Identification of unexpected unlabeled N,N-dimethylamide formation in the synthesis of deuterated fragment of ribociclib by a HATU-mediated coupling reaction

Article abstract of DOI:10.1002/jlcr.3690

Starting from N,N-dimethylamine and D₂O, deuterated fragment of ribociclib was synthesized for use as a mass spectroscopy internal standard. Furthermore, systematic studies on D₀ (unlabeled material) formation during the amidation reaction were performed, leading to the identification of a coupling reagent, HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate), as main cause. Finally, an alternative route was designed using EDCI/HOBT as coupling reagents to produce the desired deuterated compound without D₀ residue.

Full text of DOI:10.1002/jlcr.3690

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Identification of unexpected unlabeled N,N-dimethylamide formation in
the synthesis of deuterated fragment of ribociclib by a HATU-mediated
coupling reaction
Kuo Gai, Baomin Liu and Yinsheng Zhang*
Jiangsu Key Laboratory of Targeted Antiviral Research, Chia Tai Tianqing Pharmaceutical
Group Co., LTD, 699-8 Xuanwu Ave. Building #9, Nanjing 210023, Jiangsu Province, China
*Correspondence to : email: zys@cttq.com; tel: 01186-025-68551568;
Address: Jiangsu Key Laboratory of Targeted Antiviral Research, Chia Tai Tianqing
Pharmaceutical Group Co., Ltd, 699-8 Xuanwu Ave. Building #9, Nanjing 210023, Jiangsu
Province, China.
Abstract
Starting from N,N-dimethylamine and D2O, deuterated fragment of ribociclib was
synthesized for use as an mass spectroscopy (MS) internal standard. Furthermore, systematic
studies on D0 (unlabeled material) formation during the amidation reaction were performed,
leading
to
the
identification
of
a
coupling
reagent,
HATU
(O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate), as main
cause. Finally, an alternative route was designed using EDCI/HOBT as coupling reagents to
produce the desired deuterated compound without D0 residue.
Keywards: HATU, amidation, deuteration, N,N-dimethylamide, internal standard
Introduction
The N,N-dimethylamide motif is widely distributed in marketed drugs, such as bambuterol,
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camazepam, camostat, cariprazine, fimasartan, loperamide and ribociclib (Figure 1). The
most common method for synthesis of the N,N-dimethylamide moiety could be dehydration
condensation reaction of N,N-dimethylamine with carboxylic acid in the presence of
condensation reagent. 1-4 In one of our projects, a key intermediate of ribociclib was required
for use as a deuterated internal standard in further drug development.5, 6 Then, a synthetic
approach of HATU-mediated coupling reaction was designed (Scheme 1), and 3 was
successfully synthesized in 88% yield.7 However, trace amounts of 3’ (Mw = 292) was
detected in the product 3 based on MS analysis, which didn’t meet the criteria for use as a
high-qualified deuterated internal standard because of the D0 residue.7 Therefore, a thorough
investigation was performed in order to explore the cause of D0 formation, and the result is
reported herein.
Figure 1. Marketed drugs containing N,N-dimethylamide motif
Scheme
1. Synthesis of 3 by HATU-mediated coupling reaction. Reagents and conditions: (a) [D6] dimethylamine
hydrochloride 2, HATU, DIPEA, DMF, r.t., 1 h, 88% yield.
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Results and discussions
Firstly, we suspected that our own-prepared [D6]dimethylamine hydrochloride 2 might
contain trace amounts of [D0]dimethylamine due to the uncompleted deuteration since we
started with unlabeled [D0]dimethylamine hydrochloride to synthesize the [D6]dimethylamine
hydrochloride via a 3-step synthetic sequence: N-nitroso formation, base-catalyzed H-D
exchange and DCl-promoted hydrolysis as depicted in Scheme 2.
Scheme 2. Synthesis of [D6] dimethylamine hydrochloride (2) and control experiment. Reagents and
conditions: (a) NaNO2, 37% HCl, r.t., 17 h, 22% yield; (b) CH3ONa, D2O, 80 oC, 5 h, 2 cycles, 50% yield;
(c) 36% DCl, 90 oC, 16 h, 48% yield.
However, when 2 was subjected to MS analysis, no D0 molecular weight (Mw = 46) was
detected (see Scheme 2A). Also, the control experiment by acylation of 2 with benzoyl
chloride showed no D0 formation (Mw = 149) based on MS (see Scheme 2B). Repeated
preparation of deuterated N,N-dimethylamine 2 was conducted for a few times, and the
results that D0 was detected in the synthesis of 3 were identical after coupling with 1 using
HATU as a coupling reagent and N,N-dimethyl formamide (DMF) as a solvent (Table 1,
entry 1). Therefore, we speculated that the unexpected [D0] dimethylamine formation could
result from an intermediate produced during the process of HATU-mediated coupling
reaction, which rules out the possibility of starting material source 2.
Next, the possibilities of [D0]dimethylamine coming from solvents and bases were
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investigated. We wondered if there was [D0]dimethylamine residue in the solvent
N,N-dimethyl formamide (DMF). Thus, dicloromethane (DCM) was used as solvent instead
of DMF. However, trace amount of 3’ (D0) was still detected in the reaction mixture (entry 2).
The result ruled out the possibility of dimethylamine-contaminated DMF. Similarly, we
switched N,N-diisopropylethylamine (DIPEA) to N-methylmorpholine (NMM) as base to
find out whether dimethylamine residue existed in DIPEA (entry 3). Using NMM as a base,
the reaction became sluggish, and more impurities were observed in the reaction mixture.
However, LC-MS analysis still indicated the D0 formation in the synthesis of 3. .
Table 1. Reaction conditions screeninga
aThe
reaction was performed with 1 (0.1 mmol), 2 (0.15 mmol), reagent (0.12 mmol), base (0.2 mmol) in
c
solvent (1 mL) at r.t. for 1 h.b purchased from J&K, Lot: LOB0Q86 purhcased from Alfa Aesar, Lot:
5019F05S; dThe percentage was calculated based on the intensity relative to D6.
Based on the screening study above, we concluded that the issue could be attributed to
the use of HATU. To test this hypothesis, an alternative choice of coupling reagents, namely
EDCI and HOBT, were chosen instead of HATU (entry 4). Indeed, no D0 formation was
observed by MS. Furthermore, to test whether this batch of HATU (purchased from Energy
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