Chapter+7+-+Cyclization+of+the+Ugi+Product


 * Chapter 7: Cyclization of the Ugi Product into 2, 5-Diketopiperazines**

Cyclization of the Ugi product is the final step in synthesis of 2, 5 diketopiperazines. Many methods for performing this cyclization have been investigated including the use of different acidic solvent solution-phase systems (Hulme 1998) as well as solid-phase systems (Enzo 2001, Scott 1996). The initial step involves the removal of the BOC-protecting group to give an intermediate amine. The resulting amine undergoes an intramolecular transamidation attack driven by the formation of a six membered ring.
 * 7.1 Introduction**


 * 7.2 Experimental**

Trifluoroacetic acid (TFA) was purchased from Aldrich (Milwaukee, WI). Deuterated chloroform with 0.03% v/v TMS was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). Concentrated aqueous (12M) hydrochloric acid and methanol were purchased from Fisher Scientific (Fair Hill, NJ). 1,2-dichloroethane was purchased from EM Scientific (Cherry Hill, NJ). The Ugi product used in this experiment was prepared from piperonal, 5-MFA, Boc-Gly-OH, and t-butyl isocyanide.The product was prepared in our lab at Drexel University (Holsey 2007A).
 * 7.2.1 Materials and Reagents**


 * 7.2.2 Synthesis**


 * 7.2.2.1 Cyclization using a 10% TFA/CDCl3 solution** (Holsey 2007B)

To a 1mL volumetric flask was added 100uL TFA and diluted to mark with deuterated chloroform. An HNMR spectrum was taken of the starting solution upon transfer to an NMR tube. To this NMR tube was added Ugi Product (50mg, 0.1mmol) (38) (Holsey 2007A). The reaction was monitored via NMR at specific intervals for one week. The NMR tube containing the reaction mixture was placed in a water bath at 65°C to reflux in between NMR experiments. To a 5 dram vial was added the contents of the NMR tube. The NMR tube was rinsed twice with deuterated chloroform (2mL), and each wash was added to the 5 dram vial. To the 5 dram vial was added distilled water (5mL). The 5 dram vial was shaken vigorously and allowed to sit so that the organic and water layers could separate. The water layer was removed and the organic layer was rinsed twice more (2mL distilled water) and dried with magnesium sulfate (0.50 g) The organic layer was filtered into a round bottom flask and evaporated solvent via high vacuum. One milliliter of deuterated chloroform was added to the round bottom flask, and the contents were transfered to an NMR tube.


 * 7.2.2.2 Cyclization using a 10% HCl/MeOD solution** (Holsey 2007C)

To a 1mL volumetric flask was added 100uL HCl and diluted to mark with deuterated methanol. An HNMR spectrum was taken of the starting solution upon transfer to an NMR tube. To this NMR tube was added Ugi Product (38) (50mg, 0.1mmol)(Holsey 2007A). The reaction was monitored via NMR at specific intervals for 150 minutes. CDCl3 (20uL) was added to aid in the dissolution of the Ugi product (38) as there was some difficulty in the Ugi product completely dissolving in methanol. The NMR tube was placed in a water bath at 70°C to reflux in between NMR experiments.


 * 7.2.2.3 Cyclization using a 10% TFA/DCE solution** (Holsey 2007D)

To a 1mL volumetric flask was added 100uL TFA and diluted to mark with DCE. An HNMR spectrum was taken of the starting solution upon transfer to an NMR tube. To this NMR tube was added Ugi Product (50mg, 0.1mmol) (38) (Holsey 2007A). The reaction was monitored via NMR at specific intervals for twenty four hours. NMR tube was placed in a water bath at 81°C to reflux in between NMR experiments. The contents of NMR tube were evaporated. CDCl3 (500uL) was added to tube for NMR analysis.


 * 7.2.3 Instrumentation**

All HNMR spectra were taken on a 300 MHz Varian Inova at room temperature to see whether the desired diketopiperazine (39) was formed. Proton NMR spectra were taken with 16 scans and a 3.74 second acquisition time.


 * 7.2.4 Molecular Docking Experiments**

All molecular docking experiments were performed by colleague Sean Gardner (Gardner 2007). The experiments were run using the THINK software provided by the Find-A-Drug company (Find a Drug 2006). The SMILES code for the molecule was obtained from an online SMILES translator (NIH 2007).


 * 7.2.4.1 Procedure**

The uncyclized forms of the compounds were run to testing docking ability in both their Boc-protected as well as de-protected form, with the chirality specified at the variable aromatic group as with R or S, and inclusion of the t-butyl isocyanide. The molecules were all opened in THINK, then run as a 3D site search with the settings of 4 centers, and a tolerance value of 1 and 0.5.


 * 7.3 Results and Discussion**


 * 7.3.1 Cyclization using a 10% TFA/CDCl3 (DCE) solution**

Ten minutes after adding the Ugi crystals to the solution, the appearance of new peaks in the regions of of the t-butyl isocyanide group protons and the 5-MFA methyl protons is observed ([|Figure 7.1]).


 * Figure 7.1** HNMR spectra of reaction ten minutes after adding TFA solution.

A new peak at 1.60 ppm appears indicating the formation of tert-butyl trifluoroacetate (Lundt 1978). After twenty four hours, less shifting of the 5-MFA methyl protons is observed; however, the intensity of the peak decreases. The t-butyl group protons of the BOC group at 1.45 ppm disappears completely, indicating full conversion to t-butyl trifluoroacetate ([|Figure 7.2]).


 * Figure 7.2** HNMR spectra of reaction twenty four hours after adding TFA solution.

An HNMR spectrum was taken of the reaction after one week. The methyl protons nearly disappeared completely from the reaction ([|Figure 7.3]).


 * Figure 7.3** HNMR spectra of reaction one week after adding TFA solution.

This is an unexpected occurrence but was observed in other experiments performed by my colleague Khalid Mirza (Bradley 2007B, Mirza 2007). Reacting TFA with 5-MFA in a 50% TFA solution did not explain why this occurred in the Ugi product (Mirza 2007B) because the methyl groups did not disappear in this case.

After separating the aqueous and organic layers, and NMR spectra of the organic layer did not show evidence of the desired cyclized product. Similar results were obtained for the 10% TFA/DCE solution.


 * 7.3.2 Cyclization using a 10% HCl/MeOD solution**

The appearance of new peaks was observed for 10% HCl/MeOD as it was for 10% TFA/DCE and 10% TFA/CDCl3 thirty minutes after adding 10% HCl/MeOD; however, the disappearance of the methyls weren’t observed over time ([|Figure 7.4]).


 * Figure 7.4** HNMR spectra of reaction 30 minutes after adding HCl solution.

Some new peaks appeared at 1.3 ppm. We were not able to attribute these peaks to any compounds. We were unable to understand why this is occurring with TFA as Mirza's experiment (Mirza 2007C) as described above did destroy the methyl group.

Using the THINK software (Treweren 2007), the isolated uncyclized boc-protected Ugi product docked in a receptor site of malarial enoyl reductase. The Ugi products were also tested for docking using FlexX (BioSolveIt 2007) by Tan Tsu Soo, an online collaborator (Bradley 2007C) from the Bioinformatics Group at Nanyang Polytechnic (Soo 2007) in Singapore. The de-protected Ugi products (R and S) that were prepared dock in a similar fairly shallow location, different from the triclosan site (Kuo 2003) as shown in figures 7.5 and 7.6.
 * 7.3.3 Docking Experiments**
 * Figure 7.5** Uncyclized Boc- protected Ugi product docking with Malaria Enoyl Reductase.


 * Figure 7.6** Uncyclized De-Protected Ugi product docking with Malaria Enoyl Reductase

Despite boc de-protection, the Ugi Adduct did not cyclize into a 2, 5-diketopiperazine in either acidic system. Further information about the BOC de-protected intermediate will be necessary to understand why the system is not cyclizing. Although cyzlization was unsuccessful, the uncyclized Ugi product did dock using the THINK software, indicating that our compounds may be potential targets for malarial enoyl reductase inhibitors.
 * 7.4 Conclusion**


 * 7.5 Reference List**

BioSolveIt http://www.biosolveit.de/software/ 2007 Bradley, JC [|www.usefulchem.blogspot.com] 2007A Bradley, JC http://usefulchem.blogspot.com/2007/03/disappearing-methyls.html 2007B Bradley, JC http://usefulchem.blogspot.com/2007/04/open-source-science-expands-with-tan.html 2007C [|Enzo, P., et al. 2, 6-Diketopiperazines from amino acids, from solution-phase to solid-phase organic synthesis”. Comb. Chem., 3 (5), 453 -460, 2001]. DOI: 10.1021/cc0000904 S1520-4766(00)00090-0

Find A Drug [|www.find-a-drug.org] 2006 Gardner, S http://usefulchem.wikispaces.com/D-EXP005 2007 Holsey, A. http://usefulchem.wikispaces.com/exp062 2007A Holsey, A http://usefulchem.wikispaces.com/Exp065 2007B Holsey, A **http://usefulchem.wikispaces.com/Exp072 2007**C Holsey, A http://usefulchem.wikispaces.com/Exp079 2007D [|Hulme, C. The solution phase synthesis of diketopiperazine libraries via the Ugi reaction: novel application of armstrong’s convertible isonitrile” Tet. Letters 39, 1113, 1998] Kuo, M., et al. Targeting Tuberculosis and Malaria Through Inhibition of Enoyl Reductase. J. Biol. Chem. 278(23), 20851, 2003 [|Lundt BF, Johansen NL, Volund A, and Markussen J. Removal of t-butyl and t-butoxycarbonyl protecting groups with trifluoroacetic acid. Mechanisms, biproduct formation and evaluation of scavengers.” Int J Pept Protein Res.12(5), 258, 1978] Mirza, K. [|www.usefulchem.wikispaces.com/exp067] 2007A Mirza, K. [|www.usefulchem.wikispaces.com/exp091] 2007B Mirza, K. [|www.usefulchem.wikispaces.com/exp070] 2007C National Institute of Health http://cactus.nci.nih.gov/services/translate/ 2007 [|Scott, B. and Siegmund, A. Solid phase organic synthesis (SPOS): A novel route to diketopiperazines and diketomorpholines”. Molecular Diversity 1(2), 125, 1996] DOI:10.1007/BF01721328 SDBS, http://www.aist.go.jp/RIODB/SDBS/cgi-bin/direct_frame_top.cgi?lang=eng 2007 Soo, T. http://www.nyp.edu.sg/SCL/scl_big.html 2007 Treweren Consultants http://www.treweren.com/software.htm 2007

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