Fragment-based domain shuffling approach for the synthesis of pyran-based macrocycles

Article abstract of DOI:10.1073/pnas.1015255108

Complexity and the presence of stereogenic centers have been correlated with success as compounds transition from discovery through the clinic. Here we describe the synthesis of a library of pyran-containing macrocycles with a high degree of structural complexity and up to five stereogenic centers. A key feature of the design strategy was to use a modular synthetic route with three fragments that can be readily interchanged or "shuffled" to produce subtly different variants with distinct molecular shapes. A total of 352 macrocycles were synthesized ranging in size from 14- to 16-membered rings. In order to facilitate the generation of stereostructure-activity relationships, the complete matrix of stereoisomers was prepared for each macrocycle. Solid-phase assisted parallel solution-phase techniques were employed to allow for rapid analogue generation. An intramolecular nitrile-activated nucleophilic aromatic substitution reaction was used for the key macrocyclization step.

Full text of DOI:10.1073/pnas.1015255108

Fragment-based domain shuffling approach for
the synthesis of pyran-based macrocycles
Eamon Comer1, Haibo Liu1, Adrien Joliton, Alexandre Clabaut, Christopher Johnson,
Lakshmi B. Akella, and Lisa A. Marcaurelle2
Chemical Biology Platform, Broad Institute, Cambridge, MA 02142
Edited by Stuart L. Schreiber, Broad Institute, Cambridge, MA, and approved January 28, 2011 (received for review November 16, 2010)
Complexity and the presence of stereogenic centers have been
correlated with success as compounds transition from discovery
through the clinic. Here we describe the synthesis of a library
of pyran-containing macrocycles with a high degree of structural
complexity and up to five stereogenic centers. A key feature of
the design strategy was to use a modular synthetic route with
three fragments that can be readily interchanged or “shuffled” to
produce subtly different variants with distinct molecular shapes.
A total of 352 macrocycles were synthesized ranging in size from
14- to 16-membered rings. In order to facilitate the generation
of stereostructure-activity relationships, the complete matrix of
stereoisomers was prepared for each macrocycle. Solid-phase
assisted parallel solution-phase techniques were employed to
allow for rapid analogue generation. An intramolecular nitrile-
activated nucleophilic aromatic substitution reaction was used
for the key macrocyclization step.
diversity ∣ library ∣ stereochemistry ∣ high throughput
t has been demonstrated that compounds with increased com-
I
plexity and one or more stereogenic centers have molecular
attributes that contribute to their success throughout the drug
discovery process, including increased solubility (1) and greater
selectivity (2, 3). The vast majority of small-molecule screening
collections however are heavily populated with sp2-rich com-
pounds (3, 4), perhaps due, in part, to the abundant methods
for their synthesis (5). Although natural products have served
to augment screening collections as a source of complexity, struc-
tural analogs of natural products are generally difficult to access.
In order to facilitate downstream discovery, an up-front invest-
ment should be made to devise synthetic pathways that will
yield compounds that are poised for optimization (6–8). As a step
toward this goal, we have developed a modular synthesis of a
small-molecule library with features aimed to increase the prob-
ability of success in the optimization phases of drug or probe dis-
covery. To this end, our approach was to prepare a library of
compounds consisting of three fragments that could be readily
modified, interchanged, or shuffled (9, 10) to produce distinct
structural variants (Fig. 1). Such an approach should not only
yield useful structure-activity relationships of initial library mem-
bers but allow rapid access to derivatives for follow up medicinal
chemistry efforts.
Fig. 1. Fragment-based DS strategy for the synthesis of pyran-based macro-
cycles.
linear (B), and aromatic (C) by design (Fig. 1). The cyclic pyran
unit (1 or 2), which contains three stereogenic centers, originated
from the chiral pool, whereas the linear component, a β-methoxy
γ-amino acid (3), containing two stereogenic centers, was synthe-
sized using an asymmetric aldol reaction (20). The aromatic
component (3- or 5-cyano-2-fluorobenzoic acid, 4-o or 4-p) were
both obtained from commercial sources. The pyran fragment
(A) is a bis-nucleophile, whereas the linear (B) and aromatic
(C) components each contain an electrophile suitable to react
with (A), as well as functionality that would react with each other.
Thus, pyran 1 could react with acids 3 and 4 to produce macro-
In designing a small-molecule library with increased structural
complexity accessible by a domain shuffling (DS) strategy, we
were inspired by the numerous pyran-containing macrocycles
found in nature, such as rapamycin (11), bryostatin (12), and
spongistatin (13), among others (14–18), which contain a high
ratio of sp3-hybridized atoms and stereogenic centers. In light of
their unique structural features (19), macrocycles in general have
been the focus of a number of library synthesis efforts (9, 20–27),
although only a few libraries centered on pyran-containing
macrocycles (24–26). Initially, we chose to target 16-membered
rings using an intramolecular nucleophilic aromatic substitution
(SNAr) reaction (20, 21) as the key macrocyclization step. The
three fragments used in the preparation of the macrocyclic library
represent three different structural types, namely, cyclic (A),
Author contributions: E.C., H.L., and L.A.M. designed research; E.C., H.L., A.J., A.C., and C.J.
performed research; L.B.A. performed computational work; E.C., H.L., C.J., L.B.A., and
L.A.M. analyzed data; and E.C., H.L., L.B.A., and L.A.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1E.C. and H.L. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: lisa@broadinstitute.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1015255108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1015255108
PNAS ∣ April 26, 2011 ∣ vol. 108 ∣ no. 17 ∣ 6751–6756

cycle ABC-5. Alternatively, pyran 2 can react with acids 3 and
4 to give the subtly different macrocycle ACB-5. In order to
access valuable stereostructure-activity relationships (20, 28–30)
directly from primary screening, we planned to synthesize all 32
stereoisomers for each macrocycle.
was achieved using diisobutylaluminum hydride at 0 °C (60–95%
yield). Finally, TBDPS protection (82–100%) and nosyl
deprotection (82–100%) led to the desired amine 2. This reaction
sequence was successfully applied to the synthesis of all eight
stereoisomers of pyran 2.
Results and Discussion
Synthesis of β-Hydroxy-γ-Amino Acids 3a-b. With the synthesis of
pyrans 1 and 2 complete, we focused our attention to the prepara-
tion of the acyclic fragment, β-methoxy-γ-amino acid 3a-b. The
synthesis of 3 initiated from material prepared previously in
our group, namely β-hydroxy-γ-amino acids 10, accessible via
asymmetric syn- and anti-aldol methodologies (20). As shown
in Scheme 2, β-hydroxy-γ-amino acids 10 could be selectively
methylated with NaH and MeI in THF at 0 °C to afford amino
acids 3 in 77–98% yield.
Synthesis of Pyran Fragments 1 and 2. The synthesis of pyran
fragments 1 and 2 initiated with C-glycoside 6 (Scheme 1), which
itself was derived from tri-O-acetyl D- and L-glycals according to
methods previously developed in our laboratory (31). Methyla-
tion of 6 with Meerwein’s reagent in the presence of Proton
Sponge gave rise to the corresponding methyl ether in 69–94%
yield, which in turn was hydrogenated under standard conditions
(Pd/C, MeOH, H2) to afford pyrans 7 in 94–100% yield. The
resulting pyran ring systems served as a common precursor to
fragments 1 and 2. For the synthesis of pyrans 1, the ester was
reduced with lithium borohydride in the presence of a catalytic
amount of lithium triethylborohydride (32). Oxidation of the
resultant alcohol with Dess–Martin periodinane, followed by
reductive amination with methylamine in the presence of sodium
borohydride afforded the desired secondary amine 1 in 64–96%
yield. This strategy was successful in generating all eight stereo-
isomers of pyran 1.
The synthesis of pyrans 2 began with the HF-pyridine-
mediated removal of tert-butyldiphenylsilane (TBDPS) protect-
ing group of 7. The resulting primary alcohol was converted to
the nosyl protected secondary amine 9 in 32–72% yield through
a Mitsunobu reaction with N-methyl-2-nitrobenzenesulfonamide.
Chemoselective ester reduction in the presence of nosyl group
Parallel Solution-Phase Synthesis of Pyran-Containing Macrocycles.
With all stereoisomers of pyrans 1 and 2 and γ-amino acids 3
in hand, we began to investigate methods for the union of these
chiral fragments en route to macrocycles ABC-5 and ACB-5.
Although the efficiency of solid-phase synthesis for the prepara-
tion of large compound libraries has been well documented
(33, 34), polymer-assisted parallel solution-phase synthesis offers
several advantages (35, 36). Benefits include rapid analogue
generation, reduced optimization time, and ease of reaction
monitoring. We thus settled on a parallel solution-phase synthesis
strategy utilizing silica-supported reagents and scavengers.
As shown in Scheme 3 we set out to produce a total of 128
products derived from monomers 1–3, comprised of 32 stereoi-
somers and 2 regioisomers for each ring system. Acylation of
pyran amines 1 and 2 with excess acid 3 in the presence of
silica-bound dicyclohexylcarbodiimide (Si-DCC) and hydroxy-
benzotriazole (HOBt) proceeded smoothly to afford the corre-
sponding amides. Residual acid was removed along with HOBt
through the addition of a silica-bound tetraalkylammonium
carbonate (Si-CO3). Treatment with TFA in dichloromethane
(DCM) efficiently removed both the tert-butoxycarbonyl (Boc)
and TBDPS groups to provide the desired amino alcohols (11
and 12) along with a minor amount of the corresponding trifluor-
oacetyl esters. The latter underwent methanolysis in the presence
of DCM∶MeOH (3∶1) and Si-CO3 to afford the deprotected
amino alcohols. Selective acylation of the amino alcohols with
either the ortho- or para-regioisomer of benzoic acid 4 provided
the corresponding amide SNAr precursors. Again, the reaction
products were treated with Si-CO3 to remove excess benzoic acid
and then with silica-bound carboxylic acid (Si-CO2H) to remove
any residual amine or potential products resulting from O-acyla-
tion (no bisacylation was observed).
OTBDPS CO2Me
Stereochemistry
1 C4 C5
5
1
C
O
6a: R
6b: S
6c: R
6d: S
R
S
S
R
R
R
R
R
HO
4
6a-d
ent-6a-d
1. Me3OBF4, Proton Sponge,
CH2Cl2, 69-94%
2. H2, Pd/C, MeOH, 94-100%
OTBDPS CO2Me
O
MeO
7a-d
ent-7a-d
LiBH4, LiB(Et)3H,
Et2O, 71-100%
1. HF/pyr, THF, 78-90%
2. NsNHMe, DIAD, PS-
PPh3, THF, 32-72%
Traditionally intramolecular SNAr reactions have been
achieved using a nitro-activated aromatic component, however,
given the toxicities associated with nitro-containing compounds
(37), we instead chose to explore a nitrile-activated variant given
the pharmaceutical relevance of aryl-nitriles (38). During initial
feasibility studies, we found that nitrile-activated substrates
underwent SNAr cycloetherification with high efficiency. Aryl
fluorides derived from 11 and 12 were treated with cesium car-
bonate and heated to 110 °C for 4 h to affect macrocyclization.
OH
Ns
Me
OTBDPS
O
CO2Me
N
O
MeO
MeO
8a-d
ent-8a-d
9a-d
ent-9a-d
1. DIBAL, THF, 0 oC, 60-95%
2. TBDPSCl, Et3N, DMAP,
82-100%
3. PhSH, K2CO3, DMF,
82-100%
1. DMP, CH 2Cl2
2. MeNH2, NaBH4,
EtOH, 64-96%
Me
Me
NaH, MeI, THF, 0 oC
77-98%
3
Me
Me
HO2C
N
2
Me
NH
HO2C
N
HO
Boc
MeO
Boc
Me
OTBDPS
OTBDPS
O
NH
10a-b
ent-10a-b
3a-b
ent-3a-b
O
Stereochemistry
C2 C3
MeO
MeO
1a-d
10a: R
10b: R
S
R
2a-d
ent-2a-d
ent-1a-d
Scheme 1. Synthesis of pyran fragments 1 and 2 (only one stereoisomer
Scheme 2. Preparation of chiral β-methoxy-γ-amino acid 3 (only one stereo-
shown).
isomer shown).
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Comer et al.

Me
O
O
Me
N
O
F
NC
B
OMe
Me
N
C
Me
Me
NC
Boc
O
Me
HO2C
N
A
CO2H
O
N
H
H
N
OMe Me
4-o/p
3
OMe
N
OTBDPS
O
Me
Me
HO
MeO
O
1. Si-DCC, HOBt, DIPEA,
CH2Cl2 then Si-CO3
1. Si-DCC, HOBt, DIPEA,
ABC-5-o/p
CH2Cl2 then Si-CO3, Si-CO2H
MeO
MeO
2. Cs2CO3, DMF, 110 o
C
2. TFA, CH2Cl2 then
a
b
c
Si-CO3, 3:1 DCM:MeOH
1
11
then Si-CO3, CH2Cl2
32 stereoisomers
CN
OMe
Me
F
B
Me
C
OMe
Me
N
Me
NH
NC
Boc
Me
OTBDPS HO2C
N
A
Me
O
CO2H
NH
O
Me
O
OMe Me
3
4-o/p
O
N
O
Me
OH
N
O
1. Si-DCC, HOBt, DIPEA,
CH2Cl2 then Si-CO3
1. Si-DCC, HOBt, DIPEA,
O
MeO
CH2Cl2 then Si-CO3, Si-CO2H
MeO
2. Cs2CO3, DMF, 110 o
then Si-CO3, CH2Cl2
C
2
MeO
12
2. TFA, CH2Cl2 then
ACB-5-o/p
Si-CO3, 3:1 DCM:MeOH
a
c
b
A (16) X B (4) X C (2) = 128 compounds
32 stereoisomers
Scheme 3. Parallel solution-phase synthesis of pyran-containing macrocycles ABC-5 and ACB-5.
For difficult macrocyclization reactions, aryl fluoride hydrolysis
was occasionally observed. Fortunately, the corresponding phe-
nol impurity could easily be removed upon treatment with
Si-CO3. Although intramolecular SNAr reactions of nitrile-acti-
vated compounds have been reported (39), the work described
here represents an example of a macrocyclization through this
method.
Following completion of the four-step solution-phase synthesis
the crude library products were subjected to mass-directed HPLC
purification and analyzed by ultraperformance liquid chromato-
graphy-mass spectrometry (UPLC-MS). As shown in Table 1,
the overall average purity of the library members ranged from
81–90% with the purity of macrocycles ABC-5 (entries 1 and 2)
being slightly greater than the corresponding ACB-5 isomers
(entries 3 and 4). The average isolated yield for the 128-mem-
bered library after HPLC purification was 22% over four steps
with an overall pass rate of 95%.*
HPLC purification provides ample material (approximately
6 mg) for future screening studies (the SI Appendix details full
purity and yield analysis). Little differences were observed
throughout the series between the ortho- and para-aromatic
analogues, with the main determinants for success being the
order of the fragments (ABC vs. ACB) and targeted ring size.
Using this parallel synthesis strategy, typically 48-library members
could be produced over the course of 1 wk by one chemist.
Physicochemical Properties and Shape Diversity of Library Members.
We next examined various properties of the DS library as com-
pared to two structurally distinct compound sets, namely natural
products (AnalytiCon Discovery) and the National Institutes of
Health Molecular Library Small Molecule Repository (MLSMR)
(Table 3). The MLSMR serves to represent a typical small-
molecule screening collection, because it is primarily comprised
of commercial vendor libraries (>90%) (4). Not surprisingly,
products resulting from the DS library more closely resemble
natural products as compared to MLSMR compounds, having
an increased number of stereogenic centers (>4) and greater
structural complexity (Fsp3 ¼ 0.59). The degree of ring fusion
is also higher with fewer aromatic ring systems than typical
screening compounds. Whereas the oxygen count for the DS
library is comparable to natural products, the nitrogen count is
similar to that of MLSMR compounds. As an aside, it is interest-
ing to note the differences in certain properties for natural pro-
ducts of different origins. Plant-derived natural products have
a higher number of oxygen atoms, as well as stereogenic centers,
as compared to microbial natural products. Lastly, all physico-
The modularity of the DS strategy allows for fragments to
be easily interchanged thus facilitating rapid analogue synthesis
including variations in ring size. To demonstrate this, commer-
cially derived γ-amino acids 13 and 14, along with β- and α-amino
acids 15 and 16 (Fig. 2), were utilized for the preparation of 14- to
16-membered macrocycles 17–20 (Fig. 3).
As shown in Table 2, the efficiency of synthesis to access 16-
membered macrocycles derived from γ-amino acids 13 and 14 was
universally high in terms of purity (84–94%) with all compounds
having purities >75% (entries 1–8). On average, the ABC macro-
cycles (ABC-17 and ABC-18, entries 1, 2, 5, and 6) were isolated
in higher yield than the corresponding ACB isomers (entries 3, 4,
7, and 8). In contrast, the shuffled 15-membered macrocycles
ACB-19 performed better in terms of overall purity and pass rate
than their ABC counterparts (entries 9–12). Finally, the 14-mem-
bered compounds were isolated with similar purities (81–89%)
for both the shuffled and nonshuffled systems (entries 13–16),
although low yields were obtained for ACB-20-p(entry 16). The
overall purity for the 224-membered sublibrary is satisfying at
87% given the complexity of the systems involved, especially
considering that no attempts were made to tailor reaction condi-
tions to each ring size. The overall average yield of 29% after
Table 1. Average purity and yield analysis for the formation of
16-membered macrocycles ABC-5 and ACB-5
Entry Product Ring size Stereoisomers % purity* % pass rate† % yield‡
1
2
3
4
5
ABC-5-o
ABC-5-p
ACB-5-o
ACB-5-p
Total
16
16
16
16
32
32
32
32
128
90
90
86
81
87
100
100
94
84
95
29
16
26
18
22
*Average purity was determined by UPLC monitoring at UV 210 nm.
†Pass rate is defined as the percentage of compounds with purity >75%.
‡Average isolated yield over four steps after HPLC purification
(theoretical yield ¼ 45 μmol).
*Library members with purity values <75% can be subjected to
a second round of
purification to access the full matrix of stereoisomers.
Comer et al.
PNAS
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∣
vol. 108
∣
no. 17
∣
6753

Ph Boc
N
Boc
N
HO2C
HO2C
Me
Me
13 (& ent-13)
14
Me Boc
N
Me
Me
HO2C
Me
HO2C
N
Boc
15 (& ent-15)
16 (& ent-16)
Fig. 2. Amino acids 13–16 (one enantiomer shown).
chemical properties affecting permeability and solubility (e.g.,
molecular weight, ALogP, rotatable bonds, topological polar
surface area) for the DS library are within an acceptable range
for a discovery library (40, 41).
Finally, in order to visualize the structural diversity obtained
by the DS strategy, a molecular-shape-based diversity analysis
derived from normalized principal moments of inertia ratios (42)
was applied (Fig. 4). This simplistic representation categorizes
molecular shape into three distinct topologies: rod-, disc-, and
sphere-like character. It is gratifying to see the shape diversity
brought about by the change in connectivity from ABC to ACB.
Visual inspection shows the “nonshuffled” ABC scaffolds prefer
the lower-right part of the triangle, whereas the shuffled ACB
scaffolds occupy the upper-left part of the triangle. Varying ring
size also contributed to the overall shape diversity.
Fig. 4. Principal moments of inertia (PMI) analysis of the DS library products.
PMI values (x, y, and z) were computed on the minimum energy conformer
for each compound and normalized PMI ratios are plotted in the triangular
scatter plot. The ABC (red) vs. ACB (blue) skeletons display significant shape
diversity.
quent optimization processes. A total of 352 macrocyles were
prepared with increased structural complexity as compared to a
typical screening collection. The library is modular and fragments
can be readily shuffled or interchanged to afford analogues with
distinct molecular shapes and ring sizes. The library anticipates a
successful discovery phase by enabling rapid analogue synthesis
both through the use of modular fragments and rapid solid-phase
assisted parallel solution-phase techniques. The complete matrix
of stereoisomers was prepared for each macrocycle. A single
synthetic route afforded the collection of complex macrocycles
with an average yield of 25% (over four steps) and an average
purity of 87%.
Conclusions
We have developed a library of complex pyran-containing macro-
cycles with features that facilitate the initial discovery and subse-
a
c
a
c
b
b
ring size
CN
O
R
Materials and Methods
Me
N
Me
N
O
Details for the synthesis and characterization of all monomers are provided
in the SI Appendix. SiliaBond® functionalized silica gels (Si-DCC, Si-CO3, and
Si-CO2H) were purchased from SiliCycle. A Metler Toledo MiniBlock® and
MiniBlock® XT were used for parallel synthesis. Compound purity and
identity were determined by UPLC-MS (Waters). Purity was measured by
UV absorbance at 210 nm. Identity was determined on a single quad mass
spectrometer by positive electrospray ionization. Compound purification
NC
O
R
N
Me
O
Me
O
O
N
O
O
16
MeO
MeO
8-16 stereosiomers
8-16 stereosiomers
ABC-17-o/p R = H
ACB-17-o/p R = H
ACB-18-o/p R = Ph
ABC-18-o/p R = Ph
Table 2. Average purity and yield analysis for macrocycles
ABC-17-20 and ACB-17-20
Entry Product Ring size Stereoisomers % purity* % pass rate† % yield‡
CN
Me
N
O
Me
N
Me
Me
O
NC
Me
Me
N
1
2
3
4
5
6
7
8
ABC-17-o
ABC-17-p
ACB-17-o
ACB-17-p
ABC-18-o
ABC-18-p
ACB-18-o
ABC-18-p
ABC-19-o
ABC-19-p
ACB-19-o
ACB-19-p
ABC-20-o
ABC-20-p
ACB-20-o
ACB-20-p
Total
16
16
16
16
16
16
16
16
15
15
15
15
14
14
14
14
8
8
8
90
84
93
94
91
86
91
92
70
72
92
90
89
88
89
81
87
100
100
100
100
100
100
100
100
63
51
47
25
35
40
37
23
28
31
24
28
30
21
17
14
6
O
O
O
O
O
N
O
15
8
MeO
MeO
16
16
16
16
16
16
16
16
16
16
16
16
224
16 stereosiomers
16 stereosiomers
ABC-19-o/p
ACB-19-o/p
9
CN
O
O
Me
10
11
12
13
14
15
16
17
63
Me
N
O
Me
100
100
100
100
100
94
N
NC
Me
N
Me
O
O
Me
N
O
O
14
O
MeO
MeO
16 stereosiomers
16 stereosiomers
95
29
ABC-20-o/p
ACB-20-o/p
*Average purity was determined by UPLC monitoring at UV 210 nm.
†Pass rate is defined as the percentage of compounds with
purity >75%.
A (16) X B (7) X C (2) = 224 compounds
Fig. 3. Additional library members with varying ring size (only one stereo-
‡Average isolated yield over four steps after HPLC purification
(theoretical yield ¼ 45 μmol).
isomer shown).
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Comer et al.

Table 3. Mean values of different properties for DS library as compared to natural products and MLSMR
Property
DS library (n ¼ 352)
NPs (plant/microbial)* (n ¼ 3;486∕1;149)
MLSMR† (n ¼ 337;890)
Molecular weight
ALogP
457
1.4
95
1.5
3.0
5.4
2.8
0.27
4.3
0.59
500∕404
1.6∕2.2
154∕116
6.9∕6.7
0.09∕0.86
9.7∕6.5
1.9∕1.9
0.35∕0.30
8.0∕4.1
0.55∕0.54
357
2.9
86
5.3
2.9
3.0
1.3
0.75
0.4
0.30
TPSA
‡
Rotatable bonds
Nitrogen count
Oxygen count
Ring fusion degree
Aromatic to ring bonds
Stereogenic centers
Fsp3
TPSA, topological polar surface area.
*Natural products (NPs) used for this analysis were obtained from the online database for AnalytiCon Discovery (www.ac-discovery.com).
†Compounds contained within the 2010 MLSMR were used for this analysis.
‡Rotatable bonds are defined as single bonds between heavy atoms that are not in a ring and not terminal.
was performed using a mass-directed approach on a Waters Autopurification
System equipped with a ZQ mass spectrometer. Removal of organic solvents
at intermediate stages of the synthesis was achieved using an EZ-2 Genevac
centrifugal evaporator (or with the HT-24 Genevac workstation). HPLC-pur-
ified samples were concentrated using a VirTis 25L Genesis EL freeze dryer.
Acylation. The crude amino alcohols, cyano-fluorobenzoic acid 4-o or 4-p
(0.022 g, 0.135 mmol), Si-DCC (200 mg, 0.93 mmol∕g), and DIEA (0.016 mL,
0.090 mmol) were combined in 2% dimethylformamide (DMF)/DCM (3.0 mL),
and stirred at room temperature overnight. In cases where acylation is slow,
additional Si-DCC (200 mg) and a solution of HOBt (5 mg) in DMF/DCM
(1.0 mL) and DIEA base (6 μL) were added. After acylation was deemed
complete, reactions were scavenged with Si-CO3 (400 mg per reaction) and
Si-CO2H (200 mg per reaction) for 30 min and then filtered and evaporated
on a Genevac for 4 h.
Amide Coupling. To a set of 16 test tubes equipped with stir bars was added
Si-DCC (200 mg, 0.93 mmol∕g), 1.0 mL solution of amine (20 mg, 0.045 mmol)
in DCM, 1.0 mL solution of acid (0.077 mmol) in DCM. To the reaction mixture
was then added 2.0 mL solution of HOBt and diisopropylethylamine (DIEA)
in DCM, 0.038 mM and 0.135 mM, respectively. After 16 h, LC-MS showed
complete coupling. Each reaction was scavenged with Si-CO3 (400 mg,
0.63 mmol∕g) for 30 min, filtered and dried on a Genevac for 3 h. The crude
reaction products were taken on directly to the next step.
SNAr Macrocyclization. All crude products from above were dissolved in DMF
(4.0 mL) and heated at 110 °C with Cs2CO3 (approximately 200 mg per reac-
tion) for 4 h. Reaction mixtures were filtered through Celite, washed with
DCM, and solvents removed on a Genevac. Crude products were dissolved
in DCM and treated with Si-CO3 (400 mg) and Si-CO2H (200 mg) for
30 min, and then filtered through Celite and concentrated on a Genevac.
The crude products were purified by mass-directed preparative HPLC, dried
via lyophilization, and analyzed by LC-MS.
Boc/TBDPS Removal. Each reaction was treated with 0.2 mL TFA in 1.0 mL DCM
for 3 h, then solvent was removed on a Genevac overnight. The samples
were diluted with DCM (1.0 mL) and coevaporated for 30 min on a Genevac.
The crude product was then treated with Si-CO3 (400 mg, 063 mmol∕g) in
MeOH/DCM (4.0 mL, 1∶3 vol∕vol) to hydrolyze the TFA-ester. After filtration,
solvents were removed on a Genevac for 1.5 h, followed by coevaporation
with DCM (1.0 mL x2). Both Boc and TBDPS were removed cleanly based
on LC-MS data. Crude products were carried on to the next step without
purification.
ACKNOWLEDGMENTS. This work was funded in part by the National Institute
of General Medical Sciences sponsored Center of Excellence in Chemical
Methodology and Library Development (Broad Institute, P50 GM069721),
as well as the National Institutes of Health (NIH) Genomics-Based Drug
Discovery U54 Grants Discovery Pipeline RL1CA133834 (administratively
linked to NIH Grants RL1HG004671, RL1GM084437, and UL1RR024924).
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