Isotope labeled 'HEA/HEE' moiety in the synthesis of labeled HIV-protease inhibitors - Part II

Article abstract of DOI:10.1002/jlcr.913

[²H₅]-Amprenavir and [²H ₅]-saquinavir have been prepared from a common labeled precursor (1S, 2S)-(1-oxiranyl-2-[²H₅]phenylethyl)-carbamic acid tert-butyl ester, 1. Both of these compounds are in the 'HEA' class of HIV protease inhibitors. [²H₅]-Indinavir, a representative of the 'HEE' group of protease inhibitors, has also been synthesized. In the case of indinavir, 1S-(2,2-dimethyl-8, 8a-dihydro-3aH-indeno-[1,2-d]-oxazol-3R-yl)-2- oxiranylmethyl-3-[²H₅]phenylpropan-1-one, 11, provided the [phenyl-²H₅]-HEE core structure for synthesis of the desired labeled compound. Copyright

Full text of DOI:10.1002/jlcr.913

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 179–193.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.913
Research Article
Isotope labeled ‘HEA/HEE’ moiety in the synthesis
of labeled HIV-protease inhibitors}Part II
I. Victor Ekhato1,*,y, Yuan Liao2 and Mihaela Plesescu3
1 Bristol-Myers Squibb Company, Pharmaceutical Research Institute, 5 Research
Parkway, Wallingford, CT 06492, USA
2 Pfizer Global Research and Development, Ann Arbor, MI, USA
3 Millennium Pharmaceuticals, Cambridge, MA 023139, USA
Summary
[2H5]-Amprenavir and [2H5]-saquinavir have been prepared from a common labeled
precursor (1S, 2S)-(1-oxiranyl-2-[2H5]phenylethyl)-carbamic acid tert-butyl ester, 1.
Both of these compounds are in the ‘HEA’ class of HIV protease inhibitors. [2H5]-
Indinavir, a representative of the ‘HEE’ group of protease inhibitors, has also
been synthesized. In the case of indinavir, 1S-(2,2-dimethyl-8, 8a-dihydro-3aH-
indeno-[1,2-d]-oxazol-3R-yl)-2-oxiranylmethyl-3-[2H5]phenylpropan-1-one, 11, pro-
vided the [phenyl-2H5]-HEE core structure for synthesis of the desired labeled
compound. Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: protease inhibitor; anti-HIV; AIDS; D5-HEA/HEE isostere
Introduction
The hydroxyethylamine (HEA) isostere in amprenavir1 and saquinavir,2 and
the hydroxyethylene (HEE) substructure in indinavir3 have been specifically
labeled with [2H5]. Labeled versions of all three protease inhibitors were
needed for analytical method development and validation.
The synthetic approach we previously employed for [2H5]-DPH 153893 and
[2H5]-DPH 1406624,5 was used to make the two hydroxyethylamine (HEA)
peptide mimetics (see substructures in Figure 1), [2H5]-amprenavir and [2H5]-
saquinavir. In particular, [2H5]-amprenavir and [2H5]-saquinavir were
synthesized by following a sequence of reactions attaching appropriate
residues to the key intermediate, (1S, 2S)-(1-oxiranyl-2-[2H5]phenylethyl)-
carbamic acid tert-butyl ester, 1.
*Correspondence to: I. Victor Ekhato, Bristol-Myers Squibb Company, Pharmaceutical Research Institute,
5 Research Parkway, Wallingford, CT 06492, USA. Email: ihoezo.ekhato@bms.com
Copyright # 2005 John Wiley & Sons, Ltd.
Received 15 July 2003
Revised 8 September 2004
Accepted 14 November 2004

180
I. V. EKHATO ET AL.
HO H
H
N
N
H
HO
H
hydroxyethylene isostere
(HEE)
hydroxyethylamine isostere
(HEA)
Figure 1. Substructures of HIV protease inhibitors
To make [2H5]-indinavir with deuterium similarly incorporated in the
hydroxyethylene (HEE) isostere (see Figure 1) we required a different epoxide
intermediate. We chose 1S-(2,2-dimethyl-8, 8a-dihydro-3aH-indeno[1,2-d]-
oxazol-3R-yl)-2-oxiranylmethyl-3-[2H5]-phenyl-propan-1-one, 11, as the best
intermediate from which to synthesize [2H5]-indinavir. Compound 11 was
prepared from commercial [2H5]-bromobenzene.
In this paper, we report the details of the sequence of reactions from
compound 1, which contains the [2H5]-HEA moiety, to [2H5]-amprenavir and
[2H5]-saquinavir. We also report the use of compound 11 as a [2H5]-HEE
precursor for the preparation of [2H5]-indinavir.
Results and discussion
The three-step sequence from (1S, 2S)-(1-oxiranyl-2-[2H5]phenylethyl)-carba-
mic acid tert-butyl ester, 1, to 80% yield of N-(S)-3-amino-2R-hydroxyl-
4-[2H5]phenylbutyl)-N-isobutyl-4-nitrobenzenesulfonamide, 2, shown in
Scheme 1, was described in an earlier paper.5 It involved the reaction of
isobutylamine with the epoxide 1, formation of the 4-nitrobenzenesulfonamide
derivative followed by the removal of the Boc group to yield the
methanesulfonic acid salt, 2.
[2H5]-AMPRENAVIR
From 2 only two further reaction steps were required to complete making
[2H5]-amprenavir, 4. These two steps involved the transformation of 2 to the
(S)-3-hydroxytetrahydrofuran carbamoyl derivative 3 and the reduction of the
nitro-group to the amine (see Scheme 1). We attempted to make compound 3
by two literature procedures.6,7 The method whereby the chloroformate
derivative of (S)-3-hydroxytetrahydrofuran is made by treating (S)-3-hydro-
xytetrahydrofuran with phosgene, followed by the addition of compound 2,
did not yield the desired carbamoyl compound 3. The second approach, using
(S)-3-tetrahydrofuranylsuccinimidyl carbonate, a reagent that other workers
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LABELED HIV-PROTEASE INHIBITORS}PART II
181
2H
2H
O
2H
2H
O
O
2H
2H
N
2H
2H
O
O
2H
Et3N, CH2Cl2,
87 %
O
H
O
O
H
2H
S
MsOH.H2N
N
H
BocHN
HO
O
NO2
H
2
1
2H
2H
2H
2H
2H
O
2H
2H
2H
O
2H
(i) 10 % Pd/C
H2, MeOH
H
O
O
O
O
H
2H
S
O
O
O
N
N
S
68 %
H
H
HO
O
N
H
N
NH2
H
HO
NO2
4 [2H5]-Amprenavir
3
Scheme 1. Conversion of labeled epoxide 1 into amprenavir
have employed to convert amines to carbamoyl products in good yields,7,8 was
smoothly executed. We made the reagent, 3-(S)-tetrahydrofuranylsuccinimidyl
carbonate, from 3-(S)-hydroxytetrahydrofuran and N,N-disuccinimidyl car-
bonate, according to the method of Ghosh.8 Compound 2 was reacted with 3-
(S-)-tetrahydrofuranylsuccinimidyl carbonate in the presence of triethylamine,
and the reaction afforded 3 in 87% yield.
Catalytic hydrogenation of 3 in the presence of Pd/C gave a product which,
after purification by flash chromatography on a Biotage silica gel column,
afforded a 60% overall yield of [2H5]-amprenavir, 4.
[2H5]-SAQUINAVIR
Prior to our study, the Hoffmann–La Roche group had reported the synthesis
of saquinavir in several isotope labeled forms, including deuterium.9
[2H5]-Saquinavir was made through the incorporation of penta-deuterated
2-quinolinoyl end-cap. Hexa-deuteroquinaldine was used in the two proce-
dures, and one of the methods afforded 72.9 atom % [2H5] in the final product.
The second method gave 63.3 atom % [2H5] product. Our earlier work5 had
indicated that insertion of the [2H5]-labeled HEA fragment with very high
deuterium incorporation (>99 atom %) into PIs can be achieved using
(1S, 2S)-(oxiranyl-2-[2H5]-phenylethyl)-carbamic acid tert-butyl ester, 1.
We have extended this approach to make [2H5]-saquinavir. Scheme 2 shows
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182
I. V. EKHATO ET AL.
CONHtBu
H
H
2H
2H
2H
2H
HN
H
2H
2H
2H
2H
2H
H
2H
H
CONHtBu
H
H
H
CONHtBu
H
(i) MsOH, i-PrOH
(ii) aq. NaHCO3
H
H
i-PrOH, reflux
1
BocHN
N
H
H2N
N
H
78 %
78 %
HO
HO
6
5
2H
2H
2H
2H
2H
H
(i) N-Cbz-L-asparagine, HOBt, NEM, DCC
(ii) EtOH, 10 % Pd/C, H2
CONHtBu
H
O
H
H
H
N
(iii) Quinaldic acid, HOBt, NEM, DCC
N
N
N
H
H
HO
CONH2
O
51 %
9 [2H5]-Saquinavir
Scheme 2. Conversion of labeled epoxide 1 into saquinavir
our sequence of reactions from (3S)-decahydroisoquinoline carboxamide and
(1S, 2S)-(1-oxiranyl-2-[2H5]phenylethyl)-carbamic acid tert-butyl ester, 1, to
[2H5]-saquinavir. Compound 1 reacted with (3S)-decahydroisoquinoline
carboxamide in refluxing isopropanol to provide 5. The following reactions,
which included removing the Boc group from 5, gave compound 6 in 78%
yield.
Subsequent steps involved the reaction of 6 with N-Cbz-L-asparagine
to furnish {1-[1-[2H5]benzyl-3-(3-tert-butylcarbamoyl-octahydro-isoquinolin-
2-yl)-2-hydroxyl-propylcarbamoyl]-2-carbomoyl-ethyl]-carbamic acid benzyl
ester, Pd/C catalyzed hydrogenolysis of the Cbz group to provide 2-amino-
N-[1-[2H5]benzyl-3-(3-tert-butylcarbamoyl-octahydro-isoquinolin-2-yl)-2-hydro-
propyl]-succinamide, and coupling to quinaldic acid in the presence of HOBt
and DCC, giving N-[1-[2H5]benzyl-3-(3-tert-butylcarbamoyl-octahydroisoquino-
lin-2-yl)-2-hydroxy-propyl]-2-[(quinoline-2-carbamoyl)-amino]-succinamide, 9.
N-Ethylmorpholine was used as acid scavenger in the reaction forming ([2H5]-
saquinavir) 9 in 73% overall yield. This sequence was modeled on an earlier
literature synthesis of saquinavir.10
[2H5]-INDINAVIR
Since indinavir belongs to the ‘HEE’ transition state mimetic group of HIV-
PIs, it required a different [2H5]-epoxide intermediate to make the [2H5]-labeled
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LABELED HIV-PROTEASE INHIBITORS}PART II
183
product. 1S-(2,2-Dimethyl-8, 8a-dihydro-3aH-indeno-[1,2-d]-oxazol-3R-yl-2-
oxiranylmethyl-3-[2H5]-phenylpropan-1-one, 11, was selected as a starting
structure for the phenyl-[2H5]-labeled HEE class of protease inhibitors, such as
indinavir.
The chosen synthesis of [2H5]-indinavir is shown in Scheme 3. To start,
[2H5]-hydrocinnamoyl chloride was prepared from commercially available [2H5]-
bromobenzene. Compound 10 was made by the condensation of (1S,2R)-
aminoindanol and [2H5]-hydrocinnamoyl chloride. The desired [2H5]-epoxide
11 was readily assembled from 10 by the use of a literature procedure,11
involving sequential stereoselective allylation and epoxidation reactions.
Opening the epoxide ring of 11 to provide 12 required the reagent
N-tert-butyl-4-N-dimethylethylcarbamoyl-2S-piperazine carboxamide, which
was made from 2-(S)-piperazine carboxylic acid according to a literature
O
tBuHNOC
H
2H
Xc
2H
NH
2H
2H
O
H
(i) LiHMDS, allyl bromide
(ii) aq. NaHCO3, NIS
N
Boc
COXc (iii) THF, NaOMe
2H
i-PrOH
2H
2H
72 %
2H
2H
11
2H
10
tBuHNOC
H
O
tBuHNOC
H
O
N
Xc
2H
H
H
HO
HCl.HN
N
Xc
2H
2H
(i) MeOH-HCl, 0 oC
87 %, 11 to 13
H
H
HO
2H
N
Boc
2H
2H
13
2H
2H
2H
2H
12
tBuHNOC
N
H
O
N
N
H
2H
H
H
HO
2H
N
(i) KHCO3, NaI, 3-picolyl chloride
64 %, 13 to 14
HO
2H
O
N
2H
2H
14 [2H5]-Indinavir
Xc
=
Scheme 3. Preparation of labeled epoxide 11 and its conversion into indinavir
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184
I. V. EKHATO ET AL.
process.12 Reaction of 11 with this reagent in isopropyl alcohol at 808C
furnished {4-[4-benzyl-5-(2,2-dimethyl)-8,8a-dihydro-3aH-indeno[1,2-o]oxazol-
3-yl)-2-hydro-5-oxo-pentyl]-3-tert-butylcarbamoylpiperazin-1-yl}-carbamic tert-
butyl ester, 12. Compound 12 was de-blocked with a solution of hydrogen
chloride in methanol to give 13 in 87% yield from 11. The synthetic
sequence was completed by the displacement reaction between 13 and 3-picolyl
chloride in the presence of NaI and K2CO3, to give [2H5]-indinavir in 40%
overall yield.
Salient spectral features
The insertion of a [2H5]-HEA subunit to make [2H5]-amprenavir leads
to simplification of the proton NMR spectrum of the product. Figure 2 shows
the aromatic regions of the proton NMR spectra of amprenavir and
[2H5]-amprenavir. Three sets of resonances at d 7.52 (d), 7.30–720 (m)
and 6.66 (d), representing a total of nine protons in the ratio of (2:5:2),
were observed for the two benzene ring systems in amprenavir. The multiplet
0.04
7.5
0.13
0.05
7.2
6.9
6.7
6.5
7.3
7.1
6.8
6.6
6.4
7.7
7.6
7.4
7.0
0.05
7.5
0.05
7.7
7.6
7.4
7.3
7.2
7.1
7.0
6.9
6.8
6.7
6.6
6.5
ppm
Figure 2. Aromatic region of 1H-NMR spectra of amprenavir and [2H5]-
amprenavir
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LABELED HIV-PROTEASE INHIBITORS}PART II
185
at d 7.30–7.20 (m) was assigned to the five protons in the phenyl substituent
of the ‘HEA’ moiety of amprenavir. In [2H5]-amprenavir, where the
[phenyl-2H5] moiety has been incorporated, the 1H-NMR spectrum
showed only two pairs of resonances at d 7.52 and d 6.66, for a total
of 4 protons in the ratio of (1:1). This pattern is characteristic of protons
in a para-substituted benzene ring system as in the 4-amino-benzenesulfonyl
group. As would be expected, the multiplet at chemical shift d 7.30–7.20 (m)
due to the phenyl protons was absent in the proton NMR spectrum
of the [phenyl-2H5]-amprenavir. Similar results were seen in [2H5]-saquinavir
and [2H5]-indinavir, but were less obvious due to overlap of resonances
of the additional aromatic groups present in each of those com-
pounds.
Proton-decoupled 13C-NMR spectra were similarly instructive as to
the pattern of deuterium incorporation. The deuterium–carbon coupl-
ing constant (23.8 Hz) observable in the triplet resonance of a proton
decoupled 13C NMR spectrum was seen in [2H5]-amprenavir. Three
triplet resonances were observed in the ratio of (2:2:1) with chemical shifts
of 129.07, 128.23 and 126.02 ppm, respectively. Five additional single lines at
150.74, 137.45, 129.52, 126.26 and 114.11 ppm were attributable to the 4-
amino-benzenesulfonyl-group, and these completed the expected number of
aromatic carbons.
Mass spectrometry provided additional structural information. Figure 3
gives portions of the mass spectra of [2H5]-amprenavir, [2H5]-indinavir and
[2H5]-saquinavir. All three compounds were determined to contain greater
than 99 atom % [2H5]. Neither the [2H2] nor [2H3] species was detectable, and
the [2H4] species was found to be present in less than 0.5% abundance. An
alternate reported synthesis of [2H5]-saquinavir9 indicated two different
preparations of the material to consist of (2H6:2H5:2H4) in the ratio of
5.5:72.9:21.6 and (2H6:2H5:2H4:2H3) in the ratio of 2.8:63.3:27.3:6.6, respec-
tively. In that earlier report, the loss or scrambling of deuterium during
synthesis was suggested to lead to the variable composition of labeled species
in the products.
Details from full scan ESI/MS(+) and MS/MS product ion spectra
of m/z 506 from amprenavir and m/z 511 from [2H5]-amprenavir are
given in the experimental section. A summary of ESI/MS/MS (+)
and (ꢀ) CAD ion fragmentation are shown in Diagram 1. The fragmenta-
tion ions (m/z) from [2H5]-amprenavir are shown in parentheses.
An examination of the fragmentation pattern of [2H5]-amprenavir relative to
amprenavir clearly establishes the location of penta-deuteration as in the
phenyl group of the ‘HEA’ isostere. This result also provides further
confirmation for the absence of scrambling or loss of deuterium during
synthesis.
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186
I. V. EKHATO ET AL.
2H
2H
511.3
2H
2H
O
2H
O
H
O
O
S
O
N
N
H
H
HO
NH2
m/z
502 504 506
508 510 512
514
516 518 520
619.4
tBuHNOC
H
O
N
N
N
H
H
H
HO
2H
N
2H
HO
2H
2H
2H
m/z
610 612 614
616 618 620
622 624 626 628
2H
2H
2H
676.4
2H
O
2H
H
H
H
CONHtBu
H
N
N
N
N
H
H
H
HO
O
CONH2
m/z
668
670
672
674
676
678
680
682
684
Figure 3. MS TOF ESI (+) of [2H5]-Pls
Conclusion
We have developed a sequence for synthesizing penta-deuterated forms of
clinical HIV-protease inhibitors. By making [2H5]-amprenavir, [2H5]-saquina-
vir and [2H5]-indinavir, a potentially general route to [2H5]-HIV-PIs in both
the HEA and HEE classes has been demonstrated. Labeled epoxide
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LABELED HIV-PROTEASE INHIBITORS}PART II
187
-H2O
m/z 245
(m/z 250)
m/z 263
(m/z 268)
O
O
H
O
O
S
O
N
N
H
H
HO
NH2
m/z 418
(m/z 423)
-2H
m/z 373
(m/z 378)
O
O
H
O
O
S
O
N
N
H
H
HO
NH2
m/z 156
(m/z 156)
-2H
m/z 416
(m/z 421)
Diagram 1. ESI/MS/MS (+) CAD and ESI/MS/MS (ꢀ) CAD Ion
Fragmentation of Amprenavir
intermediates were critical to the approach, and these compounds were readily
made from commercially available [2H5]-bromobenzene. Very high deuterium
incorporation is achievable by the approach we have described, and the
absence of scrambling and/or loss of deuterium during synthesis is
noteworthy. We believe this method of synthesis provides access to
inexpensive, high quality [2H5]-labeled PIs for clinical research and mass
spectrometry applications.
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 Kieselgel 60
(230 m) silica gel. Flash chromatographic separations were done using Biotage
Flash System and pre-packed (90 g) silica gel cartridges. TLC visualization
reagents included (10% iodine + 10% AcOH) in 40% aqueous KI. 1H NMR
spectra were recorded at 300, 400 or 500 MHz in CDCl3. Chemical shifts are
given in ppm relative to tetramethylsilane (TMS). 13C-NMR spectra were
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188
I. V. EKHATO ET AL.
recorded at 100 MHz and chemical shifts given in ppm with CDCl3 reference
set at 77.0 ppm. Only the most important IR absorption (cmꢀ1) and the
molecular ions and/or base peaks in mass spectra are given. HPLC analyses
were completed using various columns, as specified in the experimental
section. HRMS data were acquired on an LCT-TOF spectrometer (Micromass
Ltd., Manchester, UK), in ESI mode, with a resolving power of ꢁ5000.
Capillary voltage and sample voltage were at 3 and 25 kV. A desolvation
temperature of 2508C and a source temperature of 1208C was used for all
HRMS experiments.
{3S-[(4-Amino-benzenesulfonyl)-isobutylamino]-1Sꢀ[2H5]-benzyl-2R-hydro-
xylpropyl]-carbamic acid tetrahydrofuran-3-yl ester ([2H5]-amprenavir) 4
To a stirred solution of 2 (1.82 g, 3.5 mmol) and triethylamine (906 ml,
6.47 mmol) in dichloromethane (15 ml) was added 3-(S)-tetrahydrofuranyl-
succinimidyl carbonate8 (840 mg, 3.66 mmol) in dry dichloromethane (8 ml).
After 3 h at room temperature, the reaction mixture was diluted with
dichloromethane (30 ml) and washed successively with aqueous NaHCO3
(2 ꢂ 20 ml), brine (30 ml) and dried on MgSO4. The solvent was evaporated
and a solution of the crude product in dichloromethane (10 ml) was applied to
a column of silica gel. The compound was eluted with 30–45% ethyl acetate in
1
hexane to afford 3 (856.0 mg, 87%). H NMR (400 MHz, CDCl3) d 0.87 (dd,
6H), 1.59 (brs, 1H), 1.87 (m, 2H), 2.10 (m, 1H), 2.79 (m, 4H), 3.18 (m, 2H),
3.64 (d, 1H), 3.79 (m, 4H), 4.84 (d, 1H) 4.13 (brs), 7.94 (d, 2H), and 8.34
(d, 2H). Compound 3 was hydrogenated for 3 h at room temperature in
methanol (60 ml) containing 10% Pd/C (250 mg) and filtered through a pad of
Celite. The Celite was rinsed with methanol (30 ml), and the combined filtrate
and washings was concentrated to a small volume. The material was applied to
a column of silica gel and eluted with 2–5% ethanol in dichloromethane to
give a solid. Crystallization of the solid from methanol yielded 4 [2H5]-
amprenavir, (549 mg, 68%). HPLC on a Waters XTerra MS C184.6 ꢂ 250 mm,
5 m column eluted with the solvent combination of A (0.01 M ammonium
acetate): B (acetonitrile); under gradient conditions: time 0–12 min, 40–60% B;
time 12–18 min, 60–40% B; flow rate 1 ml/min, and peak detection at
UV 254 nm, gave retention time of 8.74 min at greater than 99% chemical
purity. MS acquired on an LCT-TOF spectrometer yield 511.4 (M+H)+;
[2H5] - 99.9 at%). ESI/MS(+) yielded molecular ion at m/z 511 (M+H)+.
MS/MS at CE 45 eV yielded ion m/z 423, and 250. The corresponding ESI/
MS(+) for unlabeled amprenavir yielded m/z 506 (M+H)+. MS/MS product
ion at CE 45 eV produced m/z 418 and 245. ESI/MS(ꢀ) for [2H5]-amprenavir
yielded m/z 569 [M+Ac]ꢀ, 509, 421. MS/MS of m/z 421 yielded ions at
378 and 156. The corresponding ESI/MS(ꢀ) for unlabeled amprenavir gave
564 [M+A]ꢀ, 504, 416. MS/MS of m/z 416 gave product ion at m/z 373 and
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LABELED HIV-PROTEASE INHIBITORS}PART II
189
1
156. ½aꢃ2D0 þ 8:228 (c ¼ 0:49% in MeOH). H NMR (500 MHz, CDCl3) d 0.87
(dd, 6H), 1.80 (m, 1H), 1.92 (m, 1H) 2.07 (m, 1H), 2.76 (dd, 1H), 2.95 (m, 3H),
3.12 (dd, 1H), 3.60 (d, 1H), 3.75 (dd), 3.83 (brs), 4.12 (m, 1H), 4.84 (d, 1H),
5.10 (brs), 6.67 (d, 2H) and 7.53 (d, 2H). 13C NMR (100 MHz, CDCl3) 156.01,
150.74, 137.45, 129.52, 129.07 (tr), 128.23 (tr), 126.26, 126.02 (tr), 114.11,
75.30, 73.24, 72.55, 66.92, 58.83, 55.04, 53.76, 50.83, 35.34, 32.74, 27.30, 20.17,
and 19.90.
[1-[2H5]-Benzyl-3-(3-tert-butylcarbamoyl-octahydro-isoquinolin-2-yl)-2-hydro-
xypropyl]-carbamic acid tert-butyl ester 5
A mixture of (1S, 2S)-(oxiranyl-2-[2H5]-phenyl-ethyl)-carbamic acid tert-butyl
ester 1 (2.5 g, 9.31 mmol) and (3S, 4aS, 8aS)-N-tert-butyl-decahydroisoquino-
line-3-carboxamide (2.7 g, 11.32 mmol) in isopropyl alcohol (20 ml) was
refluxed for 4 h and cooled to room temperature. The reaction mixture was
concentrated to a residue and it was applied to a column of silica gel in a
minimum volume of 2.5% EtOH in CH2Cl2. The column was first eluted with
dichloromethane (400 ml) followed by 2.5% EtOH in CH2Cl2 to yield pure
1
compound as a foamy solid 5 (3.70 g, 78%). H NMR (500 MHz, CDCl3) d
5.88 (s, 1H), 4.83 (d, 1H), 3.85–3.58 (m, 3H), 3.02 (t, 1H), 2.90 (m, 2H), 2.65
(dd, 1H), 2.60 (d, 1H), 2.26 (m, 2H), 1.94 (q, 1H), 1.80–1.60 (m, 1H), 1.50–1.20
(m), 1.32 (s, 18H).
2-(3-Amino-2-hydro-4-[2H5]phenyl-butyl)-decahydro-isoquinoline-3-carboxylic
acid tert-butylamide 6
Methanesulfonic acid (521 ml, 8.03 mmol) in isopropyl acetate (5 ml) was added
dropwise with stirring to 5 (3.60 g, 7.17 mmol) in isopropyl acetate (25 ml)
maintained at 858C. After 40 min, additional methanesulfonic acid (521 ml,
8.03 mmol) was added. TLC (silica gel, CH2Cl2:EtOH:NH4OH; 90:10:0.25 v/
v/v) after 1 h indicated the absence of the fast running starting compound. The
mixture was cooled to room temperature, diluted with ethyl acetate (30 ml)
and washed with a solution of NaHCO3 (2 ꢂ 15 ml), brine (30 ml) and dried
over magnesium sulfate. Solvent was evaporated and crude material was
1
crystallized from methanol to afford 6 (2.27 g, 78%). H NMR (500 MHz,
CDCl3) d 6.18 (s, 1H), 3.65 (m, 1H), 3.20 (brs, 1H), 3.08 (m, 2H), 2.87 (dd,
1H), 2.70 (dd, 1H) 2.65 (dd, 1H), 2.47 (d, 1H), 2.33 (m, 2H), 1.87 (q, 1H), 1.79–
1.60 (m, 3H), 1.56–1.20 (m), 1.33 (s, 9H).
N-[1-[2H5]Benzyl-3-(3-tert-butylcarbamoyl-octahydroisoquinolin-2-yl)-2-hydro-
xypropyl]-2-[(quinoline-2-carbamoyl)-amino]-succinamide ([2H5]-saquinavir) 9
Cbz-L-Asparagine (498 mg, 1.86mmol), HOBt (253mg 1.86 mmol), 4-ethyl-
morpholine (237ml, 1.86mmol), dicyclohexylcarbodiimide DCC (424.9mg,
2.05mmol) in anhydrous THF (20 ml) was stirred under nitrogen atmosphere,
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I. V. EKHATO ET AL.
and 6 (760mg, 1.86 mmol) was added. After 72 h the reaction mixture was
concentrated to a paste and ethyl acetate (30 ml) was added. The mixture was
washed with saturated NH4Cl (2 ꢂ 25ml), water (2 ꢂ 25ml), saturated K2CO3
(30ml), and finally brine (30ml). The solution was evaporated on a rotary
evaporator to give crude {1-[1-[2H5]benzyl-3-(3-tert-butylcarbamoyl-octahydro-
isoquinolin-2-yl)-2-hydroxylpropylcarbamoyl]-2-carbamoylethyl]-carbamic acid
benzyl ester. The material was dissolved in absolute ethanol (30 ml) and
hydrogenated in the presence of 10% Pd/C (200 mg) at room temperature
overnight. The reaction mixture was filtered through a pad of Celite, and the filtrate
was concentrated to a solid residue. A solution of the material in minimum
dichloromethane was applied to a column of silica gel and the column was eluted
with 3–5% ethanol in CH2Cl2 to afford 2-amino-N0-[1-[2H5]benzyl-3-(3-tert-
butylcarbamoyl-octahydro-isoquinolin-2-yl)-2-hydropropyl]-succinamide
(817.5 mg, 84%).
Quinaldic acid (218.3 mg, 1.26 mmol), 1-hydroxybenzotriazole, HOBt
(170.3 mg, 1.26 mmol), dicyclohexylcarbodiimide, DCC (260.1 mg, 1.26
mmol), and N-ethyl-morpholine (NEM, 160.4 ml, 1.26 mmol) were added in
succession to 2-amino-N0-[1-[2H5]benzyl-3-(3-tert-butylcarbamoyl-octahydro-
isoquinolin-2-yl)-2-hydro-propyl]-succinamide (650 mg, 1.26 mmol) in dry
THF (25 ml) at 58C. After stirring for 20 h ethyl acetate (30 ml) was added
and the reaction mixture filtered. The filtrate was washed with water (20 ml),
10% aqueous K2CO3 (20 ml), brine (30 ml) and dried over sodium sulfate. The
solution was concentrated to a solid residue that was taken up in a minimum
volume of ethyl acetate, applied to a column of silica gel, and eluted with 4–
8% ethanol in CH2Cl2 to give a solid.
The recovered material was triturated with ether–petroleum ether to
give N-[1-[2H5]benzyl-3-(3-tert-butylcarbamoyl-octahydroisoquinolin-2-yl)-2-
hydroxypropyl]-2-[(quinoline-2-carbamoyl)-amino]-succinamide([2H5]-saqui-
navir, free base) 9 (520 mg, 61%). HPLC analysis was done with a
Waters XTerra MS C18 4.6 ꢂ 250 mm, 5 m column. It was eluted with
solvent combination of A (0.01 M ammonium acetate): B (acetonitrile)
under gradient conditions: time 0–12 min, 40–60% B; time 12–18 min, 60–
40% B at a flow rate of 1 ml/min, and the peak was detected by UV at 254 nm,
giving a retention time of 12.7 min in >98% chemical purity. MS acquired
on an LCT-TOF spectrometer gave 676.4 (M+H)+; [2H5]-99.9 atom%).
Optical rotation ½aꢃ2D0 ꢀ 50:328 (c ¼ 0:42% in MeOH). MS gave 676 (M+H)+
1
and others at m/z 338, 288 and 279. H NMR (400 MHz, CDCl3) d 9.07
(d, 1H), 8.26 (d, 1H), 8.16 (d, 1H), 8.12 (d, 1H), 7.85 (d, 1H), 7.67 (tr, 1H),
7.61 (tr, 1H), 7.12 (brd, 1H), 6.13 (brd, 1H), 5.71 (brs, 1H), 4.82 (m, 1H),
4.24 (m, 1H), 3.89 (d, 1H), 3.03 (brd, 1H), 2.94–2.26 (m, 2H), 2.30 (brs, 1H),
1.95–1.6 (m, 5H), 1.38 (2H), and 1.29 (s, 9H). 13C NMR (100 MHz, CDCl3)
173.71, 173.04, 170.52, 164.60, 148.80, 146.52, 137.68, 137.35, 130.17, 130.03,
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191
129.32, 128.79 (tr), 128.13, 127.79 (tr), 127.60, 125.60 (tr), 118.67, 70.71, 70.16,
59.13, 58.92, 54.52, 50.86, 50.25, 37.88, 35.75, 34.56, 33.20, 30.70, 30.65, 28.72,
26.13, 25.84, and 20.76.
1S-(2,2-Dimethyl-8,8a-dihydro-3aH-indeno[1,2-d]oxazol-3R-yl)-2-oxiranylmethyl-
3-[2H5]-phenyl-propan-1-one 11
Lithium bis(trimethysilyl)amide (LiHMDS) (29.6 ml, 29.6 mmol) was
added slowly to 10 (8.65 g, 26.48 mmol) in anhydrous THF (120 ml)
and stirred at ꢀ208C under an argon atmosphere. After 1 h, allyl bromide
(3.52 ml, 40.75 mmol) was added. The mixture was stirred for additional 1 h
and quenched with saturated aqueous NH4Cl solution (20 ml). The mixture
was partitioned between water and ethyl acetate (300 ml, 1:2 v/v). The organic
portion was separated and the aqueous phase was further extracted with
ethyl acetate (2 ꢂ 120 ml). The organic portions were combined and washed
with water (120 ml), brine (200 ml) and dried over magnesium sulfate.
The crude material was applied as a solution in dichloromethane to a silica
gel column and the compound was eluted with 10–20% EtOAc in hexane to
give 2-[2H5]benzyl-1-(2,2-dimethyl-8,8a-dihydro-3aH-indeno[1,2-d]oxazol-3-
yl)-pent-4-en-1-one (6.5 g, 67%). To a solution of the material (1.97 g,
5.34 mmol) in isopropyl acetate (30 ml) containing 2.0 M aq NaHCO3
(5.40 ml, 10.8 mmol) was added N-iodosuccinimide (2.08 g, 9.24 mmol).
After stirring the reaction mixture at room temperature for 1h, 10% aq
sodium thiosulfate solution (8 ml) was added, and the organic phase was
separated. The organic portion was further washed with water (3 ꢂ 15 ml),
dried over anhydrous Na2SO4, and filtered. Sodium methoxide (0.5 M solution
in THF, 21.5 ml, 10.68 mmol) was added to the filtrate. The reaction mixture
was stirred for 30 min, and diluted with ethyl acetate (30 ml). It was washed
with water (3 ꢂ 20 ml), brine (20 ml) and dried over anhydrous sodium sulfate.
The solution was concentrated to a residue and the product was crystallized
1
from ether–petroleum ether to give 11 (1.32 g, 64.4%). H NMR (500 MHz,
CDCl3) d 7.16 (m, 2H), 6.88 (tr, 1H), 6.22 (d, 1H), 5.54 (d, 1H), 4.81 (tr, 1H),
3.46 (dd, 1H), 3.06 (m, 1H), 2.83 (m, 2H), 2.51 (m, 1H), 2.16 (m, 1H), 1.67 (s,
3H), and 1.43 (s, 3H).
1-[2-Hydoxy-4-(2-hydroxyindan-1-ylcarbamoyl)-5-[2H5]phenylpentyl]-pipera-
zine-2-carboxyl acid tert-butylamide 13
A mixture of 11 (800 mg, 2.09 mmol) and N-tert-butyl-4-N-dimethylethylcar-
bamoyl-2S-piperazine carboxamide (627 mg, 2.19 mmol) in isopropyl alcohol
(20 ml) was heated at 808C. After 6 h the reaction was cooled to room
temperature and concentrated on a rotary evaporator to a residue. The
material was applied to a column of silica gel as a solution in dichloromethane
and eluted with 35% ethyl acetate in hexane to yield pure fractions. The
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I. V. EKHATO ET AL.
combined fractions were concentrated to a solid residue 12, dissolved in
dichloromethane (20 ml), and methanolic hydrogen chloride {prepared at 0–
58C under nitrogen atmosphere by addition of acetyl chloride (7 ml) to dry
methanol (20 ml)} was added slowly with stirring at room temperature. After
the Boc group was cleaved, as judged by TLC analyses (5% EtOH/CH2Cl2),
the solvent was evaporated, and the residue was further azeotroped with
toluene. The product was dissolved in a minimum volume of methanol, and
diethyl ether was added slowly until the solid hydrochloride product
separated. The precipitate was collected by filtration and dried under vacuum
1
at 808C to afford 13 (1.20 g, 87%). H NMR (400 MHz, CD3OD) d 7.29 (m,
1H), 7.19 (m, 3H), 5.22 (d, 1H), 4.354 (m 2H), 4.13 (tr, 1H), 4.02 (d, 1H), 3.74
(d, 1H), 3.59–3.38 (m, 4H), 3.13–2.99 (m, 5H), 2.87 (d, 1H), 2.77 (q, 1H), 1.90
(q, 1H), 1.52 (q, 1H), and 1.35 (s, 9H).
1-[2-Hydroxy-4-(2-hydroxy-indan-1-ylcarbamoyl)-5-[2H5]-phenylpentyl]-4-pyridin-
3-ylmethyl-piperazine-2-carboxylic acid tert-butylamide, (½2H5ꢃ-indinavir) 14
To a suspension of 13 (786 mg, 1.39 mmol) and K2CO3 (584 mg, 6.97 mmol)
in dry THF/DMF (1:1 v/v, 30 ml) was added catalytic KI (ffi10 mg) and
3-picolyl chloride (275 mg, 1.68 mmol). After stirring for 76 h at room
temperature the mixture was diluted with ethyl acetate (60 ml) and transferred
to a separatory funnel. The mixture was washed with water (2 ꢂ 40 ml),
brine (50 ml), dried over magnesium sulfate and concentrated to a solid
residue. The solid product was dissolved in 2% MeOH in CH2Cl2 and applied
to a column of silica gel. The compound was eluted with 5–10%
EtOH/CH2Cl2 containing 0.01% NH4OH. The fractions containing pure
product were combined and concentrated to a residue. The material
crystallized from ethyl acetate to afford 14 [2H5]-indinavir (637 mg,
64%). HPLC analysis was completed using a Waters XTerra MS C18
4.6 ꢂ 250 mm, 5 m column. The column was eluted with a solvent combination
of A (0.01 M Ammonium acetate): B (Acetonitrile); under gradient conditions:
time 0–12 min, 40–60% B; time 12–18 min, 60–40% B; at a flow rate of
1 ml/min. The peak was detected by UV at 254 nm and gave a retention time
of 7.0 min, with >97% chemical purity. MS acquired on an LCT-TOF
spectrometer gave 619.4(M+H)+; [2H5]-99.9 at%. Optical Rotation ½aꢃD20
þ
1
8:968 (c ¼ 0:60% in MeOH). H NMR (400 MHz, CDCl3) d 8.54 (m, 2H),
7.74 (brs, 1H), 7.59 (d, 1H), 7.28-7.7.10 (m), 5.91 (d, 1H), 5.26 (d, 1H), 4.25 (d,
1H), 3.79 (brs, 2H), 3.49 (brs, 2H), 3.16 (s, 1H), 3.03–2.49, 2.33 (m, 1H), 1.95
(m, 1H), 1.57 (m, 2H), 1.34 (s, 9H). 13C NMR (100 MHz, CDCl3) 175.06,
169.47, 150.49, 149.05, 140.58, 140.41, 139.79, 136.84, 132.51, 128.88 (tr),
128.14 (tr), 127.92, 126.76, 125.88 (tr), 125.16, 124.02, 123.44, 72.98, 65.86,
64.31, 61.50, 60.18, 57.47, 54.70, 52.68, 51.14, 48.02, 46.51, 39.54, 39.23, 38.12
and 29.03.
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LABELED HIV-PROTEASE INHIBITORS}PART II
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Acknowledgements
The authors are grateful to Dr. Gerald Miwa and Shimoga Prakash,
(Millenium Pharmaceuticals) for their support. I.V.E. gratefully acknowledges
D.R. Schroeder, S. Huang, Julie Nielsen, and A. Ranasinghe, (DAS, Bristol-
Myers Squibb Wallingford, CT) for analytical support. We wish also to thank
Richard E. Gedamke, (AR&D, Bristol-Myers Squibb Princeton, NJ) for the
MS determination of atom % D.
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Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 179–193