Base-Mediated Oxidative Degradation of Secondary Amides Derived from p-Amino Phenol to Primary Amides in Drug Molecules

Article abstract of DOI:10.1016/j.xphs.2020.07.028

One of the most common functional groups encountered in drug molecules is the amide, and the most common degradation pathway for amides is base-mediated hydrolysis to its constituent amine and carboxylic acid. Herein, we report for the first time, a base-

Full text of DOI:10.1016/j.xphs.2020.07.028

Journal of Pharmaceutical Sciences 109 (2020) 3394-3403
Contents lists available at ScienceDirect
Journal of Pharmaceutical Sciences
journal homepage: www.jpharmsci.org
Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Base-Mediated Oxidative Degradation of Secondary Amides
Derived from p-Amino Phenol to Primary Amides in Drug
Molecules
,
Kausik K. Nanda a *, Anthony Ginnetti b, W. Peter Wuelfing a
a Discovery Pharmaceutical Sciences, MRL, Merck & Co., Inc., West Point, PA 19486, USA
b Discovery Chemistry, MRL, Merck & Co., Inc., West Point, PA 19486, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
One of the most common functional groups encountered in drug molecules is the amide, and the most
common degradation pathway for amides is base-mediated hydrolysis to its constituent amine and
carboxylic acid. Herein, we report for the first time, a base-mediated oxidative degradation pathway of
secondary amides to primary amides. This transformation also represents a novel synthetic methodology,
reported for the first time in this work, in transforming secondary amides to primary amides without
using any oxidative reagents. The introduction of this mechanism into the pharmaceutical literature is
important given that the mechanism and required reactants are present to carry out the chemistry in
dosage forms.
Received 20 May 2020
Revised 30 June 2020
Accepted 24 July 2020
Available online 3 August 2020
Keywords:
Drug degradation
Oxidative degradation
Amide degradation
Oxidation
© 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Introduction
In a recent drug development effort in our laboratories, a drug
substance containing an amide functionality showed an unusual
Degradation of the active pharmaceutical ingredient (API) in
drug products (DP) impacts the safety and efficacy of drugs, which
prompts pharmaceutical scientists to study and understand
degradation pathways. The most common degradation pathways
operating on API in DP are hydrolytic and oxidative.1 Mechanisti-
cally, oxidative degradation pathways are often more complex than
hydrolytic pathways. The commonly recognized reactive species in
oxidative degradation are alkoxy, hydroxy or hydroperoxy radicals,
peroxides, metal ions and singlet oxygen.1,2 Oxidation involving a
radical species follows a one-electron pathway, whereas peroxides
invoke a two-electron oxidation mechanism. Typically, the role of
metal ions in oxidative degradation is to catalyze the oxidation of
peroxides to peroxy/hydroxy/alkoxy radicals, which in turn oxidize
the API.1,2 It is also known that some oxidative degradation path-
ways involve electron transfer from transition metal ions.3,4
Recently we have shown a novel oxidative pathway involving
iron(III), where certain class of drug molecules can be oxidized in
the absence of peroxides.5
degradation product under accelerated stress in the solid state as
well as in solution forced stress studies under basic (NaOH) con-
ditions. The expected degradation products from an amide are
typically the corresponding carboxylic acid and amine, which are
formed under acid- or base-catalyzed hydrolysis. But, in this case,
the observed degradation product was a primary amide (Scheme
1).
To the best of our knowledge, this degradation pathway for
amides has not been reported in the drug degradation literature.
Also, to the best of our knowledge, conversion of a secondary amide
to a primary amide, effected under basic conditions without any
added oxidizing agent, has not been reported in the organic
chemistry literature. In this paper, we report the preliminary
findings elucidating the mechanism of this unique degradation
pathway.
Materials
Reagents and solvents, used for the synthesis and reactivity
studies, are reagent grade and purchased from commercial sources.
Solvents and trifluoracetic acid, used in chromatography and mass
spectrometry, are HPLC grade and purchased from commercial
sources.
* Corresponding author.
E-mail address: kausik_nanda@merck.com (K.K. Nanda).
https://doi.org/10.1016/j.xphs.2020.07.028
0022-3549/© 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

K.K. Nanda et al. / Journal of Pharmaceutical Sciences 109 (2020) 3394-3403
3395
Table 2
Mobile Phase Gradient Used in the LC/MS Analysis of Forced Stress Experiments.
Time (Minute)
%A (0.05% TFA in H2O)
%B (0.05% TFA in MeCN)
0
3.5
4
4.01
5
95
0
0
95
95
5
100
100
5
5
Scheme 1. Unusual degradation of a secondary amide to primary amide.
Acquity UPLC BEH C18 column (1 cm ꢁ 500 mm, 1.7
Waters Corp. (Milford, MA) and eluted with a linear gradient
delivered at a flow rate of 300 L/min by an Acquity Ultra-
mm) from
m
Experiment and Analysis
Performance Liquid Chromatography system (Waters Corp., Mil-
ford, MA). Mobile phases consisted of water/acetonitrile/TFA at a
ratio of 99.95%, 0%, and 0.05% (v:v:v) for solvent A and a ratio of 0%,
99.95%, and 0.05% (v:v:v) for solvent B. The following elution con-
ditions were set: 5% of solvent B for 0.1 min, followed by a linear
increase of B from 5 to 99% within 2 min. Column temperature was
maintained at 50 ꢀC. An SQD detector (Waters Corp., Milford, MA)
was used coupled to the UPLC system. The mass spectrometer was
equipped with an electrospray ion source. The capillary was set to
3.0 kV. The cone and extraction voltages were 20.0 eV and 3.0 eV,
respectively. Source and desolvation temperatures were 120 ꢀC and
400 ꢀC, respectively.
Synthetic compounds were purified by normal phase flash silica
chromatography using an automated purification system (ISCO)
using Redisep® disposable flash cartridges with peak detection at
254 nm. Alternatively, compounds were purified by preparative
reversed-phase HPLC using a Gilson 215 liquid handler equipped
with a Waters Sunfire® C18 column (150 ꢁ 30 mm I.D.) with a
linear 40 mL/min gradient over 20 min (90:10 to 5:95H2O con-
taining 0.1% trifluoroacetic acid:MeCN) and detection at 215 nm.
NMR spectra were recorded at 500 MHz for 1H on a Varian spec-
trometer in the stated solvent. The chemical shifts are given in ppm,
referenced to the deuterated solvent signal or tetramethylsilane.
The solution state forced stress experiments were carried out by
adding one part 1 N aqueous NaOH to nine part (by volume) sub-
strate solution (~0.1 mg/mL) in 1:1H2OeMeCN, and heating the
resulting solution at 60 ꢀC. Oxidative forced stress experiments
with azobisisobutyronitrile (AIBN) and hydrogen peroxide (H2O2)
were performed as described elsewhere.6
Reversed-phase chromatography for the analysis of the forced
stress experiments were carried out on an Agilent 1100 HPLC
(Agilent Technologies, Santa Clara, CA) equipped with a quaternary
pump, heated column compartment, diode array detector, and
autosampler. The chromatographic conditions included a Supelco
Ascentis Express C18 column of dimensions 100 mm ꢁ 3 mm and
2.7
min; injection volume was 5
m
m particle size held at 45 ꢀC; flow rate was maintained at 1 mL/
m
L; tray temperature was ambient;
detector wavelength was 254 nm; mobile phase A consisted of a
mixture of 0.05% trifluoroacetic acid (TFA) in water; mobile phase B
consisted of 0.05% TFA in acetonitrile. The mobile phase gradient is
shown in Table 1.
For the LC/MS analysis of the forced stress experiments, an
Agilent 1100 series HPLC with a quaternary pump, diode array
detector and autosampler was used to separate the components of
the reaction mixture prior to MS analysis. The chromatographic
conditions used in the experiment included: Supelco Ascentis Ex-
press C18 column of dimensions 100 mm ꢁ 3 mm and 2.7
particle size held at 45 ꢀC; flow rate maintained at 0.9 mL/min;
injection volume of 5
L; tray temperature at 25 ꢀC; detection
mm
Synthesis
m
wavelength at 254 nm with spectrum scan from 190 to 400 nm.
Mobile Phase A consisting of 0.05% TFA in water; and Mobile Phase
B consisting of 0.05% TFA in MeCN. The mobile phase gradient is
shown in Table 2. A Thermo Scientific LCQ Fleet MS was used in
series with the Agilent 1100 HPLC for mass detections. The LCQ
Fleet MS used ESI operated in positive ion mode. Capillary tem-
perature was set to 250 ꢀC, the source voltage set to 4.5 kV and
source current set to 100
mA.
For the LC/MS analysis of synthetic reactions and isolated syn-
thetic products, samples were injected (0.5
mL) onto a Waters
tert-Butyl 4-(4-((4-hydroxyphenyl)carbamoyl)phenyl)piperazine-1-
carboxylate (1A)
A mixture of 4-(4-(tert-butoxycarbonyl)piperazin-1-yl)benzoic
acid (250 mg, 0.816 mmol), 4-aminophenol (223 mg, 2.04 mmol),
Table 1
Mobile Phase Gradient Used in the Reversed-Phase Chromatography Analyzing
Forced Stress Experiments.
Time (Minute)
%A (0.05% TFA in H2O)
%B (0.05% TFA in MeCN)
diisopropylethylamine (428 mL, 2.45 mmol), and HATU (388 mg,
1.02 mmol) in DMF (4 mL) was stirred at rt. After 16 h, the mixture
was added to H2O and extracted with 2ꢁ EtOAc. The combined
organic layers were washed with water and saturated aqueous
NaCl, dried with MgSO4, filtered, and concentrated under reduced
pressure to afford crude 1A.
0
5
7
7.1
10
95
0
0
95
95
5
100
100
5
5

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K.K. Nanda et al. / Journal of Pharmaceutical Sciences 109 (2020) 3394-3403
N-(4-hydroxyphenyl)-4-(piperazin-1-yl)benzamide dihydrochloride
(1B)
(10 mL) was added sodium triacetoxyborohydride (1.36 g,
6.40 mmol) and the resulting mixture was stirred at rt. After
16 h, a saturated solution of aqueous sodium bicarbonate was
added. The resulting mixture was extracted with 2ꢁ EtOAc. The
combined organic layers were washed with saturated aqueous
NaCl, dried with MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by column chro-
matography over silica gel using 0e100% ethyl acetate gradient
in hexanes to afford 3A.
HCl gas was bubbled through a mixture of tert-butyl 4-(4-((4-
hydroxyphenyl)carbamoyl)phenyl)piperazine-1-carboxylate (1A,
209 mg, 0.526 mmol) in diethyl ether at 0 ꢀC for 20 s. The mixture
was allowed to warm to room temperature while stirring for 16h,
and then concentrated to afford crude 1B.
4-(4-Benzylpiperazin-1-yl)-N-(4-hydroxyphenyl)benzamide (1)
To a mixture of N-(4-hydroxyphenyl)-4-(piperazin-1-yl)benza-
mide dihydrochloride (1B, 190 mg, 0.513 mmol) and benzaldehyde
(1 mL, 9.87 mmol) in DMF (1 mL) was added sodium triacetox-
yborohydride (218 mg, 1.026 mmol) and the resulting mixture was
stirred at rt. After 16 h, a saturated solution of aqueous sodium
bicarbonate was added. The resulting mixture was extracted with
2ꢁ EtOAc. The combined organic layers were washed with satu-
rated aqueous NaCl, dried with MgSO4, filtered, and concentrated
under reduced pressure. The residue was purified by preparative
reversed-phase HPLC to afford the title compound as white solid.
4-(4-Benzylpiperazin-1-yl)benzoic Acid (3B)
To a mixture of ethyl 4-(4-benzylpiperazin-1-yl)benzoate (3A),
1.04 g, 3.20 mmol) in 1:1 MeOH:THF (32 mL) was added 1 M
aqueous sodium hydroxide (6.40 mL, 6.40 mmol) and the resulting
mixture was stirred at rt. After 16 h, 1 M aqueous HCl (6.40 mL,
6.40 mmol) was added and the resulting mixture was concentrated
under reduced pressure to afford 3B along with 2 equivalents of
sodium chloride.
ESI MS m/z: 388.4 ([M þ H]þ). 1H NMR (500 MHz, DMSO‑d6)
d 9.70
(s, 1H), 9.17 (s, 1H), 7.83 (d, J ¼ 8.9 Hz, 2H), 7.50 (d, J ¼ 8.9 Hz, 2H),
7.34 (d, J ¼ 4.6 Hz, 4H), 7.31e7.23 (m, 1H), 6.98 (d, J ¼ 9.0 Hz, 2H),
6.71 (d, J ¼ 8.9 Hz, 2H), 3.54 (s, 2H), 3.31e3.26 (m, 4H), 3.17 (d,
J ¼ 5.3 Hz, 2H).
4-(4-Benzylpiperazin-1-yl)-N-(3-hydroxyphenyl)benzamide (3)
A mixture of 4-(4-benzylpiperazin-1-yl)benzoic acid diso-
dium chloride (3B, 209 mg, 0.506 mmol), 3-aminophenol
(138 mg, 1.26 mmol), diisopropylethylamine (442
mL,
2.53 mmol), and HATU (240 mg, 0.632 mmol) in DMF (1.7 mL)
was stirred at rt. After 16 h, the mixture was purified by pre-
parative reversed-phase HPLC to afford 3 as white solid. ESI MS
m/z: 388.5 ([M þ H]þ). 1H NMR (500 MHz, DMSO‑d6)
d 9.78 (s,
1H), 9.32 (s, 1H), 7.84 (d, J ¼ 8.9 Hz, 2H), 7.35 (d, J ¼ 4.5 Hz,
5H), 7.30e7.24 (m, 1H), 7.14 (d,
J ¼ 8.0 Hz, 1H), 6.99 (d, J ¼ 8.9 Hz, 2H), 6.46 (d, J ¼ 7.9 Hz, 1H),
J
¼
8.0 Hz, 1H), 7.08 (t,
3.54 (s, 2H), 3.29 (m, 4H), 2.53 (m, 4H).
4-(4-Benzylpiperazin-1-yl)-N-(4-methoxyphenyl)benzamide (2)
Compound 2 was prepared according to the procedure from 4-
(4-benzylpiperazin-1-yl)-N-(4-hydroxyphenyl)benzamide (1) us-
ing 4-methoyxaniline. ESI MS m/z: 402.5 ([M þ H]þ). 1H NMR
(500 MHz, DMSO‑d6)
d
9.80 (s, 1H), 7.85 (d, J ¼ 9.0 Hz, 2H), 7.65 (d,
J ¼ 9.1 Hz, 2H), 7.35 (d, J ¼ 4.6 Hz, 4H), 7.27 (q, J ¼ 4.7 Hz, 1H), 6.99
(d, J ¼ 9.0 Hz, 2H), 6.90 (d, J ¼ 9.1 Hz, 2H), 3.74 (s, 3H), 3.54 (s, 2H),
3.31e3.26 (m, 4H), 2.52 (m, 4H).
4-(4-Benzylpiperazin-1-yl)benzamide (4)
A mixture of 4-(4-benzylpiperazin-1-yl)benzoic acid disodium
chloride (3B, 106 mg, 0.257 mmol), ammonium bicarbonate (41 mg,
0.513 mmol), pyridine (42
mL, 0.513 mmol), and BOC-anhydride
(119 L, 0.513 mmol) in dioxane (0.64 mL) was stirred at rt. After
m
16 h, the mixture was concentrated under reduced pressure. The
residue was purified by column chromatography over silica gel
using 0e10% MeOH gradient in CH2Cl2 to afford 4 as solid. ESI MS m/
z: 296.3 ([M þ H]þ). 1H NMR (500 MHz, Chloroform-d)
d 7.73 (d,
J ¼ 8.9 Hz, 2H), 7.40e7.33 (m, 4H), 7.30 (dd, J ¼ 5.8, 2.7 Hz, 1H), 6.89
(d, J ¼ 8.9 Hz, 2H), 3.59 (s, 2H), 3.37e3.30 (m, 4H), 2.66e2.58 (m,
4H).
Ethyl 4-(4-benzylpiperazin-1-yl)benzoate (3A)
To a mixture of ethyl 4-(piperazin-1-yl)benzoate (750 mg,
3.20 mmol) and benzaldehyde (1.62 mL, 16.0 mmol) in DMF

K.K. Nanda et al. / Journal of Pharmaceutical Sciences 109 (2020) 3394-3403
3397
tert-Butyl 4-(4-(methoxycarbonyl)-3-(trifluoromethyl)phenyl)
piperazine-1-carboxylate (5A)
stirred at rt. After 4 h, conc HCl (96
the resulting mixture was concentrated under reduced pressure to
m
L, 1.17 mmol) was added and
A mixture of methyl 4-bromo-2-(trifluoromethyl) benzoate
(300 mg,1.06 mmol), tert-butyl piperazine-1-carboxylate, XPhos Pd
GII (167 mg, 0.212 mmol), and cesium carbonate (863 mg,
2.65 mmol) in toluene (5.3 mL) was stirred at 120 ꢀC. After 16 h, The
mixture was concentrated under reduced pressure. The residue was
purified by column chromatography over silica gel using 0e100%
ethyl acetate gradient in hexanes to afford 5A as white solid.
afford 5D.
4-(4-Benzylpiperazin-1-yl)-N-(4-hydroxyphenyl)-2-
(trifluoromethyl)benzamide dihydrochloride (5)
To a mixture of N-(4-hydroxyphenyl)-4-(piperazin-1-yl)-2-(tri-
fluoromethyl)benzamide dihydrochloride (5D,149 mg, 0.340 mmol)
and benzaldehyde (0.726 mL, 7.16 mmol) in DMF (0.7 mL) was added
sodium triacetoxyborohydride (144 mg, 0.680 mmol) and the
resulting mixture was stirred at rt. After 16 h, a saturated solution of
aqueous sodium bicarbonate was added. The resulting mixture was
extracted with 2ꢁ EtOAc. The combined organic layers were washed
with saturated aqueous NaCl, dried with MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by
column chromatography over silica gel using 0e10% MeOH gradient
in CH2Cl2 containing 0.1% NH4OH to afford white solid. The solid was
4-(4-(tert-Butoxycarbonyl)piperazin-1-yl)-2-(trifluoromethyl)
benzoic Acid (5B)
To
a
mixture of tert-butyl 4-(4-(methoxycarbonyl)-3-(tri-
(5A, 227 mg,
fluoromethyl)phenyl)piperazine-1-carboxylate
0.584 mmol) in 1:1 MeOH:THF (5.8 mL) was added 1 M aqueous so-
dium hydroxide (1.169 mL, 1.17 mmol) and the resulting mixture was
stirred at 70 ꢀC. After 7 h, 1 M aqueous HCl (1.17 mL, 1.17 mmol) was
added and the resulting mixture was concentrated under reduced
pressure to afford 5B along with 2 equivalents of sodium chloride.
dissolved in 1:1 CH2Cl2:MeOH and conc. HCl (84 mL, 5.84 mmol) was
added. The resulting mixture was concentrated under reduced
pressure to afford 5 as white solid. ESI MS m/z: 456.3 ([M þ H]þ). 1H
NMR (500 MHz, DMSO‑d6)
d 11.23 (s, 1H), 10.09 (s, 1H), 7.64 (dd,
tert-Butyl 4-(4-((4-hydroxyphenyl)carbamoyl)-3-(trifluoromethyl)
phenyl)piperazine-1-carboxylate (5C)
A mixture of 4-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(tri-
fluoromethyl)benzoic acid disodium chloride (5B, 287 mg,
0.584 mmol), 4-aminophenol (127 mg, 1.17 mmol), diisopropyle-
J ¼ 6.4, 2.8 Hz, 2H), 7.54 (d, J ¼ 8.5 Hz,1H), 7.51e7.47 (m, 3H), 7.45 (d,
J ¼ 8.9 Hz, 2H), 7.33e7.24 (m, 2H), 6.72 (d, J ¼ 8.8 Hz, 2H), 4.39 (d,
J ¼ 4.9 Hz, 2H), 4.03 (d, J ¼ 13.4 Hz, 2H), 3.38 (d, J ¼ 11.7 Hz, 2H), 3.30
(t, J ¼ 12.5 Hz, 2H), 3.23e3.07 (m, 2H).
thylamine (306 mL, 1.75 mmol), and HATU (278 mg, 0.730 mmol) in
DMF (2.92 mL) was stirred at rt. After 16 h, the mixture was poured
4-(4-Benzylpiperazin-1-yl)-N-(4-hydroxyphenyl)-2-
into water, and the resulting mixture was extracted with 2ꢁ EtOAc.
The combined organic layers were washed with saturated aqueous
NaCl, dried with MgSO4, filtered, and concentrated under reduced
pressure. The residue was purified by column chromatography over
silica gel using 0e100% ethyl acetate gradient in hexanes to afford 5C.
methoxybenzamide dihydrochloride (6)
Compound 6 was prepared according to the procedure for 4-(4-
benzylpiperazin-1-yl)-N-(4-hydroxyphenyl)-2-(trifluoromethyl)
benzamide dihydrochloride (5) using methyl 4-bromo-2-
methoxybenzoate. ESI MS m/z: 418.4 ([M
(500 MHz, DMSO‑d6)
þ
H]þ). 1H NMR
d
11.00 (s, 1H), 9.64 (s, 1H), 7.75 (d, J ¼ 9.3 Hz,
N-(4-hydroxyphenyl)-4-(piperazin-1-yl)-2-(trifluoromethyl)
benzamide dihydrochloride (5D)
A mixture of tert-butyl 4-(4-((4-hydroxyphenyl)carbamoyl)-3-
(trifluoromethyl)phenyl)piperazine-1-carboxylate (5C, 272 mg,
1H), 7.63 (dd, J ¼ 6.5, 2.8 Hz, 2H), 7.53e7.45 (m, 5H), 6.72 (d,
J ¼ 8.8 Hz, 2H), 6.66 (d, J ¼ 5.7 Hz, 2H), 4.40 (d, J ¼ 5.0 Hz, 2H), 4.04
(d, J ¼ 13.4 Hz, 2H), 3.97 (s, 3H), 3.38 (d, J ¼ 11.9 Hz, 2H), 3.28 (t,
J ¼ 12.1 Hz, 2H), 3.19e3.07 (m, 2H).
0.584 mmol) and TFA (450 mL, 5.84 mmol) in CH2Cl2 (5.8 mL) was

3398
K.K. Nanda et al. / Journal of Pharmaceutical Sciences 109 (2020) 3394-3403
4-(4-Benzylpiperazin-1-yl)-N-(4-hydroxyphenyl)-3-
4-(4-Benzylpiperazin-1-yl)-3-fluorobenzoic Acid Hydrochloride
methoxybenzamide dihydrochloride (7)
(8A)
Compound 7 was prepared according to the procedure for 4-(4-
benzylpiperazin-1-yl)-N-(4-hydroxyphenyl)-2-(trifluoromethyl)
benzamide dihydrochloride (5) using methyl 4-iodo-3-
To a mixture of 3-fluoro-4-(piperazin-1-yl)benzoic acid hy-
drochloride (231 mg, 0.886 mmol) and benzaldehyde
(0.449 mL, 4.43 mmol) in DMF (4.4 mL) was added sodium
triacetoxyborohydride (376 mg, 1.77 mmol) and the resulting
mixture was stirred at rt. After 16 h, the mixture was purified
by preparative reversed-phase HPLC to afford the product as
white solid.
methoxybenzoate. ESI MS m/z: 418.4 ([M
þ
H]þ). 1H NMR
(500 MHz, DMSO‑d6) 11.17 (s, 1H), 9.90 (s, 1H), 7.71e7.63 (m, 2H),
d
7.57 (d, J ¼ 8.3 Hz, 1H), 7.54e7.42 (m, 6H), 7.00 (d, J ¼ 8.3 Hz, 1H),
6.74 (d, J ¼ 8.8 Hz, 2H), 4.48e4.34 (m, 2H), 3.88 (s, 3H), 3.62 (d,
J ¼ 11.9 Hz, 2H), 3.38 (d, J ¼ 11.1 Hz, 2H), 3.29e3.06 (m, 4H).
Fig. 1. Structures of molecules synthesized.

K.K. Nanda et al. / Journal of Pharmaceutical Sciences 109 (2020) 3394-3403
3399
Results
0.56
0.42
0.28
0.14
0.00
A typical forced degradation experiment run with 5 in 0.1 N
NaOH at 60 ꢀC for 4 h shows the formation of the unexpected
degradation product 9 (Fig. 2 and Scheme 2). LC/MS of the peaks at
4.049 min and 4.484 min corresponds to the degradation product 9
and parent 5, respectively, as shown in Scheme 2.
Fig. 3 plots the base-mediated degradation of compounds 1, 5, 6,
7 and 8 over 24 h (time points: 4, 8, 12 and 24 h). Note that com-
pounds 1, 7 and 8 degrade completely between 12 h and 24 h time
points. The effect of varying concentration of NaOH on the degra-
dation of 1 is shown in Fig. 4. The amount of degradation product
formed increases linearly with increase in the concentration of
NaOH (0e0.1 N).
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Minutes
Fig. 2. Chromatogram from basic forced stress experiment, run in 0.1 N NaOH at 60 ꢀ
for 4 h, of 5.
C
Identity of degradation products was established using the
observed mass from LC/MS analysis of the basic forced stress ex-
periments confirming the amide and not the carboxylic acid product.
In one case, an authentic sample of 4 was synthesized and its UV
spectrum, mass and retention time from chromatography were
compared with those from the degradation product formed in the
basic forced stress of 1 (Scheme 3). UV spectrum, mass and chro-
matographic retention time of the degradation product in Scheme 3
are found to be identical to those of 4. Fig. 5 shows the overlay of
chromatograms of the basic forced stress experiment of 1, compound
4, and the spiking of the basic forced stress experiment of 1 with 4.
4-(4-Benzylpiperazin-1-yl)-3-fluoro-N-(4-hydroxyphenyl)
benzamide bis(2,2,2-trifluoroacetate) (8)
A mixture of 4-(4-benzylpiperazin-1-yl)-3-fluorobenzoic acid
hydrochloride (8A, 75 mg, 0.214 mmol), 4-aminophenol (70 mg,
0.641 mmol), diisopropylethylamine (187 mL,1.07 mmol), and HATU
(102 mg, 0.267 mmol) in DMF (1 mL) was stirred at rt. After 16 h,
the mixture was purified by preparative reversed-phase HPLC to
afford the title compound as off-white solid. ESI MS m/z: 406.4
([M þ H]þ). 1H NMR (500 MHz, Methanol-d4)
d
7.74 (dd, J ¼ 23.2,
11.0 Hz, 2H), 7.60e7.51 (m, 5H), 7.43 (d, J ¼ 8.8 Hz, 2H), 7.17 (t,
J ¼ 8.5 Hz, 1H), 6.79 (d, J ¼ 8.9 Hz, 2H), 4.40 (s, 2H), 3.78e3.35 (m,
5H), 3.31e3.01 (m, 3H).
Discussion
Structures of molecules synthesized are shown collectively in
Fig. 1.
One of the most common structural motifs in drug molecules is
the amide functional group, and the hydrolytic cleavage is a
Scheme 2. Basic forced stress reaction of 5.
90
80
70
60
50
40
30
20
10
0
1
5
6
7
8
0
5
10
15
20
25
hour
Fig. 3. Base-mediated degradation in 0.1 N NaOH at 60 ꢀC.

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K.K. Nanda et al. / Journal of Pharmaceutical Sciences 109 (2020) 3394-3403
humidity studies. In the process of evaluating the stability of a
35
30
25
20
15
10
5
development candidate containing a secondary amide group, in our
laboratories, we encountered unexpected results. Under both basic
solution forced stress studies on the API and accelerated stress solid
state studies on the formulated API, a primary amide degradation
product was formed, instead of the corresponding carboxylic acid
(Scheme 1). To study this unusual degradation pathway further, we
synthesized molecules with similarities to the development com-
pound (secondary amide) but utilized commonly available syn-
thons to build a variety of structures to probe the mechanism as
shown in Fig. 1.
0
0
0.02
0.04
0.06
0.08
0.1
0.12
The degradation profile resulting from basic forced stress of 1 is
quite different from 2 and 3 e while compound 1 degrades to yield
the degradate 4, 2 and 3 do not degrade at all under the same
condition (Scheme 5). Furthermore, compound 1 does not degrade
[NaOH] (N)
Fig. 4. Effect of the concentration of NaOH on the rate of degradation of 1. Datapoints
collected at 5.5 h.
Scheme 3. Basic forced stress reaction of 1.
0.180
0.135
0.090
0.045
0.000
(Basic forced stress experiment with 1) + compound 4
Compound 4
Basic forced stress experiment with 1
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
Minutes
Fig. 5. Overlay of chromatograms from the basic forced stress experiment of 1, compound 4 and spiked forced stress experiment of 1 with 4.
prevalent degradation pathway for amides in drug molecules. Hy-
drolysis of the amide functional group results in the constituent
carboxylic acid and amine, and the hydrolysis is usually mediated
by base, as shown in Scheme 4 for a secondary amide.
under acidic forced stress conditions (with 0.1 N HCl).
This differential reactivity of 1 from 2 and 3 indicates that a free
phenolic hydroxy group at the p-position (to the amide function-
ality) on the phenyl ring is essential for this degradation pathway to
Scheme 4. Base-mediated hydrolysis of secondary amides.
Susceptibility to hydrolytic degradation of drug molecules is
determined by basic/acidic forced degradation experiments1 and
also by accelerated stress solid state elevated temperature and
operate e methylation of the phenolic hydroxy group in 2 or
moving the phenolic hydroxy group from the p-to m-position in 3
shuts down the degradation pathway.

K.K. Nanda et al. / Journal of Pharmaceutical Sciences 109 (2020) 3394-3403
3401
Running the basic forced stress experiment on 1 under N2-at-
mosphere almost completely suppresses the formation of the
degradate 4, indicating participation of molecular oxygen in the
degradation pathway. Moreover, under oxidative forced stress
conditions, using AIBN or H2O2, 1 does not degrade to 4.
Based on the above observations, we propose a degradation
mechanism as shown in Scheme 6. The first step in this mechanistic
pathway is the base-mediated deprotonation of the p-hydroxy
hydroperoxide E. Elimination of H2O2 from E, followed by hydro-
lysis, gives the primary amide.
In an effort to understand the electronic effects on the rate of
degradation, compounds 5e8 were synthesized, where the central
phenyl ring has been substituted with the electron-donating -OMe
group or the electron-withdrawing eCF3 or eF groups. Assuming
first order kinetics, the rate of degradation has been determined as
shown in Fig. 6, and the results are tabulated in Table 3. The rates of
Scheme 5. Reactivities of 1 under basic and acidic forced stress, and 2 and 3 under basic forced stress.
group on the phenyl ring. One of the resonance forms of phenolate
A is the carbanion B, which then transfers an electron to dissolved
molecular oxygen (triplet electronic state, 3O2) to form the C-
centered radical, C and superoxide anion.7 Spin inversion of the
superoxide anion, followed by combination with C forms the per-
oxy anion D.8,7 Protonation of the peroxy anion D gives the
degradation have been found to be 6 < 5 < 1 ¼ 7 ¼ 8. All three
compounds, 1, 7 and 8, are seemingly unaffected by the electronic
effects of substituents on the central phenyl ring e 1 is unsub-
stituted, 7 is substituted with an electron-donating -OMe, and 8 has
an electron-withdrawing eF substituent. Interestingly, 5 and 6,
showing different rates of degradation from each other and lower
Scheme 6. Proposed mechanism for the oxidative degradation of secondary amides to primary amides under basic conditions.

3402
K.K. Nanda et al. / Journal of Pharmaceutical Sciences 109 (2020) 3394-3403
5
4.5
4
y = -0.0166x + 4.6
R² = 0.9712
1
5
6
7
8
y = -0.036x + 4.591
R² = 0.9576
y = -0.1264x + 4.7695
R² = 0.9342
3.5
3
y = -0.1333x + 4.7126
R² = 0.9646
y = -0.1281x + 4.7001
R² = 0.9775
2.5
0
5
10
15
20
25
hour
Fig. 6. First order rate plot for degradation of Compounds 1, 5, 6, 7, and 8.
rates of degradation than 1, 7 and 8, contain the electron-
withdrawing eCF3 group and electron-donating -OMe group,
respectively. These data do not point towards a trend where the
rate of degradation is dependent on the electronics of the central
phenyl ring. However, from a spatial point of view (i.e., the position
of the substituents), electronics of the substituents proximal to the
amide group (electron-withdrawing eCF3 in 5 and electron-
donating -OMe in 6) appear to influence the rate of degradation,
whereas electronics of the substituents distal to the amide group
(neutral eH in 1, electron-donating -OMe in 7, and electron-
withdrawing eF in 8) do not affect the rate. More work will be
required to better understand this effect.
Conclusions
In this work, we showed a novel oxidative degradation pathway
for amide functional groups in drug molecules, under basic con-
ditions in solution state and under accelerated stress on solid state
formulated API. A series of molecules have been synthesized to
probe the degradation mechanism. Although data from this work
indicate effect of electronic factors on the rate of degradation, more
work is needed to arrive at a better understanding of such factors.
This work also presents a novel methodology in organic synthesis,
whereby secondary amides can be transformed into synthetically
valuable primary amides without the use of oxidizing reagents.
Amides are present in many drug molecules9 and are useful
synthons in organic transformations.10e13Thus, a new methodology
involving easy conversion of secondary or tertiary amides to their
corresponding primary amides has important implication in phar-
maceutical degradation chemistry and synthetic organic chemistry.
In published literature, these transformations have been accom-
plished with the use of oxidizing reagents.14e17 A recent publica-
tion18 reports the transformation of tertiary amides to primary
amides using (NH4)2CO3. However, as of the time of publication, we
are unaware of any report in published literature detailing the
transformation of a secondary amide to the corresponding primary
amide without using any oxidative reagents. The novel degradation
pathway reported in this paper can be used as a new methodology
for the synthesis of primary amides under mild basic conditions.
Furthermore, this transformation establishes the utility of 4-
hydroxyphenyl as an easily removable protecting group for pri-
mary amides in organic synthesis.
Acknowledgment
We thank Shawn Stachel for helpful discussions during the
preparation of the manuscript.
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3.6 ꢁ 10ꢂ2
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