Synthesis of Shld Derivatives, Their Binding to the Destabilizing Domain, and Influence on Protein Accumulation in Transgenic Plants

Article abstract of DOI:10.1021/acs.jmedchem.9b00497

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 were supposedly i

Full text of DOI:10.1021/acs.jmedchem.9b00497

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Article
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
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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
bols@chem.ku.dk
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
1
2
3,4
The compound Shld (or Shield1, Figure 1) is a truncated synthetic analogue 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
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magnitude selectivity over the wild type. 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
6
7
8
engineered cell and has been used in mammalian cells, parasites and plants. 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
9
fundamentally different structure. 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
1
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cross the cuticles in hydration shells via aqueous polar pores. 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
OMe
1-3
MeO
OMe

B
A fragment
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C
B fragment
O


OR
N
MeO
OMe
Shld
O
O
O
O
4-7
OMe


N
OMe
B
C
O
MeO
OMe
OMe
O
8
-12
C fragment

B
C
R
A
B
A
B
O
O
O
O
OMe
OMe
MeO
MeO

R
13-34
35
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
12
trapped inside in the more basic cytosol (Figure 2B). 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
3
identified as very important for DD binding (Figure 1), 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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Cell
membrane
Plant
cuticle
Cell walls
Apoplasm
(pH 5.5)
Cytoplasm
(pH 7.5)
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A
B
Neutral
Neutral
Neutral
Vacuole
pH 5.5)
RCOOH
RCOOH
RCOOH
(
NR H
3
RCOO
NR3
RCOO
NR3
C
NR3
NR3
NR H
NR H
3
3
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.
1
Indeed the ethylmorpholino-group of Shld is believed to be outside the receptor, 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
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of stereochemistry at the stereocenter led to a 100-fold decrease in affinity. 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.
O
OH
O
O
O
O
N
O
FmocCl
NaHCO3
38
N
COOH
DCC, DMAP
CH2Cl2
O
N
H
H
OH
O
Fmoc
O
O
N
COOH
acetone/H O
2
O
Fmoc
OH
94%
83%
O
OH
37
39
N
O
36
Scheme 1. Synthesis of B-C fragment 39
Synthesis of the derivatives was carried out as follows: The C-fragment 38 was prepared
2
enantiomerically pure as previous described. Resolution of inexpensive racemic pipecolic acid
1
3
gave the tartrate 36. 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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X3
1) n-BuLi (4 equiv.)
X3
Koga base
X2
X2
O
2)
O
I
THF, -78 °C
X1
OH
X1
OH
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0: X , X =
, X = H 54%, S:R 10:1
1
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3
4
1: X = X = X = F
45%, S:R 1:1
1
2
3
4
2: X = X = H, X = Br
52%, S:R 16:1
1
3
2
O
1) RI, K CO3
O
2
2) LiOH
HO
O
RO
O
O
O
3) EtI, BuLi
Koga base
OH
OH
4
3
4
4
4
4
4: R = CH CH3
44% (over 3 steps)
8% (over 3 steps)
16% (over 3 steps)
18% (over 3 steps)
2
5: R = CH CH CH
3
2
2
6: R = CH (CH ) CH
3
2
2 6
7: R = CH CH(CH )
2
3 2
Scheme 2. Synthesis of fragment A precursors 40-42 and 44-47. In the synthesis of 45 Cs CO was used in
2
3
place of K CO . In the synthesis of 46-47 the alkyl bromides rather than the iodides were used. Koga base is
2
3
1
3
N ,N -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
5
4% 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
1) n-BuLi (4 equiv.)
O
O
Koga base
O
O
O
O
O
2)
OH
OH
OH
1
1
1
1
1
1
1
1
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R1
R2
R1
R2
THF, -78 °C
48: R , R = Me
(79%, S:R 1:1)
(72%, S:R 1:1)
(60%, S:R 1:1)
1
2
4
9: R , R =
1 2
5
0: R , R =
1
2
5
1: R , R =
(40%, S:R 1:1)
(39%, S:R 1:1)
1
2
5
2: R = H, R = isopropyl
1
2
O
O
1
) EtOH
AcCl
1) DBU
CHCl3
O
O
O
O
O
O
52
OEt
OMs
Rac
OH
2
) MsCl
DMAP
2) LiOH
H O/MeOH
2
53
54
2
8% (over 4 steps)
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
O
O
O
O
O
O
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R
Cl
N
DBU
CH Cl
N
H
N
Fmoc
O
O
O
O
O
O
R
O
2
2
3
9
N
O
N
O
N
O
O
O
O
O
O
O
9
: R =
5: R =
O
1
: R =
83%
72%
76%
75%
Br
O
O
O
O
O
O
2: R =
10: R =
72%
6
: R =
8
5%
7%
F
F
F
3: R =
O
O
O
O
O
O
7
: R =
97%
11: R =
4
O
4
8%
O
O
4: R =
O
O
O
O
8
: R =
12: R =
89%
O
90%
72%
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.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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Alkylation with an aldehyde, isobutyraldehyde, was also attempted. This gave alcohol 52 in
3
9% yield as a single diastereomeric pair (Scheme 3) in accordance with the reaction going through
1
4
a cyclic transition state. 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
1
3
1
(
Scheme 3). The configuration of 54 was determined from the C- H 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
o
chloride at 40 C. This led to smooth amide bond formation and resulted in 12 Shld derivatives in
4
7-97% 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
2
reported 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 pK values (Scheme 5). The idea behind these derivatives was to investigate if
a
the ion-trapping mechanism described above (Figure 2, B) could work for these compounds.
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Normally a protected version of the acidic group was used. Thus alkylation of the phenolic position
2
of 55 with tert-butyl bromoacetate afforded 57 in 99% yield. Similar alkylation with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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.
2
The O-substituted phenols 57, 58, 59, 60 and 61 was coupled to 56 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
1
6
ester, followed by addition of water affording the phosphoric acid analogue 17. 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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1
2
3
4
5
6
7
8
9
O
O
O
N
56
O
O
O
OH
OR
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HO
HO
O
COOH
N
O
RX, K CO3
DCC, DMAP (excess)
CH2Cl2
2
O
O
DMF
O
O
X = Br
X = Cl
O
O
OR
55
O
O
O
O
O
5
7: R =
99%
83%
22: R =
80%
O
N
O
TFA
1
8: R =
92%
OH
N
5
8: R =
23: R =
70%
90%
NaN3
ZnCl2
H
N
20: R =
N
N N
59: R =
Si
69%
73%
24: R =
Si
H
62%
65%
X = Cl
TBAF
21: R =
O
P
O
P
O
O
60: R =
25: R =
58%
99%
X = Cl
O
O
1) TMSI
2) H O
2
O
P
OH
1
7: R =
OH
O
O
O
61: R =
42%
62: R =
42%
O
O
X = Br
TFA
1
9: R =
53%
OH
Scheme 5. Synthesis of acidic Shld analogues. 59 was synthesized using DMAP and Et N in CH Cl .
3
2
2
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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2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
cycloaddition with sodium azide facilitated by ZnCl in i-PrOH, conditions reported by Vorona et
2
1
7
al. affording the tetrazole analogue 20 in 90% yield (Scheme 5).
O
O
O
N
56
O
O
O
OH
OR
HO
HO
O
COOH
N
O
RBr, K CO
DCC, DMAP (excess)
CH2Cl2
2
3
O
O
DMF
O
O
O
O
OR
55
O
O
N
N
63: R =
26%
26%
13: R =
26: R =
70%
O
O
64: R =
78%
93%
N
O
N
H
O
TFA
H
1
6: R =
NH2
O
O
65: R =
56%
17%
27: R =
14: R =
55%
79%
N
H
O
N
H
O
TFA
NH2
N
N
66: R =
15: R =
68%
Scheme 6. Synthesis of basic Shld analogues.
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1
2
3
4
5
To explore the effect of variations of pK in the base of Shld and the potential of the vacuole
a
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
6% 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
8% yields, respectively. Deprotection of the Boc-group of 26 and 27 were carried out with TFA in
6
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
K CO to give 69 in 89% yield. Coupling with 56 in the usual way gave 31 in 90% yield. Sulphur
2
3
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
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4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
N
56
O
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH
OR
HO
HO
O
COOH
N
O
RX, K CO
DCC, DMAP (excess)
CH2Cl2
2
3
O
O
DMF
O
O
O
O
OR
5
5
O
O
O
O
O
6
7
R =
82%
76%
28
29
R =
R =
75%
X = Br
O
68
R =
33%
90%
X = Cl
6
9
R =
89%
31
R =
R =
X = Cl
O
O
N
N
7
0
R =
60%
33%
30
33
90%
X = Cl
O
O
O
S
S
7
1
R =
R =
R =
53%
51%
X = Cl
m-CPBA
O
S
34
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
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1
2
3
4
5
carbamide 72 in 80% yield. Yet again, coupling to 56 using the developed DCC/DMAP conditions
afforded the carbamide analogue 32 in 70% yield.
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
N
O
N
5
6
O
O
NH
O
NH
O
O
O
COOH
O
O
DCC, DMAP (excess)
1) TFA, CH Cl2
2
N
O
O
CH Cl2
2)
2
O
HO
O
O
HO
N
Cl
70%
O
O
O
O
N
N
H
O
K CO DMF
2
3,
80%
O
O
O
O
32
7
2
65
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
O
O
N
5
6
O
O
O
35
O
COOH
OH
DCC, DMAP (excess)
O
O
N
O
CH Cl2
2
O
O
55%
O
O
Scheme 9. Synthesis of truncated Shld analogue 35.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
1) TFA, CH Cl rt
2
2,
OH
O
O
27
2) DIPEA
7
3
O
O
HO
O
OH
N
O
O
O
O
O
O
O
O
N
O
O
O
N
O
O
OH
H
O
FluoresceinNHS
79%
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 K value of 7.49 ± 1.21 nM was obtained (Figure
i
3
, right). With the fluorescence polarity assay established we determined the binding of the Shld
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1
2
3
4
5
derivatives to the DD protein in vitro. The modifications in residue A gave the K values shown in
i
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1 & 2.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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–
8
EGFP mutation 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 (E ) was determined as the percentage of RDDKeGFP stabilization compared to Shld
rel
Figure 4. Accumulation of RDDKeGFP in transgenic Arabidopsis treated with Shld derivatives 29,30 &
32
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8
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
Table 1 & 2). Erel was calculated as (I -I )/(I -I ), where I is the relative intensity from the
x u shld u x
derivative investigated, Ishld is the intensity from Shld and I is the intensity in untreated plant.
u
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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 K about 100 fold greater than Shld (Table 1). The relative affinity of Shld, 2 and 3
i
(pK = 8.1, 6.2 and 4.5) reflects the impact of the substituents on the electron density in the aromatic
i
R1
N
R2
O
O
O
O
O
O
N
O
Compound
R -group
R -group
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
K (shld)/K
Erel
100%
0%
0%
0%
50.0%
36.0%
5.8%
10.5%
0%
1
2
i
i
Shld
3,4,5-(MeO) C H -
Et
Et
Et
Et
Et
Et
Et
Et
1.0
3
6
2
1
2
2-Naphtyl-
4-BrC H -
0.00
0.01
0.00
0.54
0.54
0.00
0.20
0.21
0.03
0.05
0.07
0.00
6
5
3
*
3,4,5-F C H -
3
6
2
4
4-EtO-3,5-(MeO) C H -
4-PrO-3,5-(MeO) C H -
4-OktO-3,5-(MeO) C H -
4-iBuO-3,5-(MeO) C H -
2
6
2
5
6
7
2 6 2
2
6
2
2
6
2
8
9
*
*
3,4,5-(MeO) C H -
2-allyl
3
6
2
3,4,5-(MeO) C H -
1-cyclohexenyl
1-cyclopentenyl
1-cyclobutenyl
Isobutylene
0%
0%
18.8%
2.2%
3
6
2
1
1
0*
1*
3,4,5-(MeO) C H -
3
6
2
3,4,5-(MeO) C H -
3
6
2
1
2
3,4,5-(MeO) C H -
3 6 2
Table 1. Dissociation constants (K ) of shld derivatives modified in the A-residue binding to His-MBP-
i
DD-FKBP12 determined by fluorescence polarization and relative accumulation of DD-EGFP (E ) induced
rel
by the derivatives 1-12 compared to Shld. E =(I -I )/(I -I ), where I , I and I are the fluorescence
rel
x
u
shld
u
x
shld
u
intensity of DD-EFGP in the presences of the derivatives investigated, Shld or untreated plant, respectively.
Compound is 1:1 mixture of diastereomers
*
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3
4
5
ring (sum = -0.03, 0.23 and 0.74) according to the equation pK = -4.5sum + 7.7, where sum is the
i
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
sum of the Hammett constants (i.e.  (OMe) = 2 x 0.12-0.27;  (Br) = 0.23 ;  (F) = 2 x
sum
sum
sum
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
.34+0.06). The very high affinity of the trimethoxyphenyl group has been ascribed to formation of
3
19
two hydrogen bonds. Yet, fluorine can also act as hydrogen bond acceptor 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
2
0
one of the benzene rings in the C-fragment (Figure 5). 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
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
For compounds 1-7 the E value generally follows the results of the in vitro tests very
rel
well. Compounds 1-3, which have close to no affinity in vitro show 0% of the increase in
2
1
accumulation of RDDKeGFP 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 K is very high. As discussed above the poor
i
efficacy of 6 in vitro could be related to the assay and its solubility.
Similarly the R substituted derivatives 8-12 (Table 1) generally decreased in affinity
2
with increasing size: Allyl (8) had 4 times the K of Shld, cyclobutenyl (11) a 15-fold lower
i
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
2
to a wrong geometry as the -carbon is sp 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).
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1
2
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O
O
O
N
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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8
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0
O
O
O
O
OR
Entry Cmp
R-group
-CH CH N(CH CH ) CH
2
Type pKa
K (nM)
K (Shld)/K
Erel
74%
70%
100%
11%
35%
18%
84%
77%
18%
89%
15%
58%
10%
83%
19%
2%
21%
113%
126%
62%
107%
86%
76%
80%
i
i
i
1
2
13
14
Shld
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
base 10.5 8.78 ± 1.05
base 9.8 4.08 ± 0.37
base 7.8 7.49 ± 1.21
base 6.3 56.0 ± 30.5
base 5.1 150 ± 55.0
acid 0.5 228 ± 33.2
acid 3.2 18.7 ± 3.90
acid 4.4 38.0 ± 5.05
acid 4.7 37.2 ± 2.34
acid 10.0 30.5 ± 11.4
0.85
1.84
1.0
2
2
2
2 2
-CH CH NH
2
2
2
3
4
5
6
7
8
9
-CH CH N(CH CH ) O
2 2 2 2 2
-CH C(CHCH) N
0.13
0.05
0.03
0.40
0.20
0.20
0.24
0.07
0.24
0.00
0.47
0.00
0.07
0.08
0.45
0.45
0.13
0.82
0.27
0.91
0.03
2
2
-CH CH -p-C H -NH
2 2 6 4 2
-PO H
3 2
-CH COOH
2
-CH CH CH COOH
2 2 2
-CH C(=N)(N=N)NH
2
10
11
12
-H
-CH COOC(CH )
neutral
neutral
neutral
neutral
neutral
neutral
neutral
neutral
neutral
neutral
neutral
neutral
neutral
neutral
109 ± 18.2
31.2 ± 7.47
1650 ± 906
16.0 ± 3.31
> 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
2
3 3
-CH CN
2
1
1
1
1
1
1
1
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
4
-Si(CH ) C(CH )
3 2 3 3
-PO(OEt)2
-CH CH -p-C H -NHCOOC(CH )
3 3
2
2
6
4
-CH CH NHCOOC(CH )
2
2
3 3
-CH COC H
-CH COCH
2
6
5
2
3
-CON(CH CH ) O
2
2 2
-CH CCH
2
-CH CH NHCON(CH CH ) O
2
2
2
2 2
-CH SCH
-CH SO CH
See scheme 9
2
3
2
2
3
Table 2. Dissociation constants (K ) of Shld derivatives modified at the C-residue binding to His-MBP-
i
DD-FKBP12 determined by fluorescence polarization and relative accumulation of RDDKeGFP (Erel)
induced by the derivatives 13-35. pK values listed are not the pK values of the compound but of similar,
a
a
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 pK values. The primary amine analogue 14 (Table
a
2, entry 2) showed the strongest binding towards the protein (K = 4.08 nM) of all the tested
i
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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2
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4
5
6
7
8
9
0
1
5 (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 pK bases are more protonated in the buffer (pH 8.0) suggesting that having an
a
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 (K = 18.7 nM) of the five while the arylphosphoric acid 17 (entry 6) was the weakest with a
i
5
-10 fold decrease in binding affinity (K = 228 nM), compared to the other acid analogues. Since
i
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 (E of 84%, 77% and 89%
rel
respectively), while the tetrazole analogue 20 and the phosphate 17 had a lower effect (E of 18%).
rel
It should be noted that the higher pK acids 19 and 21 are superior at accessing the plant cell
a
compared to Shld (E 89% and 77% respectively) when taking into account their receptor binding
rel
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1
2
3
4
5
affinity is lower Shld (K (Shld)/K = 0.24 and 0.20 respectively). For 21 there could be an influence
i
i
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of this compound being of smaller size and possible more likely superior at penetrating the cuticle.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
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8
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1
2
3
4
5
6
7
8
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1
2
3
4
5
6
7
8
9
0
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
1
3 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 (E = 113%, 126% and
rel
1
07% 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 (E 80%). It is probable that the effectiveness of the compound is a result of the more
rel
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
1
affinities which appears to contradict the statement 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
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Experimental
General information.
Air and water sensitive reactions were carried out under nitrogen. All commercial available
1
chemicals and solvents were used as received. H NMR spectra were measured on a Bruker
1
3
instrument with cryo-probe at 500 MHz. C 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 CDCl (D 99.8%, referenced to δ = 7.26 ppm (CHCl ) and δ = 77.16 ppm
3
H
3
C
(CDCl )) and DMSO-d (D 99.8%, referenced to δ = 2.50 ppm (DMSO-d ) and δ = 39.52 ppm
3
6
H
5
C
(
DMSO-d )). Fluorine NMR were carried out in CDCl with a lock tube containing TFA
6 3
(
referenced to δ = -76.55 ppm). Coupling constants (J) was given in Hertz (Hz). CDCl was
F
3
passed through activated Al O (basic) prior to use. Anhydrous solvents were collected from an IT
2
3
(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 (SiO ) with particle size 40-63 µm from ROTH. Optical rotation was
2
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
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1
2
3
4
5
3
6
000 RS with an AcclaimTM RSLC 120 C18 column (2.2 µm, 120 Å, 2.1 x 100 mm) connected to a
7
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
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
uncorrected. Koga’s base is N ,N -bis((R)-1-phenyl-2-(piperidin-1-yl)ethyl)propane-1,3-diamine
2
2
1
and was prepared as previously described. For simplicity, the integrals in H 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 CH Cl (4 mL) was added thionyl
2
2
chloride (0.25 mL, 3.4 mmol) and the solution was heated to reflux and kept at this temperature for
hours. The contents of the reaction vessel were concentrated in vacuo and redissolved in
anhydrous CH Cl (3 mL). In a separate flask B, a solution of 39 (100 mg, 0.136 mmol) in
2
2
2
anhydrous CH Cl (5 mL) was added DBU (22 µL, 0.15 mmol). After 1 hour TLC analysis
2
2
indicated complete cleavage of Fmoc. To flask B, Et N (0.1 mL, 0.7 mmol) was added followed by
3
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% Et N) to yield
3
1
1
(80 mg, 83%). H NMR (600 MHz, CDCl , mixture of rotamers, 1:0.4, A:B): δ 7.83 – 7.79 (m,
3
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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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
5
0
.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,
.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,
.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 +
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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
13
(
t, J = 7.3 Hz, 3H, A), 0.84 (t, J = 7.3 Hz, 1.2H, B) ppm. C NMR (151 MHz, CDCl , mixture of
3
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),
1
1
1
1
49.10 (B), 148.99 (A), 147.62 (B), 147.43 (A), 141.73 (A), 141.63 (B), 137.90 (B), 137.34 (A),
33.82 (A), 133.71 (B), 133.64 (A), 133.41 (B), 132.61 (A), 132.59 (B), 129.92 (B), 129.47 (A),
28.89 (B), 128.59 (A), 127.87 (A), 127.86 (B), 127.81 (B), 127.78 (A), 127.01 (A), 126.37 (B),
26.36 (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),
11.51 (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),
6.84 (A), 26.60 (B), 25.57 (A), 24.55 (B), 21.11 (A), 20.80 (B), 12.72 (B), 12.55 (A) ppm. HRMS
1
(
2
+
+
(MALDI/FTICR) m/z: [M + Na] Calcd for C H N O Na 731.3667; Found 731.3664.
43 52 2 7
(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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Page 27 of 87
Journal of Medicinal Chemistry
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The reaction was carried out as described for 1, using 42 (100 mg, 0.41 mmol) and anhydrous THF
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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2
2
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2
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3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
as solvent. Purification was performed by flash column chromatography (30% acetone in toluene +
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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7
8
9
0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
% Et N) yielding 2 as a slightly yellow oil (79 mg, 72%). H NMR (600 MHz, CDCl , mixture of
3
3
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),
.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),
.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,
7
5
(
=
3
6
1
1
5
.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,
.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),
.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,
.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,
1
3
.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. C NMR
(126 MHz, CDCl , mixture of rotamers, 1:0.25, A:B): δ 172.25 (B), 172.06 (A), 170.66 (A), 170.42
3
(
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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Journal of Medicinal Chemistry
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2
4.47 (B), 21.05 (A), 20.79 (B), 12.52 (B), 12.40 (A). HRMS (MALDI/FTICR) m/z: [M + H]+
+
Calcd. for C H BrN O 737.2796; Found 737.2776.
39 50
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0
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(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% Et N) yielding 3 as a
3
1
slightly yellow oil (103 mg, 97%). H NMR (500 MHz, CDCl , mixture of rotamers (1:0.20) and
3
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),
.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,
H, 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,
5
(
1
8
2
H, 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),
.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),
.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
0
1
3
or B) ppm. C NMR (126 MHz, CDCl , mixture of rotamers (1:0.20) and diastereoisomers (1:1,
3
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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Page 29 of 87
Journal of Medicinal Chemistry
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=
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
F
6
7
(
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),
33.60 (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),
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9
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1
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4
4
4
5
5
5
5
5
5
5
5
5
5
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0
1
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0
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2
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7
8
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0
1
(
5
5
7.84 (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),
4.28 (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),
2
2
1
8.31 (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),
1
9
1.05 (A or B), 21.04 (A or B), 12.37 (A or B), 12.34 (A or B). F NMR (470 MHz, CDCl ) δ -
3
+
32.53 – -132.73 (m), -161.10 – -161.42 (m) ppm. HRMS (MALDI/FTICR) m/z: [M + H] Calcd
+
for C H F N O 713.3408; Found 713.3405.
3
9
48
3
2
7
(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% Et N) yielding 4 as a
3
1
colorless oil (99 mg, 89%). H NMR (500 MHz, CDCl , mixture of rotamers (1:0.33).Only major
3
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),
.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
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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