Stable Isotope Labeled Organic Molecules Clearsynth Research Center Torronto & Hyderabad

1. Stable Isotope Labeled Organic molecules: Applications in
Pharmaceutical Industry
Punit K Bhardwaj, T.V.R.S.Sastry and Shashwati Pal
Clearsynth Labs Ltd. Research Centre, Plot No. 177, IDA Mallapur, Hyderabad-500076
Deuterium was discovered in 1932 by Harold Clayton Urey and for which he was awarded
Nobel Prize in 1934. Deuterium occurs in nature to the extent of about 1 part in 6400. The most
important compound containing deuterium is Heavy water or deuterium oxide (D2O). It is
obtained primarily by isolation from ordinary water via distillation. Its concentration ranges from
0.0156% in sea water to 0.0139% in fresh water. Because of the use of deuterium oxide as a
moderator in nuclear reactors, its demand increased in 1950’s leading to development of several
methods of isolating D2O on very large scale. The ready availability of heavy water to the
scientific community lead to expanding interest in this field. Till date several methods have been
reported for the synthesis of the deuterium labelled compounds. These compounds are used as:
Tools to study reaction mechanisms via Kinetic isotope effect
Polanyi (1932) and Eyring (1933) independently predicted and postulated that the protonated and
deuterated compounds should react at different rates. The covalent carbon-hydrogen bond is
known to be substantially weaker than identical carbon-deuterium bond. For the C-D bond the
zero point energy is lower as compared to C-H bond, this results in a higher activation energy
and a slower rate of dissociation for C-D bond. This rate effect is the primary deuterium isotope
effect (DIE) and is expressed as kH/kD. The deuterium-carbon bond is typically six to nine times
more stable than the hydrogen-carbon bond. This, differential bond-strength has many
consequences and this property is useful in the study of mechanistic pathway of both catalytic as
well as non-catalytic reactions. These studies require used of deuterated reagents and solvents.
Deuterated drugs
Another application of Deuterium Kinetic isotopic effect is in the medicinal chemistry, also
referred to as deuterium medicinal chemistry. This drug discovery method is based on the
observation that several compounds in the phase I metabolism involving oxidation, undergo
breaking of carbon-hydrogen bonds. If the C-H bond is replaced by C-D bond, then due to DKIE
the breaking of carbon-deuterium bond can be more difficult which results in the decrease of the
rate of metabolism. Several examples have been reported in literature, where the replacement of
hydrogen with deuterium in drug molecules can lead to significant alterations in their
metabolism and thereby causing beneficial changes in their pharmacokinetics profile, leading to
less-frequent dosing. Such replacement may also have the effect of lowering toxicity by reducing
the formation of a toxic metabolites. The key determinant for a beneficial effect is that the
breaking of the C-H bond should be the rate determining step. This field of modulating the drug
metabolism, pharmacokinetics and toxicity has exploded in the last 10-15 years. Nearly 100
patents have been filed and several have been granted in which this isotopic switch has provided

2. clinically significant results. Concert Pharma is at present the front runner in this field, followed
by Auspex, Protia, Deuteria and Deuterx. The most advanced compound of Concert Pharma is
CTP-499, which is a potentially first-in-class treatment for diabetic nephropathy. Its phase II trial
is complete. Lately, all big Pharma companies have entered the fray. This is clear from the patent
literature, as the major pharmaceutical companies now also claim deuterated versions of their
new molecules in their ongoing patent applications. This strategy has become an additional tool
in the kit of the drug hunters and as a consequence the demand of the deutrated building blocks
has increased. Several companies like CDN isotopes, Clearsynth Labs, Cambridge isotopes etc.
provide labelled reagents, intermediates, building blocks or final APIs for such studies.
Deuterated drugs collectively are expected to be worth at least US$1 billion.
Reference standards in mass spectrometry
The detection and quantification of drugs and their metabolites in biological tissues and fluids is
very important process in Pharmacology, Forensic science, drug discovery and clinical diagnosis
etc. The adaptation of stable isotope labeled analogs as the internal standards in mass
spectrometric methods has greatly improved the accuracy in the quantification of drugs and their
metabolites in biological media. Their use offsets the matrix effects, which can interfere with the
quantification. The stable isotope labelled compounds generally display same retention and
ionization behavior in LC/MS, but differ in their mass. If this mass difference between the
analyte and labelled analog is selected to be large enough to keep signal overlap, as a result of
the natural isotope pattern, as low as possible, quantitative determination is possible. In addition,
use of isotope dilution methods for analysis offers high sensitivity and rapid quantification of
stable isotopes in various samples.
Commonly used stable isotopes for the preparation of internal standards are:
Element Isotope
Hydrogen Deuterium, 2
H
Carbon 13
C
Nitrogen 15
N
Oxygen 17
O, 18
O
The stable isotope labeled analog should be isotopically pure, it should co-elute with the analyte
and it should possess an adequate number of isotopic atoms (usually more than 2) at appropriate
locations in the molecular framework (to avoid scrambling).
One of the key usage of stable isotope labelled reference standards is in the evaluation of
bioavailability during the bioequivalence studies. For the approval of the generic formulation it is
mandatory to undertake bioequivalence studies. The bioavailability of the brand-name drug is
then compared to that of the generic drug in each individual. When these comparisons
demonstrate a relatively similar extent of absorption and a similar maximum drug concentration,
bioequivalence is achieved and the generic drug is proven to be as safe and effective as the
brand-name version. Some of the examples of reference standards are:

3. Canagliflozin D6 Lorcaserin Sulfate D4 Regorafenib D3
Market trends
The Global stable isotope-labeled compounds market is estimated to be $220 Million which is
divided into segments namely, Pharmaceuticals, Forensics, Food Authentication, Environmental
Sciences, Semiconductors, and Petroleum, Geology, Hydrology and Miscellaneous ones.
The pharmaceutical industry accounted for the largest share in 2013 and is the fastest-
growing segment of this market. The large share and high growth of this segment is attributed to
the increasing number of clinical trials, advent of novel quantifying techniques. On the basis of
applications, pharmaceutical segment is further classified into clinical diagnostics,
pharmaceutical research, and internal standards. Stable isotope labeled reagents commanded
largest chunk ~80% of the overall U.S. stable isotope labeled compounds market in 2013,
whereas stable isotope labeled APIs holds only ~20% of the market, in terms of value. However,
stable isotope labeled APIs market is expected to be the faster growing segment in the next five
years due to their increasing use in drug discovery and development process. The U.S. stable
isotope labeled compounds market was valued at $110 million in 2013 and is expected to reach
$127.52 million by 2018.
Out of the different stable isotope-labeled compounds comprising deuterium, carbon 13, nitrogen
15 and oxygen 18 the deuterium-enriched compounds segment accounted for the largest share in
2013 and is the fastest-growing segment of this market. This is because of factors like easy
availability, wide range of applications, and low costs.
Challenges and prospects
The cost of stable isotopes and key starting materials required for stable isotope labelled
analogue preparation is huge. In addition, the high labeling and isotopic purity evaluation costs
further add up to the cost of stable isotope-labeled compounds. Consequently, most of the crucial
isotopes required in studies are priced beyond the budgets. However availability of skilled work
force, infrastructure and key starting materials in countries like India, Canada and Norway may
provide the Global pharmaceutical industry to access the stable isotope labeled analogues at
much lower costs. Presently, India is the largest producer of heavy water in the world and is the
only country using multiple technologies for its production. The advantage of easy availability of

4. high grade heavy water in India is expected to be used by Indian stable isotope industry to its
benefit. India has the potential to become an international key player in this industry.