.20H, A),

5.33−5.24 (m, 2.12H, A), 5.21−5.14 (m, 1.02H, A), 4.74−4.64 (m,

0.35H), 4.58−4.45 (m, 2.47H, A), 4.18−4.02 (m, 4.36H, A), 3.98−

3.62 (m, 16.72H, A), 3.39−3.32 (m, 0.75H, A), 3.09 (d, J = 9.6 Hz,

0.04H), 3.01−2.91 (m, 0.36H), 2.78 (td, J = 2.9, 13.3 Hz, 0.59H, A),

2.65−2.52 (m, 1.03H, A), 2.50−2.33 (m, 1.46H, A), 2.33−2.21 (m,

1.12H, A), 2.15−1.77 (m, 4.52H, A), 1.76−1.48 (m, 7.00H, A),

1.48−1.04 (m, 6.93H, A), 1.03−0.84 (m, 1.22H, A), 0.80−0.68 (m,

0.85H, A), 0.65−0.49 (m, 0.54H). ¹³C NMR (126 MHz, CDCl₃,

mixture of rotamers; major rotamer indicated with an A): δ 172.40

(A), 172.35, 170.74, 170.63 (A), 170.48, 158.95, 158.72, 158.56 (A),

153.53, 153.34 (A), 153.24, 152.56, 152.31 (A), 152.23, 149.09,

148.97 (A), 147.60, 147.40 (A), 142.31, 141.85 (A), 141.60, 137.80,

137.69 (A), 134.81 (A), 134.73, 134.22, 133.64 (A), 133.56 (A),

133.47, 133.42, 133.36 (A), 133.15, 129.84, 129.67 (A), 120.39 (A),

120.32, 120.24, 119.28, 118.64 (A), 118.60, 117.92, 117.69 (A),

117.27, 117.09 (A), 114.36, 114.03 (A), 113.46, 113.29 (A), 112.84,

111.95 (A), 111.90, 111.83, 111.52, 111.43 (A), 108.28 (A), 107.74,

106.17, 106.06 (A), 105.68, 75.74, 75.70 (A), 72.41, 72.34 (A), 69.13,

68.96 (A), 68.93, 68.85 (A), 68.71 (A), 60.96, 60.85 (A), 56.47,

56.23, 56.16 (A), 56.07, 56.03 (A), 55.99, 55.95 (A), 55.90, 55.76,

55.11, 55.06 (A), 52.29, 52.16 (A), 43.75 (A), 41.56, 41.49 (A),

41.27, 39.55, 38.24, 38.07 (A), 37.92, 33.09, 32.90 (A), 31.59, 31.30,

31.10 (A), 30.78 (A), 30.59, 26.96, 26.91 (A), 26.84, 26.71 (A),

26.55, 26.34 (A), 26.29 (A), 26.21, 25.67 (A), 24.46, 21.10 (A),

21.04, 20.80. HRMS (m/z): (ESI⁺) calculated for C₄₇H₆₂NO₁₀[M +

H]⁺: 800.43682, found 800.43680. HPLC (70−80% solvent B, 1.5

mL/min, 20 min): t_R= 12.18 min, purity (220 nm) = 95%.

(S)-(R)-1-(3-(Allyloxy)phenyl)-3-(3,4-dimethoxyphenyl)-

propyl 1-((S)-2-(3-(But-3-en-1-yloxy)-4,5-dimethoxyphenyl)-2-

cyclohexylacetyl)piperidine-2-carboxylate (28). The substrate

24 (289 mg, 0.63 mmol, 1.00 equiv) was applied to general procedure

G with 14 (207 mg, 0.63 mmol, 1.00 equiv), 4-(1-pyrrolidinyl)-

pyridine (380 mg, 2.52 mmol, 4.00 equiv), and EDC (135 mg, 0.69

mmol, 1.10 equiv) in dry toluene (50 mL). After stirring at room

temperature for 4 h, 28 (287 mg, 59%) was obtained as a light yellow

resin after puriﬁcation by ﬂash column chromatography (CH/EA =

1

4:1 + 1% MeOH). TLC (CH/EA = 3:1, v/v): R_f= 0.39. H NMR

(500 MHz, CDCl₃, mixture of rotamers; major rotamer indicated with

an A): δ 7.28 (t, J = 7.9 Hz, 0.29H), 7.19 (t, J = 8.1 Hz, 0.10H), 7.10

(t, J = 7.9 Hz, 0.60H, A), 6.96−6.90 (m, 0.61H, A), 6.89−6.85 (m,

0.32H), 6.80−6.73 (m, 2.04H, A), 6.71−6.58 (m, 2.51H, A), 6.52−

6.48 (m, 1.41H, A), 6.43−6.39 (m, 1.10H, A), 6.08−5.98 (m, 0.91H,

A), 5.94−5.78 (m, 1.16H, A), 5.57 (dd, J = 5.6, 8.0 Hz, 0.72H, A),

5.48−5.45 (m, 0.60H, A), 5.43−5.36 (m, 1.01H, A), 5.31−5.23 (m,

1.01H, A), 5.19−5.03 (m, 2.03H, A), 4.73−4.68 (m, 0.28H), 4.58−

4.46 (m, 2.39H, A), 4.06−4.01 (m, 0.62H), 4.01−3.91 (m, 2.26H, A),

3.87−3.80 (m, 8.47H, A), 3.79−3.76 (m, 2.56H, A), 3.69 (s, 1.87H,

A), 3.39−3.33 (m, 0.73H, A), 2.98−2.93 (m, 0.30H), 2.79 (td, J =

2.9, 13.3 Hz, 0.59H, A), 2.63−2.34 (m, 4.30H, A), 2.31−2.24 (m,

0.96H, A), 2.14−2.03 (m, 1.63H, A), 1.99−1.79 (m, 2.18H, A),

1.71−1.51 (m, 5.98H, A), 1.47−1.06 (m, 6.21H, A), 1.02−0.85 (m,

0.99H, A), 0.79−0.70 (m, 0.71H, A), 0.64−0.53 (m, 0.44H, A). ¹³C

NMR (126 MHz, CDCl₃, mixture of rotamers; major rotamer

indicated with an A): δ 172.81, 172.39 (A), 172.33, 170.73, 170.62

(A), 170.48, 158.95, 158.71, 158.55 (A), 153.48, 153.26 (A), 153.15,

152.55, 152.39 (A), 152.31, 149.08, 148.96 (A), 147.60, 147.39 (A),

142.31, 141.83 (A), 141.58, 137.62, 137.51 (A), 137.45, 134.72 (A),

134.58, 134.19, 133.61 (A), 133.56 (A), 133.41, 133.34 (A), 133.13,

129.82, 129.63 (A), 120.37 (A), 120.31, 120.23, 119.27, 118.64 (A),

118.55, 117.91, 117.68 (A), 117.22, 117.04 (A), 114.35, 114.00 (A),

113.45, 113.31 (A), 112.82, 111.95 (A), 111.90, 111.82, 111.51,

111.44 (A), 107.68 (A), 107.21, 106.07, 105.97 (A), 105.55, 75.72,

75.65 (A), 68.92, 68.84 (A), 68.62, 68.46 (A), 68.41, 60.95, 60.84

(A), 56.48, 56.25, 56.18 (A), 56.06, 56.02 (A), 55.98, 55.93 (A),

55.90, 55.80, 55.14, 55.05 (A), 52.27, 52.14 (A), 43.74 (A), 41.56,

41.51 (A), 41.24, 39.54, 38.23, 38.04 (A), 37.91, 33.93 (A), 33.10,

32.89 (A), 31.61, 31.57, 31.29, 31.08 (A), 30.77 (A), 30.58, 26.90

(A), 26.70 (A), 26.55, 26.33 (A), 26.28 (A), 26.28, 26.21, 25.68 (A),

25.63, 24.46, 21.10 (A), 21.03, 20.81. HRMS (m/z): (ESI⁺)

calculated for C₄₆H₆₀NO₉[M + H]⁺: 770.42626, found 770.42648.

HPLC (75−80% solvent B, 1.5 mL/min, 20 min): t_R= 10.99 min,

purity (220 nm) = 97%.

(S)-(R)-1-(3-(Allyloxy)phenyl)-3-(3,4-dimethoxyphenyl)-

propyl 1-((S)-2-(3-(3-(Allyloxy)propoxy)-4,5-dimethoxyphen-

yl)-2-cyclohexylacetyl)piperidine-2-carboxylate (30). The sub-

strate 26 (545 mg, 1.08 mmol, 1.00 equiv) was applied to general

procedure G with 14 (355 mg, 1.08 mmol, 1.00 equiv), 4-(1-

pyrrolidinyl)pyridine (640 mg, 4.32 mmol, 4.00 equiv), and EDC

(228 mg, 1.19 mmol, 1.10 equiv) in dry toluene (75 mL). After

stirring at room temperature for 4 h and performing the standard

workup procedure, the crude product was reacted with acetic

anhydride (166 mg), pyridine (53 μL), and DMAP (8 mg) in

DCM (10 mL) to convert unreacted 14 into the corresponding

acetate and thereby facilitating the puriﬁcation of 30. 30 (522 mg,

59%) was obtained as a colorless resin after puriﬁcation by ﬂash

column chromatography (CH/EA = 4:1). TLC (CH/EA = 3:1, v/v):

1

R_f= 0.40. H NMR (500 MHz, CDCl₃, mixture of rotamers; major

rotamer indicated with an A): δ 7.30−7.26 (m, 0.19H), 7.20−7.16

(m, 0.10H), 7.09 (t, J = 7.9 Hz, 0.56H, A), 6.96−6.89 (m, 0.55H, A),

6.88−6.84 (m, 0.29H), 6.79−6.73 (m, 1.88H, A), 6.70−6.56 (m,

2.55H, A), 6.52−6.48 (m, 1.38H, A), 6.42 (s, 0.55H, A), 6.40−6.37

(m, 0.54H), 6.07−5.97 (m, 0.92H, A), 5.93−5.84 (m, 0.93H, A), 5.79

(dd, J = 6.2, 7.6 Hz, 0.27H), 5.55 (dd, J = 5.6, 8.1 Hz, 0.68H, A), 5.46

(d, J = 5.4 Hz, 0.55H, A), 5.42−5.35 (m, 1.09H, A), 5.29−5.22 (m,

2.01H, A), 5.16−5.11 (m, 0.99H, A), 4.70 (d, J = 5.6 Hz, 0.27H),

4.57−4.46 (m, 2.25H, A), 4.10−3.91 (m, 5.04H, A), 3.86−3.79 (m,

8.36H, A), 3.77−3.74 (m, 2.42H, A), 3.67−3.53 (m, 3.89H, A),

3.37−3.33 (m, 0.71H, A), 2.95 (d, J = 9.7 Hz, 0.30H), 2.78 (td, J =

2.8, 13.4 Hz, 0.54H, A), 2.62−2.50 (m, 0.80H, A), 2.48−2.33 (m,

1.22, A), 2.30−2.23 (m, 0.94H, A), 2.12−1.79 (m, 6.01H, A), 1.70−

1.52 (m, 5.93H, A), 1.44−1.06 (m, 6.25H, A), 1.03−0.82 (m, 1.01H,

A), 0.79−0.68 (m, 0.70H, A), 0.63−0.52 (m, 0.37H). ¹³C NMR (126

MHz, CDCl₃, mixture of rotamers; major rotamer indicated with an

A): δ 172.74, 172.34 (A), 172.30, 170.68, 170.59 (A), 170.44, 158.89,

158.66, 158.49 (A), 153.41, 153.26 (A), 153.15, 152.64, 152.40 (A),

152.33, 149.02, 148.89 (A), 147.53, 147.33 (A), 142.28, 141.81 (A),

141.53, 137.42, 137.35 (A), 134.95 (A), 134.90, 134.22, 133.57 (A),

133.55 (A), 133.47, 133.35, 133.30 (A), 133.09, 129.78, 129.72,

129.58 (A), 120.33 (A), 120.26, 120.18, 119.21, 118.57 (A), 118.49,

117.85, 117.63 (A), 116.88, 116.82 (A), 114.32, 113.95 (A), 113.39,

113.24 (A), 112.74, 111.89 (A), 111.83, 111.75, 111.44, 111.36 (A),

107.66 (A), 107.02, 105.71, 105.62 (A), 105.29, 76.82, 75.64, 75.60

(A), 71.95 (A), 68.86, 68.78 (A), 67.01 (A), 66.91, 66.29, 66.19 (A),

66.14, 60.87, 60.76 (A), 56.40, 56.16, 56.08 (A), 56.01, 55.97 (A),

(S)-(R)-1-(3-(Allyloxy)phenyl)-3-(3,4-dimethoxyphenyl)-

propyl 1-((S)-2-(3-(2-(Allyloxy)ethoxy)-4,5-dimethoxyphenyl)-

2-cyclohexylacetyl)piperidine-2-carboxylate (29). The substrate

25 (508 mg, 1.04 mmol, 1.00 equiv) was applied to general procedure

G with 14 (341 mg, 1.04 mmol, 1.00 equiv), 4-(1-pyrrolidinyl)-

pyridine (628 mg, 4.15 mmol, 4.00 equiv), and EDC (223 mg, 1.14

mmol, 1.10 equiv) in dry toluene (75 mL). After stirring at room

temperature for 4 h and performing the standard workup procedure,

the crude product was reacted with acetic anhydride (234 mg),

pyridine (75 μL), and DMAP (11 mg) in DCM (15 mL) to convert

unreacted 14 into the corresponding acetate and thereby facilitating

the puriﬁcation of 29. 29 (524 mg, 63%) was obtained as a colorless

resin after puriﬁcation by ﬂash column chromatography (CH/EA =

1

5:1). TLC (CH/EA = 3:1, v/v): R_f= 0.14. H NMR (500 MHz,

CDCl₃, mixture of rotamers; major rotamer indicated with an A): δ

7.29 (t, J = 7.8 Hz, 0.30H), 7.21−7.17 (m, 0.10H), 7.11 (t, J = 7.9 Hz,

0.58H, A), 6.97−6.90 (m, 0.67H, A), 6.89−6.85 (m, 0.34H, A),

6.81−6.73 (m, 2.13H, A), 6.72−6.57 (m, 2.88H, A), 6.54−6.49 (m,

1.51H, A), 6.47−6.39 (m, 1.23H, A), 6.09−5.98 (m, 1.01H, A),

5.96−5.85 (m, 0.98H, A), 5.82−5.77 (m, 0.30H), 5.60−5.54 (m,

3335

https://dx.doi.org/10.1021/acs.jmedchem.0c02195

J. Med. Chem. 2021, 64, 3320−3349

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

55.92, 55.88 (A), 55.74, 55.08, 55.02 (A), 52.23, 52.08 (A), 43.70

(A), 41.51, 41.44 (A), 41.18, 39.50, 38.20, 38.00 (A), 37.87, 33.24,

33.05, 32.84 (A), 31.53, 31.24, 31.02 (A), 30.72 (A), 30.54, 29.88

(A), 26.86 (A), 26.66 (A), 26.51, 26.28 (A), 26.24, 26.23 (A), 26.17,

25.64 (A), 24.56, 24.42, 21.06 (A), 21.02, 20.77. HRMS (m/z):

(ESI⁺) calculated for C₄₈H₆₃NO₁₀Na [M + Na]⁺: 836.43442, found

836.43489. HPLC (70−100% solvent B, 0.8 mL/min, 10.50 min): t_R

= 6.84 min, purity (220 nm) = 95%.

6.71−6.63 (m, 3H), 6.39−6.36 (m, 1H), 6.32 (br s, 1H), 5.73−5.67

(m, 2H), 5.64 (dd, J = 6.4, 8.1 Hz, 1H), 5.46−5.43 (m, 1H), 4.45 (dd,

J = 5.7, 12.5 Hz, 1H), 4.34 (dd, J = 3.9, 12.6 Hz, 1H), 4.20−4.13 (m,

1H), 4.07−4.00 (m, 1H), 3.94 (d, J = 13.8 Hz, 1H), 3.88−3.80 (m,

6H), 3.54 (br s, 3H), 3.32 (d, J = 9.9 Hz, 1H), 2.72 (td, J = 2.8, 13.4

Hz, 1H), 2.60−2.50 (m, 1H), 2.49−2.40 (m, 3H), 2.31−2.25 (m,

1H), 2.16−2.05 (m, 2H), 1.95−1.84 (m, 2H), 1.71−1.52 (m, 6H),

1.46−1.07 (m, 6H), 0.93−0.83 (m, 1H), 0.79−0.68 (m, 1H). ¹³C

NMR (126 MHz, CDCl₃): δ 172.0, 171.0, 158.3, 154.1, 152.7, 148.9,

147.3, 140.8, 134.5, 133.8, 133.4, 130.4, 129.3, 127.1, 120.9, 120.3,

116.1, 112.1, 111.9, 111.3, 107.6, 104.3, 75.9, 71.4, 63.2, 56.3, 55.9,

55.8, 55.2, 52.1, 43.6, 41.5, 36.6, 32.8, 31.3, 30.6, 28.1, 26.9, 26.6,

26.2, 26.2, 25.6, 21.0. HRMS (m/z): (ESI⁺) calculated for

C₄₄H₅₆NO₉[M + H]⁺: 742.39496, found 742.39492. HPLC (70−

80% solvent B, 1.5 mL/min, 20 min): t_R= 10.13 min, purity (220

nm) = 99%. Data for macrocycle 32-(E): TLC (CH/EA = 5:1, v/v):

Macrocycles 31-(Z) and 31-(E). The substrate 9 (357 mg, 0.47

mmol, 1.00 equiv) was applied to general procedure B with Grubbs

second generation catalyst (40 mg, 0.05 mmol, 0.10 equiv) in DCM

(1000 mL, 0.5 mM). After workup procedure a, 31-(Z) (16 mg, 5%,

white solid) and 31-(E) (132 mg, 38%, white solid) were obtained as

pure isomers after puriﬁcation by ﬂash column chromatography (CH/

EA = 5:1) and semipreparative HPLC (70−100% solvent B). Another

product fraction was obtained as a mixture of E/Z isomers (77 mg,

22%) and not further puriﬁed. Data for macrocycle 31-(Z): TLC

(CH/EA = 3:1, v/v): R_f= 0.29. ¹H NMR (500 MHz, CDCl₃): δ 7.15

(t, J = 7.7 Hz, 1H), 6.84−6.81 (m, 1H), 6.80−6.75 (m, 3H), 6.74 (d,

J = 1.9 Hz, 1H), 6.72−6.68 (m, 1H), 6.32−6.29 (m, 2H), 6.06−5.99

(m, 1H), 5.89−5.79 (m, 2H), 5.50 (d, J = 5.4 Hz, 1H), 4.65−4.54 (m,

2H), 4.09−4.03 (m, 1H), 3.88 (s, 3H), 3.85 (s, 3H), 3.84−3.78 (m,

4H), 3.62 (s, 3H), 3.56−3.51 (m, 1H), 3.27 (d, J = 9.3 Hz, 1H),

2.69−2.62 (m, 1H), 2.58−2.49 (m, 2H), 2.33−2.27 (m, 1H), 2.19−

2.10 (m, 1H), 2.09−1.92 (m, 2H), 1.89−1.82 (m, 1H), 1.72−1.60

(m, 5H), 1.57−1.48 (m, 1H), 1.48−1.28 (m, 3H), 1.24−1.18 (m,

1H), 1.18−1.07 (m, 2H), 0.98−0.88 (m, 1H), 0.81−0.71 (m, 1H).

¹³C NMR (126 MHz, CDCl₃): δ 171.8, 171.0, 158.8, 154.7, 154.0,

149.0, 147.5, 141.9, 135.1, 133.8, 133.7, 130.9, 129.5, 127.5, 120.4,

120.4, 117.3, 112.1, 111.5, 108.2, 107.4, 103.6, 76.0, 66.3, 62.5, 56.4,

56.3, 56.2, 56.1, 56.0, 52.7, 43.8, 42.5, 39.3, 33.3, 31.4, 30.9, 26.8,

26.7, 26.5, 25.7, 21.1. HRMS (m/z): (ESI⁺) calculated for

C₄₃H₅₄NO₉[M + H]⁺: 728.37931, found 728.37972. HPLC (70−

80% solvent B, 1.5 mL/min, 20 min): t_R= 9.19 min, purity (220 nm)

= 99%. Data for macrocycle 31-(E): TLC (CH/EA = 3:1, v/v): R_f=

0.21. ¹H NMR (500 MHz, CDCl₃): δ 7.17 (t, J = 7.9 Hz, 1H), 6.84−

6.81 (m, 1H), 6.80−6.76 (m, 2H), 6.71−6.65 (m, 3H), 6.42−6.39

(m, 1H), 6.27 (d, J = 1.8 Hz, 1H), 5.86 (ddd, J = 5.9, 7.4, 15.5 Hz,

1H), 5.70−5.60 (m, 2H), 5.49−5.44 (m, 1H), 4.63 (ddd, J = 1.1, 6.7,

25.9 Hz, 1H), 4.40−4.37 (m, 2H), 3.97−3.90 (m, 1H), 3.86 (s, 3H),

3.84 (s, 3H), 3.82 (s, 3H), 3.53 (s, 3H), 3.30 (d, J = 9.9 Hz, 1H),

2.88−2.79 (m, 1H), 2.64−2.46 (m, 2H), 2.29−2.22 (m, 1H), 2.13−

2.02 (m, 2H), 1.94−1.82 (m, 2H), 1.72−1.54 (m, 6H), 1.48−1.38

(m, 1H), 1.37−1.23 (m, 3H), 1.17−1.06 (m, 2H), 0.94−0.84 (m,

1H), 0.78−0.67 (m, 1H). ¹³C NMR (126 MHz, CDCl₃): δ 172.2,

171.1, 158.7, 154.2, 153.0, 148.9, 147.3, 141.7, 134.0, 133.5, 133.4,

129.7, 129.5, 129.0, 120.6, 120.3, 116.9, 113.6, 111.9, 111.3, 107.4,

104.3, 75.8, 71.4, 69.2, 56.3, 56.2, 56.0, 55.8, 55.4, 52.2, 43.6, 41.7,

38.0, 32.9, 31.3, 30.6, 26.8, 26.6, 26.2, 25.6, 20.8. HRMS (m/z):

(ESI⁺) calculated for C₄₃H₅₄NO₉[M + H]⁺: 728.37931, found

1

R_f= 0.32. H NMR (500 MHz, CDCl₃): δ 7.15 (t, J = 7.9 Hz, 1H),

6.83−6.75 (m, 3H), 6.66−6.61 (m, 2H), 6.57−6.23 (m, 3H), 5.70

(dt, J = 6.8, 15.6 Hz, 1H), 5.56−5.44 (m, 3H), 4.50−4.44 (m, 1H),

4.35−4.28 (m, 2H), 4.09−4.03 (m, 1H), 4.02−3.96 (m, 1H), 3.85−

3.84 (m, 6H), 3.79−3.53 (m, 6H), 3.35 (d, J = 9.9 Hz, 1H), 2.99 (td,

J = 2.7, 13.3 Hz, 1H), 2.56−2.48 (m, 1H), 2.47−2.37 (m, 2H), 2.34−

2.22 (m, 2H), 2.12−2.03 (m, 2H), 1.92−1.82 (m, 2H), 1.72−1.56

(m, 6H), 1.49−1.38 (m, 1H), 1.37−1.21 (m, 3H), 1.17−1.06 (m,

2H), 0.94−0.83 (m, 1H), 0.81−0.70 (m, 1H). ¹³C NMR (126 MHz,

CDCl₃): δ 172.4, 170.9, 158.5, 149.0, 147.5, 141.4, 136.1, 133.5,

133.3, 132.9, 129.5, 126.1, 120.4, 119.7, 117.3, 112.5, 112.0, 111.4,

76.3, 72.6, 68.7, 56.2, 56.1, 56.0, 55.0, 52.1, 43.6, 41.4, 37.3, 33.8,

32.9, 31.4, 30.7, 27.2, 26.7, 26.3, 26.3, 25.8, 21.1. HRMS (m/z):

(ESI⁺) calculated for C₄₄H₅₆NO₉[M + H]⁺: 742.39496, found

742.39481. HPLC (70−80% solvent B, 1.5 mL/min, 20 min): t_R

9.40 min, purity (220 nm) = 99%.

=

Macrocycles 33-(Z) and 33-(E). The substrate 11 (170 mg, 0.21

mmol, 1.00 equiv) was applied to general procedure B with Grubbs

second generation catalyst (18 mg, 0.02 mmol, 0.10 equiv) in DCM

(450 mL, 0.5 mM). After workup procedure a, 33-(Z) (11 mg, 7%,

colorless resins) and 33-(E) (91 mg, 56%, colorless resin) were

obtained after puriﬁcation by ﬂash column chromatography (CH/EA

= 3:1). Data for macrocycle 33-(Z): TLC (CH/EA = 2:1, v/v): R_f=

0.29. ¹H NMR (500 MHz, CDCl₃): δ 7.17 (t, J = 7.9 Hz, 1H), 6.87−

6.84 (m, 1H), 6.83−6.79 (m, 1H), 6.79−6.75 (m, 1H), 6.66−6.63

(m, 2H), 6.53−6.50 (m, 1H), 6.37 (s, 2H), 5.91 (ddd, J = 5.3, 6.8,

11.9 Hz, 1H), 5.87−5.81 (m, 1H), 5.49 (dd, J = 5.6, 8.7 Hz, 1H), 5.45

(d, J = 5.1 Hz, 1H), 4.63 (dd, J = 6.9, 11.9 Hz, 1H), 4.54 (ddd, J =

6.0, 12.2, 19.4 Hz, 2H), 4.23 (dd, J = 5.6, 12.7 Hz, 1H), 4.16−4.05

(m, 2H), 3.93 (d, J = 13.6 Hz, 1H), 3.87−3.84 (m, 6H), 3.80−3.68

(m, 2H), 3.58 (s, 6H), 3.34 (d, J = 10.1 Hz, 1H), 2.75 (td, J = 3.0,

13.3 Hz, 1H), 2.58−2.51 (m, 1H), 2.45−2.38 (m, 1H), 2.35−2.29

(m, 1H), 2.14−2.06 (m, 1H), 2.04−1.95 (m, 1H), 1.92−1.85 (m,

2H), 1.71−1.58 (m, 6H), 1.46−1.08 (m, 6H), 0.91−0.82 (m, 1H),

0.79−0.70 (m, 1H). ¹³C NMR (126 MHz, CDCl₃): δ 172.5, 170.8,

158.7, 153.2, 149.0, 147.5, 141.6, 135.4, 133.6, 130.8, 129.7, 127.8,

120.5, 120.1, 116.0, 112.3, 112.0, 111.5, 105.8, 76.0, 73.4, 69.1, 66.8,

64.3, 56.1, 56.1, 56.0, 55.1, 52.0, 43.6, 40.9, 36.3, 32.9, 31.5, 30.7,

27.1, 26.8, 26.4, 26.3, 25.7, 21.2. HRMS (m/z): (ESI⁺) calculated for

C₄₅H₅₈NO₁₀[M + H]⁺: 772.4055, found 772.4050. HPLC (70−80%

solvent B, 1.5 mL/min, 20 min): t_R= 9.64 min, purity (220 nm) =

99%. Data for macrocycle 33-(E): TLC (CH/EA = 2:1, v/v): R_f=

0.20. ¹H NMR (500 MHz, CDCl₃, mixture of rotamers 0.58:1, A/B):

δ 7.30−7.27 (m, 0.30H, A), 7.16−7.11 (m, 0.99H, A+B), 6.90−6.87

(m, 0.37H, A), 6.81−6.67 (m, 3.52H, A+B), 6.65−6.61 (m, 1.26H, A

+B), 6.52−6.45 (m, 2.58H, A+B), 5.83 (dt, J = 5.6, 17.0 Hz, 1.00H, A

+B), 5.75 (dt, J = 5.1, 16.0 Hz, 0.59H, B), 5.66−5.63 (m, 0.65H, B),

5.51−5.46 (m, 0.63H, B), 5.41 (dd, J = 5.5, 8.5 Hz, 0.62H, B), 4.80−

4.76 (m, 0.41H, A), 4.64−4.59 (m, 0.62H, B), 4.55−4.50 (m, 0.61H,

B), 4.45−4.38 (m, 0.74H, A), 4.28−4.24 (m, 0.72H, A), 4.16 (dd, J =

4.8, 10.1 Hz, 0.41H, A), 4.12−4.00 (m, 2.61H, A+B), 3.98−3.83 (m,

10.46H, A+B), 3.67−3.52 (m, 5.57H, A+B), 3.39 (d, J = 9.9 Hz,

0.61H, B), 3.01−2.93 (m, 0.99H, A+B), 2.71 (ddd, J = 5.4, 9.1, 14.3

Hz, 0.36H, A), 2.61 (ddd, J = 7.1, 8.8, 14.1 Hz, 0.37H, A), 2.51−2.38

728.37955. HPLC (70−80% solvent B, 1.5 mL/min, 20 min): t_R

8.37 min, purity (220 nm) = 99%.

=

Macrocycles 32-(Z) and 32-(E). The substrate 10 (304 mg, 0.40

mmol, 1.00 equiv) was applied to general procedure B with Grubbs

second generation catalyst (34 mg, 0.04 mmol, 0.10 equiv) in DCM

(800 mL, 0.5 mM). After workup procedure a, 32-(Z) (50 mg, 17%,

colorless resin) and 32-(E) (62 mg, 21%, colorless resin) were

obtained as pure isomers after puriﬁcation by ﬂash column

chromatography (CH/EA = 5:1). Another product fraction was

obtained as a mixture of E/Z isomers (118 mg, 40%) and not further

puriﬁed. Note: The NMR spectra of compounds 32-(E) and 32-(Z)

show a peak broadening for the ortho and meta protons and carbon

signals of the 3,5-dimethoxyaryl ring. This might result out of a slow

interconversion of the C2-symmetrical nuclei of the aryl ring and the

attached methoxy groups, because the connected macrocycle linker

probably hinders the rotation of the aryl ring. The six equivalent

methoxy protons of 32-(Z)therefore show a broad singlet at 3.54 ppm

with an integral of only three protons. Data for macrocycle 32-(Z):

TLC (CH/EA = 5:1, v/v): R_f= 0.37. ¹H NMR (500 MHz, CDCl₃): δ

7.15 (t, J = 7.9 Hz, 1H), 6.86−6.82 (m, 1H), 6.80−6.75 (m, 2H),

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(m, 1.30H, B), 2.35 (d, J = 10.1 Hz, 0.37H, A), 2.29−2.23 (m, 1.26H,

A+B), 2.12−2.05 (m, 0.93H, A+B), 1.96−1.85 (m, 1.65H, A+B),

1.79−1.62 (m, 6.10H, A+B), 1.49−0.77 (m, 8.30H, A+B), 0.66−0.57

(m, 0.34H, A), −0.14 (qd, J = 3.7, 12.7 Hz, 0.34H, A), −0.22 −

−0.31 (m, 0.34H, A). ¹³C NMR (126 MHz, CDCl₃, mixture of

rotamers 2:3, A/B): δ 174.21 (A), 172.57 (B), 171.69 (A), 170.80

(B), 159.55 (A), 158.08 (B), 153.20 (A), 153.01 (B), 149.13 (A),

149.01 (B), 147.65 (A), 147.49 (B), 143.64 (A), 142.06 (B), 136.64

(A), 135.80 (B), 133.90 (A), 133.49 (B), 133.36 (A), 133.20 (B),

132.16 (A), 131.80 (B), 129.91 (A), 129.60 (B), 126.85 (B), 125.04

(A), 120.43 (B), 120.39 (A), 119.95 (A), 119.34 (B), 115.96 (B),

113.19 (B), 112.02 (B), 111.89 (A), 111.54 (A), 111.46 (B), 111.01

(A), 105.75 (B), 104.73 (A), 76.08 (B), 75.73 (A), 72.82 (A), 72.40

(B), 71.83 (A), 71.13 (B), 71.09 (A), 70.03 (B), 68.23 (A), 67.87

(B), 56.32 (A), 56.08 (B), 56.03 (A), 55.99 (B), 55.95 (B), 54.91

(B), 51.93 (A), 43.65 (B), 42.31 (A), 41.44 (B), 39.47 (A), 38.69

(A), 37.54 (B), 32.83 (B), 32.12 (A), 31.71 (A), 31.25 (B), 30.75

(B), 29.57 (A), 27.14 (B), 26.83 (A), 26.72 (B), 26.51 (A), 26.30

(B), 26.26 (B), 26.14 (A), 26.09 (A), 25.79 (B), 23.94 (A), 21.08

(B), 20.47 (A). HRMS (m/z): (ESI⁺) calculated for C₄₅H₅₈NO₁₀[M

+ H]⁺: 772.40552, found 772.40455. HPLC (70−80% solvent B, 1.5

mL/min, 20 min): t_R= 8.24 min, purity (220 nm) = 99%.

Macrocycle 34-(E). The substrate 12 (125 mg, 0.15 mmol, 1.00

equiv) was applied to general procedure B with Grubbs second

generation catalyst (13 mg, 0.015 mmol, 0.10 equiv) in DCM (310

mL, 0.5 mM). After workup procedure a, 34-(E) (53 mg, 44%) was

obtained as a white solid after puriﬁcation by ﬂash column

chromatography (CH/EA = 4:1). The corresponding Z-alkene was

only observed in trace amounts during the reaction control via LC−

MS and could not be isolated. TLC (CH/EA = 4:1, v/v): R_f= 0.26.

Note: The¹H signals of the oleﬁn protons of compound34-(E)overlap

each other in diﬀerent NMR solvents (CDCl₃, THF-d₈, DMSO-d₆,

C₆D₆). Finally, the (E)-conﬁguration of the double bond was

determined via the coupling constant of the¹³C satellite signals of

one oleﬁn proton (see spectra in the Supporting Information, 512

scans for¹H spectra in C₆D₆). ¹H NMR (500 MHz, C₆D₆): δ 7.08 (t, J

= 7.9 Hz, 1H), 6.74−6.70 (m, 5H), 6.64−6.59 (m, 2H), 6.52−6.47

(m, 1H), 5.91−5.87 (m, 1H), 5.84 (dd, J = 5.3, 8.1 Hz, 1H), 5.56−

5.48 (m, 2H), 4.33−4.24 (m, 2H), 4.13−4.07 (m, 1H), 3.99−3.94

(m, 1H), 3.91−3.86 (m, 1H), 3.70−3.64 (m, 2H), 3.55 (s, 3H),

3.46−3.43 (m, 9H), 3.35−3.28 (m, 3H), 2.97−2.90 (m, 1H), 2.60−

2.51 (m, 3H), 2.33−2.28 (m, 1H), 2.25−2.21 (m, 1H), 2.08−1.99

(m, 3H), 1.91−1.85 (m, 1H), 1.77−1.73 (m, 1H), 1.67−1.62 (m,

2H), 1.56−1.52 (m, 1H), 1.44−1.28 (m, 4H), 1.20−1.14 (m, 4H),

0.97−0.91 (m, 1H), 0.87−0.78 (m, 1H). ¹³C NMR (126 MHz,

C₆D₆): δ 172.2, 170.7, 158.2, 154.3, 150.3, 148.7, 143.1, 138.0, 134.1,

133.7, 131.6, 129.8, 127.6, 120.9, 118.5, 115.1, 114.7, 113.3, 112.8,

107.0, 75.8, 70.8, 70.1, 67.5, 66.2, 56.1, 55.8, 55.7, 55.3, 52.2, 43.9,

41.9, 39.3, 33.1, 31.6, 31.3, 30.9, 27.1, 26.8, 26.7, 26.7, 26.0, 21.2.

HRMS (m/z): (ESI⁺) calculated for C₄₆H₆₀NO₁₀[M + H]⁺:

786.42117, found 786.42139. HPLC (70−80% solvent B, 1.5 mL/

min, 20 min): t_R= 8.98 min, purity (220 nm) = 99%.

(m, 0.16H, A), 4.49−4.35 (m, 1.05H, A+B), 4.27 (d, J = 13.2 Hz,

0.85H, B), 3.87−3.82 (m, 7.03H, A+B), 3.79 (s, 0.61H, A), 3.74 (s,

5.06H, A+B), 3.50 (d, J = 10.2 Hz, 0.79H, B), 3.20 (td, J = 2.6, 13.1

Hz, 0.85H, B), 2.93 (td, J = 3.5, 12.8 Hz, 0.14H, A), 2.63−2.50 (m,

0.35H, A), 2.42−2.17 (m, 3.57H, A+B), 2.15−2.09 (m, 0.30H, A),

1.88 (d, J = 12.4 Hz, 0.97H, A+B), 1.76−1.46 (m, 8.06H, A+B),

1.43−1.31 (m, 3.08H, A+B), 1.28−1.11 (m, 3.00H, A+B), 0.98−0.83

(m, 1.20H, A+B), 0.80−0.68 (m, 0.84H, B), 0.41−0.31 (m, 0.15H,

A), 0.10 − −0.01 (m, 0.14H, A). ¹³C NMR (126 MHz, CDCl₃,

mixture of rotamers 1:7, A/B): δ 172.92 (A), 172.70 (B), 170.94 (A),

170.52 (B), 159.55 (A), 159.48 (B), 154.20 (A), 153.78 (B), 152.24

(A), 150.79 (B), 149.07 (A), 148.94 (B), 147.60 (A), 147.36 (B),

142.95 (B), 141.39 (A), 137.59 (A), 137.27 (B), 134.28 (A), 133.57

(B), 133.34 (A), 133.24 (B), 129.86 (A), 129.62 (B), 129.42 (B),

129.32 (A), 127.50 (A), 127.05 (B), 121.05 (A), 120.65 (B), 120.27

(A), 119.51 (B), 118.17 (A), 117.00 (B), 111.99 (B), 111.88 (A),

111.51 (A), 111.45 (B), 110.16 (B), 110.10 (A), 109.26 (A), 108.37

(B), 104.79 (B), 103.77 (A), 75.84 (A), 74.91 (B), 70.67 (A), 68.86

(B), 68.14 (B), 68.08 (A), 61.12 (A), 60.75 (B), 56.63 (A), 56.21

(A), 56.05 (B), 55.98 (B), 55.88 (B), 55.57 (A), 54.25 (B), 52.04

(B), 44.29 (B), 41.44 (B), 39.29 (A), 38.24 (B), 38.03 (A), 32.36

(B), 31.45 (A), 30.73 (B), 29.80 (A), 26.96 (A), 26.65 (B), 26.52

(B), 26.25 (A), 26.18 (B), 26.11 (B), 25.97 (B), 24.11 (A), 21.14

(B), 20.59 (A). HRMS (m/z): (ESI⁺) calculated for C₄₃H₅₄NO₉[M

+ H]⁺: 728.37931, found 728.37963. HPLC (70−80% sSolvent B, 1.5

mL/min, 20 min): t_R= 7.15 min, purity (220 nm) = 99%.

Macrocycles 36-(Z) and 36-(E). The substrate 28 (224 mg, 0.29

mmol, 1.00 equiv) was applied to general procedure B with Grubbs

second generation catalyst (25 mg, 0.03 mmol, 0.10 equiv) and 1,4-

benzoquinone (4 mg, 0.03 mmol, 0.10 equiv) in DCM (1 mM, 300

mL). After workup procedure b, 36-(Z) (28 mg, 13%, white solid)

and 36-(E) (115 mg, 53%, white solid) were obtained as product

isomers after puriﬁcation by ﬂash column chromatography (CH/EA =

4:1) and semipreparative HPLC (80−100% solvent B). Data for

1

macrocycle 36-(Z): TLC (CH/EA = 2:1, v/v): R_f= 0.39. H NMR

(500 MHz, CDCl₃, mixture of rotamers 0.16:1, A/B): δ 7.29 (t, J =

7.9 Hz, 0.15H, A), 7.15 (t, J = 8.3 Hz, 0.85H, B), 7.08−7.06 (m,

0.14H, A), 6.94−6.88 (m, 0.32H, A), 6.81−6.74 (m, 2.73H, A+B),

6.69−6.61 (m, 2.75H, A+B), 6.56−6.51 (m, 0.22H, A), 6.43−6.40

(m, 0.80H, B), 6.31−6.28 (m, 0.81H, B), 6.21−6.19 (m, 0.13H, A),

6.02−5.95 (m, 0.15H, A), 5.93−5.78 (m, 1.87H, A+B), 5.55−5.46

(m, 1.77H, B), 4.62−4.42 (m, 0.65H, A), 4.34−4.23 (m, 2.52H, A

+B), 4.12−4.00 (m, 1.79H, B), 3.88−3.82 (m, 6.69H, A+B), 3.80−

3.76 (m, 3.00H, A+B), 3.64 (s, 2.42H, B), 3.37 (d, J = 10.0 Hz,

0.84H, B), 2.81−2.71 (m, 1.73H, A+B), 2.66−2.54 (m, 0.38H, A),

2.52−2.34 (m, 3.01H, A+B), 2.31−2.24 (m, 1.02H, A+B), 2.20−2.06

(m, 1.21H, A+B), 1.92−1.84 (m, 1.64H, A+B), 1.80−1.53 (m, 7.03H,

A+B), 1.50−1.24 (m, 3.83H, A+B), 1.19−1.07 (m, 2.59H, A+B),

0.92−0.82 (m, 1.05H, A+B), 0.78−0.69 (m, 0.82H, B), 0.30−0.21

(m, 0.13H, A), 0.14−0.05 (m, 0.18H, A). ¹³C NMR (126 MHz,

CDCl₃, mixture of rotamers 1:4, A/B): δ 173.61 (A), 172.24 (B),

171.00 (A), 170.59 (B), 159.41 (A), 158.87 (B), 154.38 (A), 153.91

(B), 151.13 (B), 150.74 (A), 149.10 (A), 148.97 (B), 147.60 (A),

147.43 (B), 142.76 (A), 141.89 (B), 137.23 (B), 133.59 (B), 133.40

(B), 131.91 (B), 129.86 (B), 129.79 (A), 129.49 (B), 128.10 (B),

127.12 (A), 120.99 (A), 120.51 (B), 120.48 (B), 120.33 (A), 116.22

(B), 113.71 (A), 111.97 (B), 111.82 (A), 111.51 (A), 111.42 (B),

110.32 (B), 109.29 (A), 107.24 (B), 104.84 (B), 103.51 (A), 76.23

(A), 75.88 (B), 67.70 (A), 67.06 (B), 63.94 (B), 63.08 (A), 61.01

(A), 60.83 (B), 56.29 (B), 56.19 (A), 56.07 (B), 56.02 (A), 55.93

(B), 55.26 (B), 52.14 (B), 43.97 (B), 41.53 (B), 39.62 (A), 38.21

(A), 37.62 (B), 32.94 (B), 32.55 (A), 31.61 (A), 31.27 (B), 30.76

(B), 30.30 (A), 28.41 (A), 27.94 (B), 27.12 (A), 26.71 (B), 26.40

(A), 26.31 (B), 26.21 (B), 26.07 (A), 25.85 (B), 24.23 (A), 21.05

(B), 20.88 (A). HRMS (m/z): (ESI⁺) calculated for C₄₄H₅₆NO₉[M

+ H]⁺: 742.39496, found 742.39491. HPLC (70−100% solvent B, 0.8

mL/min, 10.50 min): t_R= 5.58 min, purity (220 nm) = 95%. Data for

Macrocycle 35-(E). The substrate 27 (261 mg, 0.35 mmol, 1.00

equiv) was applied to general procedure B with Grubbs second

Generation catalyst (30 mg, 0.04 mmol, 0.10 equiv) in DCM (700

mL, 0.5 mM). After workup procedure b), 35-(E) (57 mg, 23%, white

solid) was obtained as pure product isomer after puriﬁcation by ﬂash

column chromatography (CH/EA = 5:1). In addition to compound

35-(E), another product (28 mg, 11%, white solid) could be isolated

from the RCM which, according to mass and NMR analysis, must be

an isomer, most likely an epimer of 35-(E), due to its remarkably

reduced binding to FKBP51, 12, and 12.6. Unfortunately, a proper

structure elucidation was not possible. TLC (DCM/MeOH = 20:1,

1

v/v): R_f= 0.32. H NMR (500 MHz, CDCl₃, mixture of rotamers

0.16:1, A/B): δ 7.24 (t, J = 7.8 Hz, 0.15H, A), 7.09 (t, J = 7.8 Hz,

0.85H, B), 7.04−7.01 (m, 0.14H, A), 6.92−6.87 (m, 0.30H, A),

6.81−6.74 (m, 2.59H, A+B), 6.70−6.51 (m, 4.36H, A+B), 6.47−6.43

(m, 0.84H, B), 6.16−6.13 (m, 0.14H, A), 6.08−5.80 (m, 2.39H, A

+B), 5.56−5.52 (m, 0.85H, B), 5.33 (dd, J = 4.2, 9.8 Hz, 0.88H, B),

4.88−4.80 (m, 0.85H, B), 4.78−4.65 (m, 2.35H, A+B), 4.58−4.53

1

macrocycle 36-(E): TLC (CH/EA = 2:1, v/v): R_f= 0.36. H NMR

(500 MHz, THF-d₈, mixture of rotamers 0.18:1, A/B): δ 7.20 (t, J =

7.8 Hz, 0.90H, B), 7.11−7.08 (m, 0.14H, A), 6.99−6.92 (m, 0.81H,

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B), 6.92−6.83 (m, 0.31H, A), 6.81−6.75 (m, 1.85H, B), 6.73−6.70

(m, 0.99H, A+B), 6.69 (d, 0.82H, B), 6.67−6.60 (m, 1.17H, A+B),

6.46 (d, J = 1.8 Hz, 0.80H, B), 6.37 (d, J = 1.9 Hz, 0.14H, A), 6.33 (d,

J = 1.8 Hz, 0.79H, B), 5.97−5.89 (m, 0.28H, A), 5.79 (dt, J = 4.9, 16.2

Hz, 0.15H, A), 5.72−5.61 (m, 1.69H, A+B), 5.56 (dd, J = 4.8, 9.2 Hz,

0.82H, B), 5.29−5.25 (m, 0.80H, B), 4.74 (d, J = 5.8 Hz, 0.14H, A),

4.69−4.53 (m, 2.01H, A+B), 4.24−4.17 (m, 0.14H, A), 3.98−3.92

(m, 0.16H, A), 3.89−3.81 (m, 1.75H, A+B), 3.76−3.67 (m, 13.11H,

A+B), 3.43 (d, J = 8.7 Hz, 0.87H, B), 2.84 (d, J = 9.9 Hz, 0.14H, A),

2.74 (td, J = 3.1, 13.3 Hz, 0.97H, B), 2.61−2.54 (m, 0.99H, A+B),

2.50−2.34 (m, 3.56H, A+B), 2.11−2.03 (m, 1.01H, A+B), 1.99−1.89

(m, 1.76H, A+B), 1.85−1.79 (m, 1.00H, A+B), 1.70−1.55 (m, 5.22H,

A+B), 1.46−1.37 (m, 0.94H, A+B), 1.33−0.76 (m, 8.59H, A+B),

0.55−0.45 (m, 0.25H, A). ¹³C NMR (126 MHz, THF-d₈, mixture of

rotamers 1:5, A/B): δ 172.93 (B), 172.50 (A), 171.01 (A), 170.82

(B), 160.49 (A), 158.96 (B), 155.71 (A), 154.13 (B), 153.78 (B),

152.91 (A), 150.90 (B), 149.38 (A), 149.31 (B), 143.39 (B), 143.08

(A), 140.29 (A), 138.59 (B), 135.26 (A), 134.78 (B), 134.63 (A),

134.05 (B), 130.50 (B), 130.46 (B), 130.20 (A), 128.57 (B), 128.09

(A), 121.32 (A), 121.15 (B), 118.90 (B), 118.09 (B), 117.02 (A),

114.42 (B), 113.76 (B), 113.50 (A), 113.35 (B), 112.60 (A), 108.21

(B), 108.16 (B), 104.99 (A), 76.99 (A), 76.77 (B), 70.74 (A), 68.99

(B), 68.89 (A), 68.73 (B), 68.10 (A), 60.66 (A), 60.54 (B), 57.18

(A), 56.52 (B), 56.38 (B), 56.28 (B), 56.09 (B), 53.14 (B), 44.15

(B), 42.35 (A), 42.27 (B), 40.01 (A), 39.64 (B), 39.29 (A), 34.01

(B), 33.80 (B), 33.48 (A), 32.60 (B), 32.43 (A), 31.71 (B), 31.15

(A), 30.80 (A), 28.19 (A), 28.03 (B), 27.79 (B), 27.61 (B), 27.41

(A), 27.21 (A), 26.49 (B), 25.98 (A), 22.52 (A), 22.35 (B). HRMS

(m/z): (ESI⁺) calculated for C₄₄H₅₆NO₉[M + H]⁺: 742.39496, found

742.39517. HPLC (70−100% solvent B, 0.8 mL/min, 10.5 min): t_R=

6.23 min, purity (220 nm) = 99%.

(B), 55.96 (A), 55.93 (B), 55.23 (B), 52.06 (B), 43.61 (B), 41.46

(B), 39.35 (A), 38.51 (B), 37.92 (A), 33.03 (B), 32.39 (A), 31.63

(A), 31.28 (B), 30.81 (B), 30.28 (A), 27.40 (A), 27.02 (B), 26.68

(B), 26.41 (A), 26.35 (B), 26.29 (A), 26.03 (A), 25.66 (B), 24.41

(A), 21.12 (B). HRMS (m/z): (ESI⁺) calculated for C₄₅H₅₈NO₁₀[M

+ H]⁺: 772.40552, found 772.40598. HPLC (70−100% solvent B,

0.80 mL/min, 10.50 min): t_R= 5.42 min, purity (220 nm) = 96%.

Data for macrocycle 37-(Z): TLC (CH/EA = 1:1 + 1% HCOOH, v/

v/v): R_f= 0.52. Note: At room temperature, the NMR spectra of

compound 37-(Z) show the presence of multiple rotamers (two

major and two minor rotamers). Due to this, the proton and carbon

NMR spectra become very complex and signals cannot be assigned to

speciﬁc rotamers. ¹H NMR (500 MHz, CDCl₃, mixture of rotamers):

δ 7.28−7.24 (m, 0.98H), 7.16 (dt, J = 7.9, 10.4 Hz, 0.72H), 7.01 (t, J

= 2.0 Hz, 0.15H), 6.97 (t, J = 2.0 Hz, 0.16H), 6.92 (dd, J = 2.1, 8.0

Hz, 0.29H), 6.89−6.73 (m, 3.01H), 6.72−6.66 (m, 1.03H), 6.65−

6.55 (m, 2.97H), 6.50 (d, J = 1.9 Hz, 0.36H), 6.35 (d, J = 1.9 Hz,

0.30H), 6.23 (d, J = 2.0 Hz, 0.14H), 6.01−5.74 (m, 2.45H), 5.52 (dd,

J = 5.6, 8.7 Hz, 0.36H), 5.45−5.36 (m, 1.00H), 4.79−4.57 (m,

2.38H), 4.52 (d, J = 13.7 Hz, 0.17H), 4.30−4.20 (m, 1.49H), 4.19−

3.97 (m, 2.86H), 3.89−3.75 (m, 14.09H), 3.72−3.69 (m, 0.35H),

3.65−3.60 (m, 0.30H), 3.57−3.51 (m, 0.29H), 3.39 (dd, J = 5.7, 9.4

Hz, 0.60H), 3.25 (d, J = 9.6 Hz, 0.17H), 2.93−2.83 (m, 0.55H), 2.77

(td, J = 3.1, 13.3 Hz, 0.32H), 2.68−2.51 (m, 1.32H), 2.48−2.40 (m,

0.65H), 2.38−2.19 (m, 1.39H), 2.16−2.02 (m, 1.70H), 1.98−1.75

(m, 2.19H), 1.72−1.55 (m, 6.00H), 1.47−1.27 (m, 4.00H), 1.19−

1.00 (m, 2.99H), 0.92−0.73 (m, 2.06H), 0.40−0.30 (m, 0.17H),

0.27−0.18 (m, 0.15H). ¹³C NMR (126 MHz, CDCl₃, mixture of

rotamers): δ 172.82, 172.54, 171.64, 170.86, 170.66, 170.33, 170.28,

159.47, 158.88, 158.85, 158.25, 154.47, 154.27, 153.46, 152.87,

152.39, 152.13, 151.94, 151.28, 149.12, 149.07, 148.99, 147.64,

147.58, 147.49, 147.46, 142.76, 141.95, 141.60, 138.28, 134.33,

134.12, 133.73, 133.66, 133.54, 133.47, 133.24, 130.30, 129.80,

129.70, 129.66, 129.54, 129.50, 129.10, 128.83, 128.76, 126.04,

121.11, 120.53, 120.42, 120.32, 119.77, 117.33, 116.71, 116.43,

116.37, 115.37, 113.69, 112.45, 112.03, 111.97, 111.91, 111.86,

111.80, 111.56, 111.49, 111.40, 109.81, 109.53, 108.82, 107.44,

106.54, 104.66, 104.46, 76.71, 76.41, 76.30, 75.63, 71.34, 70.69, 70.66,

70.26, 70.14, 69.83, 69.34, 68.60, 68.52, 68.16, 67.70, 67.39, 67.24,

67.11, 64.65, 64.43, 61.09, 60.95, 60.92, 56.41, 56.34, 56.26, 56.09,

56.07, 55.96, 55.69, 55.23, 54.81, 52.39, 52.16, 43.85, 41.26, 41.23,

40.05, 39.34, 38.37, 38.26, 38.09, 36.87, 33.55, 32.98, 32.78, 32.53,

31.81, 31.70, 31.58, 31.40, 31.21, 30.94, 30.81, 30.74, 30.33, 29.84,

27.46, 27.00, 26.79, 26.72, 26.59, 26.55, 26.46, 26.42, 26.35, 26.31,

26.06, 25.75, 25.49, 24.81, 24.34, 21.22, 20.99, 20.87, 20.79. HRMS

(m/z): (ESI⁺) calculated for C₄₅H₅₈NO₁₀[M + H]⁺: 772.40552,

found 772.40547. HPLC (70−100% solvent B, 0.8 mL/min, 10.50

min): t_R= 5.52 min, purity (220 nm) = 99%.

Macrocycle 38-(E). The substrate 30 (134 mg, 0.17 mmol, 1.00

equiv) was applied to general procedure B with Grubbs second

gGeneration catalyst (15 mg, 0.017 mmol, 0.10 equiv) and 1,4-

benzoquinone (2 mg, 0.017 mmol, 0.10 equiv) in DCM (1 mM, 175

mL). After workup procedure b, 38-(E) (43 mg, 33%, white solid)

was obtained as pure product isomer after puriﬁcation by semi-

preparative HPLC (85−100% solvent B). Another product fraction

was obtained as a mixture of 38-(E) and an unidentiﬁed isomer

(probably the Z-alkene due to same m/z on HPLC−MS; 75 mg, 58%,

ratio 1:4, unidentiﬁed isomer E-alkene). Unfortunately, we were not

able to obtain the unidentiﬁed isomer as a pure product by column

chromatography or semipreparative HPLC. TLC (CH/EA = 2:1, v/

v): R_f= 0.32. ¹H NMR (500 MHz, CDCl₃, mixture of rotamers

0.36:1, A/B): δ 7.29−7.26 (m, 0.22H, A), 7.14 (t, J = 7.9 Hz, 0.86H,

B), 7.03−7.01 (m, 0.23H, A), 6.89−6.87 (m, 0.24H, A), 6.87−6.84

(m, 0.24H, A), 6.80−6.72 (m, 2.87H, A+B), 6.69−6.64 (m, 2.16H, A

+B), 6.63−6.59 (m, 1.23H, A+B), 6.47−6.43 (m, 1.48H, A+B), 6.28

(d, J = 1.9 Hz, 0.21H, A), 5.95−5.89 (m, 0.39H, A), 5.87−5.80 (m,

1.12H, A+B), 5.77 (dt, J = 5.1, 16.0 Hz, 0.92H, B), 5.58 (dd, J = 5.6,

8.3 Hz, 0.75H, B), 5.51 (d, J = 5.6 Hz, 0.73H, B), 4.64−4.56 (m,

0.95H, A+B), 4.40−4.36 (m, 1.43H, A+B), 4.14−4.04 (m, 2.40H, A

+B), 4.02−3.97 (m, 1.07H, A+B), 3.96−3.90 (m, 1.70H, A+B),

Macrocycles 37-(E) and 37-(Z). The substrate 29 (201 mg, 0.25

mmol, 1.00 equiv) was applied to general procedure B with Grubbs

second generation catalyst (21 mg, 0.03 mmol, 0.10 equiv) and 1,4-

benzoquinone (3 mg, 0.03 mmol, 0.10 equiv) in DCM (1 mM, 250

mL). After workup procedure b, 37-(E) (58 mg, 30%, white solid)

and 37-(Z) (7 mg, 4%, white solid) were obtained as pure product

isomers after puriﬁcation by ﬂash column chromatography (CH/EA =

4:1) and semipreparative HPLC (80−100% solvent B). Another

product fraction was obtained as a mixture of E/Z isomers (75 mg,

39%) and not further puriﬁed. Data for macrocycle 37-(E): TLC

1

(CH/EA = 1:1 + 1% HCOOH, v/v/v): R_f= 0.52. H NMR (500

MHz, CDCl₃, mixture of rotamers 0.34:1, A/B): δ 7.28−7.23 (m,

0.40H, A), 7.19−7.15 (m, 0.78H, B), 7.05−7.02 (m, 0.23H, A),

6.91−6.87 (m, 0.28H, A), 6.86−6.72 (m, 2.99H, A+B), 6.71−6.61

(m, 2.06H, A+B), 6.57−6.55 (m, 0.71H, B), 6.43−6.37 (m, 1.34H, A

+B), 6.33−6.30 (m, 0.23H, A), 6.00 (dt, J = 4.9, 17.3 Hz, 0.32H, A),

5.93−5.90 (m, 0.17H, A), 5.87 (dt, J = 5.2, 16.5 Hz, 1.15H, B), 5.83−

5.80 (m, 0.40H, A), 5.58 (dd, J = 5.3, 8.5 Hz, 0.74H, B), 5.49 (d, J =

5.5 Hz, 0.78H, B), 4.65−4.54 (m, 1.05H, A+B), 4.53−4.42 (m,

1.57H, A+B), 4.31−4.18 (m, 0.79H, A), 4.15−4.03 (m, 2.82H, A+B),

4.00−3.88 (m, 2.20H, A+B), 3.86−3.79 (m, 11.69H, A+B), 3.74−

3.61 (m, 4.92H, A+B), 3.33 (d, J = 9.5 Hz, 0.72H, B), 2.85−2.76 (m,

0.98H, A+B), 2.69 (d, J = 9.7 Hz, 0.24H, A), 2.65−2.48 (m, 1.31H, A

+B), 2.45−2.38 (m, 0.77H, B), 2.32−2.18 (m, 1.51H, A+B), 2.10−

1.96 (m, 2.00H, A+B), 1.90−1.83 (m, 1.51H, A+B), 1.70−1.56 (m,

5.38H, A+B), 1.51−1.23 (m, 5.20H, A+B), 1.17−0.99 (m, 2.30H, A

+B), 0.94−0.85 (m, 0.96H, A+B), 0.77−0.69 (m, 0.82H, B), 0.48−

0.38 (m, 0.23H, A), 0.26−0.17 (m, 0.25H, A). ¹³C NMR (126 MHz,

CDCl₃, mixture of rotamers 1:4, A/B): δ 173.10 (A), 172.31 (B),

170.64 (B), 159.12 (A), 158.30 (B), 154.25 (A), 153.31 (B), 152.28

(B), 151.25 (A), 149.04 (A), 148.94 (B), 147.54 (A), 147.38 (B),

142.15 (B), 142.08 (A), 139.25 (A), 137.68 (B), 134.20 (A), 133.57

(B), 133.45 (A), 133.24 (B), 131.06 (B), 130.32 (A), 129.61 (A),

129.50 (B), 127.75 (B), 127.09 (A), 120.68 (A), 120.37 (B), 120.26

(A), 119.01 (B), 114.37 (B), 113.99 (A), 113.77 (A), 112.95 (B),

112.47 (A), 111.88 (B), 111.78 (A), 111.47 (A), 111.40 (B), 108.31

(B), 105.97 (B), 104.88 (A), 76.22 (A), 75.56 (B), 71.17 (A), 70.94

(A), 70.83 (B), 69.09 (A), 69.05 (B), 68.98 (B), 67.50 (B), 67.28

(A), 60.93 (B), 60.86 (A), 56.36 (B), 56.15 (A), 56.11 (A), 56.02

3338

https://dx.doi.org/10.1021/acs.jmedchem.0c02195

J. Med. Chem. 2021, 64, 3320−3349

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

3.87−3.83 (m, 6.67H, A+B), 3.82 (s, 0.81H, A), 3.81−3.79 (m,

0.93H, A), 3.78 (s, 2.32H, B), 3.66 (s, 2.67H, A+B), 3.58−3.53 (m,

0.90H, A+B), 3.35 (d, J = 9.8 Hz, 0.74H, B), 2.92 (td, J = 2.8, 13.5

Hz, 0.77H, B), 2.83−2.74 (m, 0.50H, A), 2.65−2.59 (m, 0.38H, A),

2.55−2.37 (m, 2.20H, A+B), 2.32−2.23 (m, 1.00H, A+B), 2.21−2.14

(m, 0.30H, A), 2.11−1.97 (m, 3.64H, A+B), 1.93−1.83 (m, 1.37H, A

+B), 1.72−1.58 (m, 5.21H, A+B), 1.54−1.40 (m, 1.19H, A+B),

1.36−1.25 (m, 3.38H, A+B), 1.17−1.07 (m, 2.20H, A+B), 0.95−0.83

(m, 1.22H, A+B), 0.78−0.70 (m, 0.81H, B), 0.61−0.51 (m, 0.22H,

A), 0.39−0.30 (m, 0.23H, A). ¹³C NMR (126 MHz, CDCl₃, mixture

of rotamers 1:3, A/B): δ 172.89 (A), 172.27 (B), 170.70 (B), 170.52

(A), 159.01 (A), 158.58 (B), 154.06 (A), 153.39 (B), 152.60 (B),

151.91 (A), 149.10 (A), 148.99 (B), 147.60 (A), 147.42 (B), 142.18

(B), 137.78 (A), 137.45 (B), 134.19 (A), 133.59 (B), 133.47 (A),

130.94 (A), 130.06 (B), 129.61 (A), 129.44 (B), 127.71 (B), 126.74

(A), 120.44 (B), 120.33 (A), 120.29 (A), 119.32 (B), 114.48 (B),

113.17 (A), 113.08 (A), 112.23 (B), 112.01 (B), 111.84 (A), 111.54

(A), 111.48 (B), 109.75 (A), 107.41 (B), 105.84 (B), 103.89 (A),

76.54 (A), 75.76 (B), 70.13 (A), 70.03 (B), 67.64 (B), 67.55 (A),

67.45 (A), 67.07 (A), 66.76 (A), 65.71 (B), 65.20 (B), 60.90 (A),

60.85 (B), 56.61 (B), 56.23 (A), 56.20 (A), 56.06 (B), 56.00 (B),

55.97 (B), 55.47 (A), 55.23 (B), 52.22 (B), 43.79 (B), 41.74 (B),

41.35 (A), 39.46 (A), 38.42 (B), 38.30 (A), 33.00 (B), 32.60 (A),

31.56 (A), 31.23 (B), 30.82 (B), 30.37 (A), 30.15 (A), 29.99 (B),

29.81 (A), 27.37 (A), 27.10 (B), 26.71 (B), 26.55 (A), 26.35 (B),

26.31 (B), 26.11 (A), 25.84 (B), 24.53 (A), 21.26 (B). HRMS (m/z):

(ESI⁺) calculated for C₄₆H₆₀NO₁₀[M + H]⁺: 786.42117, found

786.42154. HPLC (70−100% solvent B, 1.5 mL/min, 20 min): t_R=

5.96 min, purity (220 nm) = 97%.

Macrocycle 39. A stirred solution of 31 (E/Z-alkene mixture, 44

mg, 0.06 mmol, 1.00 equiv) in MeOH (3 mL) was degassed by

sparging with argon for 15 min. After addition of Pd/C (10 wt %, 6.4

mg, 0.006 mmol, 0.10 equiv), the solution was sparged with hydrogen

for 15 min and was then stirred under a hydrogen atmosphere at

room temperature for 45 min. The dark suspension was ﬁltered

through Celite and the solvent was removed under reduced pressure.

The crude product was puriﬁed by ﬂash column chromatography

(CH/EA = 5:1) to aﬀord 39 (20 mg, 45%) as white solid. TLC (CH/

EA = 3:1, v/v): R_f= 0.33. ¹H NMR (500 MHz, CDCl₃): δ 7.15 (t, J =

7.9 Hz, 1H), 6.79−6.71 (m, 5H), 6.70−6.66 (m, 1H), 6.33 (d, J = 1.8

Hz, 1H), 6.18−6.15 (m, 1H), 5.74 (dd, J = 5.8, 8.1 Hz, 1H), 5.48 (d, J

= 5.7 Hz, 1H), 4.38−4.32 (m, 1H), 4.20−4.12 (m, 1H), 4.07−4.01

(m, 1H), 3.95−3.90 (m, 1H), 3.88 (s, 3H), 3.86 (s, 3H), 3.85 (s, 3H),

3.77 (td, J = 3.0, 8.9 Hz, 1H), 3.56 (s, 3H), 3.28 (d, J = 9.9 Hz, 1H),

2.66−2.55 (m, 2H), 2.50−2.42 (m, 1H), 2.33−2.27 (m, 1H), 2.24−

2.16 (m, 1H), 2.16−2.06 (m, 2H), 2.00−1.83 (m, 3H), 1.76−1.52

(m, 8H), 1.46−1.25 (m, 4H), 1.18−1.09 (m, 2H), 0.95−0.84 (m,

1H), 0.80−0.69 (m, 1H). ¹³C NMR (126 MHz, CDCl₃): δ 172.1,

171.2, 159.6, 153.7, 152.4, 149.0, 147.4, 141.5, 137.0, 134.0, 132.9,

129.3, 120.4, 120.3, 114.6, 113.6, 112.1, 111.4, 108.3, 104.4, 76.0,

72.9, 69.6, 56.6, 56.5, 56.1, 56.0, 55.8, 52.5, 43.8, 41.7, 38.9, 33.2,

31.4, 30.7, 28.4, 26.9, 26.8, 26.4, 26.4, 25.7, 24.0, 21.1. HRMS (m/z):

(ESI⁺) calculated for C₄₃H₅₆NO₉[M + H]⁺: 730.39496, found

rotation of the aryl ring. ¹H NMR (500 MHz, CDCl₃): δ 7.16 (t, J =

7.9 Hz, 1H), 6.84−6.80 (m, 1H), 6.78−6.73 (m, 2H), 6.69−6.59 (m,

3H), 6.44−6.40 (m, 1H), 6.30 (br s, 1H), 5.59 (dd, J = 6.0, 8.3 Hz,

1H), 5.47−5.42 (m, 1H), 4.22−4.15 (m, 1H), 4.10−4.04 (m, 1H),

3.99−3.89 (m, 3H), 3.88−3.80 (m, 9H), 3.51 (br s, 3H), 3.33 (d, J =

9.8 Hz, 1H), 2.82 (td, J = 2.8, 13.4 Hz, 1H), 2.58 (ddd, J = 5.5, 9.6,

14.6 Hz, 1H), 2.46 (ddd, J = 6.6, 9.4, 13.9 Hz, 1H), 2.32−2.22 (m,

1H), 2.22−2.02 (m, 2H), 1.97−1.90 (m, 1H), 1.86 (d, J = 12.5 Hz,

1H), 1.76−1.50 (m, 12H), 1.45−1.38 (m, 1H), 1.35−1.23 (m, 3H),

1.19−1.06 (m, 2H), 0.95−0.83 (m, 1H), 0.79−0.68 (m, 1H). ¹³C

NMR (126 MHz, CDCl₃): δ 172.4, 171.0, 158.7, 153.9, 152.7, 149.0,

147.4, 141.4, 135.2, 133.7, 133.3, 129.4, 120.4, 120.0, 114.6, 114.2,

112.0, 111.4, 107.7, 104.6, 76.2, 72.6, 68.1, 56.4, 56.2, 56.1, 55.9, 55.1,

52.1, 43.7, 41.5, 37.2, 32.9, 31.5, 30.7, 28.9, 27.6, 27.0, 26.7, 26.3,

26.3, 25.7, 22.8, 21.1. HRMS (m/z): (ESI⁺) calculated for

C₄₄H₅₈NO₉[M + H]⁺: 744.41061, found 744.41050. HPLC (70−

80% solvent B, 1.5 mL/min, 20 min): t_R= 11.00 min, purity (220

nm) = 99%.

Macrocycle 41. The substrate 33-(E) (40 mg, 0.05 mmol, 1.00

equiv) was applied to general procedure E with RhCl(PPh₃)₃(10 mg,

0.01 mmol, 0.20 equiv) in toluene (2 mL). Additional portions of

RhCl(PPh₃)₃(2 × 0.1 equiv) and sparging with hydrogen were

applied until completion of the reaction. 41 (30 mg, 75%) was

obtained as light yellow resin after puriﬁcation by ﬂash column

chromatography (CH/EA = 2:1). TLC (CH/EA = 2:1, v/v): R_f=

0.23. ¹H NMR (500 MHz, CDCl₃, mixture of rotamers 0.63:1, A/B):

δ 7.31−7.26 (m, 0.34H, A), 7.15 (t, J = 7.9 Hz, 0.62H, B), 7.13−7.11

(m, 0.36H, A), 6.92−6.88 (m, 0.38H, A), 6.83−6.74 (m, 2.80H, A

+B), 6.72−6.63 (m, 2.10H, A+B), 6.51−6.44 (m, 1.32H, A+B), 6.36

(s, 1.23H, A+B), 5.81 (dd, J = 5.3, 8.7 Hz, 0.39H, A), 5.46 (q, J = 5.2

Hz, 1.25H, B), 4.77 (dd, J = 2.4, 7.0 Hz, 0.37H, A), 4.38 (d, J = 13.4

Hz, 0.38H, A), 4.32−4.26 (m, 0.39H, A), 4.22−4.17 (m, 0.42H, A),

4.16−4.10 (m, 0.67H, B), 4.09−3.79 (m, 12.23H, A+B), 3.75−3.60

(m, 2.77H, A+B), 3.57 (s, 4.20H, A+B), 3.47−3.41 (m, 0.43H, A),

3.34 (d, J = 9.9 Hz, 0.62H, B), 3.19−3.12 (m, 0.43H, A), 3.06−2.96

(m, 0.39H, A), 2.81 (td, J = 3.0, 13.3 Hz, 0.60H, B), 2.73−2.65 (m,

0.39H, A), 2.63−2.51 (m, 0.99H, A+B), 2.47−2.39 (m, 0.63H, B),

2.34−2.26 (m, 1.29H, A+B), 2.20 (d, J = 13.8 Hz, 0.39H, A+B),

2.12−1.99 (m, 1.60H, A+B), 1.94−1.55 (m, 10.94H, A+B), 1.51−

1.00 (m, 7.52H, A+B), 0.96−0.68 (m, 2.00H, A+B), 0.64−0.54 (m,

0.36H, A), −0.11 − −0.22 (m, 0.38H, A), −0.25 − −0.37 (m, 0.38H,

A). ¹³C NMR (126 MHz, CDCl₃, mixture of rotamers 1:2, A/B): δ

174.10 (A), 172.54 (B), 171.85 (A), 170.85 (B), 159.82 (A), 159.04

(B), 153.26 (A), 153.06 (B), 149.12 (A), 148.98 (B), 147.64 (A),

147.46 (B), 143.27 (A), 141.68 (B), 136.68 (A), 135.72 (B), 133.81

(A), 133.59 (B), 133.35 (A), 133.19 (B), 129.90 (A), 129.61 (B),

120.42 (B), 120.38 (A), 120.18 (A), 119.33 (B), 115.26 (B), 113.08

(A), 112.50 (B), 111.97 (B), 111.86 (A), 111.50 (A), 111.40 (B),

111.00 (A), 105.83 (B), 76.27 (B), 75.73 (A), 73.29 (A), 72.77 (B),

71.89 (A), 71.47 (A), 70.52 (B), 70.02 (B), 67.50 (B), 67.46 (A),

56.75 (A), 56.07 (A+B), 55.96 (B), 55.91 (B), 55.68 (A), 55.04 (B),

51.95 (B), 43.55 (B), 41.97 (A), 41.05 (B), 39.45 (A), 38.74 (A),

36.85 (B), 32.87 (B), 32.16 (A), 31.73 (A), 31.50 (B), 30.74 (B),

29.99 (A), 27.32 (A), 27.08 (B), 26.88 (A), 26.74 (B), 26.45 (A),

26.32 (B), 26.27 (B), 26.09 (A), 26.03 (A), 25.77 (A), 25.70 (B),

23.90 (A), 21.09 (B), 20.23 (A). HRMS (m/z): (ESI⁺) calculated for

C₄₅H₆₀NO₁₀[M + H]⁺: 774.42117, found 774.42144. HPLC (60−

100% solvent B, 1.5 mL/min, 20 min): t_R= 7.86 min, purity (220

nm) = 99%.

730.39464. HPLC (70−80% solvent B, 1.5 mL/min, 20 min): t_R

10.74 min, purity (220 nm) = 99%.

=

Macrocycle 40. A stirred solution of 32 (E/Z-alkene mixture, 118

mg, 0.16 mmol, 1.00 equiv) in MeOH (3 mL) was degassed by

sparging with argon for 15 min. After addition of Pd/C (10 wt %, 20

mg, 0.02 mmol, 0.10 equiv) the solution was sparged with hydrogen

for 15 min and was then stirred under a hydrogen atmosphere at

room temperature for 15 min. The dark suspension was ﬁltered

through Celite and the solvent was removed under reduced pressure.

The crude product was puriﬁed by ﬂash column chromatography

(CH/EA = 6:1) to aﬀord 40 (44 mg, 37%) as a white solid. TLC

(CH/EA = 3:1, v/v): R_f= 0.33. Note: The NMR spectra of

compound 40 shows a peak broadening for the ortho and meta proton

and carbon signals of the 3,5-dimethoxyaryl ring and the connected

methoxy groups. This might result out of a slow interconversion of

the C2-symmetrical nuclei of the aryl ring and the attached methoxy

groups, because the connected macrocycle linker probably hinders the

Macrocycle 42. The substrate 34-(E) (31 mg, 0.04 mmol, 1.00

equiv) was applied to general procedure E with RhCl(PPh₃)₃(15 mg,

0.016 mmol, 0.40 equiv) in toluene (2 mL). 42 (27 mg, 87%) was

obtained as a light brown resin after puriﬁcation by ﬂash column

chromatography (CH/EA = 3:1). TLC (CH/EA = 2:1, v/v): R_f=

1

0.44. H NMR (500 MHz, CDCl₃, mixture of rotamers 0.08:1. Only

major rotamer is reported here): δ 7.13 (t, J = 7.9 Hz, 1H), 6.77−6.62

(m, 5H), 6.52−6.48 (m, 1H), 6.41 (s, 2H), 5.50−5.44 (m, 2H),

3.97−3.84 (m, 11H), 3.69−3.65 (m, 1H), 3.60−3.48 (m, 9H), 3.36

(d, J = 9.9 Hz, 1H), 2.85 (td, J = 2.9, 13.3 Hz, 1H), 2.54−2.41 (m,

2H), 2.30−2.26 (m, 1H), 2.11−2.05 (m, 1H), 1.96−1.82 (m, 6H),

3339

https://dx.doi.org/10.1021/acs.jmedchem.0c02195

J. Med. Chem. 2021, 64, 3320−3349

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1.74−1.60 (m, 8H), 1.44−1.11 (m, 7H), 0.92−0.85 (m, 1H), 0.77−

0.71 (m, 1H). ¹³C NMR (126 MHz, CDCl₃, mixture of rotamers 1:9,

A/B): δ 173.48 (A), 172.50 (B), 172.07 (A), 170.83 (B), 158.79 (B),

153.34 (B), 153.24 (A), 149.00 (B), 147.47 (B), 142.73 (A), 141.99

(B), 136.28 (B), 133.56 (B), 133.26 (B), 129.83 (A), 129.64 (B),

120.45 (B), 120.34 (A), 118.97 (B), 114.98 (B), 112.95 (B), 112.51

(A), 112.43 (A), 111.99 (B), 111.88 (A), 111.53 (A), 111.45 (B),

106.14 (B), 105.26 (A), 76.18 (B), 76.02 (A), 70.84 (B), 70.57 (A),

70.05 (B), 69.97 (A), 67.88 (A), 67.70 (B), 67.17 (B), 56.22 (B),

56.07 (B), 55.97 (B), 55.74 (A), 55.03 (B), 51.91 (B), 43.60 (B),

41.56 (A), 41.22 (B), 39.47 (A), 38.42 (A), 37.50 (B), 34.85 (A),

32.92 (B), 31.59 (A), 31.39 (B), 30.87 (B), 30.72 (B), 30.57 (A),

27.03 (B), 26.74 (B), 26.53 (A), 26.44 (B), 26.34 (B), 26.28 (B),

25.75 (B), 25.71 (B), 21.05 (B), 20.42 (A). HRMS (m/z): (ESI⁺)

calculated for C₄₆H₆₂NO₁₀[M + H]⁺: 788.43682, found 788.43728.

HPLC (70−80% solvent B, 1.5 mL/min, 20 min): t_R= 11.19 min,

purity (220 nm) = 99%.

Macrocycle 43. The substrate 35 (E/Z-alkene mixture, 30 mg,

0.04 mmol, 1.00 equiv) was applied to general procedure E with

RhCl(PPh₃)₃(20 mg, 0.021 mmol, 0.50 equiv) in THF/MeOH (v/v

= 1:1, 10 mL). 43 (20 mg, 66%) was obtained after puriﬁcation by

ﬂash column chromatography (CH/EA = 5:1) as a white solid. TLC

(CH/EA = 2:1, v/v): R_f= 0.60. ¹H NMR (500 MHz, CDCl₃, mixture

of rotamers 0.22:1, A/B): δ 7.29 (t, J = 7.9 Hz, 0.22H, A), 7.15−7.10

(m, 0.97H, A+B), 6.93−6.90 (m, 0.18H, A), 6.84−6.79 (m, 0.29H,

A), 6.79−6.75 (m, 0.95H, A+B), 6.74−6.68 (m, 2.59H, A+B), 6.68−

6.66 (m, 0.19H, A), 6.65−6.61 (m, 1.58H, A+B), 6.48 (d, J = 1.7 Hz,

0.18H, A), 6.38 (d, J = 1.9 Hz, 0.75H, B), 6.29 (d, J = 1.8 Hz, 0.17H,

A), 5.92−5.87 (m, 0.75H, B), 5.78 (dd, J = 5.2, 8.6 Hz, 0.18H, A),

5.61 (dd, J = 6.6, 7.7 Hz, 0.82H, B), 5.53 (d, J = 5.5 Hz, 0.80H, B),

4.78 (td, J = 4.2, 11.3 Hz, 0.17H, A), 4.54−4.48 (m, 0.16H, A), 4.43−

4.40 (m, 0.17H, A), 4.25−4.09 (m, 2.98H, A+B), 4.00 (d, J = 13.7

Hz, 0.82H, B), 3.96−3.90 (m, 0.81H, B), 3.87−3.78 (m, 10.08H, A

+B), 3.59 (s, 2.43H, B), 3.33 (d, J = 10.2 Hz, 0.80H, B), 2.87−2.79

(m, 0.16H, A), 2.71−2.55 (m, 0.38H, A), 2.54−2.11 (m, 6.59H, A

+B), 1.97−1.78 (m, 3.90H, A+B), 1.77−1.49 (m, 7.01H, A+B),

1.46−1.07 (m, 6.33H, A+B), 0.99−0.92 (m, 0.37H, A), 0.88−0.80

(m, 1.17H, A+B), 0.76−0.68 (m, 0.73H, B), 0.22−0.13 (m, 0.16H,

A), 0.09−0.00 (m, 0.16H, A). ¹³C NMR (126 MHz, CDCl₃, mixture

of rotamers 1:4, A/B): δ 173.61 (A), 171.87 (B), 171.31 (A), 170.75

(B), 159.45 (A), 159.17 (B), 154.23 (A), 153.84 (B), 152.77 (B),

150.70 (A), 149.12 (A), 148.95 (B), 147.62 (A), 147.42 (B), 143.49

(A), 141.44 (B), 137.44 (A), 137.24 (B), 133.93 (A), 133.85 (B),

133.56 (B), 133.46 (A), 129.85 (A), 129.19 (B), 120.48 (A), 120.39

(B), 120.35 (B), 114.38 (B), 112.70 (A), 111.96 (B), 111.86 (A),

111.60 (A), 111.56 (A), 111.47 (B), 111.40 (B), 108.53 (B), 108.45

(A), 103.76 (B), 102.97 (A), 76.13 (A), 75.42 (B), 69.08 (A), 68.09

(B), 67.97 (A), 67.84 (B), 60.93 (A), 60.75 (B), 56.21 (B), 56.07

(B), 56.00 (B), 55.95 (B), 54.90 (B), 51.74 (B), 43.83 (B), 41.58

(B), 41.38 (A), 39.48 (A), 38.45 (A), 37.06 (B), 32.81 (B), 32.65

(A), 31.69 (A), 31.24 (B), 30.67 (B), 30.41 (A), 29.83 (A), 27.84

(B), 26.98 (A), 26.77 (B), 26.71 (A), 26.52 (B), 26.42 (A), 26.37

(A), 26.28 (B), 26.20 (B), 26.02 (A), 25.88 (B), 24.38 (B), 24.29

(A), 24.16 (A), 21.06 (B). HRMS (m/z): (ESI⁺) calculated for

C₄₃H₅₆NO₉[M + H]⁺: 730.39496, found 730.39472. HPLC (70−

80% solvent B, 1.5 mL/min, 20 min): t_R= 9.34 min, purity (220 nm)

= 99%.

0.81H, A), 3.75 (s, 2.29H, B), 3.65 (s, 2.30H, B), 3.39 (d, J = 10.1 Hz,

0.76H, B), 2.94−2.86 (m, 0.24H, A), 2.75 (td, J = 2.6, 13.3 Hz,

0.75H, B), 2.68−2.53 (m, 0.49H, A), 2.51−2.33 (m, 1.64H, B),

2.32−2.19 (m, 1.24H, A+B), 2.14−2.07 (m, 1.00H), 2.01−1.93 (m,

1.29H, A+B), 1.90−1.55 (m, 12.08H, A+B), 1.51−1.25 (m, 4.00H, A

+B), 1.20−1.07 (m, 2.19H, A+B), 1.03−0.82 (m, 1.59H, A+B),

0.78−0.69 (m, 0.77H, B), 0.28−0.18 (m, 0.51H, A+B). ¹³C NMR

(126 MHz, CDCl₃, mixture of rotamers 1:3, A/B): δ 174.15 (A),

172.67 (B), 170.75 (A), 170.37 (B), 159.64 (A), 159.33 (B), 154.40

(A), 153.74 (B), 152.11 (B), 151.96 (A), 149.11 (A), 148.97 (B),

147.61 (A), 147.44 (B), 142.39 (A), 141.78 (B), 139.23 (A), 137.19

(B), 133.62 (A), 133.49 (A), 133.39 (B), 133.38 (B), 129.81 (A),

129.41 (B), 120.48 (B), 120.35 (A), 120.29 (B), 113.98 (B), 113.25

(A), 113.22 (A), 112.70 (B), 112.51 (A), 112.02 (B), 111.88 (A),

111.59 (A), 111.48 (B), 107.43 (B), 104.43 (B), 104.38 (A), 76.18

(A), 75.90 (B), 71.70 (A), 68.20 (B), 67.36 (B), 67.06 (A), 60.88

(B), 56.22 (A), 56.15 (B), 56.08 (B), 56.03 (A), 55.93 (B), 55.12

(B), 52.24 (B), 44.06 (B), 41.60 (A), 41.35 (B), 39.85 (A), 38.08

(A), 37.20 (B), 32.86 (B), 32.43 (A), 31.66 (A), 31.28 (B), 30.72

(B), 30.14 (A), 28.52 (A), 28.44 (B), 28.20 (A), 27.63 (B), 27.10

(A), 26.70 (B), 26.68 (B), 26.37 (A), 26.25 (B), 26.13 (B), 25.98

(A), 25.82 (B), 24.15 (A), 22.19 (B), 21.98 (A), 21.02 (B), 20.83

(A). HRMS (m/z): (ESI⁺) calculated for C₄₄H₅₈NO₉[M + H]⁺:

744.41061, found 744.40994. HPLC (70−100% solvent B, 0.8 mL/

min, 10.5 min): t_R= 6.32 min, purity (220 nm) = 98%.

Macrocycle 45. The substrate 37-(E) (30 mg, 0.04 mmol, 1.00

equiv) was applied to general procedure E with RhCl(PPh₃)₃(18 mg,

0.02 mmol, 0.50 equiv) in THF/MeOH (v/v = 1:1, 10 mL). 45 (25

mg, 83%) was obtained as a white solid after puriﬁcation by

semipreparative HPLC (80−100% solvent B). TLC (CH/EA = 2:1,

1

v/v): R_f= 0.29. H NMR (500 MHz, CDCl₃, mixture of rotamers

0.48:1, A/B): δ 7.26−7.23 (m, 0.30H, A), 7.18 (t, J = 7.9 Hz, 0.59H,

B), 7.01−6.99 (m, 0.29H, A), 6.90−6.83 (m, 1.25H, A+B), 6.81−

6.73 (m, 1.93H, A+B), 6.71−6.61 (m, 2.15H, A+B), 6.60−6.56 (m,

0.78H, A+B), 6.52−6.49 (m, 0.59H, B), 6.38−6.34 (m, 0.69H, B),

6.28 (d, J = 2.0 Hz, 0.29H, A), 5.84 (dd, J = 5.5, 8.3 Hz, 0.29H, A),

5.52 (dd, J = 5.4, 8.7 Hz, 0.60H, B), 5.45−5.42 (m, 0.63H, B), 4.64−

4.60 (m, 0.28H, A), 4.53 (d, J = 13.6 Hz, 0.26H, A), 4.27−4.21 (m,

0.34H, A), 4.16−3.91 (m, 4.67H, A+B), 3.87−3.79 (m, 9.44H, A+B),

3.76−3.73 (m, 3.09H, A+B), 3.70−3.64 (m, 2.06H, A+B), 3.36 (d, J

= 9.7 Hz, 0.64H, A+B), 2.88 (td, J = 3.0, 13.1 Hz, 0.27H, A), 2.82 (td,

J = 3.0, 13.4 Hz, 0.61H, B), 2.66−2.60 (m, 0.65H, B), 2.58−2.46 (m,

0.98H, A+B), 2.43−2.37 (m, 0.63H, B), 2.31−2.25 (m, 1.13H, A+B),

2.19−2.05 (m, 1.96H, A+B), 1.96−1.77 (m, 6.13H, A+B), 1.69−1.57

(m, 4.99H, A+B), 1.52−1.25 (m, 4.86H, A+B), 1.20−1.01 (m, 3.13H,

A+B), 0.95−0.85 (m, 1.06H, A+B), 0.77−0.69 (m, 0.70H, B), 0.45−

0.37 (m, 0.26H, A), 0.28−0.19 (m, 0.26H, A). ¹³C NMR (126 MHz,

CDCl₃, mixture of rotamers 1:2, A/B): δ 172.99 (A), 172.49 (B),

170.75 (B), 159.85 (A), 159.18 (B), 154.33 (A), 153.32 (B), 152.26

(B), 151.31 (A), 149.10 (A), 148.98 (B), 147.59 (A), 147.43 (B),

141.96 (A), 141.88 (B), 139.04 (A), 137.72 (B), 134.29 (A), 133.55

(B), 133.53 (A), 133.47 (B), 129.57 (B), 120.53 (A), 120.48 (B),

120.30 (A), 119.48 (B), 114.92 (B), 114.81 (A), 113.01 (A), 111.97

(B), 111.84 (A), 111.67 (B), 111.53 (A), 111.46 (B), 110.88 (A),

108.44 (B), 106.18 (B), 104.71 (A), 76.32 (A), 75.75 (B), 71.17 (A),

71.00 (B), 70.62 (A), 69.26 (B), 69.16 (B), 67.77 (A), 67.55 (B),

60.93 (B), 56.39 (B), 56.23 (A), 56.08 (A), 56.06 (B), 56.02 (A),

55.94 (B), 55.88 (A), 55.76 (A), 55.15 (B), 52.16 (B), 43.74 (B),

41.28 (B), 39.36 (A), 38.19 (A), 37.46 (B), 32.91 (B), 32.49 (A),

31.68 (A), 31.32 (B), 30.82 (B), 30.37 (A), 27.45 (A), 26.97 (B),

26.72 (B), 26.55 (A), 26.43 (B), 26.37 (B), 26.17 (A), 26.04 (A),

25.79 (B), 25.67 (B), 24.38 (A), 21.08 (B), 21.00 (A). HRMS (m/z):

(ESI⁺) calculated for C₄₅H₆₀NO₁₀[M + H]⁺: 774.42117, found

774.42152. HPLC (70−100% solvent B, 0.8 mL/min, 20 min): t_R=

6.12 min, purity (220 nm) = 98%.

Macrocycle 44. The substrate 36-(E) (25 mg, 0.034 mmol, 1.00

equiv) was applied to general procedure E with RhCl(PPh₃)₃(16 mg,

0.017 mmol, 0.50 equiv) in THF/MeOH (v/v = 1:1, 10 mL). 44 (18

mg, 72%) was obtained as a white solid after puriﬁcation by

semipreparative HPLC (85−100% solvent B). TLC (CH/EA = 2:1,

1

v/v): R_f= 0.50. H NMR (500 MHz, CDCl₃, mixture of rotamers

0.32:1, A/B): δ 7.28 (d, J = 8.0 Hz, 0.21H, A), 7.14 (t, J = 7.9 Hz,

0.75H, B), 7.09−7.07 (m, 0.24H, A), 6.90−6.85 (m, 0.52H, A),

6.81−6.72 (m, 2.64H, A+B), 6.71−6.69 (m, 0.30H, A), 6.67−6.61

(m, 2.88H, A+B), 6.43 (d, J = 1.8 Hz, 0.75H, B), 6.23−6.20 (m,

1.01H, A+B), 5.87 (dd, J = 5.4, 8.5 Hz, 0.25H, A), 5.54−5.49 (m,

1.54H, A+B), 4.56−4.47 (m, 0.57H, A), 4.24−4.17 (m, 1.33H, A+B),

4.12−3.91 (m, 3.11H, A+B), 3.87−3.82 (m, 7.90H, A+B), 3.80 (s,

Macrocycle 46. The substrate 38-(E) (75 mg, 0.095 mmol, 1.00

equiv) was applied to general procedure E with RhCl(PPh₃)₃(44 mg,

0.048 mmol, 0.50 equiv) in THF/MeOH (v/v = 1:1, 10 mL). 46 (33

mg, 44%) was obtained as a white solid after puriﬁcation by

semipreparative HPLC (85−100% solvent B). TLC (CH/EA = 2:1,

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https://dx.doi.org/10.1021/acs.jmedchem.0c02195

J. Med. Chem. 2021, 64, 3320−3349

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1

v/v): R_f= 0.29. H NMR (500 MHz, CDCl₃, mixture of rotamers

(B), 52.25 (B), 43.77 (B), 41.21 (B), 39.80 (A), 37.93 (A), 37.62

(B), 33.09 (A), 32.85 (B), 31.56 (A), 31.19 (B), 30.68 (B), 30.52

(A), 26.79 (B), 26.72 (A), 26.58 (B), 26.44 (A), 26.20 (A+B), 26.14

(B), 25.50 (B), 24.38 (A), 20.83 (B), 20.62 (A). HRMS (m/z):

(ESI⁺) calculated for C₄₁H₅₄NO₁₀[M + H]⁺: 720.37422, found

0.55:1, A/B): δ 7.29−7.24 (m, 0.64H, A), 7.17−7.11 (m, 0.66H, B),

7.01−6.98 (m, 0.36H, A), 6.90−6.87 (m, 0.38H, A), 6.86−6.83 (m,

0.39H, A), 6.81−6.72 (m, 2.27H, A+B), 6.71−6.68 (m, 0.34H, A),

6.67−6.62 (m, 2.22H, A+B), 6.59 (d, J = 1.8 Hz, 0.35H, A), 6.50−

6.47 (m, 0.65H, B), 6.43 (d, J = 1.8 Hz, 0.64H, B), 6.29 (d, J = 1.8

Hz, 0.37H, A), 5.77 (dd, J = 5.6, 8.2 Hz, 0.37H, A), 5.52−5.48 (m,

1.35H, B), 4.65−4.61 (m, 0.35H, A), 4.52 (br d, J = 13.8 Hz, 0.36H,

A), 4.37 (br s, 0.66H, B), 4.26−4.20 (m, 0.39H, A), 4.15−4.02 (m,

2.40H, A+B), 3.97−3.88 (m, 1.27H, A+B), 3.87−3.78 (m, 10.32H, A

+B), 3.72−3.47 (m, 6.29H, A+B), 3.41 (d, J = 10.0 Hz, 0.63H, B),

3.00 (td, J = 2.7, 13.3 Hz, 0.64H, B), 2.87 (td, J = 3.1, 13.3 Hz, 0.35H,

A), 2.68−2.53 (m, 1.11H, A+B), 2.49−2.33 (m, 1.32H, A+B), 2.32−

2.25 (m, 1.00H, A+B), 2.19−1.57 (m, 15.12H, A+B), 1.51−1.02 (m,

7.50H, A+B), 0.95−0.85 (m, 1.00H, A+B), 0.79−0.70 (m, 0.63H, B),

0.48−0.39 (m, 0.34H, A), 0.18−0.09 (m, 0.36H, A). ¹³C NMR (126

MHz, CDCl₃, mixture of rotamers 1:2, A/B): δ 173.49 (A), 172.73

(B), 170.69 (A), 170.57 (B), 159.77 (A), 159.30 (B), 154.13 (A),

153.47 (B), 152.45 (B), 152.05 (A), 149.11 (A), 148.99 (B), 147.62

(A), 147.43 (B), 142.21 (A), 142.14 (B), 137.72 (A), 137.32 (B),

133.97 (A), 133.48 (B), 129.62 (A), 129.47 (B), 120.51 (B), 120.48

(A), 120.33 (A), 119.35 (B), 114.82 (B), 113.21 (A), 112.14 (A),

112.01 (B), 111.87 (A), 111.56 (B), 111.50 (B), 111.12 (B), 109.41

(A), 107.31 (B), 105.43 (B), 103.69 (A), 76.65 (A), 75.87 (B), 70.41

(A), 69.91 (B), 67.90 (A), 67.35 (A), 67.08 (B), 66.86 (A), 66.47

(B), 65.49 (B), 60.93 (A), 60.87 (B), 56.39 (B), 56.21 (A), 56.09

(A), 56.05 (B), 56.03 (B), 55.94 (B), 55.01 (B), 52.33 (B), 43.99

(B), 41.60 (A), 41.18 (A), 39.58 (A), 38.31 (A), 37.87 (B), 32.84

(B), 32.44 (A), 31.65 (A), 31.14 (B), 30.83 (B), 30.15 (B), 29.84

(B), 27.34 (A), 27.07 (B), 26.67 (B), 26.39 (A), 26.29 (B), 26.18

(B), 25.98 (A), 25.84 (B), 25.50 (B), 24.31 (A), 21.17 (B), 20.87

(A). HRMS (m/z): (ESI⁺) calculated for C₄₆H₆₂NO₁₀[M + H]⁺:

788.43682, found 788.43647. HPLC (70−100% solvent B, 0.8 mL/

min, 10.50 min): t_R= 6.35 min, purity (220 nm) = 99%.

(S)-(R)-3-(3,4-Dimethoxyphenyl)-1-(3-hydroxyphenyl)-

propyl 1-((S)-2-Cyclohexyl-2-(4-(2-hydroxy-ethoxy)-3,5-

dimethoxyphenyl)acetyl)piperidine-2-carboxylate (47). A sol-

ution of 11 (200 mg, 0.25 mmol, 1.00 equiv), diazabicyclo[2.2.2]-

octane (9 mg, 0.08 mmol, 0.30 equiv) and RhCl(PPh₃)₃(23 mg, 0.03

mmol, 0.1 equiv) in EtOH/H₂O (v/v = 9:1, 15 mL) was heated at

reﬂux. The reaction was assayed by TLC and LC−MS analysis and

was quenched by the addition of 1 M HCl solution (5 mL) at room

temperature when the reaction progress had stopped. After stirring at

room temperature for 2 h, the reaction mixture was extracted with EA

(3 × 20 mL), dried over MgSO₄, ﬁltered, and concentrated under

reduced pressure. The crude product was puriﬁed by ﬂash column

chromatography (CH/EA = 2:1 + 1% MeOH) to yield 47 (85 mg,

47%) as a white solid. TLC (CH/EA = 1:2, v/v): R_f= 0.40. ¹H NMR

(500 MHz, CDCl₃, mixture of rotamers 0.22:1, A/B): δ 7.78 (s,

0.13H, A), 7.40 (s, 0.71H, B), 7.18 (t, J = 7.8 Hz, 0.18H, A), 7.03 (t, J

= 7.9 Hz, 0.81H, B), 6.90−6.81 (m, 0.60H, B), 6.79−6.70 (m, 1.90H,

A+B), 6.69−6.58 (m, 2.16H, A+B), 6.54−6.41 (m, 3.83H, A+B),

5.80 (t, J = 6.6, 14.1 Hz, 0.17H, A), 5.57 (dd, J = 5.7, 8.0 Hz, 0.82H,

B), 5.47−5.42 (m, 0.82H, B), 4.74−4.68 (m, 0.17H, A), 4.57−4.49

(m, 0.17H, A), 4.14−3.99 (m, 2.17H, A+B), 3.95−3.88 (m, 0.92H, A

+B), 3.88−3.76 (m, 7.78H, A+B), 3.76−3.57 (m, 8.43H, A+B), 3.38

(d, J = 9.9 Hz, 0.82H, B), 3.04 (d, J = 9.7 Hz, 0.18H, A), 2.77−2.68

(m, 0.82H, B), 2.63−2.34 (m, 2.78H, A+B), 2.32−2.21 (m, 1.06H, A

+B), 2.15−2.03 (m, 1.48H, A+B), 1.98−1.78 (m, 2.80H, A+B),

1.70−1.48 (m, 6.29H, A+B), 1.46−1.04 (m, 6.77H, A+B), 1.04−0.82

(m, 1.14H, A+B), 0.80−0.55 (m, 1.25H, A+B). ¹³C NMR (126 MHz,

CDCl₃, mixture of rotamers 1:5, A/B): δ 172.79 (A), 172.55 (B),

170.56 (B), 170.36 (A), 157.02 (A), 156.67 (B), 153.37 (A), 153.01

(B), 149.01 (A), 148.89 (B), 147.52 (A), 147.35 (B), 141.53 (A),

141.48 (B), 135.18 (A+B), 134.74 (A), 134.07 (B), 133.58 (B),

133.42 (A), 129.83 (A), 129.60 (B), 120.34 (B), 120.20 (A), 118.55

(B), 118.38 (A), 115.56 (A), 115.33 (B), 113.53 (A), 112.88 (B),

111.93 (B), 111.81 (A), 111.52 (A), 111.41 (B), 106.00 (B), 105.19

(A), 77.00 (A), 75.76 (B), 75.27 (A+B), 61.45 (B), 56.38 (A), 56.22

(B), 56.02 (A), 55.99 (B), 55.93 (A), 55.90 (B), 55.77 (A), 55.19

720.37412. HPLC (50−80% solvent B, 1.5 mL/min, 20 min): t_R

9.73 min, purity (220 nm) = 99%.

=

(S)-(R)-3-(3,4-Dimethoxyphenyl)-1-(3-hydroxyphenyl)-

propyl 1-((S)-2-Cyclohexyl-2-(4-(3-hydroxy propoxy)-3,5-

dimethoxyphenyl)acetyl)piperidine-2-carboxylate (48). A sol-

ution of 12 (100 mg, 0.12 mmol, 1.00 equiv), diazabicyclo[2.2.2]-

octane (6 mg, 0.055 mmol, 0.45 equiv) and RhCl(PPh₃)₃(17 mg,

0.02 mmol, 0.15 equiv) in EtOH/H₂O (v/v = 9:1, 10 mL) was heated

at reﬂux. The reaction was assayed by TLC and LC−MS analysis and

was quenched by the addition of 1 M HCl solution (3 mL) at room

temperature when the reaction progress had stopped. After stirring at

room temperature for 20 min, the reaction mixture was extracted with

EA (3 × 20 mL), dried over MgSO₄, ﬁltered, and concentrated under

reduced pressure. The crude product was puriﬁed by ﬂash column

chromatography (CH/EA = 2:1) to yield 48 (20 mg, 22%) as a white

1

solid. TLC (CH/EA = 1:2, v/v): R_f= 0.32. H NMR (500 MHz,

CDCl₃, mixture of rotamers 0.21:1, A/B): δ 7.21 (t, J = 8.1 Hz,

0.15H, A), 7.06 (t, J = 7.8 Hz, 0.81H, B), 6.88−6.81 (m, 0.50H, A),

6.80−6.71 (m, 1.85H, A+B), 6.71−6.60 (m, 2.00H, A+B), 6.57−6.52

(m, 0.82H, B), 6.49 (s, 1.60H, B), 6.43 (s, 0.29H, A), 6.33 (t, J = 2.1

Hz, 0.79H, B), 5.81 (dd, J = 6.2, 7.6 Hz, 0.13H, A), 5.59 (dd, J = 5.7,

8.1 Hz, 0.79H, B), 5.48−5.43 (m, 0.80H, B), 4.72 (d, J = 5.5 Hz,

0.13H, A), 4.54 (d, J = 13.6 Hz, 0.11H, A), 4.15−4.04 (m, 1.98H, A

+B), 3.95−3.78 (m, 9.91H, A+B), 3.67 (s, 4.90H, A+B), 3.38 (d, J =

9.9 Hz, 0.82H, B), 3.06 (d, J = 9.6 Hz, 0.18H, A), 2.69 (td, J = 3.0,

13.3 Hz, 0.93H, B), 2.63−2.53 (m, 0.44H, A), 2.53−2.34 (m, 1.98H,

A+B), 2.32−2.22 (m, 1.00H, A+B), 2.18−2.02 (m, 1.42H, A+B),

2.00−1.86 (m, 3.86H, A+B), 1.86−1.75 (m, 0.90H, A+B), 1.74−1.49

(m, 6.10H, A+B), 1.47−0.95 (m, 7.18H, A+B), 0.95−0.58 (m, 2.51H,

A+B). ¹³C NMR (126 MHz, CDCl₃, mixture of rotamers 1:6, A/B): δ

172.77 (A), 172.48 (B), 170.76 (B), 170.47 (A), 156.88 (A), 156.65

(B), 153.36 (A), 152.99 (B), 149.14 (A), 149.02 (B), 147.65 (A),

147.48 (B), 141.68 (A), 141.51 (B), 135.98 (B), 134.56 (A), 134.00

(B), 133.66 (B), 133.51 (A), 132.28 (A), 129.94 (A), 129.69 (B),

120.40 (B), 120.28 (A), 119.03 (B), 118.69 (A), 115.65 (A), 115.53

(B), 113.46 (A), 112.77 (B), 112.06 (B), 111.93 (A), 111.62 (A),

111.52 (B), 106.44 (B), 105.37 (A), 75.74 (B), 72.30 (A), 72.20 (B),

61.47 (A), 61.34 (B), 56.41 (A+B), 56.10 (A+B), 56.01 (A+B), 55.91

(A), 55.20 (B), 52.25 (A), 43.84 (B), 41.19 (B), 39.82 (A), 38.04

(A), 37.51 (B), 33.25 (A), 32.91 (B), 32.27 (A+B), 31.64 (A), 31.28

(B), 30.76 (B), 30.66 (A), 26.82 (B), 26.70 (B), 26.58 (A), 26.37

(A), 26.31 (B), 26.25 (B), 25.62 (B), 24.50 (A), 20.90 (B), 20.72

(A). HRMS (m/z): (ESI⁺) calculated for C₄₂H₅₆NO₁₀[M + H]⁺:

734.38987, found 734.38989. HPLC (50−100% solvent B, 1.5 mL/

min, 20 min): t_R= 8.71 min, purity (220 nm) = 96%.

(S)-(R)-3-(3,4-Dimethoxyphenyl)-1-(3-hydroxyphenyl)-

propyl 1-((S)-2-Cyclohexyl-2-(3-(2-hydroxyethoxy)-4,5-

dimethoxyphenyl)acetyl)piperidine-2-carboxylate (49). A sol-

ution of 29 (233 mg, 0.29 mmol, 1.00 equiv), diazabicyclo[2.2.2]-

octane (10 mg, 0.09 mmol, 0.30 equiv), and RhCl(PPh₃)₃(27 mg,

0.03 mmol, 0.10 equiv) in EtOH/H₂O (v/v = 9:1, 30 mL) was heated

at reﬂux. The reaction was assayed by TLC and LC−MS analysis and

was quenched by the addition of 1 M HCl solution (5 mL) at room

temperature when the reaction progress had stopped. After stirring at

room temperature for 6 h, the reaction mixture was extracted with EA

(3 × 20 mL), dried over MgSO₄, ﬁltered, and concentrated under

reduced pressure. The crude product was puriﬁed by ﬂash column

chromatography (CH/EA = 1:2) to yield 49 (79 mg, 38%) as a white

1

solid. TLC (CH/EA = 1:2, v/v): R_f= 0.38. H NMR (500 MHz,

CDCl₃, mixture of rotamers; major rotamer indicated with an A): δ

7.24−7.19 (m, 0.19H), 7.12−7.06 (m, 0.88H, A), 7.00 (br s, 0.51H,

A), 6.88−6.85 (m, 0.24H), 6.84−6.72 (m, 2.21H, A), 6.70−6.54 (m,

4.27H, A), 6.52−6.43 (m, 1.25H, A), 6.23−6.20 (m, 0.74H, A),

5.84−5.77 (m, 0.10H, A), 5.62 (dd, J = 5.9, 8.1 Hz, 0.75H, A), 5.54

(t, J = 6.8 Hz, 0.13H, A), 5.48−5.41 (m, 0.93H, A), 4.75−4.62 (m,

3341

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Article

0.14H), 4.53 (d, J = 13.7 Hz, 0.09H), 4.14−3.97 (m, 2.42H, A),

3.96−3.73 (m, 13.96H, A), 3.66 (s, 2.33H, A), 3.43 (d, J = 9.9 Hz,

0.17H), 3.36 (d, J = 10.1 Hz, 0.78H, A), 3.23−3.01 (m, 0.48H, A),

3.00−2.89 (m, 0.37H), 2.57 (td, J = 3.0, 13.3 Hz, 1.05H, A), 2.49−

2.25 (m, 3.05H, A), 2.19−1.84 (m, 4.21H, A), 1.84−1.74 (m, 1.00H,

A), 1.73−1.52 (m, 6.02H, A), 1.42−1.08 (m, 8.03H, A), 0.94−0.71

(m, 3.01H, A). ¹³C NMR (126 MHz, CDCl₃, mixture of rotamers;

major rotamer indicated with an A): δ 172.80, 172.14 (A), 170.79

(A), 170.40, 156.77, 156.59, 156.40 (A), 153.78, 153.60 (A), 153.18,

152.32, 152.27, 151.80 (A), 149.10, 149.04, 148.98 (A), 147.62,

147.50, 147.44 (A), 141.75, 141.03 (A), 137.90, 137.34 (A), 134.58,

134.49 (A), 133.99, 133.63 (A), 133.59, 133.49, 129.98, 129.79 (A),

120.40 (A), 120.33, 120.28, 119.87 (A), 119.03, 118.51, 115.91,

115.91 (A), 115.75, 113.12, 112.98, 112.69 (A), 112.05 (A), 111.89,

111.57, 111.53, 111.43 (A), 109.16 (A), 109.05, 108.60, 107.15,

106.44 (A), 105.90, 75.92, 75.67 (A), 71.86, 71.76 (A), 71.57, 61.50,

61.46 (A), 61.24 (A), 61.07, 56.44, 56.32 (A), 56.09 (A), 56.03, 55.97

(A), 55.81, 55.04 (A), 54.95, 52.51, 52.17 (A), 44.15, 43.86 (A),

41.39, 41.22, 41.02 (A), 39.77, 38.07, 37.90, 36.90 (A), 33.14, 32.93,

32.82 (A), 31.66, 31.58, 31.42, 31.33 (A), 30.79, 30.73 (A), 30.55,

29.84, 26.80 (A), 26.69 (A), 26.54, 26.30 (A), 26.21 (A), 25.76, 25.66

(A), 24.45, 21.12, 20.95 (A), 20.71. HRMS (m/z): (ESI⁺) calculated

for C₄₁H₅₄NO₁₀[M + H]⁺: 720.3742, found 720.3742. HPLC (50−

100% solvent B, 0.8 mL/min, 10.50 min): t_R= 4.91 min, purity (220

nm) = 98%.

106.98, 106.64, 106.52 (A), 105.37, 75.71 (A), 67.85, 67.53 (A),

61.00, 60.96 (A), 60.80, 60.54, 60.42 (A), 56.42, 56.37 (A), 56.32,

56.05 (A), 55.98 (A), 55.94 (A), 55.84, 55.06 (A), 54.99, 52.64, 52.21

(A), 44.28, 43.83 (A), 41.49, 41.17 (A), 39.81, 38.26, 38.08, 37.47

(A), 33.15, 32.84 (A), 32.72, 32.10, 32.03 (A), 31.62, 31.29 (A),

30.71 (A), 30.53, 26.80 (A), 26.65 (A), 26.52, 26.26 (A), 26.21 (A),

25.71, 25.57 (A), 24.43, 21.16, 20.89 (A), 20.68. HRMS (m/z):

(ESI⁺) calculated for C₄₂H₅₆NO₁₀[M + H]⁺: 734.38987, found

734.39029. HPLC (50−100% solvent B, 0.8 mL/min, 10.50 min): t_R

= 5.09 min, purity (220 nm) = 96%.

(S)-(R)-3-(3,4-Dimethoxyphenyl)-1-(3-hydroxyphenyl)-

propyl 1-((S)-2-Cyclohexyl-2-(4-(2-iodoethoxy)-3,5-

dimethoxyphenyl)acetyl)piperidine-2-carboxylate (51). The

substrate 47 (76 mg, 0.11 mmol, 1.00 equiv) was applied to general

procedure C with PPh₃(36 mg, 0.14 mmol, 1.30 equiv), imidazole

(15 mg, 0.21 mmol, 2.00 equiv), and iodine (38 mg, 0.15 mmol, 1.40

equiv) in DCM (3 mL). Additional portions of PPh₃(1.30 + 0.50

equiv), imidazole (2.00 equiv) and iodine (1.50 + 0.50 equiv) were

added until completion of the reaction. 51 (41 mg, 47%) was

obtained after puriﬁcation by ﬂash column chromatography (CH/EA

= 3:1) as light yellow solid. TLC (CH/EA = 2:1, v/v): R_f= 0.36. ¹H

NMR (500 MHz, CDCl₃, mixture of rotamers 0.2:1, A:B): δ 7.23 (t, J

= 7.8 Hz, 0.19H, A), 7.07 (t, J = 7.9 Hz, 0.77H, B), 6.91−6.87 (m,

0.18H, A), 6.86−6.81 (m, 0.36H, A), 6.81−6.73 (m, 1.86H, A+B),

6.70−6.68 (m, 0.20H, A), 6.67−6.61 (m, 1.85H, A+B), 6.59−6.53

(m, 0.9H, A+B), 6.52 (s, 1.59H, B), 6.43 (s, 0.29H, A), 6.29−6.27

(m, 0.07H, A), 6.23 (t, J = 2.0 Hz, 0.79H, B), 5.81 (dd, J = 6.2, 7.7

Hz, 0.17H, A), 5.61 (dd, J = 6.0, 7.9 Hz, 0.83H, B), 5.47 (q, J = 2.0

Hz, 0.84H, B), 4.72 (d, J = 5.6 Hz, 0.14H, A), 4.54 (d, J = 13.7 Hz,

0.16H, A), 4.23−4.11 (m, 2.01H, A+B), 3.97−3.89 (m, 0.98H, A+B),

3.88−3.79 (m, 6.93H, A+B), 3.68 (s, 4.79H, A+B), 3.50 (s, 0.11H,

A), 3.42−3.27 (m, 2.81H, A+B), 3.06 (d, J = 9.6 Hz, 0.14H, A), 2.66

(td, J = 2.9, 13.3 Hz, 0.86H, B), 2.63−2.52 (m, 0.39H, A), 2.52−2.33

(m, 1.91H, A+B), 2.32−2.23 (m, 1.05H, A+B), 2.19−2.03 (m, 1.58H,

A+B), 1.96−1.83 (m, 2.10H, A+B), 1.83−1.49 (m, 7.28H, A+B),

1.47−1.07 (m, 7.28H, A+B), 1.05−0.82 (m, 1.38H, A+B), 0.82−0.57

(m, 1.21H, A+B). ¹³C NMR (126 MHz, CDCl₃, mixture of rotamers

1:8, A/B): δ 172.73 (A), 172.32 (B), 170.74 (B), 170.48 (A), 156.52

(A), 156.43 (B), 153.46 (A), 153.08 (B), 149.15 (A), 149.02 (B),

147.48 (B), 141.79 (A), 141.45 (B), 135.38 (B), 135.30 (A), 134.92

(A), 134.58 (B), 133.64 (B), 133.48 (A), 130.01 (A), 129.72 (B),

120.44 (B), 120.28 (A), 119.38 (B), 118.94 (A), 115.64 (A+B),

113.47 (A), 112.58 (B), 112.06 (B), 111.93 (A), 111.63 (A), 111.50

(B), 106.60 (B), 105.39 (A), 75.74 (B), 73.84 (B), 73.75 (A), 56.57

(B), 56.51 (A), 56.12 (B), 56.04 (B), 55.15 (B), 52.23 (B), 43.89

(B), 41.21 (B), 39.84 (A), 38.06 (A), 37.55 (B), 33.28 (A), 32.88

(B), 31.65 (A), 31.28 (B), 30.77 (B), 30.69 (A), 26.78 (B), 26.71 (A

+B), 26.60 (A), 26.37 (A), 26.31 (B), 26.25 (B), 25.66 (B), 24.51

(A), 20.91 (B), 20.72 (A), 2.51 (A), 2.36 (B). HRMS (m/z): (ESI⁺)

calculated for C₄₁H₅₃INO₉[M + H]⁺: 830.27598, found 830.27529.

HPLC (50−80% solvent B, 1.5 mL/min, 20 min): t_R= 16.75 min,

purity (220 nm) = 98%.

(S)-(R)-3-(3,4-Dimethoxyphenyl)-1-(3-hydroxyphenyl)-

propyl 1-((S)-2-Cyclohexyl-2-(3-(3-hydroxypropoxy)-4,5-

dimethoxyphenyl)acetyl)piperidine-2-carboxylate (50). A sol-

ution of 30 (257 mg, 0.32 mmol, 1.00 equiv), diazabicyclo[2.2.2]-

octane (11 mg, 0.10 mmol, 0.30 equiv), and RhCl(PPh₃)₃(29 mg,

0.03 mmol, 0.10 equiv) in EtOH/H₂O (v/v = 9:1, 30 mL) was heated

at reﬂux. The reaction was assayed by TLC and LC−MS analysis and

was quenched by the addition of 1 M HCl solution (5 mL) at room

temperature when the reaction progress had stopped. After stirring at

room temperature for 2 h, the reaction mixture was extracted with EA

(3 × 20 mL), dried over MgSO₄, ﬁltered, and concentrated under

reduced pressure. The crude product was puriﬁed by semipreparative

HPLC (50−100% solvent B) and ﬂash column chromatography

(CH/EA = 1:1) to yield 50 (36 mg, 16%) as a white solid. Note: The

poor yield can partly be explained by the esteriﬁcation of one of the

hydroxy groups with TFA after the puriﬁcation by semipreparative

HPLC (the solvents H₂O and MeCN contain 0.1% TFA each). To us,

it is unclear why this happened, since the product fraction was freeze-

dried immediately after the puriﬁcation process (no standing in

solution overnight and no solvent removal on a rotary evaporator at

elevated temperatures in a water bath!). TLC (CH/EA = 1:2, v/v): R_f

1

= 0.36. H NMR (500 MHz, CDCl₃, mixture of rotamers; major

rotamer indicated with an A): δ 7.30 (br s, 0.52H, A), 7.19 (t, J = 7.8

Hz, 0.15H), 7.06 (t, J = 7.9 Hz, 0.84H, A), 6.87−6.81 (m, 0.47H),

6.79−6.70 (m, 2.06H, A), 6.69−6.61 (m, 2.27H, A), 6.60−6.47 (m,

2.99H, A), 6.45−6.41 (m, 0.33H), 6.32−6.30 (m, 0.73H, A), 5.82

(dd, J = 6.0, 7.7 Hz, 0.13H), 5.60 (dd, J = 5.6, 8.1 Hz, 0.74H, A), 5.52

(dd, J = 4.8, 8.6 Hz, 0.13H), 5.47−5.43 (m, 0.90H, A), 4.74−4.70 (m,

0.12H), 4.52 (d, J = 13.5 Hz, 0.12H), 4.17−4.13 (m, 0.38H), 4.11−

4.00 (m, 1.69H, A), 3.95−3.88 (m, 1.07H, A), 3.87−3.74 (m, 12.73H,

A), 3.73−3.64 (m, 2.83H, A), 3.44 (d, J = 10.0 Hz, 0.15H), 3.37 (d, J

= 10.0 Hz, 0.74H, A), 3.10−2.92 (m, 1.01H, A), 2.68 (td, J = 2.9, 13.3

Hz, 0.77H, A), 2.60−2.53 (m, 0.42H), 2.50−2.34 (m, 1.63H, A),

2.31−2.22 (m, 1.47H, A), 2.13−2.03 (m, 1.47H, A), 1.97−1.86 (m,

3.40H, A), 1.85−1.78 (m, 0.77H, A), 1.72−1.53 (m, 6.00H, A),

1.45−1.36 (m, 0.76H, A), 1.34−1.07 (m, 4.95H, A), 0.93−0.84 (m,

0.93H, A), 0.79−0.71 (m, 0.90H, A). ¹³C NMR (126 MHz, CDCl₃,

mixture of rotamers; major rotamer indicated with an A): δ 173.03,

172.97, 172.54 (A), 170.71 (A), 170.44, 170.28, 157.06, 156.88,

156.62 (A), 153.63, 153.28 (A), 153.11, 152.65, 152.45, 152.20 (A),

149.03, 148.98, 148.93 (A), 147.55, 147.44, 147.38 (A), 142.07,

141.63 (A), 141.36, 137.50, 137.44, 137.29 (A), 134.29, 133.90 (A),

133.63 (A), 133.47, 129.90, 129.69 (A), 120.37 (A), 120.24, 118.90

(A), 118.78, 117.84, 115.74, 115.53 (A), 115.17, 112.98, 112.74 (A),

112.32, 111.96 (A), 111.83, 111.50, 111.42 (A), 107.97 (A), 107.31,

(S)-(R)-3-(3,4-Dimethoxyphenyl)-1-(3-hydroxyphenyl)-

propyl 1-((S)-2-Cyclohexyl-2-(4-(3-iodoprop-oxy)-3,5-

dimethoxyphenyl)acetyl)piperidine-2-carboxylate (52). The

substrate 48 (41 mg, 0.06 mmol, 1.00 equiv) was applied to general

procedure C with PPh₃(28 mg, 0.11 mmol, 1.90 equiv), pyridine (13

μL, 0.17 mmol, 3.00 equiv), and iodine (23 mg, 0.09 mmol, 1.60

equiv) in toluene (3 mL). Additional portions of PPh₃(1.5 + 0.7

equiv) and iodine (1.00 + 0.50 equiv) were added until completion of

the reaction. 52 (18 mg, 38%) was obtained as a light yellow solid

after puriﬁcation by ﬂash column chromatography (CH/EA = 3:1).

1

TLC (DCM/MeOH = 50:1, v/v): R_f= 0.21. H NMR (500 MHz,

CDCl₃, mixture of rotamers 0.16:1, A/B): δ 7.22 (t, J = 7.8 Hz,

0.16H, A), 7.07 (t, J = 7.8 Hz, 0.83H, B), 6.88 (d, J = 7.7 Hz, 0.15H,

A), 6.86−6.81 (m, 0.32H, A), 6.80−6.72 (m, 1.87H, A+B), 6.71−

6.67 (m, 0.19H, A), 6.67−6.62 (m, 1.87H, A+B), 6.57−6.51 (m,

2.51H, A+B), 6.42 (s, 0.31H, A), 6.31 (s, 0.76H, B), 6.17 (t, J = 1.9

Hz, 0.81H, B), 5.81 (dd, J = 6.1, 7.7 Hz, 0.14H, A), 5.61 (dd, J = 6.0,

8.0 Hz, 0.85H, B), 5.49−5.44 (m, 0.83H, B), 4.73 (d, J = 5.5 Hz,

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0.14H, A), 4.55 (d, J = 13.6 Hz, 0.14H, A), 4.03−3.96 (m, 2.03H, A

+B), 3.96−3.89 (m, 0.93H, A+B), 3.89−3.80 (m, 7.03H, A+B), 3.69

(s, 5.09H, A+B), 3.48 (t, J = 6.8 Hz, 0.32H, A), 3.43 (t, J = 6.8 Hz,

1.71H, B), 3.37 (d, J = 10.1 Hz, 0.86H, B), 3.07 (d, J = 9.6 Hz, 0.14H,

A), 2.65−2.55 (m, 1.10H, A+B), 2.51−2.42 (m, 0.99H, A+B), 2.41−

2.32 (m, 0.89H, A+B), 2.32−2.25 (m, 1.05H, A+B), 2.24−2.02 (m,

3.46H, A+B), 1.96−1.48 (m, 9.94H, A+B), 1.45−0.97 (m, 6.81H, A

+B), 0.95−0.83 (m, 0.95H, A+B), 0.82−0.70 (m, 1.02H, A+B),

0.69−0.58 (m, 0.18H, A). ¹³C NMR (126 MHz, CDCl₃, mixture of

rotamers 1:4, A/B): δ 172.76 (A), 172.23 (B), 170.78 (B), 170.50

(A), 156.51 (A+B), 153.63 (A), 153.30 (B), 149.15 (A), 149.00 (B),

147.47 (B), 141.78 (A), 141.32 (B), 135.90 (B), 134.35 (B), 133.65

(B), 133.49 (A), 130.00 (A), 129.70 (B), 120.44 (B), 120.28 (A),

119.63 (B), 118.88 (A), 115.72 (B), 115.60 (A), 113.47 (A), 112.60

(B), 112.08 (B), 111.92 (A), 111.63 (A), 111.49 (B), 106.69 (B),

105.45 (A), 75.73 (B), 72.92 (B), 72.74 (A), 56.61 (B), 56.51 (A),

56.13 (B), 56.03 (B), 55.94 (A), 55.15 (B), 52.19 (B), 43.89 (B),

41.15 (B), 39.82 (A), 38.08, 37.36 (B), 34.49 (A), 34.42 (B), 33.32

(A), 32.87 (B), 31.64 (A), 31.29 (B), 30.75 (B), 30.70 (A), 26.77

(B), 26.72 (B), 26.66 (A), 26.62 (A), 26.38 (A), 26.32 (B), 26.24

(B), 25.67 (B), 24.52 (A), 20.93 (B), 20.73 (A), 3.59 (A), 3.36 (B).

HRMS (m/z): (ESI⁺) calculated for C₄₂H₅₅NIO₉[M + H]⁺:

844.2916, found 844.2906. HPLC (60−100% solvent B, 1.5 mL/

min, 20 min): t_R= 7.46 min, purity (220 nm) = 98%.

(S)-(R)-3-(3,4-Dimethoxyphenyl)-1-(3-hydroxyphenyl)-

propyl 1-((S)-2-Cyclohexyl-2-(3-(2-iodoethoxy)-4,5-

dimethoxyphenyl)acetyl)piperidine-2-carboxylate (53). The

substrate 49 (88 mg, 0.12 mmol, 1.00 equiv) was applied to general

procedure C with PPh₃(42 mg, 0.16 mmol, 1.30 equiv), imidazole

(17 mg, 0.24 mmol, 2.00 equiv) and iodine (44 mg, 0.17 mmol, 1.40

equiv) in DCM (5 mL). Additional portions of PPh₃(0.50 equiv),

imidazole (0.50 equiv), and iodine (0.50 equiv) were added until

completion of the reaction. 53 (74 mg, 73%) was obtained as white

solid after puriﬁcation by semipreparative HPLC (70−100% solvent

B). TLC (DCM/MeOH = 20:1, v/v): R_f= 0.45. ¹H NMR (500 MHz,

CDCl₃, mixture of rotamers; major rotamer indicated with an A): δ

7.24−7.20 (m, 0.21H), 7.13−7.05 (m, 0.90H, A), 6.89−6.83 (m,

0.51H), 6.80−6.73 (m, 1.83H, A), 6.71−6.60 (m, 1.92H, A), 6.57−

6.47 (m, 2.78H, A), 6.43−6.41 (m, 0.13H, A), 6.30−6.25 (m, 0.66H,

A), 5.82 (t, J = 7.0 Hz, 0.13H), 5.63−5.56 (m, 0.86H, A), 5.49−5.44

(m, 0.85H, A), 4.78−4.73 (m, 0.15H), 4.52 (d, J = 13.6 Hz, 0.13H),

4.30−4.23 (m, 0.35H), 4.19−4.05 (m, 1.73H, A), 3.99−3.90 (m,

0.73H, A), 3.89−3.75 (m, 10.85H, A), 3.70−3.65 (m, 2.20H, A),

3.46−3.36 (m, 1.26H, A), 3.34−3.25 (m, 1.75H, A), 3.11 (d, J = 9.6

Hz, 0.04H), 3.06 (d, J = 9.9 Hz, 0.13H), 2.98 (td, J = 2.6, 13.4 Hz,

0.14H), 2.70−2.50 (m, 1.19H, A), 2.48−2.23 (m, 2.75H, A), 2.14−

2.02 (m, 1.10H, A), 1.93−1.84 (m, 1.38H, A), 1.82−1.52 (m, 7.08H,

A), 1.47−1.09 (m, 5.95H, A), 0.95−0.85 (m, 0.85H, A), 0.81−0.59

(m, 1.14H, A). ¹³C NMR (126 MHz, CDCl₃, mixture of rotamers;

major rotamer indicated with an A): δ 173.47, 173.41, 172.96 (A),

170.32 (A), 170.14, 170.02, 158.82, 158.50, 156.52, 156.45 (A),

156.45, 153.86, 153.50 (A), 151.70, 151.38 (A), 149.03, 148.91 (A),

147.55, 147.37 (A), 141.99, 141.51, 141.23 (A), 137.81 (A), 133.86,

133.62 (A), 133.52 (A), 133.39, 133.26, 130.00, 129.77 (A), 120.37

(A), 120.32, 120.25, 119.28 (A), 115.73, 115.63 (A), 115.22, 113.36,

112.60 (A), 112.46, 111.92 (A), 111.82, 111.48, 111.42 (A), 108.82

(A), 108.26, 108.03, 107.25 (A), 106.82, 106.02, 75.87 (A), 70.26

(A), 70.19, 69.99, 61.33 (A), 61.18, 56.49 (A), 56.34, 56.22, 56.05

(A), 56.00, 55.96, 55.93 (A), 55.70, 54.99 (A), 52.79, 52.47 (A),

44.32, 44.11 (A), 41.26, 41.12, 40.91 (A), 38.17, 38.01, 37.33 (A),

33.13, 32.77 (A), 31.63, 31.31, 31.22 (A), 30.69 (A), 30.56, 26.70

(A), 26.65, 26.57 (A), 26.44, 26.24, 26.18 (A), 26.05 (A), 25.64,

25.52 (A), 20.99, 20.76 (A), 20.51, 1.70, 1.62, 1.55 (A). HRMS (m/

z): (ESI⁺) calculated for C₄₁H₅₃INO₉[M + H]⁺: 830.27596, found

830.27584. HPLC (70−100% solvent B, 0.8 mL/min, 10.50 min): t_R

= 4.45 min, purity (220 nm) = 98%.

procedure C with PPh₃(19 mg, 0.07 mmol, 1.50 equiv), imidazole (8

mg, 0.12 mmol, 2.50 equiv), and iodine (22 mg, 0.09 mmol, 1.80

equiv) in DCM (5 mL). 54 (33 mg, 85%) was obtained as a light

yellow solid after puriﬁcation by ﬂash column chromatography (CH/

EA = 2:1). TLC (CH/EA = 2:1, v/v): R_f= 0.33. ¹H NMR (500 MHz,

THF-d₈, mixture of rotamers; major rotamer indicated with an A): δ

8.39 (s, 0.18H), 8.33 (s, 0.03H), 8.23 (s, 0.10H), 8.16 (s, 0.54H, A),

7.15 (t, J = 7.8 Hz, 0.23H), 7.05 (t, J = 7.8 Hz, 0.11H), 6.99 (t, J = 7.8

Hz, 0.63H, A), 6.85−6.75 (m, 1.54H, A), 6.73−6.65 (m, 1.54H, A),

6.64−6.55 (m, 3.65H, A), 6.53−6.51 (m, 0.65H, A), 6.32−6.26 (m,

0.64H, A), 5.79 (dd, J = 5.9, 7.7 Hz, 0.24H, A), 5.54 (dd, J = 5.2, 8.3

Hz, 0.76H, A), 5.43−5.40 (m, 0.63H, A), 5.35 (d, J = 4.8 Hz, 0.12H),

4.89−4.85 (m, 0.21H), 4.53 (d, J = 13.5 Hz, 0.20H), 4.08 (d, J = 13.5

Hz, 0.71H, A), 4.04−3.98 (m, 0.50H), 3.96−3.83 (m, 1.52H, A),

3.78−3.72 (m, 7.03H, A), 3.70 (s, 1.11H, A), 3.66−3.61 (m, 3.98H,

A), 3.51 (d, J = 9.9 Hz, 0.80H, A), 3.41 (td, J = 1.2, 6.8 Hz, 0.49H),

3.36−3.25 (m, 1.55H, A), 3.14 (d, J = 9.8 Hz, 0.20H), 2.90 (dd, J =

11.7, 14.4 Hz, 0.11H), 2.77 (td, J = 2.8, 13.3 Hz, 0.63H, A), 2.59−

2.32 (m, 2.00H, A), 2.29−2.20 (m, 1.60H, A), 2.17−2.02 (m, 3.04H,

A), 1.95−1.85 (m, 1.98H, A), 1.84−1.76 (m, 0.55H), 1.70−1.54 (m,

5.16H, A), 1.51−1.06 (m, 7.20H, A), 0.97−0.77 (m, 1.65H, A),

0.74−0.64 (m, 0.35H). ¹³C NMR (126 MHz, THF-d₈, mixture of

rotamers; major rotamer indicated with an A): δ 173.23, 172.85 (A),

172.61, 171.26, 171.17 (A), 171.05, 159.05, 158.60 (A), 158.49,

154.91, 154.68 (A), 154.59, 153.63, 153.46 (A), 153.40, 150.92,

150.85 (A), 149.38, 149.18 (A), 143.69, 143.21 (A), 143.00, 139.18,

139.11 (A), 139.07, 135.68, 134.94 (A), 134.86, 134.74 (A), 134.54,

130.37, 130.30 (A), 130.15, 121.22 (A), 121.11, 118.58, 117.97 (A),

117.76, 116.02, 115.42 (A), 115.33, 114.69, 114.56 (A), 114.47,

114.09, 113.93 (A), 113.85, 113.77, 113.39, 113.33 (A), 109.14 (A),

108.61, 107.78, 107.72 (A), 107.00, 77.55, 76.36 (A), 76.11, 69.81,

69.71 (A), 60.77, 60.71 (A), 60.66, 56.84, 56.56, 56.41 (A), 56.38

(A), 56.34, 56.27 (A), 55.92, 55.71, 55.50 (A), 53.15, 52.95 (A),

44.44 (A), 42.67, 42.57 (A), 42.50, 40.16, 39.42, 39.34 (A), 39.09,

34.92, 34.86 (A), 34.72, 33.84, 33.64 (A), 32.50, 32.22, 32.10 (A),

31.58 (A), 31.43, 27.79 (A), 27.72 (A), 27.60, 27.50, 27.43 (A), 27.37

(A), 27.32, 26.74 (A), 26.64, 25.98, 22.15 (A), 22.04, 21.99, 3.03,

2.98 (A), 2.93. HRMS (m/z): (ESI⁺) calculated for C₄₂H₅₅INO₉[M

+ H]⁺: 844.29161, found 844.29172. HPLC (70−100% solvent B, 0.8

mL/min, 10.50 min): t_R= 4.94 min, purity (220 nm) = 96%.

Macrocycle 55. The substrate 51 (41 mg, 0.05 mmol, 1.00 equiv)

was applied to general procedure D with K₂CO₃(14 mg, 0.10 mmol,

2.00 equiv) in MeCN (c = 1 mM, 50 mL). 55 (19 mg, 54%) was

obtained as a white solid after puriﬁcation by ﬂash column

chromatography (CH/EA = 5:1). TLC (CH/EA = 2:1, v/v): R_f=

0.42. ¹H NMR (500 MHz, CDCl₃): δ 7.17 (t, J = 7.9 Hz, 1H), 6.78−

6.69 (m, 5H), 6.66 (dd, J = 2.0, 8.2 Hz, 1H), 6.33 (d, J = 1.8 Hz, 1H),

6.04−5.98 (m, 1H), 5.89 (t, J = 6.1 Hz, 1H), 5.58 (d, J = 5.0 Hz, 1H),

4.47−4.35 (m, 3H), 4.22−4.15 (m, 1H), 4.04−3.97 (m, 1H), 3.88 (s,

3H), 3.85 (s, 3H), 3.84 (s, 3H), 3.56 (s, 3H), 3.29 (d, J = 10.0 Hz,

1H), 2.69−2.61 (m, 1H), 2.53−2.41 (m, 2H), 2.38−2.32 (m, 1H),

2.22−2.01 (m, 2H), 2.01−1.89 (m, 2H), 1.76−1.52 (m, 6H), 1.48−

1.28 (m, 4H), 1.15 (td, J = 9.3, 12.3 Hz, 2H), 0.94−0.71 (m, 2H). ¹³C

NMR (126 MHz, CDCl₃): δ 171.6, 171.0, 159.0, 154.1, 152.7, 149.0,

147.4, 141.6, 136.6, 134.0, 133.7, 129.2, 120.3, 119.8, 115.6, 113.8,

112.0, 111.4, 107.8, 103.4, 75.4, 71.7, 69.4, 56.5, 56.1, 56.1, 56.0, 52.7,

44.1, 41.5, 39.6, 33.3, 30.8, 30.7, 26.8, 26.7, 26.4, 26.4, 25.8, 21.2.

HRMS (m/z): (ESI⁺) calculated for C₄₁H₅₂NO₉[M + H]⁺:

702.36366, found 702.36395. HPLC (70−10% solvent B, 0.8 mL/

min, 10.50 min): t_R= 4.81 min, purity (220 nm) = 98%.

Macrocycle 56. The substrate 52 (16 mg, 0.02 mmol, 1.00 equiv)

was applied to general procedure D with K₂CO₃(5 mg, 0.04 mmol,

2.00 equiv) in MeCN (c = 0.5 mM, 40 mL). 56 (11 mg, 81%) was

obtained after puriﬁcation by ﬂash column chromatography (CH/EA

= 4:1) as a white solid. TLC (CH/EA = 2:1, v/v): R_f= 0.43. ¹H NMR

(500 MHz, CDCl₃): δ 7.14 (t, J = 7.8 Hz, 1H), 6.78−6.66 (m, 6H),

6.38−6.34 (m, 1H), 6.25−6.21 (m, 1H), 5.76 (t, J = 6.9 Hz, 1H),

5.51 (d, J = 5.4 Hz, 1H), 4.44−4.30 (m, 2H), 3.94−3.83 (m, 9H),

3.77 (s, 3H), 3.68 (s, 3H), 3.25 (d, J = 9.8 Hz, 1H), 2.59−2.43 (m,

3H), 2.31 (d, J = 13.5 Hz, 1H), 2.24−2.17 (m, 1H), 2.13−2.07 (m,

(S)-(R)-3-(3,4-Dimethoxyphenyl)-1-(3-hydroxyphenyl)-

propyl 1-((S)-2-Cyclohexyl-2-(3-(3-iodopropoxy)-4,5-

dimethoxyphenyl)acetyl)piperidine-2-carboxylate (54). The

substrate 50 (34 mg, 0.05 mmol, 1.00 equiv) was applied to general

3343

https://dx.doi.org/10.1021/acs.jmedchem.0c02195

J. Med. Chem. 2021, 64, 3320−3349

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

3H), 2.01−1.94 (m, 1H), 1.88 (d, J = 12.4 Hz, 1H), 1.68−1.56 (m,

6H), 1.42−1.28 (m, 4H), 1.18−1.09 (m, 2H), 0.91−0.84 (m, 1H),

0.77−0.70 (m, 1H). ¹³C NMR (126 MHz, CDCl₃): δ 172.0, 171.2,

159.3, 153.4, 152.3, 149.0, 147.4, 140.9, 136.8, 133.9, 133.5, 129.2,

120.8, 120.3, 115.8, 112.6, 112.0, 111.4, 107.7, 104.5, 76.3, 69.7, 65.3,

56.7, 56.3, 56.1, 56.0, 55.5, 52.5, 43.9, 41.6, 37.6, 33.2, 31.3, 30.8,

30.4, 27.0, 26.7, 26.4, 26.4, 25.8, 21.1. HRMS (m/z): (ESI⁺)

calculated for C₄₂H₅₄NO₉[M + H]⁺: 716.37931, found 716.38054.

HPLC (70−80% solvent B, 1.5 mL/min, 20 min): t_R= 9.56 min,

purity (220 nm) = 99%.

1.71−1.61 (m, 6H), 1.49−1.41 (m, 1H), 1.38−1.29 (m, 3H), 1.17−

1.09 (m, 2H), 0.94−0.86 (m, 1H), 0.78−0.70 (m, 1H). ¹³C NMR

(126 MHz, CDCl₃): δ 207.8, 172.5, 171.0, 158.1, 154.1, 153.0, 149.1,

147.5, 142.3, 134.4, 133.9, 133.7, 129.9, 120.6, 120.4, 113.9, 113.7,

112.0, 111.5, 107.7, 104.5, 76.0, 73.2, 67.2, 56.5, 56.2, 56.1, 56.0, 55.3,

52.3, 43.8, 41.5, 39.4, 38.2, 32.9, 31.3, 30.8, 26.9, 26.7, 26.4, 25.7,

21.0. HRMS (m/z): (ESI⁺) calculated for C₄₃H₅₄NO₁₀[M + H]⁺:

744.37422, found 744.37440. HPLC (60−70% solvent B, 1.5 mL/

min, 20 min): t_R= 13.33 min, purity (220 nm) = 99%.

Macrocycle 60. The substrate 35-(E) (32 mg, 0.04 mmol, 1.00

equiv) was applied to general procedure H with Pd(OAc)₂(1 mg,

0.004 mmol, 0.1 equiv), 1,4-benzoquinone (6 mg, 0.05 mmol, 1.20

equiv), and aqueous HBF₄(48 wt %, 8 μL, 0.06 mmol, 1.40 equiv) in

MeCN/H₂O (v/v = 7:1, 3 mL). Additional portions of Pd(OAc)₂(3

× 0.1 equiv), 1,4-benzoquinone (2 × 1.20 equiv), and aqueous HBF₄

(4 × 3.00 equiv) were added until completion of the reaction. 60 (15

mg, 45%, white solid) was obtained after puriﬁcation by semi-

preparative HPLC (70−100% solvent B) as single product isomer.

TLC (CH/EA = 1:1, v/v): R_f= 0.63. ¹H NMR (500 MHz, CDCl₃): δ

7.19 (t, J = 7.9 Hz, 1H), 6.83−6.80 (m, 1H), 6.78−6.75 (m, 1H),

6.74−6.70 (m, 1H), 6.68 (d, J = 1.8 Hz, 1H), 6.64−6.61 (m, 2H),

6.45 (d, J = 1.8 Hz, 1H), 6.13−6.09 (m, 1H), 5.57 (dd, J = 5.9, 8.2

Hz, 1H), 5.52 (d, J = 5.5 Hz, 1H), 4.57−4.42 (m, 4H), 4.08 (d, J =

13.8 Hz, 1H), 3.86−3.84 (m, 6H), 3.67 (s, 3H), 3.63 (s, 3H), 3.43 (d,

J = 10.2 Hz, 1H), 3.41−3.33 (m, 1H), 2.83−2.67 (m, 2H), 2.52−2.44

(m, 1H), 2.41−2.33 (m, 1H), 2.30 (d, J = 14.0 Hz, 1H), 2.15−2.03

(m, 1H), 1.96−1.74 (m, 3H), 1.73−1.59 (m, 6H), 1.49−1.40 (m,

1H), 1.37−1.22 (m, 3H), 1.20−1.08 (m, 2H), 0.94−0.83 (m, 1H),

0.80−0.70 (m, 1H). ¹³C NMR (126 MHz, CDCl₃): δ 207.6, 173.1,

170.1, 157.9, 154.0, 151.3, 149.0, 147.4, 142.3, 137.7, 133.3, 133.3,

129.8, 121.0, 120.5, 113.4, 113.0, 111.9, 111.5, 107.9, 105.4, 75.6,

73.5, 65.0, 60.8, 56.2, 56.1, 55.9, 55.0, 52.4, 44.3, 41.2, 38.6, 37.5,

32.8, 31.3, 30.7, 26.6, 26.5, 26.2, 26.0, 25.8, 20.8. HRMS (m/z):

(ESI⁺) calculated for C₄₃H₅₃NO₁₀Na [M + Na]⁺: 766.35617, found

766.35619. HPLC (70−100% solvent B, 0.8 mL/min, 10.5 min): t_R=

3.96 min, purity (220 nm) = 97%.

Macrocycle 57. The substrate 53 (49 mg, 0.06 mmol, 1.00 equiv)

was applied to general procedure D with K₂CO₃(16 mg, 0.12 mmol,

2.00 equiv) in MeCN (c = 1 mM, 60 mL). 57 (17 mg, 42%) was

obtained as a white solid after puriﬁcation by semipreparative HPLC

1

(85−100% Solvent B). TLC (CH/EA = 2:1, v/v): R_f= 0.38. H

NMR (500 MHz, CDCl₃): δ 7.14 (t, J = 7.9 Hz, 1H), 6.78−6.74 (m,

3H), 6.72−6.69 (m, 1H), 6.65−6.61 (m, 3H), 6.28−6.24 (m, 1H),

5.64 (dd, J = 5.5, 7.8 Hz, 1H), 5.58 (d, J = 5.5 Hz, 1H), 4.51−4.47

(m, 2H), 4.45−4.40 (m, 1H), 4.27 (dt, J = 4.3, 11.6 Hz, 1H), 4.11 (d,

J = 13.7 Hz, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.68 (s, 3H), 3.64 (s,

3H), 3.44 (d, J = 10.3 Hz, 1H), 2.89−2.82 (m, 1H), 2.44−2.38 (m,

2H), 2.26−2.13 (m, 2H), 1.90−1.78 (m, 3H), 1.72−1.58 (m, 6H),

1.47−1.30 (m, 4H), 1.21−1.10 (m, 2H), 0.92−0.85 (m, 1H), 0.79−

0.71 (m, 1H). ¹³C NMR (126 MHz, CDCl₃): δ 172.4, 170.8, 159.1,

153.9, 151.3, 149.0, 147.4, 142.0, 138.4, 133.6, 129.4, 120.4, 119.9,

115.8, 113.9, 111.9, 111.4, 111.1, 105.5, 75.1, 68.5, 67.4, 60.7, 56.1,

56.0, 54.4, 51.8, 44.1, 41.6, 37.9, 32.7, 31.0, 30.8, 26.7, 26.4, 26.2,

26.2, 26.0, 20.8. HRMS (m/z): (ESI⁺) calculated for C₄₁H₅₂NO₉[M

+ H]⁺: 702.36366, found 702.36404. HPLC (70−100% solvent B, 0.8

mL/min, 10.50 min): t_R= 4.54 min, purity (220 nm) = 99%.

Macrocycle 58. The substrate 54 (32 mg, 0.04 mmol, 1.00 equiv)

was applied to general procedure D with K₂CO₃(11 mg, 0.08 mmol,

2.00 equiv) in MeCN (c = 0.3 mM, 125 mL). 58 (7 mg, 26%) was

obtained as a white solid after puriﬁcation by semipreparative HPLC

(85−90% solvent B). TLC (CH/EA = 2:1, v/v): R_f= 0.35. ¹H NMR

(500 MHz, CDCl₃, mixture of rotamers 0.1:1. Only major rotamer is

reported here: δ 7.16−7.12 (m, 1H), 6.82−6.79 (m, 1H), 6.78−6.75

(m, 2H), 6.71 (d, J = 1.9 Hz, 1H), 6.63−6.61 (m, 2H), 6.51 (d, J =

1.8 Hz, 1H), 6.30−6.27 (m, 1H), 5.58−5.52 (m, 2H), 4.47−4.43 (m,

1H), 4.36−4.31 (m, 1H), 4.17−4.12 (m, 1H), 4.10−4.03 (m, 2H),

3.86−3.84 (m, 6H), 3.74 (s, 3H), 3.66 (s, 3H), 3.41 (d, J = 10.2 Hz,

1H), 2.76 (td, J = 2.6, 13.3 Hz, 1H), 2.43−2.26 (m, 6H), 2.20−2.15

(m, 2H), 1.88 (br d, J = 12.6 Hz, 1H), 1.80−1.75 (m, 1H), 1.71−1.58

(m, 6H), 1.41−1.30 (m, 3H), 1.22−1.11 (m, 3H), 0.90−0.84 (m,

1H), 0.78−0.70 (m, 1H). ¹³C NMR (126 MHz, CDCl₃, mixture of

rotamers 1:10. Only major rotamer is reported here: δ 172.4, 170.7,

159.7, 154.0, 151.8, 149.0, 147.4, 141.8, 137.5, 133.8, 133.5, 129.5,

121.2, 120.5, 117.1, 112.5, 112.0, 111.4, 108.3, 104.6, 75.4, 66.8, 65.0,

60.9, 56.1, 56.1, 56.0, 54.8, 52.0, 44.1, 41.3, 37.0, 32.7, 31.0, 30.7,

29.8, 26.7, 26.6, 26.3, 26.2, 25.9, 20.9. HRMS (m/z): (ESI⁺)

calculated for C₄₂H₅₄NO₉[M + H]⁺: 716.37931, found 716.38019.

HPLC (70−100% solvent B, 0.8 mL/min, 10.50 min): t_R= 5.17 min,

purity (220 nm) = 96%.

Macrocycles 61a and 61b. The substrate 36-(E) (35 mg, 0.047

mmol, 1.00 equiv) was applied to general procedure H with

Pd(OAc)₂(1.0 mg, 0.005 mmol, 0.10 equiv), 1,4-benzoquinone

(6.1 mg, 0.056 mmol, 1.20 equiv), and aqueous HBF₄(48 wt %, 12

μL, 0.066 mmol, 1.40 equiv) in MeCN/H₂O (v/v = 7:1, 4 mL). The

two product isomers 61a (19 mg, 53%, white solid) and 61b (5 mg,

14%, white solid) were obtained after puriﬁcation by semipreparative

HPLC (70−100% solvent B). Data for macrocycle 61a: TLC (CH/

1

EA = 1:1, v/v): R_f= 0.36. H NMR (500 MHz, CDCl₃, mixture of

rotamers 0.18:1, A/B): δ 7.30−7.26 (m, 0.12H, A), 7.16−7.11 (m,

0.88H, B), 7.05−7.01 (m, 0.15H, A), 6.90−6.87 (m, 0.16H, A),

6.86−6.83 (m, 0.16H, A), 6.81−6.75 (m, 1.04H, A+B), 6.74−6.66

(m, 2.91H, A+B), 6.64−6.60 (m, 1.81H, A+B), 6.41 (d, J = 1.8 Hz,

0.86H, B), 6.23 (d, J = 2.0 Hz, 0.15H, A), 5.88 (t, J = 2.0 Hz, 0.85H,

B), 5.85 (dd, J = 4.9, 8.6 Hz, 0.17H, A), 5.55−5.49 (m, 1.78H, A+B),

4.60−4.54 (m, 0.31H, A), 4.51−4.41 (m, 0.98H, A+B), 4.40−4.34

(m, 0.17H, A), 4.34−4.28 (m, 1.19H, A+B), 4.27−4.22 (m, 0.87H,

B), 4.17−4.12 (m, 0.94H, A+B), 4.11−3.98 (m, 3.04H, A+B), 3.87−

3.83 (m, 6.21H, A+B), 3.81 (s, 0.49H, A), 3.68 (s, 2.60H, B), 3.54 (s,

2.59H, B), 3.40 (d, J = 10.3 Hz, 0.86H, B), 3.15−3.08 (m, 0.85H, B),

3.08−3.01 (m, 0.88H, B), 2.94−2.87 (m, 1.60H, A+B), 2.85−2.79

(m, 0.87H, B), 2.70−2.62 (m, 0.96H, A+B), 2.46−2.38 (m, 1.01H, A

+B), 2.34−2.26 (m, 1.75H, A+B), 2.23−2.10 (m, 1.05H, A+B),

1.93−1.88 (m, 0.84H, B), 1.82−1.74 (m, 0.86H, A+B), 1.72−1.53

(m, 6.86H, A+B), 1.50−1.26 (m, 3.92H, A+B), 1.22−1.10 (m, 2.79H,

A+B), 1.05−0.97 (m, 0.32H, A), 0.91−0.82 (m, 1.04H, A+B), 0.78−

0.70 (m, 0.89H, B), 0.32−0.15 (m, 0.31H, A). ¹³C NMR (126 MHz,

CDCl₃, mixture of rotamers 1:6, A/B): δ 208.68 (B), 206.66 (A),

173.92 (A), 172.38 (B), 170.76 (A), 170.40 (B), 159.26 (A), 158.94

(B), 154.57 (A), 154.03 (B), 151.51 (B), 151.30 (A), 149.11 (A),

148.95 (B), 147.60 (A), 147.42 (B), 141.94 (B), 139.30 (A), 137.24

(B), 133.84 (A), 133.51 (B), 133.35 (B), 129.83 (A), 129.32 (B),

120.64 (B), 120.49 (B), 120.34 (A), 113.68 (A), 113.63 (B), 112.89

(A), 112.43 (A), 112.03 (B), 111.84 (A), 111.69 (B), 111.58 (A),

Macrocycle 59. The substrate 31-(E) (27 mg, 0.04 mmol, 1.00

equiv) was applied to general procedure H with Pd(OAc)₂(0.5 mg,

0.002 mmol, 0.05 equiv), 1,4-benzoquinone (4 mg, 0.04 mmol, 1.00

equiv), and aqueous HBF₄(48 wt %, 10 μL, 0.05 mmol, 1.40 equiv)

in MeCN/H₂O (v/v = 7:1, 800 μL). Additional portions of

Pd(OAc)₂(0.05 equiv) and 1,4-benzoquinone (0.30 equiv) were

added until completion of the reaction. 59 (14 mg, 52%, white solid)

was obtained after puriﬁcation by ﬂash column chromatography

(CH/EA = 4:1) as a single product isomer. TLC (CH/EA = 2:1, v/

1

v): R_f= 0.31. H NMR (500 MHz, CDCl₃): δ 7.21 (t, J = 7.9 Hz,

1H), 6.88−6.85 (m, 1H), 6.78−6.74 (m, 2H), 6.72−6.70 (m, 1H),

6.69−6.65 (m, 2H), 6.44−6.42 (m, 1H), 6.29 (d, J = 1.8 Hz, 1H),

5.64 (dd, J = 5.5, 8.1 Hz, 1H), 5.51−5.47 (m, 1H), 4.50 (dt, J = 6.7,

11.4 Hz, 1H), 4.44−4.36 (m, 3H), 3.94 (d, J = 13.9 Hz, 1H), 3.87 (s,

3H), 3.84 (s, 3H), 3.80 (s, 3H), 3.52 (s, 3H), 3.35 (d, J = 9.9 Hz,

1H), 2.86−2.79 (m, 3H), 2.60−2.46 (m, 2H), 2.33−2.27 (m, 1H),

2.18−2.07 (m, 2H), 1.99−1.92 (m, 1H), 1.88 (d, J = 12.4 Hz, 1H),

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111.45 (B), 107.67 (B), 107.13 (A), 104.83 (A), 104.55 (B), 75.80

(A), 75.72 (B), 66.35 (A), 64.52 (B), 63.93 (A), 63.80 (B), 60.89

(A), 60.76 (B), 56.22 (A), 56.07 (B), 55.92 (B), 55.07 (B), 52.19

(B), 44.60 (B), 44.18 (B), 43.65 (A), 42.31 (A), 41.66 (B), 41.41

(A), 41.22 (B), 39.79 (A), 38.52 (A), 37.76 (B), 32.78 (B), 32.48

(A), 31.59 (A), 31.25 (B), 30.66 (B), 30.14 (A), 27.12 (A), 26.69

(B), 26.42 (B), 26.32 (A), 26.23 (B), 26.08 (B), 25.97 (A), 25.76

(B), 24.14 (A), 20.92 (B), 20.82 (A). HRMS (m/z): (ESI⁺)

calculated for C₄₄H₅₆NO₁₀[M + H]⁺: 758.38987, found 758.39061.

HPLC (70−100% solvent B, 0.8 mL/min, 10.50 min): t_R= 3.64 min,

purity (220 nm) = 99%. Data for macrocycle 61b: TLC (CH/EA =

1:1, v/v): R_f= 0.51. ¹H NMR (500 MHz, CDCl₃, mixture of rotamers

0.21:1, A/B): δ 7.32 (t, J = 7.9 Hz, 0.20H, A), 7.19 (t, J = 7.9 Hz,

0.80H, B), 7.03−7.00 (m, 0.20H, A), 6.98−6.95 (m, 0.21H, A),

6.91−6.87 (m, 0.22H, A), 6.85−6.82 (m, 0.80H, B), 6.81−6.75 (m,

1.78H, A+B), 6.71−6.59 (m, 3.20H, A+B), 6.38 (d, J = 1.8 Hz,

0.75H, B), 6.25−6.22 (m, 0.94H, A+B), 5.88 (dd, J = 5.5, 8.3 Hz,

0.20H, A), 5.53−5.46 (m, 1.56H, B), 4.61−4.49 (m, 1.63H, A+B),

4.40 (d, J = 15.1 Hz, 0.72H, B), 4.18−4.11 (m, 0.19H, A), 4.05−3.98

(m, 2.53H, A+B), 3.88−3.83 (m, 6.76H, A+B), 3.80 (s, 0.68H, A),

3.78 (s, 2.34H, B), 3.67 (s, 2.38H, B), 3.37 (d, J = 10.0 Hz, 0.77H, B),

3.18 (dt, J = 7.0, 16.5 Hz, 0.20H, A), 3.05−2.98 (m, 0.78H, B), 2.87−

2.80 (m, 0.19H, A), 2.70−2.34 (m, 7.46H, A+B), 2.31−2.01 (m,

4.54H, A+B), 1.91−1.81 (m, 1.54H, A+B), 1.76−1.52 (m, 6.83H, A

+B), 1.45−1.24 (m, 4.25H, A+B), 1.19−1.10 (m, 2.57H, A+B),

0.92−0.83 (m, 1.39H, A+B), 0.78−0.69 (m, 0.85H, B), 0.36−0.21

(m, 0.40H, A). ¹³C NMR (126 MHz, CDCl₃, mixture of rotamers 1:4,

A/B): δ 208.85 (B), 208.08 (A), 173.22 (A), 172.33 (B), 170.71 (A),

170.54 (B), 158.47 (A), 158.20 (B), 154.57 (A), 153.73 (B), 151.99

(B), 151.07 (A), 149.15 (A), 149.00 (B), 147.69 (A), 147.49 (B),

142.85 (A), 142.21 (B), 137.31 (B), 134.08 (A), 133.69 (B), 133.32

(B), 130.11 (A), 129.80 (B), 121.62 (B), 121.50 (A), 120.50 (B),

120.33 (A), 114.22 (B), 113.27 (B), 112.02 (B), 111.88 (A), 111.59

(A), 111.54 (A), 111.48 (B), 107.61 (B), 105.15 (B), 104.39 (A),

76.01 (A), 75.61 (B), 74.04 (B), 73.00 (A), 69.84 (A), 67.78 (B),

60.92 (B), 56.24 (B), 56.08 (B), 55.96 (B), 54.97 (B), 52.07 (B),

43.97 (B), 41.33 (A), 41.26 (B), 39.63 (A), 38.37 (A), 37.15 (B),

35.49 (B), 34.65 (A), 32.81 (B), 32.60 (A), 31.64 (A), 31.27 (B),

30.73 (B), 30.19 (A), 27.15 (A), 26.70 (B), 26.48 (A), 26.30 (B),

26.21 (B), 26.00 (A), 25.76 (B), 24.89 (B), 24.28 (A), 23.35 (A),

21.07 (B). HRMS (m/z): (ESI⁺) calculated for C₄₄H₅₆NO₁₀[M +

H]⁺: 758.38987, found 758.39032. HPLC (70−100% solvent B, 0.8

mL/min, 10.50 min): t_R= 4.32 min, purity (220 nm) = 97%.

Macrocycle 62. The substrate 37-(E) (38 mg, 0.05 mmol, 1.00

equiv) was applied to general procedure H with Pd(OAc)₂(1.1 mg,

0.005 mmol, 0.1 equiv), 1,4-benzoquinone (6.4 mg, 0.06 mmol, 1.20

equiv), and aqueous HBF₄(48 wt %, 13 μL, 0.07 mmol, 1.40 equiv)

in MeCN/H₂O (v/v = 7:1, 5 mL). Additional portions of Pd(OAc)₂

(0.10 equiv), 1,4-benzoquinone (1.20 equiv), and aqueous HBF₄

(2.00 equiv) were added until completion of the reaction. 62 (13 mg,

33%, white solid) was obtained after puriﬁcation by semipreparative

HPLC (80−90% solvent B) as a single product isomer. TLC (CH/EA

= 1:1, v/v): R_f= 0.39. ¹H NMR (500 MHz, CDCl₃, mixture of

rotamers 0.24:1, A/B): δ 7.29−7.26 (m, 0.16H, A), 7.17 (t, J = 7.9

Hz, 0.83H, B), 7.00−6.97 (m, 0.19H, A), 6.92−6.89 (m, 0.22H, A),

6.87−6.84 (m, 0.25H, A), 6.83−6.73 (m, 2.79H, A+B), 6.71−6.63

(m, 2.75H, A+B), 6.59−6.52 (m, 0.29H, A), 6.39−6.34 (m, 1.00H, A

+B), 6.27−6.23 (m, 0.77H, B), 5.80 (dd, J = 5.3, 8.4 Hz, 0.18H, A),

5.56 (dd, J = 5.9, 8.3 Hz, 0.80H, B), 5.46 (d, J = 5.5 Hz, 0.79H, B),

5.43−5.40 (m, 0.26H, A), 4.65−4.59 (m, 0.24H, A), 4.52 (d, J = 13.7

Hz, 0.21H, A), 4.41−4.10 (m, 6.04H, A+B), 4.03 (dt, J = 3.4, 10.9

Hz, 0.88H, A+B), 3.98−3.90 (m, 2.18H, A+B), 3.85 (d, J = 4.0 Hz,

6.61H, A+B), 3.80 (d, J = 10.6 Hz, 3.16H, A+B), 3.64 (s, 2.40H, B),

3.35 (d, J = 9.8 Hz, 0.80H, B), 3.14−3.03 (m, 1.00H, A+B), 2.90−

2.81 (m, 1.11H, A+B), 2.72−2.56 (m, 3.02H, A+B), 2.52−2.36 (m,

1.96H, A+B), 2.32−2.25 (m, 1.09H, A+B), 2.13−2.04 (m, 1.09H, A

+B), 1.97−1.76 (m, 3.05H, A+B), 1.72−1.57 (m, 5.97H, A+B),

1.48−1.25 (m, 4.99H, A+B), 1.21−1.09 (m, 2.19H, A+B), 0.95−0.83

(m, 1.09H, A+B), 0.79−0.70 (m, 1.00H, A+B), 0.39−0.30 (m, 0.17H,

A), 0.15−0.06 (m, 0.15H, A). ¹³C NMR (126 MHz, CDCl₃, mixture

of rotamers 1:3, A/B): δ 207.98 (B), 207.43 (A), 173.36 (A), 172.31

(B), 158.75 (B), 158.45 (A), 154.51 (A), 153.75 (B), 151.81 (B),

151.24 (A), 149.13 (A), 149.00 (B), 147.63 (A), 147.46 (B), 142.19

(A), 141.90 (B), 139.39 (A), 137.75 (B), 134.26 (A), 133.76 (A),

133.64 (B), 133.50 (B), 133.46 (B), 129.74 (A), 129.54 (B), 120.93

(A), 120.49 (B), 120.36 (B), 119.09 (A), 113.82 (A), 113.51 (A),

113.41 (B), 112.82 (B), 112.23 (A), 112.01 (B), 111.85 (A), 111.56

(A), 111.47 (B), 108.84 (B), 108.65 (A), 105.84 (B), 105.10 (A),

76.25 (A), 75.73 (B), 70.97 (A), 70.85 (B), 70.33, 69.60 (B), 63.80

(A), 62.72 (B), 60.99 (B), 60.94 (A), 56.40 (B), 56.33 (A), 56.24

(A), 56.08 (B), 56.04 (B), 55.96 (B), 55.70 (A), 55.29 (B), 54.84

(A), 52.56 (A), 52.26 (B), 44.27 (A), 43.93 (B), 41.38 (B), 41.33

(A), 39.53 (A), 39.06 (A), 38.57 (A), 38.39 (A), 38.27 (B), 37.69

(B), 32.99 (B), 32.84 (A), 32.40 (A), 31.64 (A), 31.41 (A), 31.34

(B), 30.85 (A), 30.78 (B), 30.22 (A), 27.41 (A), 26.79 (B), 26.71

(B), 26.46 (A), 26.37 (B), 26.28 (B), 26.00 (A), 25.64 (B), 24.33

(A), 21.05 (B), 20.94 (A). HRMS (m/z): (ESI⁺) calculated for

C₄₅H₅₈NO₁₁H [M + H]⁺: 788.40044, found 788.40094. HPLC (70−

100% solvent B, 0.8 mL/min, 10.5 min): t_R= 4.16 min, purity (220

nm) = 98%.

Macrocycle 63. To a stirred solution of 31-(E) (51 mg, 0.07

mmol, 1.00 equiv) in acetone (1 mL) at 0 °C was added N-

methylmorpholine N-oxide (13 mg, 0.11 mmol, 1.50 equiv). The

mixture was treated with OsO₄(2.5 wt % in tBuOH, 144 μL, 0.014

mmol, 0.20 equiv) and H₂O (1 mL). The resulting mixture was

stirred at room temperature overnight. Then the reaction mixture was

diluted with EA (10 mL), layers separated, and the organic phase

extracted with H₂O (2 × 5 mL). The organic phase was dried over

MgSO₄and concentrated under reduced pressure. The crude product

was puriﬁed by ﬂash column chromatography (CH/EA = 2:3 and

DCM/MeOH = 50:1) to aﬀord the desired product 63 (14 mg, 26%,

white solid) as a mixture of diastereomers (dr = 34:66, determined via

HPLC). Note: Attempts to separate the diastereomers via semi-

preparative HPLC failed. TLC (DCM/MeOH = 20:1, v/v): R_f= 0.26.

¹H NMR (500 MHz, CDCl₃, mixture of diastereomers 0.5:1, A/B): δ

7.23−7.13 (m, 1.54H, A+B), 6.89−6.83 (m, 1.15H, A+B), 6.84−6.80

(m, 1.63H, A+B), 6.80−6.75 (m, 3.05H, A+B), 6.75−6.68 (m, 3.63H,

A+B), 6.41−6.36 (m, 0.95H, B), 6.33−6.30 (m, 0.91H, A), 6.26−

6.22 (m, 0.96H, B), 5.80−5.71 (m, 1.60H, A+B), 5.51−5.44 (m,

1.54H, A+B), 4.37 (dd, J = 3.0, 11.9 Hz, 0.50H, A), 4.23 (dd, J = 4.7,

10.3 Hz, 1.00H, B), 4.19−4.09 (m, 2.36H, A+B), 4.06−3.99 (m,

2.45H, A+B), 3.92−3.83 (m, 16.74H, A+B), 3.79 (dd, J = 3.6, 10.4

Hz, 1.20H, B), 3.72−3.67 (m, 1.61H, A+B), 3.60 (s, 1.30H, A), 3.52

(s, 2.84H, A+B), 3.34−3.28 (m, 1.49H, A+B), 3.00 (br s, 0.95H, A

+B), 2.74−2.61 (m, 2.44H, A+B), 2.55−2.46 (m, 1.49H, A+B),

2.36−2.26 (m, 1.61H, A+B), 2.17−2.05 (m, 2.85H, A+B), 1.96−1.85

(m, 2.76H, A+B), 1.76−1.54 (m, 10.94H, A+B), 1.46−1.27 (m,

6.55H, A+B), 1.21−1.09 (m, 2.93H, A+B), 0.98−0.86 (m, 1.45H, A

+B), 0.85−0.71 (m, 1.44H, A+B). ¹³C NMR (126 MHz, CDCl₃,

mixture of diastereomers 1:2, A/B): δ 172.05 (A), 171.75 (B), 171.26

(B), 171.21 (A), 159.21 (A), 159.10 (B), 154.23 (B), 153.67 (A),

153.28 (B), 152.13 (A), 149.02 (A+B), 147.43 (A+B), 142.19 (B),

141.74 (A), 135.30 (A), 134.97 (B), 134.55 (B), 134.38 (A), 133.85

(A+B), 129.54 (B), 129.48 (A), 121.20 (B), 120.76 (A), 120.47 (B),

120.42 (A), 115.36 (B), 115.03 (A), 114.40 (B), 113.19 (A), 112.16

(B), 112.08 (A), 111.47 (A+B), 108.12 (A), 107.71 (B), 106.26 (B),

104.28 (A), 76.03 (B), 75.87 (A), 74.58 (B), 73.67 (A), 71.21 (A),

69.72 (A), 69.59 (B), 69.52 (B), 69.09 (A), 68.65 (B), 57.20 (B),

56.83 (B), 56.79 (A), 56.32 (A), 56.28 (B), 56.10 (A+B), 56.00 (A

+B), 55.74 (A), 52.71 (B), 52.40 (A), 44.01 (B), 43.74 (A), 41.71

(A), 41.49 (B), 39.55 (B), 38.54 (A), 33.35 (B), 33.10 (A), 31.52

(B), 31.45 (A), 30.79 (B), 30.76 (A), 27.05 (A), 26.94 (B), 26.71 (A

+B), 26.50 (B), 26.43 (A+B), 26.37 (A), 25.68 (A), 25.57 (B), 21.07

(B), 21.03 (A). HRMS (m/z): (ESI⁺) calculated for C₄₃H₅₆NO₁₁[M

+ H]⁺: 762.38479, found 762.38530. HPLC (50−60% solvent B, 1.5

mL/min, 20 min, 220 nm): t_R,1= 15.37 min (34%), t_R,2= 15.96 min

(66%).

Macrocycles 64a and 64b. To a stirred solution of 35-(E) (35

mg, 0.05 mmol, 1.00 equiv) in acetone (1 mL) at 0 °C was added N-

methylmorpholine N-oxide (8 mg, 0.07 mmol, 1.50 equiv). The

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mixture was treated with OsO₄(2.5 wt % in tBuOH, 100 μL, 0.01

mmol, 0.20 equiv) and H₂O (1 mL). The resulting mixture was

stirred at room temperature overnight. Then the reaction mixture was

diluted with EA (10 mL), layers separated, and the organic phase

extracted with H₂O (2 × 5 mL). The organic phase was dried over

MgSO₄and concentrated under reduced pressure. The crude product

was puriﬁed by ﬂash column chromatography (CH/EA = 2:3) and

semipreparative HPLC (70−100% solvent B) to aﬀord the desired

product diastereomers 64a (8 mg, 22%) and 64b (7 mg, 18%) as

white solids. Data for macrocycle 64a: TLC (CH/EA = 1:2 + 1%

HCOOH, v/v/v): R_f= 0.24. ¹H NMR (500 MHz, CDCl₃, mixture of

rotamers 0.1:1. Only the major rotamer is reported here: δ 7.16 (t, J =

7.9 Hz, 1H), 6.79−6.72 (m, 4H), 6.65−6.60 (m, 2H), 6.41 (d, J = 1.8

Hz, 1H), 5.90−5.87 (m, 1H), 5.61 (dd, J = 6.4, 7.8 Hz, 1H), 5.53 (d, J

= 5.5 Hz, 1H), 4.40−4.31 (m, 2H), 4.25 (dd, J = 3.0, 10.2 Hz, 1H),

4.20 (dd, J = 3.5, 9.6 Hz, 1H), 4.00 (br d, J = 13.9 Hz, 1H), 3.86 (s,

3H), 3.85 (s, 3H), 3.74 (s, 3H), 3.59 (s, 3H), 3.36 (d, J = 10.3 Hz,

1H), 3.11 (br s, 1H), 2.55 (t, J = 13.2 Hz, 1H), 2.48−2.41 (m, 1H),

2.37−2.24 (m, 2H), 2.19−2.11 (m, 1H), 1.91−1.84 (m, 2H), 1.75−

1.52 (m, 9H), 1.40−1.11 (m, 7H), 0.90−0.81 (m, 2H), 0.76−0.68

(m, 1H). ¹³C NMR (126 MHz, CDCl₃, mixture of rotamers 1:10.

Only the major rotamer is reported here: δ 172.0, 170.7, 158.3, 153.9,

151.9, 149.0, 147.5, 141.7, 137.1, 134.3, 133.5, 129.5, 121.0, 120.3,

113.9, 111.9, 111.6, 111.4, 108.0, 104.7, 75.3, 74.1, 69.9, 69.7, 67.9,

60.9, 56.1, 56.0, 56.0, 54.7, 51.8, 43.9, 41.6, 37.2, 32.7, 31.2, 30.7,

26.7, 26.6, 26.2, 26.2, 25.8, 21.0. HRMS (m/z): (ESI⁺) calculated for

C₄₃H₅₆NO₁₁[M + H]⁺: 762.38479, found 762.38489. HPLC (50−

100% solvent B, 0.8 mL/min, 10.50 min): t_R= 4.86 min, purity (220

nm) = 95%. Data for macrocycle 64b: TLC (CH/EA = 1:2 + 1%

HCOOH, v/v/v): R_f= 0.42. ¹H NMR (500 MHz, CDCl₃): δ 7.15 (t,

J = 7.9 Hz, 1H), 6.79−6.72 (m, 4H), 6.65−6.61 (m, 2H), 6.37 (d, J =

1.8 Hz, 1H), 5.72−5.68 (m, 1H), 5.61 (t, J = 7.2 Hz, 1H), 5.50 (d, J =

5.4 Hz, 1H), 4.41 (ddd, J = 4.7, 8.6, 20.4 Hz, 2H), 4.27−4.19 (m,

2H), 4.13 (dd, J = 4.5, 9.8 Hz, 1H), 4.08−4.04 (m, 1H), 3.99−3.93

(m, 1H), 3.87 (s, 3H), 3.85 (s, 3H), 3.80 (s, 3H), 3.54 (s, 3H), 3.33

(d, J = 10.2 Hz, 1H), 2.58 (br s, 2H), 2.47−2.40 (m, 2H), 2.36−2.25

(m, 2H), 2.19−2.10 (m, 1H), 1.93−1.85 (m, 2H), 1.71−1.53 (m,

6H), 1.41−1.31 (m, 2H), 1.25−1.11 (m, 4H), 0.88−0.82 (m, 1H),

0.76−0.68 (m, 1H). ¹³C NMR (126 MHz, CDCl₃): δ 171.8, 170.6,

158.8, 153.7, 151.6, 148.9, 147.4, 141.4, 137.5, 134.1, 133.5, 129.4,

121.0, 120.4, 114.2, 112.0, 111.5, 111.4, 109.0, 104.9, 75.7, 72.8, 72.1,

70.0, 66.6, 61.0, 56.1, 55.9, 55.9, 54.9, 51.9, 43.9, 41.5, 37.2, 32.8,

31.4, 30.7, 26.7, 26.5, 26.3, 26.2, 25.7, 21.0. HRMS (m/z): (ESI⁺)

calculated for C₄₃H₅₆NO₁₁[M + H]⁺: 762.38479, found 762.38465.

HPLC (50−100% solvent B, 0.8 mL/min, 10.50 min): t_R= 5.49 min,

purity (220 nm) = 98%.

assay (FPA) was performed according to Kozany et al.⁵¹Brieﬂy, the

ﬂuorescent tracer FT (chemical structure and tracer dissociation

constants shown in the Supporting Information) was diluted in the

assay buﬀer (150 mM NaCl, 20 mM HEPES, pH 8.0, 0.002% Triton

X-100) to a concentration 11.9× the ﬁnal concentration (11.9 nM

solution used to perform FPA with FKBP12.6, −51FK1, and

−52FK1; 5.95 nM solution used to perform FPA with FKBP12).

The competitive ligand was dissolved in DMSO to a concentration

62.5× the ﬁnal concentration. This was used for a 1:1 serial dilution in

DMSO. Every sample of this serial dilution was diluted by a factor of

6.25 in the assay buﬀer supplemented with the ﬂuorescent tracer

(11.9 nM or 5.95 nM, respectively) to achieve a 10× concentrated

mixture of the ﬂuorescent tracer and competing ligand (add 2 μL of

competitive ligand to 10.5 μL of ﬂuorescent tracer). Protein aliquots

(45 μL) at 1.11× the ﬁnal concentration in the assay buﬀer were

transferred into black 384-well assay plates (no. 3575, nonbinding

surface, Corning Life Sciences). A mixture (5 μL) of the ﬂuorescent

tracer and competitive ligand were added to the protein. After

incubation at room temperature for 30 min, the ﬂuorescence

polarization was measured with a plate reader Tecan Genios Pro at

excitation/emission values of 535/590 nm. The competition curves

were analyzed using Prism 6.0 (GraphPad Software). For the analysis

of K_dvalues, data were ﬁtted to the equation provided in Kozany et al.

(Supporting Information, Appendix 3).⁵¹At least two independent

measurements were performed for each compound. K_dvalues

represent mean values the weighted standard deviation which, in

addition to the standard deviation (SD), also take the errors from the

K_dﬁtting curves into account.

2

weighted SD = SD²+ error_Fit1+ error_Fit2+ ...

Isothermal Titration Calorimetry Experiments. ITC experi-

ments were performed with a MicroCal PEAQ-ITC isothermal

titration calorimeter (Malvern). All experiments were conducted at 25

°C. For the ITC experiment of SAFit1, a 10 μM solution of

FKBP51FK1 formulated in 20 mM HEPES pH 8.0, 20 mM NaCl, and

0.5% DMSO was placed in the sample cell of the PEAQ-ITC

instrument. The syringe was ﬁlled with a 100 μM solution of SAFit1

formulated in the same buﬀer. After equilibration, the compound was

titrated into the sample cell by 18 injections of 2 μL each. Due to the

limiting solubility, compound 64a was measured in a reverse setup

injecting the protein into a solution of the ligand. Therefore, a 10 μM

solution of 64a formulated in 20 mM HEPES pH 8.0, 20 mM NaCl,

and 5% DMSO was placed in the sample cell of a PEAQ-ITC

instrument. The syringe was ﬁlled with a 100 μM solution of

FKBP51FK1 formulated in the same buﬀer. After equilibration, the

compound was titrated into the sample cell by 18 injections of 2 μL

each. The obtained data were analyzed using the provided software

package and ﬁtted to a one-site binding model. All titrations were

performed as triplicates, and errors were calculated as weighted

standard deviations which, in addition to the standard deviation, also

take the errors from the ﬁtting curves into account (see formula above

at the FP assay procedure).

Fluorescence Polarization Assay for the Binding of Labeled

Ligands to FKBP Target Proteins. The ﬂuorescence polarization

assay was performed according to Kozany et al.⁵¹For ﬂuorescence

polarization assays, the ﬂuorescent ligand FT (chemical structure

shown in Supporting Information) was diluted from a DMSO stock in

a HEPES buﬀer (150 mM NaCl, 20 mM HEPES, pH 8.0, 0.002%

Triton X-100) at double the concentration required for the ﬁnal

sample. The target protein was also diluted in this assay buﬀer at

double the highest concentration required for the ﬁnal sample. This

protein stock was used for a 1:1 serial dilution in a black 384-well

assay plate (no. 3575, nonbinding surface, Corning Life Sciences).

Finally, each protein dilution (25 μL) was mixed with the dilution of

the ﬂuorescent ligand (25 μL). After incubation at room temperature

for 30 min, the ﬂuorescence polarization was measured with a plate

reader Tecan Genios Pro at excitation/emission values of 535/590

nm. For FKBP12, 12.6, −51FK1, and −52FK1, the binding assays

were performed in triplicates in the plate format. The binding curves

were analyzed by using Prism 6.0 (GraphPad Software). For the

analysis of K_dvalues, data were ﬁtted to the equation provided in

Kozany et al. (Supporting Information, Appendix 2).⁵¹Dissociation

constants of the ﬂuorescent ligand FT for FKBP12, 12.6, −51FK1 and

−52FK1 are summarized in the Supporting Information.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02195.

■

sı

Further experimental details: crystallographic data

collection and reﬁnement statistics; X-ray structures of

56, 33-(Z), 35-(E), and 63 in complex with

FKBP51FK1; superposition of X-ray structures with

SAFit2 and linear SAFit-like ligands; ITC traces of

SAFit1 and 64a; additional synthetic representations and

explanations; analytical data of intermediate compounds;

additional information and binding curves from FP

assay; and NMR spectra and HPLC traces of ﬁnal

compounds (PDF)

Competitive Fluorescence Polarization Assay for Binding to

FKBP Target Proteins. The competitive ﬂuorescence polarization

3346

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J. Med. Chem. 2021, 64, 3320−3349

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

ABBREVIATIONS USED

Molecular formula strings (CSV)

■

CH, cyclohexane; DCC, N,N′-dicyclohexylcarbodiimide;

DMAP, 4-(dimethylamino)pyridine; DIPEA, N,N-diisopropy-

lethylamine; EDC, N-(3-(dimethylamino)propyl)-N′-ethylcar-

bodiimide hydrochloride; EA, ethyl acetate; (ESI+), electro-

spray ionization; FKBP, FK506-binding protein; HATU, 1-

[bis(dimethylamino)-methylene]-1H-1,2,3-triazolo[4,5-b]-

pyridinium 3-oxid hexaﬂuorophosphate; HRMS, high-resolu-

tion mass spectrometry; ITC, isothermal titration calorimetry;

LC−MS, liquid chromatography−mass spectrometry;

LiHMDS, lithium bis(trimethylsilyl)amide; n-BuLi, n-butyl-

lithium; NMO, 4-methylmorpholine N-oxide; NMR, nuclear

magnetic resonance; SD, standard deviation; TBAF, tetrabu-

tylammonium ﬂuoride; TBDPSCl, tert-butyl(chloro)-

diphenylsilane; TFA, triﬂuoroacetic acid; TLC, thin-layer

chromatography

Accession Codes

Atomic coordinates for the X-ray structures of compounds 33-

(Z), 35-(E), 55, 56, 63, and 64a in complex with FKBP51 (14-

140)-A19T (PDB 7A6W (33-(Z)), 7B9Z (35-(E)), 7AWX

(55), 7A6X (56), 7BA0 (63), and 7B9Y (64a)) are available

from the RCSB Protein Data Bank (www.rcsb.org). The

authors will release the atomic coordinates and experimental

data upon article publication.

AUTHOR INFORMATION

■

Corresponding Author

Felix Hausch − Department Chemistry and Biochemistry,

Clemens-Schöpf-Institute, Technical University Darmstadt,

64287 Darmstadt, Germany; orcid.org/0000-0002-

3710-8838; Phone: +49-6151-16-21245; Email: hausch@

tu-darmstadt.de

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Michael Bauder − Department Chemistry and Biochemistry,

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Christian Meyners − Department Chemistry and

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Patrick L. Purder − Department Chemistry and Biochemistry,

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Stephanie Merz − Department Chemistry and Biochemistry,

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Wisely Oki Sugiarto − Department Chemistry and

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Andreas M. Voll − Department Chemistry and Biochemistry,

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Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c02195

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by the DFG Grant HA5565-5/1, the

LOEWE cluster TRABITA, and the Pioneer Fund (ENTEGA/

TU Darmstadt). Diﬀraction data have been collected on

BL14.1 at the BESSY II electron storage ring operated by the

Helmholtz-Zentrum Berlin, Germany. We would particularly

like to acknowledge the help and support of Jan Wollenhaupt

during the experiment. We acknowledge the Paul Scherrer

Institut, Villigen, Switzerland, for provision of synchrotron

radiation beamtime at beamline PX of the Swiss Light Source

(SLS) and would like to thank the beamline scientists for

assistance. The synchrotron MX data was collected at beamline

P13 operated by EMBL Hamburg at the PETRA III storage

ring (DESY, Hamburg, Germany). We would like to thank

David von Stetten for the assistance in using the beamline.

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https://dx.doi.org/10.1021/acs.jmedchem.0c02195

J. Med. Chem. 2021, 64, 3320−3349

Article Doi

Structure-Based Design of High-Affinity Macrocyclic FKBP51 Inhibitors

DOI: 10.1021/acs.jmedchem.0c02195

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Full text of DOI:10.1021/acs.jmedchem.0c02195

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