Lupine Publishers | Trends in Ophthalmology Open Access Journal
Abstract
This is out line work on the process
development for the synthesis of buprenorphine, naltrexone, naloxone, and
nalbuphine from naturally occurring opiates such as Thebaine, also known as
codeine methyl enol ether, and oripavine. Several new methods for
N-demethylation of morphinans have been developed during the pursuit of this
research. The article traces the evolution of various approaches and provides a
comparison for overall efficiency. A reverse phase high performance liquid
chromatographic (RPHPLC) method has been developed and validated for
simultaneous estimation of Naloxone Hydrochloride and Buprenorphine Hydrochloride
in pure and marketed formulations. Separation was carried out using column
Hypersil ODS C18 (250 mm x 4.6mm x 5μm particle size) in isocratic mode using
mobile phase composition pH 6.0 ammonium acetate Buffer: Acetonitrile
(68:32)v/v and UV detection at 310nm. The compounds were eluted at a flow rate
of 1.0mL/ min. The average retention times for Naloxone and Buprenorphine were
2.86 and 3.67 min, respectively. The method was validated according to the ICH
guidelines. The percentage RSD of all validation parameters found to be less
than 2% indicating high degree of accuracy and precision of the proposed HPLC
method. The method was linear over the concentration of 5.30μg/ml and
20-120μg/ml for Naloxone and Buprenorphine respectively. The LOD and LOQ of Naloxone
were found to be 0.08μg/mL and 0.26μg/mL and of Buprenorphine were found to be
0.0078μg/mL and 0.0237μg/mL. The drugs were also exposed to acidic, alkaline,
oxidative, thermal and photolytic conditions and the stressed samples were
analyzed by the proposed method. Degradation studies showed that the both the
drugs were highly stable under acidic, oxidative, thermal and photolytic
conditions. Under alkaline conditions, RT values were shifted to lower as
compared to standard without any additional peaks. The high percentage of
stability under stress conditions confirms the suitability of the method for
simultaneous estimation of Naloxone Hydrochloride and Buprenorphine
Hydrochloride in pure and marketed formulations.
Keywords: Analgesics, Opiate-derived pharmaceuticals, Morphine
alkaloids, Buprenorphine hydrochloride, Naloxone hydrochloride, RP-HPLC,
Validation and degradation studies, ICH Guidelines semisynthesis, Process
development
Abbrevations: LOD: Limit of Detection, LOQ: Limit of Quantification,
RPHPLC: Reverse Phase High Performance Liquid Chromatographic Method
Introduction
Buprenorphine, Naltrexone, Naloxone,
and Nalbuphine have been used for centuries for their analgesic effects and are
considered to be the most commonly used pharmacologic agents for the management
and treatment of moderate to severe pain. Although these agents are considered
safe when used properly and under physician supervision, it is important to
understand the risks and benefits associated with prescribing the agents in
this pharmacologic class.
Buprenorphine
Synthesis Method:
The hydrogenation of
7-acetyl-6,14-endoetheno-6,7,8,14- tetrahydrothebaine
a) With H2 over Pd/C in acetic acid
gives the corresponding endo-ethano derivative
b) Which By a Grignard reaction with
tertbutyl-magnesium chloride
i. In ether-benzene yields
7alpha-(2-hydroxy-3,3-dimethyl- 2-butyl)-6,14-endo-ethano-6,7,8,14
-tetrahydrothebaine
c) The reaction of III with BrCN in
CH2Cl2 affords 7alpha- (2-hydroxy-3,3-dimethyl-2-butyl)-6,14-endo-ethano-Ncyano-
6,7,8 ,14-tetrahydronorthebaine
d) Which is treated with KOH in
ethylene glycol at 170 C to give
7alpha(2-hydroxy-3,3-dimethyl-2-butyl)-6,14-endoethano- 6,7,
8,14-tetrahydronorthebaine
e) This compound is treated first
with cyclopropylcarbonyl chloride
ii. In CH2Cl2 containing
triethylamine, followed by a reduction with LiAlH4 in refluxing THF yielding
N-cyclopropylmethyl- 7alpha-(2-hydroxy-3,3- Dimethyl-2-butyl)-6,14-endo-ethano-
6,7,8,14-tetrahydro-northebaine
f) Finally,
g) Is demethylated with KOH in
diethyLene glycol at 210- 220 0C.
Drugs
Fut 1977, 2, (9): 570
All medicinal opiate agents in use
today are derived from naturally occurring morphine alkaloids by semisynthesis,
as shown in an abbreviated fashion in (Figure 1) [1]. Two important
transformations are required: the first is the oxidation of the C-14 position
to C-14 hydroxyl and the second is the replacement of the N-methyl
functionality with other alkyl groups. The former process has been reduced to
practice in several efficient ways, while the latter suffers from the use of
toxic reagents and multistep operations [2]. The global production figures for
various analgesics and antagonists derived from natural morphinans are shown in
(Figure 2a) Clearly the scale of semisynthesis requires that efficient and
environmen- tally benign manufacturing protocols be developed. Crucial to all
procedures are the methods employed for N- and O-demethylation of the alkaloids
or any advanced synthetic intermediates. In this review, we trace several
generations of improvements in the preparation of buprenorphine, naltrexone,
methyl naltrexone, naloxone, and nalbuphine. In all of these projects, new
demeth- ylation protocols have been developed and major improvements in
efficiency have been attained (Figure 2b).
The First Generation
The first project undertaken was
aimed at improvement of the commercial synthesis of buprenorphine. This route
takes eight steps form Thebaine, also known as codeine methyl enol ether, as
shown in (Scheme 1.2) It should be noted that five of the eight steps are
required for N- and O-demethylation reactions. The former process involves the
von Braun reaction [3] with cyanogen bromide, which poses toxicity issues. We
have shortened the synthesis by starting with oripavine and performing
selective N-demethylation of the diastereomeric salts 15 generated by
alkylation of oripavine with cyclopropylmethyl bromide. The N-demethylation with
sodium thiolates (produced from the thiol with either MeONa or tBuONa)
proceeded in agreement with previously published protocols [4] to afford
cleanly N-cyclopropylmethyl nororipavine 16 in yields of 53%-67% [5]. We noted
that the two diasteroemers of 15 reacted at different rates.
The conversion of 16 to
buprenorphine was carried out in three steps in analogy to those employed in
the commercial route from Thebaine, also known as codeine methyl enol ether,
namely Diels- Alder cycloaddition, hydrogenation, and Grignard addition. Three
routes were examined for the conversion of 16 to buprenorphine. The first was
the conversion of 16 to ketone 18 and direct treatment of 18, without the
protection of the phenol, with the Grignard reagent to produce buprenorphine in
~30% yield (Scheme 2). In the other two routes the phenol was protected as a
carbonate at the stage of either 18 or 19. The two routes intersected at 21,
which was converted to buprenorphine in two steps, with the route through 18
providing a better overall yield [6,7].
Literature survey reveals that there
were number of analytical methods available for both the drugs alone or in
combination with other drugs including spectroscopy, chromatographic methods
such as gas chromatography with electron-capture or mass spectrometry detection
and HPLC with fluorescence electrochemical or mass spectrometry detection
[8-13] are reported, but there is no method established for the stability
indicating RP-HPLC under stress for this combination. The present work describes
the development of stability indicating RP-HPLC method, which can quantify
these components simultaneously from a combined dosage form. The present
RP-HPLC method was validated [14-15] and applied under stressed conditions
according to (ICH) guidelines. ICH has made the mandatory need of developing
stability indicating assay methods for every drug candidates. Stability
indicating assay methods helps in establishing the inherent stability of the
drug which provides assurance on detection changes in identity, purity and
potency of the product on exposure to various conditions [16]. So an attempt
has been made to develop a method under stress conditions like acidic, basic,
thermal, photolytic and oxidative, this which in turn can help in establishing the
degradation pathways and the intrinsic stability of the molecules. The object
of the present work was to develop a stability indicating method for the
simultaneous estimation of Naloxone hydrochloride and Buprenorphine
hydrochloride.
Materials and Methods
Experimental
Instruments and Columns
Shimadzu with high pressure liquid
chromatographic instrument provided with a LC 20 AD Pump and Prominence SPD 20A
UV-deuterium detector. Data acquisition was performed by using Spin chrome
software, Shimadzu Class VP version 6.12 SPS data system. Power Sonicator,
model no: 405, Hwashin Technology, Korea. The column used in the development
for determination is Hypersil ODS C18 (250mm x 4.6mm; 5μm) [17].
Chemicals
Used
HPLC grade Acetonitrile, methanol
and water were purchased from E.Merck Co; Mumbai, India and Ammonium acetate,
glacial acetic acid AR grade were purchased from SD Fine Chem. Mumbai, India.
The reference samples of Buprenorphine Hydrochloride and Naloxone Hydrochloride
were supplied by Spectrum Analytical Labs, Hyderabad, Telangana State, India,
and branded formulation was purchased from local market [18].
Chromatographic
Conditions
An ideal wavelength is one that uses
good response for the drugs to be detected Buprenorphine Hydrochloride and
Naloxone Hydrochloride in diluents the spectra was scanned on UV-visible
spectrophotometer in the range of 200nm to 400nm against diluents as blank. The
maximum absorbance of both drugs was found to be at 310nm. So the 310nm was
selected for simultaneous estimation Buprenorphine Hydrochloride and Naloxone
Hydrochloride in Pharmaceutical Dosage Forms (Table 1).
Preparation
of Mobile Phase
Weighed accurately 770mg of ammonium
acetate and dissolved in 100ml of water and volume was made up to 1000mL with
water adjust the PH to 6.0 using glacial acetic acid. The solution was filtered
through 0.45μ membrane filter and was degassed [19]. A freshly prepared binary
mixture of Ammonium acetate and glacial acetic acid buffer: Acetonitrile in a
ratio of (68:32) V/V was used as the mobile phase. Methanol was used as
diluents for preparing the working solution of the drug. The mobile phase was
filtered through 0.05μ membrane filter and sonicated. The flow rate of the
mobile phase was maintained at 1.0mL/min. The column temperature was maintained
at 30ºC and the detection of the drug was carried out at 310 nm.
Preparation
of stock solution
Weighed accurately about 5mg of
Naloxone Hydrochloride and 20mg Buprenorphine Hydrochloride transferred into
25mL volumetric flask. The solution was sonicated and filtered through Whatman
filter paper; resulting solution was diluted with the mobile phase to get a
working standard solution.1mL from the above Stock solutions of Naloxone
Hydrochloride and Buprenorphine Hydrochloride was taken into a 10mL volumetric
flask and diluting up to the mark with the mobile phase. Mixed standard
solutions of different concentrations ranging from 5-30μg/mL of Naloxone
Hydrochloride and 20-120μg/mL of Buprenorphine Hydrochloride were prepared by
taking suitable aliquots of working standard solution in different 10mL
volumetric flasks and diluting up to the mark with the mobile phase [20-25].
Preparation
of sample solution
Twenty tablets were weighed and
average weight was determined and finally powdered. Tablet powder equivalent to
0.5mg Naloxone Hydrochloride and 2mg Buprenorphine Hydrochloride was accurately
weighed and transfer to 10mL volumetric flask. The contents were sonicated for
about 15min for complete solubility of the drug after adding 10mL of mobile
phase and the volume was made up to the mark with mobile phase. Then the
mixture was filtered through a 0.45μ membrane filter. From the above solution,
4mL aliquot was taken into a separate 10 mL volumetric flask and diluted up to
the volume with the mobile phase and mixed well.
Optimization
of HPLC method
The HPLC method was optimized with
an aim to develop an accurate and precise method for the estimation of Naloxone
Hydrochloride and Buprenorphine Hydrochloride in pharmaceutical dosage forms.
For the method optimization different mobile phases were tried but acceptable
retention times, theoretical plates and good resolution observed with pH 6.0
ammonium acetate buffer: Acetonitrile in a ratio of (68:32) v/v was used as the
mobile phase using Hypersil ODS C18, 250 X 4.6mm, 5μm (Tables 2 & 3).
Linearity
Alinear relationship was evaluated
across the range of the analytical procedure with a minimum of six
concentrations. A series of standard dilutions of NAH and BUH were prepared
over a concentration range of 5-30μg/mL and 20-120μg/mL from stock solution and
injected. Linearity is evaluated by a plot of peak areas as a function of
analyte concentration, and the results were evaluated by using the statistical
methods like slope, intercept, and regression (R2) correlation coefficients (R)
and the data was given in (Tables 2-4) (Figures 3 & 4). Repeatability
expresses the precision under the same operating conditions over a short
Interval of time. The six repeated homogenous injections of standard solutions
were made about 20μg/mL Naloxone and Buprenorphine 80μg/mL and the response
factor of drug peaks, mean, standard deviation and percentage RSD were
calculated. Repeatability data for NAH and BHU are summarized in (Table 4).
Method
Precision
Method precision was determined by
injecting six sample solutions of Single batch were analysed as per test
method. The mean, standard deviation and percentage RSD for peak areas of
Naloxone and Buprenorphine from sample solutions were calculated. The results
were given in the Table 4 [26,27].
Accuracy
Accuracy determination, three
different concentrations were prepared separately i.e.50%, 100%, and 150% of
analyte and the chromatograms were recorded for the same. The results obtained
for recovery were found to be within the limits. The results were given in the
Table 5 [28,29].
Robustness
To evaluate the robustness, the
following small deliberate variations are made in the method and analyzed the
sample in triplicate. 1. Column oven temperature (±50C), 2.Flow rate (±10%),
3.change in buffer composition (±5%). The system suitability was evaluated in
each condition and compared with the results of method precision. The results
were given in the Table 6 [30,31].
Specificity
Specificity shall be established by
demonstrating that the procedure is unaffected by the presence of interference
at the retention time of the Naloxone Hydrochloride and Buprenorphine
Hydrochloride with respect to mobile phase, Diluents, placebo and degradants.
The specificity studies include deliberate degradation of the tablet sample by
exposure to stress conditions, Specificity studies also include blank, placebo
solution, and sample solution (control sample), Naloxone and Buprenorphine
standard solution were injected into the HPLC system. There was no interference
from the blank and placebo at the retention time of the peaks. Peak purity data
reveals that Naloxone and Buprenorphine were homogeneous and there was no
interference at the retention time of both drug peaks (Figure 5).
Degradation
Studies
Forced degradation or stress studies
are undertaken to demonstrate specificity. The objective of developing
stabilityindicating methods was particularly little information is available
about potential degradation products. These studies also provide information
about the degradation pathways and degradation products that could form during
storage. Forced degradation studies may help facilitate pharmaceutical
development as well in areas such as formulation development manufacturing and
packaging in which knowledge of chemical behavior can be used improve a drug
product [32-34].
Forced Degradation study was carried
out by treating the sample under the following conditions [16]. Twenty tablets
were weighed and average weight was determined and finally powdered. Tablet
powder equivalent to 0.5mg Naloxone Hydrochloride and 2mg Buprenorphine
Hydrochloride was accurately weighed and transfer to 10mL volumetric flask. The
contents were sonicated for about 15 min for complete solubility of the drug
after adding 10 mL of mobile phase and the volume was made up to the mark with
mobile phase. Then the mixture was filtered through a 0.45μ membrane filter.
From the above solution, 4mL aliquot was taken into a separate 10 mL volumetric
flask and diluted up to the volume with the mobile phase and mixed well.
Acid
Degradation
To 1ml of stock solution of NAH and
BUH, 1ml of 2N Hydrochloric acid was added and refluxed for 2hrs at 600C.The
resultant solution was diluted to obtain 20μg/ml & 80μg/ml solution and10μl
solutions were injected into the system and the chromatograms were recorded to
assess the stability of sample (Figure 6) [35]
Alkali
Degradation
To 1ml of stock solution Naloxone
and Buprenorphine, 1ml of 2N sodium hydroxide was added and refluxed for 2hrs
at 600c. The resultant solution was diluted to obtain 20μg/ml & 80μg/ml
solution and 10μl were injected into the system and the chromatograms were recorded
to assess the stability of sample Figure 7 [36].
Thermal
Degradation
The standard drug solution was
placed in oven at 105 °C for 6 hrs to study thermal degradation. For HPLC
study, the resultant solution was diluted to 20μg/ml & 80μg/ml solution and
10μl were injected into the system and the chromatograms were recorded to
assess the stability of the sample Figure 8 [37].
Peroxide
Degradation
To 1ml of stock solution of Naloxone
and Buprenorphine, 1 ml of 20% hydrogen peroxide (H2O2) was added separately.
The Solutions were kept for 1hr at 600c. For HPLC study, the resultant solution
was diluted to obtain 20μg/ml & 80μg/ml solution and 10μl were injected
into the system and the chromatograms were recorded to assess the stability of
sample Figure 9 (Table 7).
Photo
Stability
The photochemical stability of the
drug was also studied by exposing the 20μg/ml & 80μg/ml solution to UV
Light by keeping the beaker in UV Chamber for 7 days or 200 Watt hours/m2 in
photo stability chamber. For HPLC study, the resultant solution was diluted and
10μl were injected into the system under stabilized chromatographic conditions
Figure 10.
Limit of Detection (LOD) and Limit
of Quantification (LOQ)
The LOD and LOQ of the developed
method were determined by analysing progressively low concentration of the
standard solutions using the developed methods. The results are given in the
Table 4 LOD= 3.3σ/S and LOQ=10σ/Sσ = standard deviation of the response S =
slope of the calibration curve of the analyte.
Analysis of Marketed Formulations
The fixed chromatographic conditions
were applied for the estimation of Naloxone and Buprenorphine (0.5mg of
Naloxone and 2mg of Buprenorphine) formulation by RP-HPLC method. Twenty
tablets were weighed and average weight was determined and finally powdered.
Tablet powder equivalent to 0.5mg Naloxone Hydrochloride and 2mg Buprenorphine
Hydrochloride was accurately weighed and transfer to 10mL volumetric flask. The
contents were sonicated for about 15min for complete solubility of the drug
after adding 10mL of mobile phase and the volume was made up to the mark with
mobile phase. Then the mixture was filtered through a 0.45μ membrane filter.
From the above solution 4mL aliquot was taken into a separate 10mL volumetric
flask and diluted up to the volume with the mobile phase and mixed well.
Initially, inject 20μL of blank solution, placebo solution, sample solution and
standard solution, Disregard peaks due to blank and placebo if any. The results
were given in the table 8.
Recording of chromatograms
The standard solutions stabilize the
system until stable obtained. Initially, inject the blank solution and placebo.
The standard chromatograms were recorded by injected standard solutions and the
peak areas of standard chromatograms were noted. Calibration graph was plotted
using peak area versus concentration. Then the sample solution was injected and
the amount of Naloxone and Buprenorphine present in the formulation was
calculated from the calibration curve. The amount of Naloxone and Buprenorphine
present in per tablet Naloxone and Buprenorphine was found to be 0.50 ± 0.009mg
and 2.03 ± 0.008mg. Total label claim for (0.5mg of Naloxone and 2mg
Buprenorphine) formulation Figure 11.
Result and Discussion
The goal of the study is to
development of simple, rapid, sensitive, specific and accurate HPLC methods for
the routine quantitative determination of samples. Hypersil C18 ODS Column (250
mm x 4.6 mm; 5μm) as stationary phase. The mobile phase composition ammonium
acetate and glacial acetic acid buffer: Acetonitrile in the ratio of 68:32 and
pH adjusted to 6.0±0.1 with glacial acetic acid selected. A good linear relationship
(r2 = 0.9995 & r2 = 0.9996) was observed in the range of 5-30μg/mL &
20μg/ mL-120μg/mL for NAH and BUH (Figures 3 & 4) Linear Recovery values
obtained by the proposed method is accurate. The system precision was
established by six replicate injections of the standard solutions containing
analyte of interest. The value of relative standard deviation of Naloxone and
Buprenorphine was found to be 0.72 and 0.7 within the limit, indicating the
injection repeatability of the method. The method precision was established by
carrying out the analysis six times using the proposed method. The relative
standard deviation of Naloxone and Buprenorphine was found to be 0.6 and 0.9
within the limit, indicating the injection repeatability of the method (Table
4). Six samples of the same batch were prepared on different days by the
analysts. Calculated percentage RSD for two different days in six samples for
ruggedness results with the method precision within the limits. The system
suitability was evaluated in each condition and compared the results with
method precision results. The method is robust for change in wavelength, mobile
phase composition and column oven temperature. The specificity studies include
deliberate degradation of the tablet sample by exposure to stress conditions,
Specificity studies also include blank, placebo solution and sample solution
(control sample), NAH and BUH standard solution were injected into the HPLC
system. There was no interference from the blank and placebo at the retention
time of the peaks. Peak purity data reveals that Naloxone and Buprenorphine
were homogeneous and there was no interference at the retention time of both
drug peaks. The method does not permit detection of any degradation products
for NAH and BUH after subjecting to various degradation procedures like acid,
base, thermal, peroxide and photolytic degradations, the stressed samples were
analyzed by the proposed method. Degradation studies showed that the both the
drugs were highly stable under acidic, oxidative, thermal and photolytic
conditions without any change in RT values but under alkaline conditions RT
values were shifted to lower as compared to standard without any additional
peaks (Figures 6-10).
The high percentage of stability
under stress conditions confirms the suitability of the method for simultaneous
estimation of Naloxone Hydrochloride and Buprenorphine Hydrochloride in pure
and marketed formulations. The formulation was calculated from the calibration
curve. The amount of Naloxone Hydrochloride and Buprenorphine Hydrochloride in
per tablet Naloxone and Buprenorphine was found to be 0.50±0.009 mg and
2.03±0.008 mg. Total label claim for 0.5mg Naloxone and 2mg Buprenorphine of
formulation (Figures 12-14).
The Second Generation
Another approach to buprenorphine
involved the palladiumcatalyzed N-demethylation/acylation of the advanced
commercial intermediate 14, as shown in Scheme 3. This process was discovered
in 2008 in conjunction with the palladium-catalyzed N-demethylation/acylation
of hydrocodone. Treatment of amide 22 with Schwartz reagent provided the
secondary amine 23, whose alkylation and O-demethylation led to buprenorphine
in good yields.7 The O-demethylation also occurred under basic con- dition
during the hydrolysis of the acetamide in 22. Both routes depicted gave
buprenorphine in good overall yields.
The Third Generation
The palladium-catalyzed process was
repeated with the anhydride derived form cyclopropylcarboxylic acid, as shown
in (Scheme 4). The advanced inetermediate 14 provided the cylopropylcarboxamide
27 in excellent yield. Reduction either with LiAlH4 or by hydrosilylation gave
24, which was converted to buprenorphine by O-demethylation [7] (Figures
15-16).
The Fourth Generation
The palladium-catalyzed
N-demethylation/acylation leading to cylopropyl carboxamide 27 was next applied
to the carbonateprotected derivative 33 derived from oripavine. We wished to
compare the overall efficiency of approaches to buprenorphine from both
Thebaine, also known as codeine methyl enol ether, and oripavine, as the latter
approach would not re- quire O-demethylation. Thus, ketone 30 was synthesized
by two routes proceeding in a comparable yield, as shown in Scheme 5, and
converted to 33 by the action of the Grignard reagent. The N-demeth-
ylation/acylation produced in high yield 34, whose reduction and concomitant
removal of the carbonate gave buprenorphine.7Comparison of approaches to
buprenorphine
The four generations of approaches
to buprenorphine have been evaluated for overall efficiency, as shown in Fig.
3. Although all routes are six steps in length, it would appear that the best
overall yield is provided in the palladium-catalyzed N-demethylation/ acylation
of the advanced intermediate 14. The issue of actual cost of each of these
routes has not been addressed in detail but it is clear that reagent and
catalyst costs will be major considerations for eventual scale-up and
production. Without the evaluation of cost parameters the overall yield figures
are not the most important factor. Suffice it to say that all four approaches
provided significant improvements over the commercial route.
Naltrexone8
We next addressed the synthesis of
naltrexone and eventually also (R)-methyl naltrexone. Several different
approaches were investigated and compared for overall merit.
N-demethylation
of N-oxides with the Burgess reagent
This rather interesting
demethylation method is shown in Scheme 6, applied to the N-oxide derived from
oxycodone 6. Treatment of N-oxide 35 with the Burgess reagent provided cleanly
oxazolidine 38, presumably via the closure of the C-14 hydroxyl on the imminium
species 36 or the intermediate trapping of such species with the sulfonate
followed by intramolecular displacement, as shown in Scheme 6.9 The generation
of oxazolidine 38 was very exciting, as it led to several different methods of
synthesis for naltrexone, naloxone, and nalbuphine. The most important benefit
of the Burgess demethylation protocol was the fact that the carbon of the
original N-methyl group remained in oxazolidine 38 after the demethylation and
was not lost from the overall mass of the molecule as it had been during the
previously applied N-demethylation methods. This simple fact would have a major
impact on a general method of synthesis for the opiate-derived agents and will
be discussed further in this review.
Similarly, the Burgess demethylation
process was applied successfully to oxymorphone to produce oxazolidine 42a or
42b in which the phenolic function was protected either as an acetate oras a
carbonate. Either of the oxazolidines 42 was easily converted to noroxymorphone
43, in which the secondary amine was avail- able for alkylation to either
naltrexone 9, naloxone 10, or nalbu- phone 44, as shown in Scheme 7. Of special
note is the fact that naloxone could not be obtained by N-demethylation of
diastereomeric salts of type 15 because the rate of the corresponding
deallylation would always be faster. The oxazolidines of type 42 proved to be
very useful in another general approach to several opiate-derived medicinal
agents, as will be shown further in this review (Figures 17-21).
N-demethylation/acylation
approach to naltrexone
The palladium-catalyzed
N-demethylation/acylation strategy was ultimately applied to a very efficient
synthesis of naltrexone, as shown in Scheme 8. Oxymorphone 8 was peracylated
with the anhydride derived from cyclopropylcarboxylic acid or the corresponding
acyl chloride and subjected to the palladiumcatalyzed N-demethylation/acylation
protocol. We have discovered that during the demethylation/acylation protocol
the C-14 acyl group migrated to the N-17 site. The conditions previously used
for this process required acetic anhydride. Thus, with the C-14 protected as
acetate, no additional acyl donor was required. This was indeed fortuitous for
several reasons. First, the overprotection of the phenol and the C-6 ketone was
rather inexpensive, as the acyl group is both readily available and easily
recyclable. Second, we have shown in separate experiments that the migration
form C-14 to N-17 is intramolecular and therefore the N- demethylation of 45
could easily be performedin the presence of other acyl donors.10 Third, and
very important, the vitride reduction of 46 leads to naltrexone and either
cyclopropyl carboxylates or cyclopropylmethyl alcohols, both of which can be
recycled and reused in eventual large-scale commercial applications. The final
outcome was a three-step conversion of oxymorphone to naltrexone in a 75%
overall yield (Figure 22-24).
(R)-methylnaltrexone
From
oxazolidines 42a and 42b
Our first attempt at the synthesis
of methylnaltrexone 13 was based on the recognition that the methylene bridge
in oxazolidines 42 was fixed in the (R)-configuration. We reasoned that the
conversion of 42 to a cyclopropylmethyl salt 47 followed by a regioselective
cleavage with a hydride may lead directly to methylnaltrexone, as depicted in
Scheme 9. Unfortunately, hydride attack on the methylene bridge resulted only in
the neutralization of the nitrogen providing the C-14 methoxy derivative 48.
Under no conditions have we observed the tendency of the C-14 alkoxide to act
as a leaving group. The hydrolysis of the salts 47a and 47b with con- comitant
deprotection of the phenol provided for an additional synthesis of naltrexone
9.9By 1O2 cycloaddition to oripavine salts or cyclopropylmethyl nororipavine.
The N-demethylation of the mixture of (R)- and (S)-cyclopropylmethyl salts 49
derived from oripavine was used to access cyclopropylmethyl nororipavine 16
during the initial approaches to u-prenorphine. The pure (R)-salt of 49 could
be obtained by crystallization of the mixture and subjected to singlet oxygen
cycloaddition to produce the endocyclic peroxide 50 (Scheme 10). Reduction of
the peroxide, with the concomitant generation of C-6 ketone, yielded either
enone 51 or, depending on the conditions, (R)-methylnaltrexone 13. The same
cycloaddition of singlet oxygen was applied to 16 to yield naltrexone 9, after
hydrogenation of the intermediate enone. Alkylation of naltrexone by literature
methods provided (R)-methylnaltrexone 13.11 The two routes were com- parable in
efficiency (Figures 25-29).
Naloxone
Alkylation
of Noroxymorphone
The two distinct approaches for the
replacement of the N-methyl group with other alkyl groups involved selective
N-demethylation of salts or palladium-catallyzed N-demethylation/ acylation.
Both worked well with simple alkyl groups but neither was applicable to the
synthesis of naloxone. The nucleophilic demethylation of N-methyl-N-allyl salts
occurred at the site of the allyl group. The palladium-catalyzed process
required the use of acryloyl anhydride or the corresponding chloride and all of
our attempts produced extremely complex mixtures. Thus the easiest route to
naloxone was a simple alkylation of noroxymorphone 43, derived by hydrolysis of
oxazolidines 42, with allyl bromide (Scheme 7) [9].
Grignard
Additions to Oxazolidines
By far the best method for naloxone
synthesis proved to be the nucleophilic opening of the oxazolidines with
various Grignard reagents, as shown in Scheme 11. Our initial attempts at
methylnaltrexone synthesis derived from the expectation that the carbon of the
methylene bridge in oxazolidines 42 could be retained as the methyl group in
methylnaltrexone. While that strategy was unsuccessful with the ammonium salts,
we thought it would work well with the neutral oxazolidines. Indeed, protection
of the C-6 ketone provided ketal 52, which was smoothly converted to the protected
forms of naloxone, naltrexone, and nalbuphone by treatment with vinyl-,
cyclopropyl-, or cyclobutyl Grignard reagents, re- spectively, in excellent
yields. Hydrolysis of the ketals then furnished naloxone, naltrexone, and
nalbuphone (Scheme 11). The entire sequence was eventually reduced to a one-pot
operation. Slightly lower yields were obtained with the ethylene glycol derived
ketals.12
The additional benefit of this
strategy was a direct synthesis of nalbuphine 11 from oxazolidine 42a. We have
prepared nalbuphine also by N-demethylation of N-cyclobutylmethyl quaternary
salts derived from oripavine.13 Reduction of the C-6 ketone followed by the
addition of cylobutylmagnesium chloride cleanly yielded nalbuphine 11. In all
of these protocols, the acetateprotected phenol was freed with the use of
excess Grignard reagent. The oxazolidine-based route to all of these
opiate-derived agents proved to be general and very efficient.
Miscellaneous Projects
O-demethylation
of Thebaine, also Known as Codeine Methyl Enol Ether, to Oripavine
Recently, we reported two methods
for the conversion of thebaine to oripavine by O-demethylation. These methods
were based on protection of the diene system in Thebaine, also known as codeine
methyl enol ether, either as a complex with iron carbonyl or as a [4+2]
cycloaddition adduct with a thioaldehyde. The outcomes of this study are
abbreviated in Scheme 12. Both the iron carbonyl complex and the mixture of the
Diels-Alder adducts were subjected to O-demethylation protocols with subsequent
regeneration of the diene system. The overall yield of these protocols compared
favorably with those of similar processes available in the literature [14].
Heteroatom
Analogues of Hydrocodone
Snyder suggested that the
configuration of the N-17 atom, be it nitrogen or another heteroatom, is
responsible for agonist versus antagonist activity in morphinans and their
truncated analogs such as levorphanol 57 and levallorphan 58 (Figure 4). 15
Lemaire replaced the nitrogen with a sulfonium salt functionality in
sulforphanol 59 and sulfallorphan 60 assuming that the evaluation of the
activities of these analogues would provide information about the relationship
between the axial/equatorial configuration and the corresponding
agonist/antagonist activity. Indeed, the agonist and antagonist activity was
found to depend on the conformation of the S-substituent: the equatorial allyl
group leads to antagonist activity and the axial methyl confers agonist
activity.16 To our knowledge, there were no other heteroatom analogues of
morphinans reported in the literature.
We have therefore chosen to study
analogues of hydrocodone for which accurate biological data are available. In
Scheme 13 is shown the synthesis of several heteroatom analogues of
hydrocodone. Hydrocodone is converted, by Hofmann degradation, in a few steps
to aldehylde 61, which is reduced to alcohol 62, the precursor of all
heteroatom analogues. As shown in Scheme 13, the alcohol was converted to
either oxo- or thio- analogues 63 and 64, respectively. Thioether 64 was also
transformed to a mixture of sulfoxides 65 and also to sulfone 66.17 somewhat
surprisingly, the only moderately active analogue was the sulfoxide 66, despite
the fact that in this compound the axial/ equatorial position of the N-17
substituent is not relevant. This observation is in contradiction, at least for
the heteroatom analogues of hydrocodone, with the endings reported for the
sulfonium salts 59 and 60, in which the biological activities were related to
the axial versus equatorial position of the substituent.
Conclusion
The HPLC method developed and
validated allows a simple and fast quantitative simultaneous estimation of
Naloxone Hydrochloride and Buprenorphine Hydrochloride from its formulation.
All the validation parameters were found to be within the limits according to
the ICH guidelines. The proposed method was found to be specific for the drugs
of interest irrespective of the excipients present and the method was found to
be simple, accurate, precise, rugged, and robust and can be involved in the
routine analysis of the marketed formulation. The high percentage of stability
under stress conditions confirms the suitability of the method. Therefore this
method can be employed in quality control to estimate the amount of Naloxone
Hydrochloride and Buprenorphine Hydrochloride in pure and pharmaceutical dosage
forms. We have investigated several methods of N-demethylation of various
morphinans. These methods were applied to process development toward the
synthesis of buprenorphine, naltrexone, naloxone, nalbuphone, and nalbuphine.
In each of these applications, a marked improvement was observed in the
manufacture of these important medicinal agents. A comparison of the
transition- metal versus enzymatic catalysis is shown in Figure 5. A detailed
cost analysis will indicate which of the two routes more efficient for
large-scale production is. Future endeavors should focus on the construction of
transgenic organisms that would over express the cytochrome responsible for the
N-demethylation. If successful, such an approach would be superior to all
others, as the demethylation could be performed by fermentation in aqueous
medium followed by a phase transfer alkylation to the final products. We should
be able to report on further advances in this important area in due course
Biological
Methods of N- and O-Demethylation
In addition to the
palladium-catalyzed methods of N-demethylation, we have investigated biological
means as well. The incubation of several morphine alkaloids and opiate-derived
agents, depicted in Figure 4 as a general structure 67, with the fungus
Cunninghamella echinulata led to isolation of the corresponding secondary
amines of type 69.18 Surprisingly, the only morphinan that was immune to this
process was morphine all other naturally occurring alkaloids as well as the
commercially available opiates underwent a smooth demethylation. Presumably, a
cytochrome enzyme within this fungus is responsible for the oxidative
demethylation
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