A R T I C L E S
Carlson et al.
reduced pressure. Flash chromatography on a silica gel column (3 ×
10 cm), eluting with 1% MeOH/CHCl3 (200 mL) and 3% MeOH/CHCl3
(500 mL), gave the desired product (1.02 g, 1.35 mmol, 89.2%) as a
the further analysis of the substantial stability generated by these
minimal building blocks. Second, optimization of chemical
inducers of protein dimerization or other polyvalent ligands must
account for the apparent ability of the molecular structure to
stabilize conformational states that reduce the accessibility of
the ligand to the target binding sites. The frequency of
methotrexate-like aromatic or heteroaromatic functionality in
drug molecules suggests that a folding propensity may appear
in dimeric ligands based on other pharmacophores. If so, this
sharpens the design challenge of balancing the conformationally
accommodating characteristics of flexibility against the entropic
and folding-inhibition benefits of rigidity.
Finally, although the value of Keq for bis-MTX is quite large,
a value even 100-fold smaller would still exert a substantial
influence on the binding profile of a biological or synthetic
dimerizer. This is particularly true when the kinetic aspects of
dimerization and signaling are taken into account. A discrete
conformational equilibrium introduces a separation between
binding thermodynamics and kinetics: while the concentration
dependence of ligand binding is governed by the effective Ka1
(Ka1/Keq), the off rate will depend on Ka1 itself. In the case of
cell surface receptors, the duration of binding determines the
effective area a monomeric ligand-receptor complex can search
for a dimerization partner and therefore the probability of
initiating a signal.46 This phenomenon may be significant in
regulating cell-surface receptor dimerization in vivo. Investiga-
tions into conformational regulation by biological ligands, the
molecular basis for bis-MTX folding, and the implementation
of this system in the study of other inducible dimerization
processes are ongoing.
1
flaky colorless solid. TLC Rf ) 0.64 CHCl3/MeOH (9:1). H NMR
(CDCl3) δ 1.25 (s, 16 H), 1.45 (s, 4 H), 1.95 (m, 2 H), 2.23 (m, 6 H),
3.06 (d, 4 H, J ) 7 Hz), 3.73 (s, 6 H), 4.90 (q, 2 H, J ) 6, 8 Hz), 5.10
(s, 4 H), 5.80 (d, 2 H), 6.00 (bt, 2 H), 7.34 (m, 10 H).
4-[N-Methyl-N-(trifluoroacetyl)amino]benzoic Acid (2). 2 was
synthesized as described by Rosowsky et al.47 Yield (56.3%). Mp 175-
176 °C. TLC Rf ) 0.29 Hex/EtOAc (1:3). 1H NMR (MeOH-d4) δ 3.29
(s, 3 H), 7.45 (d, 2 H, J ) 9 Hz), 8.08 (d, 2 H, J ) 9 Hz).
1,12-Di-(4-(4S)-methoxycarbonyl-4-[4-(methyl(trifluoroacet-
yl)amino)benzoylamino]butyrylamino)dodecane (3). Acetyl chloride
(0.060 mL, 0.838 mmol) was mixed with MeOH (5 mL), and the
resulting methanolic HCl was used to dissolve compound 1 (0.316 g,
0.419 mmol). The solution was flushed with Ar before the addition of
10% Pd/C (0.062 g) catalyst. The mixture was stirred vigorously, and
the system was flushed with H2. The reaction was complete by TLC
after 2.5 h, whereupon it was diluted with MeOH (20 mL) and filtered
through Celite. The filtrate was stripped of solvent under reduced
pressure and dried under high vacuum to yield a greenish white solid
(0.229 g, 0.410 mmol, 97.9%). To this material were added DCC (0.338
g, 1.64 mmol) and 4-[N-methyl-N-(trifluoroacetyl)amino]benzoic acid
(2) (0.355 g, 1.4 mmol), dissolved in dry DMF (5 mL) and 1-hydroxy-
benzotriazole hydrate (0.111 g, 0.820 mmol) under an Ar atmosphere
at 0 °C. An additional amount of dry DMF (4 mL) and N-methylmor-
pholine (0.09 mL, 0.820 mL) was added, and after 3 h the reaction
was allowed to warm to room temperature. After 20 h, no starting
material was visible by TLC using ninhydrin stain. The addition of
water resulted in the precipitation of dicyclohexyl urea, which was
removed by filtration and washed with cold DMF. The filtrate was
evaporated to dryness under reduced pressure, yielding a yellowish
white solid (1.03 g). Flash chromatography on a silica gel column (3
× 12 cm), eluting with 4:1:1 Hex/EtOAc/MeOH, 4:4:1 Hex/EtOAc/
MeOH, and 1:3:1 Hex/EtOAc/MeOH, yielded the desired product
Methods
1
1
(0.344 g, 0.364 mmol, 88.8%) as a white solid. H NMR (CDCl3) δ
Materials. H NMR spectra were obtained using a Varian VAC-
1.21 (s, 16 H), 1.44 (s, 4 H), 2.22 (m, 4 H), 2.41 (m, 4 H), 3.21 (m, 4
H), 3.38 (s, 6 H), 3.78 (s, 6 H), (q, 2 H), 7.37 (d, 2H, J ) 8 Hz), 8.00
(d, 2 H, J ) 8 Hz).
300 spectrometer. Reverse-phase HPLC was preformed on a Spectra
Physics SP8800 HPLC pump connected to a Kratos Spectroflow 757
detector set at 302 nm using binary gradients formed from solvent A
(H2O + 0.1% v/v TFA) and solvent B (acetonitrile + 0.08% TFA).
Analytical HPLC was performed using a Waters Spheris Orb C8 column
(4.6 × 250 mm), while preparative HPLC was performed using an
Alltech Econosphere C8 column (10 × 250 mm). A two-part linear
gradient (min/% B, 0/30, 20/40, 25/30) was used for analytical and
preparative HPLC with flow rates of 1.0 and 6.0 mL/min, respectively.
Analytical TLC was performed on aluminum-backed silica gel (grade
60) plates obtained from EM Science. Flash chromatography and MPLC
were performed with grade 60, 230-400 mesh silica gel from EM
Science. Anhydrous DMA and DMF were purchased from Aldrich
Chemical Co. and used without further purification. All other chemicals
used were purchased from Aldrich or Acros and were used without
further purification. All other solvents were reagent grade and used as
received.
2,4-Diamino-6-(bromomethyl)pteridine (4). Bromine (0.169 mL,
3.30 mmol) was slowly added, over 20 min, to a stirring solution of
triphenylphosphine (0.902 g, 3.44 mmol) in cold (0 °C) dry DMA (2.7
mL) under an Ar atmosphere. The reaction mixture was maintained
under Ar and allowed to stand for 30 min. In one proportion, 2,4-
diamino-6-(hydroxylmethyl)pteridine (0.210 g, 1.09 mmol) was added
to the reaction mixture, which was then allowed to warm to room
temperature. The reaction was allowed to proceed for 27 h before
initiating the subsequent final coupling reaction in the same vessel.
1,12-Di-[4-(4S)-carboxy-4-{4-[(2,4-diaminopteridin-6-ylmethyl)-
methyl-amino]-benzoylamino}butyrylamino]dodecane (5) (Bis-
MTX). Compound 3 (0.3443 g, 0.364 mmol) was dissolved in a mixture
of EtOH (11.1 mL), H2O (5.5 mL), and 2.0 N NaOH (1.1 mL) and
stirred. Once the starting material had disappeared by TLC (3 h), 10%
AcOH/H2O (10 mL) was added and the mixture was acidified to pH
5.5 by addition of 1 N HCl. The acidic solution was extracted with
CHCl3 (6 × 30 mL) and EtOAc (2 × 30 mL). The organic fractions
were combined, dried over MgSO4, and evaporated under reduced
pressure to yield a foamy white solid (0.223 g), used without further
purification. The material was taken up in DMA (3 mL) and added to
the reaction mixture of compound 4 along with diisopropylethylamine
(0.190 mL, 1.09 mmol). Upon addition of base the reaction was heated
(45 °C) and maintained under Ar for 25 h. The resulting black mixture
was poured into a large volume of 0.33 N NaOH (60 mL) and the
Synthesis. 1,12-Di-(4-benzyloxycarbonylamino-4-(4S)-methoxy-
carbonyl-butyrylamino)dodecane (1). Cbz-L-glutamic acid R-methyl
ester (1.34 g, 4.52 mmol) and Et3N (1.9 mL, 9.03 mmol) were added
to a stirring solution of 1,12-diaminododecane (0.303 g, 1.51 mmol),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.34 g,
4.52 mmol), and 4-(dimethylamino)pyridine (0.193 g, 1.58 mmol) in
CH2Cl2 (20 mL) under an Ar atmosphere. After 18 h the reaction
mixture was diluted with CH2Cl2 (20 mL) and washed with saturated
NaHCO3 (20 mL), 5% KH2PO4 (20 mL), H2O (20 mL), and brine (10
mL). The organic layer was dried (Na2SO4) and concentrated under
(46) Pearce, K. H., Jr.; Cunningham, B. C.; Fuh, G.; Teeri, T.; Wells, J. A.
(47) Rosowsky, A.; Freisheim, J. H.; Bader, H.; Forsch, R. A.; Susten, S. S.;
Biochemistry 1999, 38, 81-89.
Cucchi, C. A.; Frei, E., 3rd. J. Med. Chem. 1985, 28, 660-667.
9
1506 J. AM. CHEM. SOC. VOL. 125, NO. 6, 2003