Synthesis and Characterization of the Selective, Reversible PKCβ Inhibitor (9 S)-9-[(Dimethylamino)methyl]-6,7,10,11-tetrahydro-9 H,18 H-5,21:12,17-dimethenodibenzo[ e,k]pyrrolo[3,4- h][1,4,13]oxadiazacyclohexadecine-18,20(19 H)-dione, Ruboxistaurin (LY333531)

Article abstract of DOI:10.1021/acschemneuro.8b00196

The demonstrated role of PKC_β in mediating amphetamine-stimulated dopamine efflux, which regulates amphetamine-induced dopamine transporter trafficking and activity, has promoted the research use of the selective, reversible PKC_β inhibitor (9S)-9-[(dimethylamino)methyl]-6,7,10,11-tetrahydro-9H,18H-5,21:12,17-dimethenodibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecine-18,20(19H)-dione, ruboxistaurin. Despite the interest in development of ruboxistaurin as the mesylate monohydrate (Arxxant) for the treatment of diabetic retinopathy, macular edema, and nephoropathy, several crucial details in physicochemical characterization were erroneous or missing. This report describes the synthesis and full characterization of ruboxistaurin free base (as a monohydrate), including X-ray crystallography to confirm the absolute configuration, and of the mesylate salt, isolated as a hydrate containing 1.5 mol of water per mole.

Full text of DOI:10.1021/acschemneuro.8b00196

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Article
Synthesis and Characterization of the Selective, Reversible
#
PKC Inhibitor (9S)-9-[(Dimethylamino)methyl]-6,7,10,11-
tetrahydro-9H,18H-5,21:12,17-dimethenodibenzo[e,k]pyrrolo[3,4-h]
[1,4,13]oxadiazacyclohexadecine-18,20(19H)-dione, Ruboxistaurin (LY333531)
Anita H. Lewin, Larry E Brieaddy, Jeffrey R. Deschamps, Gregory H.
Imler, S. Wayne Mascarella, P. Anantha Reddy, and F. Ivy Carroll
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00196 • Publication Date (Web): 29 Aug 2018
Downloaded from http://pubs.acs.org on August 31, 2018
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Synthesis and Characterization of the Selective, Reversible PKCβ Inhibitor (9S)ꢀ9ꢀ
[(Dimethylamino)methyl]ꢀ6,7,10,11ꢀtetrahydroꢀ9H,18Hꢀ5,21:12,17ꢀ
dimethenodibenzo[e,k]pyrrolo[3,4ꢀh][1,4,13]oxadiazacyclohexadecineꢀ18,20(19H)ꢀdione,
Ruboxistaurin (LY333531)
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Anita H. Lewin,1* Larry Brieaddy,1 Jeffrey R. Deschamps,2 Gregory H. Imler,2§ S. Wayne
Mascarella,1 P. Anantha Reddy,1 and F. Ivy Carroll1
1 Research Triangle Institute, P.O. Box 12194, Research Triangle Park, NC, 27709 United States
2 United States Naval Research Laboratory (NRL), Laboratory for Biosensors and Biomaterials,
Center for Biomolecular Science and Engineering, Washington, DC, 20375 United States
§ ASEE Postdoctoral Fellow
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ABSTRACT
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The demonstrated role of PKCβ in the mediating amphetamineꢀstimulated dopamine
efflux, which regulates amphetamineꢀinduced dopamine transporter trafficking and activity, has
promoted the research use of the selective, reversible PKCβ inhibitor (9S)ꢀ9ꢀ
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[(dimethylamino)methyl]ꢀ6,7,10,11ꢀtetrahydroꢀ9H,18Hꢀ5,21:12,17ꢀ
dimethenodibenzo[e,k]pyrrolo[3,4ꢀh][1,4,13]oxadiazacyclohexadecineꢀ18,20(19H)ꢀdione,
ruboxistaurin. Despite the interest in development of ruboxistaurin, as the mesylate monohydrate
(Arxxant), for the treatment of diabetic retinopathy, macular oedema, and nephoropathy, several
crucial details in physicochemical characterization were erroneous or missing. This report
describes the synthesis and full characterization of ruboxistaurin free base (as a monohydrate),
including X-ray crystallography to confirm the absolute configuration, and of the mesylate salt,
isolated as a hydrate containing 1.5 moles of water per mole.
KEYWORDS: Addiction, Parkinson’s disease, attention deficit disorder, schizophrenia,
psychostimulants, Arxxant, ruboxistaurin
1.
INTRODUCTION
The dopamine transporter (DAT), which has been implicated in neurodegenerative and
psychiatric disorders such as Parkinson's disease,1, 2 attention deficit/hyperactivity disorder,2 and
schizophrenia,3 has been identified as a primary target of psychostimulants such as amphetamine,
and is responsible for amphetamine addiction.4 DAT trafficking and activity are regulated by a
complicated protein network involving multiple protein kinases and DATꢀinteracting proteins5
including protein kinase C (PKC). In particular, it has been shown that amphetamineꢀstimulated
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dopamine efflux is mediated by PKCβ,6 which regulates amphetamineꢀinduced DAT trafficking
and activity.4 Due to the important role of PKCβ, the preparation of (9S)ꢀ9ꢀ
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[(dimethylamino)methyl]ꢀ6,7,10,11ꢀtetrahydroꢀ 9H,18Hꢀ5,21:12,17ꢀ
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dimethenodibenzo[e,k]pyrrolo[3,4ꢀh][1,4,13]oxadiazacyclohexadecineꢀ18,20(19H)ꢀdione,
ruboxistaurin (LY333531, 17 in Scheme 1), a selective, reversible inhibitor of PKCβ, was carried
out. Since the properties of the product isolated by our Laboratory were somewhat different than
those reported, the structure was confirmed by X-ray crystallography. Additionally, the mesylate
salt (Arxxant) was prepared and characterized. Herein we report our results.
2.
RESULTS AND DISCUSSION
The synthesis of ruboxistaurin (17 in Scheme 1), as the hydrochloride salt, was first
described in 19967 in a publication in which its cellular activity was compared to that of
staurosporin. A followꢀup publication from the same laboratory provided more detailed synthetic
information and described the monohydrate of ruboxistaurin;8 a patent in 20009 described several
salts and formulations, including a mesylate salt that was described in greater detail in the
chemical literature.10 Our synthesis of ruboxistaurin (17) was carried out following the patent
literature9 (Scheme 1) with most of the intermediates being characterized by 1H NMR analysis.
Triphenhylmethyl protection of the hydroxyl functionality of commercially procured (2R)ꢀ2ꢀ
oxiranemethanol (1) with triphenhylmethyl chloride, followed by reaction of the resulting (2S)ꢀ2ꢀ
[(triphenylmethoxy)methyl]oxirane (2) with vinylmagnesium bromide gave (2S)1ꢀ
triphenylmethoxyꢀ4ꢀpentenꢀ2ꢀol (3), which was Oꢀalkylated with allyl bromide to give 1,1',1''ꢀ
[[[(2S)ꢀ2ꢀ(2ꢀpropenꢀ1ꢀyloxy)ꢀ4ꢀpentenꢀ1ꢀyl]oxy]methylidyne]trisbenzene (4). Ozonolysis of 4,
followed by reduction with sodium borohydride, afforded (3S)ꢀ3ꢀ(2ꢀhydroxyethoxy)ꢀ4ꢀ
(triphenylmethoxy)ꢀ1ꢀbutanol (5) that was converted to the corresponding bismesylate 6 by
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treatment with methylsulfonyl chloride. Cesium carbonateꢀmediated coupling of 6 with 3,4ꢀdiꢀ
1Hꢀindolꢀ3ꢀylꢀ1ꢀmethylꢀ1Hꢀpyrroleꢀ2,5ꢀdione (11) that was prepared by converting 3,4ꢀdichloroꢀ
2,5ꢀfurandione (7) to the corresponding Nꢀmethyl imide 8 by treatment with methylamine to give
8, followed by reaction of 8 with the Grignard reagent 10, prepared by reaction of 1Hꢀindole (9)
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Scheme 1. Synthesis of Ruboxistaurin (17)
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with ethylmagnesium bromide, gave (9S)ꢀ6,7,10,11ꢀtetrahydroꢀ19ꢀmethylꢀ9ꢀ
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[(triphenylmethoxy)methyl]ꢀ9H,18Hꢀ5,21:12,17ꢀ dimethenodibenzo[e,k]pyrrolo[3,4ꢀ
h][1,4,13]oxadiazacyclohexadecineꢀ18,20(19H)ꢀdione (12). Saponification of 12 using ethanolic
potassium hydroxide to give 13, followed by amination with hexamethyldisilazane gave the
corresponding imide analog 14 that was Oꢀdeprotected by refluxing in a mixture of 6 N
hydrochloric acid and ethanol to give (9S)ꢀ6,7,10,11ꢀtetrahydroꢀ9ꢀ(hydroxymethyl)ꢀ9H, 18Hꢀ
5,21:12,7ꢀdimethenodibenzo[e,k]pyrrolo[3,4ꢀh][1,4,13]oxadiazacyclohexadecineꢀ18,20(19H)ꢀ
dione (15). Conversion of the hydroxyl functionality of 15 to the corresponding mesylate 16 by
treatment with methylsulfonic anhydride, followed by displacement of the mesyl group by
dimethylamine, gave ruboxistaurin (17) as a red solid with elemental analysis consistent with the
molecular formula C28H28N4O3• H2O and with 1H NMR spectrum (in deuterated dimethyl
sulfoxide) consistent with expectation. Specifically, the total area of the resonances in the
aromatic region (δ 7 – 7.5 ppm) corresponded to 10 protons, and resonances in the aliphatic
region (δ 1.8 – 4.5 ppm) had a total area corresponding to 17 protons that included a sharp
singlet resonance (δ 2.03) with area corresponding to 6 protons, as expected for the N,Nꢀ
dimethylamino group. To our surprise, these 1H NMR data were completely different from the
reported 1H NMR spectrum8 which listed no resonances corresponding to the Nꢀmethyl groups
(expected to be singlet(s) with area corresponding to 6H at δ ~2 ppm) but did report two doublet
resonances (δ 7.82 and 7.78), with areas corresponding to 1H each, not seen in our 1H NMR
spectrum.
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As part of the development of ruboxistaurin for the treatment of diabetic retinopathy,
macular oedema, and nephoropathy, the mesylate monohydrate (Arxxant), which is reported to
have better aqueous solubility than the corresponding hydrochloride, was identified as the
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optimal salt form for clinical studies in a salt selection study.10 This salt was characterized by
polarizing microscopy, hygroscopicity, differential scanning calorimetry (DSC), thermal
gravimetric analysis (TGA), and Xꢀray powder diffraction. 10 Surprisingly, neither the melting
point, nor the optical rotation, was reported. Although not explicitly stated, the basis for
describing the salt as a monohydrate appears to be the TGA data, which show a weight loss of ca
2.7% between 30–75 °C, consistent with the loss of a mole of water. The DSC data for
ruboxistaurin mesylate monohydrate showed a broad endotherm (140–155 °C) that was
described as being associated with water loss, followed by a larger endotherm at 260–265 °C,
said to represent the melt. 10 The TGA data were interpreted as indicating decomposition of the
salt around 270 °C.
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Our preparation of the mesylate salt of 17, which followed the literature,10 gave a roseꢀ
colored solid that, after drying under vacuum for 24 hours, had elemental analysis consistent
with a hydrate containing 1.5 moles of water per mole of 17 mesylate, not with the salt being a
monohydrate, as reported.10 This salt had a sharp melting point of 244–245 °C, significantly
lower than that reported for the monohydrate (based on DSC), consistent with it being a different
hydrate than the reported10 monohydrate. These results imply that the sample isolated in this
study may have crystallized as a 1:1 mixture of a monohydrate and a dihydrate. Engel et al. 10
had reported multiple hydrated forms of the hydrochloride salt of 17, but not of the mesylate salt;
it should be noted, however, that the hygroscopicity determination for ruboxistaurin
monohydrate demonstrated absorption of 0.6 moles of water at ca 12% relative humidity (RH)
and the desorption isotherm showed ca 0.5 moles of water to remain at 5% RH.10 Taken together
with the results of elemental analysis obtained by us it would appear that without drying at 60
°C10 ruboxistaurin mesylate contains an average of 1.5 mols of water per mole.
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Since neither the melting point nor the optical rotation for the free base 17 was reported,8 it
was decided to confirm the structure and stereochemistry of our synthetic product by X-ray
crystallography. The red crystals of the free base 17, obtained from methanol, were used for the
analysis (Figure 1). Based on the method of Hooft comparing intensities of Bijovet pairs (69% of
the pairs were measured)11 using Bayesian statistics of the data, there is a small chance that the
Xꢀray structure could be a racemic twin. However, this possibility was refuted by the observed
optical rotation of a methanolic solution of the mesylate salt of 17. Examination of the
asymmetric center in the Xꢀray structure, showed the absolute stereochemistry to be S, in
agreement with the absolute configuration of the starting material 1.
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Figure 1. X-Ray Structure of Ruboxistaurin (17).
Displacement ellipsoids are at the 50% level. One of the two molecules in the symmetric unit has
been omitted for clarity.
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At the suggestion of a reviewer, Eli Lilly was contacted and eventually a copy of a 1H
NMR spectrum of their ruboxistaurin (in DMSOꢀd6) along with a 1H–13C correlation spectrum
was provided. The 1H NMR was identical to that reported in this study.
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3.
CONCLUSIONS
This study provides thorough characterization of the selective, reversible PKCβ inhibitor
ruboxistaurin (17) and its mesylate salt. The structure of ruboxistaurin, which was prepared
following the literature procedure,8 and was found to exhibit a 1H NMR spectrum distinctly
different than reported, was characterized by X-ray crystallography. The red solid, which based
on elemental analysis was a monohydrate, had the expected structure; examination of the
asymmetric center showed the absolute stereochemistry to be S, as expected. Further
characterization was obtained by interpretation of the 1H NMR spectrum; the optical rotation was
also determined. The mesylate salt of ruboxistaurin, reported as a monohydrate, was
characterized by us as a hydrate containing 1.5 moles of water per mole of 17 mesylate. Both the
optical rotation and the melting point of ruboxistaurin mesylate hydrated with 1.5 moles of
water/mole have been determined.
4.
METHODS
Instrumentation. 1H NMR spectra were acquired on a Bruker Avance 300 MHz
spectrometer, or a 500 MHz NMR spectrometer. Lowꢀresolution LCꢀMS data were obtained
using a PerkinElmer API 150 EX mass spectrometer outfitted with an ESI (turbospray) source,
coupled to a PerkinElmer 200 Series liquid chromatography system. Melting points were
determined using a MELꢀTEMP II capilaty melting point apparatus. Optical rotations were
measured on an AutoPol III polarimeter from Rudolph Research. Elemental analyses were
carried out by Atlantic Microlab Inc., Norcross, GA
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Materials. Chemicals were reagentꢀgrade, obtained from commercial sources, and used
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without purification.
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Synthesis of ((9S)ꢀ9ꢀ[(Dimethylamino)methyl]ꢀ6,7,10,11ꢀtetrahydroꢀ9H,18Hꢀ
5,21:12,17ꢀdimethenodibenzo[e,k]pyrrolo[3,4ꢀh][1,4,13]oxadiazacyclohexadecineꢀ
18,20(19H)ꢀdione, (Ruboxistaurin, 17) Mesylate
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(2S)-2-[(Triphenylmethoxy)methyl]oxirane (2). To a solution of triphenylmethyl chloride (50.7
g, 0.182 mol) in CH2Cl2 (125 mL), at 0ꢀ5 °C, was slowly added Et3N (18.4 g, 0.182 mol)
followed by (2R)ꢀ2ꢀoxiranemethanol (1) (15.0 g, 0.202 mol). After stirring under N2, at RT for
24 h the mixture was quenched with sat'd NH4Cl (150 mL) and H2O (50 mL). The organic phase
was separated, dried (Na2SO4) and concentrated. To the residual oil was added EtOH and the
solvent was evaporated until solids appeared. The collected solids were recrystalized from iꢀ
PrOH (180 mL) to afford 2 as a white solid (35.6 g, 62%); m.p. 96.5–97.0 °C, (Lit12 94ꢀ96)[α]D =
ꢀ3.77° (c 0.770, MeOH) (Lit12 ꢀ9.7, 1.0 CHCl3).1H NMR (CDCl3, 300 MHz) δ 2.7 (dAB, 2H,
OCH2CH), 3.20 (d, 1H, OCH), 3.3 (dAB, 2H, CH2OTr), 7.32–7.37 (m, 15H, TrH)
(2S)-1-Triphenylmethyoxy-4-petnene-2-ol (3). To a solution of vinylmagnesium bromide in THF
(110 mL, 1.0 M, 0.110 mol), at ꢀ40 °C, was added CuI (0.590 g, 0.0031 mol). After stirring
under N2, for 15 min, a solution of (2S)ꢀ2ꢀ[(triphenylmethoxy)methyl]oxirane (2) (19.42 g,
0.0614 mol) in THF (90 mL) was added. The mixture was stirred at ꢀ 40 °C for 1 h, then the bath
temperature was allowed to rise to ꢀ10 °C and the reaction was quenched with sat'd NH4Cl (100
mL). EtOAc (150 mL) and H2O (150 mL) was added and the organic phase was separated,
washed with NH4OH/H2O (1:1), dried (Na2SO4), and concentrated in vacuo to afford 3 as a light
yellow oil (21.1 g, 100%). 1H NMR (CDCl3, 300 MHz) δ 2.28 (quintuplet, 2H, CHCH2CH2),
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3.18 (ddAB, 2H, CH2OTr), 3.85 (m, 1H, OCH), 5.10 (dd, 2H, =CH2), 5.9 ((quintuplet, 1H,
=CH), 7.26ꢀ7.49 (m, 15H, TrH).
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1,1',1''-[[[(2S)-4-Methyl-2-(2-propen-1-yloxy)-4-penten-1-yl]oxy]methylidyne]trisbenzene (4).To
a solution of (2S)ꢀ1ꢀtriphenylmethyoxyꢀ4ꢀpetneneꢀ2ꢀol (3) (10.06 g, 0.0292 mol) in THF (40
mL) was added a solution of tꢀBuOK (3.46 g, 0.0308 mol) in THF (40 mL). The reaction mixture
was stirred at 45 °C for 45 min, cooled to RT and a solution of allyl bromide (8.83 g, 0.0730
mol) in THF (10 mL) was slowly added. After stirring for an additional 1 h the reaction was
quenched with sat'd NH4Cl and EtOAc was added. The organic phase was separated, dried
(Na2SO4) and concentrated in vacuo to give of an orange oil (10.5 g). The oil was
chromatographed on SiO2, eluting with hexane/EtOAc (9:1), to afford 4 (8.82 g, 79%) as a light
yellow oil; [α]D = ꢀ7.63° (c 0.760, MeOH) (Lit8 ꢀ10.07 ° 0.98, MeOH), Rf = 0.63, SiO2,
hexane/EtOAc (9:1). 1H NMR (CDCl3, 300 MHz) δ 2.4 (m, 2H, CHCH2CH2), 3.18 (m, 2H,
CH2OTr), 4.07 (t, 1H, OCH), 4.10 (dAB, 2H, OCH2), 5.04 (m, 4H, (=CH2)2), 5.8 ((quintuplet,
1H, =CH), 6.0 (quintuplet, 1H, =CH), 7.24–7.53 (m, 15H, TrH).
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(3S)-3-(2-Hydroxyethoxy)-4-(triphenylmethoxy)-1-butanol (5). To a solution of (S)ꢀ1ꢀ
triphenylmethyoxyꢀ[[[2ꢀ(2ꢀpropenyloxy)ꢀ4ꢀpentenyl]oxy]methyne] (4) (19.6 g, 0.0509 mol) in
MeOH/CH2Cl2 (1:1) (120 mL) was added a solution of Sudan red indicator (0.387 g, 0.00102
mol) in CH2Cl2/EtOH (3:1) (6 mL). The reaction mixture was cooled to ꢀ45 °C and ozone was
bubbled through the mixture until a reddishꢀorange colored solution persisted. This color change
usually was seen after 2 h of bubbling ozone into the reaction mixture. Nitrogen was then
bubbled into the mixture for 15 min and the solution was then added to an iceꢀchilled solution of
NaBH4 (4.27 g, 0.112 mol) in 0.5 N NaOH (50 mL). The reaction mixture was stirred for 17 h,
allowing the bath to come to RT. The mixture was neutralized (pH = 6ꢀ7) with 1 N HCl; brine
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and EtOAc were then added and the mixture was stirred for 15 min. The organic phase was
separated, dried (Na2SO4) and concentrated to yield an orange oil (22.0 g) that was purified by
column chromatography (SiO2, EtOAc) to afford 5 as a thick orange oil (12.0 g, 60%).
(S)-3-(2-Hydroxyethoxy)-4-(triphenylmethoxy)-1-butanol Methanesulfonate (6). To an iceꢀchilled
solution of (3S)ꢀ3ꢀ(2ꢀhydroxyethoxy)ꢀ4ꢀ(triphenylmethoxy)ꢀ1ꢀbutanol (5) (14.2 g, 0.0362 mol) in
CH2Cl2 (200 mL) was added Et3N (11.3 g, 0.112 mol). After 15 min of stirring, a solution of
methanesulfonyl chloride (11.1 g, 0.0969 mol) in CH2Cl2 (50 mL) was added and the mixture
was stirred at bath temperature for 2 h. The mixture was diluted with CH2Cl2 and washed with
water and sat'd NH4Cl. The organic phase was separated, dried (Na2SO4) and concentrated to
give an orange oil. Addition of mixtures of heptane/EtOAc to the oil and concentration of the
solution in vacuo (with no heat), resulted in the formation of a solid that was filtered and dried in
vacuo to afford 6 as a beige solid (16.7 g, 85%). 1H NMR (CDCl3, 300 MHz) δ 1.98 (m, 2H,
CHCH2CH2), 3.03 (s, 3H, S CH3)), 3.05 (s, 3H, S CH3)), 3.23 (dd, 2H, CH2OTr), 3.8 (dAB, 2H,
OCH2), 4.0 (t, 1H, OCH), 4.38 (m, 4H, (CH2O)2), 7.29–7.49 (m, 15H, TrH). Note: This product
was reacted with 11 within hours of its preparation.
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3,4-Dichloro-1-methylmaleimide (8). To a solution of 3,4ꢀdichloro2,5ꢀfurandione (7) (25.0 g,
0.150 mol) and potassium acetate (22.1 g, 0.225 mol) in acetic acid (30 mL) was added
methylamine HCl (15.2 g, 0.225 mol) and the mixture was stirred at reflux for 4 h. After cooling
to RT the thick mass was slowly poured into iceꢀchilled 10% NaHCO3 solution. The product was
extracted with ether. The combined extract was washed with brine, dried (Na2SO4) and
concentrated to yield an orange solid (20.2 g) that was purified by column chromatography
(SiO2, hexane/EtOAc (9:1)) to give 8 as a white solid (18.3 g, 68%); mp. 82–83 °C (lit13 82ꢀ83).
1H NMR (CDCl3, 300 MHz) δ 3.05 (CH3).
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3,4-di-1H-Indol-3-yl-1-methyl-1H-pyrrole-2,5-dione(11). A solution of indole (9) (39.1 g, 0.330
mol) in toluene (200 mL) was added to a solution of EtMgBr (330 mL, 1.0 M, 0.330 mol) in
THF, under N2, at RT. The mixture was heated to 60 °C for 1 h and a solution of 3,4ꢀdichloroꢀ1ꢀ
methylꢀ1ꢀHꢀpyrroleꢀ2,5ꢀdione (8) (16.2 g, 0.090 mol) in toluene (200 mL) was added. After
stirring at 90 °C for 17 h the reaction mixture was cooled to RT and sat'd NH4Cl (300 mL) was
added, followed by water and EtOAc. The organic phase was separated, dried (Na2SO4) and
concentrated to give a thick red oil. Ether was added and the heterogeneous mixture was stirred
for15 min, then filtered to get 11 (9.6 g). The mother liquor was subjected to column
chromatography on SiO2, eluting with hexane/EtOAc (1:1), to afford additional 11 (4.5 g) for a
total yield of 14.1 g (46%); Rf = 0.48, SiO2, hexane/EtOAc (1:1), m/z for C21H15N3O2 calcd:
341.12; found: 341. 1H NMR (DMSOꢀd6, 300 MHz) δ 3.03 (s, 3H, CH3), 6.61 (m, 2H, ArH),
6.78 (d, 2H, ArH), 6.96 (m, 2H, ArH), 7.35, (d, 2H, ArH), 7.73 (d, 2H, (ArH), 11.66 (d, 2H,
NH).
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(9S)-6,7,10,11-Tetrahydro-19-methyl-9-[(triphenylmethoxy)methyl]-9H,18H-5,21:12,17-
dimethenodibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecine-18,20(19H)-dione (12).
To a suspension of cesium carbonate (16.9 g, 0.0519 mol) and 3,4ꢀdiꢀ1Hꢀindolꢀ3ꢀylꢀ1ꢀmethylꢀ
1Hꢀpyrroleꢀ2,5ꢀdione (11) (8.07 g,0.0236 mol) in DMF (500 mL) at 100 °C was added a solution
of (S)ꢀ3ꢀ(2ꢀhydroxyethoxy)ꢀ4ꢀ(triphenylmethoxy)ꢀ1ꢀbutanol methanesulfonate (6) (16.7 g,
0.0304 mol) in DMF (200 mL). The mixture was stirred at 100 °C for 17 h, cooled to 50 °C and
Hyflo (15 g) was added. The resulting mixture was stirred at 50 °C for 15 min then filtered. The
filter cake was washed with DMF and the filtrate was concentrated in vacuo. To the residue was
added CH3CN and the solution was concentrated. This process was repeated two more times,
then a slurry of the solid in CH3CN was chilled for 2 h. The solids were collected by filtration
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and dried to give 12 as a reddish violet solid (12.26 g, 58%) m.p. 205–208 °C, Rf = 0.63, SiO2,
hexane/EtOAc (6.5:3.5).
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(9S)-6,7,10,11-Tetrahydro-9-[(triphenylmethoxy)methyl]-5,21:12,17-dimetheno-9H-
dibenzo[e,k]furo[3,4-h][1,4,13]oxadiazacyclohexadecine-18,20-dione (13). A suspension of
(9S)ꢀ6,7,10,11ꢀtetrahydroꢀ19ꢀmethylꢀ9ꢀ[(triphenylmethoxy)methyl]ꢀ9H,18Hꢀ5,21:12,17ꢀ
dimethenodibenzo[e,k]pyrrolo[3,4ꢀh][1,4,13]oxadiazacyclohexadecineꢀ18,20(19H)ꢀdione (12)
(18.37 g, 0.0263 mol) and KOH (14.7 g, 0.263 mol) in EtOH (200 mL) was stirred at gentle
reflux for 22 h. The mixture was cooled to RT and treated with CH2Cl2 (200 mL) and water (350
mL). The organic phase was separated and treated with 20% citric acid (300 mL). Water was
added and the organic phase was separated and dried (Na2SO4). Concentration gave a thick oil
that was dissolved in EtOH and the volatiles were evaporated This was repeated two more times,
then a slurry of the solid residue in EtOH was chilled for 1 h. The solid was collected by
filtration, washed with EtOH and dried to give 13 as a purple solid (14.7 g, 82%); mp 223–225
°C, Rf = 0.44, SiO2, hexane/EtOAc (65:35). 1H NMR (DMSOꢀd6, 300 MHz) δ 2.2 (m, 2H,
CHCH2CH2), 3.14 (d, 2H, CH2), 3.6 (dAB 2H, CH2)), 3.8 (m 1H, CH), 4.3 (m, 4H, (CH2) 2),
7.21–7.37 (m, 19H, ArH+TrH), 7.46 (d, 1H, ArH), 7.7.61(d, 1H, ArH), 7.71 (d, 2H, ArH), 7.9
(dd, 2H, ArH).
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(9S)-6,7,10,11-Tetrahydro-9-[(triphenylmethoxy)methyl]-9H,18H-5,21:12,17-
dimethenodibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecine-18,20(19H)-dione (14).
To a solution of (9S)ꢀ6,7,10,11ꢀtetrahydroꢀ9ꢀ[(triphenylmethoxy)methyl]ꢀ5,21:12,17ꢀdimethenoꢀ
9Hꢀdibenzo[e,k]furo[3,4ꢀh][1,4,13]oxadiazacyclohexadecineꢀ18,20ꢀdione (13) (7.90 g, 0.0115
mol) in DMF (75 mL) was added a premixed solution of hexamethyldisilazane (18.6 g, 0.115
mol) in MeOH (2.5 mL). The reaction mixture was heated at 80 °C for 5 h. Analysis by TLC
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(SiO2, hexane/EtOAc (6.5:3.5)) showed no starting material remaining. The reaction mixture was
cooled to RT, diluted with CH2Cl2 (200 mL) and quenched with 1 N HCl (80 mL). Water was
added and the organic phase was separated, dried (Na2SO4) and concentrated in vacuo. To the
residue was added EtOH and the mixture was reꢀconcentrated. More EtOH was added and the
slurry was chilled with ice and allowed to stand for 30 min. The dark mixture was filtered to get
14 (4.66 g). The mother liquor was subjected to column chromatography (SiO2, hexane/EtOAc
(6.5:3.5)), to give additional 14 (1.82 g). The solids were combined for a total of 6.48 g (82%) of
14 as reddishꢀviolet solid; mp 145–151 °C (softened at 142 °C), Rf = 0.43, SiO2, hexane/EtOAc
(6.5:3.5).
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(9S)-6,7,10,11-Tetrahydro-9-(hydroxymethyl)-9H, 18H-5,21:12,7-
dimethenodibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecine-18,20(19H)-dione
(15). A suspension of (9S)ꢀ6,7,10,11ꢀtetrahydroꢀ9ꢀ[(triphenylmethoxy)methyl]ꢀ9H,18H,ꢀ
5,21:12,17ꢀdimethenodibenzo[e,k]pyrrolo[3,4ꢀh][1,4,13]oxadiazacyclohexadecineꢀ18,20(19H)ꢀ
dione (14) (6.48 g, 0.00947 mol) in EtOH (70 mL) and 6 N HCl (70 mL) was heated at reflux for
2 h. The mixture was cooled to RT and filtered. The solid cake was washed with water and with
a small amount of CH2Cl2. The purple solids were dried under high vacuum to yield 15 as a
purple solid (3.74 g, 89%); Rf = 0.50, SiO2, EtOAc, mp >260 °C. 1H NMR (DMSOꢀd6, 300
MHz) δ 2.04 (dAB, 2H, CHCH2CH2), 3.3 (m, 4H, CH2) 2), 3.75 (dAB, 2H, CH2)), 3.8 (m 1H,
CH), 4.0 (dAB, 2H, CH2), 4.7 (b, 1H, CH), 7.09–7.22 (m, 4H, ArH), 7.35 (m, 4H, ArH), 7.8 1(m,
2H, ArH), 10.82 (s, 2H, NH).
(9S)-6,7,10,11-Tetrahydro-9-{[(methylsulfonyl)oxy]methyl}-9H,18H-5,21:12,7-
dimethenodibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecine-18,20(19H)-dione (16).
To THF (80 mL) was added (9S)-6,7,10,11ꢀtetrahydroꢀ9ꢀ(hydroxymethyl)ꢀ9H, 18Hꢀ5,21:12,7ꢀ
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dimethenodibenzo[e,k]pyrrolo[3,4ꢀh][1,4,13]oxadiazacyclohexadecineꢀ18,20(19H)ꢀdione
(15) (1.08 g, 0.00245 mol), pyridine (0.87 g, 0.0110 mol) and methanesulfonic acid anhydride
(1.28 g, 0.00735 mol). After stirring for 90 min at 70 °C the mixture was cooled to RT and brine
(75 mL) and 1 N HCl (25 mL) was added. The mixture was extracted with EtOAc and the
organic extract was dried (Na2SO4) and concentrated in vacuo to give a purple solid (1.42 g). The
solid was chromatographed (SiO2, EtOAc/hexane (7:3)) to afford 16 as a purple solid (0.75 g,
59%); Rf = 0.50, SiO2, EtOAc/hexane (7:3), m/z calcd for C27H25N3O6S: 519.57; found: 519. 1H
NMR (DMSOꢀd6, 300 MHz) δ 2.2 (dAB, 2H, CHCH2CH2), 3.24 (s, 3H, CH3), 3.7 (m, 2H,
CH2)), 4.0 (m, 1H, CH), 4.26 (m, 4H, (CH2)2), 4.45 (d, 2H, CH2), 7.16–7.24 (m, 4H, ArH), 7.53–
7.60 (m, 4H, ArH), 7.83–7.90 (m, 2H, ArH), 11.0 (s, 2H, NH).
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(9S)-9-[(Dimethylamino)methyl]-6,7,10,11-tetrahydro-9H,18H-5,21:12,17-
dimethenodibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecine-18,20(19H)-dione,
Ruboxistaurin (17). To a solution of Me2NH in DMF (30 mL, 5.7 M, 0.171 mol) was added (9S)ꢀ
6,7,10,11ꢀtetrahydroꢀ9ꢀ{[(methylsulfonyl)oxy]methyl}ꢀ9H,18Hꢀ5,21:12,7ꢀ
dimethenodibenzo[e,k]pyrrolo[3,4ꢀh][1,4,13]oxadiazacyclohexadeceneꢀ18,20(19H)ꢀdione (16).
(2.67 g, 0.00514 mol) and the mixture was placed in a sealed reaction vessel. The mixture was
stirred at 60 °C for 22 h, cooled to RT and concentrated in vacuo. To the residue was added
MeOH (2x), the mixture was concentrated and the resulting solids were triturated with 10%
NaHCO3, filtered, washed with water and dried under high vacuum overnight to yield a red solid
(2.1 g, 88%). Solids from previous runs were combined (4.60 g) and dissolved in MeOH (1500
mL). The solution was concentrated to 800 mL and allowed stand overnight at RT. The
precipitated solid was collected by filtration (2.94 g of a red solid) and the ML was concentrated
to 300 mL. After standing overnight a second crop (0.257 g) of red solid was collected. The ML
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was concentrated and the residue was chromatographed (SiO2, MeOH/CH2Cl2 (1:1)) to afford
additional red solid (0.46 g). Analysis of the three solids by TLC and 1H NMR indicated they
were all the same. The solids were combined and dried under vacuum for two days to afford 17
as a red solid (3.64 g); mp = 235–239 °C. Rf = 0.41, SiO2, CHCl3/MeOH/ NH4OH/CH2Cl2;
(40:10:0.1:50) m/z calcd for C28H28N4O3: 468: found: 468. 1H NMR (DMSOꢀd6) δ 1.80–2.30 (m,
4H, (CH2)2), 2.03 (s, 6H, N(CH3)2), 3.42 (bm, 1H, OCH), 3.57 (m, 1H, NCH), 3.83 (bm, 1H,
NCH), 4.05–4.38 (m, 4H, (CH2)2), 7.06–7.24 (m, 4H, ArH), 7.41–7.46 (m, 4H, ArH), 7.80 (m,
2H, ArH). Anal. calcd for C28H28N4O3• H2O: C, 69.12; H, 6.21; N, 11.51; found: C, 69.3; H,
5.79; N, 11.37.
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Ruboxistaurin (17) Mesylate. To a slurry of ruboxistaurin (17) (3.42 g, 0.00703 mol) in acetone
(90 mL) was added a solution of methanesulfonic acid (0.68 g, 0.00703 mol) in H2O (10 mL)
and the heterogeneous mixture was stirred at RT for 15 min. The solids were collected by
filtration and were rinsed with acetone (25 mL). The rosy red solids were dried under highꢀ
vacuum for 24 h to yield ruboxistaurin mesylate (3.00 g, 72%); mp 244–245 °C, Rf = 0.68, SiO2,
CH2Cl2/CHCl3MeOH/NH4OH (50/40/10/0.1), m/z calcd for C28H28N4O: 468; found: 469 (M+H);
[α]633 = ꢀ19.0 (c 0.345, MeOH). Anal. for C29H32N4SO6• 1.5H2O calcd: C, 58.87; H, 5.96; N,
9.47; found: C, 58.60, H, 5.86; N, 9.31. Solubility: water, >4 mg/mL; very soluble in DMSO and
MeOH.
Xꢀray Crystal Data on (9S)ꢀ9ꢀ[(Dimethylamino)methyl]ꢀ6,7,10,11ꢀtetrahydroꢀ
9H,18Hꢀ5,21:12,17ꢀdimethenodibenzo[e,k]pyrrolo[3,4ꢀh][1,4,13]oxadiazacyclohexadecineꢀ
18,20(19H)ꢀdione, Ruboxistaurin (17)
Singleꢀcrystal Xꢀray diffraction data on (9S)ꢀ9ꢀ[(dimethylamino)methyl]ꢀ6,7,10,11ꢀtetrahydroꢀ
9H,18Hꢀ5,21:12,17ꢀdimethenodibenzo[e,k]pyrrolo[3,4ꢀh][1,4,13]oxadiazacyclohexadecineꢀ
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18,20(19H)ꢀdione, Ruboxistaurin (17), were collected using Cu Kα radiation and a Bruker
Photon 100 area detector. The crystal was prepared for data collection by coating with high
viscosity microscope oil. The oilꢀcoated crystal was mounted on a microꢀmesh mount (MiteGen,
Inc.), transferred to the diffractometer and a data set was collected at 150 °K. The 0.315 × 0.074
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× 0.067 mm3crystal was monoclinic in space group P21, with unit cell dimensions a = 9.2036(2)
Å, b = 22.0461(5) Å, c = 11.8589(3) Å,
α = 90°, β = 90.0670(10)°, and γ = 90°. Data were
89.2% complete to 67.67° θ (~0.80 Å) with an average redundancy of 2.43. The final anisotropic
full matrix leastꢀsquares refinement on F2 with 636 variables converged at R1 = 8.89%, for the
observed data and wR2 = 21.34% for all data. The structure was solved by direct methods and
refined by fullꢀmatrix least squares on F2 values using the programs found in the SHELXL suite
(Bruker, SHELXL v2014.7, 2014, Bruker AXS Inc., Madison, WI). Corrections were applied for
Lorentz, polarization, and absorption effects. Parameters refined included atomic coordinates and
anisotropic thermal parameters for all nonꢀhydrogen atoms. The H atoms were included using a
riding model. Complete information on data collection and refinement is available in the
supplemental material.
Assignment of absolute configuration was confirmed using Bayesian statistics of the Bijvoet
pairs. Results of this analysis were: P2(true) = 1.000, P3(true) = 0.952, P3(racꢀtwin) = 0.058, and
P3(false) = 0.9x10ꢀ8. These results confirm that the absolute configuration has been properly
assigned (i.e. both P2(true) and P3(true) are ~ 1) and no (or very minimal) racemic twinning is
present (P3(racꢀtwin) = 0.058). It should be noted that neither the Flack parameter nor Hooft(y)
parameter was definitive even though the Bayesian statistics are clear.
Atomic coordinates have been deposited with the Cambridge Crystallographic Data Centre
(deposition number CCDC 1583495). Copies of the data can be obtained, free of charge, on
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application to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK [fax: +44(0)ꢀ1223ꢀ336033 or
eꢀmail: deposit@ccdc.cam.ac.uk.
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SUPPORTING INORMATION
Crystal data and structure refinement for 17; Atomic coordinates (×104) and equivalent isotropic
displacement parameters (Å2 × 103) for 17. U(eq) is defined as one third of the trace of the
orthogonalized Uij tensor; Bond lengths [Å] and angles [°] for 17; Isotropic displacement
parameters (Å2 × 103) for 17; Hydrogen coordinates (×104) for 17; Torsion angles [°] for 17.
AUTHOR INFORMATION
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Corresponding Author
Telephone: 1ꢀ919ꢀ541ꢀ6691
Fax: 1ꢀ919ꢀ541ꢀ8868
Email: ahl.emeritus@rti.org
ORCID
Anita H. Lewin: orcid.org/0000ꢀ0001ꢀ6020ꢀ322X
Author Contributions
The research described in this manuscript was directed by A.H.L., S.W.M., P.A.R, H.H.S., and
F.I.C.; the synthesis of 17 was performed by L.B.; X-ray crystallography was by J.D. and G.H.I.;
the manuscript was written by A.H.L. All authors have given approval to the final version of the
manuscript.
Funding
The research was supported by National Institute on Drug Abuse Contract No.
HHSN2712013000007C. The Xꢀray crystallographic work was supported by NIDA through
Interagency Agreement (IAA) ADA17001 with the Naval Research Laboratory (NRL).
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ACKNOWLEDGEMENT
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The authors thank Mr. Leonard L. Winneroski and Mr. Robert D. Boyer at Eli Lilly for providing
authentic NMR spectra of Ruboxistaurin for comparison.
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ABBREVIATIONS
DAT, dopamine transpoter; PKC, protein kinase C; 1H NMR, proton nuclear magnetic
resonance; DSC, differential scanning calorimetry; TGA, thermal gravimetric analysis; Et3N,
triethyamine; CH2Cl2, methylene chloride; RT, room temperature; NH4Cl, ammonium chloride;
H2O, water; Na2SO4, sodium sulfate; EtOH, ethanol; iꢀPrOH, isopropanol; m.p., melting point;
CuI, cuprous iodide; THF, tetrahydrofuran; EtOAc, ethyl acetate; NH3, ammonia; s, singlet; d,
doublet; dd, doublet of doublets; m, multiplet; tꢀBuOK, potassium t-butoxide; NaBH4, sodium
borohydride; NaOH, sodium hydroxide; HCl, hydrochloric acid; CDCl3, deuterochloroform;
EtMgBr, ethylmagnesium bromide; DMF, dimethylformamide; CH3CN, acetonitrile; KOH,
potassium hydroxide; DMSOꢀd6, perdeuterated dimethylsulfoxide; SiO2, silica gel; Me2NH,
dimethylamine; ML, mother liquor.
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Ruboxistaurin
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52x52mm (300 x 300 DPI)
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