A synthetic library of cell-permeable molecules

Article abstract of DOI:10.1021/ja0023377

Small molecules that induce or stabilize the association of macromolecules have proven to be useful effectors of a wide variety of biological processes. To date, all examples of such chemical inducers of dimerization have involved known ligands to well-ch

Full text of DOI:10.1021/ja0023377

398
J. Am. Chem. Soc. 2001, 123, 398-408
A Synthetic Library of Cell-Permeable Molecules
Kazunori Koide,† Joshua M. Finkelstein, Zachary Ball,§ and Gregory L. Verdine*
Contribution from the Department of Chemistry and Chemical Biology, Institute of Chemistry and
Cell Biology, HarVard UniVersity, 12 Oxford Street, Cambridge, Massachusetts 02138
ReceiVed June 28, 2000
Abstract: Small molecules that induce or stabilize the association of macromolecules have proven to be useful
effectors of a wide variety of biological processes. To date, all examples of such chemical inducers of
dimerization have involved known ligands to well-characterized proteins. The generality of this approach could
be broadened by enabling the discovery of heterodimerizers that target known macromolecules having no
established ligand, or heterodimerizers that produce a novel biologic response in screens having no predetermined
macromolecular target. Toward this end, we report the construction of a diversified library of synthetic
heterodimerizers consisting of an invariant ligand that targets the FK506-binding protein (AP1867) attached
to 320 substituted tetrahydrooxazepines (THOXs). The THOX components were generated by a combination
of liquid- and solid-phase procedures employing sequential Mitsonobu displacements to join two structurally
diversified olefin-containing monomers, followed by ruthenium-mediated olefin metathesis to effect closure
of the seven-membered ring. The 320 resin-bound THOX ligands were coupled in parallel to AP1867, and the
products were released from the resin to yield candidate heterodimerizers in sufficient yield and purity to be
used directly in biologic testing. A representative panel of 25 candidate heterodimerizers were tested for their
ability to pass through the membrane of human fibrosarcoma cells, and all were found to possess activity in
this tissue culture system. These studies pave the way for further studies aimed at using small-molecule inducers
of heterodimerization to effect novel biological responses in intact cells.
Introduction
Small molecules that induce or stabilize the association of
macromolecules hold tremendous promise as biological effec-
tors. Such “chemical inducers of dimerization” (CIDs) are well
known in Nature.1,2 For example, the anti-cancer agents adria-
mycin3 and etoposide4 act by stabilizing a catalytic intermediate
having topoisomerase II bound to its nicked DNA substrate.
The immunosuppressive agent FK506 induces the dimerization
of FK506-binding protein (FKBP) and calcineurin (Cn), thereby
abrogating the latter’s protein phosphatase activity (Figure
1A).5,6 The immunosuppressive agent cyclosporin A (CsA) also
blocks Cn activity by an induced dimerization mechanism, but
in this case the partner protein is cyclophilin.5,7 Yet a third small-
molecule immunosuppressant, rapamycin, induces the associa-
tion of FKBP to the FK506/rapamycin associated protein
(FRAP).8-10
Figure 1. Small molecules can induce the dimerization of macromol-
ecules. (A) The macrolide natural product FK506 induces the dimer-
ization of two proteins, the FK506-binding protein (FKBP) and
calcineurin (Cn). (B) Synthetically linked dimers of FK506 induce the
homodimerization of a protein having FKBP fused to a partner A. (C)
Synthetically linked dimers of FK506 and cyclosporin A (CsA) induce
the heterodimerization of two fusion proteins, one of which has an
FKBP domain and the other of which has a cyclophilin (CyP) domain.
(D) Heterodimerizers having the FK506 analogue AP1867 linked to a
diversity element can be used to induce the dimerization of a fusion
protein with diverse intracellular targets. A and B represent proteins
to which FKBP and CyP are fused in a contiguous polypeptide chain
by ligation of the corresponding coding DNA sequences. A and B
typically possess a function that is useful in a biologic context, such
as DNA binding, transcriptional activation, fluorescence, nuclear export
or import, or intracellular signaling.
* To whom correspondence should be addressed. Telephone: 617-495-
5333. Fax: 617-495-8755. E-mail: verdine@chemistry.harvard.edu.
URL: http://glviris.harvard.edu.
† Present address: Department of Chemistry, University of Pittsburgh,
Pittsburgh, PA 15260.
§ Present address: Department of Chemistry, Stanford University, Palo
Alto, CA 94305.
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10.1021/ja0023377 CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/28/2000

Cell-Permeable Candidate Heterodimerizers
J. Am. Chem. Soc., Vol. 123, No. 3, 2001 399
Results and Discussion
controlled manner, Schreiber and Crabtree have demonstrated
the ability to engineer a variety of inducible signal transduction
processes into cells (Figure 1B,C).11-15 Numerous variations
on the general theme of using induced dimerization to generate
biological responses have been reported, thus underscoring the
broad scope of this strategy.16-34
Invariant Receptor-Ligand Pair. The invariant ligand of
our candidate heterodimerizers should bind with high affinity
and specificity to an intracellular receptor, without disrupting a
function that is essential for cell survival. Preferentially, the
ligand structure should bias toward cell permeability and be
synthetically accessible. To satisfy these criteria, we chose
AP1867, a synthetic analogue of the natural products FK506
and rapamycin.19
The foregoing examples of synthetic “dimerizers” all involve
bifunctional molecules having two ligand moieties linked
together, each of which binds a known receptor. An exciting,
and as-yet unexplored possibility is to induce the association
of one known macromolecule with an unknown one, thereby
creating biological relationships that Nature may not have
explored. Furthermore, dimerizing two known macromolecules
can present a challenge when a ligand is known for one partner
but not the other. Both these lines of research could be pursued
by the construction of bifunctional small-molecule libraries
having an invariant ligand attached to a diversified ligand whose
structure varies among the members of the library (Figure 1D).
Here we report the construction of the first such diversified
library of synthetic candidate heterodimerizers.
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AP1867 associates with wild-type FKBP (Kd ) 67 nM) but
has a much higher affinity (Kd ) 94 pM) for an engineered
mutant of FKBP having a mutation of Phe 36 to Val (FKBP*).19
This difference in affinity for FKBP* vs FKBP makes it possible
to target AP1867 to the engineered protein selectively, even in
the presence of high concentrations of the wild-type endogenous
FKBP. A synthetic homodimer of AP1867 readily permeates
mammalian cells, thus increasing the likelihood that het-
erodimerizers containing this moiety would also be cell-
permeable.19
Design of the Core Structure of the Diversified Ligand.
Our design strategy considered various aspects of receptor-
ligand interactions, cell physiology, and feasibility of solid-phase
synthesis to provide final products having sufficient purity.
Taking these issues into account, we selected the tetrahydro-
oxazepine (THOX) core shown in Scheme 1. This semirigid
core presents three diversity elements, two on the ring and one
on a side chain. The three stereogenic centers on the THOX
unit can be independently varied via asymmetric synthesis, thus
giving rise to all eight possible stereoisomers.
The presence of uncharged heteroatoms on the THOX unit
at physiological pH balances the hydrophobicity of the molecule,
thus favoring solubility in aqueous media and transit across cell
membranes. From a synthetic standpoint, the THOX unit
provides independent attachment points to both the solid support
and the invariant ligand, and the modular structure of THOX
suggests a convergent synthesis. Finally, we were intrigued by
the possibility that the N-O bond of THOX might be reduc-
tively cleaved to convert the semirigid cyclic library to a more
flexible acyclic library containing additional diversity sites.35
We envisioned producing the THOX system (Scheme 1) by
coupling two independently diversifiable monomers through
Mitsunobu chemistry,36 followed by ring-closing olefin meta-
thesis (RCM)37,38 to produce exclusively the heterocoupled
product having cis double bond geometry. The chemical
sensitivity of AP1867 suggested it would be prudent to attach
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400 J. Am. Chem. Soc., Vol. 123, No. 3, 2001
Koide et al.
Scheme 1
Scheme 2a
Scheme 3a
a Reagents and conditions: (a) EtO2CsNdNsCO2Et, Ph3P, THF,
0 f 23 °C, 1 h, 66%; (b) 0.05 equiv of Cl2Ru(PCy3)2dCHPh, CH2Cl2
(0.02 M), 1 h, 23 °C; 55% isolated yield at 100% conversion; (c)
EtO2CsNdNsCO2Et, Ph3P, THF, 0 f 23 °C, 1 h, quantitative; (d) 2
× 0.05 equiv of Cl2Ru(PCy3)2dCHPh, CH2Cl2 (0.1 M), 23 °C, 21 h;
45% isolated yield at 100% conversion.
a Reagents and conditions: (a) n-BuLi in hexanes, THF; R1CH2COCl,
THF, -78 f 23 °C, 30 min, quantitative; or R1CH2CO2H, 2-chloro-
1-methylpyridinium iodide or DCC, DMAP, Et3N, 0 f 23 °C, then 23
°C, 1.5 h, 79-94%; (b) 5, n-Bu2BOTf, iPr2NEt, 0 °C, 20 min; acrolein,
CH2Cl2, -78 f 0 °C, 40 min, 55-79%; (c) LiBH4, Et2O, 0 °C, 5 min,
81-90%; (d) TBSCl, Et3N, CH2Cl2, 0 f 23 °C, 8 h, 80-89%; (e)
PhthNOH, Ph3P, EtO2CsNdNsCO2Et, 0 f 23 °C, 45 min, 67-71%;
(f) MeNH2, EtOH, CH2Cl2, 23 °C, 15 min; 4-nitrobenzenesulfonyl
chloride, 2,4,6-collidine, CH2Cl2, 0 °C, 15 min, 98% (crude); (g) HF-
pyridine, THF, 0 °C, then 23 °C, 10 h, 65-73%. DMAP, 4-(dimethyl-
this moiety to THOX in the penultimate synthetic step, leaving
only cleavage of the complete heterodimerizer from the solid
support, an operation that can be carried out under mild
conditions.
4
amino)pyridine; TBS, SitBuMe2; Phth, phthalimide; Ns, 4-nitroben-
Model Study for the RCM Reaction Step. The most critical
step of our synthetic scheme is RCM to form the cis endocyclic
olefin. To our knowledge, no literature precedent exists for olefin
metathesis on substituents containing an N-O bond in the newly
formed ring; hence, we were uncertain as to whether the relative
rates of the RCM step would be significantly influenced by the
relative stereochemistry at the sp3 centers. To address these
issues experimentally, we coupled racemic sulfonylalkoxyamine
6 to racemic homoallylic alcohol 3 and subjected the entire
mixture of stereoisomers to RCM (Scheme 2). With 5 mol %
catalyst, cyclization was complete within 1 h at room temper-
ature. The 1:1 mixture of diastereomers obtained revealed the
lack of a significant stereochemical influence on the rate of
RCM. These results indicate that the THOX ring is formed
rapidly enough for combinatorial applications, and furthermore
is capable of producing all possible stereoisomers.
We also examined RCM on the coupling product of 6 and
racemic allylic alcohol 9, leading to the corresponding product
containing a six-membered ring. Although this RCM process
also produced a 1:1 mixture of diastereomers, the reaction was
sluggish, requiring 21 h to achieve completion. We therefore
chose the THOX system for initial library development.
Asymmetric Synthesis of Monomers. To avoid the need
for the deconvolution of complex stereochemical mixtures
zenesulfonyl.
during library screening, we decided to employ only stereo-
chemically defined substrates in the library synthesis. To set
the relative and absolute stereochemistry of the R1 monomer,
we selected the Evans chiral auxiliary-directed aldol reaction
(Scheme 3).39,40 Known oxazolidinone 12 was deprotonated with
n-butyllithium and then coupled with commercially available
acid chlorides to generate imides 5 in quantitative yield.
Alternatively, the oxazolidinone auxiliary was coupled to
carboxylic acids using 2-chloro-1-methylpyridinium iodide or
dicyclohexylcarbodiimide (DCC)/triethylamine/catalytic 4-(di-
methylamino)pyridine, in 79-94% yield.
The imides were then condensed with acrolein under standard
conditions to furnish â-hydroxyl imides 13 in 55-79% yield.
In all cases, the main diastereomer accounted for greater than
95% of the isolated product following column chromatography.
1,3-Diols 14, prepared by chemoselective reduction of imides
13, were regioselectively protected as the tert-butyldimethylsilyl
(TBS) ethers to afford secondary alcohols 15.
(39) Evans, D. A.; Bartroli, J.; Shih, T. L. J. Am. Chem. Soc. 1981, 103,
2127-2129.
(40) Ager, D. J.; Prakash, I.; Schaad, D. R. Chem. ReV. 1996, 96, 835-
875.

Cell-Permeable Candidate Heterodimerizers
J. Am. Chem. Soc., Vol. 123, No. 3, 2001 401
Scheme 4a
compounds were subjected to Mitsunobu displacement by
N-hydroxyphthalimide, and the resulting products were con-
verted by the aforementioned one-pot procedure to the sulfo-
nylalkoxyamines 21a-c. Deprotection of the trityl group was
accomplished by treatment of sulfonylalkoxyamines 21a-c with
formic acid in diethyl ether to give primary alcohols 24a-c.
A late-diversification scheme was used to generate additional
monomers. Trityl-protected bromide 25 was chemoselectively
converted to the corresponding N-alkoxyphthalimide 26 in 74%
yield (Scheme 5).43 Despite the electrophilicity of the N-
alkoxyphthalimide moiety, we found it feasible to carry out
efficient displacement of the bromide with a variety of nucleo-
philes in the presence of the phthalimide. The displacement
products 27a-d were then carried forward to the corresponding
primary alcohols 29a-d.
a Reagents and conditions: (a) TrtCl, pyridine, CH2Cl2, 0 °C, 2 h,
91%; (b) PhthNOH, Ph3P, EtO2CsNdNsCO2Et, 0 f 23 °C, 45 min,
72-97%; (c) LiOH, H2O2, MeOH/THF/H2O (2:3:1), 46 °C, 7 h, 98%;
(d) 22, 2-chloro-1-methylpyridinium iodide, iPr2NEt, CH2Cl2, 0 °C, then
23 °C, 15 min; NH(CH2CO2Et) or MeNH2, 0 °C, then 23 °C, 15 min,
58% to quantitative; (e) MeNH2, EtOH, CH2Cl2, 23 °C, 15 min;
2-nitrobenzenesulfonyl chloride, 2,4,6-collidine, CH2Cl2, 0 °C, 15 min,
71-83% and 98% (crude); (f) formic acid/Et2O (1:1), ethanethiol or
mercaptoacetic acid, 23 °C, 2 h, 29-75%. Trt, trityl; 2Ns, 2-nitroben-
zenesulfonyl.
For both unfunctionalized and functionalized R1 monomers,
the overall yields based on the starting carboxylic acids or acid
chlorides were 5-19% (Figure 2). The entire reaction sequence
could be carried out in parallel on four monomers on a 10-50
g scale.
Synthesis of the R2 Monomers. Just as with the R1 mono-
mers, the R2 monomer series was designed to incorporate a
variety of aliphatic, aromatic, and masked acidic functional
groups (Figure 3). The 16 R2 monomers were prepared enantio-
selectively using the Brown procedure for asymmetric allylation
of aldehydes.44 Bromobenzene derivatives were further elabo-
rated by the Heck reaction to furnish cinnamate derivatives
3g and 3h (Scheme 6).45,46 Methyl benzoate 3e was converted
to amide 3f via hydrolysis, followed by standard amide
formation.
Scheme 5a
Library Synthesis. With these building blocks in hand, we
undertook the parallel synthesis of a THOX library, aiming to
produce 6 µmol of each compound. To track the efficiency of
the library synthesis, we randomly chose 40 out of the total
320 library members47 and analyzed the entire course of the
synthesis by HPLC and LC-MS analysis of a small portion of
the resin removed after each reaction step.
The 20 R1 monomers were condensed with a 38-75-µm
polystyrene-based trityl chloride resin48,49 in dichloromethane
and pyridine to form trityl ethers 4 (Scheme 7).50 Prior to library
synthesis, resin-bound azide 29a (Figure 2) was converted to
a Reagents and conditions: (a) TrtCl, pyridine, CH2Cl2, 0 °C, 2 h,
88%; (b) PhthNOH, Ph3P, EtO2CsNdNsCO2Et, 0 f 23 °C, 45 min,
74%; (c) nucleophile, base, DMF, 50% to quantitative; (d) MeNH2,
EtOH, CH2Cl2, 23 °C, 15 min; 2-nitrobenzenesulfonyl chloride, 2,4,6-
collidine, CH2Cl2, 0 °C, 15 min, 26-93%; (e) formic acid/Et2O (1:1),
mercaptoacetic acid, 23 °C, 2 h, 42-82%.
The secondary alcohols were then converted to N-alkoxy-
phthalimides 16 under Mitsunobu conditions in 67-71%
yield.41,42 The phthalimides were converted to the corresponding
4-nitrobenzenesulfonyl derivatives 17 by a one-pot procedure
(methylamine, followed by 4-nitrobenezenesulfonyl chloride and
collidine) to generate a nucleophilic alkoxyamine. Treatment
of the TBS ethers with HF-pyridine gave primary alcohols 18.
R1 monomers bearing functionalized side chains were syn-
thesized by a slightly altered sequence (Schemes 4 and 5). For
certain applications,16 it would be desirable to have carboxylic
acid or amide functionality in the diversified ligand. The
synthesis of such monomers diverted from the common scheme
at the 1,3-diol stage by protection of the primary hydroxyl as a
trityl ether 19, saponification of the aryl ester, and amide
formation to give benzamides 23b,c. The trityl-protected
(43) The alkyl bromide with one less carbon underwent undesired
cyclopropane formation under mildy basic conditions and was unsuitable
for R1 monomer synthesis: 1H NMR (400 MHz, CDCl3, 23 °C) δ ) 7.44-
7.30 (m, 5H, Ar), 5.68 (d, 1H, J ) 7.0 Hz, -CHPh), 4.76 (dq, 1H, J ) 7.0,
6.6 Hz, -NCH), 3.18 (m, 1H, sCHCdO), 1.16-1.13 and 1.04-1.00 (m,
4H, -CH2CH2-), 0.90 (d, 3H, J ) 6.6 Hz, -NCHCH3); CI+ HRMS calcd
for C14H19N2O3 (M + NH4+) 263.1396, found 263.1406.
(44) Jadhav, P. K.; Bhat, K. S.; Perumal, T.; Brown, H. C. J. Org. Chem.
1986, 51, 432-439.
(45) Heck, R. F. Palladium-Catalyzed Vinylation of Organic Halides;
John Wiley & Sons: New York, 1982; Vol. 27.
(46) Horwell, D. C.; Nichols, P. D.; Ratcliffe, G. S.; Roberts, E. J. Org.
Chem. 1994, 59, 4418-4423.
(47) In addition to the 320 THOX library members reported here, 40
additional compounds lacking the THOX core were synthesized by reacting
the 20 R1 monomers with two commercially available alcohols, ethyl
6-hydroxyhexanoate and N-(2-hydroxyethyl)phthalimide. Since these mono-
mers have no olefin, they were not subjected to RCM, and are therefore
open-chain alkoxyamines:
(41) The byproduct in this step resulted from SN2′ attack to yield the
following compound:
(48) Chen, C. X.; Randall, L. A. A.; Miller, R. B.; Jones, A. D.; Kurth,
M. J. Tetrahedron 1997, 53, 6595-6609.
(49) Chen, C. X.; Randall, L. A. A.; Miller, R. B.; Jones, A. D.; Kurth,
M. J. J. Am. Chem. Soc. 1994, 116, 2661-2662.
(42) N-Hydroxy 4-nitrobenzenesulfonamide failed to react with the
alcohol under Mitsunobu conditions.

402 J. Am. Chem. Soc., Vol. 123, No. 3, 2001
Koide et al.
Figure 2. Structures of R1 monomers.
Figure 3. Structures of R2 monomers.
the corresponding acetamide by treatment with neat thiolacetic
were activated by treatment with di-tert-butyl azodicarboxylate
or diisopropyl azodicarboxylate and triphenylphosphine and then
coupled to the alkoxysulfonamide nitrogen of the resin-bound
R1 monomers under Mitsunobu conditions, thereby producing
320 dienes 2.52 For all substrates examined, the RCM reaction
was found to produce a single product with complete conversion.
acid (RN3 f RNHAc).51 The 16 homoallyl alcohols (Figure 3)
(50) We experienced lower isolated chemical yields after intramolecular
cyclization on the resin at high loading level (approximately 0.8 mmol/g).
Although the loading capacity of the commercially available trityl chloride
resin is greater than 0.8 mmol/g, we adjusted the stoichiometry so that the
loading level of the R1 monomers was at 0.2 mmol/g and the rest of trityl
chloride was reacted with ethanol or methanol in the presence of triethyl-
amine.
(51) Peters, S.; Bielfeldt, T.; Meldal, M.; Bock, K.; Paulsen, H.
Tetrahedron Lett. 1992, 33, 6445-6448.

Cell-Permeable Candidate Heterodimerizers
J. Am. Chem. Soc., Vol. 123, No. 3, 2001 403
Scheme 6a
a Reagents and conditions: (a) (-)-B-methoxydiisopinocamphenyl-
borane, allylmagnesium bromide, Et2O, 0 f 23 °C, 1 h, then 30, -78
f 23 °C, 3 h; NaOH, MeOH, and 30% H2O2, 23 °C, 72 h, 58-73%;
(b) 0.035 equiv of Pd(OAc)2, 0.13 equiv of (o-Tol)3P, 10 equiv of
methyl acrylate, Et3N, NMP, 90 f 110 °C, 4.5 h, 43%; (c), (d) see
Scheme 4 (c), (d).
Figure 4. HPLC analysis of the final library compounds. Solid-phase
reactions were monitored on a Hewlett-Packard 1100 HPLC, outfitted
with an Eclipse XDB C8 column (4.6 mm × 150 mm, 5 µm). Samples
were eluted using a gradient of 35 f 100% acetonitrile in water
containing 0.1% formic acid over 13 min (1.5 mL/min), and the UV
absorbance was monitored at 220 (shown), 240, and 254 nm.
Of the 6 µmol of the resin-bound THOX library, 5 µmol was
set aside at this stage for further library development, and 1
µmol was carried forward to generate the heterodimerizer
library.
The deprotection of the nitrobenzenesulfonyl groups is
noteworthy: under the standard conditions [thiophenol and 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) in 1-methyl-2-pyrroli-
dinone (NMP)], the 2-nitrobenzenesulfonyl group was removed
without any detectable side products.53,54 In contrast, treatment
of resin-bound library members having the 4-nitrobenzenesulfo-
nyl group with the same reagents gave mostly side products,
which arose by attack of thiophenol at aryl carbons other than
the desired sulfamido-bearing carbon.55
We speculated that it might be possible to cleave the
4-nitrobenzenesulfonyl amide bond intramolecularly by includ-
ing an additional nucleophilic center in the alkanethiol reagent.
After screening several alkanethiols, we found that ethylene 1,2-
dithiol or â-mercaptoethanol and DBU in methylene chloride
gave the desired secondary alkoxyamine as the sole reaction
product. Although we observed Michael addition products upon
treatment of the cinnamate derivatives (THOX compounds
containing R2 monomers 3g and 3h) with thiol and DBU in
NMP, subsequent â-elimination induced by DBU in NMP
regenerated the original conjugated olefin cleanly.
membered ring was found to retard the coupling reaction
significantly. Increasing the concentration of the activated acid
3-fold and allowing the reaction to proceed for 18 h circum-
vented this problem. Under these modified conditions, the
condensation was driven to completion.
Deprotection of the Fmoc group under standard conditions
(20% piperidine in DMF) liberated primary amines 34. The
amines were then coupled with AP1867 in the presence of
HATU and diisopropylethylamine to provide resin-bound het-
erodimeric compounds 35. Treatment of the resin with 1%
trifluoroacetic acid (TFA) and 5% triisopropylsilane in meth-
ylene chloride for 1 min at room temperature released the 320
compounds into solution. Reverse-phase HPLC analysis of the
final products revealed the purity of the crude products as
released from the resin to be in the range of 60-80% (Figure
4). This level of purity satisfied our criteria for direct biological
testing of the crude synthetic heterodimerizers.
Cell Permeability of the Candidate Heterodimerizers. For
synthetic heterodimerizers to be useful in biological assays with
intracellular targets, they must be cell-permeable. To assess the
cell permeability of these compounds, we employed a published
procedure.17,18,20 This assay uses cells that express two FKBP*
fusion proteins: one containing the ZFHD1 DNA-binding
domain and the other containing the p65 acidic transcriptional
activator domain (Figure 5). The small-molecule homodimerizer
AP1889 brings these two protein components together, thereby
activating transcription of the downstream encoding secreted
alkaline phosphatase (SEAP). Upon activation, the SEAP is
In the next step, the secondary alkoxyamines were condensed
with Fmoc-6-aminohexanoic acid in the presence of O-(7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate (HATU) and diisopropylethylamine to generate
Fmoc-protected amines 33.56 The presence of an aromatic ring
adjacent to the carbon bearing the nitrogen in the seven-
(52) Use of diethyl azodicarboxylate led to ethylation of the nitrogen to
the extent of 1-3%; the ethyl donor is presumably ethanol, generated by
partial decomposition of the diethyl azodicarboxylate.
(53) Wuts, P. G. M.; Northuis, J. M. Tetrahedron Lett. 1998, 39, 3889-
3890.
(54) Fukuyama, T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett. 1995, 36,
6373-6374.
(55) 1H NMR and LC-MS analysis indicates that the side products may
have the following structures:
(56) Albericio, F.; Bofill, J. M.; El-Faham, A.; Kates, S. A. J. Org. Chem.
1998, 63, 9678-9683.

404 J. Am. Chem. Soc., Vol. 123, No. 3, 2001
Koide et al.
Scheme 7a
i
i
a Reagents: (a) Trt-Cl resin, pyridine, DCM then methanol or ethanol, pyridine, CH2Cl2; (b) R2 monomers 3, Ph3P, PrO2CsNdNsCO2 Pr or
t
tBuO2CsNdNsCO2 Bu, THF; (c) Cl2Ru(PCy3)2dCHPh, CH2Cl2; (d) RSH, DBU, CH2Cl2 or NMP; (e) Fmoc-6-aminohexanoic acid, HATU, iPr2NEt,
NMP; (f) piperidine, DMF; (g) AP1867, HATU, Pr2NEt, NMP; (h) TFA, Pr3SiH, CH2Cl2. Fmoc, 9-Fluorenylmethoxycarbonyl.
i
i
Figure 5. Assay for the cell permeability of synthetic heterodimerizers.
(A) A synthetic homodimerizer, AP1889, induces the dimerization of
two mutant FKBP domains (FKBP*), one linked to a DNA binding
domain (DBD) and the other linked to a transcriptional activation
domain (AD). The induced dimerization leads to recruitment of the
basal transcriptional machinery and activation of the secreted alkaline
phosphatase (SEAP) gene. The amount of SEAP secreted into the
growth medium was determined as described (not shown).11 (B) A cell-
permeable synthetic heterodimerizer containing only one FKBP*-
targeting ligand competing with the homodimerizer for FKBP*, leading
to suppression of SEAP activation.
Figure 6. Permeability of synthetic heterodimerizers to human
fibrosarcoma cells. The vertical axis corresponds to SEAP activity in
arbitrary units. Lane 1: Induced activation of the SEAP gene by the
homodimerizer AP1889 at a concentration of 10 nM. Lanes 2-7:
Suppression by six different members of the heterodimerizer library at
100 nM in the presence of 10 nM AP1889. All assays were run in
triplicate.
secreted by the cells into the medium, where its presence can
be measured quantitatively using a fluorogenic substrate.
If a library molecule passes into the cell, it will compete with
AP1889, thereby disrupting the complex that activates SEAP
expression. Thus, loss of SEAP expression indicates that the
library member is cell-permeable. A randomly chosen subset
of the library comprising 25 heterodimerizers was screened
separately at 500 nM concentration in the presence of 10 nM
AP1889. At these high concentrations, each of the 25 com-
pounds strongly inhibited SEAP expression. None of the tested
compounds exhibited significant cytotoxicity, as determined by
visual inspection of the adherent cells. Next, six of the most
polar library members were screened in triplicate at a concentra-
tion of 100 nM in the presence of 10 nM AP1889, and again
each gave strong inhibition of SEAP expression (Figure 6).
These data provide a clear indication that the THOX-based
heterodimerizers are cell-permeable and thus suitable for use
in biological screens.
No hard and fast rules exist for selecting the variant ligand in
such a system, and in principle there exist a number of scaffolds
that would serve this purpose. We elected to use the THOX
system for its structural novelty in the cyclic form, its convert-
ibility to a linear form through reduction of the N-O bond,
and the opportunity it presents to use stereochemistry as a
diversity element. Furthermore, the presence of the olefin in
the THOX ligands should enable further functionalization, for
example through reduction, epoxidation, or dihydroxylation. In
the present work, we have described variation of both the
stereochemistry and the attached functionality at two points in
the THOX system. Straightforward elaborations of the general
strategy outlined here should permit variation of the linkage
between the variant and invariant ligand, with respect to length,
functionality, and attachment position. This would have the
beneficial effect of systematically altering the composite surface
presented by the variant component and the protein to which it
is bound (here, FKBP*).
An important criterion for any variant ligand in a hetero-
dimerization system is that it must have a tendency to produce
cell-permeable compounds when fused to the invariant ligand.
The issue of cell permeability has seldom been analyzed
systematically for components of combinatorial libraries. Here
Conclusion
Here we have described the first example of a library
constructed for the purpose of inducing the association of two
proteins, only one of which has a known small-molecule ligand.

Cell-Permeable Candidate Heterodimerizers
J. Am. Chem. Soc., Vol. 123, No. 3, 2001 405
Purification of the residue by column chromatography (10 f 20%
EtOAc in hexanes) afforded imide 5 in quantitative yield.
we have analyzed a sample of the candidate heterodimerizers,
representing a broad range of hydrophobicities, and found all
of the tested components to be freely permeable to fibrosarcoma
cells at a concentration of 100 nM. What remains to be
demonstrated is whether any of the 320 library members
reported in this study will function as heterodimerizers. Efforts
along these lines are ongoing in a variety of biological assay
systems. Several promising “hits” have already been identified
in an assay that reports the ability of the molecules to induce
nuclear export (J.M.F. and G.L.V., unpublished results).
Method B. A mixture of oxazolidone 12 (32.61 g, 184.0 mmol),
carboxylic acid (184.0 mmol), and 2-chloro-1-methylpyridinium iodide
(61.13 g, 239.2 mmol) in CH2Cl2 (350 mL) was cooled to 0 °C and
treated with triethylamine (56.4 mL, 404.8 mmol) and 4-(dimethyl-
amino)pyridine (4.50 g, 36.8 mmol). After the reaction mixture was
stirred at 23 °C for 1.5 h, hexanes (350 mL) were poured into it. The
resulting slurry was passed through a pad of silica gel, eluted with
40% EtOAc in hexanes. The eluted solution was concentrated in vacuo,
and the resulting solid was recrystallized from EtOAc-hexanes or the
resulting residue was purified by column chromatography (79-94%).
Preparation of Allyl Alcohols. Nonfunctionalized Side Chains.
A solution of imide 5 (40.00 mmol) in CH2Cl2 (80 mL) was treated
with di-n-butylboron trifluoromethanesulfonate (42.0 mL, 1.0 M in CH2-
Cl2) at 0 °C. After the solution was stirred at the same temperature for
20 min, diisopropylethylamine (7.66 mL, 44.0 mmol) was added
dropwise. After being stirred at 0 °C for 20 min, the resulting mixture
was cooled to -78 °C. Acrolein (4.81 mL, 72.0 mmol), which had
been passed through a pad of neutral alumina, was added to the solution,
and the mixture was stirred at -78 °C for 20 min, followed by 0 °C
for 20 min. The mixture was then quenched with phosphate buffer (60
mL, pH 7.0) and MeOH (300 mL), followed by 30% H2O2 (40 mL) at
0 °C. After the mixture was stirred at 0 °C for 1 h, the organic solvents
were removed in vacuo. The resulting aqueous residue was extracted
with EtOAc (200 mL × 2), and the combined organic layers were
washed with saturated aqueous NH4Cl/30% NH3 (5:1, 200 mL) and
brine (100 mL), dried over Na2SO4, filtered, and concentrated.
Purification of the residue by column chromatography (10 f 25%
EtOAc in hexanes) afforded allyl alcohols 13 (67-79%).
Functionalized Side Chains. To a solution of imide 5 (169.2 mmol)
in CH2Cl2 (178 mL) were added diisopropylethylamine (32.4 mL, 177.6
mmol) and then di-n-butylboron trifluoromethanesulfonate (177.6 mL,
1.0 M in CH2Cl2) at -78 °C. The solution was stirred at 0 °C for 30
min before being cooled to -78 °C. Acrolein (20.3 mL, 304.5 mmol)
was then slowly added to the solution at -78 °C, and the resulting
solution was stirred at the same temperature for 20 min and at -50 °C
for 20 min. The reaction was quenched by the addition of pH 7.0 buffer
(254 mL), MeOH (900 mL), and 30% H2O2 (169 mL) at -50 °C and
stirred at 0 °C for at least 1 h. The organic solvents were removed in
vacuo, and the resulting aqueous residue was extracted with EtOAc
(750 mL × 2). The same workup as above afforded allyl alcohols 13
(55-76%).
Preparation of Diols 14. A solution of allyl alcohol 13 (20.0 mmol)
in Et2O (70 mL) was treated with lithium borohydride (12.0 mL, 2.0
M in THF) at 0 °C in the air and stirred at the same temperature for 5
min. The reaction mixture was then quenched with saturated aqueous
NH4Cl (30 mL) and 30% NH3 (10 mL). After the solution was stirred
at 23 °C for 1 h, the organic solvents were removed in vacuo, and the
aqueous residue was extracted with EtOAc (100 mL × 2). The
combined organic layers were then washed with brine (100 mL × 1),
dried over Na2SO4, filtered, and concentrated. Purification of the residue
by column chromatography (0.3% MeOH and 30 f 60% EtOAc in
hexanes) afforded diol 14 (81-90%) as an oil and imide 12 (90%) as
a solid.
Preparation of Silyl Ethers 15. A solution of diol 14 (10.0 mmol)
and triethylamine (1.74 mL, 12.5 mmol) in CH2Cl2 (20 mL) was treated
with tert-butyldimethylsilyl chloride (1.73 g, 11.5 mmol) at 0 °C and
stirred at 23 °C for 8 h. The reaction mixture was then quenched with
phosphate buffer (20 mL, pH 7.0) and concentrated in vacuo to remove
the organic solvent. The resulting aqueous residue was extracted with
Et2O (100 mL × 1), and the organic layer was washed with brine (50
mL × 1), dried over Na2SO4, filtered, and concentrated. Purification
of the residue by column chromatography (4 f 8% EtOAc in hexanes)
afforded silyl ether 15 (80-89%) as an oil.
Experimental Section
General Techniques. All reactions were carried out under an argon
atmosphere with dry, freshly distilled solvents under anhydrous
conditions, unless otherwise noted. Tetrahydrofuran (THF) and diethyl
ether (Et2O) were distilled from sodium-benzophenone, and methylene
chloride (CH2Cl2) was distilled from calcium hydride. Yields refer to
chromatographically and spectroscopically (1H NMR) homogeneous
materials, unless otherwise stated.
All reactions were monitored by thin-layer chromatography carried
out on 0.25-mm E. Merck silica gel plates (60F-254) using UV light,
2.4% phosphomolybdic acid/1.4% phosphoric acid/5% sulfuric acid in
water, or 0.2% ninhydrin in ethanol and heat as developing agents.
TSI silica gel (230-400 mesh) was used for flash column chromatog-
raphy. Preparative thin-layer chromatography (PTLC) separations were
carried out on 0.50-mm E. Merck silica gel plates (60F-254).
Optical rotations were measured on a 241 polarimeter (Perkin-Elmer),
and infrared spectra were taken with a 5ZDX FT-IR spectrophotometer
(Nicolet). NMR spectra were recorded on Inova500, Inova500B,
Mercury400, or Mercury400B (Varian) or AM300 (Bruker) instruments
and calibrated using a solvent peak or tetramethylsilane as an internal
reference. The following abbreviations are used to indicate the
multiplicities: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
br, broad. High-resolution mass spectra were obtained by using an SX-
102A mass spectrometer (JEOL) using a mass resolution of 5000 for
CI+ and 10 000 for ES+/ES-.
Polystyrene-based trityl chloride resin was obtained from NovaBio-
chem (200-400 mesh). Solid-phase reactions were monitored on a
Hewlett-Packard 1100 HPLC, outfitted with an Eclipse XDB C8 column
(4.6 mm × 150 mm, 5 µm). Samples were eluted using a gradient of
35 f 100% acetonitrile in water containing 0.1% formic acid over 13
min (1.5 mL/min), and the UV absorbance was monitored at 220, 240,
and 254 nm.
LC-MS spectra were obtained using a Micromass Platform LCZ
mass spectrometer interfaced with a Waters 2690 Alliance HPLC,
outfitted with a Waters Symmetry C18 column (2.1 mm × 50 mm, 3.5
µm). Samples were eluted using a gradient of 15 f 100% acetonitrile
in water containing 0.1% formic acid over 10 min, and the UV
absorbance, positive APCI, and negative APCI were collected.
Abbreviations. Ac, acetyl; Bn, benzyl; Bu, butyl; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; DMF, dimethylforamide; Et, ethyl;
Et2O, ethyl ether; EtOAc, ethyl acetate; EtOH, ethanol; Fmoc, 9-fluo-
renylmethoxycarbonyl; Me, methyl; MeOH, methanol; NMP, 1-methyl-
2-pyrrolidinone; 2Ns, 2-nitrobenzenesulfonyl; 4Ns, 4-nitrobenzenesulfo-
nyl; Ph, phenyl; Phth, phthaloyl; TBS, tert-butyldimethylsilyl; TFA,
trifluoroacetic acid; Tf, trifluoromethanesulfonyl; THF, tetrahydrofuran;
Trt, triphenylmethyl.
(A) Preparation of Nitrobenzenesulfonyl Alkoxyamides 18a-c,
24a-c, and 29a-d (R1 Monomers). Preparation of Imide 5. Method
A. A solution of oxazolidone 12 (7.09 g, 40.0 mmol) in THF (80 mL)
was cooled to -78 °C and treated with n-butyllithium (26.3 mL, 1.6
M in hexanes). After the solution was stirred at the same temperature
for 10 min, an acid chloride (46.0 mmol) was added, and the resulting
solution was warmed to 23 °C over 30 min. The reaction mixture was
then quenched with saturated aqueous NaHCO3 (80 mL) and concen-
trated in vacuo to remove the organic solvents. The resulting aqueous
residue was extracted with Et2O (200 mL × 2), and the combined
organic layers were washed with water (100 mL × 1) and brine (100
mL × 1), and then dried over Na2SO4, filtered, and concentrated.
Preparation of Trityl Ethers 19 and 25. A solution of diol 14 (25.6
g, 96.9 mmol) and pyridine (10.2 mL, 126 mmol) in CH2Cl2 (200 mL)
was treated with trityl chloride (30.3 g, 107 mmol) at 0 °C and stirred
at the same temperature for 2 h. The reaction mixture was then quenched
with EtOH (2 mL) and poured into Et2O/EtOAc (800 mL/400 mL).
The resulting mixture was washed with aqueous KHSO4 (0.3 M, 100

406 J. Am. Chem. Soc., Vol. 123, No. 3, 2001
Koide et al.
mL × 1), water (70 mL × 1), and brine (70 mL × 1). The organic
layer was dried over Na2SO4, filtered, and concentrated. Purification
of the residue by column chromatography (8 f 20% EtOAc in hexanes)
afforded the trityl ether (88-91%) as an oil.
Na2SO4, filtered, and concentrated. Purification of the residue by column
chromatography (1% triethylamine and 10% EtOAc in hexanes)
afforded methylenedioxyphenol ether 27c (1.58 g, 50%) as an oil.
Preparation of N-Methylsulfonamide 27d. To N-methyl p-tolu-
lenesulfonamide (18.97 mmol) was added potassium tert-butoxide in
THF (1.0 M, 15.2 mL) at 0 °C, and the resulting solution was stirred
at 23 °C for 30 min. A solution of alkyl bromide 26 (7.97 mmol) and
4-(dimethylamino)pyridine in DMF (10 mL) and THF (5 mL) was
added to the solution at 23 °C, and the mixture was stirred at the same
temperature for 16 h. The organic solvent was removed in vacuo, and
the resulting solution was extracted with EtOAc (150 mL × 2). The
combined organic layers were washed with brine (150 mL), dried over
MgSO4, filtered, and concentrated. Purification of the residue by column
chromatography (10 f 30% EtOAc in hexanes) afforded N-methyl-
sulfonamide 27d (5.76 g, quantitative) as an oil.
Preparation of Benzoic Acid 22. A solution of ethyl ester 19 (19.5
g, 38.5 mmol) in THF/MeOH (270 mL/180 mL) was treated with
aqueous LiOH (77 mL, 2.0 M) and aqueous H2O2 (18 mL, 30%) at 0
°C and stirred at 46 °C for 12 h. After the reaction solution was cooled
to 0 °C, aqueous sodium sulfite was added to the solution until a KI
test was negative. The organic solvents were evaporated in vacuo, and
EtOAc (400 mL) and NaH2PO4 (250 mL, 1.0 M) were added to the
resulting aqueous mixture. After the layers were separated, the aqueous
layer was extracted with EtOAc (200 mL × 1). The combined organic
layers were washed with water (100 mL × 1) and brine (100 mL ×
1), dried over Na2SO4, filtered, and concentrated. Purification of the
residue by column chromatography (70% EtOAc in hexanes) afforded
benzoic acid 22 (18.0 g, 98%) as a colorless oil.
Preparation of Benzamides 23b,c. To the mixture of benzoic acid
22 (4.89 g, 10.2 mmol) and 2-chloro-1-methylpyridinium iodide (3.92
g, 15.3 mmol) in CH2Cl2 (26 mL) was added diisopropylethylamine
(5.34 mL, 30.7 mmol) at 0 °C, and the resulting mixture was stirred at
23 °C for 15 min. The mixture was then cooled to 0 °C and treated
with diethyl iminodiacetate (5.00 mL, 27.9 mmol) or methylamine (6.13
mL, 8.0 M in EtOH). The resulting mixture was stirred at 23 °C for 15
min and then poured into EtOAc (150 mL). The organic layer was
washed with water (40 mL × 1), aqueous NaH2PO4 (40 mL, 1.0 M),
and brine (40 mL × 1), dried over Na2SO4, filtered, and concentrated.
Purification of the residue by column chromatography (30 f 40%
EtOAc in hexanes for 23c and 55 f 60% EtOAc in hexanes for 23b)
afforded 23c (quantitative) as an oil or 23b (58%) as a white powder.
Preparation of N-Alkoxyphthalimides 16, 20a-c, and 26. A
solution of the allyl alcohol (1.00 mmol), N-hydroxyphthalimide (196
mg, 1.20 mmol), and triphenylphosphine (315 mg, 1.20 mmol) in THF
(6.0 mL) was treated with diethyl azodicarboxylate (189 µL, 1.20 mmol)
at 0 °C and warmed to 23 °C. After the solution was stirred at the
same temperature for 45 min, the solvent was removed in vacuo and
the residue passed through a plug of silica gel, eluted with 20% EtOAc
in hexanes. The eluant was concentrated and the residue purified by
column chromatography (EtOAc in hexanes) to afford the N-alkoxy-
phthalimide (67-97%).
Preparation of Azide 27a. A solution of alkyl bromide 26 (6.38
mmol) in DMF (15 mL) was treated with sodium azide (31.9 mmol)
and tetrabutylammonium iodide (3.21 mmol), and the resulting mixture
was stirred at 23 °C for 12 h. EtOAc (250 mL) was poured into the
solution, and the mixture was washed with water (200 mL). The aqueous
layer was extracted with EtOAc (200 mL), and the combined organic
layers were washed with brine (250 mL), dried over Na2SO4, filtered,
and concentrated. Purification of the residue by column chromatography
(20% EtOAc in hexanes) afforded azide 27a (3.32 g, 91%) as an oil.
Preparation of Malonate 27b. A solution of sodium hydride (60
wt %, 40.3 mmol) in DMF (10 mL) was treated with diethyl malonate
(50.1 mmol) at 0 °C and stirred at 23 °C for 30 min. A solution of
alkyl bromide 26 (9.35 mmol) and tetrabutylammonium iodide (4.74
mmol) in DMF (10 mL) was then added at 23 °C, and the mixture was
stirred at the same temperature for 12 h. EtOAc (200 mL) was then
added, and the mixture was washed with brine (100 mL). The aqueous
layer was extracted with EtOAc (200 mL), and the combined organic
extracts were washed with aqueous brine (3 × 300 mL), dried over
Na2SO4, filtered, and concentrated. Purification of the residue by column
chromatography (20 f 30% EtOAc in hexanes) afforded malonate 27b
(7.65 g, crude) as an oil. A portion of the material was purified by
PTLC for further characterization. After deprotection of the phthalimide
and reprotection with 2-nitrobenzensulfonyl, the isolated yield was 53%
for three steps.
Preparation of N-Alkoxysulfonamides 17, 21a-c, and 28a-d. A
solution of the N-alkoxyphthalimide (1.00 mmol) in CH2Cl2 (4.0 mL)
was treated with methylamine (0.38 mL, 8.0 M in EtOH) and stirred
at 23 °C for 15 min in the air. The solvents were then evaporated in
vacuo, and the residue was azeotroped with benzene 3 times to remove
methylamine. The residue was dissolved in CH2Cl2 (4.0 mL), and 2,4,6-
collidine (172 µL, 1.30 mmol) was added to the solution. The resulting
mixture was cooled to 0 °C and treated with 2- or 4-nitrobenzene-
sulfonyl chloride (266 mg, 1.20 mmol). After being stirred at the same
temperature for 15 min, the reaction mixture was quenched with
diethylamine (10 µL, 0.1 mmol) and then poured into EtOAc (20 mL).
The organic layer was washed with aqueous KHSO4 (10 mL, 0.1 M)
and then brine, dried over Na2SO4, filtered, and concentrated. Purifica-
tion of the residue by column chromatography (EtOAc in hexanes)
afforded the N-alkoxysulfonamide (26-93% and 98% (crude)).
Preparation of Alcohols 18. A solution of N-alkoxysulfonamide
17 (1.00 mmol) in THF (5.0 mL) in a polypropylene vessel was treated
with HF-pyridine (0.6 mL) at 0 °C and stirred at 23 °C for 10 h. The
reaction mixture was diluted with Et2O (5 mL) and quenched with
saturated NaHCO3 solution. After removal of the organic solvents in
vacuo, the resulting aqueous residue was extracted with EtOAc (50
mL × 2). The combined organic layers were washed with aqueous
KHSO4 (50 mL, 0.1 M), water, and brine and then dried over Na2SO4,
filtered, and concentrated. Purification of the residue by column
chromatography (0.2% MeOH and 20 f 30% EtOAc in hexanes)
afforded primary alcohol 18 (65-73%).
(2S,3S)-18c: Rf ) 0.23 (30% EtOAc in hexanes); [R]24 -102.5°
D
(c ) 1.0, benzene); IR (thin film) λmax ) 3500 (br, NH), 3220 (br,
OH), 1532, 1349, 1172 cm-1; 1H NMR (500 MHz, 1% MeOD in C6D6,
23 °C) δ ) 7.88 (ddd, 2H, J ) 9.0, 2.0, 2.0 Hz, SO2Ar), 7.73 (ddd,
2H, J ) 9.0, 2.0, 2.0 Hz, SO2Ar), 7.18 (dd, 2H, J ) 7.2, 7.0 Hz, Ph
meta), 7.14-7.11 (m, 2H, Ph ortho), 7.04 (t, 1H, J ) 7.2 Hz, Ph para),
5.74 (ddd, 1H, J ) 17.5, 10.2, 7.7 Hz, sCHdCH2), 5.26 (dd, 1H, J )
17.5, 1.5 Hz, sCHdCH2 cis), 5.19 (dd, 1H, J ) 10.2, 1.5 Hz, sCHd
CH2 trans), 4.71 (dd, 1H, J ) 7.7, 7.5 Hz, sCHONHSO2Ar), 3.66
(dd, 1H, J ) 11.5, 4.0 Hz, sCH2OH), 3.47 (dd, 1H, J ) 11.5, 5.5 Hz,
sCH2OH), 2.74 (dd, 1H, J ) 13.5, 4.5 Hz, sCH2Ph), 2.55 (dd, 1H, J
) 13.5, 10.0 Hz, sCH2Ph), 2.03 (m, 1H, sCHCH2OH); 13C NMR
(125 MHz, C6D6, 23 °C) δ ) 150.5, 142.4, 140.1, 134.7, 129.4, 129.3,
128.7, 128.2, 126.5, 124.0, 121.2, 89.2, 59.8, 46.1, 33.2, one peak was
obscured by benzene; ES- HRMS calcd for C18H19N2O6S (M - H+)
391.0964, found 391.0948.
Preparation of Alcohols 24a-c and 29a-c. A solution of the
N-alkoxysulfonamide (1.00 mmol) in Et2O (2.5 mL) was treated with
formic acid (2.5 mL) and either ethanethiol (1.50 mmol) or mercap-
toacetic acid (2.00 mmol) at 23 °C. After the solution was stirred at
the same temperature for 2 h, the organic solvents were removed in
vacuo. Purification of the residue by column chromatography (0.3%
MeOH, 30 f 60% EtOAc in hexanes) afforded the primary alcohols
(29-82%).
Preparation of Methylenedioxyphenol Ether 27c. After a mixture
of 3,4-methylenedioxyphenol (9.83 mmol) and cesium carbonate (7.30
mmol) in DMF (5 mL) was stirred at 23 °C for 1 h, a solution of alkyl
bromide 26 (4.78 mmol) in DMF (5 mL) was added to the solution at
23 °C and stirred at the same temperature for 12 h. EtOAc (250 mL)
was then added, and the organic layer was washed with brine (250 mL
× 2) and aqueous potassium carbonate (0.1 M, 100 mL), dried over
(2S,3S)-24a: Rf ) 0.33 (60% EtOAc in hexanes); [R]24D +81.3° (c
) 1.0, benzene); IR (thin film) λmax ) 3550 (br, NH), 3270 (br, OH),
1713 (CdO), 1542, 1366, 1280, 1179 cm-1; 1H NMR (500 MHz, 1%
MeOD in C6D6, 23 °C) δ ) 8.09 (d, 2H, J ) 8.1 Hz, Ar ortho to
ester), 7.97 (d, 1H, J ) 8.0 Hz, -SO2Ar), 7.06 (d, 2H, J ) 8.1 Hz, Ar
meta to ester), 6.88 (d, 1H, J ) 8.0 Hz, -SO2Ar), 6.72 (dd, 1H, J )

Cell-Permeable Candidate Heterodimerizers
J. Am. Chem. Soc., Vol. 123, No. 3, 2001 407
8.0, 8.0 Hz, -SO2Ar), 6.54 (dd, 1H, J ) 8.0, 8.0 Hz, -SO2Ar), 5.55
(ddd, 1H, J ) 17.5, 10.0, 8.0 Hz, sCHdCH2), 5.17 (d, 1H, J ) 17.5
Hz, sCHdCH2 cis), 5.12 (d, 1H, J ) 10.0 Hz, sCHdCH2 trans),
4.63 (dd, 1H, J ) 8.0, 8.0 Hz, sCHONHSO2Ar), 4.12 (q, 2H, J ) 7.5
Hz, sOCH2CH3), 3.57 (dd, 1H, J ) 11.5, 3.5 Hz, sCH2OH), 3.32
(dd, 1H, J ) 11.5, 4.5 Hz, sCH2OH), 2.54 (dd, 1H, J ) 13.7, 4.5 Hz,
sCH2Ar), 2.48 (dd, 1H, J ) 13.7, 10.0 Hz, sCH2Ar), 1.85 (m, 1H,
sCHCH2OH), 1.02 (t, 3H, J ) 7.5 Hz, sOCH2CH3); 13C NMR (125
MHz, C6D6, 23 °C) δ ) 166.2, 148.4, 145.7, 144.5, 134.4, 134.3, 132.7,
132.2, 130.5, 130.0, 129.8, 129.5, 127.0, 125.3, 121.6, 89.1, 60.7, 59.6,
45.8, 33.2, 14.2; ES+ HRMS calcd for C21H24N2O8SNa (M + Na+)
487.1151, found 487.1159.
(S)-3g: Rf ) 0.15 (20% EtOAc in hexanes); [R]25 -11.3° (c )
D
1.0, benzene); IR (thin film) λmax ) 3500 (br, OH), 1704 (CdO), 1638,
1
1322, 1195, 1175 cm-1; H NMR (400 MHz, 1% MeOD in C6D6, 23
°C) δ ) 7.75 (d, 1H, J ) 16.0 Hz, sCHdCHCO2CH3), 7.37 (s, 1H,
Ar ortho sCHdCHCO2CH3 and sCHOH), 7.21 (d, 1H, J ) 7.2 Hz,
Ar para sCHOH), 7.06-6.99 (m, 2H, J ) 7.2 Hz, Ar meta and para
sCHdCHCO2CH3), 6.43 (d, 1H, J ) 16.0 Hz, sCHdCHCO2CH3),
5.75 (m, 1H, sCHdCH2), 5.01-4.95 (m, 2H, sCHdCH2), 4.54 (dd,
1H, J ) 5.6, 1.6 Hz, CHOH), 3.86 (s, 3H, sCHdCHCO2CH3), 2.47-
2.31 (m, 2H, sCH2CHdCH2); 13C NMR (100 MHz, C6D6, 23 °C) δ
) 166.9, 145.5, 144.9, 134.7, 134.7, 128.9, 127.1, 126.0, 118.4, 118.0,
73.1, 51.4, 44.3, one peak was obscured by benzene; CI+ HRMS calcd
for C14H20NO3 (M + NH4+) 250.1443, found 250.1431.
(C) Solid-Phase Library Synthesis. Preparation of Trityl Ethers
4. To the polystyrene trityl chloride resin (4.00 g, 8.10 mmol) in CH2-
Cl2 (8.0 mL) in a polypropylene tube was added a solution of the
primary alcohol (1.00 mmol) and pyridine (1.97 mL, 24.3 mmol) in
CH2Cl2 (14.0 mL) at 0 °C. After the reaction vessel was shaken at 23
°C for 24 h, the resin was washed with DMF (20 mL × 2) and CH2Cl2
(20 mL × 3) before it was dried in vacuo. The completion of the
reaction was confirmed by monitoring the solution by thin-layer
chromatography and noting the absence of the starting alcohol. The
resin was then treated with a solution of MeOH (1.4 mL) or EtOH
(1.4 mL) and pyridine (1.97 mL, 24.3 mmol) in CH2Cl2 (14.0 mL) at
23 °C for 24 h. The resin was washed with DMF (20 mL × 2) and
CH2Cl2 (20 mL × 3) and dried in vacuo. The resulting loading level
was estimated to be 0.20 mmol/g (144 mg of the resin was treated
with the cleaving solution and 10.3 mg of (2S,3R)-24b was isolated,
which indicates that the actual loading level for this compound was
approximately 0.16 mmol/g).
Preparation of the Acetamide. To resin-bound azide 29a (508 mg,
0.13 mmol) was added thiolacetic acid (4.0 mL). After the reaction
vessel was shaken at 23 °C for 1 h, the resin was washed with DMF
(5 mL × 3) and CH2Cl2 (5 mL × 3) before it was dried in vacuo.
LC-MS analysis confirmed the presence of the desired product in high
purity (expected mass 401.1, experimental (m/z)+ ) 402.1 for M +
H+).
Splitting the Resin. A slurry of the resin was prepared in 40% CH2-
Cl2 in NMP, and a portion of the mixture was distributed into each
reaction vessel. As a result, each contained approximately 30 mg (6.0
µmol) of the resin.
(2S,3S)-29a: Rf ) 0.30 (50% EtOAc in hexanes); [R]24D +94.4° (c
) 1.5, benzene); IR (thin film) λmax ) 3550 (br, NH), 3260 (br, OH),
1
2099 (N3), 1722, 1542, 1392, 1360, 1178 cm-1; H NMR (500 MHz,
1% MeOD in C6D6, 23 °C) δ ) 8.01 (dd, 1H, J ) 7.7, 1.0 Hz, -SO2-
Ar), 6.84 (dd, 1H, J ) 7.7, 1.0 Hz, -SO2Ar), 6.77 (ddd, 1H, J ) 7.7,
7.7, 1.0 Hz, -SO2Ar), 6.52 (ddd, 1H, J ) 7.5, 7.7, 1.0 Hz, -SO2Ar),
5.45 (ddd, 1H, J ) 17.2, 10.0, 8.2 Hz, sCHdCH2), 5.13 (dd, 1H, J )
17.2, 1.0 Hz, sCHdCH2 cis), 5.08 (dd, 1H, J ) 10.0, 1.0 Hz, sCHd
CH2 trans), 4.50 (dd, 1H, J ) 8.2 Hz, sCHONHSO2Ar), 3.59 (dd,
1H, J ) 11.5, 4.5 Hz, sCH2OH), 3.40 (dd, 1H, J ) 11.5, 5.0 Hz,
sCH2OH), 2.66-2.62 (m, 2H, sCH2N3), 1.43 (br m, 1H, sCHCH2-
OH), 1.32-1.23 and 1.21-1.12 (m, 4H, sCH2CH2CH2N3); 13C NMR
(125 MHz, C6D6, 23 °C) δ ) 134.5, 134.3, 132.8, 132.2, 128.5, 125.1,
120.8, 89.1, 61.0, 51.3, 43.4, 41.4, 26.5, 24.1; ES+ HRMS calcd for
C14H19N5O6SNa (M + Na+) 408.0945, found 408.0962.
(B) Preparation of Homoallyl Alcohols 3 (R2 Monomers). To a
solution of (+)-B-methoxydiisopinocamphenylborane (21.17 g, 66.9
mmol) in Et2O (45 mL) was added a solution of allylmagnesium
bromide (60.8 mL, 1 M in Et2O) dropwise at 0 °C, and the flask was
then allowed to warm to 23 °C over 1 h. The mixture was cooled to
-78 °C and treated with methyl 4-formylbenzoate (9.99 g, 60.8 mmol).
The reaction mixture was allowed to warm to 23 °C over 3 h, at which
time the solvents were removed in vacuo. A 1:1 mixture of aqueous
NaOH (3.0 M) and MeOH (150 mL) was added to the flask at 0 °C,
30% H2O2 (20 mL) was then added dropwise, and the mixture was
stirred at 23 °C for 72 h. The MeOH was then removed in vacuo, and
additional H2O (100 mL) and aqueous NaOH (50 mL, 3.0 M) were
added to the flask. The mixture was washed with EtOAc (50 mL × 2),
and the aqueous extracts were acidified to pH 2 with concentrated HCl
and extracted with EtOAc (100 mL × 2). The combined organic layers
were then washed with brine (70 mL × 2) and concentrated to give
the homoallyl alcohol (58-73%).
Preparation of Dienes 2. (i) For Homoallyl Alcohols R2 ) 3a-c.
To the solution of di-tert-butyl azodicarboxylate (737 mg, 3.20 mmol)
and Ph3P (800 mg, 3.05 mmol) in THF (2.90 mL) at -10 °C was added
an aliphatic alcohol (2.31 mmol) in THF (2.90 mL). After being stirred
at the same temperature for 1 min, 240 µL of the solution was added
to each starting material (20 reactions for each R2 monomer). The
resulting mixture was shaken at 23 °C for 30 min, and the resin was
washed with DMF (2 mL × 2), CH2Cl2 (2 mL × 2), and THF (2 mL
× 1) and dried in vacuo.
The subsequent ester hydrolysis and amide formation for 3f was
accomplished by the same procedure as for ethyl ester 19.
(S)-3f: Rf ) 0.53 (100% Et2O); [R]25 -12.9° (c ) 1.0, benzene);
D
IR (thin film) λmax ) 3500 (br, OH), 1744 (CdO), 1642, 1209, 1191
1
cm-1; H NMR (500 MHz, 1% MeOD in C6D6, 23 °C) δ ) 7.48 (d,
1H, J ) 7.7 Hz, Ar ortho to amide), 7.05 (d, 1H, J ) 7.7 Hz, Ar meta
to amide), 5.61 (ddd, 1H, J ) 18.0, 7.5, 7.5 Hz, sCHdCH2), 4.92-
4.88 (m, 2H, sCHdCH2 cis and trans), 4.31-4.28 (m, 3H, sCHOH
and sNCH2CO2Et), 3.98 (s, 2H, sNCH2CO2Et), 3.89-3.76 (m, 4H,
sOCH2CH3), 2.25-2.15 (m, 2H, sCH2CHdCH2), 0.88-0.77 (m, 6H,
sOCH2CH3); 13C NMR (125 MHz, C6D6, 23 °C) δ ) 172.1, 171.4,
169.2, 169.0, 147.2, 134.8, 134.1, 127.3, 126.1, 117.6, 72.8, 61.1, 61.0,
60.3, 51.8, 50.0, 47.9, 44.0, 13.9; ES+ HRMS calcd for C19H25NO6Na
(M + Na+) 386.1580, found 386.1565.
Preparation of Cinnamate Esters. To a solution of 3i (7.36 g, 32.4
mmol), palladium(II) acetate (0.18 g, 0.82 mmol), and triorthotolyl
phosphine (0.74 g, 2.42 mmol) in degassed NMP (50 mL) were added
degassed methyl acrylate (29.2 mL, 323.9 mmol) and triethylamine
(6.70 mL, 48.1 mmol). After the mixture was heated to 90 °C for 1.5
h, and then to 110 °C for 2 h, palladium(II) acetate (0.084 g, 0.37
mmol) and triorthotolyl phosphine (0.55 g, 1.82 mmol) were added,
and the suspension was stirred at 110 °C for 1 h. Et2O (400 mL) and
water (250 mL) were poured into the reaction mixture, and the layers
were separated. The organic layer was washed with brine (250 mL),
dried with MgSO4, and concentrated. Purification of the residue by
column chromatography (15 f 20% EtOAc in hexanes) afforded
cinnamate 3g (3.25 g, 43%) as an oil.
(ii) For Benzylic Homoallyl Alcohols R2 ) 3d-h. To the solution
of a benzylic alcohol (1.94 mmol) and Ph3P (560 mg, 2.13 mmol) in
THF (6.93 mL) at -50 °C was added diisopropyl azodicarboxylate
(482 µL, 2.33 mmol). After the solution was stirred at the same
temperature for 1 min, a 300-µL aliquot was added to each starting
material (20 reactions for each R2 monomer). The resulting mixture
was shaken at 23 °C for 20 min, and the resin was washed with DMF
(2 mL × 2), CH2Cl2 (2 mL × 2), and THF (2 mL × 1) and dried in
vacuo.
Preparation of Tetrahydrooxazepines 31. (i) For R1 ) 18a-c,
R2 ) 3a-d. A solution of bis(tricyclohexylphosphine)benzylidene
ruthenium(IV) dichloride (182 mg, 222 µmol) in degassed CH2Cl2 (26.4
mL) was prepared, and 0.5 mL of the solution was added to each
reaction vessel (48 reactions) at 23 °C. The sealed vessels were shaken
at 23 °C for 1 h before the resin was washed with DMF (2 mL × 2),
CH2Cl2 (2 mL × 2), and THF (2 mL × 1) and dried in vacuo.
(ii) For R1 ) 18a-c, R2 ) 3e-h and R1 ) 24a-c, R2 ) 3a-h
and R1 ) 29a-d, R2 ) 3a-h. A solution of bis(tricyclohexylphos-
phine)benzylidene ruthenium(IV) dichloride (4.29 g, 5.21 mmol) in
degassed CH2Cl2 (145 mL) was prepared, and 0.5 mL of the solution
was added to each reaction vessel (272 reactions) at 23 °C. The sealed

408 J. Am. Chem. Soc., Vol. 123, No. 3, 2001
Koide et al.
vessels were shaken at 23 °C for 1 h, and the resin was washed with
DMF (2 mL × 2), CH2Cl2 (2 mL × 2), and THF (2 mL × 1) before
it was dried in vacuo.
After this step, 5 mg (ca. 1.0 µmol) of the resin from each well was
transferred to another 96-well plate, and the compound was released
from solid support by 1% TFA/5% triisopropylsilane in CH2Cl2 at 23
°C for 1 min.
Synthesis of Amides 35. To a mixture of AP1867 (749 mg, 1.08
mmol) and O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflu-
orophosphate (493 mg, 1.30 mmol) in CH2Cl2 (10.1 mL) and NMP
(15.4 mL) was added diisopropylethylamine (0.45 mL, 2.59 mmol) at
0 °C. After the solution was stirred at 23 °C for 5 min, a 70-µL aliquot
was added to each well. The reaction vessels were shaken at 23 °C for
3 h, and the resin was washed with CH2Cl2 (0.2 mL × 3), DMF (0.2
mL × 3), CH2Cl2 (0.2 mL × 3), and THF (0.2 mL × 1) and dried in
Vacuo.
Preparation of Alkoxyamines 32. For the remaining steps, each
well contained approximately 5 mg of the starting material resin.
(i) For R1 ) 18a-c, R2 ) 3a-f (72 Compounds). A solution of
ethanedithiol (0.50 mL, 6.0 mmol) and DBU (0.45 mL, 3.0 mmol) in
CH2Cl2 (12.0 mL) was prepared at -78 °C, 130 µL of the solution
was added to each vessel, and the resulting mixture was shaken at 23
°C for 10 min before the resin was washed with DMF (2 mL × 2) and
CH2Cl2 (2 mL × 2). The same procedure was repeated three times,
and then the resin was dried in vacuo.
(ii) For R1 ) 18a-c, R2 ) 3g,h (24 Compounds). A solution of
â-mercaptoethanol (210 µL, 3.0 mmol) and DBU (225 µL, 1.5 mmol)
in CH2Cl2 (6.0 mL) was prepared at -78 °C, 195 µL of the solution
was added to each vessel, and the resulting mixture was shaken at 23
°C for 10 min before the resin was washed with DMF (2 mL × 2) and
CH2Cl2 (2 mL × 2). The same procedure was repeated three times. A
solution of DBU in NMP (0.50 mL, 0.25 M) was then added to the
resin, and the resulting mixture was shaken for 20 min before the resin
was washed with NMP (0.5 mL × 1). The same procedure was repeated
until Michael adducts were not detected by HPLC analysis (three times).
The resin was then washed with DMF (2 mL × 2) and CH2Cl2 (2 mL
× 2) and dried in vacuo.
Synthesis of Primary Alcohols 36. A solution of 1% TFA and 5%
triisopropylsilane in CH2Cl2 (0.18 mL) was passed through the resin
three times at 23 °C for 1 min each. The eluted solution was collected
in 96-well plates, and the organic solvents were evaporated under the
stream of nitrogen gas. The residue was then dried in vacuo.
(D) Determination of the Permeability of the Heterodimeric
Compounds to Mammalian Cells. Cell Lines and Tissue Culture.
The human HT1080 fibrosarcoma cell line 41-5 was obtained from
ARIAD Gene Therapeutics, Inc. The 41-5 cell line contains a
retrovirally integrated Z12-IL2-SEAP reporter and is stably transfected
with the CMV-driven transcription factor expression construct pCEN-
Fv3p65/Z1Fv3/Neo.20 The cells were propagated in minimal essential
medium (GIBCO) containing 10% (vol/vol) fetal calf serum, non-
essential amino acids, penicillin/streptomycin, and Geneticin.
Assay. HT1080 cells plated in a 96-well dish (1 × 104 cells/well)
in 200 µL of medium were treated with both AP1889 (final concentra-
tion, 10 nM) and a library compound (final concentration, 100 nM).
After incubation for 21 h, the cells were viable, and 100 µL of the
medium from each well was transferred to another 96-well dish. The
transferred medium was then incubated at 67 °C for 1.5 h and cooled
to 23 °C. A 100-µL aliquot of 4-methylumbelliferyl phosphate solution
(1.2 mM, pH 10.0) was added to each well, and the resulting mixture
was incubated at 37 °C for 8 h. The fluorescence intensity was recorded
using a Titertek Fluoroskan II (ICN) with an excitation wavelength of
355 nm and an emission wavelength of 460 nm.11
(iii) For R1 ) 24a-c and 29a-d, R2 ) 3a-h (224 Compounds).
A solution of thiophenol (2.78 mL, 27.0 mmol) and DBU (2.03 mL,
13.5 mmol) in NMP (54 mL) was prepared at 0 °C, 195 µL of the
solution was added to each vessel, and the mixture was shaken at 23
°C for 30 min. The resin was then washed with NMP (0.5 mL × 1),
and the same procedure was repeated twice. The resin was then washed
with DMF (0.2 mL × 2) and CH2Cl2 (0.2 mL × 2) and dried in vacuo.
Preparation of Amides 33. (i) For R1 ) 18a-c, 24a-c, and 29a-
d, R2 ) 3a-c (120 Compounds). Fmoc-6-aminohexanoic acid (915
mg, 2.59 mmol) and O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-
uronium hexafluorophosphate (1.08 g, 2.85 mmol) were dissolved in
NMP (21.6 mL), and diisopropylethylamine (0.99 mL, 5.70 mmol) was
added to the solution at 0 °C. A 70-µL aliquot of the resulting solution
was added to each well, and the mixture was shaken at 23 °C for 1 h.
The resin was then washed with DMF (0.2 mL × 2), CH2Cl2 (0.2 mL
× 2), and THF (0.2 mL × 1) and dried in vacuo.
Acknowledgment. We are grateful to Dr. Andrew Tyler,
Ms. Jennifer Lynch, Dr. Reza Fathi, and Mr. Michael Reitman
for assistance with mass spectrometry. We thank Dr. Michael
Gilman, Dr. Roy Pollock, Dr. Leonard Rozamus, and the
scientists at ARIAD pharmaceuticals for providing us with
human fibrosarcoma cells, an original sample of AP1889,
synthetic protocols for AP1867, and helpful discussions. K.K.
is the Merck Postdoctoral Fellow of the Cancer Research Fund
of the Damon Runyon-Walter Winchell Foundation (DRG-
1493). J.M.F. is the recipient of a National Research Service
Award from the NIH (5T32 GMO7598-22). K.K. and J.M.F.
contributed equally to this work. This research was supported
by the Harvard Institute of Chemistry and Cell Biology, the
NIH (No. PO1 CA78048), and Merck and Company, Inc.
(ii) For R1 ) 18a-c, 24a-c, and 29a-d, R2 ) 3d-h (200
Compounds). Fmoc-6-aminohexanoic acid (2.33 g, 6.60 mmol) and
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate (2.64 g, 6.93 mmol) were dissolved in NMP (9.2 mL) and CH2-
Cl2 (10.0 mL), and diisopropylethylamine (2.3 mL, 13.2 mmol) was
added to the solution at 0 °C. A 91-µL aliquot of the resulting solution
was added to each well, and the mixture was shaken at 23 °C for 18
h. The resin was then washed with DMF (0.2 mL × 2), CH2Cl2 (0.2
mL × 2), and THF (0.2 mL × 1) and dried in vacuo.
Supporting Information Available: Spectral characteriza-
tion data for all intermediates produced by solution-phase
synthesis and LC-MS traces of 40 of the final heterodimeric
compounds (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
Preparation of Primary Amines 34. A 150-µL aliquot of piperidine
in DMF (150 µL, 20% v/v) was added to each well at 23 °C and drained
slowly at the same temperature over 5 min. The same procedure was
repeated three times, and the resin was then washed with DMF (0.2
mL × 3), CH2Cl2 (0.2 mL × 3), and toluene (0.2 mL × 1) and dried
in vacuo.
JA0023377