Synthesis of a labeled inhibitor of HIV-1 attachment: 1-(4- benzoylpiperazin-1-yl)-2-(4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridinyl-3-yl-[U- 14C]ethane-1,2-dione, BMS-488043-14C

Article abstract of DOI:10.1002/jlcr.1856

BMS-488043 is a potent inhibitor of the interaction between HIV-1 gp120 and the host cell receptor CD4. We prepared the carbon-14-labeled version to support preclinical studies of the compound. It was prepared from ethyl [U- ¹⁴C]oxalyl chloride

Full text of DOI:10.1002/jlcr.1856

Research Article
Received 18 August 2010,
Revised 14 October 2010,
Accepted 3 November 2010
Published online 29 December 2010 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1856
Synthesis of a labeled inhibitor of HIV-1
attachment: 1-(4-benzoylpiperazin-1-yl)-
2-(4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridinyl-
3-yl-[U-14C]ethane-1,2-dione, BMS-488043-14C
Ã
I. Victor Ekhato and J. Kent Rinehart
BMS-488043 is a potent inhibitor of the interaction between HIV-1 gp120 and the host cell receptor CD4. We prepared the
carbon-14-labeled version to support preclinical studies of the compound. It was prepared from ethyl [U-14C]oxalyl chloride
by a sequence using Friedel–Crafts acylation reaction. The overall radiochemical yield was 82% with a purity of 499.9%.
Keywords: HIV-1; attachment inhibitor; gp120-CD4 interaction inhibitor
spectra were recorded at 300, 400 or 500 MHz. Chemical shifts
are given in ppm relative to tetramethylsilane (TMS). Agilent
Introduction
The emergence of resistance, mutant strains, adverse drug side-
effects and drug toxicities from long-term use, are all major
challenges to the sustained efficacy of current antiretroviral
therapies and the goal of eradicating HIV.1 Although combina-
tion drug therapies, referred to as HAART, and other treatment
modalities aimed at these challenges have undeniably reduced
mortality and extended the lives of patients,2 the inability to
sustain the control of viral replication3 eventually results in
treatment failure. There is therefore need for new classes of
antiretroviral drugs with distinct mechanisms of action that will
improve the options available to these patients. BMS-488043 is a
novel HIV-CD4 attachment inhibitor.4 It acts by interfering in the
gp120 binding to cellular CD4 receptors.5 The conformational
change induced by the pre-CD4 bound envelope prevents
gp120-CD4 interactions and the downstream events leading to
HIV-1 infection and viral replication. BMS-488043, in Figure 1,
has demonstrated proof of concept antiviral efficacy for this
mechanism in the clinic and the safety profile in HIV-infected
subjects was favorable.6 Further preclinical characterization of
BMS-488043 required the carbon-14 isotopomer and we have
synthesized it in a short high-yielding sequence from ethyl
[U-14C]oxalyl chloride. Herein, we are discussing the details of
the preparation (Scheme 1).
1100 HPLC System including solvent degasser, pump, auto-
mated injector, and a variable UV detector connected to IN/US
BetaRam model 4 Flow Detector with a 0.25 mL detector cell
was used in the analyses of compounds. The HPLC column was
Waters Xterra Ms C8, 5 m, 4.6 Â 250 mm. Elution Solvent System
A = 10 mM Ammonium acetate, and B = Acetonitrile under
Gradient Condition of 0–8 min, 30–60% B; 8–12 min, 60–30% B,
and a Flow rate of 1.0 mL/min, with detection by UV at 254 and
280 nm. Radiochemical Counting was performed on a Packard
574 Liquid Scintillation Analyzer using Beckman Readi-Solv MP
cocktail. LC/MS analysis was performed on a Finnigan LXQ Mass
Spectrometer System, LC/MS method: Generic-B20 (1p ESI full
mass). Ethyl [U-14C]oxalyl chloride (3.70 GBq, 100 mCi) was
purchased from Amersham Biosciences UK Limited.
2-(4,7-Dimethoxy-1H-pyrrolo[2,3-c]pyridine-3-yl)-2-[U-14C]
oxoacetic acid 3
4,7-Dimethoxy-1H-pyrrolo[2,3-c]pyridine hydrochloride (1.22 g,
5.65mmol) was added to a stirred suspension of anhydrous
aluminum chloride (2.26 g, 16.95 mmol) in nitromethane (25 mL)
under nitrogen atmosphere. The mixture was stirred for 8 min
and ethyl [U-14C]oxalyl chloride (100mCi, Sp. Act. 115 mCi/mmol,
diluted with ‘cold’ to 653 mg, (532.2mL) 5.65mmol)in dry
dichloromethane (2 mL) was added in one portion. After the
reaction was stirred for 30min at room temperature, it was
quenched by pouring carefully onto 20% ammonium acetate
Experimental
All reactions were carried out under an atmosphere of argon
unless otherwise specified. Solvents were commercial grade and
used without purification or drying. Column chromatography
was carried out on Merck Kiesegel 60 (230 m) silica gel. Flash
chromatographic separations were performed on a Biotage
Flash System using a pre-packed silica gel cartridge. TLC
visualization reagents included (10% iodine plus 10% AcOH) in
40% aqueous KI and Cerium sulfate in 10% Sulfuric acid. 1H NMR
Department of Chemical Synthesis, Bristol-Myers Squibb Company, Princeton, NJ
08543, USA
*Correspondence to: I. Victor Ekhato, Department of Chemical Synthesis, Bristol-
Myers Squibb Company, Rt. 206/Provinceline Road, Princeton, NJ 08543, USA.
E-mail: ihoezo.ekhato@bms.com
J. Label Compd. Radiopharm 2011, 54 289–291
Copyright r 2010 John Wiley & Sons, Ltd.

I. V. Ekhato and J. K. Rinehart
solution in water. The mixture was stirred for additional 10min connected to C18 separation column. After equilibration with
and extracted with methylene chloride (3Â 20mL). Evaporation water (1000 mL) the compound was eluted by step-gradient
of the organic extract gave a solid 2, indicated by HPLC to be 498% method with 15, 30, 45 and 60% acetonitrile in water (500mL)
radiochemically pure and determined by counting to be each time. The radioactive fractions (at 45% solvent effluent) were
ꢀquantitative (499.6 mCi) yield. 1H NMR (400 MHz, CDCl3) d checked by HPLC method and the pure fractions were combined.
1.37 (tr, 3H), 3.90 (s, 3H), 4.02 (s, 3H), 4.36 (q, 2H, J= 7.1 Hz), 7.45 Methanol and toluene were added to aid the evaporation of
(s, 1H), 8.15 (d, 1H, J= 3.4 Hz), 9.74 (brs, 1H). HPLC (Waters Xterra MS acetonitrile under high vacuum at room temperature. A white
C8, 5 micron 4.6 Â 250 mm column, Solvent system A = 10 mM solid separated after the organic solvent was evaporated. It was
Ammonium acetate, B = Acetonitrile, Gradient Run 0–8 min 30–60% collected by filtration, dried under high vacuum overnight to
B, 8–12 min 60–30% B, Flow rate 1 mL/min at UV detection 254 nm, give 4 [14C]-BMS-488043 (661mg, 28.15 mCi, Sp.Act. 42.6mCi/mg,
elution at Retention time of 7 min, 98.6% chemical purity. 18.0mCi/mmol, 83.5%). 1H-NMR (400MHz, CDCl3), d 1.56 (s),
The material 2 was dissolved in methanol (50 mL) and while 3.51(br), 3.76 (br), 3.93 (s, 3H), 4.04 (s, 3H), 7.42, 7.46, 8.01 (d, 1H),
stirring 0.1M NaOH (12mL) was added. After the hydrolysis 9.25 (brs, 1H). By HPLC (conditions as in foregoing experiment)
was completed, as indicated by HPLC, the organic solvent was compound co-eluted with authentic reference at Retention time
evaporated completely under reduced pressure, the remaining of 6 min, Purity was determined to be 499.9%.
aqueous phase was washed with ethyl acetate (2 Â 30 mL) and the
organic portion was discarded. Acidification of the aqueous portion Results and discussion
with 1N HCl (12 mL) yielded solid that was collected and dried
In designing the current synthesis we wanted to be able to take
advantage of known intermediates from the existing work in the
HIV attachment program.5 From this perspective, the bridging of
the two halves of BMS-488043 with a label from elements of
under high vacuum to give 3 (2.27 g, Sp. Act. 43.19 mCi/mg,
98.17 mCi, 98.17%). 1H-NMR (400 MHz, DMSO-d6) d 3.82 (s, 3H), 3.99
(s, 3H), 7.45 (s, 1H), 8.17 (d, 1H, J= 3.4 Hz), 13.05 (brs, 1H).
oxalyl chloride was considered favorable. It is known that 4,7-
dimethoxy-1H-pyrrolo[2,3-c]pyridine BMS-544347
1-(4-Benzoylpiperazin-1-yl)-2-(4,7-dimethoxy-1H-pyrrolo
[2,3-c]pyridin-3-yl)[U-14C]ethane-1,2-dion 4, [14C]-BMS-488043
1 could be
C-acylated with ethyl oxalyl chloride to produce ethyl 2-(4,7-
dimethoxy-1H-pyrrolo[2,3-c]pyridine-3-yl)-2-oxoacetate 2. The free
acid 3 from this product could then be coupled to phenyl(piper-
azin-1-yl)methanone to afford BMS-488043. While developing the
reaction sequence outlined above we encountered conditions
that would not give acceptable yields of labeled product. A more
practical sequence that includes changes to the isolation protocol
had to be developed. The Friedel–Crafts reaction of ethyl oxalyl
chloride with 4,7-dimethoxy-1H-pyrrolo-[2,3-c]pyridine 1 to make
the ethyl pyrrolo-oxoacetic ester according to the process
reported in a patent application5 produced product yields less
than ideal for the preparation of labeled material. The reaction of
these compounds in dichloromethane resulted in extensive
polymerization which complicated product recovery. However,
with nitromethane as the reaction medium, the reaction
proceeded smoothly to provide the desired product in excellent
yield. It was ascertained in our studies that about 5% v/v
To a suspension of 3 (780 mg, 2.16mmol, 33.69 mCi, Sp. Act.
43.19 mC/mg) and HATU (1.232g, 3.24mmol, 1.5 eq) in dry DMF:
DCM (1:1 v/v), (20 mL) was added a mixture of DMAP (791.6mg,
6.48mmol, 3.0 equiv), and 5, BMS-19848, (734.5mg, 3.24mmol).
The mixture was stirred for 4 h under nitrogen at room
temperature and concentrated to a residue. This material was
applied to a pre-column packed with C18 silica gel and it was
Figure 1. HIV-CD4 attachment inhibitor.
Scheme 1. Preparation of [14C]-BMS-488043.
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 289–291

I. V. Ekhato and J. K. Rinehart
dichloromethane, which we would need to transfer the labeled
reagent, did not adversely affect the yield of the desired product.
The optimized reaction conditions involved mixing compound 1
and aluminum chloride in anhydrous nitromethane and, reacting
with ethyl [U-14C]oxalyl chloride, added at room temperature as a
solution in DCM, to furnish the -pyrrolo[2,3-c]pyridine 3-yl)-2-
[U-14C]oxoacetic acid ethyl ester 2 in 99.6% radiochemical yield.
The ester grouping was removed by hydrolysis and acidification
to provide the free acid 3 in a total yield of 98% for the two steps.
The free acid 3 was activated with HATU and treated with 5, BMS-
196848, in the presence of 3 eq of DMAP in a reaction which
proceeded quantitatively to the desired product. However, the
isolation of product was burdensome and recovery was poor. It
seemed that the product might not be amenable to the
extraction method used for isolation and therefore we examined
the use of a C18-column to separate the reaction debris and
isolate the pure product in a few fractions. In the event, the
product separated as a solid upon the concentration of pure
fractions under reduced pressure, allowing us to collect the
product by filtration. The foregoing procedure afforded 84% yield
of 4 [14C]-BMS-488043. The overall reaction yield from ethyl
[U-14C]oxalyl chloride was found to be 82%. All of the compounds
were identified by a combination of chromatographic compar-
ison with authentic material where available, or with the
appropriate products from pre-labeling studies as reference,
and by matching the spectroscopic data of references with those
of the labeled versions.
Acknowledgements
I. V. E gratefully acknowledges BMS, Department of Chemical
Synthesis for its support. We also want to thank Doris Chen, BMS
PR&D Engineering Technologies and Prashant Deshpande,
BMS Process Dev. Chem., New Brunswick, NJ for the supply
of Reference BMS-488043, BMS-573483 and the intermediate
BMS-544347.
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J. Label Compd. Radiopharm 2011, 54 289–291
Copyright r 2010 John Wiley & Sons, Ltd.
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