Application Note Part 2

Automated synthesis
planning and execution
With SYNTHIA™ Retrosynthesis Softwareand Synple Automated Synthesizer

The Life Sciencebusiness of Merck
operates as
MilliporeSigma in theU.S. and Canada.

Application Note

Automated synthesis planningand execution with SYNTHIA™
retrosynthesis software andSynple Automated Synthesizer
Part 2: Analysis of time-savings using automatedtechnologies compared with traditional methods
Introduction

Synthesis of C6

Enabling technologies such as SYNTHIA™ retrosynthesis
software for route planning and Synple’s automatedcartridge-based synthesis technology can be used tostreamline the drug discovery process by accelerating
synthesis design and execution.

The first compound selected (C6), a fluoroprolinebased compound, was part of an earlier collaborationof Synple. In the resulting publication1, it was shownthat the compound can be made rapidly with the Synple
instrument in 3 steps. When submitted to SYNTHIA™
using the preset Discovery search configuration, thesoftware was able to suggest a 2-step route consistingof an alkylation followed by an amide coupling. Using
the custom search configuration that was developed topromote Synple-automatable reactions, SYNTHIA™ wasable to propose an alternative 2-step route starting witha reductive amination instead of the alkylation reactionthat is not available using the Synple platform.

In Part 1, we introduced how the two platforms can beused together through a custom set of search parameters
in SYNTHIA™ that allows it to plan routes that can beexecuted using Synple’s automated synthesizer. Herein,
we will present a detailed comparison of the synthesesof two compounds using automated technology or
traditional methods to highlight the time-savings enabledby the use of SYNTHIA™ and Synple together.

Figure 1. Compounds synthesized manually and using Synple.

Scheme 1. Synthesis routes of C6 calculated by SYNTHIA executed
manually and with Synple automation

The first route based on the Discovery configuration wasexecuted via typical synthesis in round-bottom flasks.
Alkylation of the amine proved challenging under basic
conditions due to the facile elimination of the halide. A
reaction screening was performed in order to identify
the appropriate base. It was discovered that the optimal
conditions were potassium carbonate with 5 equivalentsof the halide. For the second step, a short literaturesearch was performed to find the best coupling reagent
for these substrates. Using HATU yielded the desired
compound after a short purification step.
The second route based on the Synple search
configuration was carried out exclusively on the Synple
platform. Execution of the first reaction required first
filling the solvent reservoirs, setting up the reaction on
the automated synthesizer and then evaporating thesolvent from the final product. All together this required
only a few minutes of manual work. The second step
was set up in the same manner, and the resulting target
compound did not require any additional purification.
Both the routes proposed by SYNTHIA™ reduced the total
step count to get to the target over the original route.
The route proposed using the Discovery configurationof SYNTHIA™ required 7.25 hours of manual labor to
execute using traditional batch synthesis in a roundbottom flask (Table 1). This did not include waitingfor delivery of the starting materials or reaction time.
The same compound was obtained via the Synple
configuration route with only 30 minutes of manual laborand a similar yield as the manually executed route. Using
the SYNTHIA™-Synple combination saved 6.75 hours ofmanual labor, which a chemist could instead spend onother high-value activities.
Table 1. Manual time to execute SYNTHIA™-planned
syntheses of compound C6 using either Traditional or
Synple-enabled workflow. Time is given in ‘hands-on’
working time, not including reaction time
Traditional

SynpleAlkylation

Reductive
Amination

Literature Search

0.75 h0h

Reaction Optimization

4.5 h0h

Reaction Set-up

0.25 h

0.25 hWork-up

0.5 h0h

Purification

0h0h

Amide
Coupling

Amide
Coupling

Literature Search

0.5 h0h

Reaction set-up

0.5 h0.25 hWork-up

0.5 h0h

Purification

1.0 h0h7.25 h
(49% yield)

0.5 h(46% yield)

Step 1

Step 2

Total Working TimeIn addition to the laboratory execution, there are othertime-consuming tasks that are important to consider
when analyzing the time savings gained by using tools
such as SYNTHIA™ and Synple to accelerate research.
These include things like literature searches forreaction conditions and vendor searches for commercialcompounds. To consider these non-lab tasks, we analyzed
the total working time required to access C6 via threedifferent approaches: a) Traditional synthesis workflow
using neither SYNTHIA™ nor Synple, b) Route planning
using SYNTHIA™ but traditional lab execution, and c)
Combination of route planning using SYNTHIA™ andautomated execution with Synple. This is summarizedin Table 2.
To determine the planning time required to design thesynthesis, two chemists independently planned routes tothe target molecule – one using only traditional literaturesearching tools and the other using SYNTHIA™ in addition
to traditional tools. The first approach via traditional
literature search alone required about 2 hours of thechemist’s time, followed by an additional 1 hour to identify
and order starting materials and related chemicals fromvarious vendors. The second approach, which included
using SYNTHIA™ to plan the routes, shortened the time
for the literature search to 1.25 hours. Since the overall
route had already been proposed by the software,
only searches for reaction conditions were necessary.
SYNTHIA™ allows for commercially available starting
materials to be viewed directly within the platform. As aresult, the time to select and procure starting materials
was significantly reduced by 45 minutes. In the third
approach using SYNTHIA™ and Synple, the overall
time was further reduced since the reaction conditions
were already programmed in Synple and no additional
research by the chemist was necessary. Considering allof the required labor together, SYNTHIA™ was able toreduce the manual time to get to the purified compoundby 24%, while the combination of SYNTHIA™ and theSynple platform was able to reduce the manual time by93% over a traditional workflow.
Table 2. Overall time required to access C6LiteraturesearchMaterial
preparation/Ordering

Synthesis

Total

Full
traditional
workflow

2h1h

8.5 h*

11.5 hSYNTHIA™+
traditionalsynthesis

1.25 h

0.25 h

7.25 h8.75 hSYNTHIA™ +Synple0h-24%
0.25 h*Estimated based on 3-step published path1

0.5 h0.75 h-93%

Synthesis of C2
A second target C2 was selected for synthesis
representing a more complex scaffold. This compound
was patented as an antithrombotic agent and wassynthesized in 6 steps2.
The target was run on SYNTHIA™ using both theDiscovery configuration and the Synple configuration.
The routes proposed using the Discovery configuration
employed several complex steps, while the Synple
configuration proposed a straightforward 4-step route.
Because of this, the Synple route was chosen to be
executed using both traditional and automated synthesis
workflows (Scheme 2).

Table 3. Hands-on time to execute SYNTHIA™-planned
syntheses of compound C2 using either Traditional or
Synple-enabled workflow. Time is given in ‘hands-on’working time, not including reaction time
TraditionalSynple

Step 1: Reduction
Literature Search

0.5 h

Reaction Set-up

0.75 hWork-up

0.5 h

Purification

0h

Step notavailableon Synple

Step 2: Amide Coupling
Literature Search

0.25 h0h

Reaction Set-up

0.75 h

0.25 hWork-up

0.5 h0h

Purification

0h0hStep 3: Boc Deprotection

Scheme 2. Synthetic route for C2 as proposed by SYNTHIA™’s Synple
configuration. Conditions for traditional execution: a. LiAlH4, THF, 5h,60°C, 71%; b. HATU, NMM, DMF, 18h, 65°C, 61%; c. HCl, MeOH, rt,2h, quant; d. TsCl, NMM, MeCN, 5h, rt, 74%. Conditions for automatedexecution with Synple: b. Amide coupling capsule, MeCN, rt, 16h, 57%;
c. Boc-cleavage capsule, DME, rt, 2 h, quant.; d. Amide coupling capsule,
MeCN, rt, 16h, 36%.

Because the carbamate reduction step (Scheme 2, a)
was not available on Synple, this step had to be executed
manually and resulted in a 71% yield. The subsequentamide coupling and Boc deprotection steps gave similaryields for both traditional and automated methods. The
last step was an amide coupling which generated theproduct in a 74% yield when executed manually. For the
automated execution, the intermediate amine had to be
pre-treated to remove the salt before running the Synple
method. This required additional time and contributed to
the lower 36% yield for this step. This is not completely
unsurprising considering the Synple cartridge system isdesigned to provide quick access to compounds with awide substrate scope, but is not necessarily optimized todeliver the highest yield for a particular substrate.
The route proposed by SYNTHIA™ and executed using
the traditional workflow was able to provide C2 in 32%
overall yield with 8 hours of total working time. Theautomated route took only 5.5 hours of manual working
time, albeit yielding slightly less material. The majorityof the time spent on the synthesis was due to the manual
first step. This demonstrates that introducing Synple
automation can save time even in a partially automated,hybrid workflow.

Literature Search0h0hReaction Set-up

0.25 h

0.25 hWork-up

0.75 h0h

Purification

0h0hLiterature Search0.5 h0h

Reaction Set-up

1.0 h0.75 h*Work-up

0.5 h0h

Purification

1.0 h

1.0 h

8h (32% yield)

5.5h (15% yield)

Step 4: Amide Coupling

Total Working Time*includes pretreatment of amine

The same analysis was performed for C2 that was done
for C6 to consider the total working time for each method
including any additional time required for literaturesearches and material ordering (Table 4). In this case,using SYNTHIA™ was able to cut down 6.5 hours ofwork and Synple another 3.25 hours of work, resulting
in a total 61% reduction in working time using thecombined technologies.
Table 4. Overall time required to access C2
LiteraturesearchMaterial
preparation/Ordering

Synthesis

Total

Full
traditional
workflow

2.5 h

1.5 h

12 h*

16 h

SYNTHIA™+
traditional
synthesis

1.25 h

0.25 h

8.0 h

SYNTHIA™ +Synple0.5 h

0.5 h-40%

0.25 h*Estimated based on 6-step published path2

5.5 h

6.25 h-61%

Summary
In both cases, the use of SYNTHIA™ and Synple was able
to reduce the overall time required to access the target
molecules. In the case of C6, the use of SYNTHIA™
alone created a modest time-savings of 24%, but thecombination of SYNTHIA™and Synple reduced over90% of the time required to obtain the final molecule.
In the case of the more complex target, C2, the useof SYNTHIA™ resulted in a more significant 40% timesavings. Although only 3 of the 4 steps were able tobe automated, using Synple for the synthesis execution
reduced another 21% of the overall working time.
In summary, we were able to show significant timesavings by combining SYNTHIA™ and Synple. This
study has determined that each platform separately
can have varying potential for time optimization. Using
the combination of technologies enhances the synthesis
workflow and allows the chemist to spend time on otherhigh-value activities.

Material List
4-fluoropyrrolidine-2-carboxylic acid

ENAH3045A016(Chloromethyl)cyclopropane

184667

Furfurylamine

F20009Cyclopropanecarboxaldehyde

272213

HATU

4454607-Amino-1,2,4,5-tetrahydro-3-Boc-3benzazepine-3-carboxylic acid

SY3432447954

3-(Boc-amino)tetrahydrofuran-3-carboxylic acid

SY3432448312

5-Bromo-2-thiophenecarboxylic acid467944

Synple 2 Automated Synthesizer

SYNPLE-SC002

Reagent Cartdige – Reductive AminationSYNPLE-R001

Reagent Cartridge – Amide FormationSYNPLE-A011

Reagent Cartridge – Boc deprotectionSYNPLE-B011

References
1. MacMillan, A. E.; Wu, W. W. X.; Nichols, P. L.; Wanner, B.
M.; Bode, J. W. external pageA Vending Machine for Druglike Molecules – Automated Synthesis of Virtual Screening
Hits, Chem. Sci. 2022, 13, 14292-14299
2. Han, Z.; Gerlach, K.; Krishnamurthy, D.; Matthes, B.;
Nar, H.; Priepke, H.; Schuler-Metz, A.; Senanayake, C.
H.; Sieger, P.; Tang, W.; Wienen, W.; Xu, Y.; Yee, N. K.
Synthesis of 3-aminotetrahydrofuran-3-carboxylic acidderivatives for use as medicaments. WO2008080891, 2007.

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