Synthesis of stable isotope–labeled chloroquine and amodiaquine and their metabolites

Article abstract of DOI:10.1002/jlcr.3721

Anti-malaria drugs chloroquine and amodiaquine and their metabolites were synthesized to incorporate ¹³C and ¹⁵N starting from U-¹³C–labeled benzene to give M?+?7 isotopomers. Chloroquine and its metabolites were prepared

Full text of DOI:10.1002/jlcr.3721

Wu Ruilian (Orcid ID: 0000-0002-1899-183X)
Lawrie Ken (Orcid ID: 0000-0002-8061-3696)
Maxwell Brad (Orcid ID: 0000-0001-8507-2078)
Synthesis of Stable Isotope Labeled Chloroquine, Amodiaquine and Their Metabolites
Ruilian Wu,a Robert F. Williams,a L. A. “Pete” Silks,a and Jurgen G. Schmidtb*
Anti-malaria drugs chloroquine, amodiaquine and their metabolites were stable isotope labeled with
13C and 15N starting from uniformly 13C labeled benzene and 15N labeled potassium nitrate to give
M+7 isotopomers. Chloroquine, and its metabolites were prepared from 7-chloro-1,2,3,4-
tetrahydroquinolin-4-one through addition to the carbonyl with the corresponding amines; and the
amodiaquine and its metabolites were prepared from 4,7-dichloroquinoline in a selective aryl-
halogen exchange with the corresponding amines.
Keywords: chloroquine, amodiaquine, uniformly 13C labeled benzene, mass spectral standards
aBioenergy and Biome Sciences, Group-B11, Bioscience Division, Los Alamos National Laboratory, Los
Alamos, NM 87545
bInorganic, Isotope and Actinide Chemistry, Group-IIAC, Chemistry Division, Los Alamos National
Laboratory, Los Alamos, NM 87545, now at Bioenergy and Biome Sciences, Group-B11, Bioscience
Division, Los Alamos National Laboratory, Los Alamos, NM 87545
Introduction
Chloroquine, discovered in 1934,1,2 has traditionally been used to prevent and treat malaria in areas
where malaria is known to be sensitive to the pharmaceutical,3 although certain types of malaria
have developed wide spread resistance to chloroquine, thus requiring patients to be given
alternative approaches or additional medications. Chloroquine is on the World Health Organization’s
list of Essential Medicines, as the most effective and safe medicine needed in arena. In addition, its
availability in a generic version makes chloroquine a readily affordable drug in developing countries.4
Chloroquine has also found use for treatment of amebiasis and rheumatic diseases. Significantly, it
has also been studied as anti-retroviral and anti-cancer agents.3
Amodiaquine,5 structurally very similar to chloroquine, has also been used for treatment of malaria
where resistance to chloroquine is significant. However, there is some notable side effects with the
use of amodiaquine, such as hepatotoxicity.6 In general, antimalarial drugs of this class have shown
to cause severe side effects.7, 8 Therefore, diagnostic monitoring of chloroquine and amodiaquine and
their metabolites by quantitative mass spectrometry has found recent increased attention. Importantly,
stable isotope labeled reference samples have become the gold standard for facilitate accurate
measurements and method development for mass spectrometry detection and quantification.9,10
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We have been interested in the synthesis of stable isotope labeled compounds since the 1980’s,
these include the syntheses of sulfur and nitrogen mustard metabolites and polyaromatics.9,10 We
now report on the synthesis of stable isotope labeled chloroquine, amodiaquine and their metabolic
products.
Previous Synthetic Routes to Chloroquine
Chloroquine and related compounds are traditionally made by Conrad-Limpach synthesis,11,12 shown
in Scheme 1:
Scheme 1. Conrad-Limpach synthesis of chloroquine
Condensation of m-chloroaniline with diethyl oxaloacetate to give diethyl (E)-2-((3-
chlorophenyl)imino)succinate 1 was followed by pyrolytic cyclization to give ethyl 7-chloro-4-
hydroxyquinoline-2-carboxylate 2a and ethyl 5-chloro-4-hydroxyquinoline-2-carboxylate 2b.
Saponification of ester 2a to its acid 7-chloro-4-hydroxyquinoline-2-carboxylic acid 3 followed by
thermal decarboxylation provided 7-chloroquinolin-4-ol 4. Subsequent reaction of 4 with
phosphorus oxychloride gave 4,7-dichloroquinoline 5. Yields are generally high, except for the
cyclization step of diethyl (E)-2-((3-chlorophenyl)imino)succinate 1 to ethyl 7-chloro-4-
hydroxyquinoline-2-carboxylate 2a, in which the annulation closes partly into the ortho position to
the chloro group resulting in a mixture containing about 50% of the undesired 5-chloro isomer 2b.
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Chloroquine has also been constructed using the Gould-Jacobs approach,13 as shown in Scheme 2:
Scheme 2. Gould-Jacobs synthesis of chloroquine
The reaction of diethyl ethoxymethylenemalonate with m-chloroaniline forms diethyl 2-(((3-
chlorophenyl)amino)methylene)malonate 6. This product undergoes cycloaddition in refluxing
diphenyl ether to form ethyl 7-chloro-4-hydroxyquinoline-3-carboxylate 7. The presence of other
positional isomer is negligible. The saponification of ester 7 affords acid 8, and subsequent
decarboxylation gives decarboxylated product 6. Overall, this synthesis requires several very high
temperature reactions and thermal decompositions are possible.
W. S. Johnson14 improved upon synthetic methods to chloroquine as illustrated in Scheme 3:
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Scheme 3. W. S. Johnson synthesis of chloroquine
m-Chloroaniline undergoes a Michael addition with methyl acrylate in the presence of catalytic
amount of acetic acid to form methyl 3-((3-chlorophenyl)amino)propanoate 9. Reaction of 9 with
tosyl chloride gives rise to methyl 3-((N-(3-chlorophenyl)-4-methylphenyl)sulfonamido)propanoate
10. Subsequent hydrolysis affords the acid 11. Treatment of the acid 11 with phosphorus
pentachloride followed by a Friedel-Crafts reaction afforded exclusively 7-chloro-1-tosyl-2, 3-
dihydroquinolin-4(1H)-one 12. Cleavage of the tosyl group from 12 affords 7-chloro-2, 3-
dihydroquinolin-4(1H)-one 13. In the presence of an oxidizing agent such as nitrobenzene, 13
reacted with 2-amino-5-diethylaminopentane to form chloroquine.
A closely related synthetic approach starts with m-chloroaniline and acrylonitrile as shown in
Scheme 4:15
Scheme 4. Alternate to W. S. Johnson synthesis
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m-Chloroaniline is cyanoethylated by heating with acrylonitrile and a catalytic amount of cupric
acetate to give the Michael addition product 3-((3-chlorophenyl)amino)propanenitrile 14 in 60%
yield. Upon alkaline hydrolysis 14 produces the corresponding propionic acid 15, which is further
cyclodehydrated with PPA (polyphosphoric acid) to give 7-chloro-2, 3-dihydroquinolin-4(1H)-one 13
in 50% yield. In this reaction, the unwanted 5-chloro isomer did not form.
Results and Discussion
Radioactive isotope labeled chloroquine and its metabolites have been prepared for
pharmacological studies. The synthesis of stable isotope labeled materials, however, has not enjoyed
the same level of attention, with only syntheses of deuterated side chains reported.16 The
degradation of chloroquine and amodiaquine was studied, both of them undergo de-alkylation (de-
ethylation) by P450 to form desethyl and bisdesethyl derivatives.17 The synthesis of the dealkylated
stable isotope labeled metabolites are also very scarce.16
We adapted W. S. Johnson’s method for the synthesis of stable isotope labeled (SIL) chloroquine,
amodiaquine, and their metabolites. The chloroquine series 8’, 9’ and 10’ were prepared from 7-
chloro-2,3-dihydroquinolin-4(1H)-one 7’ with the corresponding amines; while the amodiaquine
series 12’, 13’ and 14’ were prepared from 4,7-dichloroquinoline 11’ with the corresponding amines,
as shown in scheme 5 and 6.
Scheme 5. Key step to produce labeled chloroquine and its metabolites
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Scheme 6. Key step to produce labeled amodiaquine and its metabolites
The syntheses of precursors follows the synthetic route shortly outlined vide infra: Nitration of
benzene-1,2,3,4,5,6-13C6 (Scheme 7) with K15NO3 and concentrated sulfuric acid affords (nitro-
15N)benzene-1,2,3,4,5,6-13C6 1’. Nitrobenzene reacts with trichloroisocyanuric acid (TCI) and
concentrated sulfuric acid to afford 1-chloro-3-(nitro-15N)benzene-1,2,3,4,5,6-13C6 2’. Reduction of 1-
chloro-3-nitrobenzene 2’ is affected with hydrazine in the presence of montmorillonite to provide 3-
chloroaniline-1,2,3,4,5,6-13C6, 15N 3’. Reaction of 3-chloroaniline 3’ with methyl acrylate in the
presence of glacial acetic acid produces ethyl 3-((3-chlorophenyl-1,2,3,4,5,6-13C6)amino-
15N)propanoate 4’. This product is then treated with p-toluenesulfonyl chloride to yield ethyl 3-((N-
(3-chlorophenyl-1,2,3,4,5,6-13C6)-4-methylphenyl)sulfonamido-15N)propanoate 5’. Hydrolysis of the
ester with hydrochloric acid in dioxane-water, followed by acid chloride formation using oxalyl
chloride, and a subsequent Friedel-Crafts reaction affords 7-chloro-1-tosyl-1,2,3,4-
tetrahydroquinoline-4a,5,6,7,8,8a-13C6, 15N 6’. Removal of tosyl group is achieved with acetic acid
and hydrochloric acid in water at reflux to form 7-chloro-1,2,3,4-tetrahydroquinoline-4a,5,6,7,8,8a-
13
C
15N 7’.
6,
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Scheme 7. Synthesis of labeled precursors for chloroquine and metabolites
[13C,15N]Chloroquine 8’ is then formed in the reaction of 2,3-dihydroquinolin-4(1H)-one 7’ with 2-
amino-5-diethylaminopentane in nitrobenzene at 150-160oC as shown in Scheme 8.
Scheme 8. Synthesis of labeled chloroquine
For the synthesis of [13C,15N]desethylchloroquine and [13C,15N]bisdesethylchloroquine, the
corresponding amines need to be prepared (Scheme 9, 10 and 11). 5-chloropentane-2-ol 16 is
obtained by reduction of 5-chloro-2-pentanone with sodium borohydride in 95% ethanol. The
product undergoes a Mitsunobo reaction with phthalimide to form 2-(5-chloropentan-2-
yl)isoindoline-1,3-dione 17. A subsequent Finkelstein reaction with sodium iodide forms 2-(5-
iodopentan-2-yl)isoindoline-1,3-dione 18. This product serves as a common intermediate for the
syntheses of the two amines 20, 23.
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Scheme 9. Precursor synthesis for chloroquine metabolites
For the synthesis of desethylchloroquine, the reaction sequence is shown in Scheme 10, 2-(5-
iodopentan-2-yl)isoindoline-1,3-dione 18 reacts with N-ethyl-p-toluenesulfonamide in the presence
of sodium hydride in DMF to form N-(4-((7-chloroquinolin-4-yl)amino)pentyl)-N-ethyl-4-
methylbenzenesulfonamide 19. Cleavage of phthalimide group in methylamine provides N-1-ethyl-
N-1-tosylpentane-1, 4-diamine 20. Reaction of 20 with 7-chloro-2,3-dihydroquinolin-4(1H)-one 7’ in
nitrobenzene affords N-(4-((7-Chloroquinolin-4-yl-4a,5,6,7,8,8a-13C6, 15N)amino)pentyl)-N-ethyl-4-
methylbenzenesulfonamide 21. Removal of tosyl group with hydrobromic acid, acetic acid in water
provides desethylchloroquine hydrobromide 9’.
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Scheme 10. Synthesis of labeled desethylchloroquine
For the synthesis of bisdesethylchloroquine, the reactions are shown in Scheme 11. Reaction of di-t-
butyliminodicarboxylate with 2-(5-iodopentan-2-yl)isoindoline-1,3-dione 18 in the presence of
sodium hydride, in THF, provides 2-(5-di-t-butyl-iminodicarboxypentan-2-yl)isoindoline-1,3-dione 22.
Removal of phthalimide group leads to 2-(5-di-t-butyl-iminodicarboxypentan-2-yl)amine 23.
Reaction of 23 with 7-chloro-2,3-dihydroquinolin-4(1H)-one 7’ in nitrobenzene then produces tert-
butyl (tert-butoxycarbonyl)(4-((7-chloroquinolin-4-yl-4a,5,6,7,8,8a-13C6, 15N)amino)pentyl)carbamate
24. Removal of di-Boc groups using hydrogen chloride generated from acetyl chloride and ethanol
finally yields labeled bisdesethylchloroquine hydrochloride 10’.
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Scheme 11. Synthesis of labeled bisdesethylchloroquine
For the synthesis of [13C,15N]amodiaquine, [13C,15N]desethylamodiaquine and
[13C,15N]bisdesethylamodiaquine, the reactions are shown in Scheme 12. 7-Chloro-2, 3-
dihydroquinolin-4(1H)-one 7’ is converted to 4,7-dichloroquinoline 11’18, it then reacts with the
corresponding amines as shown in Scheme 12 and 13 to provide [13C,15N]amodiaquine 12’ and
[13C,15N]bisdesethylamodiaquine 14’, only one regioisomer is obtained for the synthesis of
bisdesethylamodiaquine.19, 20
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Scheme 12. Synthesis of labeled amodiaquine
Scheme 13. Synthesis of labeled bisdesethylamodiaquine
The corresponding amine precursor for desethylamodiaquine is constructed as illustrated in scheme
14.
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Scheme 14. Precursor synthesis for desethylamodiaquine
2-((Ethylimino)methyl)-4-nitrophenol 25 is prepared from 2-hydroxy-5-nitrobenzaldehyde and
ethylamine in refluxing methanol. The enamine is reduced by sodium borohydride in a basic solution
to afford 4-amino-2-(ethylamino)methylphenol 26, hydrogenation of nitro group by palladium on
carbon provides the desired amine salt 4-amino-2-((ethylamino)methyl)phenol bishydrochloride 27.
Labeled desethylamodiaquine 13’ is prepared by heating 4,7-dichloroquinoline 11’ and 4-amino-2-
((ethylamino)methyl)phenol bishydrochloride 27 in water as shown in Scheme 15.20
Scheme 15. Synthesis of labeled desethylamodiaquine
In summary, we report a concise, straightforward synthetic route to stable isotope labeled
chloroquine, amodiaquine and their metabolites, with consideration to the cost of starting materials
as well as atom and isotope efficiencies of the chemistry used. To our knowledge, these are the first
reported syntheses of [13C,15N] labeled antimalarial drugs.
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Experimental: General
(Nitro-15N)benzene-1,2,3,4,5,6-13C6 1’:
A modification of the reported procedure was used:21 A one-neck 250 mL round bottom flask
equipped with a reflux condenser was charged with benzene-1,2,3,4,5,6-13C6 (8.41 g, 100 mmol),
potassium [15N]nitrate (10.21 g, 100 mmol) and dichloromethane (60 mL) and the mixture was
cooled with an ice bath. Sulfuric acid (21.32 mL) was added dropwise to the above mixture through
an addition funnel. The mixture was stirred at room temperature overnight. Additional sulfuric acid
(25 mL) and potassium [15N]nitrate (4.08 g, 40 mmol) were added and the mixture was stirred at
room temperature overnight. The reaction progress was monitored for completion by 13C NMR. The
layers were separated and the aqueous layer was washed with dichloromethane. The combined
organic layers were washed with water, saturated sodium bicarbonate and brine, then dried over
sodium sulfate. The solvent was removed by rotary evaporation and the residue was distilled under
vacuum at 92-94oC (15 mmHg) to provide the title product as a colorless liquid (10.99 g, 84%). 1H
NMR (CDCl3, 300 Hz)  8.61-8.49 and 8.08-7.94 (bd, J = 167.4 Hz), 7.93-7.77 and 7.40-7.24 (bd, J =
163.2 Hz), 7.55-7.40 (bpent, J = 8.67 Hz). 13C NMR (CDCl3, 75 Hz)  148.37 (ddt, J = 9.73, 14.66, 67.47
Hz), 134.82 (mt, J = 55.40 Hz), 129.47 (mt, J = 56.17 Hz), 123.63 (mt, J = 62.28 Hz).
1-Chloro-3-(nitro-15N)benzene-1,2,3,4,5,6-13C6 2’:
This compound was prepared following a reported procedure as outlined below:22 A suspension of
(nitro-15N)benzene-1,2,3,4,5,6-13C6 1’ (2.37 g, 18.2 mmol) and trichloroisocyanuric acid (1.44 g, 6.20
mmol) in sulfuric acid (7.3 mL) was heated to 80oC overnight. After the addition of cold water, the
resulting solution was extracted with EtOAc. The EtOAc layer was washed with water, saturated
sodium bicarbonate and brine. It was then dried over sodium sulfate. The solvent was removed
under reduced pressure, and the residue was purified on silica gel with 10% EtOAc/hexanes to
provide 2.95 g (99%) of the title product as a light yellow liquid. 1H NMR (CDCl3, 300 Hz)  8.23
(complexed d, 1 H), 8.13 (complex d, 1 H), 7.69 (complex d, 1 H), and 7.55 (complex d, 1 H). 13C NMR
(CDCl3, 75 Hz)  149.0 (complex t), 135.5 (complex t), 134.8 (complex t), 130.5 (complex t), 124.1
(complex t) and 121.9 (complex t). HRMS for 13C6H4Cl15NO2 calculated 163.0027 (M-H), found
163.0022.
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3-Chloroaniline-13C6, 15N 3’:
This compound was prepared following a reported procedure as outlined below:23 To a stirred
suspension of montmorillonite (5.34 g) and 1-chloro-3-(nitro-15N)benzene-1,2,3,4,5,6-13C6 2’ (2.936
g, 17.84 mmol) in dry ethanol (18.0 mL) was added anhydrous hydrazine (3.5 mL, 111.5 mmol) in one
portion. The resulting reaction mixture was heated to reflux overnight. More hydrazine (1.0 mL, 32
mmol) was added and the reaction was heated for another night. The contents were filtered and
washed with ethanol, the solvent was removed under reduced pressure and the residue was purified
on silica gel with 20% EtOAc/hexanes to provide 1.787 g (74%) of the product as a light yellow liquid.
1H NMR (CDCl3, 300 Hz)  7.44-7.28 and 7.09-7.00 (m, 1 H), 7.00-6.93 and 6.91-6.74 (m, 1 H), 6.91-
6.74 and 6.54-6.38 (m, 1 H), 6.54-6.38 and 6.37-6.25 (m, 1 H), and 3.76 (br d, J = 79 Hz). 13C NMR
(CDCl3, 75 Hz)  147.8 (complex t, J = 62 Hz), 135.0 (complex t, J = 67 Hz), 130.5 (complex t, J = 58
Hz), 118.6 (complex t, J = 62 Hz) and 114.2 (complex pent, J = 67 Hz). HRMS for 13C6H6Cl15N calculated
135.0441 (M+H), found 135.0437.
Methyl 3-((3-chlorophenyl-1,2,3,4,5,6-13C6)amino-15N)propanoate 4’:
This compound was prepared according to WS Johnson procedure:14 A mixture of 3-chloroaniline-
13C6, 15N 3’ (3.349 g, 24.89 mmol), methyl acrylate (3.36 mL, 37.33 mmol), and glacial acetic acid
(0.190 mL) was heated to reflux for 24 hr, GC/MS showed a complete reaction. The reaction mixture
was loaded on silica gel, and purified with 20% ethyl acetate in hexanes to provide 4.06 g (74%) of
the title product as a colorless liquid. 1H NMR (CDCl3, 300 Hz)  7.45-7.30 and 7.15-6.94 (m, 1 H),
6.94-6.20 (m, 1 H), 6.35-6.20 and 6.50-6.40 (m, 1 H), 6.40-6.30 and 6.30-6.20 (m, 1 H), 4.20 (br d J =
82 Hz, 1 H), 3.74 (s, 3 H), 3.46 (m, 2 H), 2.65 (m, 2 H). 13C NMR (CDCl3, 75 Hz)  172.9 (s, carbonyl C),
148.9 (complex t, J = 62 Hz), 135.3 (complex t, J = 68 Hz), 130.4 (complex t, J = 58 Hz), 117.6 (complex
t, J = 62 Hz), 112.6 (complex t, J = 67 Hz), 111.5 (complex t, J = 62 Hz), 52.1 (s), 39.3 (dt, J = 9.5, 3.2
Hz), 33.7 (d, J = 0.9 Hz). HRMS calculated for 13C6C4H12Cl15NO2 (M+H) 221.0809, found 221.0810.
Methyl 3-((N-(3-chlorophenyl-1,2,3,4,5,6-13C6)-4-methylphenyl)sulfonamido-15N)-propanoate 5’:
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To a solution of methyl 3-((3-chlorophenyl-1,2,3,4,5,6-13C6)amino-15N)propanoate 4’ (4.063 g, 18.41
mmol) in pyridine (20 mL) was added tosyl chloride (3.89 g, 20.42 mmol) in one portion at room
temperature. The reaction mixture was then heated to reflux for 30 minutes. The reaction was then
cooled to room temperature and water was added. The mixture was extracted with ether, and the
organic extracts were washed twice with 1 M HCl, water and saturated sodium bicarbonate then
dried over sodium sulfate. The solvent was removed under reduced pressure and the residue was
purified on silica gel with 30% ethyl acetate in hexanes to provide 5.89 g (85%) of the title product as
a colorless liquid. 1H NMR (CDCl3, 300 Hz)  7.55-7.33 (bm, 1 H), 7.38 (d, J = 8.33 Hz, 1 H), 7.25-7.06
(bm, 1 H), 7.17 (d, J = 8.33 Hz), 6.99-6.78 (bm, 1 H), 6.70-6.51 (bm, 1 H), 3.70 (dt, J = 7.40, 3.70 Hz, 2
H), 3.51 (s, 3 H), 2.46 (dt, J = 7.40, 2.36 Hz, 2 H), 2.33 (s, 3 H). 13C NMR (CDCl3, 75 Hz)  171.4 (s,
carbonyl C), 144.1 (s), 140.5 (complex t, J = 65 Hz), 134.8 (complex t, J = 60 Hz), 131.2-126.2 (m), 52.0
(s), 40.7 (d, J = 7.3 Hz), 34.1 (d, J = 2.6 Hz), 21.8. HRMS calculated for 13C6C11H18Cl15NO4S (M+H)
375.0898, found 375.0897.
7-Chloro-1-tosyl-2,3-dihydroquinolin-4(1H)-one-4a,5,6,7,8,8a-13C6, 15N 6’:
A mixture of methyl 3-((N-(3-chlorophenyl-1,2,3,4,5,6-13C6)-4-methylphenyl)sulfonamido-
15N)propanoate 5’ (7.91 g, 21.10 mmol), concentrated hydrochloric acid (11.6 mL), water (35.0 mL),
and dioxane (97.0 mL) was heated to reflux for 5 hours. The solution was concentrated with rotary
evaporation to half of the volume, and neutralized by careful addition of solid sodium bicarbonate.
The mixture was then extracted with ether to remove any unreacted starting material. The aqueous
layer was acidified with concentrated hydrochloric acid and then extracted with ether. The organic
extracts were dried over sodium sulfate and the solvent was removed under reduced pressure to
provide the acid as gummy syrup that was used without further purification. To the crude product
obtained above was added dichloromethane (10.0 mL) followed by addition of oxalyl chloride (13.72
mL, 27.43 mmol, 2.0 M in dichloromethane) and the mixture was stirred at room temperature
overnight. The excess reagent and solvent were removed under reduced pressure. To this mixture
was added dichloromethane (105 mL). The solution was cooled with an ice bath and aluminum
chloride (3.17 g, 23.77 mmol) was added in one portion. The reaction mixture was stirred at 0oC for
5-10 minutes, and allowed to warm to room temperature for 1-2 hr, or until TLC showed a complete
reaction. The mixture was cooled to 0oC and water was slowly added. The reaction mixture was then
extracted with dichloromethane, dried over sodium sulfate and purified on silica gel with 20% ethyl
acetate in hexanes to provide 5.04 g (70% from methyl 3-((N-(3-chlorophenyl-1,2,3,4,5,6-13C6)-4-
methylphenyl)sulfonamido-15N)propanoate 5’) of the title product as colorless liquid. 1H NMR (CDCl3,
300 Hz)  8.26-8.09 (m, 1 H), 7.71-7.58 (m, 1 H), 7.62 (d, J = 8.50 Hz, 1 H), 7.58-7.49 (m, 1 H), 7.29 (d,
J = 7.95 Hz), 7.05-6.90 (m, 1 H), 4.24 (dt, J = 6.44, 3.73 Hz, 2 H), 2.48-2.36 (m, 5 H). 13C NMR (CDCl3,
75 Hz)  191.8 (d, J = 51.8 Hz, carbonyl C), 145.1 (s), 143.3 (complex t, J = 64.1 Hz), 141.1 (complex t,
J = 64.1 Hz), 136.5 (d, J = 4.15 Hz), 130.4 (s), 129.2 (complex t, J = 58.3 Hz), 127.0 (s), 126.0 (complex
t, J = 60.0 Hz), 124.2 (complex t, J = 65.0 (s), 123.8 (dt, J = 61, 7.4 Hz), 46.3 (d, J = 5.84 Hz), 36.5 (d, J =
10.8 Hz), 21.8 (s).
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7-Chloro-2, 3-dihydroquinolin-4(1H)-one-4a,5,6,7,8,8a-13C6, 15N 7’:
A mixture of 7-chloro-1-tosyl-2,3-dihydroquinolin-4(1H)-one-4a,5,6,7,8,8a-13C6, 15N 6’ (5.04 g, 14.70
mol), acetic acid (50.0 mL), concentrated hydrochloric acid (50.0 mL), and water (12.5 mL) was
heated to reflux for 5 hr. The mixture was diluted with water and neutralized with slow addition of
solid sodium carbonate. The mixture was extracted with ether, and the organic extracts were
washed with 5% potassium hydroxide, brine and dried over sodium sulfate. The solvent was
removed under reduced pressure to provide 2.57 g (93%) of the title compound as a yellow solid. 1H
NMR (CDCl3, 300 Hz)  8.08 (dt, J = 12.9, 8.33 Hz, 1 H), 8.08 (dt, J = 12.9, 8.33 Hz, 1 H), 7.06-6.94 (bm,
1 H), 6.50-6.39 (bm, 1 H), 4.49 (bd, J = 86.5 Hz, 1 H), 3.68-3.57 (m, 2 H), 3.45 (dt, J = 6.90, 3.45 Hz, 2
H). 13C NMR (CDCl3, 75 Hz)  192.5 (s, carbonyl C), 152.6 (complex t, J = 60.5 Hz), 141.4 (complex t, J =
66.7 Hz), 129.5 (complex t, J = 60 Hz), 118.8 (complex t, J = 60 Hz), 117.2 (apparent d, J = 66 Hz),
115.3 (complex t, J = 60 Hz), 42.3 (br d, J = 8.3 Hz), 38.0 (d, J = 12.2 Hz).
Chloroquine 8’:
A mixture of 7-chloro-2,3-dihydroquinolin-4(1H)-one-4a,5,6,7,8,8a-13C6, 15N 7’ (0.849 g, 4.5 mmol), 2-
amino-5-diethylaminopentane (1.118 g, 7.065 mmol) and nitrobenzene (0.456 g, 3.78 mmol) was
heated to 150-160oC (oil temp) overnight. The solvent and excess reagents were removed by
vacuum distillation at 80oC/15 mmHg, and the residue was purified on silica gel with
dichloromethane: methanol: ammonium hydroxide 80:20:1 to provide 1.21 g of the title compound
as a colorless solid (82%), the material was further purified on HPLC (Waters, XTerra C18, 5 , 4.6 x
150 mm, 1mL/min, linear gradient 98/2 to 20/80 of 0.1% TFA in water/acetonitrile for 16 minutes,
retention time 10.88 min) to get the product as trifluoroacetic acid salt. 1H NMR (D2O, 300 Hz).
8.40-8.35 and 8.00-7.90 (m, 1 H), 8.30-8.20 (m, 1 H), 8.15-8.05 and 7.60-7.50 (m, 1 H), 7.90-7.80 and
7.40-7.30 (m, 1 H), 6.85-6.70 (m, 1 H), 4.15-4.05 (m, 1 H), 3.20-3.02 (m, 6 H), 1.75 (br s, 4 H), 1.35 (d,
J = 5.25 Hz, 3 H), 1.18 (br t, J = 6.81 Hz, 6 H). 13C NMR (CDCl3, 75 Hz) 154.9 (d, J = 57.9 Hz), 142.0 (d,
J = 12.3 Hz), 138.9 (t, J = 65.1 Hz), 137.6 (dt, J = 10.5, 62.7 Hz), 126.7 (dt, J = 5.24, 59.3 Hz), 123.7 (t, J
= 58.1 Hz), 118.5 (t, J = 66.3 Hz), 114.6 (dt, J = 6.99, 60.5 Hz), 98.5, 57.4, 51.0, 49.4, 47.1, 31.7, 20.1,
18.6, 8.0. HRMS calculated for 13C6C12H26Cl15NN2 (M+H) 327.2068, found 327.2057.
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Syntheses leading to desethylchloroquine (13C6, 15N) 9’
Desethylchloroquine 9’
OH
Cl
5-Chloropentan-2-ol 16:
This compound was prepared following a reported procedure as outlined below:24 To a stirred
solution of 5-chloro-2-pentanone (12.05 g, 100 mmol) in 95% EtOH (100 mL) was added NaBH4
portionwise at 0oC. The mixture was stirred at 0oC for 30 min and allowed to warm to room
temperature and stir for 2 hr. The reaction mixture was cooled to 0oC and acetone (20.0 mL) was
added dropwise. The solvents were removed under reduced pressure and water was added to the
residue. The solution was extracted with ether and the combined extracts were dried over sodium
sulfate and evaporated. The residue was distilled (bp 74-76oC/15 mmHg) to give 9.27 g (79%) of the
title product as a colorless liquid. 1H NMR (CDCl3)  3.84 (sext, J = 6.19 Hz, 1 H), 3.58 (dt, J = 6.69,
0.88 Hz, 2 H), 2.01-1.76 (m, 2 H), 1.69-1.49 (m, 2 H), 1.22 (d, J = 6.19 Hz, 3 H). 13C NMR (CDCl3)  67.6,
45.3, 36.5, 29.1, 23.9.
O
O
N
Cl
2-(5-Chloropentan-2-yl)isoindoline-1,3-dione 17:
To a solution of 5-chloropentan-2-ol (2.69 g, 21.94 mmol), phthalimide (3.55 g, 24.14 mmol), and
triphenylphosphine (6.33 g, 24.14 mmol) in THF (50 mL) was added dropwise diisopropyl
azodicarboxylate (4.88 g, 24.14 mmol) at 0oC. The mixture was allowed to warm to room
temperature and stirred overnight. To the reaction mixture was added silica gel and slurry was
concentrated. The mixture was loaded onto a silica gel column and purified with 15% ethyl acetate
in hexanes to provide the title product (4.88 g, 88%) as a clear liquid that solidified on standing. 1H
NMR (CDCl3)  7.86-7.79 (m, 2 H), 7.74-7.67 (m, 2 H), 4.44-4.31 (m, 1 H), 3.53 (dt, J = 6.80, 1.26 Hz, 2
H), 2.30-2.15 (m, 1 H), 1.9-1.84 (m, 1 H), 1.84-1.64 (m, 2 H), 1.50 (d, J = 6.96 Hz, 3 H). 13C NMR (CDCl3)
 168.7, 134.2, 132.2, 123.4, 46.9, 44.5, 31.2, 30.0, 19.0.
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O
O
N
I
2-(5-Iodopentan-2-yl)isoindoline-1,3-dione 18:
A mixture of 2-(5-chloropentan-2-yl)isoindoline-1,3-dione 17 (6.99 g, 27.77 mmol) and sodium iodide
(6.24 g, 41.66 mmol) in acetone (75 mL) was refluxed for two days. The reaction mixture was filtered
and the solvent was removed under reduced pressure. The residue was purified on silica gel with
15% ethyl acetate in hexanes to provide the title product (9.23 g, 97%) as a clear liquid. 1H NMR
(CDCl3)  7.86-7.79 (m, 2 H), 7.74-7.67 (m, 2 H), 4.43-4.30 (m, 1 H), 3.17 (t, J = 6.96 Hz, 2 H), 2.28-2.14
(m, 1 H), 1.95-1.69 (m, 3 H), 1.49 (d, J = 6.96 Hz, 3 H). 13C NMR (CDCl3)  168.6, 134.2, 132.1, 123.4,
46.6, 34.8, 30.9, 19.0, 5.77.
O
O
N
NEtTs
N-(4-(1,3-Dioxoisoindolin-2-yl)pentyl)-N-ethyl-4-methylbenzenesulfonamide 19:
To a mixture of 2-(5-iodopentan-2-yl)isoindoline-1,3-dione (4.61 g, 13.45 mmol) 18 and N-ethyl-p-
toluenesulfonamide (3.74 g, 18.78 mmol) in DMF (50.0 mL) was added sodium hydride (0.75 g, 18.77
mmol, 60% in oil) in one portion at 0oC. The mixture was allowed to warm to room temperature and
stirred overnight. Water was added slowly and the reaction was extracted with diethyl ether, the
organic extracts were washed with 0.2 M sodium hydroxide, dried and concentrated. The residue
was purified on silica gel with 35-40% ethyl acetate in hexanes to provide the title product as a clear
liquid (3.86 g, 69%). 1H NMR (CDCl3)  7.85-7.79 (m, 2 H), 7.75-7.69 (m, 2 H), 7.66 (d, J = 8.28 Hz, 2 H),
7.24 (d, J = 8.28 Hz, 2 H), 4.41-4.26 (m, 1 H), 3.33-2.96 (m, 4 H), 2.38 (s, 3 H), 2.16-2.00 (m, 1 H), 1.82-
1.67 (m, 1 H), 1.56-1.43 (m, 2 H), 1.49 (d, J = 6.96 Hz, 3 H). 13C NMR (CDCl3)  168.7, 143.2, 137.4,
134.1, 132.2, 129.8, 127.3, 123.3, 47.2, 47.1, 43.0, 30.9, 26.2, 21.7, 18.9, 14.2.
NH2
NEtTs
N1-Ethyl-N1-tosylpentane-1,4-diamine 20:
To a mixture of N-(4-(1,3-dioxoisoindolin-2-yl)pentyl)-N-ethyl-4-methylbenzenesulfonamide 19 (3.86
g, 9.32 mmol) in ethanol (38.0 mL) was added methylamine (3.48 mL, 27.97 mmol, 33% wt in
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ethanol). The reaction mixture was heated at reflux overnight. The reaction was cooled to room
temperature, precipitates were filtered off and washed with ethanol. The filtrate was concentrated
and acidified with 1 M hydrochloric acid, then extracted with ether. The aqueous layer was then
basified with sodium hydroxide and extracted with ether. The organic extracts from the basic
extraction were concentrated to provide the title product (1.11 g, 42%) as a clear liquid. 1H NMR
(CDCl3)  7.69 (d, J = 8.55 Hz, 2 H), 7.29 (d, J = 8.55 Hz, 2 H), 3.21 (q, J = 7.23 Hz, 2 H), 3.12 (dt, J =
7.02, 2.30 Hz, 2 H), 2.88 (sext, J = 6.30 Hz, 1 H), 1.68-1.48 (m, 2 H), 1.84-1.64 (m, 2 H), 1.37-1.25 (m, 2
H), 1.10 (t, J = 7.23 Hz, 3 H), 1.05 (d, J = 6.30 Hz, 3 H). 13C NMR (CDCl3)  143.2, 137.4, 129.8, 127.3,
47.8, 46.8, 42.9, 37.1, 25.9, 24.3, 21.7, 14.3. HRMS calculated for C14H24N2O2S (M+H) 285.1637,
found 285.1627.
N-(4-((7-Chloroquinolin-4-yl-4a,5,6,7,8,8a-13C6,15N)amino)pentyl)-N-ethyl-4-
methylbenzenesulfonamide 21:
A mixture of N-1-ethyl-N-1-tosylpentane-1,4-diamine (0.462 g, 1.63 mmol) 20, 7-chloro-2,3-
dihydroquinolin-4(1H)-one (15N, 13C6) 7’ (0.193 g, 1.02 mmol) in nitrobenzene (0.088 mL, 0.852
mmol) was heated at 160-165oC overnight. The reaction solution was loaded onto silica gel and
eluted with 70% ethyl acetate in hexanes to provide 0.347 g (75%) of the title product as brown
syrup. 1H NMR (CDCl3)  8.44-8.17 (bm, 1 H), 7.83-7.54 (m, 1 H), 7.65 (d, J = 8.39 Hz, 2 H), 7.27 (d, J =
8.39 Hz, 2 H), 7.33-6.98 (m, 1 H), 6.71-6.61 (m, 1 H), 6.49-6.40 (m, 1 H), 6.33-5.81 (m, 1 H), 3.90-3.76
(m, 1 H), 3.29-3.05 (m, 4 H), 2.41 (s, 3 H), 1.81-1.66 (m, 2 H), 1.39 (d, J = 6.91 Hz, 3 H), 1.06 (t, J = 7.45
Hz, 3 H). 13C NMR (CDCl3)  143.4 (t, J = 62.8 Hz), 137.2 (d, J = 65.3 Hz), 129.7, 126.3 (dt, J = 60.0, 5.97
Hz), 123.2 (t, J = 72.1 Hz), 116.0 (t, J = 58.9 Hz), 49.3, 47.3, 43.2, 32.4, 25.5, 21.5, 20.1, 14.0. HRMS
calculated for 13C6C17H28Cl15NN2O2S (M+H) 453.1843, found 453.1826.
Desethylchloroquine 9’:
A mixture of N-(4-((7-chloroquinolin-4-yl-4a,5,6,7,8,8a-13C6, 15N)amino)pentyl)-N-ethyl-4-
methylbenzenesulfonamide 21 (0.347 g, 0.766 mmol) in 48% aqueous hydrobromic acid (2.02 mL)
and acetic acid (1.13 mL) was heated to reflux overnight. The mixture was neutralized with sodium
hydroxide and extracted with dichloromethane. The solvent was removed under reduced pressure
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and the residue was purified on silica gel with 10-15% MeOH in CH2Cl2 to provide 0.114 g (50%) of
the title product as a light yellow liquid. The product was then acidified with 2.0 mL of 1 M HCl and
the mixture was dried under reduced pressure. The residue was purified with HPLC (Waters, XTerra ,
5 , 4.6 x 150 mm, 1 mL/min, linear gradient, 98/2 to 20/80 of 0.1 % TFA in water/acetonitrile for 16
minutes, retention time 10.95 min). 1H NMR (D2O)  8.50-8.33 and 7.96-7.78 (m, 1 H), 8.22 (br t, J =
7.56 Hz, 1 H), 8.05 and 7.49 (m, 1 H), 7.96-7.78 and 7.53-7.44 (m, 1 H), 6.79 (q, J = 5.43 Hz, 1 H), 4.08
(q, J = 5.97 Hz, 1 H), 3.02 (q, J = 7.07 Hz, 4 H), 1.77 (br s, 4 H), 1.36 (d, J = 6.25 Hz, 3 H), 1.21 (t, J =
7.45 Hz, 3 H). 13C NMR (D2O )  156.3, 155.6, 154.9, 142.2, 142.0, 139.7 (complex t, J = 65.66 Hz),
138.4 (ddt, J = 4.70, 14.00, 63.03 Hz), 127.8 (dt, J = 6.30, 60.22 Hz), 124.0 ( (dt, J = 5.03, 58.64 Hz),
119.0 (complex t, J = 67.16 Hz), 116.8, 115.3 (complex t, J = 60.8 Hz), 100.0, 46.0, 42.4, 10.3. HRMS
calculated for 13C6C10H22Cl15NN2 (M+H) 299.1755, found 299.1750.
Syntheses leading to bisdesethylchloroquine (13C6, 15N) 10’
Bisdesethylchloroquine 10’
O
O
N
NBoc2
2-(5-di-tert-butyl-iminodicarboxypentan-2-yl)isoindoline-1,3-dione 22:
To a mixture of 2-(5-iodopentan-2-yl)isoindoline-1,3-dione (4.614 g, 13.45 mmol) 18 and di-tert-
butyl-iminodicarboxylate (4.08 g, 18.78 mmol) in THF (50.0 mL) was added sodium hydride (0.751 g,
18.78 mmol, 60% in oil) at 0oC. The mixture was allowed to warm to room temperature and was
then heated to reflux overnight. The mixture was partitioned between ethyl acetate and water, and
the organic extracts were washed with brine and dried over sodium sulfate. The solvent was
removed under reduced pressure and the residue was purified on silica gel with 10-15% ethyl
acetate in hexanes to provide 5.302 g (91%) of the title product as colorless liquid. 1H NMR (D2O) 
7.81 (dd, J = Hz, 2 H), 7.70 (dd, J = Hz, 2 H), 4.43 (m, 1 H), 3.55 (t, J = 7.56 Hz, 2 H), 2.14-1.99 (m, 2 H),
1.82-1.67 (m, 2 H), 1.48 (s, 9 H), 1.45 (s, 9 H), 1.47 (d, J = 6.91 Hz, 3 H). 13C NMR (CDCl3)  168.4,
152.5, 133.8, 132.0, 123.1, 82.1, 47.1, 45.9, 30.9, 28.04, 28.03, 26.4, 18.7.
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NH2
NBoc2
2-(5-di-tert-butyl-iminodicarboxypentan-2-yl)amine 23:
To a solution of 2-(5-di-tert-butyl-iminodicarboxypentan-2-yl)isoindoline-1,3-dione 22 (5.30 g, 12.26
mmol) in ethanol (50.0 mL) was added methylamine (4.60 mL, 36.78 mL, 33% wt in ethanol). The
reaction mixture was heated to reflux overnight. After the solution cooled to room temperature, the
precipitates were filtered and washed with ethanol. The filtrate was concentrated and was purified
on silica gel with 100% ethyl acetate to provide the title product (1.68 g, 56%) as a clear liquid. 1H
NMR (CDCl3)  3.56 (t, J = 7.56 Hz, 2 H), 2.92 (sext, J = 6.36 Hz, 1 H), 1.61 (sext, J = 7.51 Hz, 2 H), 1.33
(q, J = 7.23 Hz, 2 H), 1.08 (d, J = 6.30 Hz, 3 H). 13C NMR (CDCl3)  152.9, 82.4, 46.9, 46.5, 37.1, 28.3,
26.1, 23.9. HRMS calculated for C15H30N2O4 (M+H) 303.2284, found 303.2291.
Tert-butyl (tert-butoxycarbonyl)(4-((7-chloroquinolin-4-yl-4a,5,6,7,8,8a-13C6,
15N)amino)pentyl)carbamate 24:
A mixture of 7-chloro-2,3-dihydroquinolin-4(1H)-one (13C6, 15N,) 7’ (0.190 g, 1.01 mmol) and 2-(5-di-t-
butyl-iminodicarboxypentan-2-yl)amine 23 (0.487 g, 1.61 mmol) in nitrobenzene (0.086 mL) was
heated under Argon to 150-160oC overnight. The solution was loaded on silica gel and eluted with
70% ethyl acetate in hexanes to provide 0.235 g (50%) of the title product. 1H NMR (CDCl3)  8.50 (m,
1 H), 8.27-8.10 and 7.73-7.55 (m, 1 H), 8.12-7.95 and 7.55-7.40 (m, 1 H), 7.55-7.40 and 7.14-6.98 (m,
1 H), 6.42 (t, J = 5.32 Hz, 1 H), 5.39-5.24 (m 1 H), 4.82-4.67 (m, 1 H), 3.91-3.66 (m 1 H), 3.33-3.05 (m,
2 H), 1.83-1.59 (m, 2 H), 1.47 (s, 18 H), 1.33 (d, J = 6.36 Hz, 3 H). 13C NMR (CDCl3)  149.3 (t, J = 60.9
Hz), 135.0 (t, J = 65.1 Hz), 128.6 (t, J = 65.0 Hz), 125.3 (dt, J = 60.3, 5.45 Hz), 121.4 (t, J = 58.3 Hz),
117.3 (t, J = 57.5 Hz), 99.2 9d, J = 8.43 Hz), 79.6, 48.6, 40.4, 33.3, 28.5, 20.6.
Bisdesethylchloroquine 10’:
To a solution of tert-butyl (tert-butoxycarbonyl)(4-((7-chloroquinolin-4-yl-4a,5,6,7,8,8a-13C6,
15N)amino)pentyl)carbamate 24 (0.221 g, 0.47 mmol) in ethyl acetate (6.0 mL) was first added
ethanol (0.270 mL), then acetyl chloride (0.200 mL) at 0oC. The reaction was allowed to warm to
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room temperature and stir overnight. Water was added to dissolve the precipitate and layers were
separated. The aqueous layer was washed with ethyl acetate, concentrated and the residue was
purified by HPLC (Waters, Symmetry C18, 5 , 4.6 x 150 mm, 1 mL/min, linear gradient, 98/2 to
20/80 of 0.1 % TFA in water/acetonitrile for 16 minutes, retention time 10.75 min) to provide 0.198
g (84%) of the title product as a white solid. 1H NMR (D2O)  8.28-8.14 and 7.71-7.55 (m, 1 H), 8.10
(dt, J = 7.51, 2.80 Hz, 1 H), 7.73 and 7.17 (two br s, 1 H), 7.71-7.55 and 7.12-6.99 (m 1 H), 6.68 (q, J =
5.37 Hz, 1 H), 3.98 (q, J = 6.03 Hz, 1 H), 2.94 (t, J = 5.92 Hz, 2 H), 1.70 (br s, 4 H), 1.31 (d, J = 6.47 Hz, 3
H). 13C NMR (CDCl3)  162.8 (q, J = 35.8 Hz), 155.2 (d, J = 58.5 Hz), 141.9 (d, J = 13.2 Hz), 139.2
(complex t, J = 66.2 Hz), 137.9 (t, J = 62.4 Hz), 127.1 (dt, J = 60.5, 7.14 Hz), 123.9 (dt, J = 58.4, 5.19
Hz), 122.1, 118.6 (complex t, J = 68.3 Hz), 118.2, 114.9 (complex t, J = 61.6 Hz), 114.4, 110.5, 98.5,
49.6, 39.3, 31.9, 23.6, 18.7. HRMS calculated for 13C6C8H18Cl15NN2 (M+H) 271.1442, found 271.1438.
4,7-Dichloroquinoline-4a,5,6,7,8,8a-13C6, 15N 11’:
In a 3-neck 50 mL round bottom flask equipped with reflux condenser, a stirring bar and a
thermometer, iodine (0.88 g, 3.45 mmol) and phosphorus oxychloride (3.76 mL, 41.07 mmol) were
heated to 85-90oC for 30 min. To this solution was added 7-chloro-2,3-dihydroquinolin-4(1H)-one-
4a,5,6,7,8,8a-13C6, 15N 7’ (1.300 g, 6.90 mmol) in 1,2-dichloroethane (5.0 mL) dropwise via a
cannular. The reaction was heated to 85-90oC for 2 hr, and then it was added dropwise to a mixture
consisting of an aqueous solution of ammonia, ice and sodium metasulfite. The organic layer was
separated and the aqueous layer was extracted with dichloromethane. The combined organic
extracts were concentrated. The residue was purified on silica gel with 10% ethyl acetate/hexanes to
provide the title product (0.575 g, 41%) as a white solid. 1H NMR (CDCl3, 300 Hz)  8.83-8.72 (bm,
1H), 8.58-8.35 (bm, 1H), 8.00-7.78 (bm, 1H), 7.48 (t, J = 4.5 Hz, 1H), 8.00-7.78 and 7.39-7.26 (bm,
1H). 13C NMR (CDCl3, 75 Hz)  192.5 (s, carbonyl C), 152.6 (complex t, J = 60.5 Hz), 141.4 (complex t, J
= 66.7 Hz), 129.5 (complex t, J = 60 Hz), 118.8 (complex t, J = 60 Hz), 117.2 (apparent d, J = 66 Hz),
115.3 (complex t, J = 60 Hz), 42.3 (br d, J = 8.3 Hz), 38.0 (d, J = 12.2 Hz). HRMS calculated for
15
13C6C3H5Cl2 N (M+H) 205.0052, found 205.0046.
Amodiaquine 12’:
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A mixture of 4,7-Dichloroquinoline-4a,5,6,7,8,8a-13C6, 15N 11’ (0.192 g, 0.93 mmol) and 2-
((diethylamino)methyl)-4-aminophenol dihydrochloride (0.300 g, 1.12 mmol) in ethanol (3.0 mL) was
heated to reflux overnight. The solvent was removed under reduced pressure, and the residue was
adsorbed on silica gel and purified with 10-20% MeOH/CH2Cl2 to provide the crude product. This was
recrystallized from ethanol to provide 200 mg (49%) of the product as a tan solid. HPLC trace
(Waters, XTerra C18, 5 , 4.6 x 150 mm, 1 mL/Min, linear gradient, 98/2 to 40/60 of 0.1 % TFA in
water/acetonitrile for 20 minutes, retention time 15.03). 1H NMR (CD3OD)  8.52-7.20 (m, 4 H),
7.13(dd, J = 0.6, 2.2 Hz, 1 H), 7.06 (d, J = 0.8 Hz, 1 H), 6.82 (d, J = 2.3 Hz), 6.62 (t, J = 1.4 Hz, 1 H), 3.85
(s, 2H), 2.70 (q, J = 1.8 Hz, 2 H), 1.16 (t, J = 1.8 Hz, 3 H). 13C NMR (75 MHz, CDCl3)  157.8, 152.4,
150.2 (t, J = 61.6 Hz), 136.8 (t, J = 61.6 Hz), 132.0, 128.6-123.9 (complex m), 119.2 (t, J = 56.9 Hz),
117.9, 101.9, 57.0, 47.9, 11.6. HRMS calculated for 13C6C14H22Cl15NN2O (M+H) 363.1704, found
363.1719.
Syntheses leading to desethylamodiaquine (13C6, 15N) 14’
Desethylamodiaquine (13C6, 15N) 13’
OH
NEt
NO2
2-((E)-Ethylimino)methyl)-4-nitrophenol 25:
The compound was prepared following the procedure below:25 A mixture of 2-hydroxy-5-
nitrobenzaldehyde (1.67 g, 10.0 mmol), ethylamine (7.0 mL, 14.0 mmol, 2 M in THF) in THF (40.0 mL)
was heated to reflux for 3 h. The solvent was removed under reduced pressure and the crude
material was checked by 1H and 13C NMR, and used without further purification. 1H NMR (300 MHz,
CDCl3)  8.33 (s), 8.16 (dd, J = 9.25, 2.86 Hz), 6.92 (d, J = 9.25 Hz), 3.70 (dq, J = 7.31, 1.14 Hz), 1.38 (t,
J = 7.13 Hz). 13C NMR (75 MHz, CDCl3)  170.8, 163.7, 138.6, 128.6, 127.4, 119.8, 116.5, 51.9, 15.9.
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OH
NHEt
NO2
2-(Ethylamino)methyl-4-nitrophenol 26:
2-((E)-(Ethylimino)methyl)-4-nitrophenol 25 was suspended in methanol (20 mL). To this mixture was
added sodium borohydride (0.297 g, 7.85 mmol) in 0.5 mM solution of NaOH (30.0 mL) at room
temperature. The reaction mixture was stirred at room temperature overnight. The solids were
filtered, washed thoroughly with methanol and air-dried to provide 1.652 g (84% from 2-hydroxy-5-
nitrobenzaldehyde) of the title product as a yellow solid. 1H NMR (300 MHz, DMSO-d6)  7.98 (d, J =
3.07 Hz), 7.86 (dd, J = 9.32, 3.07 Hz), 6.23 (d, J = 9.32 Hz), 3.94 (s), 2.84 (q, J = 7.32 Hz), 1.16 (t, J =
7.32 Hz). 13C NMR (75 MHz, DMSO-d6)  176.4, 130.4, 127.1, 127.0, 120.0, 118.2, 48.1, 41.1, 11.4.
OH
NHEt.HCl
.
NH2 HCl
4-Amino-2-((ethylamino)methyl)phenol bishydrochloride 27:
2-(Ethylamino)methyl-4-nitrophenol 26 (1.652 g, 8.42 mmol) was dissolved in 1 M HCl(18.7mL) and
10% Pd/C (0.084 g) was added. The mixture was hydrogenated at 45-50 psi at room temperature
overnight. The mixture was filtered and the filtrate was concentrated and dried under vacuum to
provide 1.097 g (54%) of the title product as an off-white solid. 1H NMR (300 MHz, MeOH-d6)  7.44
(d, J = 2.80 Hz), 7.31 (dd, J = 8.66, 2.80 Hz), 7.02 (d, J = 8.66 Hz), 4.20 (s), 3.09 (q, J = 7.34 Hz), 1.33 (t,
J = 7.34 Hz). 13C NMR (75 MHz, MeOH-d6) 158.3, 127.6, 127.1, 123.5, 120.9, 117.7, 47.0, 44.1, 11.5.
Desethylamodiaquine (13C6, 15N) 13:
A mixture of 4,7-Dichloroquinoline-4a,5,6,7,8,8a-13C6, 15N 11’ (0.186 g, 0.907 mmol) and 4-amino-2-
((ethylamino)methyl)phenol dihydrochloride 27 (0.239 g, 0.998 mmol) in water (5.0 mL) was heated
to reflux for 1 hr. The solvent was removed under reduced pressure and the residue was adsorbed
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on silica gel then purified with 10% MeOH/CH2Cl2 to provide the crude product, which was further
purified by HPLC (Waters XTerra C18, 5 , 4.6 x 150 mm, 1 mL/min linear gradient, 98/2-20/80 of
0.1% TFA in water/acetonitrile for 16 minutes, retention time 11.03 min) to provide 0.353 g (95%) of
the title product as a white solid. 1H NMR (300 MHz, D2O)  8.55-8.40 and 8.13-8.04 (m, 1 H), 8.23-
8.13 (m, 1 H), 8.01-7.85 (m, 1H), 7.59-7.48 and 7.43-7.27 (m, 3 H), 7.07 (d, J = 9.0 Hz, 1 H), 6.73 (q, J =
6.0 Hz, 2 H), 4.21 (s, 2 H), 3.12 (q, J = 9.0 Hz, 2 H), 1.28 (t, J = 6.0 Hz, 3 H). 13C NMR (75 MHz, D2O)
156.3, 155.6, 154.9, 142.2, 142.0, 139.7 (complex t, J = 65.6 Hz), 138.4 (ddt, J = 4.70, 14.0, 63.0 Hz),
128.9, 128.7, 127.8 (dt, J = 6.30, 60.2 Hz), 123.9 (dt, J = 5.03, 58.6 Hz), 119.0 (complex t, J = 67.2 Hz),
116.8, 115.3 (complex t, J = 60.8 Hz), 100.0, 46.0, 42.4, 10.3. HRMS calculated for 13C6C12H18Cl15NN2O
(M+H) 335.1391, found 335.1386.
Bisdesethylamodiaquine (13C6, 15N) 14:
A mixture of 4,7-Dichloroquinoline-4a,5,6,7,8,8a-13C6, 15N 11’ (0.191 g, 0.93 mmol) and 4-amino-2-
(amino)methyl)phenol dihydrochloride (0.237 g, 1.12 mmol) in water (5.0 mL) was heated to reflux
overnight. The solvent was removed under reduced pressure and the residue was adsorbed on silica
gel then purified by FLC with 20% MeOH/CH2Cl2 to provide the crude product, which was further
purified by HPLC (Waters XTerra, C18, 5 , 4.6 x 150 mm 1 mL/min for 25 minutes, linear gradient
from 98/2 to 30/70 0.1% TFA in water/acetonitrile, retention time 14.03 min) to provide 0.197 g
(56%) of the title product as a white solid. 1H NMR (300 MHz, MeOH-d6)  8.62-8.53 and 7.85-7.73
(m, 1 H), 8.37-8.25 (m, 1H), 8.16-7.85 (m, 1 H), 7.60-7.50 and 7.26-7.18 (m, 1 H), 7.35 (d, J = 2.52 Hz),
7.30-7.25 m, 1 H), 7.02 (d, J = 8.55 Hz, 1 H), 6.69 (t, J = 5.06 Hz, 1 H), 4.14 (s, 2 H). 13C NMR (75 MHz,
CDCl3)  155.8, 153.5, 152.8, 151.4, 149.0 (t, J = 58.6 Hz), 137.6 (t, J = 58.6 Hz), 132.4, 129.1 (d, J = 15
Hz), 128.0-124.0 (complex m), 122.2, 119.0 (t, J = 56.7 Hz), 117.5, 102.2 (d, J = 15 Hz), 40.7. HRMS
calculated for 13C6C10H14Cl15NN2O (M+H) 307.1078, found 307.1086.
Acknowledgements: We gratefully acknowledge the support of this work from the CDC
(R‐2589‐05‐0). We would also like to thank Drs. John and Dana Barr from CDC, Emory University, and
Dr. Rudy Martinez from Spinceutica Inc (Santa Fe, NM) for their insightful contributions and to their
science and editing suggestions for the manuscript.
Conflict of Interest: The authors did not report any conflict of interest.
This article is protected by copyright. All rights reserved.

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