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Independent SARMS Lab Test

Independent RAD-140 Lab Test

Summary

Introduction:

Spectroscopic analysis of a sample of RAD-140 (testolone) Report compiled by an independent lab.

A sample of testolone was analysed using 1D proton and carbon nuclear magnetic spectroscopy. The sample was identified by comparison with literature values and found to be pure.

The sample, identified as RAD-140 by RipTech was delivered to the chemistry department at the University. The sample was in a capsule form, that, when opened, contained a fine, white powder. The chemical structure of RAD-140 (Testolone) is shown in Figure 1. It is a synthetic selective androgen receptor modulator.

N

O
NH

NC
Cl

N OH

 RAD 140 SARMS

 

 

 

 

 

Figure 1. The structure of testolone (2-chloro-4-((2-hydroxy-1-(5-(4-isocyanophenyl)-1,3,4-oxadiazol-2- yl)propyl)amino)-3-methylbenzonitrile).

Experimental:

Sample prep:

A capsule containing 100 mg of RAD-140 was delivered to the university. According to the literature, RAD- 140 is only slightly soluble in water but is soluble in chloroform. A sample of approximately 10 mg was dissolved in 0.7 ml deuterated chloroform (CDCl3) and transferred to a 5mm NMR tube for analysis.

NMR analysis

NMR spectra (1D 1H, 13C and DEPT spectra) were obtained using a Bruker Avance III 600 MHz NMR spectrometer equipped with a BBO Prodigy cryoprobe. The spectra were recorded at 303K and processed using standard Bruker software (TopSpin 3.2). The 1D spectra were recorded using a 30 degree pulse and a D1 of 2s. All spectra were referenced relative to residual solvent: 1H at 7.26 ppm and 13C at 77.16 ppm.

Results

1H NMR

1D NMR gives a diagnostic fingerprint for all the protons and carbons contained in the structure. The appearance of a peak at a specific chemical shift is indicative of the environment that the specific proton is in. The chemical shift (x-axis) is indicative of the environment that the proton is in, the integration of the peak is informative as to the number of protons in that exact same environment and the multiplicity of the peak describes how many neighbouring protons the proton of interest has. All of these factors lead to the identification of each peak and the final spectrum gives a fingerprint of the compound in its entirety.

RAD 140 NMR SARM test

Figure 2. 1D 1H NMR spectra of RAD-140 sample recorded at 303K in CDCl3.

Figure 2 shows the 1D 1H NMR spectra that was recorded for the RAD-140 sample. The peak with a chemical shift of 0 ppm corresponded to silicone (which could possibly have come from the sample preparation). A peak at 7.26 ppm was identified as the proton in the deuterated solvent. All other signals corresponded to the protons in RAD-140. The four signals between 6.6 and 8.2 ppm were identified as aromatic protons (protons situated on the two rings), the broad signal at 5.3 was identified

as the proton on the hydroxyl (-OH) functional group. Signals corresponding to the protons attached to carbons throughout the structure were also identified.

Spectra were also recorded in order to identify the carbons in the sample compound and to add weight to the confirmation of the RAD-140 identity. As with the proton 1D NMR, the 13C NMR spectrum provides diagnostic peaks. All signals that were recorded in Figure 3 were identified as belonging to RAD-140. Four aromatics carbons, two CH groups, two methyl (CH3) groups and nine quaternary carbons were identified. Their chemical shift helped with the identification as did the recording of a DEPT spectra. Distortionless Enhancement by Polarization Transfer or DEPT spectra provides a window into how many hydrogens are attached to each carbon. Carbon can have up to four atoms attached to it. DEPT is able to differentiate between one, two and three hydrogens attached to each carbon, if a carbon does not have any hydrogens surrounding it, the signal is not picked up thus comparison with the original carbon spectrum will allow for the identification of this quaternary carbon. Carbons with two hydrogens attached to it will appear as a negative peak. Figure 4 compares the DEPT spectrum with the original carbon spectrum. Fewer peaks are seen in the DEPT spectrum due to the number of quaternary carbons.

 

RAD 140 NMR SARMS test

 

Figure 3. 1D 13C spectrum of RAD-140 in CDCl3. This spectrum was recorded at 150 MHz and 303K.

RAD 140 NMR SARMS test

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Figure 4. Comparison of DEPT and 13C spectra in order to confirm identity of carbon signals.

The NMR spectra was then compared to the literature spectra available.1 As shown in Figures 5 and 6, all signals from the sample matched up to the literature values. However, there were two signals in the literature proton spectra (3.3 and 1.8 ppm) that did not correspond to any protons in the RAD-140 compound. This suggests that the literature compound was not pure and is also indicative of the purity of the sample analysed.

RAD 140 NMR SARMS test

Figure 5. Comparison of 1D 1H NMR spectra of RAD-140 from (A) literature and (B) the analysed sample.

RAD 140 NMR SARMS test

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Figure 6. Comparison of 1D 13C NMR spectra of RAD-140 from (A) literature and (B) the analysed sample. Conclusion

A capsule containing a fine, white powder, assumed to be RAD-140 was analysed. The identity of RAD-140 was confirmed with 1D proton and carbon spectroscopy. Comparison was made to the literature data available and the purity of the compound was confirmed. The very small additional peaks seen in the spectra were not identified and could be due to a noisy background.

References

  1. Miller, C.P, Shomali, M., Lyttle, C.R., O’Dea, L, Herendeen, H., Gallacher, K., Pquin, D. Compton, D. R., Sahoo, B., Kerrigan, S. A., Burge, M. S., Nickels, M., Green, J. L., Katzenellenbogen, J. A., Tchesnokow, A. and Hattersley, G., Design, synthesis, and preclinical characterization of the selective androgen reception modulator (SARM) RAD140., ACS Med Chem Lett, 2010, 2(2) 124

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