L. M. Miller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 62–66
65
Step 2: 1-Ethyl-2-(4-methoxy-benzylsulfanyl)-4,5-dimethyl-1H-imidazole (44). To
a stirred solution of 2-(4-methoxy-benzylsulfanyl)-4,5-dimethyl-1H-imidazole
(3.14 g, 12.6 mmol) in DMF (30 mL) at 0 °C was added NaH (0.56 g, 13.9 mmol).
After 15 min at 0 °C iodoethane (1.11 mL, 13.9 mmol) was added slowly, the
reaction mixture was stirred 15 min at 0 °C, then warmed to rt and stirred
overnight. The reaction was quenched with MeOH (20 mL), diluted with water
(10 mL) and extracted with EtOAc (3Â 50 mL). The organic layer was washed
with brine (50 mL), dried (MgSO4) and concentrated. The residue was purified
by column chromatography (0–5% MeOH: CHCl3 gradient) to afford the
product (2.9 g, 12.6 mmol, 83%) as an oil; 1H NMR (DMSO-d6) d 0.96 (t,
J = 14.5 Hz, 3H), 1.97–2.03 (m, 2H), 2.69 (s, 3H), 2.84 (s, 3H), 3.67 (s, 3H), 4.04
(s, 2H), 6.78 (d, J = 8.7 Hz, 2H), 7.10 (d, J = 8.7 Hz, 2H), 11.64 (s, 1H); mass
spectrum [(+) ESI], m/z 277 (M+H)+.
Step 3: 1-Ethyl-4,5-dimethyl-1H-imidazole-2-thiol (45). To a stirred solution of 1-
ethyl-2-(4-methoxy-benzylsulfanyl)-4,5-dimethyl-1H-imidazole (3.48 g,
12.6 mmol) in toluene (10 mL) was added TFA (10 mL). The reaction mixture
was heated at 100 °C for 60 h. The solution was concentrated and purified by
column chromatography (0–5% MeOH/CHCl3 gradient) to afford the product
(1.51 g, 9.66 mmol, 77%) as an oil; 1H NMR (DMSO-d6) d 1.09 (t, J = 14.3 Hz,
3H), 1.96 (s, 3H), 2.06 (s, 3H), 3.88 (q, J = 19.5 Hz, 2H), 7.90 (s, 1H); mass
spectrum [(+) ESI], m/z 157 (M+H)+.
more selective for IR instead of IGF-1R. The goal for future analogs
will be to maintain potency while achieving selectivity for IGF-1R
over IR.
Acknowledgments
We are grateful to the Discovery Analytical Chemistry depart-
ment of Wyeth Research for elemental analyses, 1H NMR, and mass
spectroscopy and Dr. John A. Butera, Dr. John Ellingboe, Ms. Ann-
ette Banker, Ms. Joan Sabalski, and Mr. Gengcheng Yang for
scientific discussions.
References and notes
1. (a) Randhawa, R.; Cohen, P. Mol. Genet. Metab. 2005, 86, 84; (b) Chng, W. J.;
Gualberto, A.; Fonseca, R. Leukemia 2006, 20, 174; (c) Strömberg, T.; Ekman, S.;
Girnita, L.; Dimberg, L. Y.; Larsson, O.; Axelson, M.; Lennartsson, J.; Hellman, U.;
Carlson, K.; Österborg, A.; Vanderkerken, K.; Nilsson, K.; Jernberg-Wiklund, H.
Blood 2006, 107, 669; (d) Breuhahn, K.; Longerich, T.; Schirmacher, P. Oncogene
2006, 25, 3787; (e) Fang, J.; Zhou, Q.; Shi, X.; Jiang, B. Carcinogenesis 2007, 28,
713.
2. Riedemann, J.; Macaulay, V. M. Endocr. Related Cancer 2006, 13, 33.
3. De Meyts, P.; Whittaker, J. Nat. Rev.: Drug Discov. 2002, 1, 769.
4. Mayer, S. C.; Banker, A. L.; Boschelli, F.; Di, L.; Johnson, M.; Kenny, C. H.;
Krishnamurthy, G.; Kutterer, K.; Moy, F.; Petusky, S.; Ravi, M.; Tkach, D.; Tsou,
H.-R.; Xu, W. Bioorg. Med. Chem. Lett. 2008, 12, 3641.
5. (a) Zhang, N.; Wu, B.; Eudy, N.; Wang, Y.; Ye, F.; Powell, D.; Wissner, A.;
Feldberg, L. R.; Kim, S. C.; Mallon, R.; Kovacs, E. D.; Toral-Barza, L.; Kohler, C. A.
Bioorg. Med. Chem. Lett. 2001, 11, 1407; (b) Wissner, A.; Tsou, H.-R.; Berger, D.
M.; Floyd, M. B., Jr.; Hamann, P. R.; Zhang, N.; Salvati, M. E.; Frost, P. U.S. Patent
6,288,082, 2001.; (c) Berger, D.; Dutia, M.; Powell, D.; Wu, B.; Wissner, A.;
Boschelli, D. H.; Floyd, M. B.; Zhang, N.; Torres, N.; Levin, J.; Du, X.;
Wojciechowicz, D.; Discafani, C.; Kohler, C.; Kim, S. C.; Feldberg, L. R.; Collins,
K.; Mallon, R. Bioorg. Med. Chem. Lett. 2003, 13, 3031; (d) Boschelli, D. H.; Wang,
Y. D.; Johnson, S. L.; Berger, D. M. U.S. Patent 6, 780, 996, 2004.
6. IGF-1R and IR kinase assays: The catalytic domains of IGF-1R or IR were cloned
described in Pautsch et al.12 into pAcG2T [BD Biosciences (San Jose, CA)]. GST-
IGF-1R and GST-IR fusion proteins were isolated by glutathione bead capture,
eluted, incubated in 20 mM Tris–HCl, pH 7.5, 0.2 M NaCl, 0.01 M MgCl2 and
7.5 mM ATP for 12 min. followed by thrombin cleavage, purification on a
glutathione column and MonoQ fractionation. IGF-1R or IR catalytic domains
phosphorylated on all three tyrosines in the activation loop as determined by
mass spectrometry were then purified on a Superdex 200 column. This ‘tris’
phosphorylated protein was used in enzyme assays at 0.6 ng per reaction in
Step 4: 2-(2-Chloro-4-nitro-phenylsulfanyl)-1-ethyl-4,5-dimethyl-1H-imidazole
(46). To
a stirred solution of 1-ethyl-4,5-dimethyl-1H-imidazole-2-thiol
(1.51 g, 9.66 mmol) in DMF (20 mL) at 0 °C was added NaH (0.425 g,
10.6 mmol). After 15 min at 0 °C, 3-chloro-4-fluoronitrobenzene (0.1.70 g,
9.66 mmol) was added slowly, the reaction mixture was stirred at 0 °C, 30 min,
warmed to rt and stirred overnight. The reaction was diluted with water (20 mL)
and extracted with EtOAc (3Â 50 mL). The organic layer was washed with brine
(50 mL), dried (MgSO4) and concentrated. The residue was purified by column
chromatography (0–10% MeOH/CHCl3 gradient) to afford the product (2.2 g,
7.06 mmol, 73%) as an oil; 1H NMR (DMSO-d6) d 1.06 (t, J = 14.5 Hz, 3H), 2.10 (s,
3H), 2.18 (s, 3H), 3.88 (q, J = 21.7 Hz, 2H), 6.61 (d, J = 8.9 Hz, 1H), 8.04 (dd, J = 2.4,
8.9 Hz, 1H), 8.29 (s, 1H); mass spectrum [(+) ESI], m/z 312 (M+H)+.
Step 5: 3-Chloro-4-(1-ethyl-4,5-dimethyl-1H-imidazol-2-ylsulfanyl)-phenylamine
(anilino-group A, 47). To
a
stirred solution of 2-(2-chloro-4-nitro-
phenylsulfanyl)-1-ethyl-4,5-dimethyl-1H-imidazole (2.20 g, 7.06 mmol) in
MeOH:H2O (5:1, 54 mL) was added iron (1.24 g, 22.3 mmol) followed by NH4Cl
(1.24 g, 34.2 mmol). The reaction mixture was heated to reflux for 2 h, cooled to
rt, diluted with water (20 mL) and EtOAc (60 mL), filtered through celite and
washed with EtOAc. The layers were separated, the organic layer was washed
with brine (60 mL), dried (MgSO4) and concentrated. The residue was purified by
column chromatography (0–5% MeOH: CHCl3) to afford the product (1.4 g,
4.97 mmol, 71%) as a solid; 1H NMR (DMSO-d6) d 0.98 (t, J = 14.5 Hz, 3H), 1.99 (s,
3H), 2.08 (s, 3H), 3.89 (q, J = 21.7 Hz, 2H), 5.47 (s, 2H), 6.68–6.74 (m, 3H); mass
spectrum [(+) ESI], m/z 282 (M+H)+.
Step 6: Methyl 4-(3-chloropropoxy)-3-methoxybenzoate (48). To a stirred solution
of methyl vanillate (2.00 g, 11.0 mmol) in acetone (50 mL) was added Cs2CO3
(3.94 g, 12.1 mmol) followed by 3-chloro-1-propyl bromide (2.16 mL,
22.0 mmol). The solution was stirred at rt for 3 days. The solution was then
diluted with H2O (50 mL) and extracted with Et2O (2Â 200 mL). The organic layer
was washed with brine, dried (MgSO4), and concentrated to afford the product
(0.290 g, 85%) as a solid; 1H NMR (DMSO-d6) d 2.10–2.20 (m, 2H), 3.74 (t,
J = 6.5 Hz, 2H), 3.77 (s, 6H), 4.12 (t, J = 6.0 Hz, 2H), 7.06 (d, J = 8.5 Hz, 1H), 7.41 (d,
J = 2.0 Hz, 1H), 7.53 (dd, J = 2.1, 8.5 Hz, 1H); mass spectrum [(+) ESI], m/z 259
(M+H)+.
50 mM Hepes, pH 7.5, 0.01 M MgCl2 with 150
(Sigma #A-8918) and peptide at a final concentration of 1
TRDIYETDYYRK-OH) for 90 min at room temperature. The final ATP
concentration was 100 M.
l
g/mL of bovine serum albumin
l
g/mL (Biotin-NH2-
l
7. (a) Jamieson, E. R.; Jacobson, M. P.; Barnes, C. M.; Chow, C. S.; Lippard, S. J. J. Biol.
Chem. 1999, 274, 12346; (b) The changes in the intrinsic fluorescence of the
protein (at 1 lM) were measured in the absence and presence of various
Step 7: Methyl 4-(3-chloropropoxy)-5-methoxy-2-nitrobenzoate (49). To a cooled
flask of HNO3 (12 mL) at 0 °C was slowly added methyl 4-(3-chloropropoxy)-3-
methoxybenzoate (2.75 g, 10.6 mmol) portion wise over 15 min. The solution
was stirred at this temperature for 0.5 h and then at rt for 1 h. The solution was
cooled back down to 0 °C and diluted H2O (ꢀ10 mL). The mixture was stirred for
0.5 h, and a yellowish orange oily solid came out. The mixture was extracted with
Et2O, and the resulting organic layer washed with brine, dried (MgSO4), and
concentrated to afford the product (3.03 g, 94%); 1H NMR (DMSO-d6) d 2.12–2.21
(m, 2H), 3.73 (t, J = 6.5 Hz, 2H), 3.78 (s, 3H), 3.88 (s, 3H), 4.20 (t, J = 6.1 Hz, 2H),
7.28 (s, 1H), 7.63 (s, 1H); mass spectrum [(+) ESI], m/z 326 (M+Na)+.
Step 8: Methyl 2-amino-4-(3-chloropropoxy)-5-methoxybenzoate (50). To a stirred
solution of methyl 4-(3-chloropropoxy)-5-methoxy-2-nitrobenzoate (3.03 g,
9.98 mmol) in MeOH:H2O (5:1, 48 mL) was added iron (2.23 g, 39.9 mmol)
followed by NH4Cl (2.13 g, 39.9 mmol). The solution was heated to reflux
overnight. The solution was cooled to rt and diluted with EtOAc (100 mL) and
H2O (30 mL). The mixture was filtered through celite, and the filtrate layers were
separated. The organic layer was washed with brine, dried (MgSO4), and
concentrated. The residue was purified with Biotage Flash 40 (10–30% EtOAc/
petroleum ether gradient) to afford the product (2.07 g, 76%); 1H NMR (DMSO-
d6) d 2.11–2.19 (m, 2H), 3.60 (s, 3H), 3.70 (s, 3H), 3.73 (t, J = 6.5 Hz, 2H), 4.00 (t,
J = 6.1 Hz, 2H), 6.35 (s, 1H), 6.37 (s, 2H), 7.09 (s, 1H); mass spectrum [(+) ESI], m/z
274 (M+H)+.
Step 9: (E)-Methyl 4-(3-chloropropoxy)-2-[(dimethyl-amino)methyleneamino]-5-
methoxybenzoate (51). To a flask with methyl 2-amino-4-(3-chloropropoxy)-5-
methoxy-benzoate (2.07 g, 7.56 mmol) was added DMFÁDMA (12 mL). The
solution was heated to reflux for 4 h. The solvent was remove via high vacuum,
and the resulting residue was passed through florasil, eluting with CH2Cl2, and
then concentrated toafford the product (2.47 g, 99%); 1HNMR (DMSO-d6) d2.08–
2.17 (m, 2H), 2.83–2.95 (br s, 6H), 3.64 (s, 3H), 3.68 (s, 3H), 3.73 (t, J = 6.5 Hz, 2H),
4.07 (t, J = 6.0 Hz, 2H), 6.44 (s, 1H), 7.11 (s, 1H), 7.43 (s, 1H); mass spectrum [(+)
ESI], m/z 329 (M+H)+.
concentrations (0–2200 nM) of compound 2, using an excitation wavelength of
295 nm and emission at 347 nm. The fluorescence emission spectra of the
protein were recorded from 310 to 580 nm using the Jobin Yvon Spex
fluorometer. The reaction buffer contained 50 mM Hepes, pH 7.5, and 10 mM
MgCl2. Increasing concentrations of the inhibitor quenched the fluorescence
and the changes were used to determine the binding affinity.
8. (a) Xu, W.; Dwyer, B.; Moy, F.; Mayer, S.; Boschelli, F.; Tkach, D.; Bean, K.;
Mosyak, L.; Wilhelm, J. Presented at the 5th Annual Meeting of Protein Kinase
Targets & Structure-Based Design, Boston, MA, June 2007; poster session.; (b)
RCSB Protein Data Bank (PDB) deposition number 3F5P.
9. IGF1-R dependent proliferation assay: Full-length IGF1-R was cloned from a
human placenta cDNA library (Invitrogen) and inserted into pIRESneo2 (BD
Biosciences). IL-3 dependent FDCP1 myeloid cells (ATCC) were transformed
with or an IGF1-R expression clone pIRESneo2-IGFR clone (NM_000875). G418
resistant cells were selected by limiting dilution. IGF1-R expressing clones that
acquired IL-3 independence in the presence of IGF1 (10 ng/mL) were identified
from these clones. For 96-well proliferation assays, 2000 cells were plated per
well on day 1 with 10 ng/mL IGF1, compound was added on day 2, and relative
cell number was determined by addition of CellTiter-Glo (Promega). IC50s were
determined using model 63 of the LSW Excel plug-in.
10. Experimental: The synthesis of compound 29 described here serves as
representative example of the synthetic methodology used in this paper.
Step 1: 2-(4-Methoxy-benzylsulfanyl)-4,5-dimethyl-1H-imidazole (43). To
a
a
stirred solution of 4,5-dimethyl-1H-imidazole-2-thiol (2.00 g, 15.6 mmol) in
NMP (50 mL) was added DIEA (2.72 mL, 16.4 mmol) and 5-methoxybenzyl
chloride (2.72 mL, 16.4 mmol). The reaction mixture was stirred at rt
overnight, diluted with water, and the precipitate was collected via vacuum
filtration to give the desired product (3.14 g, 12.6 mmol, 81%) as a white solid;
1H NMR (DMSO-d6) d 1.97 (2, 6H), 3.67 (s, 3H), 4.06 (s, 2H), 6.78 (d, J = 8.7 Hz,
2H), 7.14 (d, J = 8.7 Hz, 2H), 11.67 (s, 1H); mass spectrum [(+) ESI], m/z 249
(M+H)+.