Shield-1

Synthesis of Shld derivatives, their binding the Destabilizing Domain and influence on protein accumulation in transgenic plants
Frederik Praestholm Jorgesen, Daniel Madsen, Morten Meldal, Jakob
Valdbjørn Olsen, Morten Petersen, Jeppe Granhøj, and Mikael Bols
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00497 • Publication Date (Web): 06 May 2019
Downloaded from http://pubs.acs.org on May 6, 2019

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Synthesis of Shld derivatives, their binding the Destabilizing Domain and influence on protein accumulation in transgenic
plants

Frederik Præstholm Jørgensen, Daniel Madsen, Morten Meldal, Jacob Valdbjørn Olsen, Morten Petersen, Jeppe Granhøj and Mikael Bols*

Departments of chemistry & biology, University of Copenhagen, Universitetsparken 5, 2100

Copenhagen Ø, Denmark

[email protected]

Abstract
A series of 35 analogues of Shld with modifications in the A-residue and the C-residues were prepared and investigated for binding to FKBP and GFP accumulation in transgenic plants. The modifications investigated explored variations that was supposedly inside or outside the receptor binding site with the latter being important by influencing the overall polarity of the compounds in order to improve the absorption in plants. The binding of the new compounds to the destabilizing domain was determined using a fluorescence polarization competition assay and the GFP expression in engineered Arabidopsis thaliana was studied. The results showed that modifications of the C-building block phenol with acidic, basic and neutral groups led to better ligands with some being better that Shld in the plant. Generally small, polar substituents showed the best GFP accumulation.

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Introduction
The compound Shld (or Shield1, Figure 1)1 is a truncated synthetic2 analogue3,4 of the natural compounds Rapamycin and FK506, that present strong affinity for FK binding proteins, but only when the binding site is mutated. Shld binds to a F36V mutant of FKBP12 with a 3 orders of magnitude selectivity over the wild type.5 Shld is employed to stabilize destabilized protein domains attached to a protein of interest – when Shld binds the protein-conjugate it is not degraded. Hence, Shld can be used to induce the accumulation of a protein-of-interest in a genetically engineered cell and has been used in mammalian cells,6 parasites7 and plants.8 In a project where we needed to use Shld in plants it became apparent that Shld might not have an optimal structure for absorption in plants. The propensity of which a small organic compound like Shld penetrates a plant cell can be very different from what is observed for mammalian cells, due to the plant cell’s fundamentally different structure.9 To protect the plant from environmental stress and water loss, the outer-layer of cells (epidermis) is covered by a hydrophobic wax layer called the cuticle, which small molecules applied on the leaf must penetrate. This penetration is heavily impacted by the size of the molecule.10,11 For the ionic species this tendency is not as pronounced as they are believed to cross the cuticles in hydration shells via aqueous polar pores.10 The acid/base properties of the compound is also important as it after having passed the cuticle, has to cross the slightly acidic (pH

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R

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10

Afragment

MeO
OMe
OMe



B

C
1-3

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12


N


Bfragment
O

OR

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19

Shld

O

N

O

O

O

O



OMe

OMe

MeO

OMe
OMe
MeO



O
OMe

B
C

4-7

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23
24

A
B

O
Cfragment

R



O

B
8-12

C

O

A
B

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R
O

OMe

13-34
MeO

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OMe
MeO
35

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Figure 1. Structure of Shld and target compounds 1-35. The structure is conveniently divided into fragments A-C.

5.5) cell wall or apoplasm before crossing the cell membrane (Figure 2). This is potentially beneficial for acidic compounds as they more readily penetrate from the outside and become trapped inside in the more basic cytosol (Figure 2B).12 The reverse is true for the basic compounds such as Shld (Figure 2C), which more readily penetrate the cell-membrane from the inside. However such compound can accumulate in the acidic vacuoles inside the cell and be slowly released complicating matters further. A neutral compound obviously penetrates the cell without being affected of the unusual pH phenomena (Figure 2A).

The purpose of the present study was to investigate whether a Shld derivative with better properties for plant cell penetration could be found. The Shld A fragment has previously been identified as very important for DD binding (Figure 1),3 but only a limited number of Shld structures have been investigated. We have therefore decided to explore modifications of the A-

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Plant cuticle

Cell walls

Cell membrane

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9
Apoplasm
(pH 5.5)
Cytoplasm
(pH 7.5)

10 A

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13
Neutral
Neutral
Neutral

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B
RCOOH

C
NR3

RCOOH

RCOO

NR3

NR3H

RCOOH

RCOO

NR3

NR3H

Vacuole (pH 5.5)

NR3H

NR3

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Figure 2. Schematic representation of organic compounds access to a plant cell (bottom). A) Diffusion of neutral lipophilic compound. B) Diffusion of organic carboxylic acid as protonated form, followed by deprotonation in the cytoplasm (ion-trapping). C) Diffusion of deprotonated amine into the vacuole where it is protonated (ion-trapping).

fragment (Figure 1, compounds 1-12). Furthermore the above mentioned pH conditions in the plant cell made it obvious to investigate compounds that were acidic rather than a base such as Shld. Indeed the ethylmorpholino-group of Shld is believed to be outside the receptor,1 and thus replacing this moiety with different acidic (or basic) groups was an obvious choice. Therefore the series of Shld derivatives 13-35 (Figure 1) with a wide range of different acidic and basic substituents were prepared. In this paper we describe the synthesis of these compounds, their receptor binding as measured by a fluorescence polarization assay developed to the purpose and their protein stabilization when applied to transgenic plants. We find that some of the derivatives have improved efficacy compared to Shld.

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Results and discussion
Previous modifications of the A-fragment (Figure 1) found that removal of the three methoxy- substituents on the aromatic ring led to a 20-fold decrease in affinity to the mutant, while a change of stereochemistry at the stereocenter led to a 100-fold decrease in affinity.3 These observation prompted synthesis of two sets of derivatives. In the first set, we changed the aromatic group by either making it electronically different in derivatives 1-3 or changing its steric impact in derivatives 4-7 (Figure 1). In a second set of derivatives, 8-12, we varied the ethyl substituent – this was based on molecular modelling studies which indicated that the size of this substituent could be expanded slightly.

26
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28

N

O

OH

O

O

O

29 O O

30
31

H
NCOOH
H
FmocCl NaHCO3
38
DCC, DMAP

N
O

32
33
34

O

O
OH O

OH

OH
acetone/H2O

83%
NCOOH Fmoc
CH2Cl2 94%
Fmoc
O

O

35 37 39

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36
N

O

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Scheme 1. Synthesis of B-C fragment 39

Synthesis of the derivatives was carried out as follows: The C-fragment 38 was prepared enantiomerically pure as previous described.2 Resolution of inexpensive racemic pipecolic acid gave the tartrate 36.13 Direct Fmoc protection of 36 gave 37 in 83% yield (Scheme 1), which subsequently was esterified with 38 using DCC and DMAP to give 39 in 94% yield. Compound 39 is the B-C fragment of the molecule and, after removal of the Fmoc group with DBU, it was ready, to be coupled with fragment A acids. The fragment A acids that were precursors for 1-3 were prepared as outlined in scheme 2. Using the enantioselective alkylation method described by Stivala and Zakarian,Error! Bookmark not defined. 2-

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X2

X3

O

1) n-BuLi (4 equiv.)
Koga base 2)
I

X2

X3

O

9
10
11
12
13
X1
OH

40: X1, X2 =

THF, -78 °C

, X3 = H
X1

54%, S:R 10:1
OH

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21
22
23

HO

O
41: X1 = X2 = X3 = F
42: X1 = X3 = H, X2 = Br

1)RI, K2CO3
2)LiOH
O
45%, S:R 1:1 52%, S:R 16:1

O
RO

O

24
25
O
OH
3)EtI, BuLi Koga base

O

OH

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43

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34
44: R = CH2CH3
45: R = CH2CH2CH3 46: R = CH2(CH2)6CH3 47: R = CH2CH(CH3)2
44% (over 3 steps) 8% (over 3 steps) 16% (over 3 steps) 18% (over 3 steps)

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Scheme 2. Synthesis of fragment A precursors 40-42 and 44-47. In the synthesis of 45 Cs2CO3 was used in place of K2CO3. In the synthesis of 46-47 the alkyl bromides rather than the iodides were used. Koga base is N1,N3-bis((R)-1-phenyl-2-(piperidin-1-yl)ethyl)propane-1,3-diamine.

naphtylacetic acid was ethylated using butyl lithium and Koga’s base giving the S-derivative 40 in 54% yield. The stereoselectivity was 10:1. From the 3,4,5-trifluorophenylacetic acid this reaction gave the ethyl derivative 41 in 45% yield, but with no stereoselectivity. Similar ethylation of 4- bromophenylacetic acid gave the S-ethyl derivative 42 in 52% yield and with a stereoselectivity of 15:1. The stereochemical purity of the products was determined by comparing the optical rotation with literature values of enantiopure samples.

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O

O

O

O

OH

1)n-BuLi (4 equiv.) Koga base
2)O
R1 R2 THF, -78 °C

O

O

O

O

OH

R1 R OH
2

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48: R1, R2 = Me

49: R1, R2 = 50: R1, R2 = 51: R1, R2 =
52: R1 = H, R2 = isopropyl

O
(79%, S:R 1:1) (72%, S:R 1:1) (60%, S:R 1:1) (40%, S:R 1:1)
(39%, S:R 1:1)

O

28
29
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31
32

52
1)EtOH AcCl

2)MsCl DMAP
O

O

O

OEt

OMs
1)DBU CHCl3

2)LiOH H2O/MeOH
O

O

O

OH

33
34
35
36
53

Rac
28% (over 4 steps)
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Scheme 3. Synthesis of 48-52 and 54

Modification of the substituents of the trimethoxyphenyl group was done by taking advantage of the commercial availability of 4-hydroxy-3,5-dimethoxyphenylacetic acid 43 (Scheme 2). This compound was alkylated with ethyl iodide and potassium carbonate giving the 4-ethoxy 3,5- dimethoxyphenyl acetic ethyl ester. The ester was hydrolysed with LiOH to the corresponding acid that was then ethylated using Stivala and Zakarian’s method to give the S-ethyl derivative 44. The yield was 44% over the three steps. Similarly, this transformative sequence was performed with propyl iodide, octyl iodide and isobutyl iodide giving the 4-propoxy acid 45 in 8% yield, the 4- octyloxy acid 46 in 16% yield and the isobutoxy acid 47 in 18% yield (Scheme 2).

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O

O

O

7 O O O

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13
14

N
Fmoc

O

O

O

DBU CH2Cl2

N
H

O

O

O
O

R Cl

R

N

O

O

O

O

15
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20

N

O

39

N

O

N

O

21
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25
26

1: R =

83%

5: R =

72%

O

O
O

9: R =

76%

O

O
O

27
28
Br

O

O

29 2: R = O O

30
31
72%

6: R =

O
10: R =

O

32 85%

33
F 75%

34 F O
35 3: R = O O

36
37
38
39

97%
F

O
7: R =

47%

O

11: R =
O

O

40 O O 48%

41 4: R =
O
O

42
43
44
45

89%
O
8: R =

90%

O

12: R =
O

O

46 72%

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48
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56
57
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59
60
Scheme 4. Synthesis of Shld analogues 1-12

The A-fragments for 8-12 were made by alkylation of the 3,4,5-trimethoxyphenylacetic acid with various carbonyl derivatives (Scheme 3). Reaction with acetone gave alcohol 48 in 79% yield. Similarly, alkylation of the phenylacetic acid with butyl lithium, Koga’s base and cyclohexanone, cyclopentantone or cyclobutanone gave 49, 50, and 51 in 72, 60, and 40% yield, respectively. None

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of the reactions displayed any enantioselectivity presumably, because the crossed Claisen-aldol adduct equilibrates in the strong base.

Alkylation with an aldehyde, isobutyraldehyde, was also attempted. This gave alcohol 52 in 39% yield as a single diastereomeric pair (Scheme 3) in accordance with the reaction going through a cyclic transition state.14 The alcohol was eliminated by first converting the acid to the ethyl ester and mesylating the alcohol to obtain 53 (Scheme 3). Subsequent anti-elimination with DBU and reconversion to the acid with LiOH gave unsaturated acid 54 in 28% yield over these 4 steps (Scheme 3). The configuration of 54 was determined from the 13C-1H coupling constant between the vinyl proton and the carbonyl carbon. It was 6.9 Hz while model compounds E-diphenyl acrylic acid had a coupling of 7.3 Hz, while the Z-isomer had 12.5 Hz.15

The synthesis of Shld analogues 1-12 was completed as shown in Scheme 4. The Fmoc protective group was removed from B-C-fragment 39 using DBU and the resulting amine was reacted with acid chlorides formed from each of the acids 40-47 formed via reaction with thionyl chloride at 40oC. This led to smooth amide bond formation and resulted in 12 Shld derivatives in 47-97% yield. Since the acid chlorides formed from 41 & 48-51 were racemic, 3 & 8-11 were 1:1 diastereomeric mixtures and were tested as such (see below).

The analogues with C-fragment modifications were made using the synthesis strategy recently reported2 where the fragments are assembled in order from A to C (i.e. A+B → AB + C →ABC). The phenol group substituent was attached to the C-fragment 55 (Scheme 5) that was then attached to the AB fragment (56) by esterification. Depending on the derivative some late state deprotection/
or modification of the side chain was necessary. First a series of acidic analogues were prepared with a broad range of pKa values (Scheme 5). The idea behind these derivatives was to investigate if the ion-trapping mechanism described above (Figure 2, B) could work for these compounds.

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Normally a protected version of the acidic group was used. Thus alkylation of the phenolic position of 552 with tert-butyl bromoacetate afforded 57 in 99% yield. Similar alkylation with chloroacetonitrile gave 58 in 83% yield. To silyl protect the phenol of 55, tert-butyldimethylsilyl chloride was used together with DMAP and triethylamine in dichloromethane to obtain 59 in 69% yield. Phosphorylation of 55 was performed with diethylchlorophosphate to acquire the arylphosphoric ester 60 in 58% yield. Finally, alkylation of 55 with tert-butyl 4-bromobutanoate afforded 61 in 42% yield.

The O-substituted phenols 57, 58, 59, 60 and 61 was coupled to 562 using DCC and DMAP. This gave the protected Shld analogues 22, 23, 24, 25 and 62 in 80%, 70%, 62%, 58% and 42% yield respectively (Scheme 5). These products were then modified/deprotected as follows: The tert- butyl analogues 22 and 62 were treated with TFA in dichloromethane to afford the carboxylic acid analogues 18 and 19 in 92% and 53% yield, respectively (Scheme 5). The silyl protected phenol 24 was deprotected using TBAF under slightly acidic conditions to furnish the phenol derivative 21. Deprotection of the ethyl groups of the phosphoric ester 25 was performed using trimethylsilyl iodide (TMSI) as described by Blackburn and Ingleson, by first generating the O-TMS phosphoric ester, followed by addition of water affording the phosphoric acid analogue 17.16 It turned out that the ester hydrolysis with TMSI was exceptionally prone to formation of byproducts. Initially, addition of TMSI was performed at -78 ºC and the reaction allowed to heat to ambient temperature overnight before hydrolysis with water. LC-MS and NMR revealed that a mixture of two products

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O

O

O

N

56

O

O

O

10 OH OR

11
HO HO O COOH
N

12
13
14

O

55
RX, K2CO3
DMF

O
DCC, DMAP (excess) CH2Cl2

O

O

O

O

OR

15
16

O

O

O

17
18
19
20
21
22
23
24
25
26

57: R =

58: R =

X = Br

X = Cl

O

O

N

99%

83%

22: R =

18: R =

23: R =

O

O

O

OH

N

H
N

80%

92%

70%

TFA

NaN3 ZnCl2

27
28
29
20: R =
N
NN
90%

30
59: R = Si 69% 24: R = Si 62%

31
32
X = Cl

TBAF

33
34
35
36

60: R =

O
P

O

73%
21: R =

25: R =
H

O
P

O
65%

58%

37 X = Cl O O 1) TMSI

38
39
40
41

17: R =
O
OH
P
OH

99%
2) H2O

42 O O

43
44
45
46
47
48
49
61: R =

X = Br
O
42%
62: R =

19: R =

O
O

OH
42%

53%

TFA

50
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53
54
55
56
57
58
59
60
Scheme 5. Synthesis of acidic Shld analogues. 59 was synthesized using DMAP and Et3N in CH2Cl2.

were obtained, where one was the desired deprotected phosphoric acid 17, while the other was a sideproduct resulting from hydrolysis of one of the five methoxy groups present in the structure. To avoid hydrolysis of the methoxy groups, it was attempted to keep the reaction at -78 ºC for 5 hours

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19

before addition of water. Unfortunately, this resulted in incomplete deprotection of the phosphoric ester. Eventually, it was found that adding TMSI at -40 ºC and slowly heating the reaction to ambient temperature over 4 hours before addition of water afforded the desired phosphoric acid 17 in quantitative yield. The nitrile analogue 23 was converted into the acidic tetrazole by a 1,3-dipolar cycloaddition with sodium azide facilitated by ZnCl2 in i-PrOH, conditions reported by Vorona et al.17 affording the tetrazole analogue 20 in 90% yield (Scheme 5).

20
21

O
O

O

22 56 O

23
24
25
26
27
28
29

O

HO

55

OH

RBr, K2CO3
DMF

O

HO

OR

N

OCOOH
DCC, DMAP (excess) CH2Cl2
O

O

O
O

N

O

O

OR

30
31
32
O
O

O

33
34
35
63: R =
N
26%
13: R =
N
70%

36 O O

37
38
39
40
41
64: R =

N
H

O
26%
26: R =

16: R =

N
H

NH2

O
78%

93%

TFA

42
O O

43
44
45
46
47
48
65: R =

N
H

O
56%
27: R =

14: R =

N
H

NH2

O
55%

79%

TFA

49
50
51
52
53

66: R =
N

17%

15: R =
N

68%

54
55
56
57
58
59
60
Scheme 6. Synthesis of basic Shld analogues.

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59
60

To explore the effect of variations of pKa in the base of Shld and the potential of the vacuole trapping hypothesis (figure 2, C), four other basic Shld analogues were prepared: A piperidine, a pyridine, a primary amine and an aromatic amine (Scheme 6). The latter two had to protected with Boc-groups during the synthesis. In order to prepare the piperidine analogue the alkylation of the phenolic position of 55 were carried out using potassium carbonate and N-(2-bromoethyl)piperidine affording 63 in 26% yield. Similar alkylation of 55 with Boc-protected p-(2-bromoethyl)aniline, with Boc-protected 2-bromoethylamine and with 4-(bromomethyl)pyridine gave 64, 65 and 66 in 26% yield, in 56% yield and in 17% yield, respectively (Scheme 6). These four O-alkylated phenols were then coupled to 56 using DCC and DMAP giving 13, 26, 27, and 15 in 70%, 78%, 55% and 68% yields, respectively. Deprotection of the Boc-group of 26 and 27 were carried out with TFA in dichloromethane to afford the aniline derivative 16 and the primary amine 14 in 93% and 79% respectively (Scheme 6).

A series of neutral analogues with differing size and polarity were also synthesized. Two ketone containing analogues were prepared (Scheme 7) by alkylation of the phenolic position of 1 with bromoacetophenone and chloroacetone affording 67 and 68 in 82% and 76%, respectively. Subsequent DCC coupling with 56 furnished Shld analogues 28 and 29 in 75% and 33%, respectively. An alkyn derivative was also prepared by alkylation with propargyl chloride and K2CO3 to give 69 in 89% yield. Coupling with 56 in the usual way gave 31 in 90% yield. Sulphur containing Shld analogues were synthesized: Alkylation of 1 was performed using chloromethyl methyl sulfide to give 71 in 33% yield (Scheme 7). Coupling to 56 afforded the sulfide analogue 39 in 53% yield and finally oxidation using m-chloroperbenzoic acid (m-CPBA) of the sulfide gave the corresponding sulfone 34 in 51% yield.

Neutral analogues resembling the shape of Shld were also prepared by replacing the amine with

14

1
2
3
4
5
6
7
8
9
10

OH

OR

O

O

O

N

56

O

O

O

11
12
13
14
15

O
HO

55

RX, K2CO3 DMF

O
HO
OCOOH
DCC, DMAP (excess) CH2Cl2

O

O
N

O

O

OR

16
17
O
O
O

18 O O

19
20
21
67
R =
X = Br
82%
28
R =
75%

22 O O

23
24
25
26
27
28
29
68

69
R =
X = Cl

R =
X = Cl
76%

89%
29

31
R =

R =
33%

90%

30
31

70

R =
O
N 60%

30

R =
O
N 90%

32
33
34
X = Cl
O
O

35
36
71
R =
X = Cl
S 33%
33
R =
S 53%

37
38
39
40
41
42

34

R =

O

O
S 51%
m-CPBA

43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 7. Synthesis of neutral Shld analogues with differing size and polarity

a carbamide or carbamate. Reaction of 55 with 4-morpholinecarbonyl chloride under basic conditions gave 70 in 60% yield, which was subjected to DCC-coupling with 56 affording the carbamate 30 in 90% yield (Scheme 7). To closer mimic the structure of Shld the ethyl linker attaching the morpholine and the phenolic position was included in the structure which was done by using the precursor for the primary amine analogue 65 as a starting point (Scheme 8). Deprotection with TFA and reaction with 4-morpholinecarbonyl chloride and potassium carbonate in DMF gave

15

1
2
3
4
5
6
7
8
9
10

carbamide 72 in 80% yield. Yet again, coupling to 56 using the developed DCC/DMAP conditions afforded the carbamide analogue 32 in 70% yield.

11 O O

12
13
14
15

O
O

NH

O

N

NH
O
O

N

56

O

O

O

16
17

O

O

O

COOH

18 1) TFA, CH2Cl2 DCC, DMAP (excess) N

19
20
21
22
23

HO
2)
O

NCl O
K2CO3, DMF

HO
CH2Cl2 70%

O

O

O

O

O

O

N
H

O

N

O

24
25
26

OO
80%

OO

32

27 65 72

28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Scheme 8. Synthesis of Shld analogue with morpholine scaffold but as a carbamide unable to act as a base in

the aqueous buffer

The hypothesis that small molecules more readily penetrate the cuticle led us to prepare a smaller truncated Shld analogue. The synthesis was simply carried out by coupling of commercially available 3-(3,4-dimethoxyphenyl)propanol with 56 giving 35 in 55% yield (Scheme 9).

O

43
44
O
O

56

45
O

46
47
N
O
O
35

48
49
50

O

OH
O COOH
DCC, DMAP (excess)

N

51
52
53
54
55
56
57
58

O
CH2Cl2 55%

O

O

O

O

O

59
60
Scheme 9. Synthesis of truncated Shld analogue 35.

16

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3
4
5
6
7
8
9
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11
12

In order to test the in vitro binding of the new compounds a fluorescence polarization competition assay was devised. A fluorescein labeled version of the known SLF compound has been widely used in fluorescence polarization assays for high throughput screening studies of

13
14
15

27

1)TFA, CH2Cl2, rt

O
O

O

OH

16
17
18
19
20
21
22
23
24
25
26
27

HO
2)DIPEA
O OH

O
O
O
O
FluoresceinNHS
79%

O

N

O

O

O

O

N

O

O
73

O
O

N
H

O

O

O

OH

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31
32
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34
35
36
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55
56
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58
59
60
Scheme 10. Synthesis of a Fluorescein-labeled Shld derivative 73

inhibitors towards the FKBPs.18 For the binding studies of the DD protein, we synthesized an adapted fluoresceinated version of Shld as outlined in scheme 10. From the ester 27 the Boc group was removed with TFA and the resulting amine was coupled with commercial 6-carboxyfluorescein succinimide (6-Fluorescein-NHS, Scheme 10) in the presence of Hünigs base to give the fluorescein-labeled Shld conjugate 73 in 79 % yield.

A binding curve was generated in a saturation binding study of the fluorescent probe 73 with a maltose-binding protein fused DD protein (MBP-DD, Figure 3 left). A binding constant of 3.01 ± 1.02 nM was observed, which were in the expected area for binding to the DD.3,18 In order to identify the binding affinity of ligands for the DD protein, the binding of the novel fluorescent probe (73) was tested in competition with Shld. A Ki value of 7.49 ± 1.21 nM was obtained (Figure 3, right). With the fluorescence polarity assay established we determined the binding of the Shld

17

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60

derivatives to the DD protein in vitro. The modifications in residue A gave the Ki values shown in Table 1 & 2.

Figure 3. Fluorescence polarization anisotropy versus His-MBP-DD-FKBP12 added to fluorescent ligand 73 (left) & Shld replacement of fluorescent probe 73 from His-MBP-DD-FKBP12 (right)

The effect of the compounds in transgenic plants of species Arabidopsis with the 35S::DD– EGFP mutation8 was also investigated. Plants were sprayed with solutions of 1-35 and immunoblot analyses revealed various levels of accumulation of RDDKeGFP (Figure 4). The results were compared to RDDKeGFP accumulation from Shld and untreated plants and the value relative effectiveness (Erel) was determined as the percentage of RDDKeGFP stabilization compared to Shld

Figure 4. Accumulation of RDDKeGFP in transgenic Arabidopsis treated with Shld derivatives 29,30 &

32

18

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6
7
8
9
10
11
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15
16
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19
20
21
22

(Table 1 & 2). Erel was calculated as (Ix-Iu)/(Ishld-Iu), where Ix is the relative intensity from the derivative investigated, Ishld is the intensity from Shld and Iu is the intensity in untreated plant.

The naphtyl derivative 1 bound with about three hundred fold less affinity than Shld while the trifluorophenyl derivative 3 bound 4000 fold less. The 4-bromophenyl compound 2 was slightly better with a Ki about 100 fold greater than Shld (Table 1). The relative affinity of Shld, 2 and 3 (pKi = 8.1, 6.2 and 4.5) reflects the impact of the substituents on the electron density in the aromatic

R1

23
24
25
R2

O
N

O

O

26
27

N
O
O

28
29
30
31
O

O

32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Compound
Shld
1
2
3*
4
5
6
7
8*
9*
10*
11*
12
R1-group 3,4,5-(MeO)3C6H2-
2-Naphtyl- 4-BrC6H5-
3,4,5-F3C6H2-
4-EtO-3,5-(MeO)2C6H2- 4-PrO-3,5-(MeO)2C6H2-
4-OktO-3,5-(MeO)2C6H2- 4-iBuO-3,5-(MeO)2C6H2-
3,4,5-(MeO)3C6H2- 3,4,5-(MeO)3C6H2- 3,4,5-(MeO)3C6H2- 3,4,5-(MeO)3C6H2- 3,4,5-(MeO)3C6H2-
R2-group Et
Et
Et
Et
Et
Et
Et
Et
2-allyl
1-cyclohexenyl 1-cyclopentenyl 1-cyclobutenyl
Isobutylene
Ki
7.49 ± 1.21 2101 ± 1820
635 ± 248
>10000
13.9 ± 3.63 13.9 ± 1.53 1581 ± 1132 37.8 ± 4.16 35.0 ± 7.82 217 ± 32.3 151 ± 21.8 104 ± 8.27
>10000
Ki(shld)/Ki 1.0 0.00 0.01 0.00 0.54 0.54 0.00 0.20 0.21 0.03 0.05 0.07 0.00
Erel 100%
0%
0%
0%
50.0%
36.0%
5.8%
10.5%
0%
0%
0%
18.8%
2.2%

47
48
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51
52
53
54
55
56
57
58
59
60
Table 1. Dissociation constants (Ki) of shld derivatives modified in the A-residue binding to His-MBP- DD-FKBP12 determined by fluorescence polarization and relative accumulation of DD-EGFP (Erel) induced by the derivatives 1-12 compared to Shld. Erel=(Ix-Iu)/(Ishld-Iu), where Ix, Ishld and Iu are the fluorescence intensity of DD-EFGP in the presences of the derivatives investigated, Shld or untreated plant, respectively. * Compound is 1:1 mixture of diastereomers

19

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7
8
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55
56
57
58
59
60

ring (sum = -0.03, 0.23 and 0.74) according to the equation pKi = -4.5sum + 7.7, where sum is the sum of the Hammett constants (i.e. sum(OMe) = 2 x 0.12-0.27; sum(Br) = 0.23 ; sum(F) = 2 x 0.34+0.06). The very high affinity of the trimethoxyphenyl group has been ascribed to formation of two hydrogen bonds.3 Yet, fluorine can also act as hydrogen bond acceptor19 so the very poor binding of 3 compared to Shld or 2 does not fit well with this explanation. An alternative explanation more consistent with our results would be that this aromatic ring is involved in a interaction whose strength would depend on electron density. However, an x-ray structure of a Shld-analogue bound to F36V-FKBP indicates only a intramolecular T-shape - interaction with

Figure 5. The interaction of shld with the DD (F36V) mutant of FKBP. A) Illustrates the tight fit of a shld- analogue in the binding site of FKBP, B) Presents the important T- stacking of the trimethoxyphenyl with the carboxymethyloxyphen-3-yl group of the shld-analogue likely to facilitate preorganization of the ligand. C) Shows the exact and tight fitting of the ethyl group and the pipercolic acid at the bottom of the binding pocket. The volume surrounding the ethyl group does not appear to allow favorable modifications as seen from the crystal structure perspective. The models were made from the PDB structure 1BL420

one of the benzene rings in the C-fragment (Figure 5).20 Perhaps the huge difference in binding affinity of the various aromatic derivatives is a result of different conformations of the ligand due to a difference in strength of this interaction. This is supported by previous NMR experiments, which indicated that different Shld analogues are bound in different conformations.4

20

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The 4-O-alkylated derivatives 4-7 exhibited decreasing levels of binding with size of the substituent. The ethyl and propyl derivatives 4 and 5 had only slightly reduced affinity, while isobutyl derivative 7 was 4 times less potent than Shld (Table 1). A very big drop in binding was observed for the octyl derivative 6, which binds 200 times weaker than Shld. The crystal structure of F36V-FKBP shows there is plenty of space for substituents in this area, which suggests that the low binding of 6 could be related to its lipophilicity in the assay.

For compounds 1-7 the Erel value generally follows the results of the in vitro tests very well. Compounds 1-3, which have close to no affinity in vitro show 0% of the increase in accumulation of RDDKeGFP21 compared to Shld, while 4, 5 and 7 show in vivo effectiveness that very closely mimics the in vitro values. An exception is the octyl derivative 6 which has 5% of the effectiveness of Shld in the plant – yet in vitro the Ki is very high. As discussed above the poor efficacy of 6 in vitro could be related to the assay and its solubility.

Similarly the R2 substituted derivatives 8-12 (Table 1) generally decreased in affinity with increasing size: Allyl (8) had 4 times the Ki of Shld, cyclobutenyl (11) a 15-fold lower affinity, cyclopentenyl (10) a 20-fold lower affinity and cyclohexenyl (9) a 25-fold lower affinity. Only the isobutylene compound 12 was significantly different by having an extremely low affinity. An explanation for these results is that the protein affinity for 8-11 fit more and more poorly into the protein cavity with increased bulk of the substituent. The very low binding of 12 is probably due to a wrong geometry as the -carbon is sp2 hybridized. There is comparatively poor agreement between the protein accumulation in plant and in vitro binding constants for these compounds. Compound 8-10 have Erel of 0% yet particularly 8 shows relatively good in vitro binding. For compound 11 there is however a rough agreement between the in vitro and in vivo data (Table 1).

21

1
2
3
4
5
6
7
8
9
10

O

O

O

N

11 O
O O

12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32

Entry Cmp
113
214
3Shld
415
516
617
718
819
920
1021
1122
1223
1324
1425
O

O

R-group
-CH2CH2N(CH2CH2)2CH2
-CH2CH2NH2
-CH2CH2N(CH2CH2)2O
-CH2C(CHCH)2N
-CH2CH2-p-C6H4-NH2
-PO3H2
-CH2COOH
-CH2CH2CH2COOH
-CH2C(=N)(N=N)NH
-H
-CH2COOC(CH3)3
-CH2CN
-Si(CH3)2C(CH3)3
-PO(OEt)2
OR

Type pKa Ki (nM) Ki(Shld)/Ki Erel
base 10.5 8.78 ± 1.05 0.85 74%
base 9.8 4.08 ± 0.37 1.84 70%
base 7.8 7.49 ± 1.21 1.0 100%
base 6.3 56.0 ± 30.5 0.13 11%
base 5.1 150 ± 55.0 0.05 35%
acid 0.5 228 ± 33.2 0.03 18%
acid 3.2 18.7 ± 3.90 0.40 84%
acid 4.4 38.0 ± 5.05 0.20 77%
acid 4.7 37.2 ± 2.34 0.20 18%
acid 10.0 30.5 ± 11.4 0.24 89%
neutral 109 ± 18.2 0.07 15%
neutral 31.2 ± 7.47 0.24 58%
neutral 1650 ± 906 0.00 10%
neutral 16.0 ± 3.31 0.47 83%

33
34
35
36
37
38
39
40
41
42
43
15
16
17
18
19
20
21
22
23
24
26 -CH2CH2-p-C6H4-NHCOOC(CH3)3
27 -CH2CH2NHCOOC(CH3)3
28 -CH2COC6H5
29 -CH2COCH3
30 -CON(CH2CH2)2O
31 -CH2CCH
32 -CH2CH2NHCON(CH2CH2)2O
33 -CH2SCH3
34 -CH2SO2CH3
35 See scheme 9
neutral neutral neutral neutral neutral neutral neutral neutral neutral neutral
> 10000 109 ± 15.1 91.8 ± 13.7 16.6 ± 2.37 16.8 ± 5.84 56.9 ± 11.8 9.09 ± 0.97 28.1 ± 8.38 8.19 ± 2.25 261 ± 59.8
0.00
0.07
0.08
0.45
0.45
0.13
0.82
0.27
0.91
0.03
19%
2%
21%
113%
126%
62%
107%
86%
76%
80%

44
45
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47
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49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Dissociation constants (Ki) of Shld derivatives modified at the C-residue binding to His-MBP- DD-FKBP12 determined by fluorescence polarization and relative accumulation of RDDKeGFP (Erel) induced by the derivatives 13-35. pKa values listed are not the pKa values of the compound but of similar, simpler, compounds containing the same functional group.

The test results of the C-residue modifications are shown in Table 2, where they are listed according to their acid-base properties and their pKa values. The primary amine analogue 14 (Table 2, entry 2) showed the strongest binding towards the protein (Ki = 4.08 nM) of all the tested

22

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56
57
58
59
60

compounds in the in vitro assay, providing a twofold increase in binding affinity compared to Shld. The piperidine derivative 13 (entry 1) gave a binding affinity similar to Shld, whereas the pyridine 15 (entry 4) and aniline 16 (entry 5) derivative afforded a 7-fold and 20-fold decrease in binding affinity respectively compared to Shld. The significant change in binding affinity for the base analogues suggests that the phenolic substituent does indeed influence the binding towards the protein. The higher pKa bases are more protonated in the buffer (pH 8.0) suggesting that having an positive charge in this area is favorable. The in vivo assay only 13 and 14 provided stabilization and accumulation of the RDDKeGFP protein similar to Shld (Erel 70% and 74% respectively) and overall the protein accumulation crudely follows in vitro binding.

The acid analogues 18-20 (entry 6-10) generally showed weaker (than Shld) and individually similar binding affinities (in vitro). The short-chained carboxylic acid 18 is the strongest binding ligand (Ki = 18.7 nM) of the five while the arylphosphoric acid 17 (entry 6) was the weakest with a 5-10 fold decrease in binding affinity (Ki = 228 nM), compared to the other acid analogues. Since all these derivatives are negatively charged at pH 8 and the phosphate particularly so the results suggest that negative charge cause repulsion from the protein much in line with positive charge causing stronger binding. On the other hand in the plant based assay, the five acid analogues generally show much better protein accumulation than anticipated based on receptor binding which suggest that the ion-trapping mechanism (Figur 2B) is working with these derivatives. Best stabilization of the RDDKeGFP protein was observed for 18, 19 and 21 (Erel of 84%, 77% and 89% respectively), while the tetrazole analogue 20 and the phosphate 17 had a lower effect (Erel of 18%). It should be noted that the higher pKa acids 19 and 21 are superior at accessing the plant cell compared to Shld (Erel 89% and 77% respectively) when taking into account their receptor binding

23

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affinity is lower Shld (Ki(Shld)/Ki = 0.24 and 0.20 respectively). For 21 there could be an influence of this compound being of smaller size and possible more likely superior at penetrating the cuticle.

The neutral analogues 22-35 (entries 11-24) had a surprisingly large span in binding affinities given the supposition that the modification is outside the receptor binding site. The most potent derivatives, 25,29-30,32 and 34, have polar and typically small substituents and a binding as Shld or twofold lower. In contrast the derivatives with the bulky, lipophilic derivatives 24 and 26 (entry 13 and 15) showed the weakest in vitro binding of the entire assay. The other bulky tert-butyl ester or carbamates 22 and 27 also gave relatively poor binding affinities. In the plant assay GFP accumulation roughly followed receptor affinity. The best analogues 29-30 (entry 18-19) and the carbamide 32 (entry 21) exhibited better protein accumulation that Shld (Erel = 113%, 126% and 107% respectively). In contrast, the large and bulky groups gave the low GFP protein. Overall the results show that Shld’s ability to act as a base is not required neither for binding to the receptor or for biological uptake in the plant.

The truncated analogue 35 showed poor receptor binding (261 nM), yet very respectable GFP accumulation (Erel 80%). It is probable that the effectiveness of the compound is a result of the more effective penetration of the cuticle by a small compound such as 35.

Conclusions

In conclusion this study has shown that the electron rich nature of aromatic group in Shld is important for binding and that only minor modifications in the A-fragment are allowed to retain affinity. Modifications in the C-fragment had a surprisingly large variation in receptor binding affinities which appears to contradict the statement1 that the phenolic substituent is not related to binding. The results showed that it is beneficial for receptor binding to have a positive charge in this

24

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60

area while negative charge is bad. Strongly basic derivative 14 had twice the affinity of Shld. In the plant Shld derivatives with acidic and basic groups did not show better protein accumulation that the parent. In contrast analogous with small neutral substituents, 29, 30 and 32, gave slightly better GFP production than Shld. This is probably due to better penetration. This was supported by the observation that the simplified Shld analogue 35 despite being a comparatively weak binder, showed 80% of the effect of Shld in plants.

Experimental

General information.
Air and water sensitive reactions were carried out under nitrogen. All commercial available chemicals and solvents were used as received. 1H NMR spectra were measured on a Bruker instrument with cryo-probe at 500 MHz. 13C NMR was measured on the same instrument but at 126 MHz. 19F NMR was recorded on a Bruker instrument with inverse probe at 470 MHz. NMR solvents used were CDCl3 (D 99.8%, referenced to δH = 7.26 ppm (CHCl3) and δC = 77.16 ppm (CDCl3)) and DMSO-d6 (D 99.8%, referenced to δH = 2.50 ppm (DMSO-d5) and δC = 39.52 ppm (DMSO-d6)). Fluorine NMR were carried out in CDCl3 with a lock tube containing TFA (referenced to δF = -76.55 ppm). Coupling constants (J) was given in Hertz (Hz). CDCl3 was passed through activated Al2O3 (basic) prior to use. Anhydrous solvents were collected from an IT (Innovative Technology) installation of the model PS-MD-05. Thin-layer chromatography (TLC) was performed on precoated (silica 60) aluminum plates with fluorescence indicator. Flash column chromatography was on silica (SiO2) with particle size 40-63 µm from ROTH. Optical rotation was measured on an Anton Paar MCP 300 polarimeter with a 50 x 5 mm cuvette. UPLC-MS (Ultra Performance Liquid Chromatography – Mass Spectrometry) was performed on a Dionex UltiMate

25

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3000 RS with an AcclaimTM RSLC 120 C18 column (2.2 µm, 120 Å, 2.1 x 100 mm) connected to a Bruker micrOTOF Q-III mass spectrometer. High resolution mass spectrometry (HR-MS) was on a FT-ICR spectrometer using either matrix assisted laser desorption ionization (MALDI) with dithranol as matrix or electrospray ionization (ESI+) with methanol + 1% TFA. Melting points are uncorrected. Koga’s base is N1,N3-bis((R)-1-phenyl-2-(piperidin-1-yl)ethyl)propane-1,3-diamine and was prepared as previously described.22 For simplicity, the integrals in 1H NMR of rotamers (when both rotamers are distinguishable) are reported as the sum of the major- and minor contributor (e.g. for rotamer ratio of 1:0.7), 1 proton reported as 1H and 0.7H instead of 0.59H and 0.41H). Tested compounds were analyzed with LCMS confirming > 95% purity.

General procedure for amide bond formation. (R)-3-(3,4-dimethoxyphenyl)-1-(3-(2- morpholinoethoxy)phenyl)propyl (S)-1-((S)-2-naphtylbutanoyl)piperidine-2-carboxylate (1)

To flask A, a solution of 40 (100 mg, 0.47 mmol) in anhydrous CH2Cl2 (4 mL) was added thionyl chloride (0.25 mL, 3.4 mmol) and the solution was heated to reflux and kept at this temperature for 2 hours. The contents of the reaction vessel were concentrated in vacuo and redissolved in anhydrous CH2Cl2 (3 mL). In a separate flask B, a solution of 39 (100 mg, 0.136 mmol) in anhydrous CH2Cl2 (5 mL) was added DBU (22 µL, 0.15 mmol). After 1 hour TLC analysis indicated complete cleavage of Fmoc. To flask B, Et3N (0.1 mL, 0.7 mmol) was added followed by the contents of flask A. The resulting solution was left stirring 30 min before it was concentrated in vacuo and subjected to flash column chromatography (30% acetone in toluene + 1% Et3N) to yield 1 (80 mg, 83%). 1H NMR (600 MHz, CDCl3, mixture of rotamers, 1:0.4, A:B): δ 7.83 – 7.79 (m, 1.4H, A + B), 7.77 – 7.68 (m, 3.8H, A + B), 7.66 (d, J = 1.8 Hz, 0.4H, B), 7.50 – 7.44 (m, 1.8H, A + B) 7.44 – 7.39 (m, 2H, A), 7.38 (dd, J = 8.5, 1.8 Hz, 0.4H, B), 7.31 (t, J = 7.9 Hz, 0.4H, B), 6.95 (d, J = 7.7 Hz, 0.4H, B), 6.93 – 6.89 (m, 0.4H, B), 6.90 – 6.85 (m, 0.4H, B), 6.82 – 6.74 (m, 2.4H,

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A + B), 6.70 (dd, J = 8.2, 2.1 Hz, 0.4H, B), 6.70 – 6.64 (m, 2H, A), 6.65 (s, 1.4H, A + B), 6.62 – 6.58 (m, 1H, A), 6.23 (d, J = 7.6 Hz, 1H, A), 5.82 (dd, J = 7.8, 6.0 Hz, 0.4H, B), 5.62 (dd, J = 8.2, 5.6 Hz, 1H, A), 5.54 (d, J = 4.2 Hz, 1H, A), 4.69 (d, J = 5.7 Hz, 0.4H, B), 4.64 (d, J = 13.9 Hz, 0.4H, B), 4.14 (t, J = 5.5 Hz, 0.8H, B), 3.96 – 3.91 (m, 3H, A), 3.87 (s, 1.2H, B), 3.86 (s, 4.2H, A + B), 3.84 (s, 4H, A), 3.77 – 3.69 (m, 5.6H, A + B), 3.55 (t, J = 7.1 Hz, 0.4H, B), 2.86 – 2.81 (m, 0.8H, B), 2.74 – 2.69 (m, 2H, A), 2.69 – 2.47 (m, 8.8H, A + B), 2.45 – 2.36 (m, 1H, A), 2.33 – 2.26 (m, 1.4H, A + B), 2.25 – 2.15 (m, 1.4H, A + B), 2.14 – 2.06 (m, 0.4H, B), 2.04 – 1.95 (m, 1H, A),
1.96– 1.90 (m, 0.4H, B), 1.91 – 1.83 (m, 1H, A), 1.84 – 1.75 (m, 1.4H, A + B), 1.69 – 1.60 (m, 2H, A), 1.57 – 1.52 (m, 1.4H, A + B), 1.49 – 1.39 (m, 1.8H, A + B), 1.28 – 1.14 (m, 1.8H, A + B), 0.91 (t, J = 7.3 Hz, 3H, A), 0.84 (t, J = 7.3 Hz, 1.2H, B) ppm. 13C NMR (151 MHz, CDCl3, mixture of rotamers, 1:0.4, A:B): δ 172.70 (B), 172.41 (A), 170.77 (A), 170.67 (B), 159.09 (B), 158.60 (A), 149.10 (B), 148.99 (A), 147.62 (B), 147.43 (A), 141.73 (A), 141.63 (B), 137.90 (B), 137.34 (A), 133.82 (A), 133.71 (B), 133.64 (A), 133.41 (B), 132.61 (A), 132.59 (B), 129.92 (B), 129.47 (A), 128.89 (B), 128.59 (A), 127.87 (A), 127.86 (B), 127.81 (B), 127.78 (A), 127.01 (A), 126.37 (B), 126.36 (A), 126.32 (B), 126.21 (A), 125.95 (B), 125.87 (B), 125.80 (A), 120.36 (A), 120.27 (B), 119.21 (B), 118.90 (A), 114.36 (B), 114.06 (A), 113.16 (B), 113.01 (A), 111.95 (A), 111.80 (B), 111.51 (B), 111.45 (A), 76.94 (B), 76.05 (A), 66.98 (A + B), 65.98 (B), 65.68 (A), 57.81 (B), 57.71 (A), 56.08 (A + B), 56.00 (B), 55.96 (A), 55.88 (B), 54.25 (B), 54.16 (A), 52.18 (A), 51.30 (B), 50.89 (A), 43.77 (A), 39.80 (B), 38.15 (B), 38.08 (A), 31.67 (B), 31.29 (A), 28.51 (B), 28.34 (A), 26.84 (A), 26.60 (B), 25.57 (A), 24.55 (B), 21.11 (A), 20.80 (B), 12.72 (B), 12.55 (A) ppm. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C43H52N2O7Na+ 731.3667; Found 731.3664.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-morpholinoethoxy)phenyl)propyl (S)-1-((S)-2-(4- bromophenyl)butanoyl)piperidine-2-carboxylate (2)

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The reaction was carried out as described for 1, using 42 (100 mg, 0.41 mmol) and anhydrous THF as solvent. Purification was performed by flash column chromatography (30% acetone in toluene + 1% Et3N) yielding 2 as a slightly yellow oil (79 mg, 72%). 1H NMR (600 MHz, CDCl3, mixture of rotamers, 1:0.25, A:B): δ 7.46 – 7.42 (m, 0.5H, B), 7.39 – 7.35 (m, 2H, A), 7.17 – 7.12 (m, 2H, A), 7.13 – 7.08 (m, 0.5H, B), 6.92 (d, J = 7.7 Hz, 0.25H, B), 6.89 – 6.83 (m, 1H, A), 6.82 – 6.77 (m, 2.5H, A + B), 6.69 – 6.64 (m, 4H, A + B), 6.56 (d, J = 7.6 Hz, 1H, A), 5.82 – 5.77 (m, 0.25H, B), 5.67 (dd, J = 8.1, 5.8 Hz, 1H, A), 5.47 (d, J = 4.7 Hz, 1H, A), 4.57 (d, J = 13.6 Hz, 0.25H, B), 4.53 (d, J = 5.0 Hz, 0.25H, B), 4.11 (t, J = 5.7 Hz, 0.5H, B), 4.06 (t, J = 5.7 Hz, 1H, A), 4.05 (t, J = 5.7 Hz, 1H, A), 3.85 (br s, 7.5H, A + B), 3.80 – 3.76 (m, 1H, A), 3.75 – 3.71 (m, 5H, A + B), 3.63 (t, J = 7.2 Hz, 1H, A), 3.33 (t, J = 7.1 Hz, 0.25H, B), 2.82 – 2.77 (m, 2.5H, A + B), 2.72 (td, J = 13.4, 3.0 Hz, 1H, A), 2.59 – 2.56 (m, 5H, A + B), 2.57 – 2.49 (m, 1.75H, A + B), 2.44 (ddd, J = 14.0, 9.7, 6.5 Hz, 1H, A), 2.33 – 2.25 (m, 1H, A), 2.25 – 2.22 (m, 0.25H, B), 2.12 – 2.01 (m, 2.75H, A + B),
1.97– 1.87 (m, 1H, A), 1.78 – 1.68 (m, 1.25H, A + B), 1.68 – 1.59 (m, 2H, A), 1.59 – 1.52 (m, 1.5H, A + B), 1.41 (qt, J = 12.8, 3.8 Hz, 1H, A), 1.31 – 1.16 (m, 1.5H, A + B), 1.02 (tdd, J = 12.9, 5.5, 3.7 Hz, 0.25H, B), 0.86 (t, J = 7.3 Hz, 3H, A), 0.77 (t, J = 7.3 Hz, 0.75H, B) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers, 1:0.25, A:B): δ 172.25 (B), 172.06 (A), 170.66 (A), 170.42 (B), 159.07 (B), 158.81 (A), 149.06 (B), 148.98 (A), 147.59 (B), 147.42 (A), 141.70 (A), 141.50 (B), 139.42 (B), 138.83 (A), 133.69 (A), 133.32 (B), 132.13 (B), 131.87 (A), 129.98 (A), 129.89 (B), 129.70 (A), 129.42 (B), 120.99 (B), 120.84 (A), 120.27 (A), 120.23 (B), 119.13 (B), 118.98 (A), 114.30 (B), 114.06 (A), 113.16 (A + B), 111.84 (A), 111.75 (B), 111.46 (A + B), 77.00 (B), 76.14 (A), 67.03 (A + B), 65.96 (B), 65.80 (A), 57.79 (A + B), 56.04 (A), 55.97 (B), 55.96 (B), 55.95 (A), 55.85 (B), 54.24 (B), 54.22 (A), 52.18 (A), 50.42 (B), 50.04 (A), 43.68 (A), 39.76 (B), 38.13 (A), 38.05 (B), 31.62 (B), 31.30 (A), 28.38 (B), 28.26 (A), 26.80 (A), 26.70 (B), 25.52 (A),

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24.47 (B), 21.05 (A), 20.79 (B), 12.52 (B), 12.40 (A). HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd. for C39H50BrN2O7+ 737.2796; Found 737.2776.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-morpholinoethoxy)phenyl)propyl (2S)-1-(2-(3,4,5- trifluorophenyl)butanoyl)piperidine-2-carboxylate (3)

The reaction was carried out as described for 1, using 41 (100 mg, 0.46 mmol). Purification was performed by flash column chromatography (25% acetone in toluene + 1% Et3N) yielding 3 as a slightly yellow oil (103 mg, 97%). 1H NMR (500 MHz, CDCl3, mixture of rotamers (1:0.20) and diastereoisomers (1:1, A:B). Only major rotamer for both diastereomers are reported here): δ 7.26 (t, J = 7.9 Hz, 1H, A or B), 7.17 (t, J = 7.9 Hz, 1H, A or B), 6.93 (dd, J = 8.2, 6.6 Hz, 4H, A + B),
6.93– 6.88 (m, 1H, A or B), 6.87 – 6.86 (m, 1H, A or B), 6.84 (ddd, J = 8.1, 2.6, 0.9 Hz, 1H, A or B), 6.82 – 6.76 (m, 3H, A + B), 6.71 – 6.62 (m, 6H, A + B), 5.75 (dd, J = 7.9, 5.8 Hz, 1H, A or B), 5.70 (dd, J = 8.0, 5.9 Hz, 1H, A or B), 5.49 (d, J = 4.9 Hz, 1H, A or B), 5.45 (d, J = 5.1 Hz, 1H, A or B), 4.11 (t, J = 5.7 Hz, 2H, A or B), 4.07 (t, J = 5.7 Hz, 2H, A or B), 3.86 (s, 3H, A or B), 3.85 (s, 9H, A + B), 3.77 (br d, J = 13.4 Hz, 2H, A + B), 3.75 – 3.72 (m, 8H, A + B), 3.63 (t, J = 7.2 Hz, 1H, A or B), 3.62 (t, J = 7.2 Hz, 1H, A or B), 3.20 (td, J = 13.1, 2.9 Hz, 1H, A or B), 2.86 – 2.81 (m, 1H, A or B), 2.81 (t, J = 5.7 Hz, 2H, A or B), 2.79 (t, J = 5.7 Hz, 2H, A or B), 2.61 – 2.55 (m, 8H, A + B), 2.55 – 2.42 (m, 4H, A + B), 2.34 – 2.27 (m, 2H, A + B), 2.26 – 2.19 (m, 1H, A or B), 2.14 – 2.00 (m, 4H, A + B), 1.95 (ddt, J = 13.8, 9.8, 6.4 Hz, 1H, A or B), 1.76 – 1.59 (m, 6H, A + B), 1.59 – 1.48 (m, 2H, A + B), 1.44 (qt, J = 13.3, 4.2 Hz, 1H, A or B), 1.36 – 1.22 (m, 2H, A + B), 0.95 (qt, J = 12.9, 4.2 Hz, 1H, A or B), 0.88 (t, J = 7.3 Hz, 3H, A or B), 0.87 (t, J = 7.3 Hz, 3H, A or B) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers (1:0.20) and diastereoisomers (1:1, A:B). Only major rotamer for both diastereomers are reported here): δ 171.94 (A or B), 171.48 (A or B), 170.63 (A or B), 170.47 (A or B), 159.03 (A or B), 158.92 (A or B), 152.48 – 150.20 (m, JC-

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F = 250.7 Hz, A + B), 149.05 (A or B), 149.00 (A or B), 147.53 (A or B), 147.46 (A or B), 141.61 (A or B), 141.59 (A or B), 138.86 (dt, J = 250.9, 15.0 Hz, A + B), 136.85 – 136.02 (m, A + B), 133.60 (A + B), 129.79 (A or B), 129.65 (A or B), 120.30 (A or B), 120.27 (A or B), 119.07 (A or B), 118.86 (A or B), 114.10 (A or B), 113.97 (A or B), 113.34 (A or B), 113.31 (A or B), 112.22 (ddd, J = 34.5, 16.3, 5.1 Hz), 111.85 (A or B), 111.83 (A or B), 111.46 (A or B), 111.44 (A or B), 76.52 (A or B), 76.21 (A or B), 67.10 (A or B), 67.09 (A or B), 65.95 (A or B), 65.89 (A or B), 57.84 (A or B), 57.77 (A or B), 56.07 (A or B), 56.06 (A or B), 55.99 (A or B), 55.93 (A or B), 54.28 (A or B), 54.25 (A or B), 52.47 (A or B), 52.37 (A or B), 49.56 (A or B), 49.52 (A or B), 43.77 (A or B), 43.59 (A or B), 38.15 (A or B), 37.92 (A or B), 31.43 (A or B), 31.29 (A or B), 28.31 (A or B), 28.15 (A or B), 26.81 (A or B), 26.66 (A or B), 25.55 (A or B), 25.29 (A or B), 21.05 (A or B), 21.04 (A or B), 12.37 (A or B), 12.34 (A or B). 19F NMR (470 MHz, CDCl3) δ – 132.53 – -132.73 (m), -161.10 – -161.42 (m) ppm. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C39H48F3N2O7+ 713.3408; Found 713.3405.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-morpholinoethoxy)phenyl)propyl (S)-1-(((S)-2-(4- ethoxy- 3,5-dimethoxyphenyl)butanoyl)oxy)piperidine-2-carboxylate (4)

The reaction was carried out as described for 1, using 44 (167 mg, 0.635 mmol). Purification was performed by flash column chromatography (40% acetone in toluene + 1% Et3N) yielding 4 as a colorless oil (99 mg, 89%). 1H NMR (500 MHz, CDCl3, mixture of rotamers (1:0.33).Only major rotamer is reported here): δ 7.26-7.22 (m, 2H), 7.19 ‒ 7.11 (m, 2H), 6.77 (d, J = 7.9 Hz, 1H), 6.66 ‒ 6.63 (m, 1H), 6.40 (s, 2H), 5.60 (dd, J = 8.2, 5.5 Hz, 1H), 5.46 (d, J = 5.1 Hz, 1H), 4.12 (s, 2H), 3.99 (q, J = 7.1 Hz, 2H), 3.65 (2xs, 6H), 3.79 ‒ 3.72 (m, 5H), 3.67 (s, 6H), 3.57 (dd, J = 7.9, 6.3 Hz, 1H), 2.82 (dt, J = 13.3, 3.1 Hz, 2H), 2.65 ‒ 2.38 (m, 8H), 2.30 (d, J = 13.8, 1H), 2.17 ‒ 2,02 (m, 1H), 1.74 ‒ 1.50 (m, 8 H), 1.41 (tt, J = 13.5, 4.1 Hz, 1H), 1.32 (t, J = 7.1, 3H), 0.9 (t, J = 7.3

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Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers (1:0.33). Only major rotamer is reported here): δ 172.77, 170.70, 153.50, 149.01, 147.57, 142.06, 141.57, 135.81, 135.24, 133.64, 129.69, 129.17, 129.17, 128.36, 125.43, 120.34, 114.01, 113.27, 111.87, 111.43, 105.17, 76.01, 68.90, 67.01, 65.90, 57.77, 56.80, 56.00, 54.19, 52.17, 50.95, 43.55, 38.36, 31.43, 28.54, 27.03, 25.51, 21.10, 15.71, 12.74 ppm. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C43H59N2O10+ 763.41553; Found 763.41553.

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(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-morpholinoethoxy)phenyl)propyl dimethoxy-4-propoxyphenyl)butanuyl)oxy)piperidine-2-carboxylate (5)
(S)-1-(((S)-2-(3,5-

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The reaction was carried out as described for 1, using 45 (75 mg, 0.266 mmol). Purification was performed by flash column chromatography (40% acetone in toluene + 1% Et3N) yielding 5 as a colorless oil (82 mg, 72%). 1H NMR (500 MHz, CDCl3, mixture of rotamers (1:0.25). Only major rotamer is reported here): δ 7.14 (dd, J = 9.0, 7.6 Hz, 1H), 6.76 (d, J = 8.2 Hz, 3H), 6.67 ‒ 6.56 (m, 2H), 6.39 (s, 2H), 5.60 (dd, J = 8.2, 5.5 Hz, 1H), 5.47 ‒ 5.42 (m, 1H), 4.17 ‒ 4.06 (m, 2H), 3.9 (t, J = 6.9, 2H), 3.84 (d, J = 2.8, 2H), 3.84 (2xs, 6H), 3.73 (t, J = 4.7 Hz, 5H), 3.66 (s, 6H), 3.56 (dd, J = 7.8, 6.2 Hz, 1H), 2.80 (d, J = 5.3 Hz, 2H), 2.58 (s, 5H), 2.55 ‒ 2.41 (m, 2H), 2.35 ‒ 2.19 (m, 1H), 2.13 ‒ 2.02 (m, 1H), 1.98 ‒ 1.87 (m, 1H), 1.79 ‒ 1.71 (m, 4H), 1.60 ‒ 154 (m, 2H), 1.47 ‒ 1.34 (m, 1H), 1.34 ‒ 1.17 (m, 1H), 0.99 (td, J = 7.4, 2.0 Hz, 2H), 0.95 (t, J = 7.4 Hz, 3H), 0.89 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers (1:0.32). Only major rotamer is reported here): δ 173.72, 176.65, 158.76, 153.50, 148.96, 147.42, 136.17, 135.83, 133.61, 129.63, 120.29, 118.66, 113.94, 112.78, 111.83, 111.39, 105.27, 75.96, 75.08, 66.98, 66.74, 57.77, 56.15, 56.03, 55.95, 54.19, 52.11, 50.89, 43.50, 38.29, 31.37, 28.48, 26.98, 25.46, 23.43, 21.06, 12.69, 10.43 ppm 172.75, 170.66, 158.77, 153.51, 148.96, 147.42, 142.00, 136.18, 135.07, 133.62, 129.64, 120.30, 118.67, 113.94, 112.78, 111.83, 111.40, 105.27, 75.96, 75.08, 66.97, 65.74, 57.77, 56.15,

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56.03, 55.95, 54.19, 52.11, 50.89, 43.50, 38.28, 31.37, 28.48, 26.98, 25.46, 23.43, 21.05, 12.68, 10.43 ppm. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C44H61N2O10+ 777.43207; Found 777.43153.

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(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-morpholinoethoxy)phenyl)propyl dimethoxy-4-(octyloxy)phenyl)butanoyl)piperidine-2-carboxylate (6)
(S)-1-((S)-2-(3,5-

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The reaction was carried out as described for 1, using 46 (132 mg, 0.375 mmol). Purification was performed by flash column chromatography (40% EtOAc in toluene + 1% Et3N) yielding 6 as a brown oil (103 mg, 85%). 1H NMR (500 MHz, CDCl3, mixture of rotamers (1:0.43). Only major rotamer is reported here): δ 7.15 (m, 1H), 6.77 (m, 3H), 6.65 (m, 4H), 6.39 (s, 2H), 5.60 (dd, J=8.3, 5.4 Hz, 1H), 5.45 (d, J=5.4, 1H), 4.20 (br s, 4H), 3.94 (t, J=6.7 Hz, 2H), 3.86 (s, 3H), 3.85, (s, 3H),
3.81(s, 3H), 3.66 (s, 6H), 3.57 (dd, J=7.9, 6.5 Hz, 1H), 2.94 (br s,2H), 2.81 (td, J=13.3, 3.0 Hz, 2H), 2.73 (br s,2H), 2.56 (ddd, J=14.7, 9.8, 5.7 Hz, 3H), 2.48 (m, 2H), 2.30 (d, J=13.4 Hz, 1H), 2.22 (m, 1H), 2.07 (m, 4H), 1.93 (m, 2H), 1.71 (m, 4H), 1.40 (m,2H), 1.28 (m,6H), 0.88 (m,6H) ppm. 13C NMR (126 MHz, CDCl3): δ 172.84, 170.73, 153.58, 149.03, 147.49, 135.14, 134.84, 133.64, 129.75, 129.24, 124.49, 120.36, 114.26, 114.08, 113.30, 111.89, 111.45, 105.32, 75.97, 73.57, 57.52, 56.50, 56.20, 56.08, 56.01, 52.18, 50.95, 43.57, 38.37, 32.01, 31.45, 30.27, 29.56, 29.46, 29.45, 28.55, 27.02, 26.01, 22.50, 22.82, 21.09, 14.26, 12.73 ppm. (1 signal less due to overlapping CH2). HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C49H71N2O10+ 847.51032; Found 847.50834.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-morpholinoethoxy)phenyl)propyl (S)-1-(((S)-2-(3,5- dimethoxy-4-isobutoxyphenyl)butanuyl)oxy)piperidine-2-carboxylate (7)

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The reaction was carried out as described for 1, using 47 (167 mg, 0.630 mmol). Purification was performed by flash column chromatography (40% acetone in toluene + 1% Et3N) yielding 7 as a colorless oil (55 mg, 47%). 1H NMR (500 MHz, CDCl3, mixture of rotamers (1:0.28). Only major rotamer is reported here): δ 7.18 ‒ 7.12 (m, 1H), 6.81 ‒ 6.74 (m, 2H), 6.66 ‒ 6.61 (m 2H), 6.38 (s, 2H), 5.60 (dd, J = 8.1, 5.4 Hz, 1H), 5.45 (d, J = 4.1 Hz, 1H), 4.12 (s, 4H), 3.88 (d, J = 9.8 Hz, 2H), 3.85 (s, 3H), 3.84 (s, 3H), 3.80 (s, 2H), 3.74 (m, 6H), 3.70 (dd, J = 6.7, 1.7 Hz, 1H), 3.65 (s, 6H) 3.65 (dd, J = 7.9, 6.4 Hz, 1H), 3.56 (dd, J = 7.9, 6.4 Hz, 1H), 2.82 (q, J = 4.1, 3.1 Hz, 2H), 2.60 (br s, 4 H), 2.55 ‒ 2.40 (m, 2H), 2.32 ‒ 2.26 (m, 1H), 2.14 ‒ 1.98 (m, 5H), 1.74 ‒ 1.64 (m, 4H), 1.57 (t, J = 12.5 Hz, 2H), 1.02 ‒ 0.99 (m, 3H), 0.98 (dd, J = 6.7, 2.6 Hz, 6H), 0.89 (t, J = 7.3 Hz, 3H) ppm. 13C-NMR (500 MHz, CDCl3, mixture of rotamers (1:0.33). Only major rotamer is reported here): δ 172.78, 170.67, 158.74, 153.49, 148.98, 147.44, 142.04, 136.63, 134.96, 133.63, 120.67, 120.23, 118.75, 113.89, 111.85, 111.41, 107.48, 105.49, 80.08, 75.97, 66.93, 61.25, 57.76, 56.22, 56.05, 55.97, 54.20, 52.13, 50.93, 46.72, 43.51, 38.31, 31.40, 29.14, 28.48, 27.00, 25.48, 21.07, 19.40, 12.69 ppm. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C45H63N2O10+ 791.44772; Found 791.44680.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-morpholinoethoxy)phenyl)propyl (2S)-1-(3-methyl-2- (3,4,5-trimethoxyphenyl)but-3-enoyl)piperidine-2-carboxylate (8)

The reaction was carried out as described for 1, using 48 (246 mg, 0.87 mmol) and 39 (150 mg, 0.204 mmol). Purification was performed by flash column chromatography (30% acetone in toluene + 1% Et3N) yielding 8 as a slightly yellow oil (140 mg, 90%). 1H NMR (500 MHz, CDCl3, mixture of rotamers (1:0.25) and diastereoisomers (1:1, A:B). Only major rotamer for both diastereomers are reported here): δ 7.25 (t, J = 7.9 Hz, 1H, A or B), 7.20 (t, J = 7.9 Hz, 1H, A or B), 6.95 – 6.75 (m, 8H, A + B), 6.69 – 6.61 (m, 4H, A + B), 6.48 (s, 2H, A or B), 6.40 (s, 2H, A or B), 5.77 (dd, J

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= 7.7, 5.9 Hz, 1H, A or B), 5.66 (dd, J = 7.9, 5.6 Hz, 1H, A or B), 5.53 – 5.47 (m, 2H, A + B), 5.03 (s, 1H, A or B), 5.00 (s, 1H, A or B), 4.87 (s, 1H, A or B), 4.73 (s, 1H, A or B), 4.41 (s, 1H, A or B), 4.36 (s, 1H, A or B), 4.12 – 4.07 (m, 4H, A + B), 3.85 (s, 3H, A or B), 3.845 (s, 3H, A or B), 3.841 (s, 3H, A or B), 3.837 (s, 3H, A or B), 3.83 (s, 9H, A + B), 3.80 (s, 3H, A or B), 3.74 – 3.71 (m, 8H, A + B), 3.71 (s, 6H, A or B), 3.68 – 3.62 (m, 2H, A + B), 3.17 (td, J = 13.2, 2.9 Hz, 1H, A or B), 3.08 (td, J = 13.1, 3.0 Hz, 1H, A or B), 2.79 (t, J = 5.8 Hz, 2H, A or B), 2.78 (t, J = 5.8 Hz, 2H, A or B), 2.63 – 2.46 (m, 12H, A + B), 2.33 (d, J = 13.1 Hz, 2H, A + B), 2.29 – 2.14 (m, 2H, A + B), 2.12 – 1.97 (m, 2H, A + B), 1.81 – 1.52 (m, 12H, A + B), 1.49 – 1.14 (m, 4H, A + B) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers (1:0.25) and diastereoisomers (1:1, A:B). Only major rotamer for both diastereomers are reported here): δ 171.47 (A or B), 171.26 (A or B), 170.71 (A or B), 170.69 (A or B), 158.96 (A or B), 158.95 (A or B), 153.18 (A or B), 153.16 (A or B), 149.01 (A or B), 148.99 (A or B), 147.48 (A or B), 147.47 (A or B), 144.43 (A or B), 143.16 (A or B), 141.85 (A or B), 141.63 (A or B), 137.21 (A or B), 136.92 (A or B), 133.64 (A or B), 133.60 (A or B), 133.43 (A or B), 132.79 (A or B), 129.72 (A or B), 129.67 (A or B), 120.24 (A + B), 119.14 (A or B), 118.80 (A or B), 114.72 (A or B), 114.30 (A or B), 114.04 (A or B), 113.90 (A or B), 113.33 (A or B), 112.90 (A or B), 111.85 (A or B), 111.80 (A or B), 111.42 (A + B), 106.45 (A or B), 106.25 (A or B), 76.38 (A or B), 76.30 (A or B), 67.04 (A + B), 65.91 (A or B), 65.83 (A or B), 60.94 (A or B), 60.87 (A or B), 57.80 (A + B), 56.93 (A or B), 56.88 (A or B), 56.25 (A + B), 56.06 (A + B), 56.04 (A + B), 55.96 (A + B), 54.23 (A + B), 52.34 (A or B), 52.14 (A or B), 44.02 (A or B), 43.81 (A or B), 38.36 (A or B), 38.18 (A or B), 31.47 (A or B), 31.40 (A or B), 27.07 (A or B), 26.96 (A or B), 25.38 (A + B), 22.48 (A or B), 22.18 (A or B), 21.17 (A or B), 21.08 (A or B) ppm. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C43H57N2O10+ 761.4008; Found 761.4004.

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(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-morpholinoethoxy)phenyl)propyl (S)-1-(2-(cyclohex-1- en-1-yl)-2-(3,4,5-trimethoxyphenyl)acetyl)piperidine-2-carboxylate (9)

The reaction was carried out as described for 1, using 49 (136 mg, 0.419 mmol) and 39 (120 mg, 0.163 mmol). Purification was performed by flash column chromatography (30% acetone in toluene + 1% Et3N) yielding 9 as a slightly yellow oil (100 mg, 76%). 1H NMR (500 MHz, CDCl3, mixture of rotamers (1:0.20) and diastereoisomers (1:1, A:B). Only major rotamer for both diastereomers are reported here): δ 7.26 – 7.24 (m, 1H, A or B), 7.19 – 7.15 (m, 1H, A or B), 6.94 – 6.90 (m, 1H, A or B), 6.88 – 6.81 (m, 5H, A + B), 6.78 (dd, J = 8.1, 3.4 Hz, 2H, A + B), 6.71 – 6.61 (m, 4H, A + B), 6.46 (s, 2H, A or B), 6.40 (s, 2H, A or B), 5.78 (dd, J = 7.9, 5.8 Hz, 1H, A or B), 5.67 (dd, J = 8.1, 5.5 Hz, 1H, A or B), 5.58 – 5.55 (m, 1H, A or B), 5.51 (br t, J = 6.5 Hz, 2H, A + B), 5.49 – 5.44 (m, 1H, A or B), 4.31 (s, 1H, A or B), 4.29 (s, 1H, A or B), 4.13 – 4.08 (m, 4H, A + B), 3.86 (s, 3H, A or B), 3.854 (s, 3H, A or B), 3.850 (s, 3H, A or B), 3.84 (s, 3H, A or B), 3.84 (s, 9H, A + B), 3.81 (s, 3H, A or B), 3.75 – 3.72 (m, 14H, A + B), 3.72 – 3.68 (m, 2H, A or B), 3.18 (td, J = 13.3, 2.9 Hz, 1H, A or B), 3.09 (td, J = 13.1, 3.0 Hz, 1H, A or B), 2.80 (q, J = 5.9 Hz, 4H, A + B), 2.64 – 2.47 (m, 12H, A + B), 2.35 – 2.30 (m, 2H, A + B), 2.28 – 2.16 (m, 2H, A + B), 2.14 – 2.01 (m, 6H, A + B), 1.98 – 1.82 (m, 4H, A + B), 1.77 – 1.52 (m, 15H, A + B), 1.46 – 1.28 (m, 3H, A + B) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers (1:0.20) and diastereoisomers (1:1, A:B). Only major rotamer for both diastereomers are reported here): δ 171.95 (A or B), 171.71 (A or B), 170.80 (A or B), 170.73 (A or B), 158.97 (A + B), 153.12 (A or B), 153.10 (A or B), 149.04 (A or B), 149.03 (A or B), 147.51 (A or B), 147.50 (A or B), 141.87 (A or B), 141.81 (A or B), 137.06 (A or B), 136.88 (A or B), 136.81 (A or B), 135.76 (A or B), 134.09 (A or B), 133.71 (A or B),
133.65(A or B), 133.40 (A or B), 129.74 (A or B), 129.71 (A or B), 125.47 (A or B), 125.36 (A or B), 120.29 (A or B), 120.27 (A or B), 119.11 (A or B), 118.92 (A or B), 113.98 (A or B), 113.91

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(A or B), 113.39 (A or B), 113.06 (A or B), 111.89 (A or B), 111.84 (A or B), 111.45 (A + B), 106.55 (A or B), 106.40 (A or B), 76.30 (A or B), 76.27 (A or B), 67.07 (A + B), 65.93 (A or B), 65.85 (A or B), 60.97 (A or B), 60.90 (A or B), 57.82 (A + B), 57.12 (A or B), 57.07 (A or B), 56.27 (A + B), 56.12 (A + B), 56.07 (A + B), 55.99 (A + B), 54.26 (A or B), 54.25 (A or B), 52.33 (A or B), 52.11 (A or B), 43.97 (A or B), 43.80 (A or B), 38.38 (A or B), 38.32 (A or B), 31.52 (A or B), 31.40 (A or B), 28.40 (A or B), 28.30 (A or B), 27.15 (A or B), 27.05 (A or B), 25.52 (A + B), 25.48 (A or B), 25.46 (A or B), 23.15 (A or B), 23.14 (A or B), 22.43 (A or B), 22.39 (A or B), 21.23 (A or B), 21.21 (A or B) ppm. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C46H61N2O10+ 801.4321; Found 801.4324.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-morpholinoethoxy)phenyl)propyl (S)-1-(2-(cyclopent-1- en-1-yl)-2-(3,4,5-trimethoxyphenyl)acetyl)piperidine-2-carboxylate (10)

The reaction was carried out as described for 1, using 50 (100 mg, 0.32 mmol). Purification was performed by flash column chromatography (30% acetone in toluene + 1% Et3N) yielding 10 as a slightly yellow oil (80 mg, 75%). 1H NMR (500 MHz, CDCl3, mixture of rotamers (1:0.23) and diastereoisomers (1:1, A:B). Only major rotamer for both diastereomers are reported here): δ 7.25 (t, J = 7.9 Hz, 1H, A or B), 7.19 (d, J = 7.9 Hz, 1H, A or B), 6.92 (d, J = 7.6 Hz, 1H, A or B), 6.87 – 6.76 (m, 7H, A + B), 6.70 – 6.61 (m, 4H, A + B), 6.47 (s, 2H, A or B), 6.40 (s, 2H, A or B), 5.78 (dd, J = 7.6, 5.9 Hz, 1H, A or B), 5.65 (dd, J = 7.9, 5.5 Hz, 1H, A or B), 5.56 – 5.54 (m, 1H, A or B), 5.49 (br t, J = 5.6 Hz, 2H, A + B), 5.42 – 5.40 (m, 1H, A or B), 4.54 (s, 1H, A or B), 4.49 (s, 1H, A or B), 4.16 – 4.05 (m, 4H, A + B), 3.86 (s, 3H, A or B), 3.85 (s, 3H, A or B), 3.846 (s, 3H, A or B), 3.842 (s, 3H, A or B), 3.83 (s, 9H, A + B), 3.81 (s, 3H, A or B), 3.77 – 3.70 (m, 16H, A + B), 3.17 (td, J = 13.3, 2.8 Hz, 1H, A or B), 3.06 (td, J = 12.9, 2.8 Hz, 1H, A or B), 2.85 – 2.76 (m, 4H, A + B), 2.64 – 2.47 (m, 12H, A + B), 2.38 – 2.29 (m, 6H, A + B), 2.28 – 2.16 (m, 6H, A + B), 2.10

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– 1.97 (m, 2H, A + B), 1.93 – 1.84 (m, 4H, A + B), 1.76 – 1.59 (m, 5H, A + B), 1.57 – 1.50 (m, 1H, A or B), 1.47 – 1.27 (m, 3H, A + B), 1.23 – 1.10 (m, 1H, A or B) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers (1:0.23) and diastereoisomers (1:1, A:B). Only major rotamer for both diastereomers are reported here): δ 171.61 (A or B), 171.42 (A or B), 170.78 (A or B), 170.71 (A or B), 158.89 (A + B), 153.20 (A or B), 153.17 (A or B), 149.03 (A + B), 147.51 (A + B), 143.27 (A or B), 142.10 (A or B), 141.93 (A or B), 141.75 (A or B), 137.11 (A or B), 136.84 (A or B), 134.03 (A or B), 133.67 (A or B), 133.63 (A or B), 133.38 (A or B), 129.77 (A or B), 129.71 (A or B), 128.63 (A or B), 128.36 (A or B), 120.27 (A + B), 119.20 (A or B), 118.94 (A or B), 114.03 (A or B), 113.95 (A or B), 113.34 (A or B), 113.23 (A or B), 111.87 (A or B), 111.84 (A or B), 111.45 (A + B), 106.25 (A or B), 106.06 (A or B), 76.31 (A or B), 76.28 (A or B), 66.94 (A + B), 65.77 (A + B), 60.97 (A or B), 60.91 (A or B), 57.76 (A + B), 56.30 (A + B), 56.13 (A + B), 56.07 (A + B), 55.99 (A + B), 54.18 (A + B), 52.37 (A or B), 52.18 (A or B), 51.73 (A or B), 51.68 (A or B), 44.01 (A or B), 43.84 (A or B), 38.36 (A or B), 38.26 (A or B), 34.75 (A or B), 34.59 (A or B), 32.64 (A or B), 32.45 (A or B), 31.50 (A or B), 31.43 (A or B), 27.09 (A or B), 27.01 (A or B), 25.43 (A or B), 25.40 (A or B), 23.77 (A or B), 23.67 (A or B), 21.22 (A or B), 21.11 (A or B) ppm. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C45H59N2O10+ 787.4164; Found 787.4173.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-morpholinoethoxy)phenyl)propyl (S)-1-(2-(cyclobut-1-en- 1-yl)-2-(3,4,5-trimethoxyphenyl)acetyl)piperidine-2-carboxylate (11)

The reaction was carried out as described for 1, using 51 (80 mg, 0.27 mmol). Purification was performed by flash column chromatography (30% acetone in toluene + 1% Et3N) yielding 11 as a slightly yellow oil (50 mg, 48%). 1H NMR (500 MHz, CDCl3, mixture of rotamers (1:0.24) and diastereoisomers (1:0.8, A:B). Only major rotamer for both diastereomers are reported here): δ 7.27 – 7.23 (m, 1H, A), 7.21 – 7.15 (m, 1H, B), 6.94 – 6.89 (m, 1H, A), 6.89 – 6.74 (m, 6H, A + B) 6.76

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– 6.73 (m, 1H, B), 6.70 – 6.61 (m, 4H, A + B), 6.50 (s, 2H, A), 6.43 (s, 2H, B), 5.86 – 5.82 (m, 1H, A), 5.78 – 5.74 (m, 2H, A + B), 5.64 (dd, J = 8.1, 5.6 Hz, 1H, B), 5.50 (br d, J = 5.2 Hz, 1H, A), 5.47 (br d, J = 5.7 Hz, 1H, B), 4.50 (s, 2H, A + B), 4.16 – 4.09 (m, 4H, A + B), 3.86 (s, 3H, A or B), 3.85 (s, 3H, A or B), 3.848 (s, 3H, A or B), 3.846 (s, 3H, A or B), 3.84 – 3.83 (m, 9H, A + B), 3.84 – 3.80 (m, 2H, A + B), 3.81 (s, 3H, B), 3.77 – 3.73 (m, 8H, A + B), 3.72 (s, 6H, B), 3.21 (td, J = 13.3, 2.9 Hz, 1H, A), 3.02 (td, J = 13.1, 3.0 Hz, 1H, B), 2.86 – 2.78 (m, 4H, A + B), 2.66 – 2.48 (m, 16H, A + B), 2.42 – 2.29 (m, 8H, A + B), 2.28 – 1.96 (m, 4H, A + B), 1.78 – 1.50 (m, 6H, A + B), 1.38 – 1.27 (m, 2H, A + B) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers (1:0.24) and diastereoisomers (1:0.8, A:B). Only major rotamer for both diastereomers are reported here): δ 170.83 (B), 170.71 (A), 170.64 (B), 170.61 (A), 158.93 (A + B), 153.35 (A), 153.29 (B), 149.04 (A + B), 147.52 (A + B), 147.01 (B), 146.14 (A), 141.91 (B), 141.73(A), 137.16 (A), 136.93 (B),
133.66(A), 133.62 (B), 132.92 (A), 132.22 (B), 131.77 (A), 131.33 (B), 129.79 (A), 129.72 (B), 120.29 (A + B), 119.20 (A), 118.92 (B), 114.06 (A), 113.98 (B), 113.28 (A), 112.96 (B), 111.88 (A), 111.86 (B), 111.46 (A), 111.45 (B), 105.88 (A), 105.80 (B), 76.38 (A), 76.30 (B), 67.01 (A + B), 65.84 (A + B), 61.00 (A), 60.92 (B), 57.79 (A + B), 56.35 (A + B), 56.16 (A + B), 56.08 (A + B), 56.01 (A + B), 54.20 (A + B), 52.40 (A), 52.21 (B), 51.88 (A), 51.64 (B), 44.05 (A), 43.91 (B), 38.34 (B), 38.26 (A), 31.50 (B), 31.45 (A), 31.17 (A + B), 27.06 (B), 27.01 (A), 26.92 (A + B), 25.42 (B), 25.31 (A), 21.21 (A), 21.09 (B) ppm. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C44H57N2O10+ 773.40077; Found 773.39995.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-morpholinoethoxy)phenyl)propyl (S)-1-((E)-4-methyl-2- (3,4,5-trimethoxyphenyl)pent-2-enoyl)piperidine-2-carboxylate (12)

The reaction was carried out as described for 1, using 54 (115 mg, 0.41 mmol). Purification was performed by flash column chromatography (30% acetone in toluene + 1% Et3N) yielding 12 as a

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slightly yellow oil (76 mg, 72%). 1H NMR (500 MHz, CDCl3, mixture of rotamers (1:0.17). Only major rotamere is reported here): δ 7.21 (t, J = 7.8 Hz, 1H), 6.85 – 6.76 (m, 4H), 6.69 – 6.62 (m, 2H), 6.55 (s, 2H), 5.73 – 5.66 (m, 1H), 5.59 (d, J = 10.3 Hz, 1H), 5.47 (d, J = 5.5 Hz, 1H), 4.11 (t, J = 5.9 Hz, 2H), 3.90 – 3.87 (m, 1H), 3.85 (s, 3H), 3.84 (s, 6H), 3.83 (s, 3H), 3.79 (s, 6H), 3.74 (t, J = 4.7 Hz, 4H), 3.11 (td, J = 13.2, 3.1 Hz, 1H), 2.84 – 2.73 (m, 3H), 2.66 – 2.47 (m, 6H), 2.36 – 2.30 (m, 1H), 2.25 – 2.13 (m, 1H), 2.07 – 1.97 (m, 1H), 1.75 – 1.59 (m, 2H), 1.56 (d, J = 12.4 Hz, 1H), 1.40 – 1.22 (m, 2H), 1.07 (d, J = 6.6 Hz, 3H), 1.04 (d, J = 6.6 Hz, 3H). 13C NMR (126 MHz, CDCl3, mixture of rotamers (1:0.17). Only major rotamer is reported here): δ 171.47, 170.53, 158.84, 153.16, 149.01, 147.48, 141.73, 139.38, 137.68, 134.20, 133.60, 131.11, 129.76, 120.27, 118.99, 114.00, 113.18, 111.87, 111.43, 105.93, 76.37, 66.84, 65.66, 60.96, 57.71, 56.23, 56.05, 55.97, 54.13, 52.00, 45.21, 38.22, 31.39, 27.77, 26.84, 25.37, 22.91, 22.88, 21.33. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C44H59N2O10+ 775.4164; Found 775.4157.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-(piperidin-1-yl)ethoxy)phenyl)propyl (S)-1-((S)-2-(3,4,5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (13)
To a solution of 63 (50.0 mg, 125 µmol), 56 (64 mg, 0.18 mmol) and 4-(dimethylamino) pyridine

(41mg, 0.34 mmol) in anhydrous CH2Cl2 (2 mL) at 0 ºC was added N,N´-dicyclohexylcarbodiimide

(42mg, 0.20 mmol). The reaction mixture was allowed to heat to ambient temperature and left stirring overnight. The resulting suspension was filtered and the filtrate was concentrated (to approximately 0.5 mL) by passing a stream of nitrogen over the surface. A second filtration was performed and the filtrate subjected to flash column chromatography (30% acetone in toluene + 1% Et3N) to yield 13 as a colorless oil (65 mg, 70%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 7.17 – 7.11 (m, 1H), 6.93 – 6.84 (m, 1H), 6.78 – 6.74 (m, 2H), 6.65 – 6.62 (m, 2H), 6.57 (br d, J = 7.7 Hz, 1H), 6.41 (s, 2H), 5.60 (dd, J = 8.0, 5.8 Hz, 1H), 5.46 (br d, J = 5.0 Hz, 1H), 4.07 (t, J = 6.2 Hz, 2H), 3.84 (s, 3H), 3.83 (s, 3H), 3.81 – 3.79 (m,

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1H), 3.78 (s, 3H), 3.69 (s, 6H), 3.59 – 3.55 (m, 1H), 2.81 (td, J = 13.4, 2.9 Hz, 1H), 2.74 (t, J = 6.2 Hz, 2H), 2.63 – 2.39 (m, 6H), 2.30 (br d, J = 13.6 Hz, 1H), 2.13 – 1.99 (m, 2H), 1.97 – 1.86 (m, 1H), 1.73 – 1.63 (m, 3H), 1.61 – 1.56 (m, 5H), 1.47 – 1.39 (m, 3H), 1.30 – 1.20 (m, 1H), 0.89 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 172.63, 170.63, 158.91, 153.28, 148.95, 147.40, 141.83, 136.74, 135.44, 133.63, 129.60, 120.28, 118.45, 113.84, 113.03, 111.82, 111.38, 105.08, 76.04, 66.00, 60.85, 58.07, 56.09, 56.01, 55.92, 55.18, 52.11, 50.87, 43.50, 38.26, 31.34, 28.47, 26.97, 26.05, 25.47, 24.30, 21.07, 12.67. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C43H59N2O9+ 747.4215; Found 747.4211.

(R)-1-(3-(2-aminoethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-((S)-2-(3,4,5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (14)
To a solution of 27 (9.4 mg, 12 µmol) in anhydrous CH2Cl2 (0.5 mL) was added trifluoroacetic acid (50 µL, 0.66 mmol) and the reaction mixture was left stirring for 3 hours. The contents of the reaction vessel were concentrated in vacuo and the crude residue subjected to flash column chromatography (10% MeOH in CH2Cl2 + 1% Et3N) to afford 14 as a colorless oil (6.5 mg, 79%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.3. Only major rotamer is reported here) δ 7.15 (t, J = 7.9 Hz, 1H), 6.79 – 6.75 (m, 2H), 6.71 – 6.70 (m, 1H), 6.67 – 6.63 (m, 2H), 6.62 (dt, J = 7.9, 1.3 Hz, 1H), 6.41 (s, 2H), 5.62 (dd, J = 8.0, 5.6 Hz, 1H), 5.49 – 5.45 (m, 1H), 4.02 – 3.98 (m, 2H), 3.85 (s, 3H), 3.84 (s, 3H), 3.82 – 3.80 (m, 1H), 3.79 (s, 3H), 3.69 (s, 6H), 3.57 (dd, J = 7.8, 6.4 Hz, 1H), 3.11 (dd, J = 5.9, 4.4 Hz, 2H), 2.78 (td, J = 13.3, 3.0 Hz, 1H), 2.55 (ddd, J = 14.7, 10.1, 5.5 Hz, 1H), 2.46 (ddd, J = 13.9, 9.4, 6.7 Hz, 1H), 2.33 – 2.27 (m, 1H), 2.13 – 2.03 (m, 2H), 1.97 – 1.89 (m, 1H), 1.75 – 1.63 (m, 3H), 1.59 (br d, J = 13.6 Hz, 1H), 1.47 – 1.38 (m, 1H), 1.32 – 1.26 (m, 1H), 0.90 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.3. Only major rotamer is reported here) δ 172.69, 170.76, 158.96, 153.32, 149.01, 147.47, 141.99, 136.79, 135.53, 133.64, 129.67, 120.35, 118.98, 113.81, 112.89, 111.89, 111.43, 105.19, 76.05,

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69.56, 60.93, 56.17, 56.07, 55.98, 52.18, 50.94, 43.58, 41.45, 38.23, 31.43, 28.51, 26.96, 25.49, 21.06, 12.69. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C38H51N2O9+ 679.3589; Found 679.3575.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(pyridin-4-ylmethoxy)phenyl)propyl (S)-1-((S)-2-(3,4,5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (15)
To a solution of 66 (27 mg, 71 µmol), 56 (33 mg, 90 µmol) and 4-(dimethylamino) pyridine (75 mg, 0.61 mmol) in anhydrous CH2Cl2 (2 mL) at 0 ºC was added N,N´-dicyclohexylcarbodiimide (19 mg, 92 µmol). The reaction mixture was allowed to heat to ambient temperature and left stirring overnight. The resulting suspension was filtered and the filtrate was concentrated (to approximately 0.5 mL) by passing a stream of nitrogen over the surface. A second filtration was performed and the filtrate subjected to flash column chromatography (EtOAc + 1% Et3N) to yield 15 as a colorless oil (35 mg, 68%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.2. Only major rotamer is reported here) δ 8.57 – 8.51 (m, 2H), 7.32 – 7.28 (m, 2H), 7.11 (t, J = 7.9 Hz, 1H), 6.81 – 6.80 (m, 1H), 6.75 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 6.70 (d, J = 8.0 Hz, 1H), 6.61 – 6.56 (m, 3H), 6.31 (s, 2H), 5.55 (dd, J = 8.3, 5.3 Hz, 1H), 5.41 – 5.37 (m, 1H), 5.06 (d (AB system), J = 13.4 Hz, 1H), 5.03 (d (AB system), J = 13.4 Hz, 1H), 3.780 (s, 3H), 3.777 (s, 3H), 3.75 – 3.72 (m, 1H), 3.71 (s, 3H), 3.58 (s, 6H), 3.51 (dd, J = 7.9, 6.2 Hz, 1H), 2.76 (td, J = 13.3, 3.0 Hz, 1H), 2.53 – 2.45 (m, 1H), 2.45 – 2.37 (m, 1H), 2.25 – 2.19 (m, 1H), 2.08 – 1.96 (m, 2H), 1.88 (dddd, J = 13.9, 9.6, 6.7, 5.4 Hz, 1H), 1.67 – 1.60 (m, 3H), 1.53 (br d, J = 14.1 Hz, 1H), 1.36 (qt, J = 12.9, 3.8 Hz, 1H), 1.26 – 1.17 (m, 1H), 0.83 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.2. Only major rotamer is reported here) δ 172.74, 170.74, 158.29, 153.21, 150.00, 148.91, 147.38, 146.21, 142.23, 136.65, 135.32, 133.44, 129.67, 121.55, 120.21, 119.08, 114.11, 112.65, 111.74, 111.32, 104.94, 75.75, 68.14, 60.77, 55.94, 55.87, 52.07, 50.86, 43.43, 38.22, 31.28, 28.44,

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26.85, 25.32, 20.86, 12.61 ppm (1 Signal missing from OMe, Overlaying with similar OMe signal). HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C42H51N2O9+ 727.3589; Found 727.3584.

(R)-1-(3-(4-aminophenethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-((S)-2-(3,4,5-
trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (16)
To a solution of 26 (19.5 mg, 22.8 µmol) in anhydrous CH2Cl2 (1 mL) was added trifluoroacetic acid (80 µL, 1.1 mmol) and the reaction mixture was left stirring for 3 hours. The contents of the reaction vessel were concentrated in vacuo and the crude residue subjected to flash column chromatography (70% EtOAc in toluene + 1% Et3N) to afford 16 as a colorless oil (16 mg, 93%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.3. Only major rotamer is reported here) δ 7.13 (t, J = 7.9 Hz, 1H), 7.07 (d (AX system), J = 8.2 Hz, 2H), 6.80 – 6.73 (m, 3H), 6.67 – 6.61 (m, 4H), 6.57 (d, J = 7.5 Hz, 1H), 6.42 (s, 2H), 5.60 (dd, J = 7.8, 5.9 Hz, 1H), 5.47 (br d, J = 4.7 Hz, 1H), 4.08 (t, J = 7.2 Hz, 2H), 3.843 (s, 3H), 3.841 (s, 3H), 3.82 – 3.80 (m, 1H), 3.79 (s, 3H), 3.69 (s, 6H), 3.63 – 3.55 (m, 3H), 2.97 (t, J = 6.9 Hz, 2H), 2.81 (td, J = 13.4, 2.8 Hz, 1H), 2.58 – 2.49 (m, 1H), 2.47 – 2.40 (m, 1H), 2.30 (br d, J = 12.1 Hz, 1H), 2.14 – 2.03 (m, 2H), 1.98 – 1.86 (m, 1H), 1.75 – 1.64 (m, 3H), 1.61 – 1.55 (m, 1H), 1.42 (qt, J = 12.8, 3.8 Hz, 1H), 1.34 – 1.22 (m, 1H), 0.90 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.3. Only major rotamer is reported here) δ 172.65, 170.67, 158.98, 153.33, 148.99, 147.44, 145.04, 141.85, 136.79, 135.49, 133.67, 129.96, 129.65, 128.36, 120.33, 118.44, 115.42, 113.90, 113.01, 111.86, 111.42, 105.14, 76.12, 69.19, 60.90, 56.13, 56.06, 55.96, 52.15, 50.91, 43.55, 38.27, 35.09, 31.38, 28.51, 27.01, 25.52, 21.11, 12.72. HRMS (MALDI/FTICR) m/z: [M – e]+ Calcd for C44H54N2O9•+ 754.3824; Found 754.3801.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(phosphonooxy)phenyl)propyl (S)-1-((S)-2-(3,4,5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (17)

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To a solution of 25 (62 mg, 80 µmol) in anhydrous CH2Cl2 (5 mL) at -40 ºC was added trimethylsilyl iodide (23 µL, 0.16 mmol) and the reaction mixture was kept at this temperature for 1 hour and then allowed to heat to ambient temperature over the next 3 hours. The contents of the reaction vessel were concentrated in vacuo and the residue redissolved in THF (4 mL) and added water (1 mL). The resulting solution was left stirring for 1.5 hours before it was concentrated in vacuo and dried on high vacuum for several days to afford 17 as a dark yellow solid (61 mg, quantitative yield). 1H NMR (500 MHz, DMSO-d6, mixture of rotamers 1:0.3. Only major rotamer is reported here) δ 7.25 – 7.21 (m, 1H), 7.15 – 7.02 (m, 2H), 6.83 (d, J = 8.2 Hz, 1H), 6.77 – 6.73 (m, 2H), 6.64 (dd, J = 8.2, 1.7 Hz, 1H), 6.55 (s, 2H), 5.56 – 5.49 (m, 1H), 5.28 (br d, J = 4.8 Hz, 1H), 4.06 – 3.99 (m, 1H), 3.90 – 3.85 (m, 1H), 3.71 (s, 3H), 3.70 (s, 3H), 3.61 (s, 6H), 3.57 (s, 3H), 2.70 – 2.61 (m, 1H), 2.48 – 2.41 (m, 1H), 2.41 – 2.31 (m, 1H), 2.21 – 2.13 (m, 1H), 1.97 – 1.84 (m, 3H), 1.68 – 1.49 (m, 4H), 1.43 – 1.35 (m, 1H), 1.19 – 1.10 (m, 1H), 0.81 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, DMSO-d6, mixture of rotamers 1:0.3. Only major rotamer is reported here) δ 172.16, 170.27, 152.65, 151.36 (d, J = 6.2 Hz), 148.66, 147.07, 141.96, 136.24, 135.53, 133.05, 129.50, 121.19, 120.00, 119.38 (d, J = 4.3 Hz), 117.97 (d, J = 5.0 Hz), 112.17, 111.90, 105.11, 74.71, 59.82, 55.56, 55.48, 55.34, 51.53, 48.72, 42.88, 37.64, 30.48, 27.98, 26.36, 24.87, 20.55, 12.29 ppm. 31P NMR (202 MHz, DMSO-d6, mixture of rotamers 1:0.3, A:B) δ -5.08 (B), -5.15 (A). HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C36H46NO12PNa+ 738.2650; Found 738.2638.

2-(3-((R)-3-(3,4-dimethoxyphenyl)-1-(((S)-1-((S)-2-(3,4,5-trimethoxyphenyl)butanoyl) piperidine-2-carbonyl)oxy)propyl)phenoxy)acetic acid (18)
To a solution of 22 (113 mg, 151 µmol) in anhydrous CH2Cl2 (7 mL) was added trifluoroacetic acid (0.4 mL, 5.2 mmol) and the solution was left stirring at ambient temperature for 2.5 hours. The contents of the reaction vessel were concentrated in vacuo, followed by addition of toluene (7 mL). The resulting solution was concentrated in vacuo and the crude residue was purified by flash

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column chromatography (40% EtOAc in toluene + 1% formic acid) to yield 18 as a colorless oil (85 mg, 81%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.05. Only major rotamer is reported here) δ 8.58 (br s, 1H), 7.19 – 7.16 (m, 1H), 6.85 (ddd, J = 8.2, 2.6, 0.8 Hz, 1H), 6.78 (d, J = 7.9 Hz, 1H), 6.76 – 6.73 (m, 1H), 6.72 (dd, J = 2.5, 1.5 Hz, 1H), 6.69 – 6.66 (m, 2H), 6.25 (s, 2H), 5.52 – 5.47 (m, 2H), 4.70 (d, J = 16.2 Hz, 1H), 4.63 (d, J = 16.2 Hz, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.78 (s, 3H), 3.76 – 3.71 (m, 1H), 3.58 – 3.56 (m, 1H), 3.55 (s, 6H), 2.88 (td, J = 13.4, 3.1 Hz, 1H), 2.67 (ddd, J = 14.2, 9.7, 5.2 Hz, 1H), 2.55 (ddd, J = 14.2, 9.2, 6.7 Hz, 1H), 2.34 – 2.31 (m, 1H), 2.22 – 2.11 (m, 1H), 2.09 – 1.95 (m, 2H), 1.82 – 1.60 (m, 4H), 1.49 – 1.38 (m, 1H), 1.33 – 1.22 (m, 1H), 0.90 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.05. Only major rotamer is reported here) δ 173.77, 171.25, 170.17, 158.21, 153.28, 149.09, 147.56, 142.74, 136.65, 134.67, 133.44, 129.62, 120.34, 119.57, 115.82, 111.78, 111.47, 109.04, 104.95, 76.78, 65.67, 60.89, 56.06, 56.01, 56.00, 52.45, 51.22, 43.57, 38.51, 31.68, 28.30, 27.33, 25.30, 20.93, 12.61. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C38H48NO11+ 694.3222; Found 694.3216.

4-(3-((R)-3-(3,4-dimethoxyphenyl)-1-(((S)-1-((S)-2-(3,4,5-trimethoxyphenyl)butanoyl) piperidine-2-carbonyl)oxy)phenoxy)butanoic acid (19)
62 (0.134 g, 0.172 mmol) were dissolved in 4 mL DCM and trifluoroacetic acid (0.15 mL, 1.8 mmol) were added and the solution was stirred for 6 hours before 1 drop of water was added because TLC analysis showed that not all starting material was consumed. The reaction mixture was then stirred overnight and additionally trifluoroacetic acid was added (0.2 mL, 2.4 mmol) and the reaction was stirred for another 24 hours before it was concentrated in vacuo and purified by flash column chromatography (70:30:1 of toluene, EtOAc and formic acid) to yield 19 (66 mg, 53
%). 1H-NMR (500 MHz, CDCl3, mixture of rotamers, major rotamer reported, rotamer ratio 1.0:0.17): δ 7.16 (s, 1H), 6.94 (d, J=9.0 Hz, 1H), 6.86 (t, J=2.1 Hz, 2H), 6.77 (d, J=8.0 Hz, 1H),
6.65(q, J=2.0 Hz, 2H), 6.30 (s, 2H), 5.57 (dd, J=8.7, 4.6 Hz, 1H), 5.52 (d, J=6.2 Hz, 1H), 4.12 (m,

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1H), 4.03 (m, 2H), 3.85 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H), 3.83 (s, 3H), 3.78 (s, 3H), 2.91 (td, J=13.3, 3.2 Hz, 2H), 2.60 (m, 3H), 2.53 (m, 3H), 2.48 (m, 2H), 2.13 (m, 4H), 1.72 (m, 4H), 0.90 (m, 3H) ppm (Acid H not seen). 13C-NMR (126 MHz, CDCl3, mixture of rotamers, major rotamer reported): δ 173.93, 170.62, 159.19, 153.27, 149.08, 147.53, 142.50, 134.74, 133.70, 129.39, 120.33, 118.49, 113.92, 112.62, 111.82, 111.47, 105.08, 89.91, 76.08, 67.02, 60.91, 56.09, 56.02, 55.99, 52.38, 51.29, 38.49, 31.57, 31.37, 29.85, 28.36, 27.13, 25.28, 25.00, 24.81, 20.85, 12.53 ppm. HR-MS (MALDI, FT-ICR, dithranol): m/z 722.35204 [M+H+], calculated mass for (C40H52NO11+) 722.35349.

(R)-1-(3-((1H-tetrazol-5-yl)methoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-((S)-2- (3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (20)
To a suspension of 23 (13 mg, 19 µmol) and ZnCl2 (2.7 mg, 20 µmol) in i-PrOH (1.5 mL) was added sodium azide (1.5 mg, 23 µmol) and the reaction mixture was heated to 80 ºC for 3 hours. TLC analysis indicated complete conversion of 23 and the contents of the reaction vessel were partitioned between 1M HCl (10 mL) and EtOAc (10 mL) and the phases were separated. The aquous phase was extracted once more with EtOAc (10 mL). The combined organic phases were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated in vacuo to afford 20 as a colorless oil (12.4 mg, 90%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.04. Only major rotamer is reported here) δ 7.19 (t, J = 7.9 Hz, 1H), 6.91 (dd, J = 8.4, 2.6 Hz, 1H), 6.80 (d, J = 7.6 Hz, 1H), 6.77 (d, J = 8.7 Hz, 1H), 6.70 – 6.66 (m, 2H), 6.50 – 6.46 (m, 1H), 6.44 (s, 2H), 5.79 (d (AB system), J = 15.1 Hz, 1H), 5.68 (dd, J = 8.1, 5.6 Hz, 1H), 5.64 (d (AB system), J = 15.1 Hz, 1H), 5.57 (d, J = 4.7 Hz, 1H), 3.93 – 3.89 (m, 1H), 3.88 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H), 3.68 (t, J = 7.3 Hz, 1H), 3.59 (s, 6H), 2.78 – 2.71 (m, 1H), 2.59 (ddd, J = 14.5, 9.4, 5.5 Hz, 1H), 2.50 (ddd, J = 14.3, 9.2, 6.7 Hz, 1H), 2.34 (br d, J = 13.5 Hz, 1H), 2.18 – 2.06 (m, 2H), 1.98 – 1.89 (m, 1H), 1.88 – 1.81 (m, 1H), 1.79 – 1.68 (m, 2H), 1.65 (br d, J = 13.5 Hz, 1H), 1.52 – 1.41 (m, 1H), 1.24 –

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1.19 (m, 1H), 0.91 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.04. Only major rotamer is reported here) δ 174.31, 170.48, 157.37, 154.76, 153.17, 149.00, 147.47, 142.25, 136.08, 135.35, 133.44, 129.93, 121.71, 120.48, 113.44, 113.13, 111.98, 111.45, 105.30, 75.96, 61.26, 60.42, 56.24, 56.09, 56.03, 53.12, 50.86, 44.33, 37.69, 31.52, 27.61, 26.61, 25.18, 20.63, 12.41. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C38H47N5O9Na+ 740.3266; Found 740.3256.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-hydroxyphenyl)propyl (S)-1-((S)-2-(3,4,5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (21)
To a solution of 24 (20 mg, 27 µmol) and acetic acid (4 µL, 70 µmol) in anhydrous THF (0.5 mL) at 0 ºC was added tributylammonium fluoride (on silica gel, 1.5 mmol fluoride/g resin, 42 mg, 64 µmol) and the reaction mixture was left stirring for 1.5 hour. The contents of the reaction vessel were concentrated in vacuo and crude residue purified by flash column chromatography (40% EtOAc in toluene) to yield 21 as a colorless oil (11 mg, 65%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.2. Only major rotamer is reported here) δ 7.09 (t, J = 7.9 Hz, 1H), 6.77 (d, J = 8.2 Hz, 1H), 6.74 (ddd, J = 8.2, 2.5, 0.8 Hz, 1H), 6.70 – 6.68 (m, 1H), 6.67 – 6.63 (m, 2H), 6.60 (br d, J = 7.5 Hz, 1H), 6.46 – 6.42 (m, 3H), 5.62 (dd, J = 7.8, 5.9 Hz, 1H), 5.47 (br d, J = 4.7 Hz, 1H), 3.86 (s, 3H), 3.84 (s, 3H), 3.83 – 3.81 (m, 1H), 3.80 (s, 3H), 3.66 (s, 6H), 3.59 (t, J = 7.2 Hz, 1H), 2.71 (td, J = 13.3, 3.0 Hz, 1H), 2.52 (ddd, J = 14.8, 9.8, 5.5 Hz, 1H), 2.43 (ddd, J = 14.0, 9.4, 6.7 Hz, 1H), 2.28 (br d, J = 12.8 Hz, 1H), 2.13 – 2.06 (m, 1H), 2.04 – 2.00 (m, 1H), 1.92 – 1.84 (m, 1H), 1.79 – 1.73 (m, 1H), 1.71 – 1.63 (m, 2H), 1.61 – 1.55 (m, 1H), 1.42 (qt, J = 13.0, 3.6 Hz, 1H), 1.31 – 1.20 (m, 1H), 0.90 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.2. Only major rotamer is reported here) δ 172.89, 170.89, 156.62, 153.31, 148.99, 147.44, 141.57, 136.91, 135.43, 133.66, 129.71, 120.35, 118.89, 115.44, 112.54, 111.90, 111.41, 105.69,

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75.98, 60.97, 56.36, 56.07, 55.96, 52.26, 50.81, 43.73, 37.76, 31.41, 28.17, 26.85, 25.42, 20.79, 12.60. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C36H46NO9+ 636.3167; Found 636.3154.

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(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl (3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (22)
(S)-1-((S)-2-

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To a solution of 57 (154 mg, 383 µmol), 56 (140 mg, 383 µmol) and 4-(dimethylamino) pyridine (111 mg, 909 µmol) in CH2Cl2 (2 mL) at 0 ºC was added N,N´-dicyclohexyl carbodiimide (89 mg, 431 µmol). The resulting solution was allowed to heat to ambient temperature and left stirring overnight, before it was filtered and the filtrate concentrated (to approximately 0.5 mL) by passing a stream of nitrogen over the surface. A second filtration was performed and the crude filtrate was purified by flash column chromatography (30% EtOAc in toluene) to afford 22 (230 mg, 80%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 7.15 (t, J = 7.9 Hz, 1H), 6.79 – 6.73 (m, 3H), 6.66 – 6.60 (m, 3H), 6.41 (s, 2H), 5.61 (dd, J = 7.9, 5.7 Hz, 1H), 5.45 (br d, J = 4.6 Hz, 1H), 4.51 (s, 2H), 3.844 (s, 3H), 3.838 (s, 3H), 3.81 – 3.79 (m, 1H),
3.78(s, 3H), 3.69 (s, 6H), 3.59 – 3.56 (m, 1H), 2.80 (td, J = 13.4, 3.0 Hz, 1H), 2.59 – 2.48 (m, 1H), 2.47 – 2.39 (m, 1H), 2.32 – 2.26 (m, 1H), 2.12 – 2.02 (m, 2H), 1.95 – 1.87 (m, 1H), 1.75 – 1.65 (m, 3H), 1.62 – 1.56 (m, 2H), 1.47 (s, 9H), 1.31 – 1.23 (m, 1H), 0.89 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 172.69, 170.69, 168.07, 158.03, 153.32, 148.98, 147.43, 142.00, 136.77, 135.44, 133.59, 129.69, 120.29, 119.35, 113.96, 113.14, 111.82, 111.40, 105.08, 82.38, 75.86, 65.86, 60.87, 56.09, 56.03, 55.94, 52.16, 50.90, 43.56, 38.28, 31.30, 28.49, 28.15, 26.93, 25.47, 21.03, 12.69. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C42H55NO11Na+ 772.3667; Found 772.3652.

(R)-1-(3-(cyanomethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-((S)-2-(3,4,5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (23)

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To a solution of 58 (23 mg, 70 µmol), 56(28 mg, 77 µmol) and 4-(dimethylamino) pyridine (29 mg, 0.24 mmol) in anhydrous CH2Cl2 (2 mL) at 0 ºC was added N,N´-dicyclohexylcarbodiimide (16 mg, 78 µmol). The reaction mixture was allowed to heat to ambient temperature and left stirring overnight. The resulting suspension was filtered and the filtrate was concentrated (to approximately 0.5 mL) by passing a stream of nitrogen over the surface. A second filtration was performed and the filtrate subjected to flash column chromatography (40% EtOAc in toluene) to yield 23 as a colorless oil (33 mg, 70%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.2. Only major rotamer is reported here) δ 7.22 – 7.18 (m, 1H), 6.87 – 6.84 (m, 1H), 6.84 – 6.80 (m, 1H), 6.79 – 6.74 (m, 2H), 6.69 – 6.64 (m, 2H), 6.37 (s, 2H), 5.63 (dd, J = 8.3, 5.3 Hz, 1H), 5.43 (br d, J = 4.4 Hz, 1H), 4.88 (d, J = 16.0 Hz, 1H), 4.83 (d, J = 16.0 Hz, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.83 – 3.79 (m, 1H), 3.78 (s, 3H), 3.64 (s, 6H), 3.61 – 3.54 (m, 1H), 2.80 (td, J = 13.3, 3.0 Hz, 1H), 2.62 – 2.45 (m, 2H), 2.30 – 2.24 (m, 1H), 2.15 – 2.01 (m, 2H), 1.99 – 1.90 (m, 1H), 1.79 – 1.66 (m, 3H), 1.65 – 1.56 (m, 1H), 1.48 – 1.37 (m, 1H), 1.32 – 1.22 (m, 1H), 0.90 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3) δ 172.99, 171.04, 156.89, 153.35, 149.06, 147.52, 142.78, 136.81, 135.41, 133.46, 129.98, 120.88, 120.37, 115.46, 114.82, 112.20, 111.85, 111.45, 105.09, 75.56, 60.91, 56.08, 56.07, 56.00, 53.69, 52.29, 51.01, 43.66, 38.36, 31.37, 28.58, 26.84, 25.33, 20.82, 12.69. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C38H47N2O9+ 675.3276; Found 675.3270.

(R)-1-(3-((tert-butyldimethylsilyl)oxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-((S)-2- (3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (24)
To a solution of 59 (39 mg, 97 µmol), 56 (38 mg, 0.10 mmol) and 4-(dimethylamino) pyridine (32 mg, 0.26 mmol) in anhydrous CH2Cl2 (2 mL) at 0 ºC was added N,N´-dicyclohexylcarbodiimide (25 mg, 0.12 mmol). The reaction mixture was allowed to heat to ambient temperature and left stirring for 1.5 hours. The resulting suspension was filtered and the filtrate was concentrated (to approximately 0.5 mL) by passing a stream of nitrogen over the surface. A second filtration was

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performed and the filtrate subjected to flash column chromatography (5% EtOAc in CH2Cl2) to yield 24 as a colorless oil (45 mg, 62%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.3. Only major rotamer is reported here) δ 7.09 (t, J = 7.9 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.71 (ddd, J = 8.0, 2.4, 1.0 Hz, 1H), 6.70 – 6.68 (m, 1H), 6.66 – 6.62 (m, 2H), 6.57 (dt, J = 7.6, 1.2 Hz, 1H), 6.45 (s, 2H), 5.61 (dd, J = 7.5, 6.1 Hz, 1H), 5.49 – 5.46 (m, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.81 – 3.80 (m, 1H), 3.80 (s, 3H), 3.73 (s, 6H), 3.58 (br t, J = 7.2 Hz, 1H), 2.80 (td, J = 13.4, 2.9 Hz, 1H), 2.59 – 2.46 (m, 1H), 2.41 (ddd, J = 13.9, 9.5, 6.4 Hz, 1H), 2.34 – 2.29 (m, 1H), 2.13 – 2.02 (m, 2H), 1.91 (ddt, J = 12.6, 9.9, 6.3 Hz, 1H), 1.76 – 1.63 (m, 3H), 1.61 – 1.54 (m, 1H), 1.42 (qt, J = 13.0, 3.8 Hz, 1H), 1.32 – 1.20 (m, 1H), 0.97 (s, 9H), 0.90 (t, J = 7.3 Hz, 3H), 0.18 (s, 6H) ppm. 13C NMR (126 MHz, CDCl3) δ 172.57, 170.57, 155.66, 153.34, 149.00, 147.44, 141.70, 136.82, 135.55, 133.70, 129.64, 120.32, 119.58, 119.20, 118.63, 111.84, 111.41, 105.18, 75.98, 60.91, 56.18, 56.06, 55.96, 52.17, 50.88, 43.58, 38.22, 31.32, 28.48, 27.02, 25.81, 25.57, 21.18, 18.32, 12.69, -4.26. ppm. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C42H60NO9Si+ 750.4032; Found 750.4021.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-((methylthio)methoxy)phenyl)propyl (S)-1-((S)-2-(3,4,5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (25)
To a solution of 60 (211 mg, 0.50 mmol), 56 (191 mg, 0.52 mmol) and 4-(dimethylamino) pyridine (150 mg, 1.23 mmol) in anhydrous CH2Cl2 (8 mL) at 0 ºC was added N,N´-dicyclohexyl- carbodiimide (121 mg, 0.59 mmol). The reaction mixture was allowed to heat to ambient temperature and left stirring overnight. The resulting suspension was filtered and the filtrate was concentrated (to approximately 0.5 mL) by passing a stream of nitrogen over the surface. A second filtration was performed and the filtrate subjected to flash column chromatography (70% EtOAc in toluene) to yield 25 as a slightly yellow oil (224 mg, 58%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 7.21 (t, J = 7.9 Hz, 1H), 7.13 (ddt, J = 8.2, 2.3, 1.1 Hz, 1H), 7.07 – 7.05 (m, 1H), 6.80 – 6.78 (m, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.67 – 6.61 (m,

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2H), 6.44 (s, 2H), 5.64 (dd, J = 8.0, 5.6 Hz, 1H), 5.49 – 5.44 (m, 1H), 4.24 – 4.16 (m, 4H), 3.85 (s, 3H), 3.84 (s, 3H), 3.82 – 3.80 (m, 1H), 3.79 (s, 3H), 3.72 (s, 6H), 3.59 (dd, J = 7.5, 6.8 Hz, 1H), 2.79 (td, J = 13.4, 3.0 Hz, 1H), 2.56 – 2.48 (m, 1H), 2.43 (ddd, J = 14.0, 9.4, 6.6 Hz, 1H), 2.33 – 2.28 (m, 1H), 2.13 – 2.01 (m, 2H), 1.91 (dddd, J = 13.8, 9.7, 6.6, 5.6 Hz, 1H), 1.77 – 1.64 (m, 3H), 1.62 – 1.56 (m, 1H), 1.48 – 1.37 (m, 1H), 1.36 – 1.31 (m, 6H), 1.29 – 1.23 (m, 1H), 0.90 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 172.67, 170.58, 153.35, 150.80 (d, J = 6.7 Hz), 149.03, 147.50, 142.35, 136.82, 135.51, 133.44, 130.01, 122.75, 120.33, 119.47 (d, J = 4.4 Hz), 118.39 (d, J = 5.5 Hz), 111.84, 111.44, 105.15, 75.49, 64.77 (d, J = 5.6 Hz), 60.90, 56.15, 56.06, 55.98, 52.16, 50.85, 43.62, 38.25, 31.29, 28.48, 26.96, 25.51, 21.14, 16.23 (d, J = 6.6 Hz), 12.68. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C40H55NO12P+ 772.3456; Found 772.3446.

(R)-1-(3-(4-((tert-butoxycarbonyl)amino)phenethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-((S)-2-(3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (26)
To a solution of 64 (50 mg, 99 µmol), 56 (47 mg, 0.13 mmol) and 4-(dimethylamino) pyridine (36 mg, 0.29 mmol) in anhydrous CH2Cl2 (2 mL) at 0 ºC was added N,N´-dicyclohexylcarbodiimide (33 mg, 0.16 mmol). The reaction mixture was allowed to heat to ambient temperature and left stirring overnight. The resulting suspension was filtered and the filtrate was concentrated (to approximately 0.5 mL) by passing a stream of nitrogen over the surface. A second filtration was performed and the filtrate subjected to flash column chromatography (20% EtOAc in toluene) to yield 26 as a colorless oil (66 mg, 78%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 7.32 – 7.27 (m, 2H), 7.21 – 7.17 (m, 2H), 7.13 (t, J = 7.8 Hz, 1H), 6.80 – 6.72 (m, 3H), 6.66 – 6.61 (m, 2H), 6.57 (br dt, J = 7.9, 1.2 Hz, 1H), 6.42 (s, 2H), 5.59 (dd, J = 7.9, 5.7 Hz, 1H), 5.48 – 5.45 (m, 1H), 4.14 – 4.09 (m, 2H), 3.840 (s, 3H), 3.838 (s, 3H), 3.81 – 3.80 (m, 1H),
3.79(s, 3H), 3.69 (s, 6H), 3.60 – 3.56 (m, 1H), 3.02 (t, J = 7.1 Hz, 2H), 2.81 (td, J = 13.4, 2.9 Hz, 1H), 2.58 – 2.49 (m, 1H), 2.44 (ddd, J = 14.1, 9.4, 6.7 Hz, 1H), 2.33 – 2.27 (m, 1H), 2.13 – 2.03

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(m, 2H), 1.97 – 1.86 (m, 1H), 1.75 – 1.66 (m, 3H), 1.61 – 1.56 (m, 1H), 1.51 (s, 9H), 1.47 – 1.37 (m, 1H), 1.31 – 1.21 (m, 1H), 0.90 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 172.66, 170.67, 158.90, 153.32, 152.95, 148.98, 147.43, 141.88, 136.93, 136.79, 135.49, 133.65, 133.00, 129.63, 129.61, 120.32, 118.90, 118.54, 113.90, 112.96, 111.85, 111.41, 105.13, 80.52, 76.08, 68.81, 60.89, 56.12, 56.05, 55.95, 52.15, 50.89, 43.54, 38.25, 35.23, 31.38, 28.51, 28.47, 27.00, 25.51, 21.10, 12.71 ppm. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C49H62N2O11Na+ 877.4246; Found 877.4231.

(R)-1-(3-(2-((tert-butoxycarbonyl)amino)ethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1- ((S)-2-(3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (27)

A solution of 65 (40 mg, 93 µmol), 56 (38 mg, 95 µmol) and 4-(dimethylamino)pyridine (25 mg, 0.20 mmol) in anhydrous CH2Cl2 (1 mL) was cooled to 0 ºC before N,N´-dicyclohexyl carbodiimide (21 mg, 0.10 mmol) was added. The resulting solution was allowed to heat to ambient temperature and left stirring overnight, before it was filtered and the filtrate concentrated (to approximately 0.5 mL). A second filtration was performed and the filtrate was purified by flash column chromatography (5% MeOH in CH2Cl2) to afford 27 as a colorless oil (40 mg, 55%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.2. Only major rotamer is reported here) δ 7.18 – 7.13 (m, 1H), 6.79 – 6.74 (m, 2H), 6.70 – 6.68 (m, 1H), 6.67 – 6.63 (m, 2H), 6.61 (d, J = 7.6 Hz, 1H), 6.42 (s, 2H), 5.61 (dd, J = 8.0, 5.7 Hz, 1H), 5.47 (d, J = 4.6 Hz, 1H), 5.20 (br s, 1H), 4.05 – 3.96 (m, 2H), 3.85 (s, 3H), 3.84 (s, 3H), 3.82 – 3.79 (m, 1H), 3.79 (s, 3H), 3.68 (s, 6H), 3.57 (t, J = 7.1 Hz, 1H), 3.55 – 3.47 (m, 2H), 2.79 (td, J = 13.3, 2.9 Hz, 1H), 2.58 – 2.51 (m, 1H), 2.46 (ddd, J = 14.0, 9.3, 6.7 Hz, 1H), 2.33 – 2.28 (m, 1H), 2.14 – 2.02 (m, 2H), 1.98 – 1.87 (m, 1H), 1.76 – 1.67 (m, 3H), 1.62 – 1.56 (m, 1H), 1.44 (s, 10H), 1.32 – 1.23 (m, 1H), 0.90 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.2. Only major rotamer is reported here) δ 172.69,

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170.74, 158.76, 156.10, 153.34, 149.00, 147.46, 142.05, 136.86, 135.45, 133.59, 129.68, 120.33, 119.00, 113.75, 112.73, 111.86, 111.42, 105.20, 79.54, 75.98, 67.31, 60.89, 56.15, 56.05, 55.96, 52.15, 50.94, 43.56, 40.19, 38.27, 31.41, 28.53, 28.28, 26.94, 25.48, 21.04, 12.68. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C43H58N2O11Na+ 801.3933; Found 801.3923.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-oxo-2-phenylethoxy)phenyl)propyl (S)-1-((S)-2-(3,4,5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (28)
To a solution of 67 (15 mg, 37 µmol), 56 (15 mg, 41 µmol) and 4-(dimethylamino) pyridine (15 mg, 0.12 mmol) in anhydrous CH2Cl2 (2 mL) at 0 ºC was added N,N´-dicyclohexylcarbodiimide (9 mg, 44 µmol). The reaction mixture was allowed to heat to ambient temperature and left stirring overnight. The resulting suspension was filtered and the filtrate was concentrated (to approximately 0.5 mL) by passing a stream of nitrogen over the surface. A second filtration was performed and the filtrate subjected to flash column chromatography (30% EtOAc in toluene) to yield 28 as a colorless oil (21 mg, 75%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.3. Only major rotamer is reported here) δ 8.06 – 8.02 (m, 2H), 7.64 – 7.58 (m, 1H), 7.54 – 7.46 (m, 2H), 7.17 – 7.13 (m, 1H), 7.00 – 6.92 (m, 1H), 6.87 – 6.85 (m, 1H), 6.83 (ddd, J = 8.2, 2.6, 0.7 Hz, 1H), 6.79 – 6.74 (m, 1H),
6.66– 6.63 (m, 2H), 6.37 (s, 2H), 5.60 (dd, J = 8.2, 5.4 Hz, 1H), 5.45 (dd, J = 5.8, 1.6 Hz, 1H), 5.36 (s, 2H), 3.85 (s, 3H), 3.84 (s, 3H), 3.81 – 3.78 (m, 1H), 3.78 (s, 3H), 3.64 (s, 6H), 3.59 – 3.55 (m, 1H), 2.83 (td, J = 13.3, 2.9 Hz, 1H), 2.59 – 2.43 (m, 2H), 2.31 – 2.23 (m, 1H), 2.14 – 2.01 (m, 2H), 1.98 – 1.90 (m, 1H), 1.75 – 1.63 (m, 3H), 1.63 – 1.56 (m, 1H), 1.48 – 1.37 (m, 1H), 1.32 – 1.25 (m, 1H), 0.88 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.3. Only major rotamer is reported here) δ 194.55, 172.95, 170.91, 158.26, 153.33, 149.03, 147.47, 142.28, 136.76, 135.38, 134.79, 133.92, 133.61, 129.75, 128.95, 128.31, 120.35, 119.34, 114.55, 112.41, 111.84, 111.42, 105.01, 75.89, 70.88, 60.89, 56.06, 56.04, 55.98, 52.19, 50.98, 43.58,

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38.38, 31.36, 28.55, 26.95, 25.43, 20.92, 12.77. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C44H52NO10+ 754.3586; Found 754.3577.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-oxopropoxy)phenyl)propyl (S)-1-((S)-2-(3,4,5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (29)
To a solution of 68 (24 mg, 70 µmol), 56 (39 mg, 0.11 mmol) and 4-(dimethylamino) pyridine (31 mg, 0.25 mmol) in anhydrous CH2Cl2 (2 mL) at 0 ºC was added N,N´-dicyclohexylcarbodiimide (16 mg, 78 µmol). The reaction mixture was allowed to heat to ambient temperature and left stirring overnight. The resulting suspension was filtered and the filtrate was concentrated (to approximately 0.5 mL) by passing a stream of nitrogen over the surface. A second filtration was performed and the filtrate subjected to flash column chromatography (30% EtOAc in toluene) to yield 29 as a colorless oil (16 mg, 33%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.2. Only major rotamer is reported here) δ 7.19 – 7.14 (m, 1H), 6.79 – 6.73 (m, 3H), 6.68 – 6.62 (m, 3H), 6.38 (s, 2H), 5.61 (dd, J = 8.3, 5.3 Hz, 1H), 5.48 – 5.42 (m, 1H), 4.59 (s, 2H), 3.85 (s, 3H), 3.84 (s, 3H), 3.80 – 3.78 (m, 1H), 3.78 (s, 3H), 3.66 (s, 6H), 3.58 (dd, J = 7.9, 6.3 Hz, 1H), 2.81 (td, J = 13.3, 3.1 Hz, 1H), 2.55 (ddd, J = 14.2, 9.7, 5.4 Hz, 1H), 2.47 (ddd, J = 14.2, 9.4, 6.7 Hz, 1H), 2.31 – 2.29 (m, 1H),
2.27(s, 3H), 2.14 – 2.03 (m, 2H), 1.96 – 1.89 (m, 1H), 1.73 – 1.65 (m, 3H), 1.62 – 1.56 (m, 1H), 1.48 – 1.37 (m, 1H), 1.31 – 1.24 (m, 1H), 0.90 (t, J = 7.4 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.2. Only major rotamer is reported here) δ 205.47, 172.86, 170.83, 157.95, 153.32, 149.02, 147.49, 142.40, 136.76, 135.41, 133.54, 129.84, 120.33, 119.49, 114.10, 112.54, 111.84, 111.43, 105.06, 75.80, 73.17, 60.88, 56.06, 56.05, 55.97, 52.19, 50.96, 43.56, 38.35, 31.39, 28.55, 26.94, 26.72, 25.42, 20.96, 12.70 ppm. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C39H49NO10Na+ 714.3249; Found 714.3245.

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3-((R)-3-(3,4-dimethoxyphenyl)-1-(((S)-1-((S)-2-(3,4,5-trimethoxyphenyl)butanoyl)piperidine- 2-carbonyl)oxy)propyl)phenyl morpholine-4-carboxylate (30)
To a solution of 70 (35 mg, 87 µmol), 56 (47 mg, 0.13 mmol) and 4-(dimethylamino) pyridine (61 mg, 0.50 mmol) in anhydrous CH2Cl2 (2 mL) at 0 ºC was added N,N´-dicyclohexylcarbodiimide (38 mg, 0.18 mmol). The reaction mixture was allowed to heat to ambient temperature and left stirring overnight. The resulting suspension was filtered and the filtrate was concentrated (to approximately 0.5 mL) by passing a stream of nitrogen over the surface. A second filtration was performed and the filtrate subjected to flash column chromatography (80% EtOAc in toluene) to yield 30 as a colorless oil (59 mg, 90%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.3. Only major rotamer is reported here) δ 7.25 – 7.23 (m, 1H), 7.01 (ddd, J = 8.1, 2.4, 1.0 Hz, 1H), 6.98 (t, J = 2.0 Hz, 1H), 6.84 (dt, J = 7.8, 1.4 Hz, 1H), 6.77 (d, J = 8.1 Hz, 1H), 6.66 – 6.62 (m, 2H), 6.42 (s, 2H), 5.65 (dd, J = 8.2, 5.4 Hz, 1H), 5.49 – 5.45 (m, 1H), 3.848 (s, 3H), 3.845 (s, 3H), 3.82 – 3.80 (m, 1H), 3.79 (s, 3H), 3.76 – 3.73 (m, 4H), 3.70 (s, 6H), 3.68 – 3.65 (m, 2H), 3.60 – 3.53 (m, 3H), 2.80 (td, J = 13.4, 3.1 Hz, 1H), 2.55 (ddd, J = 14.5, 9.4, 5.5 Hz, 1H), 2.47 (ddd, J = 13.9, 9.4, 6.7 Hz, 1H), 2.33 – 2.28 (m, 1H), 2.14 – 2.03 (m, 2H), 1.99 – 1.93 (m, 1H), 1.75 – 1.65 (m, 3H), 1.63 – 1.56 (m, 1H), 1.48 – 1.37 (m, 1H), 1.31 – 1.23 (m, 1H), 0.90 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.3. Only major rotamer is reported here) δ 172.69, 170.67, 153.61, 153.34, 151.26, 149.01, 147.47, 141.85, 136.80, 135.50, 133.51, 129.57, 123.12, 121.28, 120.35, 120.02, 111.88, 111.43, 105.15, 75.58, 66.75, 66.66, 60.91, 56.15, 56.06, 55.97, 52.15, 50.91, 45.02, 44.25, 43.56, 38.23, 31.36, 28.49, 27.00, 25.50, 21.10, 12.71 ppm. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C41H53N2O11+ 749.3644; Found 749.3640.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(prop-2-yn-1-yloxy)phenyl)propyl (S)-1-((S)-2-(3,4,5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (31)

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To a solution of 69 (116 mg, 355 µmol), 56 (148 mg, 0.405 mmol) and 4-(dimethylamino) pyridine (101 mg, 0.827 mmol) in anhydrous CH2Cl2 (2 mL) at 0 ºC was added N,N´- dicyclohexylcarbodiimide (82 mg, 397 µmol). The reaction mixture was allowed to heat to ambient temperature and left stirring overnight. The resulting suspension was filtered and the filtrate was concentrated (to approximately 0.5 mL) by passing a stream of nitrogen over the surface. A second filtration was performed and the filtrate subjected to flash column chromatography (30% EtOAc in toluene) to yield 31 as a colorless oil (215 mg, 90%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 7.20 – 7.14 (m, 1H), 6.86 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 6.81 – 6.78 (m, 1H), 6.77 (d, J = 8.1 Hz, 1H), 6.67 – 6.63 (m, 3H), 6.41 (s, 2H), 5.62 (dd, J = 8.0, 5.6 Hz, 1H), 5.51 – 5.43 (m, 1H), 4.69 (d, J = 2.4 Hz, 2H), 3.85 (s, 3H), 3.84 (s, 3H), 3.83 – 3.77 (m, 1H), 3.79 (s, 3H), 3.69 (s, 6H), 3.58 (dd, J = 7.9, 6.5 Hz, 1H), 2.81 (td, J = 13.3, 3.1 Hz, 1H), 2.58 – 2.50 (m, 1H), 2.53 (t, J = 2.4 Hz, 1H), 2.46 (ddd, J = 14.0, 9.4, 6.6 Hz, 1H), 2.33 –
2.28(m, 1H), 2.13 – 2.04 (m, 2H), 1.98 – 1.89 (m, 1H), 1.75 – 1.65 (m, 3H), 1.63 – 1.55 (m, 1H), 1.47 – 1.38 (m, 1H), 1.31 – 1.24 (m, 1H), 0.90 (t, J = 7.4 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 172.69, 170.70, 157.66, 153.32, 149.00, 147.45, 142.04, 136.78, 135.44, 133.60, 129.67, 120.31, 119.41, 114.25, 113.21, 111.84, 111.41, 105.09, 78.65, 75.92, 75.75, 60.88, 56.10, 56.04, 55.95, 55.92, 52.15, 50.90, 43.55, 38.29, 31.33, 28.50, 26.96, 25.48, 21.05, 12.68. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C39H48NO9+ 674.3324; Found 674.3318.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-(morpholine-4-carboxamido)ethoxy)phenyl)propyl (S)-1- ((S)-2-(3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (32)
To a solution of 72 (16 mg, 36 µmol), 56 (23 mg, 63 µmol) and 4-(dimethylamino) pyridine (19

mg, 0.16 mmol) in anhydrous CH2Cl2 (2 mL) at 0 ºC was added N,N´-dicyclohexylcarbodiimide (17 mg, 82 µmol). The reaction mixture was allowed to heat to ambient temperature and left stirring

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overnight. The resulting suspension was filtered and the filtrate was concentrated (to approximately 0.5 mL) by passing a stream of nitrogen over the surface. A second filtration was performed and the filtrate subjected to flash column chromatography (EtOAc + 2% MeOH) to yield 32 as a colorless oil (20 mg, 70%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 7.16 (t, J = 7.9 Hz, 1H), 6.80 – 6.74 (m, 2H), 6.69 – 6.62 (m, 4H), 6.40 (s, 2H), 5.60 (dd, J = 8.1, 5.4 Hz, 1H), 5.46 (br d, J = 4.3 Hz, 1H), 5.18 (t, J = 5.6 Hz, 1H), 4.08 – 4.02 (m, 2H), 3.852 (s, 3H), 3.849 (s, 3H), 3.82 – 3.78 (m, 1H), 3.78 (s, 3H), 3.70 – 3.65 (m, 12H), 3.59 – 3.56 (m, 1H), 3.37 (t, J = 4.9 Hz, 4H), 2.80 (td, J = 13.4, 3.0 Hz, 1H), 2.56 (ddd, J = 14.5, 9.5, 5.6 Hz, 1H), 2.46 (ddd, J = 14.0, 9.5, 6.7 Hz, 1H), 2.33 – 2.27 (m, 1H), 2.13 – 2.04 (m, 2H), 1.97 –
1.89 (m, 1H), 1.75 – 1.65 (m, 3H), 1.61 – 1.54 (m, 1H), 1.43 (qt, J = 13.1, 3.8 Hz, 1H), 1.33 – 1.25 (m, 1H), 0.90 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3) δ 172.76, 170.76, 158.83, 157.94, 153.31, 149.03, 147.51, 142.18, 136.84, 135.41, 133.56, 129.73, 120.35, 119.07, 113.75, 112.73, 111.88, 111.44, 105.19, 76.04, 67.53, 66.66, 60.87, 56.14, 56.08, 55.99, 52.17, 50.94, 44.11, 43.59, 40.54, 38.36, 31.47, 28.50, 26.96, 25.48, 21.04, 12.70. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C43H58N3O11+ 792.4066; Found 792.4056.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-((methylthio)methoxy)phenyl)propyl (S)-1-((S)-2-(3,4,5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (33)
To a solution of 71 (30 mg, 86 µmol), 56 (41 mg, 0.11 mmol) and 4-(dimethylamino) pyridine (27 mg, 0.22 mmol) in anhydrous CH2Cl2 (2 mL) at 0 ºC was added N,N´-dicyclohexylcarbodiimide (25 mg, 0.12 mmol). The reaction mixture was allowed to heat to ambient temperature and left stirring overnight. The resulting suspension was filtered and the filtrate was concentrated (to approximately 0.5 mL) by passing a stream of nitrogen over the surface. A second filtration was performed and the filtrate subjected to flash column chromatography (20% EtOAc in toluene) to yield 33 as a colorless oil (31.5 mg, 53%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is

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reported here) δ 7.17 (t, J = 7.9 Hz, 1H), 6.83 (ddd, J = 8.1, 2.6, 0.9 Hz, 1H), 6.81 – 6.78 (m, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.67 – 6.63 (m, 3H), 6.42 (s, 2H), 5.62 (dd, J = 8.0, 5.6 Hz, 1H), 5.49 – 5.43 (m, 1H), 5.14 (d (AB system), J = 11.5 Hz, 1H), 5.13 (d (AB system), J = 11.5 Hz, 1H), 3.85 (s, 3H), 3.85 (s, 3H), 3.81 – 3.79 (m, 1H), 3.79 (s, 3H), 3.69 (s, 6H), 3.58 (dd, J = 7.8, 6.4 Hz, 1H), 2.81 (td, J = 13.3, 3.1 Hz, 1H), 2.59 – 2.50 (m, 1H), 2.46 (ddd, J = 14.0, 9.6, 6.7 Hz, 1H), 2.32 – 2.28 (m, 1H), 2.24 (s, 3H), 2.14 – 2.03 (m, 2H), 1.93 (dddd, J = 13.8, 9.8, 6.7, 5.6 Hz, 1H), 1.76 – 1.64 (m, 3H), 1.60 – 1.53 (m, 1H), 1.43 (qt, J = 12.9, 4.1 Hz, 1H), 1.32 – 1.23 (m, 1H), 0.90 (t, J = 7.4 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 172.70, 170.75, 157.24, 153.34, 149.01, 147.46, 142.04, 136.81, 135.48, 133.61, 129.64, 120.34, 119.56, 115.30, 114.11, 111.85, 111.42, 105.13, 75.90, 72.52, 60.90, 56.13, 56.06, 55.97, 52.18, 50.94, 43.59, 38.32, 31.35, 28.53, 26.97, 25.49, 21.06, 14.77, 12.70. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C38H49NO9SNa+ 718.3020; Found 718.3010.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-((methylsulfonyl)methoxy)phenyl)propyl (S)-1-((S)-2-(3,4,5- trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (34)
To a solution of 33 (17 mg, 24 µmol) in anhydrous CH2Cl2 (1 mL) at 0 ºC was added meta- chloroperbenzoic acid (19 mg, 85 µmol) and the resulting solution was stirred for 2 hours before it was subjected to flash column chromatography (40% EtOAc in toluene) to afford 34 as a colorless oil (9 mg, 51%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 7.21 (t, J = 7.9 Hz, 1H), 6.98 – 6.91 (m, 2H), 6.81 – 6.73 (m, 2H), 6.69 – 6.61 (m, 2H), 6.36 (s, 2H), 5.62 (dd, J = 8.3, 5.1 Hz, 1H), 5.42 (br d, J = 5.9 Hz, 1H), 5.07 (d (AB system), J = 12.0 Hz, 1H), 5.04 (d (AB system), J = 12.0 Hz, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.81 – 3.78 (m, 1H), 3.78 (s, 3H), 3.64 (s, 6H), 3.60 – 3.55 (m, 1H), 3.00 (s, 3H), 2.82 (td, J = 13.2, 2.8 Hz, 1H), 2.58 (ddd, J = 14.5, 9.4, 5.3 Hz, 1H), 2.53 – 2.45 (m, 1H), 2.30 – 2.24 (m, 1H), 2.16 – 2.05 (m, 2H), 2.00 – 1.91 (m, 1H), 1.77 – 1.65 (m, 3H), 1.65 – 1.58 (m, 1H), 1.49 – 1.39 (m, 1H), 1.33 –

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1.27 (m, 1H), 0.91 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 173.07, 170.99, 157.37, 153.34, 149.08, 147.55, 142.90, 136.80, 135.44, 133.44, 130.00, 121.21, 120.36, 115.51, 113.17, 111.86, 111.46, 105.09, 82.07, 75.54, 60.91, 56.09, 56.08, 56.01, 52.33, 51.03, 43.62, 38.67, 38.40, 31.40, 28.60, 26.87, 25.34, 20.87, 12.73. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C38H49NO11SNa+ 750.2919; Found 750.2903.

3-(3,4-dimethoxyphenyl)propyl (S)-1-((S)-2-(3,4,5-trimethoxyphenyl)butanoyl)piperidine-2- carboxylate (35)
To a solution of 3-(3,4-dimethoxyphenyl)-1-propanol (42.5 mg, 153 µmol), 56 (80 mg, 0.18 mmol) and 4-(dimethylamino) pyridine (145 mg, 0.76 mmol) in anhydrous CH2Cl2 (2 mL) at 0 ºC was added N,N´-dicyclohexylcarbodiimide (54 mg, 0.17 mmol). The reaction mixture was allowed to heat to ambient temperature and left stirring overnight. The resulting suspension was filtered and the filtrate was concentrated (to approximately 0.5 mL) by passing a stream of nitrogen over the surface. A second filtration was performed and the filtrate subjected to flash column chromatography (30% EtOAc in toluene) to yield 35 as a colorless oil (65 mg, 55%). 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 6.78 – 6.74 (m, 1H), 6.67 – 6.63 (m, 2H), 6.46 (s, 2H), 5.39 – 5.36 (m, 1H), 4.08 (dt, J = 10.8, 6.6 Hz, 1H), 4.02
(dt, J = 10.8, 6.6 Hz, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.82 (s, 6H), 3.80 (s, 3H), 3.81 – 3.75 (m, 1H), 3.59 (dd, J = 7.9, 6.4 Hz, 1H), 2.89 (td, J = 13.3, 3.1 Hz, 1H), 2.53 (t, J = 7.6 Hz, 2H), 2.29 – 2.20 (m, 1H), 2.16 – 2.04 (m, 1H), 1.88 – 1.78 (m, 2H), 1.76 – 1.58 (m, 4H), 1.43 (qt, J = 12.9, 3.8 Hz, 1H), 1.31 – 1.22 (m, 1H), 0.91 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3) δ 172.87, 171.26, 153.28, 149.00, 147.44, 136.76, 135.48, 133.68, 120.32, 111.83, 111.39, 105.16, 64.19, 60.88, 56.23, 56.04, 55.94, 52.30, 51.02, 43.57, 31.68, 30.45, 28.40, 26.96, 25.45, 21.12, 12.70. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C30H42NO8+ 544.2905; Found 544.2900.

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(S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)piperidine-2-carboxylic acid (37)23
To a solution of 36 (2.02 g, 7.24 mmol) in water (15 mL) was slowly added NaHCO3 (8.1 g, 96 mmol). After gas evolution had ceased acetone (40 mL) was added followed by 9- fluorenylmethoxycarbonyl chloride (3.94 g, 15.2 mmol) and the solution was left stirring for 18 hours at ambient temperature. The reaction mixture was concentrated in vacuo removing acetone and the resulting aqueous suspension was extracted with CH2Cl2. The organic phase was dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (CH2Cl2 + 1% formic acid) to yield 37 as a pale yellow solid (2.1 g, 83%). [𝛼]D25 ―34.5 (c = 1.1, CHCl3). Mp 155 – 157 °C. 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.7, A:B): δ 10.77 (br s, 1.7H, A + B), 7.79 (d, J = 7.4 Hz, 2H, A), 7.77 – 7.73 (m, 1.4H, B), 7.64 (t, J = 7.3 Hz, 2H, A), 7.57 (t, J = 7.6 Hz, 1.4H, B), 7.45 – 7.29 (m, 6.8H, A + B), 5.09 (d, J = 4.9 Hz, 1H, A), 4.79 (d, J = 4.9 Hz, 0.7H, B), 4.57 – 4.40 (m, 3.4H, A + B), 4.32 (t, J = 7.0 Hz, 1H, A), 4.25 (t, J = 6.4 Hz, 0.7H, B), 4.16 (d, J = 12.8 Hz, 0.7H, B), 4.09 (d, J = 12.5 Hz, 1H, A), 3.22 – 3.15 (m, 1H, A), 3.08 – 3.01 (m, 0.7H, B), 2.34 (d, J = 12.9 Hz, 1H, A), 2.26 (d, J = 13.2 Hz, 0.7H, B), 1.80 – 1.67 (m, 5.1H, A + B), 1.51 – 1.31 (m, 3.4H, A + B) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.7, A:B): δ 177.21 (A), 177.13 (B), 156.70 (A), 156.01 (B), 143.87 (B), 143.85 (A), 141.31 (A), 141.26 (B), 127.70 (A), 127.67 (B), 127.08 (A), 127.03 (B), 125.10 (A), 125.09 (A), 124.94 (B), 124.84 (B), 120.10 (B), 119.97 (A), 67.91 (A), 67.72 (B), 54.34 (B), 54.24 (A), 47.20 (A + B), 41.95 (A), 41.72 (B), 26.74 (B), 26.57 (A), 24.69 (A), 24.46 (B), 20.73 (A), 20.59 (B) ppm (1 extra peak (A + B) Fmoc group not completely symmetrical). HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C21H21NO4Na+ 374.1363; Found 374.1372.

1-((9H-fluoren-9-yl)methyl) 2-((R)-3-(3,4-dimethoxyphenyl)-1-(3-(2- morpholinoethoxy)phenyl)propyl) (S)-piperidine-1,2-dicarboxylate (39)24

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To a solution of 37 (402 mg, 1.14 mmol), 38 (418 mg, 1.04 mmol), 4-(dimethylamino)pyridine (12 mg, 0.1 mmol) in dry CH2Cl2 (10 mL) at 0 ºC was added N,N´-dicyclohexylcarbodiimide (236 mg, 1.14 mmol). The reaction mixture was allowed to heat to ambient temperature and was stirred at this temperature overnight. The resulting suspension was filtered and the filtrate was concentrated (to a total volume of approximately 3 mL) by passing a stream of nitrogen over the surface. A second filtration was performed before the filtrate was subjected to flash column chromatography (40% acetone in toluene + 1% Et3N) to yield 39 as a colorless oil (508 mg, 94%).. 1H NMR (500 MHz, CDCl3, mixture of rotamers 1:0.7, A:B): δ 7.77 (dd, J = 7.2, 2.3 Hz, 2H, A), 7.72 (d, J = 7.4 Hz, 1.4H, B), 7.59 (t, J = 8.4 Hz, 2H, A), 7.49 (d, J = 7.4 Hz, 0.7H), 7.45 – 7.14 (m, 9.2H, A + B), 6.92 (d, J = 7.4 Hz, 1.7H, A + B), 6.88 (s, 2H, A + B), 6.83 (d, J = 8.2 Hz, 1H, A), 6.78 (d, J = 7.6 Hz, 0.7H, B), 6.75 (s, 0.7H, B), 6.74 (s, 1H, A), 6.67 – 6.58 (m, 3.4H, A + B), 5.80 – 5.72 (m, 1.7H, A + B), 5.04 (d, J = 4.4 Hz, 1H, A), 4.90 (d, J = 4.2 Hz, 0.7H, B), 4.49 – 4.32 (m, 3.4H, A + B), 4.28 (t, J = 7.1 Hz, 1H, A), 4.17 – 4.06 (m, 4.4H, A + B), 3.99 (t, J = 5.4 Hz, 1.4H, B), 3.84 (s, 5.1H, A + B), 3.82 (s, 3H, A), 3.80 (s, 2.1H, B), 3.73 – 3.68 (m, 6.8H, A + B), 3.16 (td, J = 12.8, 2.2 Hz, 0.7H, B), 3.00 (td, J = 13.3, 2.4 Hz, 0.7H, B), 2.77 (t, J = 5.7 Hz, 2H, A), 2.69 (t, J = 5.6 Hz, 1.4H, B), 2.59 – 2.49 (m, 10.2H, A + B), 2.33 – 2.29 (m, 1.7H, A + B), 2.26 – 2.16 (m, 1.7H, A + B), 2.07 – 1.98 (m, 1.7H, A + B), 1.79 – 1.66 (m, 5.1H, A + B), 1.52 – 1.42 (m, 1.7H, A + B), 1.33 – 1.22 (m, 1.7H, A + B) ppm. 13C NMR (126 MHz, CDCl3, mixture of rotamers 1:0.7, A:B): δ 171.07 (A + B), 158.98 (A + B), 156.50 (A), 156.04 (B), 148.99 (B), 148.98 (A), 147.47 (B), 147.42 (A), 144.22 (A), 144.16 (B), 143.99 (A), 143.93 (B), 141.80 (A), 141.62 (B), 141.42 (A), 141.40 (A), 141.33 (B), 141.30 (B), 133.63 (A), 133.50 (B), 129.75 (A + B), 127.81 (A), 127.78 (B), 127.17 (A + B), 125.20 (A), 125.09 (B), 120.22 (A), 120.20 (B), 120.11 (B), 120.09 (A), 120.06 (A), 120.04 (B), 119.16 (B), 119.01 (A), 114.05 (A + B), 113.20 (B), 113.18 (A), 111.82 (A), 111.73 (B), 111.42 (A + B), 76.67 (B), 76.41 (A), 67.99 (B), 67.89 (A), 67.03 (A + B), 65.86

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(A), 65.82 (B), 57.77 (A), 57.69 (B), 56.03 (A + B), 55.92 (A + B), 55.02 (B), 54.64 (A), 54.21 (A), 54.19 (B), 47.33 (A + B), 42.12 (A), 41.99 (B), 38.22 (A), 38.19 (B), 31.35 (B), 31.32 (A), 27.17 (B), 26.96 (A), 24.93 (A), 24.68 (B), 20.94 (A), 20.81 (B) ppm (3 extra peaks (3A + 3B) Fmoc group not completely symmetrical). HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C44H51N2O8+ 735.3640; Found 735.3631.

(S)-2-(naphthalen-2-yl)butanoic acid (40)
To a solution of 2-(naphthalen-2-yl)acetic acid (230 mg, 1.24 mmol) and Koga’s base (571 mg, 127 mmol) in anhydrous THF (4 mL) at 0 ºC was added dropwise n-BuLi (2.15 mL, 2.3 M solution in hexanes, 4.94 mmol). After addition the reaction mixture was left stirring 15 min, then cooled to – 78 ºC and left at this temperature for 5 min before adding ethyl iodide (0.4 mL, 5.0 mmol). The reaction was left stirring 5 min before adding MeOH (1 mL) and letting the solution heat to ambient temperature. The reaction mixture was partitioned between 1 M aqueous HCl (20 mL) and EtOAc (20 mL). The phases were separated and the aqueous phase was extracted 4 times with EtOAc (20 mL). The combined organic phases were washed with water (30 mL) followed by brine (30 mL), dried over MgSO4, filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (15% EtOAc in heptane + 1% formic acid) to yield 40 as a white solid (190 mg, 54%). [𝛼]D25 +79.6 (c = 0.36, CHCl3) (lit.25: [𝛼]D25 +93.3 (c = 1, CHCl3) – 96% ee). Ee: 82% based on optical rotation. Mp 103 – 105 °C. 1H NMR (500 MHz, CDCl3): δ 7.82 – 7.79 (m, 3H), 7.78 – 7.74 (m, 1H), 7.50 – 7.42 (m, 3H), 3.64 (t, J = 7.4 Hz, 3H), 2.20 (dp, J = 13.6, 7.4 Hz, 1H), 1.93 (dp, J = 13.6, 7.4 Hz, 1H), 0.94 (t, J = 7.4 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3): δ 180.04, 135.89, 133.54, 132.89, 128.52, 127.96, 127.76, 127.26, 126.32, 126.05, 126.05, 53.51, 26.37, 12.27 ppm. HRMS (ESI/FTICR) m/z: [M + H]+ Calcd for C14H15O2+ 215.1067; Found 215.1073.

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(±)-2-(3,4,5-fluorophenyl)butanoic acid (41)
The reaction was carried out as described for compound 40, using 3,4,5-trifluorophenylacetic acid (250 mg, 1.31 mmol), Koga’s base (608 mg, 1.35 mmol), n-BuLi (2.3 mL, 2.3 M in hexanes, 5.29 mmol) and ethyl iodide (0.45 mL, 5.63 mmol). The reaction was quenched with MeOH 15 min after complete addition of ethyl iodide. Purification was performed by flash column chromatography (10% EtOAc in toluene + 1% formic acid) yielding 41 (170 mg, 45%). 1H NMR (500 MHz, CDCl3): δ 6.96 (dd, J = 8.3, 6.5 Hz, 2H), 3.40 (t, J = 7.7 Hz, 1H), 2.12 – 2.02 (m, 1H), 1.86 – 1.69 (m, 1H), 0.92 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3): δ 178.84, 151.32 (ddd, J = 250.4, 10.0, 4.0 Hz), 139.31 (dt, J = 251.5, 15.2 Hz), 134.63 – 134.20 (m), 112.49 (dd, J = 16.5, 5.2 Hz), 52.51, 26.52, 12.01 ppm. HRMS (ESI/FTICR) m/z: [M + H]+ Calcd for C10H10F3O2+ 219.0627; Found 219.0635.

(S)-2-(4-bromophenyl)butanoic acid (42)
The reaction was carried out as described for compound 40, using 2-(4-bromophenyl)acetic acid (770 mg, 3.58 mmol), Koga’s base (1.65 g, 3.69 mmol), n-BuLi (5.8 mL, 2.5 M in hexanes, 14.5 mmol) and ethyl iodide (1.2 mL, 15 mmol). The reaction was quenched with MeOH 15 min after complete addition of ethyl iodide. Purification was performed by flash column chromatography

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(c = 1.0,

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CHCl3) (litt.Error! Bookmark not defined.: [𝛼]D25 +49.5 (c = 1, CHCl3) – 93% ee). Ee: 88% based on optical rotation. 1H NMR (500 MHz, CDCl3): δ 7.47 – 7.43 (m, 2H), 7.21 – 7.17 (m, 2H), 3.42 (t, J = 7.7 Hz, 1H), 2.13 – 2.03 (m, 1H), 1.84 – 1.73 (m, 1H), 0.90 (t, J = 7.4 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3): δ 179.55, 137.41, 131.92, 129.98, 121.62, 52.79, 26.39, 12.13 ppm. HRMS (ESI/FTICR) m/z: [M + H]+ Calcd for C10H12BrO2+ 243.0015; Found 243.0017.

(S)-2-(4-ethoxy-3,5-dimethoxyphenyl)butanoic acid (44).

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Ethyl iodide (0.50 mL, 6.25 mmol) was added to a suspension of (4-hydroxy-3,5- dimethoxyphenyl)acetic acid (43, 0.500 g, 2.35 mmol) and potassium carbonate (1.07 g, 7.75 mmol) in 13.0 mL DMSO. The reaction was heated to 60 ºC for 5 h, after which additional ethyl iodide (0.25 mL, 3.13 mmol) was added to the reaction. After a total of 7 h TLC showed the reaction was complete, and the suspension was cooled to room temperature and diluted with 1 M aqueous HCl. The aqueous phase was extracted with EtOAc 3 x 25 mL and the combined organic phases were washed with brine, dried with NaSO4, filtered and concentrated in vacuo to a red/brown oil. The oil was purified by flash column chromatography (30% EtOAc in heptane, + 1% formic acid) to yield the 4-ethoxy ethyl ester as a red/brown oil (0.392 g, 62 %). 1H-NMR (500 MHz, CDCl3): δ 6.50 (s, 2H), 4.17 (q, J = 7.1 Hz, 2H), 4.03 (q, J = 7.1 Hz, 2H), 3.84 (s, 6H), 3.54 (s 2H), 1.35 (t, J = 7.1 Hz, 3H), 1.27 (t, J = 7.1 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3) δ 171.8, 153.6, 136.2, 129.6, 106.5, 69.0, 61.0, 56.3, 41.8, 15.7, 14.4 ppm.

Water (10 mL) was added to this ethyl ester (0.392 g, 1.46 mmol) in MeOH (5.0 mL). After a short while, lithium hydroxide monohydrate (0.323 g, 7.52 mmol) was added, and the solution was stirred overnight. It was then quenched with HCl (25 mL, 1 M). The aqueous solution was extracted with diethyl ether (3 x 25 mL) and the combined organic phases was washed with brine, dried with MgSO4, filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (40% EtOAc in toluene +1% formic acid) to yield the 4-ethoxy acid as a pale yellow oil (0.327 g, 91 %). 1H-NMR (500 MHz, CDCl3): δ 6.49 (s, 2H), 4.03 (q, J = 7.1 Hz, 2H),
3.82(s, 6H), 3.57 (s, 2H), 1.34 (t, J = 7.1, 3H) ppm. 13C-NMR (126 MHz, CDCl3) δ 177.8, 153.7, 136.3, 128.7, 106.6, 69.0, 56.2, 41.5, 15.6 ppm.

To a solution of this compound (0.192 g, 0.799 mmol) and Koga’s base (0.876 mmol) in 10 mL THF at 0 ºC was added dropwise n-BuLi (3 mL, 1.6 M in hexane, 4.8 mmol) over one hour. During

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the time of addition, the color of the solution changed from clear to pale yellow. After complete addition, the reaction mixture was stirred at additional 15 min. at 0 ºC and the cooled to -78 ºC. EtI (0.4 mL, 4.8 mmol) was added over 10 min. After 2.5 h TLC showed that the reaction was completed and was quenched with 1 mL methanol and diluted with 1 M aqueous HCl until it was acidic. The reaction turned orange. The mixture was extracted with EtOAc (3 x 45 mL), then the combined organic phase was washed twice with brine, dried with MgSO4, filtered and concentrated. The crude residue was purified with column chromatography (25% EtOAc in toluene + 1% formic acid) to yield 44 as a red/brown solid (0.167 g, 78 %). 1H-NMR (500 MHz, CDCl3): δ 6.53 (s, 2H), 4.03 (q, J = 7.1 Hz, 2H), 3.84 (s, 6H), 3.39 (t, J = 7.7 Hz, 1H), 2.09 (dq, J = 13.6, 7.5 Hz, 1H), 1.80 (dq, J = 13.6, 7.4 Hz, 1H), 1.35 (t, J = 7.1 Hz, 3H), 0.93 (t, J = 7.3 Hz, 3H) ppm. 13C-NMR (126 MHz, CDCl3): δ 177.89, 153.71, 136.47, 133.93, 105.19, 69.03, 56.29, 53.32, 26.67, 15.69, 12.29 ppm. HRMS (MALDI/FTICR) m/z: [M ‒ e]+ Calcd for C14H20O5+ 268,13053; Found 268,13046.

(S)-2-(3,5-methoxy-4-propoxyphenyl) butanoic acid (45)

1-Iodopropane (0.6 mL, 6.15 mmol) was added to a suspension of (4-hydroxy-3,5- dimethoxyphenyl)acetic acid (43, 0.5077 g, 2.36 mmol) and CsCO3 (2.46 g, 12.8 mmol) at 65 ºC and left overnight. TLC indicated complete reaction and was quenched and diluted with 1 M aqueous HCl and the aqueous phase was extracted with EtOAc (3 x 45 mL) and the combined organic phase was washed with brine, dried with MgSO4, filtered and concentrated in vacuo. The crude residue was purified with flash column chromatography (25% EtOAc in heptane, + 1% formic acid) to yield the 4-propoxy propylester as a pale oil (0.323 g, 46 %). 1H-NMR (500 MHz, CDCl3): δ 6.50 (s, 2H), 4.06 (t, J = 6.7 Hz, 2H), 3.90 (t, J = 7.0 Hz, 2H), 3.83 (2 x s, 6H), 3.54 (s, 2H), 1.76 (h, J = 7.3 Hz, 2H), 1.65 (h, J = 7.4, 2H), 0.99 (t, J = 7.4, 3H), 0.92 (t, J = 7.4, 3H) ppm.

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13C-NMR (126 MHz, CDCl3): δ 171.8, 153.6, 136.6, 129.5, 106.6, 75.3, 66.6, 56.3, 41.8, 23.5, 22.1, 10.5, 10.4 ppm.

To a solution of this propyl ester (0.323 g, 1.09 mmol), 7.5 mL methanol and 4 mL water, lithium hydroxide monohydrate was added (0.250 g, 5.84 mmol). The reaction was stirred overnight and quenched with 30 mL 1 M aqueous HCl. The aqueous phase was extracted with 3 x 25 mL EtOAc and the combined organic phase was washed with brine, dried with MgSO4, filtered and concentrated in vacuo. The crude residue was purified with column chromatography (40% EtOAc in heptane + 1% formic acid) to yield the 4-propoxy acid as a slightly yellow oil (0.141 g, 41 %). 1H-NMR (500 MHz, CDCl3): δ 6.49 (s, 2H), 3.91 (t, J = 6.9 Hz, 2H), 3.83 (s, 6H), 3.58 (s, 2H), 1.76 (h, J = 7.3 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H) ppm. 13C-NMR (126 MHz, CDCl3): δ 177.3, 153.7, 136.8, 128.5, 106.8, 75.3, 56.3, 41.4, 23.4, 10.5 ppm.

To a solution of the acid (0.141 g, 0.55 mmol), Koga’s base (0.256 g, 0.56 mmol) and dry THF, n- BuLi (1.5 mL, 1.6 M in hexane, 2.4 mmol) was added at 0 ºC over 1 hour. During the time of addition, the reaction turned from clear to pale yellow. After the addition was complete, the reaction was stirred for additional 15 min. and then cooled to -78 ºC. Iodoethane (2 mL, 2.50 mmol) was added over 10 min. After 2.5 h, the reaction was quenched with 1 mL ethanol and 1 mL water. The mixture was diluted with 1 M aqueous HCl (35 mL). The aqueous phase was extracted with 3 x 25 mL EtOAc, and the combined organic phase was washed with saturated aqueous Na2S2O3. It was then washed with brine, dried with MgSO4, filtered and concentrated in vacuo. The residue was purified with flash column chromatography (40% EtOAc in heptane +1% formic acid) to yield 45 (66.3 mg, 42 %) as a clear oil. 1H-NMR (500 MHz, CDCl3): δ 6.52 (s, 2H), 3.91 (t, J = 6.9 Hz, 2H), 3.39 (t, J = 7.7, 1H), 2.08 (m, 1H), 1.78 (m, 3H), 0.99 (t, J = 7.4, 3 H), 0.96 (t, J = 7.4, 3H) ppm. 13C-NMR (126 MHz, CDCl3) δ 177.7, 153.5, 136.7, 133.7, 105.2, 75.1, 56.2, 53.1, 26.5, 23.3, 12.1,

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(S)-2-(3,5-dimethoxy-4-octyloxyphenyl) butanoic acid (46)

To a suspension of (3,5-dimethoxy-4-hydroxyphenyl)acetic acid (43, 0.510 g, 2.36 mmol) and K2CO3 (1.32 g, 9.56 mmol) in 5 mL of dry DMF at 60 ºC, 1-bromooctane (1.7 mL, 2.02 g, 10.5 mmol) was added. The suspension was stirred for 27 hours until the TLC showed the reaction was complete. The yellow suspension was then diluted with 1 M aqueous HCl (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic phase was washed twice with brine, dried with MgSO4, filtered and concentrated in vacuo. The oil was purified with column chromatography (30% EtOAc in heptane +1% formic acid) to yield the 4-octyloxy octyl ester as a light brown oil (0.50 g, 48 %). 1H-NMR (500 MHz, CDCl3): δ 6.50 (s, 2H), 4.09 (t, J = 6.8 Hz, 2H), 3.93 (t, J =
6.8Hz, 2H), 3.83 (2 x s, 6H), 3.54 (s, 2H), 1.74 (dt, J = 14.8, 6.9 Hz, 2H), 1.62 (dt, J = 14.3, 6.7 Hz, 2H) 1.47 ‒ 1.38 (m, 2H), 1.36 ‒ 1.23 (m, 18H), 0.88 (m, 6H) ppm. 13C (126 MHz, CDCl3): δ 171.84, 153.59, 136.61, 129.36, 105.58, 73.67, 65.23, 56.26, 41.87, 32.01, 31.92, 30.24, 29.56, 29.46, 29.34, 29.33, 28.75, 26.05, 26.01, 25.82, 22.78, 14.25, 14.22 ppm.

To a solution of this compound (0.50 g) in 10 mL of water, 5 mL of MeOH and lithium hydroxide monohydrate (0.561 g, 13.3 mmol) was added. The solution was stirred for 17 hours and quenched with 50 mL of 1 M aqueous HCl. The aqueous phase was extracted with EtOAc (3 x 30 mL), and then the organic phase was washed with brine, dried with MgSO4, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (40% EtOAc in heptane +1% formic acid) to yield the 4-octyl carboxylic acid as a clear oil (0.364 g, 99 %). 1HNMR (500 MHz, CDCl3): δ 6.49 (s, 2H), 3.93 (t, J = 6.9 Hz, 2H), 3.83 (2 x s, 6H), 3.58 (s, 2H), 1.74 (dt, J = 14.7, 7.0 Hz, 2H), 1.47 ‒ 1.38 (m, 2H), 1.35 ‒ 1-22 (m, 8H), 0.87 (t, J = 7.1 Hz, 3H) ppm. 13C-NMR (126

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To a solution of the acid (0.356 g, 1.10 mmol) and Koga’s base (0.556 g, 1.2 mmol) in dry THF (10 mL), n-BuLi (2.9 mL, 1.6 M in hexane, 4.64 mmol) was added dropwise at 0 ºC over an hour. During the time of the addition the color of the solution turned from pale white to purple, then blue, green, brown and red respectively. The reaction mixture was agitated for additional 15 min. after the addition was complete. The reaction mixture was then cooled to -78 ºC. Iodoethane (0.4 mL, 4.98 mmol) was added over 10 min. and the reaction color turned green. After 5 hours, the reaction was complete and was quenched with 1 mL of methanol followed by 1 mL of water. The reaction mixture was diluted with aqueous HCl (1M, 30 mL) and extracted with EtOAc (3 x 40 mL). The combined organic phase was washed with brine, saturated aqueous Na2S2O3, dried with MgSO4 and concentrated in vacuo. The residue was purified by flash column chromatography (30% EtOAc in heptane + 1% formic acid) to yield 46 as a clear oil (0.132 g, 34 %). 1H-NMR (500 MHz, CDCl3): δ 6.52 (s, 2H), 3.93 (t, J = 6.9 Hz, 2H), 3.83 (s, 6H), 3.38 (t, J = 7.7 Hz, 1H), 2.13 ‒ 2.03 (m, 1H), 1.84 ‒ 1.71 (m, 4H), 1.45 ‒ 1.40 (m, 3H), 1.31 ‒ 1.27 (m, 6H), 0.94 (t, J = 7.4 Hz, 3H), 0.88 (t, J =
6.9Hz, 3H) ppm. 13C-NMR (126 MHz, CDCl3): δ 178.37, 153.66, 136.94, 133.76, 105.33, 73.67, 56.32, 53.37, 32.01, 30.24, 29.56, 29.46, 26.66, 26.46, 26.00, 22.82, 14.26, 12.28 ppm. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C20H32O5Na+ 375.2140; Found 375.2137.

(S)-2-(4-isobutoxy-3,5-dimethoxyphenyl)butyric acid (47)

To a suspension of (3,5-dimethoxy-4-hydroxyphenyl)acetic acid (43, 0.509 g, 2.79 mmol) and K2CO3 (2.61 g, 18.9 mmol) in 7 mL of dry DMF at 60 ºC, isobutyl bromide (1.1 mL, 1.39 g, 10 mmol) was added and the mixture stirred for 23 hours after which the TLC indicated that the reaction was complete. The reaction mixture was then diluted with 50 mL 1 M aqueous HCl and

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extracted with EtOAc (3 x 25 mL). The combined organic phase was then washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (15%EtOAc in heptane + 1% formic acid) to yield the isobutoxy isobutyl ester as a yellow oil (0.448 g, 55 %). 1H-NMR (500 MHz, CDCl3): δ 6.50 (s, 2H), 3.88 (d, J = 6.6 Hz, 2H), 3.82 (s, 6H), 3.70 (d, J = 6.8, 2H), 3.54 (s, 2H), 2.04 (n, J = 6.8 Hz, 1H), 1.92 (n, J = 6.8 Hz, 1H), 1.01 (d, J = 6.7 Hz, 6H), 0.90 (d, J = 6.7 Hz, 6H) ppm. 13C-NMR (126 MHz, CDCl3): δ 171.79, 153.51, 137.03, 129.43, 106.81, 80.28, 71.10, 56.33, 41.90, 29.17, 27.88, 19.44, 19.18 ppm.

To a solution of this ester in methanol (8 mL) and water (8 mL), lithium hydroxide monohydrate was added (0.273 g, 6.51 mmol). The suspension was stirred overnight and quenched with aqueous HCl (1 M, 50 mL) and extracted with 3 x 25 mL EtOAc. The combined organic phase was washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (30% EtOAc in heptane + 1% formic acid) to yield the 4-isobutoxy carboxylic acid as a white solid (0.265 g, 71 %). 1H-NMR (500 MHz, CDCl3): δ 6.49 (s, 2H), 3.82 (s, 6H), 3.70 (d, J = 6.7 Hz, 2H), 3.57 (s, 2H), 2.04 (n, J = 6.7, 1H), 1,01 (d, J = 6.7 Hz, 6H) ppm. 13C-NMR (126 MHz, CDCl3): δ 177.30, 153.60, 137.29, 128.38, 106.93, 80.30, 56.36, 41.38, 29.16, 19.42 ppm.

To a solution of the acid (0.265 g, 0.986 mmol) and Koga’s base (0.535 g, 0.119 mmol) in dry THF (10 mL) at 0 ºC, n-BuLi (1.58 mL, 2.5 M in hexane, 3.95 mmol) was added dropwise over an hour. After complete addition, the reaction mixture was stirred for additional 15 min before it was cooled to -78 ºC. To the cold solution, EtI (0.32 mL, 3,96 mmol) was added over 10 min. The reaction was stirred for additional 3 hours and quenched with methanol (1 mL) and water (1 mL). The solution was then diluted with 25 mL of aqueous HCl (1 M, 25 mL) and extracted with EtOAc (3 x 25 mL). The combined organic phase was then washed with brine, dried over MgSO4, filtered

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and concentrated in vacuo. The crude residue was purified by column chromatography (30% EtOAc in heptane +1% formic acid) to yield 47 as a white solid (0.153 g, 46 %). 1H-NMR (500 MHz, CDCl3): δ 6.52(s, 2H), 3.82 (2 x s, 6H), 3.70 (d, J = 6.8 Hz, 2H), 3.38 (t, J = 7.6 Hz, 1H), 2.13-2.08 (m, 2H), 1.87-1.73 (m, 1H), 1.00 (d, J=6.8 Hz, 6H), 0.92 (t, 3H) ppm. 13C-NMR (126 MHz, CDCl3): δ 177.83, 153.61, 137.36, 133.67, 105.55, 80.26, 56.40, 53.26, 29.17, 26.66, 19.43, 12.27 ppm. HRMS (ESI/FTICR) m/z: [M+H]+ Calcd for C16H25O5+ 297.16965; Found 297.16973.

3-Hydroxy-3-methyl-2-(3,4,5-trimethoxyphenyl)butanoic acid (48)
The reaction was carried out as described for compound 40, using 3,4,5-trimethoxyphenyl acetic acid (300 mg, 1.33 mmol), Koga’s base (613 mg, 1.37 mmol), n-BuLi (3.3 mL, 1.6 M in hexanes, 5.3 mmol) and anhydrous acetone (0.4 mL, 5.5 mmol). The reaction was quenched with MeOH 30 min after addition of acetone. Purification was performed by flash column chromatography (10- 50% EtOAc in toluene + 1% formic acid) yielding 48 (297 mg, 79%). 1H NMR (500 MHz, CDCl3): δ 6.63 (s, 2H), 3.84 (s, 6H), 3.84 (s, 3H), 3.55 (s, 1H), 1.40 (s, 3H), 1.15 (s, 3H) ppm. 13C NMR (126 MHz, CDCl3): δ 177.08, 153.16, 137.97, 130.43, 107.03, 72.16, 61.00, 60.55, 56.32, 29.65, 27.04 ppm. HRMS (ESI/FTICR) m/z: [M + H]+ Calcd for C14H21O6+ 285.1333; Found 285.1339.

2-(1-hydroxycyclohexyl)-2-(3,4,5-trimethoxyphenyl)acetic acid (49)
The reaction was carried out as described for compound 40, using 3,4,5-trimethoxyphenylacetic acid (300 mg, 1.33 mmol), Koga’s base (613 mg, 1.37 mmol), n-BuLi (3.3 mL, 1.6 M in hexanes, 5.3 mmol) and cyclohexanone (0.55 mL, 5.3 mmol). The reaction was quenched with MeOH 30 min after addition of cyclohexanone. Purification was performed by flash column chromatography (30% EtOAc in toluene + 1% formic acid) yielding 49 (310 mg, 72%). Mp 63 – 65 °C . 1H NMR (500 MHz, CDCl3): δ 6.64 (s, 2H), 3.84 (s, 6H), 3.83 (s, 3H), 3.57 (s, 1H), 1.82 – 1.75 (m, 1H), 1.75 – 1.64 (m, 1H), 1.63 – 1.50 (m, 4H), 1.49 – 1.37 (m, 2H), 1.29 – 1.17 (m, 2H) ppm. 13C NMR (126

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MHz, CDCl3): δ 177.49, 153.11, 137.92, 129.74, 107.11, 77.41, 77.16, 76.90, 72.84, 60.98, 59.91, 56.30, 37.58, 34.86, 25.60, 22.04, 21.68 ppm. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C17H24O6Na+ 347.14651; Found 347.14655.

2-(1-hydroxycyclopentyl)-2-(3,4,5-trimethoxyphenyl)acetic acid (50)
The reaction was carried out as described for compound 40, using 3,4,5-trimethoxyphenylacetic acid (300 mg, 1.33 mmol), Koga’s base (613 mg, 1.37 mmol), n-BuLi (3.3 mL, 1.6 M in hexanes, 5.3 mmol) and cyclopentanone (0.47 mL, 5.3 mmol). The reaction was quenched with MeOH 30 min after addition of cyclopentanone. Purification was performed by flash column chromatography (30% EtOAc in toluene + 1% formic acid) yielding 50 (228 mg, 60%). 1H NMR (500 MHz, CDCl3): δ 6.66 (s, 2H), 3.84 (s, 6H), 3.83 (s, 3H), 3.58 (s, 1H), 1.93 – 1.83 (m, 2H), 1.81 – 1.70 (m, 2H), 1.70 – 1.65 (m, 1H), 1.65 – 1.56 (m, 1H), 1.55 – 1.44 (m, 2H) ppm. 13C NMR (126 MHz, CDCl3): δ 177.99, 153.13, 137.85, 131.07, 106.76, 83.04, 60.97, 59.20, 56.29, 40.41, 37.79, 23.85, 23.46 ppm. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C16H22O6Na+ 333.1309; Found 333.1312.

2-(1-hydroxycyclobutyl)-2-(3,4,5-trimethoxyphenyl)acetic acid (51)
The reaction was carried out as described for compound 40, using 3,4,5-trimethoxyphenylacetic acid (300 mg, 1.33 mmol), Koga’s base (613 mg, 1.37 mmol), n-BuLi (3.3 mL, 1.6 M in hexanes, 5.3 mmol) and cyclobutanone (0.40 mL, 5.3 mmol). The reaction was quenched with MeOH 3 hours after addition of cyclobutanone. Purification was performed by flash column chromatography (30% EtOAc in toluene + 1% formic acid) yielding 51 (150 mg, 40%). 1H NMR (500 MHz, CDCl3): δ 6.63 (s, 2H), 3.82 (s, 6H), 3.82 (s, 3H), 3.78 (s, 1H), 2.24 – 2.17 (m, 2H), 2.08 – 2.01 (m, 1H), 2.00 – 1.92 (m, 1H), 1.92 – 1.83 (m, 1H), 1.67 – 1.55 (m, 1H) ppm. 13C NMR (126 MHz,

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3-hydroxy-4-methyl-2-(3,4,5-trimethoxyphenyl)pentanoic acid (52)
To a solution of 3,4,5-trimethoxyphenylacetic acid (2.0 g, 8.8 mmol) in anhydrous THF (15 mL) at 0 ºC was added dropwise n-BuLi (7.4 mL, 2.5 M solution in hexanes, 18 mmol). The mixture was left stirring at 0 ºC for 30 min before adding isobutyraldehyde (3.3 mL, 36 mmol). After 3 hours the reaction was allowed to warm to ambient temperature and then partitioned between 1 M aqueous HCl (100 mL) and EtOAc (100 mL). The phases were separated and the aqueous phase was extracted 3 times with EtOAc (100 mL). The combined organic phases were washed with water (200 mL) followed by brine (200 mL), dried over MgSO4, filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (40% EtOAc in toluene + 1% formic acid) to yield 52 (1.0 g, 39%). Mp 133 – 135 °C. 1H NMR (500 MHz, CDCl3): δ 6.53 (s, 2H), 4.04 (dd, J = 9.5, 2.8 Hz, 1H), 3.84 (s, 6H), 3.82 (s, 3H), 3.65 (d, J = 9.5 Hz, 1H), 1.52 (dh, J = 9.5, 6.8, 2.8 Hz, 1H), 0.95 (d, J = 6.8 Hz, 3H), 0.87 (d, J = 6.8 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3): δ 178.11, 153.57, 137.84, 131.14, 105.62, 77.42, 60.98, 56.34, 56.07, 29.20, 20.40, 14.58 ppm. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C15H22O6Na+ 321.1309; Found 321.1303.

Ethyl 4-methyl-3-((methylsulfonyl)oxy)-2-(3,4,5-trimethoxyphenyl)pentanoate (53)
To a solution of 52 (1.0 g, 3.4 mmol) in absolute EtOH (50 mL) over 3Å Molecular Sieves was added AcCl (10 mL, 141 mmol) and the reaction was cautiously stirred overnight. TLC analysis indicated complete reaction and the suspension were filtered through a pad of Celite and the filtrate concentrated in vacuo. To the solid residue was added toluene (30 mL) and the resulting suspension was concentrated in vacuo to remove remaining EtOH. To the crude solid was added pyridine (20 mL), mesyl chloride (4 mL, 52 mmol) and DMAP (1.0 g, 8.2 mmol) and the reaction was left

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stirring overnight. TLC analysis indicated formation of product but also unreacted starting material, so additional pyridine (20 mL), mesyl chloride (4 mL, 52 mmol) and DMAP (1.0 g, 8.2 mmol) was added together with CH2Cl2 (20 mL). The reaction was stirred overnight and TLC analysis indicated complete reaction. The contents of the reaction vessel was concentrated in vacuo and the crude solid purified by flash column chromatography (20% EtOAc in toluene) to yield 53 (713 mg, 53%). 1H NMR (500 MHz, CDCl3): δ 6.55 (s, 2H), 5.29 (dd, J = 11.0, 1.8 Hz, 1H), 4.19 (dq, J = 10.9, 7.1 Hz, 1H), 4.10 (dq, J = 10.9, 7.1 Hz, 1H), 3.86 (s, 6H), 3.84 (s, 3H), 3.03 (s, 3H), 1.69 (dh, J = 6.9, 1.8 Hz, 1H), 1.24 (t, J = 7.1 Hz, 3H), 1.04 (d, J = 6.9 Hz, 3H), 0.87 (d, J = 6.9 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3): δ 171.90, 153.73, 138.18, 129.83, 105.62, 88.54, 61.64, 61.00, 56.41, 54.80, 39.10, 29.20, 20.09, 15.14, 14.20 ppm. HRMS (MALDI/FTICR) m/z: [M ‒ e]+ Calcd for C18H28O8S+ 404.1499; Found 404.1494.

(E)-4-methyl-2-(3,4,5-trimethoxyphenyl)pent-2-enoic acid (54)
To a solution of 53 (713 mg, 1.8 mmol) in CHCl3 was added DBU (0.55 mL, 3.7 mmol). After 7 hours TLC analysis indicate complete reaction and the reaction mixture was concentrated in vacuo. The solid residue was dissolved in MeOH (10 mL) and water was added (10 mL) together with LiOH (monohydrate, 415 mg, 9.9 mmol) and the resulting solution was stirred overnight. The reaction mixture was partitioned between 1 M aqueous HCl (30 mL) and EtOAc (30 mL). The phases were separated and the aqueous phase was extracted 3 times with EtOAc (30 mL). The combined organic phases were washed with water (100 mL) followed by brine (100 mL), dried over MgSO4, filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (30% EtOAc in toluene + 1% formic acid) to yield 54 (265 mg, 54%). 1H NMR (500 MHz, CDCl3): δ 6.95 (d, J = 10.6 Hz, 1H), 6.39 (s, 2H), 3.87 (s, 3H), 3.84 (s, 6H), 2.48 (dh, J = 10.6, 6.6 Hz, 1H), 1.02 (d, J = 6.6 Hz, 6H) ppm. 13C NMR (126 MHz, CDCl3): δ 171.78, 154.35,

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153.06, 137.62, 130.94, 130.51, 106.88, 60.99, 56.24, 29.01, 22.27 ppm. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C15H20O5Na+ 303.1208; Found 303.1211.

Tert-butyl (R)-2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate (57)
To a solution of (R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenol (55, 423 mg, 1.47 mmol) and tert-butyl-2-bromoacetate (0.24 mL, 1.6 mmol) in DMF (5 mL) was added K2CO3 (624 mg, 4.52 mmol). The resulting suspension was left stirring for 2.5 hours before it was concentrated in vacuo and purified by flash column chromatography (30% EtOAc in toluene) to afford 57 (586 mg, 99%). 1H NMR (500 MHz, CDCl3) δ 7.27 – 7.25 (m, 1H), 6.96 (br d, J = 7.7 Hz, 1H), 6.95 – 6.91 (m, 1H), 6.82 – 6.77 (m, 2H), 6.75 – 6.70 (m, 2H), 4.66 (ddd, J = 8.2, 5.1, 3.5 Hz, 1H), 4.52 (s,
2H), 3.86 (s, 3H), 3.85 (s, 3H), 2.73 – 2.58 (m, 2H), 2.13 – 2.04 (m, 1H), 2.03 – 1.95 (m, 1H), 1.87 (d, J = 3.5 Hz, 1H), 1.48 (s, 9H) ppm. 13C NMR (126 MHz, CDCl3) δ 168.13, 158.29, 149.00, 147.35, 146.59, 134.46, 129.73, 120.33, 119.23, 113.75, 112.35, 111.90, 111.41, 82.52, 73.86, 65.80, 56.07, 55.97, 40.72, 31.74, 28.19 ppm. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C23H30O6Na+ 425.1935; Found 425.1931.

(R)-2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetonitrile (58)
To a solution of (R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenol (55, 48 mg, 0.17 mmol) and 2-chloroacetonitrile (15 µL, 0.24 mmol) in DMF (2mL) was added K2CO3 (96 mg, 0.69 mmol) and the resulting suspension was left stirring for 2 hours. The contents of the reaction vessel were concentrated in vacuo and the crude residue purified by flash column chromatography (40% EtOAc in toluene) to afford 58 as a colorless oil (42 mg, 83%). 1H NMR (500 MHz, CDCl3) δ 7.32 (t, J = 8.1 Hz, 1H), 7.07 – 7.04 (m, 1H), 7.01 – 6.99 (m, 1H), 6.89 (ddd, J = 8.1, 2.7, 0.8 Hz, 1H), 6.79 (d, J = 8.1 Hz, 1H), 6.73 (dd, J = 8.1, 1.9 Hz, 1H), 6.71 (d, J = 1.9 Hz, 1H), 4.77 (s, 2H), 4.69 (dd, J = 8.0, 5.1 Hz, 1H), 3.855 (s, 3H), 3.849 (s, 3H), 2.74 – 2.59 (m, 2H), 2.13 – 1.95 (m, 3H) ppm. 13C

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NMR (126 MHz, CDCl3) δ 156.85, 149.00, 147.37, 147.18, 134.26, 130.06, 120.75, 120.33, 115.22, 114.04, 112.61, 111.89, 111.43, 73.53, 56.05, 55.96, 53.69, 40.81, 31.68 ppm. HRMS (MALDI/FTICR) m/z: [M – e]+ Calcd for C19H21NO4•+ 327.1465; Found 327.1463.

(R)-1-(3-((tert-butyldimethylsilyl)oxy)phenyl)-3-(3,4-dimethoxyphenyl)propan-1-ol (59) To a solution of (R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl) phenol (55, 101 mg, 0.350
mmol), 4-(dimethylamino) pyridine (28 mg, 0.23 mmol) and Et3N (0.4 mL, 3 mmol) in anhydrous CH2Cl2 (1.5 mL) was added tert-butyldimethylsilyl chloride (69 mg, 0.46 mmol) and the resulting solution was left stirring for 30 min. The contents of the reaction vessel were concentrated in vacuo and the crude residue was purified by flash column chromatography (5% EtOAc in CH2Cl2) to afford 59 as a colorless oil (98 mg, 69%). 1H NMR (500 MHz, CDCl3) δ 7.20 (t, J = 7.8 Hz, 1H),
6.94– 6.92 (m, 1H), 6.85 (t, J = 2.0 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.76 (ddd, J = 8.0, 2.4, 0.9 Hz, 1H), 6.74 – 6.70 (m, 2H), 4.63 (dd, J = 7.6, 5.4 Hz, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 2.73 – 2.56 (m, 2H), 2.15 – 1.94 (m, 2H), 1.87 (br s, 1H), 0.99 (s, 9H), 0.20 (s, 6H) ppm. 13C NMR (126 MHz, CDCl3) δ 155.97, 148.96, 147.30, 146.42, 134.54, 129.56, 120.31, 119.34, 118.99, 117.75, 111.87, 111.38, 73.83, 56.04, 55.92, 40.72, 31.74, 25.74, 18.32, -4.26. HRMS (MALDI/FTICR) m/z: [M – e]+ Calcd for C23H34O4Si•+ 402.22209; Found 402.22205.

(R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenyl diethyl phosphate (60)
To a suspension of (R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenol (55, 150 mg, 0.520 mmol) and K2CO3 (410 mg, 2.97 mmol) in anhydrous DMF (2 mL) was added diethyl chlorophosphate (0.1 mL, 0.7 mmol) and the resulting suspension was left stirring for 48 hours. The contents of the reaction vessel were concentrated in vacuo and the crude residue purified by flash column chromatography (80% EtOAc in toluene) to afford 60 as a colorless oil (162 mg, 73%). 1H NMR (500 MHz, CDCl3) δ 7.29 (t, J = 7.9 Hz, 1H), 7.22 – 7.20 (m, 1H), 7.15 – 7.13 (m, 1H), 7.13

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– 7.09 (m, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.73 – 6.70 (m, 2H), 4.69 – 4.65 (m, 1H), 4.24 – 4.15 (m, 4H), 3.85 (s, 3H), 3.84 (s, 3H), 2.73 – 2.58 (m, 2H), 2.39 (br s, 1H), 2.12 – 1.93 (m, 2H), 1.34 (td, J = 7.1, 1.0 Hz, 3H), 1.33 (td, J = 7.1, 1.0 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3) δ 150.97 (d, J = 6.8 Hz), 148.98, 147.33, 147.17, 134.39, 129.82, 122.57, 120.32, 119.02 (d, J = 4.6 Hz), 117.69 (d, J = 5.1 Hz), 111.89, 111.40, 73.31, 64.72 (d, J = 6.1 Hz), 56.04, 55.94, 40.84, 31.69, 16.21 (d, J = 6.6 Hz) ppm. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C21H29O7PNa+ 447.1543; Found 447.1540.

Tert-butyl (R)-4-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)butanoate (61)
To a suspension of 55 (0.222 g, 0.770 mmol) and K2CO3 (0.634 g, 4.59 mmol) in 10 mL DMF at 90 oC, a solution of t-butyl 4-bromobutanoic acid ester (0.250 g, 1.12 mmol) in 6 mL THF added. The suspension was stirred for 19 hours before it was diluted with 1 M aq. HCl and extracted with 3 x 50 mL EtOAc. The combined organic phase was dried over MgSO4, filtered and concentrated in vacuo. The remanence was purified by flash column chromatography (4:1 of toluene and EtOAc) to yield 61 (0.1380 g, 0.320 mmol, 42 %) as a clear oil. 1H-NMR (500 MHz, CDCl3): δ 7.25 (t, J=8.1 Hz, 1H), 6.91 (m, 2H), 6.80 (m, 2H), 6.73 (dd, J=13.3, 2.0 Hz, 1H), 6.72 (d, J=2.0 Hz, 1H), 4.66 (dd, J=7.9, 5.2, 1H), 4.00 (t, J=6.2, 2H), 2.70 (ddd, J=13.8, 9.7, 5.7, 1H), 2.62 (ddd, J=14.0, 9.5, 6.5 Hz, 1H), 2.42 (t, J=7.3 Hz, 2H), 2.05 (m, 4 H), 1.45 (s, 9H) ppm. 13C-NMR (126 MHz, CDCl3): δ 172.69, 159.29, 148.98, 147.33, 146.48, 134.51, 129.67, 120.34, 118.38, 113.67, 112.25, 111.91, 111.40, 80.52, 73.96, 67.00, 56.07, 55.06, 40.71, 32.17, 31.80, 28.26, 24.93 ppm.

(R)-1-(3-(4-(tert-butoxy)-4-oxobutoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-((S)-2- (3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (62)
To a solution of 61 (0.138 g, 0.321 mmol) and 56 (0.131 g, 0.359 mmol) in DCM (5 mL) at 0o C, 4-

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dimethylaminopyridine (0.078 g, 0.64 mmol) was added and the solution was stirred for 10 minuttes. N,N ’-diisopropylcarbodiimide (55 μ L, 354 mmol) was added and the reaction mixture was slowly heated to room temperature over 1 hour and then stirred at this temperature for 6 hours before it was diluted with 25 mL DCM and washed with 5 x 25 mL of water, washed once with brine, dried over MgSO4, filtered and concentrated in vacuo. The remanence was purified by flash column chromatography (4:1 of toluene and EtOAc) to yield 62 (0.1054, 42 %) as a clear oil. 1H- NMR (500 MHz, CDCl3, mixture of rotamers and distereomers, major rotamer reported when possible, rotamer ratio: 1.0:0.46): δ 7.17 (s, 1H), 7.11 (t, 1H), 6.78 (m, 2H), 6.58 (dd, J=13.4, 2.0 Hz, 2H and s, 1H), 6.35 (s, 2H), 5.54 (dd, J=8.1, 5.6 Hz, 1H), 5.40 (d, J=5.0 Hz, 1H), 3.90 (m, 1H), 3.79 (s, 6H), 3.77 (s, 3H), 3.76 (s, 3H), 3.72 (s, 3H), 2.75 (dt, J=13.2, 2.8 Hz, 1H), 2.48 (m, 3H), 2.35 (m, 4H), 2.20 (m, 4H), 2.00 (m, 4H), 1.63 (m, 4H), 1.38 (s, 9H), 0.83 (d, J=2.3 Hz, 3H) ppm. 13C-NMR (126 MHz, CDCl3, mixture of rotamers and distereomers, major rotamer reported): δ 172.66, 170.69, 159.01, 153.33, 148.99, 147.45, 141.90, 136.80, 135.49, 133.66, 129.67, 120.33, 118.50, 113.76, 112.98, 111.86, 111.42, 105.14, 80.47, 76.07, 66.98, 60.90, 56.13, 56.06, 52.16, 50.92, 43.56, 39.76, 38.28, 32.19, 28.51, 28.25, 26.99, 25.52, 24.93, 21.10, 12.70 ppm. HR-MS (MALDI, FT-ICR, dithranol): m/z 778.41479 [M+H+], calculated mass for (C44H60NO11+) 778.41609.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-(piperidin-1-yl)ethoxy)phenyl)propan-1-ol (63)
To a solution of (R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl) phenol (55, 258 mg, 0.694 mmol) and 1-(2-bromoethyl) piperidine hydrobromide (280 mg, 1.03 mmol) in anhydrous DMF (2 mL) was added K2CO3 (482 mg, 3.49 mmol) and the resulting suspension was left stirring at 90 ºC overnight. The contents of the reaction vessel were concentrated in vacuo and the crude residue purified by flash column chromatography (30% acetone in toluene + 1% Et3N) to afford 63 as a colorless oil (94 mg, 26%). 1H NMR (500 MHz, CDCl3) δ 7.24 (t, J = 8.1 Hz, 1H), 6.92 – 6.89 (m,

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2H), 6.82 – 6.77 (m, 2H), 6.74 – 6.70 (m, 2H), 4.64 (dd, J = 7.7, 5.2 Hz, 1H), 4.08 (t, J = 6.1 Hz, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 2.74 (t, J = 6.1 Hz, 2H), 2.72 – 2.57 (m, 2H), 2.50 (br s, 4H), 2.23 (br s, 1H), 2.13 – 1.94 (m, 2H), 1.60 (p, J = 5.7 Hz, 4H), 1.47 – 1.41 (m, 2H) ppm. 13C NMR (126 MHz, CDCl3) δ 159.18, 148.96, 147.31, 146.56, 134.55, 129.60, 120.32, 118.41, 113.75, 112.27, 111.90, 111.39, 73.87, 66.01, 58.07, 56.06, 55.95, 55.19, 40.72, 31.80, 26.00, 24.30 ppm. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C24H34NO4+ 400.2482; Found 400.2484.

Tert-Butyl (R)-(4-(2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)ethyl)phenyl) carbamate (64)
To a suspension of (R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl) phenol (55, 230 mg, 0.798 mmol) and K2CO3 (700 mg, 5.06 mmol) in anhydrous DMF (2 mL) was added tert-butyl (4-(2- bromoethyl)phenyl)carbamate (366 mg, 1.22 mmol) and the resulting suspension was left stirring at 90 ºC overnight. The contents of the reaction vessel were concentrated in vacuo and the crude residue purified by flash column chromatography (20% EtOAc in toluene) to afford 64 as a colorless oil (106 mg, 26%). 1H NMR (500 MHz, CDCl3) δ 7.30 (br d, J = 8.1 Hz, 2H), 7.26 – 7.15 (m, 3H), 6.92 – 6.88 (m, 2H), 6.82 – 6.77 (m, 2H), 6.75 – 6.70 (m, 2H), 6.46 (br s, 1H), 4.64 (dd, J = 7.9, 5.2 Hz, 1H), 4.13 (t, J = 7.1 Hz, 2H), 3.85 (s, 6H), 3.04 (t, J = 7.1 Hz, 2H), 2.73 – 2.58 (m, 2H), 2.14 – 1.95 (m, 2H), 1.92 (br s, 1H), 1.51 (s, 9H) ppm. 13C NMR (126 MHz, CDCl3) δ 159.17, 152.95, 148.96, 147.31, 146.45, 136.92, 134.51, 133.00, 129.65, 129.63, 120.33, 118.93, 118.39, 113.74, 112.25, 111.90, 111.39, 80.60, 73.94, 68.87, 56.05, 55.94, 40.68, 35.27, 31.77, 28.47 ppm. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C30H37NO6Na+ 530.2513; Found 530.2508.

Tert-butyl (R)-(2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)ethyl)carbamate (65)

To a solution of (R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenol (55, 100 mg, 0.347 mmol) and tert-butyl (2-bromoethyl)carbamate (260 mg, 1.16 mmol) in DMF (2 mL) was added

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K2CO3 (280 mg, 2.03 mmol) and the resulting suspension was heated to 60 ºC 6 h before it was concentrated in vacuo. The crude residue was purified by flash column chromatography (20% EtOAc in toluene + 1% Et3N) to afford 65 as a colorless oil (84 mg, 56%). 1H NMR (500 MHz, CDCl3) δ 7.26 – 7.22 (m, 1H), 6.94 – 6.89 (m, 2H), 6.81 – 6.77 (m, 2H), 6.74 – 6.70 (m, 2H), 5.03 (br s, 1H), 4.68 – 4.63 (m, 1H), 4.00 (t, J = 5.0 Hz, 2H), 3.85 (s, 3H), 3.84 (s, 3H), 3.51 (br q, J = 5.0 Hz, 2H), 2.74 – 2.58 (m, 2H), 2.15 (d, J = 2.8 Hz, 1H), 2.12 – 2.04 (m, 1H), 2.04 – 1.93 (m, 1H), 1.44 (s, 9H) ppm. 13C NMR (126 MHz, CDCl3) δ 158.87, 156.01, 148.94, 147.30, 146.66, 134.46, 129.84, 120.30, 118.73, 113.52, 112.15, 111.89, 111.38, 79.66, 73.77, 67.22, 56.03, 55.92, 40.74, 40.21, 31.75, 28.49. HRMS (MALDI/FTICR) m/z: [M + K]+ Calcd for C24H33KNO6+ 470.1940; Found 470.1938.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(pyridin-4-ylmethoxy)phenyl)propan-1-ol (66)
To a suspension of (R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl) phenol (55, 120 mg, 0.416 mmol) and K2CO3 (115 mg, 0.832 mmol) in anhydrous DMF (2 mL) was added 4-(bromomethyl) pyridine hydrobromide (126 mg, 0.498 mmol) and the resulting suspension was left stirring overnight. The contents of the reaction vessel were concentrated in vacuo and the crude residue purified by flash column chromatography (EtOAc + 1% Et3N) to afford 66 as a colorless oil (27 mg, 17%). 1H NMR (500 MHz, CDCl3) δ 8.62 – 8.60 (m, 2H), 7.38 – 7.35 (m, 2H), 7.32 – 7.27 (m, 1H), 7.02 – 6.98 (m, 1H), 6.99 – 6.96 (m, 1H), 6.87 (ddd, J = 8.2, 2.7, 1.0 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.74 – 6.71 (m, 2H), 5.10 (s, 2H), 4.68 (dd, J = 7.9, 5.1 Hz, 1H), 3.860 (s, 3H), 3.855 (s, 3H), 2.74 – 2.59 (m, 2H), 2.14 – 1.95 (m, 3H) ppm. 13C NMR (126 MHz, CDCl3) δ 158.56, 150.07, 149.02, 147.40, 146.82, 146.44, 134.40, 129.86, 121.67, 120.34, 119.20, 113.92, 112.48, 111.93, 111.43, 73.81, 68.25, 56.09, 55.99, 40.79, 31.78 ppm. HRMS (MALDI/FTICR) m/z: [M + H]+ Calcd for C23H26NO4+ 380.18563; Found 380.18558.

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tert-butyl (R)-2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate (67)
To a solution of (R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenol (55, 43 mg, 0.15 mmol) and bromomethyl phenylketone (44 mg, 0.22 mmol) in DMF (2 mL) was added K2CO3 (96 mg, 0.69 mmol) and the resulting suspension was left stirring for 1 hour. The contents of the reaction vessel were concentrated in vacuo and the crude residue purified by flash column chromatography (30% EtOAc in toluene) to afford 67 as a white solid (50 mg, 82%). 1H NMR (500 MHz, CDCl3) δ 8.01 – 7.98 (m, 2H), 7.62 (tt, J = 7.0, 1.1 Hz, 1H), 7.53 – 7.47 (m, 2H), 7.26 (t, J = 7.7 Hz, 1H), 6.99 – 6.95 (m, 2H), 6.85 (dd, J = 8.1, 2.4 Hz, 1H), 6.78 (d, J = 7.9 Hz, 1H), 6.74 – 6.70 (m, 2H), 5.28 (s, 2H), 4.66 (dd, J = 7.8, 5.3 Hz, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 2.74 – 2.57 (m, 2H), 2.13 – 1.95 (m, 2H), 1.87 (br s, 1H) ppm. 13C NMR (126 MHz, CDCl3) δ 194.50, 158.37, 148.98, 147.33, 146.70, 134.69, 134.44, 134.04, 129.79, 128.99, 128.24, 120.33, 119.34, 113.89, 112.60, 111.89, 111.39, 73.80, 70.85, 56.06, 55.96, 40.68, 31.73 ppm. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C25H26O5Na+ 429.1672; Found 429.1669.

(R)-1-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)propan-2-one (68)
To a solution of (R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenol (55, 75 mg, 0.26 mmol) and chloroacetone (40 µL, 0.50 mmol) in DMF (2mL) was added K2CO3 (145 mg, 1.05 mmol) and the resulting suspension was left stirring for 3 hours. The contents of the reaction vessel were concentrated in vacuo and the crude residue purified by flash column chromatography (30% EtOAc in toluene) to afford 68 as a colorless oil (68 mg, 76%). 1H NMR (500 MHz, CDCl3) δ 7.26 (t, J = 7.9 Hz, 1H), 6.97 – 6.95 (m, 1H), 6.92 – 6.91 (m, 1H), 6.79 – 6.75 (m, 2H), 6.73 – 6.70 (m, 2H), 4.66 (dd, J = 8.0, 5.1 Hz, 1H), 4.53 (s, 2H), 3.85 (s, 3H), 3.84 (s, 3H), 2.71 – 2.58 (m, 2H), 2.26 (s, 3H), 2.14 – 1.93 (m, 2H) ppm. 13C NMR (126 MHz, CDCl3) δ 205.68, 158.03, 148.95, 147.31, 146.86, 134.37, 129.83, 120.30, 119.39, 113.50, 112.29, 111.88, 111.39, 73.67, 73.07, 56.02, 55.92,

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40.75, 31.71, 26.71 ppm. HRMS (MALDI/FTICR) m/z: [M – e]+ Calcd for C20H24O5•+ 344.1618; Found 344.1615.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-(prop-2-yn-1-yloxy)phenyl)propan-1-ol (69)
To a solution of (R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenol (55, 195 mg, 0.676 mmol) and propargyl bromide (1 mL, 80% in toluene, 9 mmol) in DMF (10 mL) was added K2CO3
(409mg, 2.96 mmol) and the resulting suspension was left stirring for 2.5 hours. The contents of the reaction vessel were concentrated in vacuo and the crude residue purified by flash column chromatography (30% EtOAc in toluene) to afford 69 as a colorless oil (183 mg, 89%). 1H NMR (500 MHz, CDCl3) δ 7.27 (t, J = 7.9 Hz, 1H), 6.99 – 6.95 (m, 2H), 6.89 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 6.78 (d, J = 8.2 Hz, 1H), 6.74 – 6.70 (m, 2H), 4.68 (d, J = 2.5 Hz, 2H), 4.65 (dd, J = 7.7, 5.4 Hz, 1H), 3.844 (s, 3H), 3.839 (s, 3H), 2.73 – 2.57 (m, 2H), 2.51 (t, J = 2.5 Hz, 1H), 2.13 – 1.95 (m, 3H) ppm. 13C NMR (126 MHz, CDCl3) δ 157.79, 148.90, 147.26, 146.55, 134.42, 129.61, 120.27,
119.21, 113.90, 112.63, 111.86, 111.36, 78.64, 75.65, 73.71, 55.99, 55.89, 55.84, 40.63, 31.65 ppm. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C20H22O4Na+ 349.1410; Found 349.1409.

(R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenyl morpholine-4-carboxylate (70)
To a suspension of (R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl) phenol (55, 100 mg, 0.347 mmol) and K2CO3 (334 mg, 2.42 mmol) in anhydrous DMF (2 mL) was added morpholine-4- carbonyl chloride (0.13 mL, 1.1 mmol) and the resulting suspension was left stirring overnight. The contents of the reaction vessel were concentrated in vacuo and the crude residue purified by flash column chromatography (80% EtOAc in toluene) to afford 70 as a colorless oil (83 mg, 60%). 1H NMR (500 MHz, CDCl3) δ 7.33 (t, J = 7.9 Hz, 1H), 7.17 (d, J = 7.7 Hz, 1H), 7.13 – 7.11 (m, 1H), 7.02 (ddd, J = 8.2, 2.3, 0.9 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.74 – 6.70 (m, 2H), 4.71 – 4.64 (m, 1H), 3.852 (s, 3H), 3.847 (s, 3H), 3.75 – 3.72 (m, 4H), 3.66 (br s, 2H), 3.55 (br s, 2H), 2.74 – 2.59

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(m, 2H), 2.19 (d, J = 3.3 Hz, 1H), 2.13 – 1.95 (m, 2H) ppm. 13C NMR (126 MHz, CDCl3) δ 153.82, 151.48, 148.96, 147.32, 146.59, 134.41, 129.48, 123.08, 120.86, 120.33, 119.35, 111.93, 111.39, 73.40, 66.74, 66.61, 56.05, 55.94, 44.99, 44.22, 40.72, 31.69 ppm. HRMS (MALDI/FTICR) m/z: [M – e]+ Calcd for C22H27NO6•+ 401.18329; Found 401.18332.

(R)-3-(3,4-dimethoxyphenyl)-1-(3-((methylthio)methoxy)phenyl)propan-1-ol (71)
To a solution of (R)-3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenol (55, 214 mg, 0.742 mmol) and chloromethyl methyl sulfide (70 µL, 0.84 mmol) in DMF (4 mL) was added K2CO3
(410mg, 2.97 mmol) and the resulting suspension was left stirring for 48 hours. The contents of the reaction vessel were concentrated in vacuo and the crude residue purified by flash column chromatography (20% EtOAc in toluene) to afford 71 as a colorless oil (86 mg, 33%). 1H NMR
(500 MHz, CDCl3) δ 7.28 (t, J = 8.1 Hz, 1H), 7.00 – 6.96 (m, 2H), 6.89 – 6.85 (m, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.75 – 6.71 (m, 2H), 5.15 (s, 2H), 4.67 (dd, J = 7.9, 5.2 Hz, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 2.75 – 2.58 (m, 2H), 2.25 (s, 3H), 2.16 – 1.90 (m, 3H) ppm. 13C NMR (126 MHz, CDCl3) δ 157.37, 148.98, 147.34, 146.55, 134.45, 129.67, 120.33, 119.42, 115.10, 113.64, 111.89, 111.40, 73.83, 72.50, 56.06, 55.95, 40.71, 31.75, 14.76 ppm. HRMS (MALDI/FTICR) m/z: [M – e]+ Calcd for C19H24O4S•+ 348.1390; Found 348.1388.

(R)-N-(2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)ethyl)morpholine-4- carboxamide (72)
To a solution of 65 (40 mg, 93 µmol) in anhydrous CH2Cl2 (1.5 mL) was added trifluoroacetic acid (0.20 mL, 2.6 mmol). After 1 hour TLC analysis indicated fully deprotection of Boc and the slightly yellow solution was concentrated in vacuo. The residue was dissolved in anhydrous DMF (2 mL) and Et3N (13 µL, 93 µmol) and morpholine-4-carbonyl chloride (15 µL, 0.13 mmol) was added. The reaction mixture was left stirring for 2 hours before it was concentrated in vacuo and the crude

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residue purified by flash column chromatography (2% MeOH in EtOAc + 1% Et3N) to yield 72 as a colorless oil (33 mg, 80%). 1H NMR (500 MHz, CDCl3) δ 7.25 (t, J = 8.0 Hz, 1H), 6.94 – 6.91 (m, 2H), 6.82 – 6.77 (m, 2H), 6.74 – 6.70 (m, 2H), 4.94 (t, J = 5.6 Hz, 1H), 4.66 (dd, J = 8.0, 5.0 Hz, 1H), 4.05 (t, J = 5.1 Hz, 2H), 3.853 (s, 3H), 3.848 (s, 3H), 3.68 – 3.62 (m, 6H), 3.36 – 3.30 (m, 4H), 2.71 (ddd, J = 14.1, 9.7, 5.6 Hz, 1H), 2.62 (ddd, J = 14.1, 9.4, 6.6 Hz, 1H), 2.14 (br s, 1H), 2.13 – 1.94 (m, 2H) ppm. 13C NMR (126 MHz, CDCl3) δ 158.96, 157.79, 148.99, 147.36, 146.76, 134.43, 129.74, 120.32, 118.82, 113.58, 112.13, 111.92, 111.41, 73.78, 67.43, 66.56, 56.06, 55.97, 44.06, 40.81, 40.55, 31.79. HRMS (MALDI/FTICR) m/z: [M + Na]+ Calcd for C24H32N2O6Na+ 467.2153; Found 467.2151.

Fluorescein Shield (73)

To a solution of 27 (12.2 mg, 15.7 µmol) in anhydrous CH2Cl2 (1 mL) was added trifluoroacetic acid (50 µL, 0.65 mmol). After 2 h TLC analysis indicated fully deprotection of the Boc group and the slightly yellow solution was concentrated in vacuo. The residue was dissolved in toluene (2 mL) and concentrated in vacuo, before DMF (2 mL), DIPEA (20 µL, 0.11 mmol) and FluorosceinNHS (10.2 mg, 21.6 µmol) was added. The dark yellow solution was left stirring for 1.5 h before it was concentrated in vacuo and the crude product purified by flash column chromatography (5% MeOH in CH2Cl2 + 1% formic acid) to yield 73 as a yellow solid (12.8 mg, 79%). 1H NMR (500 MHz, DMSO-d6, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 10.15 (s, 2H), 8.91 (br t, J = 5.5 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.70 (s, 1H), 7.11 (t, J = 7.9 Hz, 1H), 6.91 – 6.73 (m, 3H), 6.69 (d, J = 1.9 Hz, 2H), 6.63 – 6.50 (m, 9H), 5.49 (dd, J = 8.3, 5.3 Hz, 1H), 5.25 (br d, J = 3.9 Hz, 1H), 4.05 (t, J = 5.8 Hz, 2H), 3.98 (br d, J = 13.1 Hz, 1H), 3.84 (t, J = 7.1 Hz, 1H), 3.70 (s, 6H), 3.58 – 3.55 (m, 11H), 2.67 – 2.61 (m, 1H), 2.47 – 2.41 (m, 1H), 2.38 – 2.30 (m, 1H), 2.17 – 2.11 (m, 1H), 1.96 – 1.84 (m, 3H), 1.64 – 1.50 (m, 4H), 1.40 – 1.31 (m,

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1H), 1.16 – 1.07 (m, 1H), 0.78 (t, J = 7.3 Hz, 3H) ppm. 13C-NMR (126 MHz, DMSO-d6, mixture of rotamers 1:0.25. Only major rotamer is reported here) δ 172.12, 170.31, 170.02, 168.01, 164.84, 159.62, 158.17, 152.83, 152.62, 151.84, 148.62, 147.04, 141.99, 140.41, 136.20, 135.95, 135.51, 133.13, 129.42, 129.23, 128.30, 124.89, 122.33, 119.96, 118.13, 113.76, 112.73, 112.17, 111.87, 109.13, 105.09, 102.24, 83.30, 75.07, 65.65, 59.81, 55.50, 55.32, 55.31, 51.50, 48.78, 42.82, 37.50, 30.55, 28.02, 26.33, 24.83, 20.47, 12.27 ppm (1 signal hidden under DMSO signal. Confirmed by NMR in CDCl3). HRMS (MALDI/FTICR) m/z: [M + 2H]+ Calcd for C59H61N2O15+ 1038.41395; Found 1038.3626.

Fluorescence polarization assay for the measurement of the dissociation constant

The saturation binding measurement were performed in black, flat bottom 384-well NBS microplates (No.: 3820, Corning Life Science) on a Safire2 plate-reader (Tecan, Mannedorf, Switzerland). The Fluorescein probe 72 was measured at excitation/emission values of 485/535 nm (bandwidth = 20 and 25 nm, respectively). The fluorescent probe 72 was diluted from a DMSO stock in a HEPES buffer (20 mM HEPES, 0.01% Triton-X, pH 8), at double the final concentration required for the final assay. A 1:1 serial dilution of the His-MBP-DD was performed in HEPES buffer and each protein dilution was mixed with the diluted Fluorescein labeled probe (giving a final DMSO conc. of 1%) and transferred to the 384-well plate. After 30 minutes of incubation at ambient temperature, the fluorescence anisotropy was measured. The binding curves were analyzed using Prism 7.0 (GraphPad, La Jolla, CA, USA) and the data were fitted to a four parameter logistic curve to deduce the EC50. The error bars represent the standard deviation of experiments performed in three repetitions. The dissociation constant was determined using equation:

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Kd = [EC50] ―
[LT]
2

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where [EC50] is the total concentration of His-MBP-DD, where equal amounts of free ligand of free and bound ligand is present, and [LT] is the total concentration of fluorescent probe.

Fluorescence polarization competitive binding assay

The fluorescein labeled probe 72 and the competitive ligand was in parallel diluted from a DMSO stock solution in HEPES buffer (20 mM HEPES, 0.01% Triton-X, pH 8) to give a 20% final DMSO concentration at 40 times the final concentration (a fixed concentration of 3.6 nM for the fluorescent probe). A 1:1 serial dilution of the competitive ligand was then performed in the solution containing the probe 72. Every sample was then diluted by a factor of 10 in HEPES buffer and the mixture of fluorescent probe and competitive inhibitor was then mixed with an equal volume of the protein, which had been diluted in HEPES buffer to give two times the concentration required for the final assay (34 nM). The samples were then transferred to a black, flat bottom 384-well NBS microplates (No.: 3820, Corning Life Science) and the fluorescence anisotropy was after 30 min of incubation measured on a Safire2 plate-reader (Tecan, Mannedorf, Switzerland). The data was afterwards processed to determining the fraction bound probe (FSB) using the equation below, with AOBS being the observed anisotropy, and AF and AB is the anisotropies of the free and bound probe respectively.

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(AB ― AOBS) + AOBS ― AF

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𝐾i =
1 +
IC50 [LT](𝑓0 + 2) 2Kd(𝑓0 + 1)

+ 𝑓𝑜
𝑓0
+ Kd
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Induced accumulation of DD-GFP in transgenic plants by Shld and derivatives

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Shld and derivatives were diluted in H20 with 0.05% silwett to a final concentration of 10µM, sprayed on 28-day old Arabidopsis thaliana plants expressing the RDDKeGFP protein. After 4 hours, 5 medium leaf disks were isolated and flash frozen in liquid nitrogen. Protein was extracted in 120µL LDS buffer (Novex NuPage) and boiled for 5 min. Total protein was separated by SDS- PAGE and electro-blotted. Immunoblots were blocked for 1 hour in TBS-Tween (0.1% v/v) and 5% BSA. GFP was detected by incubation with primary anti-GFP antibody (1/5000; Torrey Pines Biolabs), followed by incubation with anti-rabbit-IgG-HRP (1/5000; Promega). The horseradish peroxidase-conjugated antibody was visualized with ECL substrate (2mM 41BPA, 500 µM luminol, 100mM Tris pH 8,8 and 1,7•10-2 H2O2) and pictures taken with a Sony A7S camera.

Ancillary information
Supporting information is available containing replacement curves of fluorescent probe 73 from His-MBP-DD-FKBP12 as measured by fluorescence polarization, NMR spectra of compounds 1- 73, LC-MS traces & spectra for compounds 1-35 and molecular formula strings and the associated biodata. The 3D models shown in Figure 5 was based on the PDB structure 1BL4. Authors will release the atomic coordinates upon article publication. Corresponding author is M. Bols ([email protected]). Non-standard abbreviation used: FKBP = FK binding protein, pKa = minus logarithm of the acidity constant, DD = destabilizing domain, MBP = maltose-binding protein, HEPES = (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

The authors thank the Danish national research council for production and technology and the University of Copenhagen for financial support.

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Shield-1

References

1Maynard-Smith, L. A.; Chen, L.-C.; Banaszynski, L. A.; Ooi, A. G. L.; Wandless, T. J. A Directed Approach for Engineering Conditional Protein Stability Using Biologically Silent Small Molecules. J. Biol. Chem. 2007, 282,
24866-24872.

2Jørgensen, F. P.; Bols, M. An Inexpensive and Scalable Synthesis of Shld. J. Org. Chem. 2018, 83, 6050–6055.

3Clackson, T.; Yang, W.; Rozamus, L. W.; Hatada, M.; Amara, J. F.; Rollins, C. T.; Stevenson, L. F.; Magari, S. R.; Wood, S. A.; Courage, N. L.; Lu, X.; Cerasoli, F.; Gilman, M.; Holt, D. A. Redesigning an FKBP–ligand Interface to Generate Chemical Dimerizers with Novel Specificity. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 10437–10442.
4Yang, W.; Rozamus, L. W.; Narula, S.; Rollins, C. T.; Yuan, R.; Andrade, L. J.; Ram, M. K.; Phillips, T. B.; van Schravenkijk, M. R.; Dalgarno, D.; Clackson, T.; Holt, D. A. Investigating Protein-Ligand Interactions with a Mutant FKBP Possessing a Designed Specificity Pocket. J. Med. Chem. 2000, 43, 1135–1142.
5Lampson, M. A.; Kapoor, T. M. Targeting Protein Stability with a Small Molecule. Cell 2006, 126, 827-829.

6Banaszynski, L.A.; Chen, L.C.; Maynard-Smith, L.A.; Ooi, A.G.; Wandless, T.J. A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules. Cell 2006, 126, 995–1004.
7a) Armstrong, C.M.; Goldberg, D.E. An FKBP Destabilization Domain Modulates Protein Levels in Plasmodium Falciparum. Nat. Methods. 2007, 4, 1007–1009. b) Herm-Götz, A., Agop-Nersesian, C., Munter, S., Grimley, J.S.; Wandless, T.J.; Frischknecht, F.; Meissner, M. Rapid Control of Protein Level in the Apicomplexan Toxoplasma Gondii. Nat. Methods. 2007, 4, 1003–1005.
8Su, L.; Li, A.; Li, H.; Chu, C.; Qiu, J. –L. Direct Modulation of Protein Level in Arabidopsis. Molecular Plant 2013, 6, 1711–1714.
9Kaschani, F.; van der Hoorn, R. Small Molecule Approaches in Plants. Curr. Opin. Chem. Biol. 2007, 11, 88-98.

10Schreiber, L. Polar Paths of Diffusion across Plant Cuticles: New Evidence for an Old Hypothesis. Ann. Bot. 2005, 95, 1069-1073.
11Schönherr, J. A Mechanistic Analysis of Penetration of Glyphosate Salts across Astomatous Cuticular Membranes Pest Manag. Sci. 2002, 58, 343-351.
12Sterling, M. T. Mechanisms of Herbicide Absorption across Plant Membranes and Accumulation in Plant Cells Weed Sci. 1994, 42, 263-276.

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13a) Moldvai, I.; Dörnyei, G.; Temesvári-Major, E.; Szántay, C. A Practical One-pot Synthesis of Weinreblike Amides of (S)- and (R)-N-Boc-pipecolic acids from (+)-Piperidine-2-carboxylic acid Org. Prep. Proced. Int. 2007, 39, 503-508 b) Ying, W.; Herndon, J. W. Total Synthesis of (+)‐Antofine and (–)‐Cryptopleurine Eur. J. Org. Chem. 2013, 3112- 3122.
14Mulzer, J.; Segner, J.; Bruntrup, G. Stereoselective Synthesis of Threo-3-hydroxycarboxylic Acids. Stereochemistry of an Aldoltype Addition under Kinetic and Thermodynamic Control. Tetrahedron Lett. 1977, 18, 4651-4654.
15Kingsbury, C. A.; Draney, D.; Sopchik, A.; Rissler, W.; Durham, D. Survey of Carbon-13-hydrogen Splittings in Alkenes. J. Org. Chem. 1976, 41, 3863-3868.
16Blackburn, G. M.; Ingleson, D. Specific Dealkylation of Phosphonate Esters using Iodotrimethylsilane. J. Chem. Soc. Chem. Commun. 1978, 870-871.
17Vorona, S.; Artamonova, T.; Zevatskii, Y.; Myznikov, L. An Improved Protocol for the Preparation of 5-Substituted Tetrazoles from Organic Thiocyanates and Nitriles. Synthesis 2014, 46, 781-786.
18Kozany, C.; März, A.; Kress, C.; Hausch, F. Fluorescent Probes to Characterise FK506-Binding Proteins. Chembiochem, 2009, 10, 1402-1410.
19Linclau, B.; Peron, F.; Bogdan, E.; Wells, N.; Wang, Z.; Compain, G.; Fontenelle, C. Q.; Galland, N.; LeQuestel, J. – Y. and Graton, J. Intramolecular OH⋅⋅⋅Fluorine Hydrogen Bonding in Saturated, Acyclic Fluorohydrins: The γ‐Fluoropropanol Motif. Chem. Eur. J. 2015, 21, 17808–17816.
20PDB: http://www.rcsb.org/3d-view/1BL4

21RDDKeGFP was used rather than DD-eGFP to reduce the leakiness of the system – see ref. 8

22Yamashita, Y.; Odashima, K.; Koga, K. Construction of Chiral Quaternary Carbon Centers by Asymmetric Alkylation of Achiral Lithium Enolates Mediated by Chiral Tetradentate Ligands: Stoichiometric and Catalytic Approaches. Tetrahedron Lett. 1999, 40, 2803–2806.
23Perlow, D. S.; Erb, J. M.; Gould, N. P.; Tung, R. D.; Freidinger, R. M.; Williams, P. D.; Veber, D. F. Use of N-Fmoc Amino Acid Chlorides and Activated 2-(Fluorenylmethoxy)-5(4H)-oxazolones in Solid-phase Peptide Synthesis. Efficient Syntheses of Highly N-Alkylated Cyclic Hexapeptide Oxytocin Antagonists Related to L-365,209. J. Org. Chem. 1992, 57, 4394-4400

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24Gopalakrishnan, R.; Kozany, C.; Gaali, S.; Kress, C.; Hoogeland, B.; Bracher, A.; Hausch, F. Evaluation of Synthetic FK506 Analogues as Ligands for the FK506-Binding Proteins 51 and 52. J. Med. Chem. 2012, 55, 4114-4122.
25Stivala, C. E.; Zakarian, A. Highly Enantioselective Direct Alkylation of Arylacetic Acids with Chiral Lithium Amides as Traceless Auxiliaries. J. Am. Chem. Soc. 2011, 133, 11936-11939.

Graphical abstract (TOC)

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R2

R3
R1


O

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

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OMe

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Shld: R1 = 3,4,5-(MeO)3Ph; R2 = Et R3 = 2-(N-morpholino)ethyl
1-12: R1 = various aryl; R2 = various alkyl; R3 =
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13-35: R1 = 3,4,5-(MeO)3Ph; R2 = Et; R3 = various acidic, basic & neutral groups