A General Strategy for the Preparation of Thalidomide-Conjugate Linkers

Article abstract of DOI:10.1055/s-0036-1588539

The synthesis of small-molecule linkers for installation of thalidomide-based conjugates is described. Linker properties have been recognized as vital to conjugate success in drug discovery and delivery systems. These small-molecule tethers act as linkages between molecules, can also aid in cell permeability, and act as solubilizing agents. This work shows our progress in synthesizing conjugates with a variety of linker characteristics. The adaptability and manipulation of these and other linkers holds potential in improving synthetic control of chemical connectivities toward therapeutic development.

Full text of DOI:10.1055/s-0036-1588539

SYNLETT
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6
©
Georg Thieme Verlag Stuttgart · New York
2017, 28, A–E
letter
A
en
Synlett
J. W. Papatzimas et al.
Letter
A General Strategy for the Preparation of Thalidomide-Conjugate
Linkers
James W. Papatzimasa◊
Evgueni Gorobetsa◊
N
N
F
F
Duncan K. Brownseya
Ranjan Maityb
Nizar J. Bahlisb
O
F
F
F
N
F
F
O
F
O
S
N
O
O
O
F
N
O
Darren J. Derksen*a
0
0
0
0
0-
0
0
2
5-
9
4
5
6-
9
2
1
F
NH
O
O
a
Department of Chemistry, University of Calgary,
500 University Drive NW, Calgary, Alberta, T2N 1N4,
Cl
2
Canada
dderksen@ucalgary.ca
Department of Hematology and Oncology, University
of Calgary, Calgary, Alberta, T2N 4N1, Canada
b
◊
These authors contributed equally
Dedicated to Victor Snieckus on the occasion of his 80th birthday
Received: 26.05.2017
Accepted after revision: 17.07.2017
Published online: 23.08.2017
A
OR
O
1
2
, R = H
, R = Linker
DOI: 10.1055/s-0036-1588539; Art ID: st-2017-r0401-l
N
O
2 2
3, R = CH CO Bn
NH
Abstract The synthesis of small-molecule linkers for installation of
thalidomide-based conjugates is described. Linker properties have been
recognized as vital to conjugate success in drug discovery and delivery
systems. These small-molecule tethers act as linkages between mole-
cules, can also aid in cell permeability, and act as solubilizing agents.
This work shows our progress in synthesizing conjugates with a variety
of linker characteristics. The adaptability and manipulation of these and
other linkers holds potential in improving synthetic control of chemical
connectivities toward therapeutic development.
O
O
B
C
D
Linker
OH
OH
O
CO2Me
CO2Me
CO2Me
CO2Me
2
O
O
N
Key words PROTAC, thalidomide, conjugates, linker, pentafluoro-
phenyl ester
OH
BnO
O
3
NH
PPh3, DEAD
O
O
O
1
2
Since the seminal reports by Bradner and Crews on the
use of thalidomide conjugation for in vivo protein degrada-
tion, our group and others have become interested in the
synthetic preparation of small-molecule conjugates incor-
H
X
N
O
F
O
O
F
F
F
F
O
3
porating this moiety (Scheme 1, A). As both linker length
N
O
NH
and composition have been shown to be essential for pre-
O
O
1–5
paring functional conjugates, we have worked to develop
a sufficiently convergent synthetic strategy to prepare mul-
tiple linkers for structure–activity relationship studies.
A recent paradigm shift in proximity-directed protein
degradation has seen the implementation of proteolysis
targeting chimeras (PROTACs) as a means of inducing pro-
Scheme 1 4-Hydroxythalidomide derivative syntheses; X = CH or
2
(
CH ) C(O)NH(CH ) (OCH CH )
2 2 2 3 2 2 3
proximity enhancement for PROTACs, linker length be-
tween targeting molecules becomes vital to effective re-
cruitment and positioning of POIs.
4,6–8
tein degradation.
PROTACs are small-molecule conju-
gates which enhance proximity between proteins of inter-
est (POIs) through bifunctional targeting conjugates to initi-
Our initial approach to thalidomide conjugates relied on
a highly linear synthesis from literature where the linker
moiety needed to be introduced prior to glutarimide intro-
4
ate protein degradation. Thalidomide derivatives have
been successfully used as targeting ligands in previously
1,2,3b,9
10
published PROTAC work.
Due to the importance of
duction (Scheme 1, B). Our own attempts to improve the
synthesis found that direct phenol alkylation of 4-hydroxy-
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–E

B
Synlett
J. W. Papatzimas et al.
Letter
thalidomide (1) led to competing N-alkylation of the glu-
tarimide moiety. We were working to make this synthesis
more convergent when elegant work from Miller showed
that the phenolic site of hydroxyl-thalidomide could be
functionalized directly using modified Mitsunobu condi-
tions (Scheme 1, C).11 This advancement allowed us to syn-
thesize our desired compounds in a much shorter time
frame by making the synthesis much more convergent.
Our strategy for coupling conjugate fragments was
heavily dependent on the use of pentafluorophenyl (Pfp)
esters (Scheme 1, D) in order to form amide bonds, which
were chosen specifically for their robust chemical proper-
O
O
O
a, b
c
H
H
N
NH2
N
HO
OPfp
R2
N
R2
5
6
2
7, R = Boc
O
d
2
8
9, R
, R = H
2
R1
=
e
=
N
O
OR1
NH
O
O
O
3
4
f, g
O
H
H
OPfp
R3
h
N
O
N
O
1
R O
3
R1
12
O
O
O
ties. Several other conjugation techniques, such as click
3
1
1
0, R = OBn
1, R = OPfp
13
i, j
chemistry and selective nucleophilic aromatic substitu-
3
2
3
tion, have also been reported in the literature.
12, R
=
k
N
The hydroxyl-thalidomide functionality (Scheme 1, A)
suffers from poor chemoselectivity in alkylation reactions
which make the synthesis of thalidomide conjugates prob-
lematic. While 1 appears stable under storage, its deriva-
tives suffer from both acidic and basic instability resulting
in cleavage of the phthalimide ring. This limitation severely
restricts the chemistry compatible for conjugation. The re-
sultant instability dictates which protecting groups could
be employed based on the sensitivity of phthalimide ring
opening during deprotection conditions. This undesirable
reactivity is likely due to the increased electrophilicty of
phthalimide carbonyls through hydrogen bonding with the
O
O
O
S
O
l, m
n, o, p
N
SO2Cl
13
O
14
H
R1
N
N
O
O
S
O
O
15
Scheme 2 Model conjugation of thalidomide linkers with secondary
amine model compound - piperidine. Reagents and conditions: a) Boc O,
2
DMAP, 99%; b) CF C(O)OC F , DIPEA, 78%; c) piperidine, DIPEA, quanti-
3
6 5
tative; d) TFA, 98% e) 4, DIPEA, 75%; f) H , Pd/C; g) CF C(O)OC F , DIPEA,
2
3
6 5
7
9% (2 steps); h) NH CH CH CH (OCH CH ) CH NHC(O)(CH ) C(O)OBn,
2 2 2 2 2 2 3 2 2 3
14
phenolic hydrogen. This requires the utilization of pro-
tecting-group chemistry stable to a variety of reactions and
yet labile under neutral conditions, leading us to benzyl es-
ters.
DIPEA, 79%; i) H , Pd/C; j) CF C(O)OC F , DIPEA, 74% (2 steps); k) piper-
2
3
6 5
idine, DIPEA, 79%; l) potassium phthalimide; m) SOCl 22% (2 steps);
n) piperidine, Et N, 53%; o) hydrazine, 94%; p) 4, DIPEA, 47%.
2
3
Central to this synthetic strategy was a short and facile
synthesis of 3 following literature precedent. First, 3-hy-
droxyphthalic anhydride was reacted with 3-amino glu-
tarimide, followed by thalidomide formation using dicyclo-
carbodiimide (DCC) in a 77% yield.11 Phenol 1 was exposed
to Mitsunobu reaction conditions to form the benzyl ester
linkage onto the thalidomide intermediate to yield 3. These
initial steps were performed on multigram scales. Thalido-
mide derivative 3 was then hydrogenated to quantitatively
deprotect the acid over Pd/C in DMF (Scheme 2). This reac-
tion was easily monitored using TLC and complete conver-
sion was observed after only one hour. The free acid was
then converted into the active Pfp ester 4 using pentafluo-
rophenyl trifluoroacetate, the products of which can be eas-
formed to reactive Pfp ester 6 using pentafluorophenyl tri-
fluroacetate. Compound 6 was then coupled to piperidine
under basic conditions to afford compound 7. Intermediate
7 was exposed to trifluoroacetic acid (TFA) to liberate the
17
terminal amine which was then exposed to 4, affording 9
in two hours at ambient temperature.18
Our second generation of linkers was designed to inves-
tigate the synthetic differences in employing longer linkers
with more polar functionalities, inspired by recent publica-
tions.1
,2,3b,9
We looked to derivatize the polyethylene glycol
(PEG) diamine 4,7,10-trioxa-1,13-tridecanediamine. While
this linker was considerably longer than the four-carbon al-
kyl linker, it was inherently much more water-soluble. Suc-
cinic anhydride was opened using benzyl alcohol and NaH
to afford a monoprotected diacid. The remaining acid was
then converted into an acid chloride in situ. The PEG di-
amine was first mono-Boc protected and then exposed to
the activated acid to afford the desired intermediate con-
sisting of two different protected terminal functionalities.
The amine pole was then deprotected using TFA, and
the newly formed primary amine was reacted with 4 to
form intermediate 10 which could also be easily purified by
15
ily purified via trituration with diethyl ether. Pfp ester 4
was found to be stable for prolonged storage and was then
available for conjugation with a variety of amine containing
16
compounds such as our model piperidine.
First-generation linkers consisted of four carbon linkag-
es following a system which had shown promising results
1
in recently published work. The primary amine of the sim-
ple building block, γ-aminobutyric acid (5) was Boc pro-
tected (Scheme 2). The remaining free acid was then trans-
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–E

C
Synlett
J. W. Papatzimas et al.
Letter
trituration with diethyl ether. The remaining benzyl ester
was hydrogenated over Pd/C in two hours to afford the free
acid which was exposed to pentafluorophenyl trifluoro-
acetate to form the respective Pfp ester 11 in one pot
Pfp ester of JQ1 (16). As this approach minimizes the
amount of byproduct formation, this strategy allows purifi-
cation by flash chromatography instead of HPLC as previ-
ously reported.1
(
Scheme 2). Intermediate 11 was reacted with piperidine to
Conjugates 9, 12, 15, and 17 were successfully formed
through a synthetic route with clear advantages over previ-
ous thalidomide-containing syntheses. One of the most
beneficial characteristics of this linker strategy is the use of
pentafluorophenyl trifluoroacetate in order to form respec-
tive Pfp esters. The ease of purification associated with sim-
ple trituration eliminates issues associated with isolation of
highly polar compounds. In addition to the ease of synthesis
and purification, this linker strategy can easily be tailored
to accommodate a broad scope of targeting molecules. The
adaptability of this synthetic strategy lends itself to the di-
verse linkers required for PROTAC synthesis. By varying the
length, hydrophobic properties, as well as linkage identity
we have developed a strategy for conjugating a broad scope
of target molecules through a variety of functional groups.
In vitro structure activity relationship studies are currently
underway.
23
afford conjugate 12 in a 79% yield.
To expand the scope of linkages beyond simple amide
bonds, sulfonamides were also explored. A Gabriel synthe-
sis of 1,4-butane sultone (13) was performed, opening the
ring to produce a straight chain linker with already estab-
lished sulfonate and protected amine terminal motifs. The
sulfonate salt was then converted into sulfonyl chloride 14,
which proved to be stable to flash chromatography (Scheme
2
). Previous attempts to open the sultone ring were suc-
cessfully performed using NH OH to yield a straight chain
4
linker with a free primary amine which was Boc-protected.
However, attempts at forming the sulfonyl chloride afforded
low yields (6%), due to rapid intramolecular recyclization of
the monoprotected amine. The Gabriel synthesis was em-
ployed as a means of introducing a doubly protected termi-
nal amine in one step. Sulfonyl chloride 14 was then ex-
posed to the model reaction conditions with piperidine to
afford the desired sulfonamide in moderate yield. The
phthalimide ring was then cleaved with hydrazine mono-
hydrate to liberate the free terminal amine, which was re-
Funding Information
This work was funded by the Multiple Myeloma Research Foundation,
NSERC, the University of Calgary, Alberta Children’s Hospital Founda-
24
acted with 4 to yield conjugate 15.
tion and Research Institute, and the Charbonneau Cancer Institute.
)(
N
N
F
O
F
F
N
Supporting Information
a, b
S
N
O
JQ1
F
Supporting information for this article is available online at
https://doi.org/10.1055/s-0036-1588539.
1
6
F
S
u
p
p
o
nrtogI
i
f
rm oaitn
S
u
p
p
ortioIgnfmr oaitn
Cl
References and Notes
c, d
e, f, g
4
(1) Winter, G. E.; Buckley, D. L.; Paulk, J.; Roberts, J. M.; Souza, A.;
N
N
Dhe-Paganon, S.; Bradner, J. E. Science 2015, 348, 1376.
N
O
H
(2) Lu, J.; Qian, Y.; Altieri, M.; Dong, H.; Wang, J.; Raina, K.; Hines, J.;
Winkler, J. D.; Crew, A. P.; Coleman, K.; Crews, C. M. Chem. Biol.
2015, 22, 755.
N
2
N
H
S
N
O
O
O
1
7
(3) (a) Fischer, E. S.; Bohm, K.; Lydeard, J. R.; Yang, H.; Stadler, M. B.;
Cavadini, S.; Nagel, J.; Serluca, F.; Acker, V.; Lingaraju, G. M.;
Tichkule, R. B.; Schebesta, M.; Forrester, W. C.; Schirle, M.;
Hassiepen, U.; Ottl, J.; Hild, M.; Beckwith, R. E. J.; Harper, J. W.;
Jenkins, J. L.; Thoma, N. H. Nature 2014, 512, 49. (b) Lebraud, H.;
Wright, D. J.; Johnson, C. N.; Heightman, T. D. ACS Cent. Sci.
2016, 2, 927.
N
O
NH
O
O
Cl
Scheme 3 Syntheses of known PROTAC 17. Reagents and conditions: a)
19
20
HCO H, quantitative; b) CF C(O)OC F , DIPEA, 56%; c) 1,4-diaminobutane,
2
3
6 5
21
DIPEA; d) 4, DIPEA, 48% over two steps; e) 4, H N(CH ) NHC(O)OC(CH ) ,
2
2 4
3 3
(4) Long, M. J. C.; Poganik, J. R.; Aye, Y. J. Am. Chem. Soc. 2016, 138,
11
22
DIPEA, 81%; f) TFA, quantitative; g) DIPEA, 16, 81%.
3
610.
5) Srinivasarao, M.; Galliford, C. V.; Low, P. S. Nat. Rev. Drug Discov.
015, 14, 203.
(6) DeRose, R.; Miyamoto, T.; Inoue, T. Pfluegers Arch. 2013, 465,
09.
(7) Corson, T. W.; Aberle, N.; Crews, C. M. ACS Chem. Biol. 2008, 3,
77.
8) Lai, A. C.; Crews, C. M. Nat. Rev. Drug Discov. 2017, 16, 101.
(
2
In order to showcase the versatility of 4 in our synthetic
strategy we employed this approach to synthesize a
PROTAC reported by the Bradner group (Scheme 3). We de-
veloped two novel synthetic routes for forming 17 via the
4
1
6
(
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–E

D
Synlett
J. W. Papatzimas et al.
Letter
(
9) (a) Remillard, D.; Buckley, D. L.; Paulk, J.; Brien, G. L.; Sonnett,
(20) Perfluorophenyl 2-{4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-
thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl}acetate
(16)
M.; Seo, H.-S.; Dastjerdi, S.; Wühr, M.; Dhe-Paganon, S.;
Armstrong, S. A.; Bradner, J. E. Angew. Chem. Int. Ed. 2017, 56,
5
738. (b) Schiedel M., Herp D., Hammelmann S., Swyter S.,
To a solution of JQ1-acid (0.055 g, 0.136 mmol) in DMF (2 mL)
was added DIPEA (0.17 mL, 0.123 g, 0.952 mmol) and
pentaflurophenyl trifluoroacetate (0.05 mL, 0.076 g, 0.272
mmol). The solution was stirred for 1 h and solvent was
removed in vacuo. The residue was purified by silica gel flash
Lehotzky A., Robaa D., Oláh J., Ovádi J., Sippl W., Jung M.; J. Med.
Chem.; 2017, DOI: 10.1021/acs.jmedchem.6b01872 (c) Lai, A. C.;
Toure, M.; Hellerschmied, D.; Salami, J.; Jaime-Figueroa, S.; Ko,
E.; Hines, J.; Crews, C. M. Angew. Chem. Int. Ed. 2016, 55, 807.
(
10) Gladysz, J. A.; Lee, S. J.; Tomasello, J. A. V.; Yu, Y. S. J. Org. Chem.
chromatography (EtOAc/hexanes/THF = 1:1:1) to afford 0.0432
1
1977, 42, 4170.
g (56%) as a yellow oil. H NMR (400 MHz, CDCl ): δ = 7.49–7.34
3
(
(
(
11) Lohbeck, J.; Miller, A. K. Bioorg. Med. Chem. Lett. 2016, 26, 5260.
12) Montalbetti, C. A. G. N.; Falque, V. Tetrahedron 2005, 61, 10827.
13) Wurz, R. P.; Dellamaggiore, K.; Dou, H.; Javier, N.; Lo, M.-C.;
McCarter, J. D.; Mohl, D.; Sastri, C.; Lipford, J. R.; Cee, V. J. J. Med.
Chem. 2017, DOI: 10.1021/acs.jmedchem.6b01781.
(m, 4 H), 4.71 (dd, J = 9.5, 4.6 Hz, 1 H), 4.08 (dd, J = 16.9, 9.5 Hz,
1 H), 3.91 (dd, J = 16.9, 4.6 Hz, 1 H), 2.73 (s, 3 H), 2.45 (d, J =
13
0.9 Hz, 3 H), 1.73 (d, J = 0.9 Hz, 3 H). C NMR (151 MHz, CDCl ):
3
δ = 167.62, 164.38, 154.65, 150.13, 141.90 140.26, 138.70,
137.03, 136.30, 132.22, 131.00, 130.97, 130.26, 129.83, 128.76,
53.57, 36.39, 14.40, 13.11, 11.85. HRMS (MALDI): m/z calcd for
(
14) Orchin, M.; Macomber, R. S.; Pinhas, A. R.; Wilson, R. M. The
Vocabulary and Concepts of Organic Chemistry; John Wiley and
Sons: Hoboken, 2005, 2nd ed.
+
[C25H16ClF N O S + H] : 567.0675; found: 567.0771.
5
4
2
(21) 2-{4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-
f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl}-N-[4-(2-{[2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}acet-
amido)butyl]acetamide (17)
(15) General Procedure for the Synthesis of 4
A solution of 3 (0.691 g, 1.64 mmol) in DMF (15 mL) was stirred
with (Pd/C (0.04 g, 10 mol%) under hydrogen for 1.5 h, filtered,
and concentrated in vacuo. The crude product (0.148 g,
To a solution of 1,4-diaminobutane (0.03 g, 0.371 mmol) in DMF
(2 mL) was added DIPEA (0.065 mL, 0.048 g, 0.371 mmol) and
16 (0.021 g, 0.0371 mmol). The mixture was stirred for 1.5 h,
concentrated in vacuo, and redissolved in DMF (1 mL). DIPEA
(0.026 mL, 0.019 g, 0.0148 mmol) and 4 (0.028 g, 0.0557 mmol)
were added and stirred overnight, The mixture was concen-
trated in vacuo and purified by silica gel flash chromatography
0
0
.45 mmol) was then redissolved in DMF (6 mL), DIPEA (0.118 g,
.16 mL, 0.92 mmol) was added with stirring, and the solution
was cooled to 0 °C. Pentafluorophenyl trifluoroacetate (0.187 g,
.12 mL, 0.67 mmol) was then added with stirring. The reaction
0
mixture was allowed to come to ambient temperature for 2 h.
The mixture was concentrated in vacuo, and purified by tritura-
tion with Et O to afford 0.175 g (79%) of 4.
(5–10% MeOH in CHCl ) to afford 0.014 g (48%) of the title com-
pound as a mixture of diastereomers. H NMR (600 MHz,
2
3
1
1
H NMR (400 MHz, CDCl ) δ = 7.99 (s, 1 H), 7.76 (dd, J = 8.4,
3
7
0
2
.3 Hz, 1 H), 7.63 (dd, J = 7.4, 0.7 Hz, 1 H), 7.26 (dd, J = 8.5,
.8 Hz, 1 H), 5.34 (d, J = 1.4 Hz, 2 H), 5.04–4.97 (m, 1 H), 2.98–
MeOD): δ = 8.34–8.29 (*m, 1 H), 8.12 (*q, J = 5.7 Hz, 1 H), 7.80
(dd, J = 8.4, 7.3 Hz, 1 H), 7.52 (d, J = 7.3 Hz, 1 H), 7.45–7.38 (m,
5 H), 5.10 (ddd, J = 12.4, 5.5, 3.1 Hz, 1 H), 4.77 (d, J = 1.7 Hz, 2 H),
4.63 (ddd, J = 9.1, 5.4, 1.1 Hz, 1 H), 3.44–3.32 (m, 4 H), 3.30–3.25
(m, 2 H), 2.86–2.77 (m, 1 H), 2.73–2.65 (m, 5 H), 2.43 (d, J =
2.5 Hz, 3 H), 2.10 (dddd, J = 10.7, 8.0, 4.9, 2.6 Hz, 1 H), 1.70–1.60
.74 (m, 3 H), 2.22–2.14 (m, 1 H). ESI-HRMS: m/z calcd for
+
[
C21H11F N O + Na] : 521.0379; found: 521.0368.
5
2
7
(
(
16) No decomposition observed after 5 weeks at –20 °C.
17) General Procedure for Coupling to 4
13
To a solution of corresponding free amine (1 equiv) in DMF was
added DIPEA (3 equiv) under stirring. A solution of 4 (1.1 equiv)
in DMF was added to the reaction mixture at ambient tempera-
ture. After 2 h the mixture was concentrated in vacuo and puri-
fied by silica gel flash chromatography to afford the title com-
pound.
(m, 7 H); * exchangeable protons.
NMR (151 MHz, CDCl ):
3
δ = 174.4, 172.77, 172.68, 171.30, 171.27, 169.91, 168.24,
167.79, 166.24, 166.21, 157.00, 156.29, 152.19, 138.23, 138.09,
137.95, 134.87, 133.53, 133.18, 132.04, 132.02, 131.96, 131.31,
129.78, 121.90, 121.87, 119.37, 118.00, 69.57, 69.55, 55.23,
50.55, 50.54, 40.21, 40.19, 40.09, 40.06, 39.84, 38.88, 38.86,
(
18) 2-[(2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
32.14, 32.12, 27.77, 27.66, 27.64, 23.63, 23.61, 14.41, 12.92,
+
yl)oxy]-N-[4-oxo-4-(piperidin-1-yl)butyl]acetamide (9)
11.62. HRMS (MALDI): m/z calcd for [C38H37ClN O S + H]
=
8
7
Purified by silica gel flash chromatography (5% MeOH in CHCl )
to afford 0.119 g (75%) as a colorless oil. H NMR (400 MHz,
785.2267; found: 785.2232.
3
1
(22) 2-{4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-
f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl}-N-[4-(2-{[2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}acet-
amido)butyl]acetamide (17)
CDCl ): δ = 8.70 (s, 1 H), 7.75 (dd, J = 8.4, 7.3 Hz, 1 H), 7.71 (t, J =
3
5.6 Hz, 1 H), 7.57 (dd, J = 7.4, 0.7 Hz, 1 H), 7.23 (dd, J = 8.5,
0.7 Hz, 1 H), 5.00–4.92 (m, 1 H), 4.73–4.60 (m, 2 H), 3.58–3.52
(
2
m, 2 H), 3.45 (ddd, J = 14.7, 7.4, 6.0 Hz, 1 H), 3.41–3.33 (m, 3 H),
To a solution of the free amine 4 linker (0.021 g, 0.052 mmol) in
DMF (1.5 mL) was added DIPEA (0.09 mL, 0.067 g, 0.5 mmol)
and 16 (0.022 g, 0.039 mmol) and stirred overnight. The reac-
tion mixture was stirred with K CO (0.021 g) for 30 min, fil-
.98–2.70 (m, 3 H), 2.42–2.36 (m, 2 H), 2.22–2.15 (m, 1 H),
.97–1.88 (m, 2 H), 1.66–1.60 (m, 2 H), 1.59–1.49 (m, 4 H).
1
1
3
C NMR (400 MHz, CDCl ): δ = 170.80, 170.42, 168.04, 166.89,
3
2
3
1
6
2
4
66.54, 154.97, 137.01, 133.61, 120.51, 118.56 117.68, 77.20,
tered, concentrated in vacuo, and purified by silica gel flash
9.00, 49.42, 46.61, 42.78, 39.12, 31.34, 30.66, 26.48, 25.55,
chromatography (5–10% MeOH in CHCl ) to afford 0.025 g (81%)
3
+
4.84, 24.54, 22.73. EIS-HRMS: m/z calcd for [C24H28N O + H] :
of the title compound as a mixture of diastereomers. Data are
identical to ref. 22.
4
7
85.2031; found: 485.2047.
(
19) 2-{4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-
(23) N-(1-{[2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl]oxy}-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)-4-oxo-
4-(piperidin-1-yl)butanamide (12)
f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl}acetic Acid (JQ1-Acid)
JQ1 (0.05g, 0.1099 mmol) was dissolved in formic acid (4.5 mL)
and stirred for 4 d. The solvent was removed in vacuo to afford a
fine yellow powder. The product was used without purification.
Purified by flash chromatography (5% MeOH in CHCl ) to afford
3
1
0.011 g (79%) as a colorless oil. H NMR (600 MHz, CDCl ):
3
δ = 9.43 (s, 1 H), 7.74 (dd, J = 8.4, 7.4 Hz, 1 H), 7.60 (t, J = 5.8 Hz,
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–E

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Synlett
J. W. Papatzimas et al.
Letter
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H), 7.55 (dd, J = 7.5, 0.7 Hz, 1 H), 7.21 (dd, J = 8.4, 0.6 Hz, 1 H),
.66 (t, J = 5.6 Hz, 1 H), 5.03–4.96 (m, 1 H), 4.65 (d, J = 2.7 Hz,
H), 3.67–3.64 (m, 4 H), 3.64–3.61 (m, 2 H), 3.58 (tt, J = 6.5,
.7 Hz, 3 H), 3.53 (td, J = 5.7, 1.8 Hz, 4 H), 3.50–3.44 (m, 2 H),
.43–3.39 (m, 2 H), 3.31 (qd, J = 6.4, 4.9 Hz, 2 H), 2.91–2.75 (m,
H), 2.66 (td, J = 6.7, 1.3 Hz, 2 H), 2.49 (t, J = 6.8 Hz, 2 H), 2.18–
.13 (m, 1 H), 1.87 (p, J = 6.4 Hz, 2 H), 1.82 (s, 1 H), 1.75 (q, J =
.3 Hz, 2 H), 1.62 (td, J = 6.9, 4.9 Hz, 2 H), 1.58–1.48 (m,
(24) 2-{[2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl]oxy}-N-[4-(piperidin-1-ylsulfonyl)butyl]acetamide (15)
Purified by flash chromatography (5% MeOH in CHCl ) to afford
3
1
0.016 g (47%) as a white solid. H NMR (400 MHz, CDCl ):
3
δ = 8.08 (s, 1 H), 7.75 (dd, J = 8.4, 7.4 Hz, 1 H), 7.67 (s, 1 H), 7.56
(dd, J = 7.3, 0.7 Hz, 1 H), 7.20 (dd, J = 8.4, 0.7 Hz, 1 H), 5.05–4.98
(m, 1 H), 4.65 (d, J = 2.3 Hz, 2 H), 3.43 (sept, J = 6.9 Hz, 2 H),
3.29–3.12 (m, 4 H), 2.97–2.89 (m, 3 H), 2.89–2.71 (m, 2 H),
2.23–2.14 (m, 1 H), 1.99–1.87 (m, 2 H), 1.74 (dt, J = 14.4, 6.9 Hz,
13
H). C NMR (600 MHz, CDCl ): δ = 172.57, 171.34, 170.13,
3
13
68.33, 166.69, 166.62, 154.61, 136.93, 133.62, 119.68, 117.33,
0.41, 70.40, 70.15, 70.06, 69.39, 68.73, 68.22, 49.35, 46.40,
2.90, 37.31, 36.49, 31.59, 31.46, 29.23, 28.97, 28.80, 26.30,
2 H), 1.67–1.53 (m, 6 H). C NMR (400 MHz, CDCl ): δ = 170.82,
3
168.00, 166.95, 166.55, 154.65, 137.10, 133.55, 120.08, 118.46,
117.64, 77.21, 68.49, 49.38, 48.51, 46.64, 38.44, 31.40, 28.27,
25.67, 23.81, 22.57, 20.55. ESI-HRMS: m/z calcd for
5.54, 24.48, 22.69. ESI-HRMS: m/z calcd for [C34H47N O
+
5
11
+
+
Na] : 724.3163; found: 724.3168.
[C24H30N O S + H] : 535.1857; found: 535.1853.
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©
Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–E