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A hitherto unreported impurity in Terazosin – elucidation of the structure, synthesis and cytotoxicity

Analysis of several batches of the 1–adrenergic blocking agent terazosin being used as a medication for treating benign prostatic hyperplasia and hypertension revealed the presence of a hitherto not reported impurity. The latter was isolated, and its structure was elucidated from NMR and Mass Spectrometry (MS) data and unambiguously confirmed by independent synthesis. This contamination, represented in 1-[4-(amino-6,7dimethoxyquinazolin-2-yl)-piperazin-1-yl]-pentane-1,2-dione 2 is likely to occur as the product of a side-reaction in the catalytic hydrogenation step during the synthesis of the drug. Biological screening showed this compound as not cytotoxic for several human tumor cell lines and non-malignant fibroblasts.


Introduction
Drugs are developed, produced and marketed to be used as medications. They have to be delivered in utmost pure form. Their purification, however, is most often very challenging since there is an increased regulatory focus on impurities. A usual and generally accepted identification threshold is 0.1% for drugs whose maximum daily dose is less than or equal to 2.0 g while for those drugs whose daily dose exceeds 2.0 g the identification threshold is 0.05% [1][2][3][4] . During a project dealing with the synthesis of postsynaptic 1-adrenergic blocking agents, we came across the drug terazosin 1 (Fig. 1) [5][6][7][8][9] .
Terazosin was first used in medical practice in 1987 to treat benign prostatic hyperplasia [10][11][12] . A quarter of all 40-year-old men already have benign prostate hyperplasia with the number for 80-year-old men being above 80%. While in stage 1, a conservative "strategy of controlled waiting" is often supported by over-the-counter phytopharmaceuticals, but in stage 2 more effective drugs have to be used, such as 1 receptor blockers including Terazosin. A second indication for Terazosin, although less widespread, is the treatment of hypertension. It is estimated that about 25% of the world's population has a too high blood pressure; by 2025 this number of patients is expected to rise to 29%, which means that about 1.5 billion people will be affected 13 .
Recently, two other possible applications of Terazosin have become known. These include the treatment of hyperhidrosis (phase 4, NCT00449683) 14-16 and a study indicating possible re-purposing of 1 to treat Parkinson's disease (PD) 8 . PD is the second most common neurodegenerative disease being first diagnosed in 1817 by the English physician James Parkinson. Nowadays, the number of patients worldwide has increased from 2.5 million in 1990 to 6.1 million in 2016. In 2016 alone, 3.2 million DALYs (disability-adjusted life years) -i.e. symptom-free years with a good quality of life -were lost worldwide due to this disease 17 . The disease recently claimed 200000 deaths per year. Recently, 1 has been repurposed 18,19 as anti-apoptotic drug targeting the human enzyme phosphoglycerate kinase 1.

Results and discussion
These data indicate that there may be an increased need for Terazosin in the future. Given the recent problem of critical impurities in several drugs, we examined some batches of Terazosin for impurities. It became apparent that individual batches contained traces (0.05-0.11%) of an impurity 2 not previously having been described in the literature. The structure of 2 was elucidated by the commercially obtained drugs which were extracted with methanol, and the extract was subjected to a repeated semi-preparative HPLC separation (RP18, CH3CN/H2O) to yield pure 2 being subject to an excellent elucidation of its structure by Mass spectrometry and NMR spectroscopy. Low-resolution ESI MS showed a quasi-molecular ion [M+H] + m/z 388.27; this finding was supported by HRMS showing m/z 388.1977 and -as a consequence -a molecular formula of C19H26O4N5. An ESI-MS fragmentation experiment gave fragments m/z 360.2 (m = 28, CO) and m/z = 290.2 (splitting off of the side chain from the piperazinyl residue).
Two carbonyl groups were detected in the 13 C NMR spectrum at  = 202.4 and 166.3 ppm, respectively. An intact piperazinyl moiety was established by the presence of the two N-CH2-groups in the respective 1 H and 13 C NMR spectra, and the two OMe groups were detected at  = 56.3 and 55.9 ppm, respectively; their unambiguous assignment was made by 2D-NOESY NMR spectroscopy. The 1 H NMR spectrum also showed the presence of a n-propyl side chain. From these results as well as extra 2D NMR spectra, the structure of 2 was deduced as depicted in Fig 1. A synthesis made independent confirmation of this proposal of 2 was designed and carried out (Scheme 1). Thus, commercially available 2-chloro-6,7-dimethoxyquinazolin-4-amine (3) was allowed to react with piperazine 20-24 for 6 hours at reflux temperature in water to yield 68% of known compound 4. The latter compound was transformed into target amide 2 by its reaction with 2-oxopentanoic acid in the presence of HOBT, EDCl and TEA to yield 50% of 2. The sample obtained by synthesis was identical in each property (IR, UV-Vis, 1 H NMR, 13 C NMR, MS) with compound 2 obtained from the extraction of the drug. However, there are some reports on ring-opening reactions under these conditions. Thus, 2-methylfuran forms 2-pentanone upon hydrogenation in the presence of Ru, Pt, Pd or a ZnO/Ni [26][27][28][29][30][31][32] . Since these reactions usually give good yields when higher pressures and/or higher reaction temperatures are used, it can be assumed that -when applying low pressure of hydrogen and room temperaturethis reaction only occurs as a minor side-reaction on a small scale. Thus, it seems highly probable that the impurity 2 observed in the end product is due to an already existing impurity 7 being formed during the synthesis of 6. Ring-opening reactions during the synthesis of 1 are known: For example, European Pharmacopoeia lists two impurities, a 5-hydroxypentanoyl compound (impurity F) and 2-hydroxy-pentanoyl compound (impurity J). The latter is related to the 2-oxo-penanoyl impurity 2 described here.

Scheme 2.
Reported synthesis of terazosin 1 and the proposed mechanism for the formation of impurity 2

Conclusion
A hitherto unknown impurity in commercial samples of Terazosin was detected, isolated and its structure was elucidated by spectroscopy and independent synthesis. This contamination, 1-[4-(amino-6,7dimethoxyquinazolin-2-yl)-piperazin-1-yl]-pentane-1,2-dione 2 is likely to occur as the product of a sidereaction in the catalytic hydrogenation step during the synthesis of the drug. Biological screening showed this compound as not cytotoxic for several human tumor cell lines and non-malignant mouse fibroblasts.

General
Melting points are uncorrected (Leica hot stage microscope), NMR spectra were recorded using the Varian spectrometers Gemini 2000 or Unity 500 (δ given in ppm, J in Hz), MS spectra were taken on a Finnigan MAT LCQ 7000 (electrospray, voltage 4.5 kV, sheath gas nitrogen) instrument. IR spectra were taken on a Perkin Elmer Spectrum Two (ATR) and UV/vis on a Perkin Elmer Lambda 14 instrument. TLC was performed on silica gel (Merck 5554); elemental analyses were performed on a Vario EL (CHNS). The solvents were dried according to usual procedures. For HPLC-DAD investigations a LaChrom D-7000 HPLC-system (from Merck-Hitachi) was used at an operating temperature of 25°C using RP-18 Superspher column (125-2 mm, 4 mm) as the stationary phase.