exp19a.JPG


Objective

To produce a diketopiperazine by a Ugi synthesis and cyclization using piperonal, 5-methylfurfurylamine, Boc-Gly-OH, and benzylisocyanide using the protocol described here with the modification that THF will be used instead of 1,2-dichloroethane in the cyclization step. This compound is not an anti-malarial target but is a close analog of the product that we wish to make once the catechol aldehyde is obtained.

Procedure

Methanol (20 mL), piperonal (73.79mg, 0.49 mmol), Boc-Gly-OH (85.85 mg, 0.49 mmol), 5-methylfurfurylamine (50µL, 0.49 mmol), and benzylisocyanide (50µL, 0.43 mmol) were added to a 50 mL Erlenmeyer flask. The flask was stoppered and the mixture was stirred for 24 hr, then evaporated on rotovap and high vac. 27 mL of THF and 3 mL of trifluoroacetic acid was added to the product and the solution was refluxed for 30 min. The crude product was taken up in methylene chloride (50 mL) and then separated by washing with distilled water. In order to recover any lost product, a backwash was also done on the water layer obtained during separation and then combined with the organic layer. The combined organic layer was dried over anhydrous MgSO4 and evaporated again by rotovap and high vac. A dark, reddish oil was obtained with a mass of 147mg. Chromatography was done to separate the diketopiperazine from any other products produced.

Results

Characterization of Ugi Reaction (resulting in 19A)

-- Video of setup with a pale yellow solution, right after starting materials were added.
-- TLCs of the starting materials (left spots) were run against the initial Ugi reaction (right spots) in 1:1 methylene chloride hexanes. Left to right: piperonal, amine, amino acid, and benzylisocyanide (Picture).
-- Video after 20 hours with a dark yellow solution (sample 19A), right before stirring ended.
-- TLCs of the starting materials (left spots) against 19A (right spots) were done in 1:1 methylene chloride hexanes. Left to right: piperonal, amine, amino acid, and benzylisocyanide (Picture).

Characterization of 19B (after Ugi reaction before cyclization)

-- H-NMR

Characterization of 19C (after cyclization and extraction)

-- TLC of piperonal against 19C was stained in anisaldehyde. Piperonal did not stain in anisaldehyde (Picture).
-- TLCs of 5-methylfurfurylamine against 19C (Picture) and Boc-Gly-OH against 19C (Picture) were stained in ninhydrin.

Characterization of 19D (after cyclization and high vac)

-- TLCs of 19D (right spots) were run against the starting materials (lefts spots) and 19C (middle spots). Solvent system was 1:1 methylene chloride/hexanes (Picture under UV light).

Characterization of 19D Fractions (first separation)

-- 19D-F1: (H-NMR).
-- 19D-F2: mass = 8.2 mg (H-NMR).
-- 19D-F3: mass = 19.4 mg (H-NMR).
-- 19D-F4: mass = 118.1 mg (H-NMR).

Characterization of 19D-F4 Fractions (second separation)

-- 19D-F4A: mass = 1.65 mg (H-NMR)
-- 19D-F4B: mass = 32.5 mg (H-NMR).
-- 19D-F4C: mass = 30.6 mg (H-NMR).
-- 19D-F4D: mass = 18.3 mg (H-NMR).
-- Ran TLC of 19D-F4B and 19D-F4C in 3:1 THF/Methylene chloride and then 0.5% methanol (Picture).
-- Ran TLCs of 19D-F4B and 19D-F4C in 0.5%, 0.75%, and 1% methanol (Picture).

Discussion

The key H-NMR peaks expected of the diketopiperazine are:
1. furan aromatic H's at 5.8 and 5.9 ppm, and methyl on furan ring at 2.2 ppm, based on the NMR of 5-methylfurfurylamine
2. methylenedioxy at 6.0 ppm
3. the glycine methylene peak is harder to predict exactly

H-NMRs of 19D Fractions

4. The diketopiperazine is most likely not in fractions 19D-F1 (unreacted piperonal), 19D-F2 (no relevant peaks), and 19D-F3 (only methylenedioxy peak)
5. The diketopiperazine could be in sample 19D-F4 because it has the relevant peaks at 5.8, 5.9, 6.0, and 2.2 ppm
6. 19D-F4 needs to be purified because of the unknown peaks at 4.2 and 6.8 ppm

H-NMRs of 19D-F4 Fractions

7. The diketopiperazine is most likely not in fractions 19D-F4A or 19D-F4D because no relevant peaks were present
8. The diketopiperazine could be in both fractions 19D-F4B and 19D-F4C because they have the relevant peaks at 5.8, 5.9, 2.2, and 6.0 ppm. Impurities are present in both fractions (peaks at 4.2 and 6.8 ppm)

Conclusion

Experiment was aborted. Although the H-NMRs of 19D-F4B and 19D-F4C appear to contain the same compounds, TLCs of the two fractions indicate (by the extensive smears - Picture) that even though the two fractions could be combined, separation from the impurities would be difficult.

Log

2006-07-20

1. 17:25] Mixed chemicals and started stirring at room temperature, stir setting 3 (Video). The 5-methylfurfurylamine and the benzylisocyanide were added by using a 500 microliter GC syringe.
2. Took a small sample (solution was pale yellow) to run TLCs.

2006-07-21

3. 1:36] Stirring was stopped to get 19A. Solution had turned a dark yellow color (Video).
4. Ran TLCs of starting components (dissolved in methanol) against the sample taken yesterday (right after stirring began). Solvent system was 1:1 methylene chloride/hexanes.
5. Ran TLCs of starting components (dissolved in methanol) against 19A. Solvent system was 1:1 methylene chloride/hexanes.
6. 15:36] Rotovap solution at 76C for 24 min.

2006-07-24

7. 10:55] Put sample on high vac at 0.3 mmHg.
8. 14:05] Sample take off high vac after heating with heat gun (sample did not appear dry). Product 19B had a mass of 0.2106 g.

2006-07-26

9. Add 27 mL THF and 3 mL TFA to 19B in a round bottom flask.
10. 12:15] Put the RBF on a heat/stir plate and turn on (high heat and stir setting 3).
11. 12:29] Reflux started (Video).
12. 12:59] Removed solution from heat and turned off heat/stir plate. Allow solution to cool.
13. 15:30] Add 50 mL methylene chloride to solution in a 500 mL separatory funnel.
14. Separated the organic layer in a separatory funnel by washing with distilled water (20 mL x 7), (85 mL x 5), and (100 mL x 4) each time. Some globs of organic layer was floating on top of water layer, more methylene chloride is needed next time to achieve better separation.
15. After each separation, the pH of the organic layer was taken until the solution became neutral.
16. Using methylene chloride, a back wash was done on the water layer to recover the globs of organic layer that failed to separate (above).

2006-07-27

17. The recovered organic layer (from yesterday) was washed 5 times with distilled water (50 mL each time) until the solution's pH was neutral.
18. Combined the organic layers and tested pH.
19. Saturated solution with anhydrous magnesium sulfate and filtered by vacuum to get 19C.
20. Ran TLCs of 19C against the starting materials (except Boc-Gly-OH) and 19B. Solvent system was 1:1 methylene chloride/hexanes. The TLCs were rerun again because the samples were not spotted enough (too faint under UV).
21. TLC of piperonal against 19C was stained in anisaldehyde.
22. TLCs of 5-methylfurfurylamine against 19C and Boc-Gly-OH against 19C were stained in ninhydrin.
23. 15:17] Rotovap 19C at 50C. Mass of round bottom flask was 65.1826g.
24. 15:40] Removed from rotovap.
25. 17:05] High vac solution at 1.5 mmHg.
26. 18:40] Removed from high vac to get 19D. Mass of crude product is 147 mg.

2006-07-28

27. Ran TLCs of 19D (dissolved in methanol) against the starting material and 19C. Solvent system was 1:1 methylene chloride/hexanes.
28. Saved small sample of 19D dissolved in CDCl3 for NMR.

2006-07-31

29. Used the chromatotron to separate 19D on a 1 mm plate. A 1:1 mix of hexanes and methylene chloride was used at first, then 5:2, then pure methylene chloride, and then finally 1:1 methanol/methylene chloride. 10 fractions were collected.

2006-08-01

30. Since some of the fractions contained large amounts of solvent (over 200 mL), steam was used to evaporate the solvent until the fractions had a 15-20mL volume.
31. TLCs were done on each fraction in 1:1 methylene chloride/hexanes. Based on the TLCs, the fractions were combined to form 6 fractions.
32. The 6 fractions were rotovaped at 50C.

2006-08-02

33. TLCs were done on the 6 fractions in 1:1 methylene chloride/hexanes.
34. Based on the TLCs, the fractions that contained similar moving spots were combined. Two fractions, containing spots that did not move, were combined. Two fractions that had the fastest moving spots were also combined. This resulted in four fractions.
35. The combined sample that had the fastest moving spots was put on roto vap at 50C.
36. 14:29] Sample was put on high vac at 1.5 mmHg for 24 hours to get 19D-F1.

2006-08-03

37. Ran TLC of 19D-F1 in 1:1 methylene chloride/hexanes.
38. Take an NMR of 19D-F1

2006-08-07

39. Based on the TLCs done (2006-08-02), the fraction with the next fastest moving spot was identified.
40. 12:07] Sample was put on roto vap at 50C for 2 minutes.
41. 12:21] Sample was put on high vac at 1 mmHg for 4hr 15min to get 19D-F2. Mass of crude product is 8.2 mg.
42. Ran TLC of 19D-F2 in 1:1 methylene chloride/hexanes.

2006-08-09

43. Based on the TLCs done (2006-08-02), the fraction with the next fastest moving spot was identified (third fastest overall).
44. Rotovap sample 19D-F3 at 50C.
45. Ran TLC of 19D, 19D-F1, 19D-F2, and 19D-F3 on the same plate in 1:1 methylene chloride to hexanes (Picture).

2006-08-10

46. 12:33] Rotovap the final fraction, containing baseline compounds, at 45C for 1.5 hr.
47. 14:48)] High vacuum the final fraction (19D-F4) at 1.1 mmHg. Mass of 19D-F4 is 118.1 mg.
48. High vac the third faction (19D-F3). Mass of 19D-F3 is 19.4 mg.

2006-08-11

49. Ran TLC of 19D, 19D-F1, 19D-F2, 19D-F3, and 19D-F4 on the same plate in 1:1 methylene chloride to hexanes (Picture).

2006-08-18

50. Used the chromatotron to separate 19D-F4 on a 1 mm plate. A 3:1 mix of hexanes and methylene chloride was used at first, then 5:1, then pure methylene chloride, then 1% methanol, 2%, 5%, 10%, and then finally pure methanol. 7 fractions were collected. Only 4 fractions had UV active compounds.

2006-08-21

51. Rotovap the four fractions (19D-F4A, 19D-F4B, 19D-F4C, and 19D-F4D).
52. Mass of 19D-F4A is 1.65 mg.
53. Mass of 19D-F4B is 32.5 mg.
54. Mass of 19D-F4C is 30.6 mg.
55. Mass of 19D-F4D is 18.3 mg.

2006-08-22

56. Ran TLC of 19D-F4, 19D-F4A, 19D-F4B, 19D-F4C, and 19D-F4D on the same plate in 1:1 methylene chloride to hexanes (Picture) and then in pure methylene chloride.

2006-08-24

57. Ran TLC of 19D-F4B and 19D-F4C in 1% methanol.

2006-08-25

58. Ran TLC of 19D-F4B and 19D-F4C in 3:1 THF/Methylene chloride and then 0.5% methanol (Picture).

2006-08-28

59. Ran TLCs of 19D-F4B and 19D-F4C in 0.5%, 0.75%, and 1% methanol (Picture).

2006-08-30

60. Ran TLC of 19D-F4B and 19D-F4C in 5% methanol.

Tags

Benzylisocyanide InChI=1/C8H7N/c1-9-7-8-5-3-2-4-6-8/h2-6H,7H2
Piperonal InChI=1/C8H6O3/c9-4-6-1-2-7-8(3-6)11-5-10-7/h1-4H,5H2
Boc-Gly-OH InChI=1/C7H13NO4/c1-7(2,3)12-6(11)8-4-5(9)10/h4H2,1-3H3,(H,8,11)(H,9,10)
5-methylfurfurylamine InChI=1/C6H9NO/c1-5-2-3-6(4-7)8-5/h2-3H,4,7H2,1H3
ugi019: InChI=1/C29H33N3O7/c1-19-10-12-22(38-19)17-32(25(33)16-31-28(35)39-29(2,3)4)26(21-11-13-23-24(14-21)37-18-36-23)27(34)30-15-20-8-6-5-7-9-20/h5-14,26H,15-18H2,1-4H3,(H,30,34)(H,31,35)
DKP019 InChI=1/C17H16N2O5/c1-10-2-4-12(24-10)8-19-15(20)7-18-17(21)16(19)11-3-5-13-14(6-11)23-9-22-13/h2-6,16H,7-9H2,1H3,(H,18,21)