Development of a Scalable Strategy for the Synthesis of PI3KÎ´

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Development of a Scalable Strategy for the Synthesis of PI3kd Inhibitors Diane Carrera, Remy Angelaud, PeiJue Sheng, Brian Safina, and Jun Li Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/op300235t • Publication Date (Web): 18 Dec 2012 Downloaded from http://pubs.acs.org on December 25, 2012

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Organic Process Research & Development

Development of a Scalable Strategy of the Synthesis of PI3kd Inhibitors; Selective and Efficient Functionalization of Purine Derivatives Diane E. Carrera,*,† PeiJue Sheng, † Brian S. Safina,‡ Jun Li‡ and Remy Angelaud† †

Small Molecule Process Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA. ‡

Discovery Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA

[email protected] RECEIVED DATE CORRESPONDING AUTHOR *E-mail: [email protected] Table of contents graphic

ABSTRACT The first generation development route used to prepare the PI3Kδ inhibitor GNE-293(3) for early toxicology studies is described. Through the use of a metal free SNAr reaction in place of a Pd catalyzed 1 ACS Paragon Plus Environment


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C-N coupling, the synthesis was both simplified and made more reproducible in preparation for scale-up by reducing the number of operations required for purification and eliminating the need for column chromatography. The utility of the recently developed reagent TMPZnCl•LiCl is highlighted by a novel method of iodination to access the key aryl halide intermediate.

INTRODUCTION The autoimmune disorder rheumatoid arthritis (RA), involving chronic inflammation of the synovial membrane lining the joints1, can lead to pain, stiffness and deformity as a result of damage to the bone, cartilage and ligaments in the patients’ joints. Up to 50% of patients do not respond to currently available treatments, such as disease-modifying-antirheumatic drugs (DMARDS) and biological therapies,2 making the search for novel pathways that target RA a significant area of unmet medical need. Recent studies have implicated the PI3Kδ lipid kinase as potentially playing a key part in the progression of RA through its role in B cell receptor mediated signaling3. As such, the development of a small molecule PI3Kδ inhibitor is an attractive goal for developing a potential treatment for rheumatoid arthritis.

Figure 1. PI3Kδ δ inhibitors from the O-linked series

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Discovery efforts at Genentech resulted in the identification of a number of potential clinical candidates with a high degree of structural homology, several examples of which are given in Figure 14. Due to the shared structural motifs found across these lead compounds, the development of a scalable process to access kilogram quantities was initiated to support IND-enabling toxicology studies and accelerate the delivery timeline for the first required batch of GMP material. A representative medicinal chemistry route to one of the PI3Kδ inhibitors investigated, GNE-026(2), is a five-step sequence combining the three key building blocks morpholinopurine 4, azetidinol 6 and ethyl-benzimizadole 9a that provides the desired product in 38% overall yield (Scheme 1). Several aspects of the synthesis needed to be addressed before proceeding with large-scale synthetic efforts. Safety concerns were raised by the use of sodium hydride and DMF in the second step and the solvent dioxane in the final C-N coupling. Of most concern was the final palladium mediated C-N coupling reaction to install ethylbenzimidazole 9a which not only used dioxane but also gave low and inconsistent levels of conversion on scales greater than 20 g, presumably due to poor dispersion of the cesium carbonate base in larger solvent volumes. Further complicating matters was the requirements of chromatography, thiosilica treatment to scavenge residual palladium metal and a final crystallization to achieve sufficient purity levels. In combination, the use of chromatography and scavenging would not only be costly but impact delivery timelines. Costs would also be increased by the use of the proprietary ligand XPhos, which was required at a high loading of 20 mol%. Finally, we felt that the overall strategy of using a transition metal that needs to be controlled down to the ppm level in the final step of the synthesis was simply unwise due to the limited opportunity it gave for control over residual palladium levels in the active pharmaceutical ingredient (API) on scale.


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Scheme 1. Medicinal chemistry route to the PI3Kδ δ inhibitor 2a

a

Reagents and conditions: (a) NBS, AcOH, 65˚C, 81%; (b) NaH, DMF, 8 ˚C to 25 ˚C, 83%; (c) 4N HCl in MeOH, DCM, 40 ˚C; (d) Isobutyryl chloride, DIPEA, DCM, 40˚C, 90%; (e) (i) Pd2(dba)3, XPhos, Cs2CO3, dioxane, 105 ˚C, 80% (ii) chromatography; (iii) Phosphonics SPM-32 thiolated silica; (iv) EtOAc, 65˚C, 79%.

RESULTS AND DISCUSSION Initial efforts to improve the efficiency of the C-N coupling reaction showed that dropping the catalyst and ligand loading to 0.5% and 2% respectively resulted in high levels of conversion (83%) after 17 h at 135 ˚C with vigorous stirring in an autoclave5. However, attempts to replace the proprietary ligand XPhos and dioxane proved unsuccessful so attention was shifted to a new synthetic strategy that would bypass the need for a palladium mediated C-N coupling reaction by installing the benzimidazole fragment through a metal-free nucleophilic aromatic substitution reaction (SNAr). Towards this end, initial experiments were undertaken to investigate the feasibility of a late stage displacement of the 2chloro substituted purine cores 7 and 8 with ethylbenzimidazole 9a in the presence of an acid or base promoter (Scheme 2). Unfortunately, under all conditions in which this transformation was attempted,


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none of the desired product was formed and the major compound observed resulted from benzimidazole displacement of the azetidinol.

Scheme 2. Attempts at a late stage SNAr reaction to install the benzimidazole fragment

By contrast, the SNAr displacement of 2-chloropurine 4 with ethylbenzimidazole 9a was found to proceed in good yield with no by-product formation (Table 1). The reactivity of this system proved to be highly temperature dependent, as no reaction was observed at ≤ 90 ˚C and conversion remained below 20% up to 120 ˚C. However, at ≥ 120 ˚C product formation was seen with several bases in polar aprotic solvents such as DMF and NMP. Cesium carbonate (entry 5, 69% yield) and tribasic potassium phosphate (entry 6, 65 % yield) both gave good levels of conversion after 24 h at 120 ˚C. Further optimization with tribasic potassium phosphate demonstrated that increasing the temperature to 160 ˚C allowed the reaction to reach full conversion in only 6 h. Pleasingly, the reaction profile was very clean and no significant amounts of degradation products were observed when the reaction was held at elevated temperatures over extended periods of time.


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Table 1. Optimization of base and temperature for benzimidazole additiona

a

entry

baseb

time (h)

temp (˚C)

11 (%)c

1

NaOtBu

24

120

0

2

KOtBu

24

120

0

3

NaOAc

24

120

5

4

K2CO3

24

120

3

5

Cs2CO3

24

120

69

6

K3PO4

24

120

65

7

K3PO4

6

120

51

8

K3PO4

6

150

87

9

K3PO4

6

160

98 b

Reaction performed with 100 mg of 4, 75 mg of 9a (1.3 equiv) and 0.5 mL NMP (5 volumes). 3.0 equivalents. c Determined by LC analysis.

Having replaced the late-stage palladium mediated C-N coupling with an early stage SNAr reaction, conditions for the halogenation and etherification steps were evaluated for compatibility with the newly installed benzimidazole fragment. The previously developed electrophilic bromination conditions utilizing N-bromosuccinimide (NBS) and acetic acid proved to be incompatible with the benzimidazole containing substrate as significant amounts of di-brominated product 13 was formed in addition to the desired mono-bromo compound 12. A wide variety of electrophilic brominating agents and reaction conditions were investigated to see if the mono-brominated product could be preferentially formed, however, in all cases where complete conversion of the starting material occurred a large amount of the dibromominated by-product was also formed (Table 2, entries 1-4). Electrophilic sources of chlorine and iodine were also examined but they proved to either have low reactivity (Table 2, entries 6-8) or be susceptible to over halogenation (Table 2, entry 5).


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Table 2. Investigations into electrophilic halogenationa

a

entry

reagentb

solventc

11 (%)d

12 (%)d

13 (%)d

1

NBS

MeCN

25

52

23

2

NBS

THF

79

21

0

3

Br2

MeCN

88

12

0

4

DBHe

MeCN

0

19

81

5

NCS

MeCN

19

64

17

6

NCS

THF

58

42

0

7

NIS

MeCN

100

0

0

8

I2

MeCN

100

0

0

Reaction performed on 100 mg scale (11) at 40 ˚C. b 1.05 equivalents. c 1.0 mL. d Determined by HPLC analysis. e Dibromohydantoin (DBH).

As a result of the susceptibility of the benzimidazole-substituted purine 11 to over halogenation upon exposure to electrophilic halogenation reagents, we decided to explore a stepwise halogenation strategy wherein the formation of an organometallic intermediate is followed by quenching to install the halogen substituent at the desired position of the purine ring. The benefit of this approach is that it offered the potential to limit over halogenation through control of base stoichiometry and judicious choice of halogenating agent. Initial deprotonation experiments with nbutyllithium (nBuLi) demonstrated that while the organolithium species could be successfully formed at low temperature6, this species was relatively unstable and gave primarily starting material when quenched with electrophiles such as DMF (Table 3, entry 1). Additional metal-lithium bases such as lithium hexamethyldisilazide (LHMDS) and lithium diisopropylamide (LDA) were also examined (Table 3, entries 2-3) but were found to give only low levels of conversion to the desired product. Having determined that aryl-lithium species were unsuitable for the desired halogenation, we considered using a magnesiate species in the hopes that it would both impart a greater degree of stability and allow us to perform the deprotonation under non-cryogenic ACS Paragon Plus Environment

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conditions. We were pleased to see that the organomagnesiate could indeed be formed through a combination of nBuLi and iPrMgCl and did successfully provide the desired brominated product in 42% yield (Table 3, entry 4). However, additional optimization of reaction temperature, base stoichiometry and halogen source failed to increase conversion and in some cases led to complete loss of reactivity (Table 3, entry 5). Recent reports in the literature7 on the effectiveness of the reagent tetramethylpiperidine (TMP) magnesium chloride lithium chloride (TMPMgCl•LiCl) led us to examine magnesiate generation with this novel base. However, though this reagent conferred the benefit of allowing magnesiate formation to occur at room temperature, more equivalents of base were required to achieve complete deprotonation and gave none of the desired product upon quenching with NBS and instead returned exclusively starting material (Table 3, entry 6). Working under the hypothesis that the excess magnesium in the reaction mixture could be causing in-situ dehalogenation of the product, we attempted to perform this reaction in a one-pot fashion by adding TMPMgCl•LiCl to a solution containing the purine starting material 11 and the brominating reagent dibromotetrachloroethane at room temperature (Table 3, entry 7). Though this procedure proved more efficient than the two-step protocol of full deprotonation followed by quenching, the highest yield obtained was only 20%.

Table 3. Halogenation via organometallationa

baseb

entry

a e

“X+”c

temp (˚C)

12

1

nBuLi

DMF

–79

0

2

LHMDS

NBS

0

19

3

LDAe

Br2Cl4Et

–20

20

4

nBuLi/iPrMgCl

NBS

–15

42

5

nBuLi/iPrMgCl

Br2

–15

0

6

TMPMgCl•LiCle

NBS

rt

0

7

TMPMgCl•LiCl f

Br2Cl4Et

rt

b

20 c

Reaction performed on 1.0 g scale (11) in 2-MeTHF, R = 2-ethylbenzimidazole. 1.1 equivalents. 1.2 -1.5 equivalents. d Determined by HPLC analysis. 1.7 equivalents were required for full deprotonation. f The base was added to a combined solution of 11 and dibromotetrachloroethane.


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Due to the incomplete levels of halogenation seen with both the lithium and magnesiate intermediates, we next turned our attention to the possibility of using a zincate intermediate in the hopes that it would be both more stable once formed and more reactive when exposed to electrophilic halogenating reagents. Initial experiments utilizing a combination nBuLi and zinc chloride (ZnCl2) proved to be very promising, giving 60% conversion to the desired product when quenched with an NBS solution (Table 4, entry 1) and further improvement was observed when the milder base LDA was used (Table 4, entry 2, 82% yield). In both of these reactions, it was found that the addition of 1.2 equivalents of tetramethylethylendiamine (TMEDA) was required to keep the aryl zincate intermediate fully solubilized throughout the course of the reaction. Transmetalation of the aryl magnesium species formed by treatment with TMPMgCl•LiCl with ZnCl2 gave even higher levels of conversion to desired bromide (Table 4, entry 3, 84% yield) and imparted the additional benefit of allowing the reaction to be run at room temperature. As the conversion with NBS was not quantitative, a number of other halogenating agents were examined for their efficiency in converting the zincate intermediate to the desired bromide. While both the chlorinated and iodinated purines were accessible using the TMPMgCl•LiCl/ZnCl2 combination with the appropriate succinimide halogenating reagent, (Table 4, entries 4-5, 20-84% yield), the greatest conversion to desired product was obtained by quenching the organozincate with a solution of bromine (Table 4, entry 6, 95% yield).


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Table 4. Halogenation through a zincate intermediate a

entry

a

baseb

“X+”c

temp (˚C)

12

NBS

–40

60

1

nBuLi/ZnCl2

2

LDA/ZnCl2

NBS

–40

82

3

TMPMgCl•LiCl/ZnCl2e

NBS

rt

84

4


NCS

rt

20

5


NIS

rt

84

6


Br2

rt

95

7

TMPZnCl•LiCl

NBS

rt

66

8

TMPZnCl•LiCl

Br2

rt

84

9

TMPZnCl•LiCl

I2

rt

98

10

TMPZnCl•LiCl

I2

rt

50f

Reaction performed on 1.0 g scale (11) in 2-MeTHF, R = 2-ethylbenzimidazole. b 1.1 equivalents. c 1.2 -1.5 equivalents. d Determined by LC analysis. e 1.7 equivalents were required for full deprotonation. f The zincate was quenched directly with an iodine solution at 0 ˚C.

While forming the zincate intermediate through a combination of TMPMgCl•LiCl and ZnCl2 proved to be an efficient method for installing the desired halogen functionality on the purine ring, the need for stepwise addition of multiple reagents and tight control of the internal temperature over a narrow range was less than desirable.

In order to simplify this operation, it was decided to test a one-step

deprotonation and formation of the zincate with the novel base TMPZnCl•LiCl8,9. Experiments revealed that direct used of this base with an NBS quench did indeed provide the desired product, albeit with lower yields than what was obtained with the successive TMPMgCl•LiCl/ZnCl2 system (Table 4, entry 7, 66% yield). It was subsequently found that near quantitative conversion could be obtained with this base simply by quenching with a cold solution of iodine in THF (Table 4, entry 10, 98% yield). In addition to the high levels of conversion, formation of the iodinated compound proved to greatly simplify product isolation with the insoluble iodinated purine precipitating directly from the crude reaction mixture. It is interesting to note that a reverse quench operation, whereby the organozincate is transferred into an iodine solution at 0 ˚C, gave much higher conversions than a direct quench of the cooled zincate with an iodine solution, which returned 50% of the reprotonated starting material (Table


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4, entry 11, 50 % yield). In both cases, quantitative deprotonation and zincate formation at the desired position on the purine ring was confirmed by 1HNMR, suggesting that exposure of the aryl iodide product to an excess of basic organometallic species facilitates de-iodination. Similar results in the subsequent etherification step were also observed in that addition of the aryl iodide 16 to a solution of the azetidine alkoxide, formed by treatment of the azetidinol 7 with potassium tert-butoxide, primarily returned the des-iodo purine 11. In contrast, slow addition of the alkoxide to a solution of aryl iodide gave quantitative conversion to the desired aryl ether 17. In accordance with the mechanism proposed electron-transfer mechanism for this type of reactivity10, this trend was not observed with the corresponding aryl bromide analog, which was stable in both the halogenation and etherification steps regardless of the method of quenching.

Scheme 3. Effect of addition order on etherification with azetidine alkoxide

Having established conditions for the metal free SNAr installation of the benzimidazole, halogenation and displacement with the azetidinol, we next embarked on a scale-up campaign of the lead PI3Kδ inhibitor GNE-293(3). The K3PO4 mediated installation of isopropylbenzimidazole 9b onto the purine core 4 furnished 116 g of the desired product 15 with >99% area purity in 78% yield following crystallization from isopropanol. Deprotonation and formation of the zincate intermediate with TMPZnCl•LiCl in THF was performed at ambient temperature in the presence of TMEDA with quantitative zincation after 30 min being confirmed by 1HNMR. Upon reverse quenching this species via slow addition into a THF solution of I2, the aryl iodide product 16 was obtained by direct ACS Paragon Plus Environment

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crystallization to give 140g (86% yield) of material with 98.5% LC area purity (LCAP). The azetidinol fragment was installed by exposure of the aryl iodide in DMF at –25 ˚C to the potassium alkoxide formed from the treatment of Boc-protected azetidinol 6 with KOtBu. Direct crystallization followed by washing with water and heptane gave the desired arylether product 17 with >99% area purity in 83% yield. Removal of the Boc protecting group using ethanol HCl allowed telescoping directly to reductive amination with diooxothianone 18. Sodium triacetoxyborohydride reduction provided 126.2 g of the desired compound 3 with >99% LCAP in 76% yield following IPA recrystallization. Overall, this first generation development route provided the desired compound in 41% overall yield in five steps with a total of five isolations and provided sufficient quantities of material to support second species toxicology studies. Additionally, it completely eliminated palladium and its accompanying requirements for column chromatography and resin treatment. Most importantly of all, however, is that this chemistry proved to be robust on batch scale, thereby providing a reliable API supply for development needs.

Scheme 4. Revised synthetic route to 3a

a

Reagents and conditions: (a) (i) K3PO4, NMP, 160 ˚C; (ii) IPA, 83 ˚C, 78%; (b) TMEDA, THF, TMPZn•LiCl, 25 ˚C then I2, 0 ˚C to 20 ˚C, 86%; (c) KOtBu, DMF, –25 ˚C to 20 ˚C;, 83% (d) (i) 2.5N HCl in EtOH, THF, 63 ˚C; (ii) NaBH(OAc)3, DIPEA, DCM, 25 ˚C, 76%;(e) IPA, 83 ˚C, 98%;


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CONCLUSION We described the process development and scale-up route used to produce the PI3Kδ inhibitor GNE293. Replacement of a Pd catalyzed C-N coupling with a metal free SNAr reaction simplified the synthesis by reducing the number of operations required for purification and eliminating column chromatography.

A novel method of iodination proceeding through a zincate intermediate was

developed to access the key aryl halide intermediate 16 and highlights the utility of the recently developed reagent TMPZnCl•LiCl as a mild base that can be used to generate highly stable organometallic intermediates that can undergo further functionalization.

EXPERIMENTAL SECTION General: Tetramethylpiperidinylzinc chloride lithium chloride complex (TMPZnCl•LiCl) was purchased from Chemetall Foote Corp. and used directly. HPLC Methods: HPLC method for monitoring the formation of 11, 15, 16, 17 and 3. Onyx Monolithic SL C18, 2 mm x 50 mm, gradient elution from 95:5 to 65:35 0.05% TFA in water/ 0.05% TFA in MeCN over 4.0 min, 0.75 mL/min flow at 35 ˚C with detection at 220 nm, 254 nm and 280 nm. HPLC retention times: 11 = 2.277 min, 15 = 2.322 min, 16 = 1.872 min, 17 = 2.161 min, 3 = 1.416 min.

4-[2-(2ethylbenzimidazol-1-yl)-9-methyl-purin-6-yl]morpholine, 11. 4-(2-chloro-9-methyl-purin-6yl)morpholine 4 (50.70 g, 199.9 mmol) and 2-ethylbenzimidazole 9a (35.06 g, 239.8 mmol) were slurried in N-methylpyridinone (253.5 mL) at ambient temperature. Tribasic potassium phosphate (134.0 g, 599.6 mmol) was added and the mixture was heated to 150 ˚C. After 17 h the reaction was complete by HPLC when the LC Area Percent (LCAP) of 4 at 254 nm was ≤ 1%. The solution was cooled to 80 ˚C then added water (1.014 L) over 60 min. As the addition took place the temperature was allowed to cooled to 60 ˚C. Once all the water had been added, the reaction was allowed to come to ambient temperature over 15 h and the product crystallized out of solution. The resulting suspension was stirred at 22 ˚C for 4 h then the solid was isolated by vacuum filtration. The cake was washed with water (250 ACS Paragon Plus Environment

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mL) and methyl-tbutylether (300 mL) and dried in a 50 ˚C vacuum oven for 15 h to afford the product 11 as a white solid (75.4 g, 104% yield, 100% LCAP11). 1H NMR (400 MHz, DMSO-d6) 8.26 (1H, s), 8.04 (1H, m), 7.64 (1H, m), 7.26 (2H, m), 4.28 (4H, bs), 3.81 (3H, s), 3.78 (4H, m), 3.27 (2H, q, J = 3 Hz), 1.34 (3H, t, J = 3 Hz). 13C NMR (100 MHz, DMSO-d6) 156.3, 153.4, 151.8, 149.7, 142.1, 141.5, 134.2, 122.8, 122.4, 118.6, 117.3, 113.3, 66.1, 29.6, 23.1, 12.1. HRMS (ESI+) calculated for C19H22N7O ([M+H]+), 364.1880, found 364.1865.

4-[2-(2-isopropylbenzimidazol-1-yl)-9-methyl-purin-6-yl]morpholine, 15. 4-(2-chloro-9-methylpurin-6-yl)morpholine 4 (100 g, 394 mmol) and 2-isopropylbenzimidazole 9b (69.5 g, 434 mmol) were slurried in N-methylpyridinone (500 mL) at ambient temperature. Tribasic potassium phosphate (160 g, 753 mmol) was added and the mixture was heated to 160 ˚C internal temperature for 8 h then cooled and held at 135 ˚C. The reaction was determined to be complete by HPLC after 15 h when the LCAP of 4 at 254 nm was ≤ 1%. The solution was cooled to 80 ˚C and the mixture was quenched by addition of a solution of monobasic sodium phosphate (43 g) in water (1.0 L). The mixture was further cooled to 60 ˚C, seeded and held at 60 ˚C for 1 h then cooled further to 25 ˚C. An additional 600 mL of water was added and the slurry was stirred at 25 ˚C for 18 h to ensure complete crystallization. The solid was isolated by vacuum filtration and the cake was washed with water (2x500 mL) and IPA (100 mL). The crude material was taken up in IPA (582 mL), heated to 83 ˚C internal temperature for 30 min and the cloudy solution was filtered while hot to remove residual inorganics. The filter was rinsed with 50 mL of hot IPA and the combined filtrates were concentrated to a total volume of 350 mL, cooled to 70 ˚C, seeded and allowed to cool to room temperature. The resulting slurry was stirred at ambient temperature for 1 h and the solids were isolated by vacuum filtration, rinsed with 50 mL of IPA and dried in 60 ˚C vacuum oven for 15 h to afford the product 15 as a white solid (116.4 g, 78% yield). 1H NMR (400 MHz, DMSO-d6) 8.28 (1H, s), 7.91 (1H, m), 7.65 (1H, m), 7.25 (2H, m), 4.28 (4H, bs), 3.92 (1H, m), 3.80 (3H, s), 3.77 (4H, m), 1.36 (6H, d, J = 6.7 Hz).

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C NMR (100 MHz, DMSO-d6) 159.8, 153.5,


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151.8, 149.8, 142.0, 141.6, 143.2, 122.7, 122.4, 118.6, 117.4, 113.0, 66.1, 29.6, 27.3, 21.8. HRMS (ESI+) calculated for C20H24N7O ([M+H]+), 378.2037, found 378.2037.

4-[8-iodo-2-(2-isopropylbenzimidazol-1-yl)-9-methyl-purin-6-yl]morpholine, 16. To a mixture of 4-[2-(2-isopropylbenzimidazol-1-yl)-9-methyl-purin-6-yl]morpholine 15 (122 g, 323 mmol), and tetramethyletheylenediamine (73.4 mL, 485 mmol) in tetrahydrofuran (610 mL) was added a solution of TMPZnCl•LiCl via addition funnel while maintaining the internal temperature below 29 ˚C. After stirring at ambient temperature for 30 min, complete deprotonation was confirmed by quenching an aliquot into D2O and analyzing by 1H NMR. The zincate solution was transferred via cannula to a solution of iodine (98.5 g, 388 mmol) in tetrahydrofuran (366 mL) at 0 ˚C while maintaining the temperature of the iodine solution below 10 ˚C. Once the transfer was complete, the reaction was let come to ambient temperature and the product began to crystallize out of solution. The slurry was stirred at ambient temperature for 1 h to ensure complete product crystallization and the solids were isolated by vacuum filtration. The cake was washed with tetrahydrofuran (120mL), saturated aqueous ammonium chloride solution (610 mL), saturated sodium thiosulfate solution (610 mL) and methyl-tbutylether (500 mL). The material was dried at 50 ˚C in a vacuum oven for 15 h to afford the desired product 16 as a white solid (139.7 g, 86% yield). 1H NMR (400 MHz, DMSO-d6) 7.91 (1H, m), 7.64 (1H, m), 7.24 (2H, m), 4.22 (4H, bs), 3.93 (1H, m), 3.77 (4H, m), 3.70 (3H, s), 1.35 (6H d, J = 8 Hz). 13C NMR (100 MHz, DMSO-d6) 159.8, 153.1, 152.8, 152.2, 149.7, 142.1, 135.1, 134.2, 123.7, 122.7, 122.4, 120.0, 118.7, 113.0, 104.13, 66.0, 32.2, 27.4, 21.8. HRMS (ESI+) calculated for C20H23IN7O ([M+H]+), 504.1003, found 504.0988.

Tert-butyl 3[2-(2-isopropylbenzimidazol-1-yl)-9-methyl-6-morpholino-purin-8-yl]oxyazetidine-1carboxylate, 17. To a solution of tert-butyl 3-hydroxyazetidine-1-carboxylate 6 (22.1 g, 127 mmol) in dimethylformamide (148 mL) was added potassium tertbutoxide (22.7 g, 196 mmol), causing an exotherm as the solution reached 37 ˚C. Once the solution had returned to ambient temperature, it was ACS Paragon Plus Environment

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slowly added to a slurry of 4-[8-iodo-2-(2-isopropylbenzimidazol-1-yl)-9-methyl-purin-6-yl]morpholine 16 (49.3 g, 98.0 mmol) in dimethylformamide (197 mL) at –25 ˚C while maintaining the internal temperature below –20 ˚C. Once the addition was complete, the slurry was let come to room temperature over 4 h and stirred at ambient temperature for an additional 1 h, at which time the reaction was determined to be complete by HPLC when the LCAP of 16 at 254 nm was ≤ 1%. Water (493 mL) was added while maintaining the temperature below 25 ˚C and the product began to crystallize out of solution. Stirred at ambient temperature for 12 h to ensure complete crystallization and the solids were isolated by vacuum filtration. The cake was washed with water (250 mL) and heptane (250 mL) and dried at 50 ˚C in a vacuum oven for 15 h to afford the desired product 17 as a white solid (44.7 g, 83% yield). 1H NMR (400 MHz, DMSO-d6) 7.84 (1H, m), 7.64 (1H, m), 7.23 (2H, m), 5.47 (1H, m), 4.33 (2H, m), 4.13 (4H, s), 4.00 (2H, m), 3.87 (1H, m), 3.75 (4H, m), 3.57 (3H, s), 1.40 (9H, s), 1.34 (6H, d, J = 6.6 Hz).

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C NMR (100 MHz, DMSO-d6) 159.1, 155.4, 152.7, 151.6, 141.9, 134.4, 122.3, 118.6,

113.5, 112.7, 98.0, 79.0, 68.4, 66.1, 45.4, 28.0, 27.4, 27.3, 21.8. HRMS (ESI+) calculated for C28H37N8O4 ([M+H]+), 549.2932, found 549.2918.

4-[3[2-(2-isopropylbenzimidazol-1-yl)-9-methyl-6-morpholino-purin-8-yl]oxyazetidin-1-yl]thiane 1,1-dioxide, 3. To a solution of tert-butyl 3[2-(2-isopropylbenzimidazol-1-yl)-9-methyl-6-morpholinopurin-8-yl]oxyazetidine-1-carboxylate 17 (66.3 g, 121 mmol) in tetrahydrofuran (663 mL) was added 2.5N hydrochloric acid in ethanol (150 mL, 363 mmol) and the solution was heated to reflux for 24 h at which point it was judged complete by HPLC when the LCAP of 17 at 254 nm was ≤ 5%. The solution was concentrated under vacuum to an oil then added dichloromethane (926 mL). The solution was cooled to 10 ˚C and diisopropylethylamine (134 mL, 764 mmol) was added portionwise to maintain the internal temperature below 20 ˚C. Next, 1,1-dioxothian-4-one 18 (62.0 g, 418 mmol) was added followed by sodium triacetoxyborohydride (91.4 g, 431 mmol) and the resulting solution was stirred at ambient temperature for 16 h at which point it was judged complete by HPLC when the LCAP of 3 at 254 nm was ≥ 84%. Added saturated sodium bicarbonate solution (463 mL) and the solution was stirred ACS Paragon Plus Environment

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for 30 min to ensure complete quenching of the excess borohydride. The layers were split and the aqueous phase was extracted with dichloromethane (250 mL) then the organic layers were combined and dried over sodium sulfate. The dichloromethane was removed via rotary evaporation then added THF (250 mL) and heated to 40 ˚C to distill off an additional 200 mL of dichloromethane. Another 250 mL of tetrahydrofuran was added and the mixture was heated to 65 ˚C to 1 h then let come to room temperature. The resulting slurry was stirred at ambient temperature for 15 h then the solids were isolated by vacuum filtration. The cake was washed with THF (100 mL) and dried at 50 ˚C in a vacuum oven for 15 h to afford 86.0 g (78% yield) of crude 3 as a pale tan solid. The solid obtained was combined with an additional 40.2 g of crude material from a previous lot, taken up in IPA (631 mL), heated to 83 ˚C internal temperature and the slurry was held at temperature for 1.5 h. The slurry was allowed to slowly come to room temperature and held with stirring for 16 h after which the solids were isolated by vacuum filtration and rinsed with 50 mL of IPA. Drying in a 50 ˚C vacuum oven for 24 h afforded the desired product 3 as a white solid (123.84 g, 98% yield). 1H NMR (400 MHz, DMSO-d6) 7.84 (1H, m), 7.64 (1H, m), 7.23 (2H, m), 5.31 (1H, m), 4.13 (4H, s), 3.87 (1H, m), 3.80 (2H, m), 3.75 (4H, m), 3.55 (3H, s), 3.20 (2H, m), 3.12 (2H, m), 2.97 (2H, m), 1.96 (2H, m), 1.85 (2H, m), 1.34 (6H, d, J = 6.8 Hz). 13C NMR (100 MHz, DMSO-d6) 159.1, 152.7, 151.7, 151.4, 148.29, 141.9, 134.2, 122.6, 122.3, 118.6, 113.4, 112.7, 67.8, 66.1, 59.5, 58.4, 47.2, 40.2, 27.3, 27.2, 26.2, 21.8. HRMS (ESI+) calculated for C28H37N8O4S ([M+H]+), 581.2653, found 581.2639. ACKNOWLEDGMENT. The authors wish to acknowledge Dr. Christine Gu, Tina Nguyen and Dr. Ila Patel for analytical support.


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REFERENCES

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Isaacs, J. D. Nat. Rev. Immunol. 2010, 10, 605-611.

2

Majithia, V.; Geraci, S. A. The American Journal of Medicine 2007, 120, 936-939.

3

(a) Cantley, L. C. Science 2002, 296, 1655-1657. (b) Rommel, C.; Camps M.; Ji H. Nat. Rev. Immunol. 2007, 7, 191-201. (c) Clayton, E.; Bardi, G.; Bell, S. E.; Chantry, D.; Downes, C. P.; Gray, A.; Humphries, L. A.; Rawlings, D.; Reynolds, H.; Vigorito, E.; Turner, M. J. Exp. Med. 2002 196, 753763. (d) Jou, S.; Carpino, N.; Takahashi, Y.; Piekorz, R.; Chao, J.; Carpino, N.; Wang, D.; Ihle, J. N. Mol. Cell. Biol. 2002, 22, 8580-8591. (e) Okkenhaug, K.; Bilancio, A.; Farjot, G.; Priddle, H.; Sancho, S.; Peskett, E.; Pearce, W.; Meek, S. E.; Salpekar, A.; Waterfield, M. D.; Smith, A. J. H.; Vanhaesebroeck, B. Science 2002, 297, 1031-1034. (f) Edwards, C. W. J.; Cambridge, G. Nat. Rev. Immunol. 2006, 6, 394-403. (g) Monach, P. A.; Benoist, C.; Mathis, D. Adv. Immunol. 2004, 82, 217248. (h) Vanhaesebroeck, B.; Ali, K.; Bilancio, A.; Geering, B.; Foukas, L. C. Trends in Biochem. Sci. 2005, 30, 194-204. 4

(a) Jun, L.; Safina, B.; Sutherlin, D.P.; Sweeney, Z. (Genentech, USA). Purine Compound Selective for PI3K P110 Delta, and Methods of Use. US 20120015931 A1, January 19, 2012. (b) Sutherlin, D. P. et al Bioorg. & Med. Chem. Lett. 2012, 22, 4296. (b) Safina, B. S. et al J. Med. Chem. 2012, 55, 5887. (c) Murray, J. M. et al J. Med. Chem. 2012, 55, 7686. 5

During the course of the reaction the internal pressure reached 1.6 bar.

6

The successful formation of the aryl-metal species was monitored and confirmed by quenching an aliquot from the reaction mixture into D2O and analyzed for disappearance of the corresponding aryl proton by 1HNMR. 7

For a review on this reagent see: (a) Haag, B.; Mosrin, M.; Ila, H.; Malakhov, V.; Knochel, P. Angew. Chem. Int. Ed. 2011, 50, 9794. (b) Ila, H.; Baron, O.; Wagner, A. J.; Knochel, P Chem, Lett. 2006 35, 2. (c) Knochel, P.; Dohle, W.; Gommerman, N.; Kneisel, F. F.; Kopp, F.; Korn, T.; Sapountzis, I.; Vu, V.A. Angew. Chem. Int. Ed. 2003, 42, 4302. For direct metalation of pyrimidines with TMPMgCl•LiCl see (d) Mosrin, M.; Knochel, P. Chem Euro. J. 2009, 15, 1468. 8

For the functionalization of purines with TMPZnCl•LiCl see: Zimdars, S.; Mollat du Jourdin, X.; Crestey, F.; Carrell, T.; Knochel, P. Org. Lett. 2011, 13, 792. 9

The reagent TMPZnCl•LiCl is commercially available from Chemetall Foote Corp. on scales ranging from 1 L to 100 L. 10

In both cases, dehalogenation is presumed to occur via a radical-chain, electron-transfer process. For a detailed mechanistic review of this process with alkoxides see: (a) Bunner, J.F. Acc. Chem. Res. 1992, 25, 2. For a discussion of this process catalyzed by metals (including lithium and zinc) see: (b) Alonso, F.; Beletskaya, I. P.; Yus, M. Chem. Rev. 2002, 102, 4009. 11

The yield is >100% due to the presence of inorganic impurities that are removed by the subsequent IPA crystallization. These inorganic impurities are indirectly detected by KF with the contaminated material containing >1000 ppm water.


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Development of a Scalable Strategy for the Synthesis of PI3KÎ´

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